Animal Welfare Information Center

Johne's Disease--Mycobacterium avium subsp. paratuberculosis: A Debilitating Enteric Disease of Ruminants


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USDA and USDA Sponsored Research


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ACCESSION NO: 0203750 SUBFILE: CRIS
PROJ NO: ALV-HABTEMARIAM AGENCY: CSREES AL.V
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 JUN 2005 TERM: 31 MAY 2008 FY: 2006
INVESTIGATOR: Habtemariam, T.; Yehualaeshet, T.
PERFORMING INSTITUTION:
Microbiology
Tuskegee University
Tuskegee , Alabama 36088
COMPARISON AND VALIDATION OF IS900 AND PUTATIVE SEQUENCE (MPTB52.16) AS A DIAGNOSTIC TOOL FOR MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: Prolonged incubation period of Mycobacterium avium subsp. paratuberculosis and individual variability in subclinical and clinical disease expression makes the diagnosis of the disease challenging. The goal of this study is to develop a more rapid and sensitive DNA-based (conventional or real-time PCR-based diagnostic test) to detect M. avium subsp. paratuberculosis in milk. The specificity and susceptibility of real-time PCR based on IS900 and putative sequence as target will be compared.
OBJECTIVES: General: Paratuberculosis (Johne's disease) is a chronic granulomatous enteric disease of mainly ruminants caused by Mycobacterium avium subsp. paratuberculosis. A prolonged incubation period and great individual variability in subclinical and clinical disease expression are characteristic of paratuberculosis. Excretion of the organism may occur for prolonged periods (1 to 2.5 years) before the onset of clinical disease (1,2). Thus, early diagnosis of infected animals is important to avoid spreading of the disease, because control is dependent on detection and culling of infected animals as early as possible. Until recently, only a few M. paratuberculosis-specific genes and antigens/epitopes have been identified, of which IS900 has been mainly documented followed by some putative sequences (3). Several PCR assays based on IS900 have been developed for the detection of M. avium subsp. paratuberculosis (4,5). PCR has been used to improve the identification of microorganisms, especially where traditional microbiological detection methods have serious limitations. The goal of this study is to develop a more rapid and sensitive DNA-based (conventional or real-time PCR-based diagnostic test) to detect M. avium subsp. paratuberculosis in milk. We will compare the specificity and susceptibility of real-time PCR based on IS900 and putative sequence as target. Specific objectives: Objective 1: To optimize the conventional PCR and real-time PCR-based diagnostic test of M. avium subsp. paratuberculosis based on IS900- insertion segment and Putative sequence, Mptb52.16. Cost analysis, including material and labor, indicated an approximately 50% higher cost per test for bacteriological culture than for the conventional and real-time PCR tests. The real-time PCR (RT-PCR) method is relatively simple and robust, and results can be achieved within 24 h. The additional advantage of DNA-based assays is the detection of nonculturable organisms where it is critical to detect all sources of infection. Objective 2: Determine the diagnostic limit of DNA-based diagnostic methods in spiked milk. Milk and fecal samples are the most common specimens for paratuberculosis diagnosis. Milk is considered to be a difficult specimen for the detection of organisms by PCR, due to the presence of large amounts of fat and calcium ions. Detectable quantities of M. avium subsp. paratuberculosis have previously been reported in the milk of both clinically infected and subclinically infected cattle with Johne's disease. Therefore, we selected to spike the milk as the first line of sample to determine the specificity and limitation of RT-PCR as a diagnostic tool. Objective 3: Compare and validate the real-time PCR using IS900- insertion and Putative sequence (Mptb52.16 ). The main goal of this objective is to examine whether any of the fragments have better diagnostic potential to diagnose M. avium subsp. paratuberculosis. Purified DNA will be analyzed by conventional and real-time PCR targeting IS900 and putative sequence to detect M. avium subsp. paratuberculosis in milk.
APPROACH: The DNA-based RT- PCR will target IS900 segment and putative sequence. The details of the sequence (AF503873, Mptb52.16), for the primers and probe synthesis will be accessed from the GenBank. Bacterial strains, which will be used in this investigation, are: Strain *Source or reference Origin M. avium, 15769 ATCC chicken M. paratuberculosis, 19698 ATCC (type strain) bovine M. paratuberculosis 43015 ATCC human M. scrofulaceum 19275 ATCC M. vaccae 15483 ATCC cow's milk *Sources of bacterial strains are from ATCC, American Type Culture Collection (Rockville, MD, USA); The strains of M. avium, scrofulaceum and M. vaccae will be used as a negative control. Bacterial suspension preparation and bacterial growth: After repeated treatment of cells in the Branson ultrasonic cleaner to disintegrate of clumps an approximate measure of cell number will be determined by using the optical density at 550 nm or hematocytometer. Extraction of mycobacterial DNA from culture and spiked milk: Due to the complex, difficult matrix involved, several strategies will be considered for the DNA extraction. To overcome this problem, organisms will be concentrated to a pellet by centrifugation and resuspended in prewarmed in PBS. DNA will be isolated from growing cultures and purified by using freeze thawing, enzymatic degradation, lysis, phenol-chloroform treatment, and isopropanol precipitation. Samples of milk (10 ml) will be spiked with 10 to 106 M. avium subsp. Paratuberculosis organisms, and the organisms will be concentrated by centrifugation and resuspended in 2 ml of PBS. Real-time PCR assay. RT-PCR will be conducted in a Smartcycler. To evaluate the usefulness of the technique we will perform a parallel study of culture detection and Ziehl-Neelsen stain of M. avium subsp. Paratuberculosis. Quantitation: The unknown targets will be evaluated by using DNA samples containing various amounts of known template (range, 101 to 106 copies). Personnel and Institutional capacity The experience of the first PI in molecular diagnosis of Mycobacterium bovis, as a PhD research work, will be potential to solve the unformatted difficulties. Tuskegee University, College of veterinary medicine, Nursing and Allied Health, has all the instrumentations (Culture facilities, conventional PCR, RT-PCR machines) to accomplish the proposed experiment. Pitfalls and Alternative Approaches One possible difficulty may be getting the DNA, which is suitable for the PCR technique. To overcome this problem, the use of different DNA extraction protocol including immunocapture will be as an alternate solution. Expected Result and Future Direction Our hypothesis is that, there is possibility of improving the diagnosis of Johne's disease in context of time, cost and accuracy. The proposal will compare and select the efficient target and protocol for the diagnosis of M. avium subsp. paratuberculosis from milk samples. Time Table Year 1: Optimize the protocol, PCR, RT-PCR, Year 2: Milk spike and validation of the procedure Year 3: Further laboratory work, prepare manuscript and design for further grant application
PROGRESS: 2006/01 TO 2006/12
Polymerase chain reaction (PCR)-based detection system has proven to be a popular and sensitive method for screening clinical samples for M. avium subsp. Paratuberculosis (MAP). The focus of this study is to compare and validate the previously reported insertion segment, IS900, to other reported repetitive sequences. Non-MAP mycobacterial species (M. fortuitum subsp. fortuitum, M. intracellulare, M. marinum, M. phlei, and M. scrofulaceum) and non mycobacterial species (Escherichia coli, Campylobacter jejuni, Yersinia pseudotuberculosis, and Streptococcus) are purchased from ATCC and included in the study. The diagnostic targets (IS900 & Mptb52.16) have been evaluated to detect MAP and both targets were equally susceptible for all Mycobacterium avium subsp. paratuberculosis strains (4 ATCC strains and 2 clinical isolates) included in the experiment. Real-time PCR and the gel analysis indicated that both sequences are found in the MAP strains. Multiplex-PCR of IS900 and putative sequence Mptb52.16 showed the presence of two distinct amplicon-bands with molecular weight of 124 and 156 bp, respectively. Abstract is sent to IAFP (International Association for Food Protection) and accepted for poster presentation. The PI contacted and met with Dr. Sara Rowe (State of Alabama, Department of Agriculture and Industries) and Dr. Arthur Hinton (USDA, ARS, SAA, Georgia) to establish future collaboration.
IMPACT: 2006/01 TO 2006/12
Johne's disease is estimated to cost the dairy industry $200 million a year. Johne's control programs have been hampered by lack of rapid and standardized diagnostic techniques with optimal sensitivity and specificity to identify MAP infected animals. Currently the National Milk Producers Federation is requesting $1.3 billion from Congress in order to wipe out the disease. Eradicating Johne's would entail precise testing to identify diseased cattle in order to eradicate the disease. PCR is the choice of diagnostic tools for such fastidious bacteria. IS900 has been used for long time as diagnostic target for MAP, there are recent reports indicating the presence of this segment in other mycobacterium species. Therefore, searching other specific target was the main focus of this project. Our preliminary results showed that targeting of Mptb52.16 sequence or use of multiplex PCR with IS900 may be promising to screen MAP from other mycobacterium species. Additionally this experiment will establish the diagnostic of MAP from milk sample. The development of this specific diagnostic protocol will be used to detect MAP from animals and also from food products. The results of this research will help us to establish other grant on the epidemiology and genetic diversity of MAP. Training: The results of this study will be disseminated to the scientific community by presenting to scientific meetings and publications.
PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
Abstract submitted to The International Association for Food Protection: Teshome Yehualaeshet, Marica Montgomery, Mica Vapner, Tsegaye Habtemariam. Comparison and Validation of IS900-sequence and Putative Sequence (Mptb52.16) as a Diagnostic Tool to Mycobacterium avium subsp. paratuberculosis. 94th IAFP Meeting, Florida, July 8-11, 2007.
PROJECT CONTACT:
Name: Yehualaeshet, T.
Phone: 334-727-8107
Fax: 334-724-4277
Email: teyehual@tuskegee.edu
URL: http://compepid.tuskegee.edu

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ACCESSION NO: 0211485 SUBFILE: CRIS
PROJ NO: ARZR-2007-01542 AGENCY: CSREES ARZR
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO : 2007-35204-18443 PROPOSAL NO: 2007-01542
START: 15 SEP 2007 TERM: 14 SEP 2010 GRANT YR: 2007
GRANT AMT: $125,000
INVESTIGATOR: Porter, M. D.
Performing Institution:
Arizona State University
Tempe , Arizona 85287
ULTRA SENSITIVE DIAGNOSTIC TESTS FOR THE EARLY DETECTION OF JOHNE'S DISEASE.
NON-TECHNICAL SUMMARY: Currently, Johne's disease can only be detected when it has reached the clinical phase, where Mycobacterium avium, subsp. Paratuberculosis (MAP), the causative agent, is present at high-levels. It is evident that there is a critical need for a diagnostic test to detect the presence of MAP at sub-clinical levels in order minimize loss of more cattle due to the spread of infection. Our goal is to obtain subclinical levels of detection (~10 MAP/mL) within one day using a surface enhanced Raman scattering (SERS) diagnostic test for Johne's disease.
OBJECTIVES: The overall goal of this project is the development of a superior immunoassay for the selective, rapid, and ultra-low level detection of MAP, which causes Johne's disease. We will continue to exploit innovations in SERS-based analytical techniques and the latest advances in the production of highly specific MAP antibodies. In order to demonstrate significant progress for the awarded one year of funding and support for a revised grant application, we have amended our specific aims as follows. Since selective ERL labels for MAP have already been created, we have reduced our effort to two specific aims, which we believe we can complete in a one-year timeframe. We will therefore optimize methods to increase sample volume and decrease total assay time with subsequent improvements in the limit of detection (LOD). These methods will then be incorporated into assays for MAP spiked in milk and feces to simulate actual field samples. Subsequently, the assay will be tested using samples collected from well-characterized cattle at the USDA National Animal Disease Center (NADC).
APPROACH: SPECIFIC AIM 1: Optimization of assay throughput by increasing sample volumes, decreasing assay time, and improving the LOD. We will explore innovative methodologies that will increase the flux of antigens to the capture surface in order to decrease the total assay time. As detailed in our full proposal, these strategies will use substrate rotation and a reduction in assay address size to improve the LOD of our assay. SPECIFIC AIM 2: Development of MAP assays in milk and feces and first validations. With the optimized assay conditions developed in specific aim 1, methodologies will be generated to perform MAP assays at the 10 MAP/mL level using spiked milk and fecal samples to mimic field samples. As with any 'real world' sample matrix, this includes identification of effective measures to minimize nonspecific adsorption. Performance will then be evaluated using a NADC cattle herd dedicated to Johne's disease research; these experiments will be performed in the blind by researchers at ASU. The level of infection of each cow has been well characterized by standard methods over an extended period and will provide an excellent reference for assay validation.
PROJECT CONTACT:
Name: Porter, M. D.
Phone: 480-727-8598
Email: Marc.Porter@asu.edu

 
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ACCESSION NO: 0405669 SUBFILE: CRIS
PROJ NO: 5325-32000-005-00D AGENCY: ARS 5325
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 13 JUN 2002 TERM: 14 MAR 2006 FY: 2006
INVESTIGATOR: Mcgarvey J A; Ravva S V; Hernlem B J
PERFORMING INSTITUTION:
Western Regional Res Center
Albany , California 94710
CONTROL OF PATHOGENS IN MANURE AND TRANSFER TO PLANT ENVIRONMENTS.
OBJECTIVES: 1. To identify factors influencing survival and re-growth of human pathogenic bacteria in a dairy system, including transfer of pathogens from manure and manure by-products to crops. 2. To identify bacterial populations in the manure environment. 3. To apply this information for designing pathogen control strategies that will be both effective and dairy-friendly.
APPROACH: Our overall approach will be to examine pathogenic and non-pathogenic bacteria presence, and their re-growth and transfer to agricultural crops during commercial dairy operations, test these hypotheses in laboratory/greenhouse studies using pathogens with bioluminescent and or antibiotic resistant markers and then evaluate laboratory based pathogen control technologies in field trials at commercial dairies. Rapid, sensitive pathogen detection methodologies that are currently available will be adopted for evaluating the life cycle of pathogen transmission on a typical California dairy. Methods for environmental detection of MAP and for pathogens in bio-aerosols and fog will be developed during these investigations. Electroflotation: Design and evaluate bench-scale prototypes and apply to dairy lagoon fluids and process samples to define contaminant removal in relation to substrate composition selection of electrode, electrolytes (chloride, nitrate, nitrite, sulfate, etc.).
FORMERLY: 5325-32000-002-00D; 5325-42000-023-00D. 5325-42000-028-00D combined into this project(4/04).
PROGRESS: 2002/06 TO 2006/03
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Americans suffer from over 75 million cases of food-borne illnesses each year, resulting in over 5,000 deaths. Financial losses associated with human and animal food-borne disease are estimated to exceed $20 billion annually. Many human and animal food-borne illnesses are caused by the consumption of foods that were contaminated by animal waste containing pathogenic bacteria including E. coli, Salmonella, Campylobacter, and Mycobacterium avium subsp. paraterculosis (MAP). Confined animal feeding operations (CAFOs), including dairies and beef feed lot operations, are often located near crops, where the animal manure is used as a low cost fertilizer. If disease-causing bacteria are present in the manure, it is possible that the crops will become contaminated, leading to food-borne illness. One way contamination may occur is from dust generated by waste handling, which may cause disease either directly by ingestion or indirectly through the contamination of crops. In addition to disease, animal waste generates large amounts of volatile organic chemicals, ammonia, methane, and hydrogen sulfide that cause nuisance odor and air quality problems in rural farming areas. To prevent foodborne illnesses associated with the use of animal waste as a fertilizer we are developing new treatment methodologies for the treatment of waste before it is composted or stored in liquid wastewater holding lagoons. These methodologies include aerobic and anaerobic digestion technologies that not only have the potential to reduce pathogens but also decrease emissions of volatile chemicals into the atmosphere. We are also evaluating the potential of pathogen transmission via aerosols using state of the art air sampling techniques. Finally, using new DNA microarray technology, we are identifying the genes in MAP bacteria that allow them to persist in the environment and cause disease. This research is administered under National Program 108 Food and Safety. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY02) Isolation of microbes from manure Characterization of pathogens in manure Assays for pathogenic E. coli in manure Year 2 (FY03) Laboratory models of manure lagoons Assays for M. a. paratuberculosis in manure Year 3 (FY04) Culture methods for M. a. paratuberculosis Factors influencing pathogen regrowth on plants Year 4 (FY05) Aeration treatment of manure lagoons Electroflotation treatment of manure lagoons Sampling microbes from aerosol and fog Year 5 (FY06) [None] 4b List other significant research accomplishment(s), if any. Testing for bacteria in dairy environments. It is unclear which disease- causing microbes are present in various places on a dairy. We characterized manure, drinking trough water and air samples from a Sonoma dairy for total aerobic bacteria, fungi, coliforms, and the pathogens E. coli O157:H7 and Salmonella. In aerosols we measured about 10,000 bacteria per liter of air, but only 1-10% of these were alive. And we were unable to detect pathogens in any samples. Thus if pathogen contamination occurs via aerosol, regrowth from extremely low levels is necessary for disease transmission. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.1 Methodology; 1.1.2 Epidemiology; 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships; and 1.2.1 Detection and Validation. MAP gene expression. Although known to be responsible for Johnes disease, MAP remains poorly characterized. Using DNA microarray technology we identified genes that may be involved in the persistence of MAP in dairy wastewater environments. This information should prove useful in preventing spread of MAP and Johnes disease. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships; and 1.2.5 Omics. Potential MAP virulence gene discovered. Because MAP is unusually slow- growing and difficult to study in the lab, little is known about how it causes disease. We have identified a gene in MAP that may be involved in virulence. We then constructed a mutant strain of MAP that has a deletion in this gene. To determine the role of this gene we will determine whether this mutant is capable of causing disease. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.1 Methodology; 1.1.2 Epidemiology; 1.1. 3 Ecology, Host Pathogen, and Chemical Residue Relationships. 4d Progress report. This Project terminated in FY06. It is replaced by Project 5325-32000-006- 00D Environmental and Genetic Factors Affecting Pathogen Persistence in Animal Waste and Transfer to Crops, which has a separate report. 5. Describe the major accomplishments to date and their predicted or actual impact. Our results on circulation of dairy wastewater for odor reduction are widely known and used among California dairy farmers and producers of circulator equipment. Wide implementation of circulation is reducing environmental impact of dairy industry by reduction of odor and other volatile compounds. It also creates a market for circulation equipment, and our data will be used by regulatory agencies for limiting emissions by dairies. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1. 1 Methodology; 1.1.2 Epidemiology; 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships; and 1.2.1 Detection and Validation. Our newly developed MAP detection method is very sensitive and specific. This method will likely be used by stakeholders in veterinary diagnostics for the improved detection of MAP. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.1 Methodology; 1.1.2 Epidemiology; 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships; and 1.2.1 Detection and Validation. We observed that pathogens grow and re-grow rapidly during the traditional methods of making compost teas supplemented with molasses, but some essential oils and natural products inhibit pathogen re-growth in compost teas. This information is being used by organic and traditional farmers for pathogen reduction. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.2 Epidemiology; and 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We have transferred the data regarding circulation of dairy wastewater lagoons to farmers and equipment manufacturers. The equipment manufacturers are now making this technology widely available. Our results on electrolytic treatment of dairy wastewater to destroy urea and pathogenic bacteria have been transferred to the public via publication in a scientific journal. Implementation of this technology would require cheap on-farm electrical power generation, which might be produced via operation of a methane power plant.
PUBLICATIONS (not previously reported): 2002/06 TO 2006/03
1. Zhong, R., Mitloehner, F., McGarvey, J.A., Ma, Y. 2005. Treatment of dairy manure with anaerobic digestion and aeration technologies for reducing gaseous emissions [abstract]. California Air Resources Board Symposia, January 26, 2005, Sacramento, CA. Paper No. 005
2. McGarvey, J.A., Miller, W.G., Sanchez, S. 2005. Comparison of circulated and stagnant dairy wastewaters [abstract]. International Conference on Environmental, Industrial and Applied Microbiology, March 15-18, 2005, Badajoz, Spain. P. 71
3. Yang, P., Ruihong, Z., McGarvey, J.A., Benemann, J. 2005. Hydrogen production from waste by anaerobic fermentation. American Society of Agricultural Engineers, July 17-20, 2005, Tampa, FL. Paper No. 056019
4. Mcgarvey, J.A., Miller, W.G., Sanchez, S., Silva, C.J. 2005. Bacterial and chemical composition of dairy wastewater [abstract]. American Society for Microbiology General Meeting, 6/5-6/9/05, Atlanta, GA. P. 008.
5. Mcgarvey, J.A., Miller, W.G., Sanchez, S., Silva, C.J. 2005. Bacterial population structure of dairy wastewaters [abstract]. International Union of Microbiological Societies, July 23-28, 2005, San Francisco, CA. Poster No. B1102.
6. Ma, Y., Zhang, R., Mitloehner, F., Mcgarvey, J.A. 2005. Reduction of gas emissions from dairy manure storages by different biological treatment strategies [abstract]. American Society for Agricultural Engineers Annual International Meeting, Tampa, FL. July 17-20, 2005. Paper No. 054054.

 
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ACCESSION NO: 0409718 SUBFILE: CRIS
PROJ NO: 5325-32000-006-00D AGENCY: ARS 5325
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 15 MAR 2006 TERM: 14 MAR 2011 FY: 2006
INVESTIGATOR: Mcgarvey J A; Ravva S V; Hernlem B J; Carter J M
PERFORMING INSTITUTION:
Western Regional Res Center
Albany , California 94710
ENVIRONMENTAL AND GENETIC FACTORS AFFECTING PATHOGEN PERSISTENCE IN ANIMAL WASTE AND TRANSFER TO CROPS.
OBJECTIVES: 1: The identification of factors contributing to the persistence and horizontal transfer of MAP and other pathogens on dairy farms. We will examine the role alternate protozoan hosts play in MAP persistence and horizontal transfer. We will also perform functional genomic analysis of MAP in the environment to elucidate clues about the strategies employed by this organism for environmental survival. 2: The development of methodologies for the reduction of pathogens in waste. Two of the most common waste treatment methodologies are aerobic and anaerobic digestion, but little is known about the effect these processes have on the microbial population structure, pathogen levels, and volatile compound missions. The bacterial community structure, chemical composition and volatile compounds emitted from animal waste, aerobic and anaerobic digesters, and holding tanks receiving effluent will be characterized. We will also analyze the effect of diet and dietary supplements on the microbial population structure, pathogen levels, and volatile compound emissions from animal waste and animal waste treatment systems. 3: Pathogen transfer from manure and CAFOs (confined animal feeding operations) to crops. We will use high volume aerosol collection and agar impaction devices to collect bacteria from aerosols near CAFOs. Aerosol samples will be evaluated for pathogens using culture and non-culture based methods. We will examine the bacterial community structure and pathogen levels on crops fertilized with waste using culture and non-culture based methods and compare them to those of plants fertilized with only chemical fertilizers. We will study the colonization of crop plants by Salmonella enterica grown in soil amended with manure spiked with pathogens using standard culture and quantitative PCR methods.
APPROACH: The goal of this project is to reduce the incidence of foodborne illnesses by reducing the levels of pathogens in animal waste and in CAFO environments. In the next five years we will identify the locations in CAFOs that serve as reservoirs for pathogenic bacteria such as MAP, develop low cost pathogen abatement strategies for contaminated animal waste, examine the effect of diet on pathogen load and evaluate the risk of pathogen transfer from CAFOs to surrounding crop plants via aerosols and from fertilizing crop plants with contaminated manure.
FORMERLY: 5325-32000-002-00D; 5325-42000-023-00D.
5325-42000-028-00D combined into this project (4/04). Replaces
5325-32000-005-00D (2/06).
PROGRESS: 2005/10 TO 2006/09
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Americans suffer from over 75 million cases of food-borne illnesses each year, resulting in over 5,000 deaths. Financial losses associated with human and animal food-borne disease are estimated to exceed $20 billion annually. Many human and animal food-borne illnesses are caused by the consumption of foods that were contaminated by animal waste containing pathogenic bacteria including E. coli, Salmonella, Campylobacter, and Mycobacterium avium subsp. paratuberculosis (MAP). Confined animal feeding operations (CAFOs), including dairies and beef feed lot operations, are often located near crops, where the animal manure is used as a low cost fertilizer. If disease-causing bacteria are present in the manure, it is possible that the crops will become contaminated, leading to food-borne illness. One way contamination may occur is from dust generated by waste handling, which may cause disease either directly by ingestion or indirectly through the contamination of crops. In addition to disease, animal waste generates large amounts of volatile organic chemicals, ammonia, methane, and hydrogen sulfide that cause nuisance odor and air quality problems in rural farming areas. To prevent foodborne illnesses associated with the use of animal waste as a fertilizer we are developing new treatment methodologies for the treatment of waste before it is composted or stored in liquid wastewater holding lagoons. These methodologies include aerobic and anaerobic digestion technologies that not only have the potential to reduce pathogens but also decrease emissions of volatile chemicals into the atmosphere. We are also evaluating the potential of pathogen transmission via aerosols using state of the art air sampling techniques. Finally, using new DNA microarray technology, we are identifying the genes in MAP bacteria that allow them to persist in the environment and cause disease. This research is administered under National Program 108 Food Safety. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY06) Characterize gene expression of MAP in vitro. Characterize bacterial communities in waste after aerobic and anaerobic digestion. Develop culture and immunomagnetic methods for detection of pathogens in aerosol/fog. Evaluate aerosol collection devices. Year 2 (FY07) Characterize gene expression of MAP in vivo. Study effect of Monensin addition on microbial population and waste chemistry in aerobic and anaerobic manure digestion. Use cytometry to characterize protozoan hosts for MAP. Validate aerosol samplers with trapping media. Validate coupled immunomagnetic enrichment with quantitative PCR for detection of MAP in aerosols. Use polymerase chain reaction with differential gradient gel electrophoresis of 16S RNA genes for characterization of predominant members of the bacterial community. Compare microbial community of soil and corn plants grown with manure versus chemical fertilizers. Year 3 (FY08) Generate green fluorescent protein-expressing MAP, infect protozoan hosts, and maintain in culture. Characterize gene expression of MAP in protozoan hosts. Compare microbial communities and profile waste chemistry in dairy manure from animals fed with low versus high protein diet. Determine susceptibility of crops to pathogen contamination and colonization by aerosol transmission of pathogens. Characterize time-of-day fluctuations in aerosol (fog) transmission of pathogens. Year 4 (FY09) Determine whether protozoan hosts facilitate multiplication of MAP. Determine whether aerobic/anaerobic digestion reduce pathogen load in animal waste. Characterize seasonal fluctuations of bacteria in aerosols. Compare culturable and non-culturable bacterial species in aerosols Compare microbial community of soil and cantaloupe plants grown with manure versus chemical fertilizers. Year 5 (FY10) Determine changes in MAP virulence caused by protozoan hosts. Track movement of bacteria from aerosols to crops. Characterize survival of bacteria on crops. 4a List the single most significant research accomplishment during FY 2006. Aerobic and anaerobic treatment of dairy waste: Dairy waste is a visible and contentious source of pathogens and volatile compound emissions. We compared the effect of aerobic and anaerobic digestion with subsequent storage in simulated wastewater lagoons on the bacterial population, chemical composition, and volatile compound emissions as compared to no treatment before storage. Our results show that both treatment methods substantially altered the bacterial population of waste, and reduced the levels of nutrients and volatile compound emissions. These methods may be adopted by CAFOs to reduce the spread of foodborne illness and decrease noxious odor. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statement 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships. 4b List other significant research accomplishment(s), if any. Testing for bacteria in dairy environments. It is unclear which disease- causing microbes are present in various places on a dairy. We characterized manure, drinking trough water and air samples from a Sonoma dairy for total aerobic bacteria, fungi, coliforms, and the pathogens E. coli O157:H7 and Salmonella. In aerosols we measured about 10,000 bacteria per liter of air, but only 1-10% of these were alive. And we were unable to detect pathogens in any samples. Thus if pathogen contamination occurs via aerosol, regrowth from extremely low levels is necessary for disease transmission. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.1 Methodology; 1.1.2 Epidemiology; 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships; and 1.2.1 Detection and Validation. MAP gene expression. Although known to be responsible for Johnes disease, MAP remains poorly characterized. Using DNA microarray technology we identified genes that may be involved in the persistence of MAP in dairy wastewater environments. This information should prove useful in preventing spread of MAP and Johnes disease. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships; and 1.2.5 Omics. Potential MAP virulence gene discovered. Because MAP is unusually slow- growing and difficult to study in the lab, little is known about how it causes disease. We have identified a gene in MAP that may be involved in virulence. We then constructed a mutant strain of MAP that has a deletion in this gene. To determine the role of this gene we will determine whether this mutant is capable of causing disease. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statements: 1.1.1 Methodology; 1.1.2 Epidemiology; 1.1. 3 Ecology, Host Pathogen, and Chemical Residue Relationships. 5. Describe the major accomplishments to date and their predicted or actual impact. Aerobic and anaerobic treatment of dairy waste. Dairy waste is a visible and contentious source of pathogens and volatile compound emissions. We compared the effect of aerobic and anaerobic digestion with subsequent storage in simulated wastewater lagoons on the bacterial population, chemical composition, and volatile compound emissions as compared to no treatment before storage. Our results show that both treatment methods substantially altered the bacterial population of waste, and reduced the levels of nutrients and volatile compound emissions. These methods may be adopted by CAFOs to reduce the spread of foodborne illness and decrease noxious odor. This accomplishment is aligned with NP108 2006-2010 Action Plan Component 1.1 Pathogens, Toxins, and Chemical Contaminants Preharvest. Specifically this research addresses Problem Statement 1.1.3 Ecology, Host Pathogen, and Chemical Residue Relationships. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). J. McGarvey, 2006. Why should you want purple photosynthetic bacteria in your wastewater lagoons? Presented to: The California Diary Campaign members and other stakeholders, Modesto, CA.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
1. Mcgarvey, J.A., Barak Cunningham, J.D., Miller, W.G. 2005. Surface sanitization of cantaloupes using thymol/cinnamaldehyde. California Melon Research Board Annual Meeting, November 20-23, 2005, Hermosilla, MX.
2. Ravva, S.V. 2006. Nutrients and wastewater components influence the survival and growth of pathogenic Escherichia coli O157:H7 in dairy lagoons [Abstract]. 2nd Federation of European Microbioilogy Societies (FEMS) Congress of European Microbiologists. Poster #P.ENV.109.
3. Ravva, S.V., Sarreal, C.Z., Duffy, B., Stanker, L.H. 2006. Survival of Escherichia coli O157:H7 and Salmonella enterica in manure waste water from dairy lagoons. Journal of Applied Microbiology. 101(2006):891-902
4. Ravva, S.V., Stanker, L.H. 2005. Real-time quantitative PCR detection of Mycobacterium avium subsp. paratuberculosis and differentiation from other mycobacteria using Sybr Green and Taqman assays. Journal of Microbiological Methods. 63(2005) 305-317.


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ACCESSION NO: 0409207 SUBFILE: CRIS
PROJ NO: 5325-32000-006-01S AGENCY: ARS 5325
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 07 MAR 2005 TERM: 30 APR 2007 FY: 2006
INVESTIGATOR: Mcgarvey J A; Zhang R; Mitloehner F
PERFORMING INSTITUTION:
Univ of California
Davis , California 95616
CHARACTERIZATION OF DAIRY WASTE MANAGEMENT STRATEGIES WITH REGARD TO PATHOGENS AND AIR QUALITY.
OBJECTIVES: The goal of this project is to compare dairy waste treatment methodologies, especially varying levels of aerobic and anaerobic treatment, as well as wastewater lagoon circulation, aeration and covering for the reduction of pathogens and volatile compound emission. These studies will provide greater understanding of how pathogens persist or are destroyed under various conditions and simultaneously elucidate the type of volatile organic compounds that are emitted during treatment. The studies will involve lab scale aerobic and anaerobic digesters, as well as on site dairy waste lagoon experiments conducted at an operating dairy, and thus real world conditions. The results of these studies will have impact on the way diary waste is treated for both human health and environmental considerations.
APPROACH: Dairy waste treatment methodologies are of concern because of human illnesses caused by pathogens within manure and because of volatile organic gas emissions which cause poor air quality in and around dairy operations. Pathogens such as E. coli O157:H7. Salmonella spp., Campylobacter spp. etc. have caused outbreaks associated with improper diary waste treatment. In addition, improper treatment of diary waste can emit volatile chemicals into the atmosphere, including volatile fatty acids (VFAs), volatile organic compounds (VOCs), and ammonia. Furthermore, improper application of diary waste to crop fields can result in the accumulation of sodium chloride, phosphate, nitrate and nitrite. These chemicals not only impact crop production, but also leach into ground water making it unsuitable for human or animal consumption. To better understand how dairy waste can be treated to eliminate pathogens, and decrease air emissions and unwanted groundwater leaching, we will initiate bench scale aerobic and anaerobic digester experiments. We will feed raw diary waste (manure and urine) into aerobic and anaerobic reactors under different parameters such as temperature, amount of dissolved oxygen, redox potential, retention time, etc. and analyze the starting material and effluent for microbial content. We will also collect gasses emitted from the waste treatment systems and analyze their chemical composition using GC/MS. We will conduct experiments with manure that has been spiked with known amounts of pathogenic bacteria including Salmonella, E. coli O157:H7, Mycobacterium avium subsp. paratuberculosis, etc. to determine the effect these methodologies have on pathogen growth and survival. Lastly we will conduct experiments on a working 800-cow dairy farm. This farm is uniquely suited for experiments on waste treatment methodologies because it has two waste lagoons that receive waste from the same source, but are run independently of each other, thus providing an experimental treatment lagoon and a control lagoon. We will treat one lagoon under conditions found to be ideal for the reduction of pathogen and volatile organic compound emission, and chemical changes as they evolve. This work will not only provide proof of principal in lab experiments but will show how these concepts correlate to real world conditions on a working dairy. Documents SCA with UC-Davis. Formerly
5325-32000-005-01S (3/06).
PROGRESS: 2005/10 TO 2006/09
Progress Report 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the University of California at Davis. Additional details of research can be found in the report for the parent CRIS 5325-32000-006-00D Environmental and Genetic Factors Affecting Pathogen Persistence in Animal Waste and Transfer to Crops. In FY06 this project was extended into a second year. This Project primarily is comparing two common types of treatment for dairy waste: aerobic and anaerobic digestion. Current work involves lab scale digesters, but plans include work with waste lagoons on site at an operating dairy. We are studying effects of the different treatments on microbial communities and chemical profile. We are especially interested in reduction of pathogen levels and volatile emissions.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
No publications reported this period.

 
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ACCESSION NO: 0403289 SUBFILE: CRIS
PROJ NO: 5325-42000-027-00D AGENCY: ARS 5325
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 25 JAN 2000 TERM: 24 JAN 2005 FY: 2005
INVESTIGATOR: Stanker L H; Binder R G; Brandon D L; Onisko B C; Wong R Y
PERFORMING INSTITUTION:
Western Regional Res Center
Albany , California 94710
DEVELOPMENT OF NEW TECHNOLOGIES FOR RAPID IDENTIFICATION OF PATHOGEN/PATHOGEN PRODUCTS.
OBJECTIVES: Develop multi-analyte biosensors (multiple members of a drug or chemical family) that may be coupled directly on-line for the detection of residues in foods and feed. The methods developed should be of direct use to federal regulatory and/or action agencies. FY01 Program Increase $269,370. 1SY Added.
APPROACH: Development of adequate sampling protocols is a critical component of any detection scheme. Methods will be developed that result in separation of pathogens from an aggregate matrix or biofilm, and simultaneously enrich and concentrate multiple pathogenic species. Extraction and concentration is essential for a biosensor that does not rely on a preenrichment culture step. Pathogens will be prepared and concentrated using techniques such as sequential filtration, gravitation, immunotrapping, ligand-receptor interactions, and cell sorting. If preentrichment is necessary, methods will be developed to shorten the incubation times required. Methods such as integrated optical sensing, mass spectrometry, sensitive fluorescence, immuno-electrochemical detection, immunomagnetic electrochemiluminesce, microchip arrays, and combinatorial chemistry will be adapted to meet these goals. Reagents, such as monoclonal antibodies, receptors, and molecular imprints will be generated to facilitate separation and detection schemes. FY01 Program Increase $269,370. 1SY Added.
PROGRESS: 2000/01 TO 2005/01
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This project addresses the need for validated methods to detect toxins and pathogens in foods. Existing surveillance systems are designed to detect occurrences of infections and intoxications resulting from consumption of foods naturally contaminated with pathogens or the toxins produced by these organisms. However, it has been of increasing concern that the intentional adulteration of food with chemical or biological agents such as bacterial and plant toxins could become a major instrument of bioterrorism. In addition, it is likely that crude, rather than purified, toxins would be used as bioweapons, and it is possible that combinations of toxins could be employed. Therefore, some data gaps related to dose response relationships must be filled. In addition, rapid, portable sample preparation techniques applicable to important food matrices are needed, particularly for new detection technologies. This research falls under National Program 108 Food Safety. Specifically this research addresses the following Problem Statements in the NP108 Action Plan: 1.2.1 Detection and Validation, 1.2.4 Processing Intervention Strategies, 1.2.5 Omics, 1.2.7 Risk Assessment, 1.2.8 Pathogenicity, 1.2.9 Food Security. The research also addresses ARS Strategic Plan Performance Measure 3.1.2: Develop and transfer to Federal agencies and the private sector systems that rapidly and accurately detect, identify, and differentiate the most critical and economically important foodborne microbial pathogens. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (FY03) Obtain Campylobacter and Mycobacteria strains and immunize mice Year 2 (FY04) New ELISA for Campylobacter strain analysis New ELISA for Mycobacteria strain analysis Year 3 (FY05) Validation of new Campylobacter and Mycobacteria strain-specific ELISAs 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Validation of new Campylobacter and Mycobacteria strain-specific ELISAs Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This project has been replaced by 5325-42000-042-00D. Please see report for 5325-42000-042-00D for future milestones. 4a What was the single most significant accomplishment this past year? Campylobacter ELISA. Food poisoning caused by Campylobacter bacteria is one of the most rapidly growing diseases in the US. Tracing outbreaks of this disease to their sources requires a special set of reagents and techniques. We created a set of novel strain-specific monoclonal antibodies for C. upsaliensis, and developed them into simple ELISA and immunoblot test formats. These assays are now being used by scientists in the Produce Safety and Microbiology Unit for characterization of isolates from disease outbreaks. 4b List other significant accomplishments, if any. MAP antibodies. Mycobacterium avium subsp. paratuberculosis (MAP) is the bacterial pathogen responsible for Johnes disease in cows and sheep, relatively recently implicated in human Crohns disease. The organism is extremely slow-growing and thus difficult to detect, isolate, and handle in the lab. We developed a panel of new monoclonal antibodies against MAP. They are currently under evaluation for immunomagnetic capture of MAP from environmental sources. If successful they will be attractive for commercial development in MAP detection. Bacterial communication compound. Bacteria make and detect unique chemicals to recognize other bacteria in their environment, enumerate them, and adjust their behaviour accordingly. Examples of this process include colonization of food and food preparation surfaces and expression of genes involved in disease virulence. To help study this process we invented a novel simplified scheme for synthesis of the bacterial quorum- sensing compound 4,5-dihydroxy-2,3-pentanedione. After complete purification and characterization the product will be analyzed by food safety microbiologists for use in environmental studies. 4d Progress report. In FY05 this Project was programmatically split from our Prion Project (CRIS 5325-32000-003-00D). Thus milestones for the Prion Project are not listed in this report, as they were in previous years. This Project expired 31 December 2004 and was replaced with bridging CRIS 5325-42000-042-00D. The bridging CRIS was subsequently redirected in FY05, upon a Program Increase and respective PDRAM, to address primarily foodborne toxins. Milestones for the bridging CRIS are described in the FY05 annual report submitted separately for CRIS 5325-42000-042-00D. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Upon a request from the Food Safety Inspection Service (FSIS) of the USDA, we developed an assay for residues of Ceftiofur, a broad spectrum cephalosporin antibiotic used in beef production. The immunoassay we created is faster and cheaper than previous bioassay methods and is now routinely employed by FSIS for regulation. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? We have developed an assay for residues of Ceftiofur, a regulated antibiotic found in beef. Our immunoassay is faster and cheaper than previous methods. This technology was transferred to FSIS, along with reagents and training/consultation. Immunoassay technology for Bowman-Birk Inhibitor, a naturally-occurring anti-nutritional component of soybeans, was patented and licensed to an immunodiagnostics manufacturer, Agdia, Inc. ( Elkhart, IN).
PUBLICATIONS (not previously reported): 2000/01 TO 2005/01
1. Brandon, D. L., Bates, A. H., Binder, R. G., Montague, W. C., Jr., Whitehand, L. C., Barker, S.A. Analysis of fenbendazole residues in bovine milk by ELISA. Journal of Agricultural Food Chemistry. 2002. v. 50. p. 5791-5796.
2. Brandon, D. L., Friedman, M. Immunoassays of soy proteins. Journal of Agricultural Food Chemistry. Oct. 2002. v. 50. p. 6635-6642.
3 . Johnson, L., Baxter, G. A., Crooks, S. R. H., Brandon, D. L., Elliott, C. T. E. Reduction of sample matrix effects-the analysis of benzimidazole residues in serum by immunobiosensor. Food Agricultural and Immunology. 2002. v. 14(4). p. 209-214.
4. Weeks, B.L., Camarero, J., Noy, A., Miller, A., Stanker, L., DeYoreo, J.J. Development of a Microcantilever-Based Pathogen Detector. Proceedings of NanoTech 2003 Conference. San Francisco, California, USA. February 23-27, 2003.
5. Stanker L. H. Ravva S. V. Survival of E. coli O157:H7 in aerated dairy manure lagoons. Proceedings of the 31st U.S. and Japan Natural Resources Protein Resources Panel Meeting, Monterey CA, USA. Nov. 12-19 2002.

 
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ACCESSION NO: 0192561 SUBFILE: CRIS
PROJ NO: CA-V*-PHR-7015-AH199 AGENCY: CSREES CALB
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2001 TERM: 30 SEP 2006 FY: 2006
INVESTIGATOR: Cullor, J. S.
PERFORMING INSTITUTION:
Population Health & Reproduction
Univ of California (Vet-Med)
Davis , California 95616
THE USE OF MICRO-COLONY PCR FOR DETECTION OF MYCOBACTERIUM PARATUBERCULOSIS FROM BOVINE MANURE.
NON-TECHNICAL SUMMARY: Detection of M. paratuberculosis in young cattle continues to be difficult using current tests. In an effort to improve the sensitivity and specificity of PCR for Johne's disease screen assays, we plan to investigate a micro-colony PCR detection method.
OBJECTIVES: Our objective is to assess and optimize the PCR confirmation of M. paratuberculosis microcolonies. The primary hypothesis to be tested is that PCR analysis can be performed on micocolonies of M. paratuberculosis appearing between 7-14 days after spirally plated on Middlebrook agar plates. The use of PCR on micro-colonies will circumvent the problems of sensitivity and PCR inhibition when this technique is used directly on broth cultures, manure or tissues.
APPROACH: Bovine manure from clinically normal cows will first be spiked with up to 1x10(8) CFU/g of M. paratuberculosis and decontaminated via the standard protocol used by the California Animal Health Food Safety Laboratory (CAHFS, Davis CA). After the decontamination step, the samples will be spirally plated onto Middlebrooks 7H10 agar plates for micro-colony morphology detection. The spiral plates will be incubated at 37 degrees centigrade and examined weekly for M. paratuberculosis growth. The resulting micro-colonies will be "picked" from the spiral plates and the DNA extracted from them. PCR amplification using an automated LightCycler and M. paratuberculosis specific primers will then be performed with results of a typical 30cycle reaction obtainable within 30 minutes. Primer concentration, MgCl concentration, annealing temperatures and cycle numbers will all be assessed and adjusted for maximum sensitivity and specificity.
PROGRESS: 2001/10 TO 2006/09
PCR analysis of microscopic colonies of Mycobacterium avium subsp. paratuberculosis from bovine manure was investigated. Bovine manure was spiked with laboratory strains of Mycobacterium avium subsp. paratuberculosis, spirally plated onto Middlebrook agar plates and screened for microscopic colonies after varying days of incubation. DNA was then extracted from the resultant microscopic colonies for PCR analysis to determine the minimal number of colonies to 'pick' and the shortest incubation time necessary to achieve positive results. Positive results could be achieved within 7-14 days after sample plating.
IMPACT: 2001/10 TO 2006/09
This technique may provide a useful compromise between the rapidity of direct PCR and the sensitivity of culture as a manure screening assay for bovine paratuberculosis.
PUBLICATIONS (not previously reported): 2001/10 TO 2006/09
No publications reported this period
PROJECT CONTACT:
Name: Cullor, J. S.
Phone: 559-688-1731
Fax: 559-686-4231
Email: jscullor@ucdavis.edu
URL: http://www.vmtrc.ucdavis.edu/dfsl/dfsl.html

 
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ACCESSION NO: 0189445 SUBFILE: CRIS
PROJ NO: CA-V*-VME-7111-CG AGENCY: CSREES CALB
PROJ TYPE : NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2001-35204-10874 PROPOSAL NO: 2001-02494
START: 15 OCT 2001 TERM: 31 OCT 2003 FY: 2005 GRANT YR: 2002
GRANT AMT: $205,000
INVESTIGATOR: Gardner, I. A.; Salman, M.; Johnson, W. O.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet-Med)
Davis , California 95616
CERTIFICATION OF DISEASE FREEDOM IN ANIMAL POPULATIONS: USE OF BAYESIAN METHODS.
NON-TECHNICAL SUMMARY: Trade in animals and animal products is in part dependent on the validity of assurances that exporting herds and countries provide about the infection status of their animals. Our objective is to develop Bayesian models that can be used to make improved inferences about freedom from infection for a single herd or multiple herds in a country/region. We will use beta distributions to model uncertainty and variability in test sensitivity and specificity, and prevalence. For the single-herd model, we will use bovine paratuberculosis as our example because of current interest in the Voluntary Johne's Disease Herd Status Program (VJDHSP) and the difficulties inherent in differentiating low prevalence from non-infected herds. For the multiple-herd model, we will use survey data on porcine reproductive and respiratory syndrome, infectious bovine rhinotracheitis and bluetongue to validate our model. There are often extensive surveillance data that can complement negative survey findings, but there are no standardized methods for incorporating these data into assessment of freedom from infection. Our final objective is to develop Bayesian methods for incorporation of non-survey data and adjustment for time-dependent changes in the quality of survey and surveillance data. We will use data on bovine spongiform encephalopathy, brucellosis and tuberculosis for these assessments. The methods developed will be adaptable to most infectious livestock diseases, including those of interest to commodity groups such as the VJDHSP and to those that are federally regulated such as brucellosis.
OBJECTIVES: To develop Bayesian methods for: (1) Certification of the status of a single herd with respect to prevalence and freedom from infection (incorporating the prior probability of infection and uncertainly and variability in test sensitivity and specificity). (2) Certification of status of a group of herds with respect to within-herd prevalence, proportion of infected herds and freedom from infection (incorporating the prior probability of infection, uncertainty and variability in test sensitivity and specificity, and the possible clustering of positive test results at the herd level). (3) Incorporation of non-survey data and adjustment for time-dependent changes in the quality of survey and surveillance data.
APPROACH: We will use beta distributions to model uncertainty and variability in test sensitivity and specificity, and prevalence. Gibbs sampling will be used to combine prior distributions and herd-level test results into a posterior prevalence distribution. For the single-herd model, we will use bovine paratuberculosis as our example because of current interest in the Voluntary Johne's Disease Herd Status Program (VJDHSP) and the difficulties inherent in differentiating low prevalence from non-infected herds. For the multiple-herd model, we will use survey data on porcine reproductive and respiratory syndrome, infectious bovine rhinotracheitis and bluetongue to validate our model.
PROGRESS: 2001/10 TO 2003/10
Trade in animals and animal products is in part dependent on the validity of assurances that exporting herds and countries provide about the infection status of their animals. We developed Bayesian hierarchical models for determining infection status and infection prevalence given testing of all or a sample of animals from a single herd with an imperfect test. Expert prior information about herd infection status, diagnostic test accuracy are incorporated into the model. Posterior versus prior probabilities are presented as a curve, summarizing the probability of infection over a range of possible prior probability values. The model has been applied to serologic data for paratuberculosis in dairy herds. For the multiple herd setting, we created a model that allows for 3 levels of inference: probability that a country (region) is free of infection, the proportion of infected herds in an infected country, and the within-herd prevalence. The model uses test results from animals sampled in a two-stage cluster sample of herds. The model was validated with simulated data and applied to surveys of Newcastle disease and porcine reproductive and respiratory syndrome virus infection. A Bayesian sample size calculator was constructed to incorporate prior test results and other information to provide guidance as to appropriate numbers for herd testing programs. Because of the complexity of calculations, we wrote a software program (BDFree) that is available at www.epi.ucdavis.edu/diagnostictests/. We have written code in WinBUGS for Bayesian inference about animal and herd prevalence of infectious diseases for a variety of different sampling and testing scenarios. In addition, we investigated methods of estimation of the intracluster correlation coefficient (ICC) for infectious animal diseases. We demonstrated that the ICC based on apparent infection status for individuals is less than or equal to the ICC based on true infection status. We developed a Bayesian model for estimating the ICC that incorporates imperfect test accuracy and applied the model to a NAHMS seroprevalence survey of ovine progressive pneumonia. Two analytical models were constructed to determine the likelihood of freedom from an infectious disease using existing surveillance data, rather than use of survey data, as in the prior scenarios. The two models were explored using Danish data from the surveillance program for infectious bovine rhinotracheitis. The relationship between within-herd prevalence and animal-level prevalence was investigated to derive a reliable estimate of a herd prevalence of a given disease. The distribution impact of the within-herd prevalence as determined by animal-level prevalence and the intracluster correlation coefficient on herd-level sensitivity and specificity was modeled. A sample size formula, dependent on herd-level sensitivity and specificity, was proposed for estimating herd-level prevalence in a two-stage sampling design. The use of a distribution for within-herd prevalence resulted in a conservative estimate of herd-level test characteristics.
IMPACT: 2001/10 TO 2003/10
An international workshop was organized by the team and held in Copenhagen, Denmark in March 2003. The aim of this workshop was to address issues and terms related to international rules and regulations for declaring a country free from a disease. Terms and definitions were agreed on by the participants. Current methods and techniques used by our research team were recognized as the most advanced available and as valid approaches for declaring a country free from a disease. An international training course was offered in November 2003 in association with the 10th ISVEE meeting in Chile. One goal of the workshop was to legitimatize the techniques that were developed and evaluated by our research team and make these techniques available to participants from many countries. Dr. Zepeda participated in the OIE working group to draft a comprehensive chapter for surveillance methods to be used by OIE members. A major component of this chapter is devoted to surveillance methods to declare a country free from a disease. Techniques that are being currently researched by our team were included in the draft of this chapter. Our team will incorporate the outcome from the assessment of these techniques in the final draft of this chapter. The utility of Bayesian methods was promoted in a leading article in a major veterinary journal. We are working with the National Pork Board to make our approach available relative to herd certification for toxoplasmosis, to those interested in the Voluntary Johnes Disease Herd Status Program relative to herd certification for paratuberculosis.
PUBLICATIONS (not previously reported): 2001/10 TO 2003/10
1. Suess EA, Gardner IA, Johnson WO. Hierarchical Bayesian model for prevalence inferences and determination of a countrys status for an animal pathogen. Prev Vet Med 2002; 55:155-171
2. Gardner IA.The utility of Bayes theorem and Bayesian inference in veterinary clinical practice and research. Aust Vet J 2002; 80:758-761
3. Hanson TE, Johnson WO, Gardner IA. Determining the infection status of a herd. J Agric Biol Environ Stat 2003; 8:469-485
4. Doherr MG, Audige Salman MD, Gardner IA. Use of animal monitoring and surveillance systems when the frequency of health-related events is near zero. Animal Disease Surveillance and Survey Systems: Methods and Applications. M.D. Salman, ed. Iowa State Press 2003, pp135-147
5. Salman MD. Improvement of survey and sampling methods to document freedom from diseases in Danish cattle population on both national and herd levels. Technical report (project 5), 2003, available at http://www.dfvf.dk/Default.asp?ID=9726
6. Johnson WO, Su CL, Gardner IA, Christensen R. Sample size calculations for surveys to substantiate freedom of populations from infectious agents. Biometrics 2004;60:165-171
7. Branscum AJ, Gardner IA, Johnson WO. Bayesian modeling of animal- and herd-level prevalences. Prev Vet Med 2004; 66:101-112
8. Chriel M, Salman M.D., Wagner B. Improvement of surveillance and sampling methods to document freedom from infectious bovine rhinotracheitis in the Danish cattle population. Prev Vet Med 2004 (submitted)
9. Branscum AJ, Gardner IA, Wagner BA, McInturff PS, Salman MD. Effect of diagnostic testing error on intracluster correlation coefficient estimation. Prev Vet Med 2004 (submitted)
10. Branscum AJ, Johnson WO, Gardner IA. Bayesian approach to sample size calculations for studies designed to estimate sensitivity and specificity. Prev Vet Med 2004 (submitted)
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-6992
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

 
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ACCESSION NO: 0204853 SUBFILE: CRIS
PROJ NO: CA-V*-VME-7474-AH224 AGENCY: CSREES CALB
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 15 APR 2005 TERM: 14 APR 2009 FY: 2006
INVESTIGATOR: Gardner, I. A.; Adaska, J. M.; Whitlock, R. H.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet-Med)
Davis , California 95616
ENVIRONMENTAL SAMPLING TO ASSESS THE BIOBURDEN OF CALIFORNIA DAIRIES NON-TECHNICAL. . . . (Note: Title exceeded the title field character limit.)
SUMMARY: There are many deficiencies in existing knowledge about the transmission and detection of Map in cattle herds, including how to most effectively use currently-available tests for diagnosis of Map infection. This project will assess persistence of the Map on dairy farms and methods for quantification of bioburden.
OBJECTIVES: 1. To quantify and evaluate the role of the environmental bio-burden of Mycobacterium avium subsp. paratuberculosis (Map) in the transmission of Johne's disease on dairy farms 2. To evaluate optimal testing strategies for detection of Map in dairy herds
APPROACH: Objective 1, numbers of Map bacteria in multiple environmental locations in dairy herds will be quantified using Herrold's egg yolk medium and the Trek ESP II system. Cultures which yield values that are very high shedders will be quantified further by serial dilution to estimate the most probable numbers of bacteria in the samples. Objective 2, a stochastic simulation model will be modified to evaluate various testing (including use of environmental samples) and sampling methods for detection of Map in dairy herds.
PROGRESS: 2006/01 TO 2006/12
Our primary objective was to quantify concentrations of Mycobacterium avium subsp. paratuberculosis (MAP), as measured by culture on Herrold's egg yolk medium (HEYM) and in liquid culture (Trek ESP II) and quantitative real-time (qrt) PCR , in environmental samples including recycled lagoon water used for flushing cow alleyways. We are investigating the utility of environmental sampling in a dairy with about 2700 lactating cows (aggregated in 12 strings), 300 dry cows and about 150 pregnant heifers. Fecal culture prevalence and ELISA seroprevalence of cows at dry-off are approximately 9 % and 5 %, respectively. In November 2005, we investigated logistical aspects of detection of cows shedding high numbers of MAP in feces. All environmental samples were positive by quantitative real-time (qrt) PCR. We did follow-up testing of 3 strings identified as having different likelihoods (high, moderate and low, respectively) of having cows that are shed high numbers of MAP. Group-level serum ELISA results correlated well with qrtPCR data. Samples from 17 ELISA positive cows within these strings were submitted for HEYM culture and qrtPCR testing in December 2005. Six cows were identified as super-shedders and repeated samples were collected from 2 cows to evaluate longitudinal changes in shedding patterns. Tissues were collected from both cows and currently are being tested. The reliability of a single environmental sample for determination of MAP status by HEYM, Trek ESP II, and qrtPCR is currently being evaluated on 2 dairies with 2 investigators following a standardized protocol. The same environments are being sampled on alternate days while cow groups are static. Results are expected by March 2006.
IMPACT: 2006/01 TO 2006/12
Cows shedding large numbers of MAP pose a tremendous risk for transmitting the organism to other cattle on the farm, and they may also contribute to passive ("pass through") fecal shedding of MAP by uninfected cows, and thus false-positive fecal culture results. Quantitative real-time PCR appears to have utility as a rapid means of quantifying MAP load in feces and in the dairy environment and hence, detecting individual high-risk cows and strings of high-risk cows.
PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
No publications reported this period
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-6992
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

 
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ACCESSION NO: 0210322 SUBFILE: CRIS
PROJ NO: CA-V*-VME-7650-H AGENCY: CSREES CALB
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 15 FEB 2007 TERM: 28 FEB 2009
INVESTIGATOR: Gardner, I. A.; Adaska, J. M.; Whitlock, R. H.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet-Med)
Davis , California 95616
ENVIRONMENTAL SAMPLING TO ASSESS THE BIO-BURDEN OFMYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS AND THE EPIDEMIOLOGY OF SUPER SHEDDER COWS ON C. . . (Note: Title exceed the title field character limit.)
NON-TECHNICAL SUMMARY: Dairy cows shedding large numbers of Mycobacterium avium subsp. paratuberculosis in feces contribute substantially to the environmental load of the organism and transmission of the agent to previously non-infected animals. This project evaluates methods to detect and quantify Mycobacterium avium subsp. paratuberculosis in cows and in the dairy herd environment.
OBJECTIVES: 1. To compare the sensitivity of fecal culture with acid-fast staining of fecal smears and ELISA testing to detect super shedder cows from a case series of fecal samples identified as TNTC on HEYM or low days to positive on liquid culture (Trek ESP II) and confirmed by IS900 PCR. 2. To assess the environmental load of MAP before and after removal of cows designated as being super shedders. 3. To evaluate optimal testing strategies for detection of MAP in dairy herds with varying disease prevalence
APPROACH: Samples collected from cows in the 2 California Johnes disease demonstration herds (1350 Holsteins and 3100 Jerseys) in addition to 400 repository samples from University of Pennsylvania will be cultured on HEYM after serial dilution and inoculation of 3 flasks per dilution (1:10, 1:100, and 1:1000) with 100 μl inoculum. Fecal smears will be stained by the Ziehl Neelsen method, air dried overnight and examined under an oil immersion lens for a count of acid-fast bacilli/ field, with results categorized based on CDC guidelines (see Appendix: Table 2). Blood samples will be tested using the IDEXX MAP ELISA and test results categorized on an ordinal scale (negative, suspect, weak positive, positive and strong positive). Information on cow demographics will be downloaded from computerized record systems and correlated with fecal smear acid-fast stain results, ELISA results and fecal culture results. Fecal slurry samples will be collected from cross-over alleyways in each of the freestall barns, as described in one of our previous studies10. Slurry samples will be cultured on the day of moving cows designated as super shedders and the day after moving these cows. Alleyways will be scraped between sampling days to minimize the risk of carryover of MAP contamination. Similar testing will be done in the strings that received super shedder cows. A stochastic simulation model will be modified to evaluate various testing and sampling methods for detection of MAP in dairy herds. Testing methods to be evaluated include 1) ELISA testing, 2) ELISA followed by fecal culture, 3) Solid media culture of individual cow fecal samples, 5) Solid media culture of pooled fecal samples, 5) PCR of individual fecal samples, 6) PCR of pooled fecal samples, 7) Solid media culture of environmental samples. Sampling methods include samples of cows from 1) all cows, 2) cows in lactation > 2, 3) dry cows, 4) cows in early lactation, 5) cows in late lactation, 6) cows with clinical signs consistent to MAP and cows designated to be culled. Specific factors that will be evaluated include herd size, herd management, within-herd prevalence, prevalence within subgroup of cows, shedding distribution of MAP in feces of infected cows, sampling methods, number of samples tested, and detection limit of testing methods. Variability and uncertainty in certain model parameters are incorporated using probability density functions and empirical data. Estimates of herd-level sensitivity, misclassification probability, herd negative predictive value, and cost-effectiveness of each testing strategy under different conditions of herd management will be obtained by Monte Carlo simulation. Outcomes from the simulation model will be validated with available field data. Sensitivity analysis will be performed to evaluate the influence of model assumptions on conclusions of the study.
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-6992
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

 
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ACCESSION NO : 0196274 SUBFILE: CRIS
PROJ NO: CALV-2003-02398 AGENCY: CSREES CALV
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2003-35204-13374 PROPOSAL NO: 2003-02398
START: 01 AUG 2003 TERM: 31 JUL 2007 FY: 2007 GRANT YR: 2003
GRANT AMT: $157,000
INVESTIGATOR: Gardner, I. A.; Johnson, W. O.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet Med)
Davis , California 95616
NOVEL METHODS FOR IMPROVED CLASSIFICATION OF HERD DISEASE STATUS WITH APPLICATIONS TO BOVINE PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: Correct classification of herd status for animal pathogens is an integral component of disease control programs, risk-factor studies, health certification programs, and risk analyses related to animal movements. Our long-term research goal is to develop new methods to assess test accuracy for animal diseases, to improve classification of herd disease status and apply these methods to herd testing for bovine paratuberculosis. First, we will develop methods that use quantitative test results in the overall assessment of herd-level sensitivity and specificity. To achieve this objective we will use ELISA values for paratuberculosis infected and non-infected cattle and develop a generalized linear model that includes animal and herd-level risk factors. Second, we will develop Bayesian methods for estimating the herd-level sensitivity and specificity of testing systems for animal diseases when there are two conditionally independent or dependent tests. We expect that Bayesian methods will be superior to traditional frequentist methods for assessing the accuracy of herd-level tests when there is no "gold standard" and where tests are conditionally dependent. We will use paired ELISA and fecal culture results from three paratuberculosis studies. We will assess cut-off dependent and independent approaches for herd classification. Methods developed in the proposed research will be applicable to many infectious animal diseases where quantitative test results are available and where herd-level interpretation of test results in the basis of classification of disease risk.
OBJECTIVES: Develop and apply methods that account for the quantitative nature of test results in the overall assessment of herd-level sensitivity and specificity. Develop and apply Bayesian methods for estimating the herd-level sensitivity and herd-level specificity of testing systems for animal diseases when there is one test or two conditionally independent or dependent tests.
APPROACH: For objective 1, we will develop a general linear mixed model that discriminates the quantitative test scores from infected and non-infected animals. The model will allow for the possibility of animal-level risk factor (covariate) information such as age, lactation number, an indicator of purchased versus raised etc., and for herd-level information such as average age, type of ownership etc. In addition, we will extend this univariate model to a multivariate model that allows for several diagnostic tests. The model will be applied to ELISA and culture data for bovine paratuberculosis. Once this initial study is completed using "gold standard" information, we will extend the problem to the situation where the true status of individual animals is unknown. This will be achieved with use of a "mixture" model that is based on weighted average of the densities of test results corresponding to infected and non-infected animals. For objective 2, we will develop and compare cutoff-based and cutoff-independent methods of assessing herd-level sensitivity and specificity. The latter method allocates a herd as positive if the posterior probability that the herd is infected, given all observed data, is larger than a specified value e.g. 95%. The methods will be applied to one test in a single population, two tests in one population, and two tests in two or more populations. We will also allow for the possibility of sequential and simultaneous use of multiple tests. Our Bayesian approach will involve Gibbs sampling, a Markov chain Monte Carlo simulation technique, that allows for incorporation of existing information about test sensitivity and specificity, and disease prevalence.
PROGRESS: 2003/08 TO 2007/07
For objective 1, we developed a Bayesian receiver-operating characteristic (ROC) analysis that can be applied to multiple correlated diagnostic tests with and without a gold standard. Simulation studies showed that the discrimination ability of the no-gold standard (NGS) method was adequate compared with the gold standard (GS) method providing that the overlap between the two distributions of test scores was not too great. We used the proposed method to analyze results of 2 serum ELISAs and fecal culture for Johne's disease (JD). Data were available from 449 cattle with a positive fecal culture and from 393 cattle from JD free herds. Log transformation of ELISA S/P ratios was necessary to achieve bivariate normality. The Parachek ELISA had a greater area under the ROC curve than the HerdChek ELISA by both GS and NGS methods. The NGS method provided adequate discrimination only when the subset of Johne's infected cattle with fecal culture scores of ≥ 3 was used (Choi et al., 2005). One limitation of the Choi approach was the need to make the test results conform to bivariate normality. To overcome this restrictive assumption, we developed a general flexible modeling framework involving mixtures of Polya trees that can be used to evaluate the accuracy of continuous diagnostic tests and to estimate the predictive probability of disease based on serologic test values. For both scenarios, we assume the true disease status of the sampled units was unknown, although the proposed models and methods can be easily modified when true disease status is known. We considered the (i) the characterization of the performance of a diagnostic test using ROC curves and area under the curve, and (ii) the determination of predictive probabilities of disease based on diagnostic test results. When true disease status is unknown, a nonparametric (or parametric) analysis requires the distributions of serologic values for the diseased and non-diseased populations to be sufficiently separated. However, with covariate information that distinguishes diseased from non-diseased individuals, data from populations that have a relatively large overlap can be handled. We evaluated data from a Johne's disease study where the performance of an enzyme-linked immunosorbent assay was evaluated (Branscum et al., 2007). For objective 2, we developed Bayesian models to estimate herd-level test characteristics based on 4 different sampling schemes: a single test case and three sequential test cases. The corresponding herd-level characteristics were calculated and compared with different sample sizes, sampling schemes, animal-level sensitivity, specificity, and cut-off values. Models were developed to incorporate animal or herd-level risk factors and models with small herd size are also considered. We compared posterior estimates of animal and herd-level characteristics for these four sampling schemes with simulated data. Two examples, one for JD anda second for Salmonella in pig herds, were used to demonstrate application of the methods in field studies (Su et al., 2006). A hierarchical modeling approach has been submitted for publication (Choi et al., 2006).
IMPACT: 2003/08 TO 2007/07
Correct classification of herd status for animal pathogens is an integral component of disease control programs, risk-factor studies, health certification programs, and risk analyses related to animal movements. This classification is primarily dependent on the interpretation of diagnostic tests results at the herd-level. Our approach is based on Bayesian methods which allow incorporation of prior information and knowledge about test performance into the current study. We have written code in the shareware program WinBUGS so that it can be used widely. Code for these problems is available to users on our website, www.epi.ucdavis.edu/diagnostictests/ We are currently working with other university faculty and USDA personnel to improve herd classification, based on within-herd prevalence estimates as part of the Voluntary Bovine Johne's Disease Control Program. This is currently being done using a frequentist approach but our goal is to eventually change this to a Bayesian approach that will allow incorporation of other sources of information about the Johne's status of the herd.
PUBLICATIONS (not previously reported): 2003/08 TO 2007/07
1. Choi YK, Gardner IA, Johnson WO, Collins MT. Bayesian modeling of ROC curves without a gold standard. Proc 85th Conf Res Work Anim Dis, Chicago 2004 (abstract 70).
2. Choi YK, Johnson WO, Collins MT, Gardner IA. Bayesian inferences of receiver operating characteristic curves in the absence of a gold standard. J Agric Biol Environ Stat 2006;11:210-229.
3. Su CL, Gardner IA, Johnson WO. Bayesian estimation of cluster-level test accuracy based on different sampling schemes J Agric Biol Environ Stat 2007;12: 250 - 271
4. Su CL, Johnson WO, Gardner IA. Hierarchical modeling and estimation of the distribution of prevalences across subpopulations based non-dichotomized serologic data in the absence of a gold-standard test. J Applied Stat 2007 (submitted).
5. Branscum AJ, Johnson WO, Hanson TE, Gardner IA. Bayesian semiparametric ROC curve estimation and disease diagnosis. Statist Med 2007 (in revision).
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-1363
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

 
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ACCESSION NO: 0211165 SUBFILE: CRIS
PROJ NO: CALV-2007-01355 AGENCY: CSREES CALV
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2007-35204-18391 PROPOSAL NO: 2007-01355
START: 15 SEP 2007 TERM: 14 SEP 2009 GRANT YR: 2007
GRANT AMT: $374,906
INVESTIGATOR: Gardner, I. A.; Whitlock, R. H.; Sweeney, R.; Hovingh, E.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet Med)
Davis , California 95616
EPIDEMIOLOGIC CHARACTERISTICS AND DETECTION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS SUPER -SHEDDER" COWS IN DAIRY HERDS."
NON-TECHNICAL SUMMARY: Patterns of transmission of Mycobacterium avium subspecies paratuberculosis (MAP) in dairy herds are inadequately understood. Our goal is to define the role of cows shedding large numbers of MAP in feces (termed super-shedders) on the transmission of MAP infection in dairy herds and the relative importance of these animals in a successful Johne's disease management plan. Expected outcomes from the study include an improved case definition of a fecal super-shedder based on host demographic characteristics and a better understanding of the development and progression of the super-shedding state. Findings from these studies will provide critical information to the success of the Voluntary Bovine Johnes Disease Control Program, including potential diagnostic strategies to rapidly and cost-effectively identify super-shedder cows, especially in large dairy herds where the cost of detection can be prohibitive. If Johne's disease is to be effectively controlled on dairy farms, we speculate that super-shedder cows must be identified and managed to reduce the risk of exposure to herd mates. From a practical perspective, the development of a prioritization plan for culling of super-shedder cows, as determined by quantitative real-time polymerase chain reaction or other rapid diagnostic methods, will provide a complementary approach to that provided using likelihood ratios based on serum enzyme-linked immunosorbent assays.
OBJECTIVES: The specific objectives of the proposed research are to: 1) describe the epidemiologic characteristics of dairy cows at the super-shedding stage of Johnes disease focusing on host demographic and environmental factors, including development of a refined case definition of a super-shedder and characterization of the clinical course of disease, and 2) evaluate the cost-effectiveness of testing strategies to facilitate detection of super-shedders in dairy herds of varying size and prevalence of disease. The first objective will be achieved through 3 aims: 1.1) to describe the distribution of Mycobacterium avium subspecies paratuberculosis (MAP) colony counts in feces of super-shedder cows, 1.2) to characterize demographic factors that discriminate super-shedder from non super-shedding cows, and 1.3) to estimate the time to super-shedding and the duration and clinical course of super-shedding. The second objective has 2 aims: 2.1) to evaluate methods to identify super-shedder cow s from individual fecal and serum samples, and 2.2) to develop cost-effective methods to identify super-shedder cows in a large dairy herd.
APPROACH: Fecal samples in the Johnes Disease Research Laboratory repository and from 15 prospectively-monitored herds in Pennsylvania, New York, Vermont, and California will be used to assess the distribution of colony counts. Serial dilutions of fecal samples classified initially as heavy shedders on Herrolds egg yolk medium (HEYM) will be performed to determine the range and variability of MAP colony counts in super-shedder cows. Cows will be followed longitudinally in some herds by fecal culture and enzyme-linked immunoassay (ELISA) to define patterns of test results and identify demographic risk factors in cows that become super-shedders compared with cows that don’t become super-shedders. Risk factors will be assessed by random-effects logistic regression. Time to and the duration of super-shedding will be described by Kaplan-Meier survival methods. Testing methods to be evaluated for sensitive detection of individual super-shedders include routine HEYM culture for early growth, liquid culture, quantitative real-time (qrt) PCR, serum and milk ELISA and acid-fast staining of fecal smears. Sensitivities will be compared by McNemars chi-square test. These detection methods will then be used in combination with environmental sampling of the dairy environment to develop testing strategies for herd-level application in a herd with an estimated prevalence of super-shedder cows of approximately 1%. The cost-effectiveness of different testing strategies will be estimated as the ratio of its cost to detection probability. Marginal cost-effectiveness analysis will be used to compare the difference in costs of 2 methods to the difference in their detection probability and decision analysis will also be used to determine the optimal testing strategy.
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-6992
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

 
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ACCESSION NO: 0182013 SUBFILE: CRIS
PROJ NO: CALV-AH-176 AGENCY: CSREES CALV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2000 TERM: 30 SEP 2004 FY: 2003
INVESTIGATOR: Gardner, I. A.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet-Med)
Davis , California 95616
QUANTITATIVE METHODS TO CERTIFY FREEDOM OF ANIMALS FROM PATHOGENS.
NON-TECHNICAL SUMMARY: If countries and regions are able to "certify" freedom from important animal pathogens, trade opportunities may increase and product export costs may decrease. To develop a Bayesian statistical approach (using Gibbs sampling) to quantification of disease freedom. The output from the model will be probability distributions that can be used to make inferences about the proportion of diseased herds, within-herd prevalence, and the probability that a country is free of disease. The research will be involve collaboration with others in the US, Australia and Switzerland.
OBJECTIVES: 1. Develop a Bayesian approach to certify disease freedom of a country/region that incorporates uncertainty in probability estimates. 2. Compare frequentist and Bayesian approaches to certify disease freedom using common data sets and to compare sample size requirements for surveys with both approaches.
APPROACH: 1. The Bayesian approach will be implemented with the Gibbs sampler, an interactive Markov-chain Monte Carlo method. The mathematical calculations will incorporate the prior probability that a country is free of disease, the uncertainty in sensitivity and specificity estimates and the possible clustering of positive test results at a herd level. The output will be a probabilistic estimate of disease freedom. 2. Frequentist and Bayesian estimates will be compared with common published data sets on porcine reproductive and respiratory syndrome and Newcastle Disease. The effect of selected prior distributions for the Bayesian approach will be evaluated. Sample sites used in frequentist calculations for surveys will be compared with estimates that we will derive using Bayesian approaches.
PROGRESS: 2000/10 TO 2004/09
Quantitative methods to certify freedom from animal pathogens (no change from what was submitted last year (see below): Quantitative approaches are needed to allow scientifically-valid inferences about freedom of animals from important pathogens that affect animal trade locally, regionally and internationally. Freedom in the context of these inferences includes a herd prevalence of a pathogen less than a threshold value (e.g. <0.2% of infected herds). In a related project, we have developed Bayesian models to make probabilistic inferences for multiple herds and for single herds. The single herd model has both a binomial sampling version (relevant to a small sample of the herd e.g. 10% of animals) and a hypergeometric version (relevant to testing of the entire herd. In the model, expert prior information about the infection status of the herd, diagnostic test accuracy (sensitivity and specificity) and within-herd prevalence are used, when such data are available. Post-test probabilities versus pre-test probabilities of infection are presented in the novel form of a curve, summarizing the probability of infection over a range of possible prior probability values. The primary objective of this study is to collect field data to validate the suitability of the Bayesian models under field conditions. The model is currently being evaluated using serologic and fecal culture data for Mycobacterium paratuberculosis infection in 29 herds in the Central Valley of California. ELISA testing of 60 adult cows in each of the selected herds has been completed. Results were interpreted as per standard procedures at the California Animal Health and Food Safety Laboratory: <0.2 = negative, 0.2 to 0.35 = suspicious, and >0.35 = positive. For the 29 herds, the median number of positive or suspicious animals out of the 60 cows tested was 3 (range, 0 to 15). Five herds had no positive cows in 60 tested. We have completed follow-up ELISA testing of all adult cows in 2 of the herds with 0/60 positive on the initial screening and found 15/332 (4.5%) and 16/1144 (1.4%) ELISA-positive/suspicious samples. Fecal samples are currently being cultured from the 31 reactors to try and unequivocally establish whether these 2 herds are infected.
IMPACT: 2000/10 TO 2004/09
Our initial findings indicated that screening of 60 lactation-2 or older cows with ELISA in large California dairy herds (median size, 700 cows) provides a reasonable balance between cost of testing and failing to detect low prevalences of M. paratuberculosis infection.
PUBLICATIONS (not previously reported): 2000/10 TO 2004/09
1. Gardner IA., T. E. Carpenter, and M.T. Collins. 2002. Risk of introduction of Mycobacterium paratuberculosis into dairy herds: effects of prevalence and test sensitivity. Presented at the 7th International Conference on Paratuberculosis, Bilboa, Spain. June, 2002.
2. Gardner I.A., T. E. Hanson, and W.O. Johnson. 2002. Bayesian inference about infection status of cattle herds with Mycobacterium paratuberculosis. Presented at the 7th International Conference on Paratuberculosis, Bilboa, Spain. June, 2002.
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-6992
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

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ACCESSION NO: 0204476 SUBFILE: CRIS
PROJ NO: CALV-AH-223 AGENCY: CSREES CALV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 APR 2005 TERM: 31 MAY 2010 FY: 2007
INVESTIGATOR: Gardner, I. A.; Adaska, J. M.; Whitlock, R. H.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet-Med)
Davis , California 95616
ECOLOGY AND DETECTION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN DAIRY HERDS.
NON-TECHNICAL SUMMARY: There are many deficiencies in existing knowledge about the transmission and detection of Map in cattle herds, including how to most effectively use currently-available tests for diagnosis of Map infection. This project will assess persistence of the Map on dairy farms and methods for quantification of bioburden.
OBJECTIVES: 1. To quantify and evaluate the role of the environmental bio-burden of Mycobacterium avium subsp. paratuberculosis (Map) in the transmission of Johne's disease on dairy farms 2. To evaluate optimal testing strategies for detection of Map in dairy herds
APPROACH: For objective 1, numbers of Map bacteria in multiple environmental locations in dairy herds will be quantified using Herrold's egg yolk medium and the Trek ESP II system. Cultures which yield values that are very high shedders will be quantified further by serial dilution to estimate the most probable numbers of bacteria in the samples. For objective 2, a stochastic simulation model will be modified to evaluate various testing (including use of environmental samples) and sampling methods for detection of Map in dairy herds.
PROGRESS: 2007/01 TO 2007/12
Our investigations are being done in collaboration with the University of Pennsylvania in a California dairy herd which currently has 3577 cows (aggregated in 14 strings). In 2005 and 2006, we identified at least 6 super-shedder cows based on limited follow-up sampling. Our working case definition for a super-shedder was based on quantitative real-time PCR results (Tetracore, Rockville, MD) and large numbers of colonies on Herrold's egg yolk medium (HEYM ) when feces were diluted 1:100 and/or 1:1000 for culture. One super-shedder cow was moved to Tulare and housed at the VMTRC while follow-up testing was done. In brief, the main findings from longitudinal follow-up from February 2006 to June 2006 were: * Tetracore qrt PCR results were mostly between 18 and 21 cycles (equivalent to 381,000 to 2.1 million CFU/gram of feces) * IDEXX ELISA was initially positive on February 27 (S/P ratio = 0.968) but declined and became negative (S/P ratio <0.25) from March 15 until euthanasia on June 22. For much of that time period, the cow was non-clinical. * Biocor ELISA was positive at all time points * Milk ELISA and Milk PCR were consistently positive after the cow calved on June 6 In our current herd test (October 2007), we ranked the 14 production strings according to qrt PCR cycle to threshold (CT)
values and the likely risk of a 1 or more super-shedder cows being present. Estimated colony counts are based on predicted values from work of our collaborators. At least 4 strings have the equivalent of >30 colony forming units (CFU)/tube and are likely to have super-shedder cows. Pen no. Description of females No.cows CT value Estimated CFU on 4 tubes of HEYM 9 Pre-dry special 240 28.7 821 12 Close-up heifers and cows 152 30.3 323 4 Cows 294 31.1 202 47 Dry cows (group 1) 197 31.8 134 48 Dry cows (group 2) 200 32.8 75 10 Fresh cows 278 32.8 75 7 Pre-dry 269 32.9 71 2 Heifers 201 34.8 23 6 Cows 300 35.0 21 3 Heifers 307 35.3 17 1 Fresh heifers 172 35.6 15 5 Cows 298 35.6 15 8 Pre-dry 285 35.7 14 11 Mixed group 357 36.1 11 We have tested 41 cows with loose feces (noted at the time of the herd test) and identified 9 with qrt PCR values from
18 to 32 (potential super-shedders). Of these 9 cows, 2 are negative on the IDEXX ELISA (S/P ratios of 0.04 and 0). Four additional possible super-shedder cows have been identified from fecal pool testing with CT values from 22 to 29. We expect to have all PCR testing completed by the end of January 2008 and have our cohort of cows (super-shedders, moderate-to-low shedders, and non-shedders) enrolled for longitudinal follow-up.
IMPACT: 2007/01 TO 2007/12
Cows shedding large numbers of MAP pose a tremendous risk for transmitting the organism to other cattle on the farm, and they may also contribute to passive ("pass through") fecal shedding of MAP by uninfected cows, and thus false-positive fecal culture results. That some fecal culture results (low and moderate shedders) might be false positives is a major paradigm shift for management of bovine paratuberculosis and also has implications for evaluation of serologic tests for MAP because most investigators use fecal culture as the gold standard. Prioritization of cows for culling based on fecal qrt PCR results rather than waiting for culture results (HEYM or Trek ESP II) has the potential to be a timely strategy for better managing cows according to their current risk of contaminating the dairy environment. In addition, we are using qrtPCR for a beef herd in the Johne's disease Demonstation Herd Project since obtaining rapid results (in addition to accuracy) is key to their utility to herd owners.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
1. Tavornpanich S, Gardner IA, Anderson RJ, Carpenter TE. Cost-effectiveness of targeted sampling methods for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds; American Journal of Veterinary Research 2006; 67: 821-828
2. Tavornpanich S, Munoz-Zanzi CA, Wells SJ, Raizman EA, Carpenter TE, Johnson WO, Gardner IA. 2007. Simulation model for evaluation of testing strategies for detection of paratuberculosis in Midwestern US dairy herds. Prev Vet Med. 2007; Aug 22; [Epub ahead of print].
3. Messam LL, Branscum AJ, Collins MT, Gardner IA. Frequentist and Bayesian approaches to prevalence estimation using examples from Johne's disease. Animal Health Research Reviews 2007 (in press)
4. Tavornpanich S, Johnson WO, Anderson RJ, Gardner IA. Herd characteristics and management practices associated with paratuberculosis seroprevalence in California diary herds. American Journal of Veterinary Research 2007 (in press).
5. Abstracts, presentations, and submitted manuscripts: Whitlock RH, Gardner IA, Mangold BL, Smith J, Sweeney RW, Schukken Y, Van Kessel J, Hovingh E, Karns J, Wolfgang D, Fyock T. Johne's disease: Mycobacterium paratuberculosis super-shedders: Detection and contribution to passive shedding (false positive fecal cultures). Proceedings of American Association of Bovine Practitioner's Meeting, St Paul, Minnesota, September 2006
6. Adaska JM, Whitlock RH, Gardner IA. Peri-parturient bacterial shedding and ELISA testing of a Johne's disease 'super-shedder' cow. American Association of Veterinary Laboratory Diagnosticians, Reno, Nevada, October 2007
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-6992
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

 
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ACCESSION NO: 0209349 SUBFILE: CRIS
PROJ NO: CALV-APHIS-05-9106 AGENCY: CSVM CALV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 19 AUG 2005 TERM: 18 AUG 2006 FY: 2007
INVESTIGATOR: Cullor, J. S.
PERFORMING INSTITUTION:
Population Health & Reproduction
Univ of California (Vet-Med)
Davis , California 95616
THE USE OF A MULTIPLEX REAL TIME PCR ASSAY FOR THE SCREENING OF MILK FILTERS AND ENVIRONMENTAL SAMPLES FOR MYCOBACTERIUM AVIUM SUBSP PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: Many existing diagnostic tests for the detection of Johne's disease lack sensitivity in the earlier stages of diseases and culture based tests are often expensive, laborious and time consuming. This project aims to develop a diagnostic method that is able to obtain results faster and analyze a larger sample weight than culture. This will be extremely helpful for the screening of dairy herds for the presence of MAP.
OBJECTIVES: This project aims to apply real-time polymerase chain reaction (PCR) methods for the diagnosis of MAP.
APPROACH: This study will: 1) Optimize a semi-quantitative multiplex real-time PCR to detect MAP and identify the presence of potential inhibitors in the samples, thus reducing the likelihood of false negative results. 2) Use the real-time PCR method to screen in-line milk filters and environmental samples (cow alleyway and wastewater lagoon) from 33 dairy herds in California, Minnesota and Pennsylvania. 3) Evaluate the use of the PCR assay on milk filters and environmental samples as a tool for identifying MAP infected herds.
PROGRESS: 2005/08 TO 2006/08
OUTPUTS: The major output of this project was to develop and validate a novel test assay to be used on milk filters to help screen and prevent the spread of bovine paratuberculosis (Johne's disease). Thus it sought to provide a product (test system) that could be of service to the cattle industry and food animal clinicians. The developed test assay was developed, optimized and characterized and used in a collaborative project with the USDA to screen milk filters from herds with high incidences of paratuberculosis. The results of the developed PCR assay where also compared with standard culture methods. The results were analyzed and disseminated to the collaborators and summarized in manuscripts for publications. PARTICIPANTS: The project formed the basis of a successful PhD thesis in Comparative Pathology. The student is now participating in a post doctoral program in the same field. Other participants include other University (collaborative) researchers from Pennsylvania and Minnesota. The Dairy Food Safety Laboratory (DFSL) in Davis performed the real-time PCR and Dr. Whitlock, in Pennsylvania cultured the samples. A total of Thirty three dairy herds (23 in California, 5 in Minnesota and 5 in Pennsylvania) were sampled and analyzed with a great amount of collaboration. TARGET AUDIENCES: The target audiences included graduate students as well as professionals in the dairy cattle industry, veterinary clinicians and regulatory personnel from the FDA and USDA.
IMPACT: 2005/08 TO 2006/08
The major impact of this project was the development and characterization of a potential test system to be used by veterinary clinicians to assess the paratuberculosis status of a given herd. The knowledge supplied by this assay may impact decision making in the screening of cattle herds for Johne's disease and may lead to changes in efforts to control the spread of this important dairy industry disease. For example, it was found that the concentration of the Johne's agent on milk filters may be too low to detect via PCR and/or culture, suggesting that higher sample numbers or greater sample volumes may need to be screened. It was also found that the developed PCR assay was as sensitive as conventional culture in screening environmental samples on the farms, and thus could lead to a change in herd sampling methods. Ultimately, a better way to screen for the Johne's agent on the farm may lead to changes in animal husbandry to prevent or limit the on-farm spread of this disease.
PUBLICATIONS (not previously reported): 2005/08 TO 2006/08
Ruzante Juliana M., Wayne L. Smith, Ian A. Gardner, Charles G. Thornton, James S. Cullor. 2006 Modified culture protocol for isolation of Mycobacterium avium subsp. paratuberculosis from raw milk. Foodborne Pathogens and Disease Vol 3 (4)
PROJECT CONTACT:
Name: Cullor, J. S.
Phone: 559-688-1731
Fax: 559-686-4231
Email: jscullor@vmtrc.ucdavis.edu

 
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ACCESSION NO: 0209312 SUBFILE: CRIS
PROJ NO: CALV-APHIS05-9706150 AGENCY: CSVM CALV
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 12 SEP 2005 TERM: 31 DEC 2010 FY: 2007
INVESTIGATOR: Ardans, A.
PERFORMING INSTITUTION:
California Animal Health and Food Safety Laboratory System (CAHFS)
Univ of California (Vet-Med)
Davis , California 95616
JOHNES DISEASE DEMONSTRATION PROJECT IN INFECTED HERDS IN CALIFORNIA.
NON-TECHNICAL SUMMARY: There is a need to not only demonstrate effectiveness of the standard disease management recommendations for Johne's Disease in very large milking herds, but also to use epidemiologic methods to determine the validity of the recommendations that do not fit into current California dairy practices. This project will study Johne's disease demonstration dairy herds in California and the factors that play a role in the infection, transmission and expansion of Mycobacterium avium subspecies paratuberculosis (MAP) within very large dairy herds.
OBJECTIVES: One of the major goals of Johne's disease control in the United States is to enroll the majority of dairy owners in control programs. Such control programs also need to focus on having the largest possible numbers of animals included. Because western states dairy owners make the majority of decisions based on economic perceptions, it is necessary to determine if Johne's disease has a significant economic impact on large western dairies. This project will test the long held belief that the aggressive culling practices used by western dairies negate any potential negative effect of Johne's disease. If a negative economic effect is demonstrated, even in the face of 'typical' western management methods, the likelihood of western dairy owners embracing Johne's disease control programs should be increased.
APPROACH: The following questions will be addressed: 1) Do the standard management recommendations for dairies effectively decrease and control the incidence and prevalence of Johne's disease, and other fecal-orally transmitted pathogens, in large California dairies? 2) What is the economic impact of Johne's disease in typical large California dairy herds with aggressive culling practices.
PROJECT CONTACT:
Name: Ardans, A. A.
Phone: 530-752-8709
Fax: 530-752-5680
Email: aaardans@ucdavis.edu

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ACCESSION NO: 0202431 SUBFILE: CRIS
PROJ NO: CALV-CDRF-03-ADJ-01- AGENCY: CSVM CALV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JAN 2003 TERM: 31 DEC 2004
INVESTIGATOR: Adaska, J. M.
PERFORMING INSTITUTION:
California Animal Health and Food Safety Laboratory System (CAHFS)
Univ of California (Vet-Med)
Davis , California 95616
PREVALENCE OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN WASTE MILK DELIVERED TO FIVE CALIFORNIA CALF RANCHES.
NON-TECHNICAL SUMMARY: Johne's disease is a chronic intestinal disease of cattle that has received much attention lately due to attempts to construct a voluntary national control program. This project examines the possibility that waste milk fed to calves on calf ranches contains Mycobacterium avium subspecies paratuberculosis (MAP) and poses a significant threat for the amplification of Johne's disease in California dairies.
OBJECTIVES: A large percentage of dairy calves in California are raised on calf ranches and the majority of those ranches feed waste milk. This waste milk is a potential source of Mycobacterium avium subspecies paratuberculosis (MAP) and therefore may be a significant risk for the amplification of Johne's disease in the California dairy herd.
APPROACH: Evaluation of the prevalence of MAP in the waste milk as it enters the calf ranch and as it is fed will determine the level of risk of MAP.
PROGRESS: 2003/01 TO 2004/12
The main objective of the project was to determine whether or not the causal organism of Johnes disease, Mycobacterium avium ssp. Paratuberculosis (MAP), was present and viable in pre- and post-pasteurized samples of hospital milk being fed to calves on calf ranches. We initially enrolled five calf ranches but one dropped out after the initial sampling period leaving data from four for analysis. Three of the calf ranches each collected milk from approximately forty dairies while the fourth collected milk from 15 dairies. All four ranches reported that they heated milk to approximately 165oF and had holding time of 2-3 minutes. We were able to identify MAP by PCR in about 5% of pre-pasteurization samples and from the same percentage of post-pasteurization samples. There was no agreement between which samples were positive pre-pasteurization and which were positive post-pasteurization. It needs to be kept in mind that PCR identifies the presence of DNA and can be positive even in samples where no viable or infectious organisms are present. By culture we have been able to identify viable organisms in 2.5% of the total samples with an equal number found in pre-pasteurization samples and post-pasteurization samples. The results of this study indicate that hospital milk collected from several dairies and fed to calves on calf ranches is a potential source of infection of calves with the causal organism of Johnes disease. We were able to isolate MAP from post-pasteurization samples indicating that the pasteurization methods currently in use on the calf ranches in this study (165oF for 2-3 minutes) may not be adequate to kill MAP in hospital milk. Because the pasteurization equipment was not evaluated or monitored during the course of the study however it is possible that technical problems with the equipment resulting in contamination of post-pasteurization samples or inadequate temperature or time of pasteurization may have resulted in inadequate killing of MAP. The significance and benefit to the dairy industry from this work is that a major question, whether dilution, by combining hospital milk from several dairies, would result in MAP being undetectable and presumably an insignificant source of infection in milk fed to calf ranch calves. Further, the study answered the question of whether or not on-farm pasteurization eliminates 100% of the viable organisms in those same milk samples. 3. Conclusions: Mycobacterium avium ssp paratuberculosis was detected by PCR from approximately 5% of hospital milk fed to calves on four California calf ranches both before and after on-farm pasteurization. In addition, viable MAP was cultured from about 2.5% of the same milk samples. The professional calf raisers and the dairies that contract with them need to consider these findings when feeding calves and consider raising pasteurization temperatures and time or using commercial milk replacer rather than hospital milk to feed the calves.
IMPACT: 2003/01 TO 2004/12
The results of this study indicate that the causal organism of Johnes disease, Mycobacterium avium ssp paratuberculosis, is present in a small percentage of waste milk delivered to large calf ranches and indicate that the organism is capable of surviving the pasteurization efforts used on those same calf ranches. These results suggest that calf ranch operators need to consider improved pasteurization methods or feeding only milk replacer to calves in order to minimize the potential transmission of Johnes disease in the calves they raise.
PUBLICATIONS (not previously reported): 2003/01 TO 2004/12
No publications reported this period
PROJECT CONTACT:
Name: Adaska, J. M.
Phone: 559-688-7543
Fax: 559-686-4231
Email: jmadaska@ucdavis.edu

 
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ACCESSION NO: 0202440 SUBFILE: CRIS
PROJ NO: CALV-CDRF03-GAI-02-D AGENCY: CSVM CALV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JAN 2003 TERM: 30 DEC 2004 FY: 2004
INVESTIGATOR: Gardner, I. A.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet-Med)
Davis , California 95616
CERTIFICATION OF CALIFORNIA CATTLE HERDS AS FREE OF JOHNE'S DISEASE USING BAYESIAN METHODS.
NON-TECHNICAL SUMMARY : The availability of rapid, low cost and accurate diagnostic tests and testing strategies is critically important to dairy producers, herd veterinarians and regulatory authorities involved in the detection and control of bovine paratuberculosis. Existing tests are considered to have only low to moderate sensitivity in subclinically infected animals, but high to very high specificity. This project investigates alternative approaches to develop more cost-effective ways to obtain information of similar diagnostic utility.
OBJECTIVES: This project will examine whether Bayesian methods can be successfully applied to analysis of Johne's Disease herd-test results, thereby better accounting for uncertainty in test performance, prevalence estimates and the herd-specific present probability of JD.
APPROACH: 1. Determine the sensitivity of pooled fecal culture relative to individual fecal culture. 2. Estimate the prevalence of infected dairy herds and within-herd prevalence based on pooled fecal culture results and individual animal results.
PROGRESS: 2003/01 TO 2004/12
The objectives of the study were to 1) estimate the sensitivity of pooled fecal culture(10 randomly-selected samples per pool) for detection of Mycobacterium avium subsp. paratuberculosis (Map) in large dairy herds and 2)assess the utility of the method for estimation of Map prevalence. A cross-sectional study design that involved random sampling of 60 cows in each of 29 California dairy herds was used. The herds ranged from 285 to 2233 cows and had no or minimal previous testing for Map. Testing of serum was done with the IDEXX ELISA and fecal samples (individual and pooled) were tested by Herrolds egg yolk agar (HEY) and Trek-ESP para-JEM system II (ESP). Bayesian methods were used to estimate animal-level prevalence and herd sensitivity of pooled fecal culture. Differences between overall animal-level prevalence based on pooled data and individual test results were compared. Sensitivity of pooled fecal culture across all herds was estimated to be 0.71 (27 positive pools of 38 pools that were Map positive). Sensitivity increased as the number of culture-positive samples in a pool increased. Pooled fecal culture detected 30% of rare (mean, 0.25 to 4 CFU/tube), 67% of low to moderate (mean, >4 to 70 CFU/tube), and 100% of high (mean, >70 CFU/tube) shedders. Detection by the ESP method was not significantly different from HEY and the mean days-to-detection of ESP was 24 compared with 12-16 weeks by HEY. Animal-level prevalence (based on fecal culture alone) was estimated to be 4% with a 95% probability interval of 2 to 6% based on pooled fecal culture results. Herd-level sensitivity ranged from 90% to 100% and was dependent on prevalence in the population and the sensitivity of pooled fecal culture. When animal-level prevalence was estimated with a combination of ELISA and fecal culture, prevalence estimates were about 2-fold higher, indicating that ELISA is detecting some truly infected animals not detected by culture.
IMPACT: 2003/01 TO 2004/12
Use of fecal pools from 10 cows was a cost-effective tool for herd screening and might provide a good estimate of the percentage of Map-infected cows in low prevalence herds with minimal amounts of previous testing. Other low cost detection methods such as environmental sampling are currently being evaluated by our group in collaboration with the University of Minnesota and the Centers for Epidemiology and Animal Health. The Bayesian approach allowed for updated estimation of the performance of the ELISA, individual fecal culture and pooled fecal culture based on expert opinion and data from the current study. The present study further demonstrated the utility of Bayesian methods for evaluation of test accuracy and estimation of paratuberculosis prevalence.
PUBLICATIONS (not previously reported): 2003/01 TO 2004/12
1. Tavornpanich, S., I.A. Gardner, R.J. Anderson, S. Shin, R.H. Whitlock, T.Fyock, J.M. Adaska, R.L. Walker, and S.K. Hietala. Evaluation of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp. paratuberculosis in large dairy herds. American Journal of Veterinary Research 2004; 65:1061-1070.
2. Crossley, B.M., F.J. Zagmutt-Vergara, T.L. Fyock. R.H. Whitlock, and I.A. Gardner. Fecal shedding of Mycobacterium avium subsp. paratuberculosis by dairy cows. VeterinaryMicrobiology 2005 (in press).
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-1363
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

 
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ACCESSION NO: 0198804 SUBFILE: CRIS
PROJ NO: CALV-UCBIO02-102 AGENCY: CSVM CALV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 28 JAN 2003 TERM: 27 JAN 2005
INVESTIGATOR: Cullor, J. S.
PERFORMING INSTITUTION:
Population Health & Reproduction
Univ of California (Vet Med)
Davis , California 95616
DIAGNOSTIC TESTING AND MANAGEMENT SCHEMES FOR THE CONTROL OF THE JOHNE'S AGENT IN MILK.
NON-TECHNICAL SUMMARY: Johne's disease is a chronic infectious and wasting bacterial disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP) and occurs worldwide. The PI has undertaken preliminary work to develop a rapid and sensitive Johne's non-invasive diagnostic and a management program for eradication of Johne's disease. This project will take the next step to incorporate the diagnostic into Dairy-BTM (Dairy Breakthrough Management - a HACCP program) and develop an interactive software program.
OBJECTIVES: The specific aim of this project is to further develop, refine and test an improved Johne's diagnostic test on both manure and milk and then integrate the test into an existing Johne's management program that is then reduced to farm-ready interactive software. This project is supported by the UC Star Biotechnology Program, RZ Syntopical Technologies and California Dairy Research Foundation.
APPROACH: The two fold approach is: Can spiral plating technology be used to generate a rapid sensitive and specific Johne's disease assay and 2) Can the Johne's spiral plate assay be incorporated into a management program, reduced to software, that is effective in preventing Johne's infection in non-impacted herds and eliminating Johne's disease in impacted herds?
PROGRESS: 2003/01 TO 2005/01
A rapid culture method for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in raw milk has been developed. The method incorporated a zwitterionic detergent that concentrates MAP bacteria (CB18) and spiral plating with microscopic colony screening. Raw bulk tank milk from a paratuberculosis negative herd was spiked with different concentrations of MAP (10(6) to 10cfu/ml) and processed using this novel method as well as the conventional method (HEYM). Time and limit of detection was recorded and compared between the two assays. Both essays were repeated independently eight times. On average, the presence of MAP in spiked milk samples could be detected between 14 and 45 days (mean=23) using the CB18/spiral plater method and between 21 and 63 days (mean=30.9) using the HEYM culture method (the time to detection between the two methods was statistically different at p<0.001). Samples containing higher concentrations of spiked MAP were detected earlier than the samples containing lower concentrations in both methods and the limit of detection did not differ statistically. A real time PCR assay using CB18 was also developed and evaluated. Bulk tank milk was spiked with 10(4) to 1cfu of MAP per ml. Different DNA extraction methods, milk volumes (5 versus 12.5ml) and PCR probes were tested and compared. The best assay method was able to detect 100% of the samples spiked with 100cfu/ml (9 out of 9), 55% of the samples spiked with 10cfu/ml (5 out of 9) and 20% of the samples spiked to 1cfu/ml (1 out of 5). Both the real time PCR and milk culture methods using CB18 are currently being tested on samples from dairy farms. The DFSL is collaborating with other UC Davis investigators who have been providing pre and post pasteurized waste milk from calf ranches in the Central Valley, as well as bulk tank milk samples from dairies.
IMPACT: 2003/01 TO 2005/01
Johne's is a contagious bacterial disease of ruminants that has significant animal health, economic, and potential human health consequences. The results thus far are providing the preliminary data for designing field testing experiments to determine sensitivity and specificity of the assay so that positive and negative predicted value versus disease prevalence can be determined. This data will eventually be used in the development of on-farm management protocols to control the spread of Jone's disease.
PUBLICATIONS (not previously reported): 2003/01 TO 2005/01
No publications reported this period
PROJECT CONTACT:
Name: Cullor, J. S.
Phone: 559-688-1731
Fax: 559-688-4231
Email: jscullor@ucdavis.edu

 
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ACCESSION NO: 0209358 SUBFILE: CRIS
PROJ NO: CALV-UMINNESOTA-04- AGENCY: CSVM CALV
PROJ TYPE: STATE PROJ STATUS: NEW
START: 01 AUG 2004 TERM: 31 JUL 2008 FY: 2007
INVESTIGATOR: Gardner, I. A.
PERFORMING INSTITUTION:
Medicine & Epidemiology
Univ of California (Vet Med)
Davis , California 95616
EVALUATION OF CRITICAL CONTROL POINTS IN YOUNGSTOCK AND ADULT DAIRY COW MGT TO REDUCE TRANSMISSION OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCUL[OSIS].
NON-TECHNICAL SUMMARY: Off-site rearing of heifers has potential to reduce exposure to M. paratuberculosis especially in infected herds that raise heifers on site and have high environmental exposures to the organism. This project will evaluate whether heifer calves reared at off-site facilities have a lower risk for developing subclinical Johne's Disease as adults.
OBJECTIVES: This project studies the hypothesis: Heifer calves that are reared at off-site facilities will have a lower risk for developing subclinical Johne's Disease (as adults) and greater longevity in the herd than heifer calves reared on-site in herds with a higher prevalence of infection.
APPROACH: The study will be conducted in 2 herds of >1000 cows that currently have a high seroprevalence (>10% ELISA positive cows) and where M. paratuberculosis has been confirmed in environmental samples. In each herd, 200 heifer calves will be randomly allocated to each treatment group (off-site or on-site rearing). Based on the required sample sizes, enrollment of calves will take approximately 12 months. Follow-up will involve annual testing of all trial heifers from 24 months of age by fecal culture and ELISA. Mortality and culling data will be recorded including the date of the vent and the associated reason. Survival analysis using Cox proportional hazards model will be used to describe the effect of off-site rearing (compared with on-site rearing) on risk and age of onset of subclinical Johne's Disease, as indicated by a positive serum ELISA or fecal culture test (two separate models) while controlling for random herd effects and other important covariates such as age at removal to off-site facility, JD status of dam (if known) and time at off-site facility. Since off-site production has an additional cost of about $1.50 per day over on-site production, a benefit-cost analysis will be required in addition to the statistical analysis.
PROGRESS: 2007/01 TO 2007/12
The goal of this study is to complete a series of prospective on-farm controlled field studies to critically evaluate the efficacy, magnitude of impact, and cost-effectiveness of several commonly recommended practices surrounding the management of young stock and adult dairy cattle, for reducing the transmission of Mycobacterium avium subspecies paratuberculosis (Map) in infected dairy herds. Studies are being done in Minnesota (objectives 1, 3, and 4) and Californian (objective 2). Project objectives are to 1. Evaluate the effect of maternity pen management (individual vs. group maternity pens) on the transmission of Map to newborn calves. 2. Evaluate the effect of off-site (vs. on-site) heifer rearing on the transmission of Map in young stock. 3. Evaluate the effect of feeding of bovine colostrum (vs. a commercial colostrum substitute) on the risk for transmission of Map in newborn calves. 4. Evaluate the effect of feeding pasteurized waste milk (vs. commercial milk replacer) to control transmission of Map in dairy calves For objective 2, we have enrolled 3 cohorts of approximately 350 heifers each in a herd where there is a high ongoing environmental burden of Map and hence, likely risk of Map infection in the newborn animals Cohort 1 was totally raised on site; cohort 2 was raised on site until about 5 months of age and then off-site in Nevada; and cohort 3 was raised totally offsite at a heifer ranch that uses milk replacer with subsequent rearing in Nevada. The study is in its early phases in terms of results. All springing heifers, aged approximately 22-24 months, have been ELISA negative and fecal culture negative when tested. Most trial animals are currently in lactation 1 and a few are in lactation 2. Our plan is to test (ELISA and fecal culture) all enrolled females by ELISA and fecal culture at dry-off at the end of lactations 1, 2, and 3 and at culling from the herd to compare time to Map seroconverson. Effects of cohort rearing method on milk production and reproductive performance will be assessed.
IMPACT: 2007/01 TO 2007/12
As we are only mid-way through to completion of these long-term projects, final results will require an additional 2 years of follow-up. Once completed, this research will improve our understanding of the importance of various modes of transmission for Map and will contribute to the development of more comprehensive Johne's disease control programs that are both scientifically sound and cost-effective.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
No publications reported this period
PROJECT CONTACT:
Name: Gardner, I. A.
Phone: 530-752-7363
Fax: 530-752-0414
Email: iagardner@ucdavis.edu

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ACCESSION NO: 0174289 SUBFILE: CRIS
PROJ NO: COL00101 AGENCY: SAES COL
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 MAR 1999 TERM: 30 JUN 2004 FY: 2004
INVESTIGATOR: Sommers, L. E.; Auld, G. W.; Burns, P. D.; Byrne, P. F.; Garry, F. B.; Kelly, E. F.
PERFORMING INSTITUTION:
Administration
Colorado State University
Fort Collins , Colorado 80523
INTERDISCIPLINARY RESEARCH IN ANIMAL, PLANT AND ENVIRONMENTAL SCIENCE.
NON-TECHNICAL SUMMARY: Interdisciplinary teams are needed to solve complex problems in agricultural production systems. This project examines Johne's disease, use of GIS for precision agriculture, reproduction in beef cows, and stress tolerance in wheat.
OBJECTIVES: Research will be conducted by interdisciplinary teams of scientists in the following areas: 1) investigate the development of new food links between local producers and organizations serving food; 2) evaluate regulation of prostaglandin synthesis and efficacy of feeding fish meal in ruminants; 3) identify physiological mechanisms and selection criteria for stress tolerance in wheat; 4) investigate the epidemiology, molecular genetics, and immunology of Johne's disease; and 5) develop techniques for improved use of spatial statistics and remote sensing in precision farming.
APPROACH: Laboratory and field experiments will be conducted by 5 teams of researchers to accomplish the stated objectives.
PROGRESS: 1999/03 TO 2004/06
I. Creating local food links - This sub-project was designed to promote local, sustainable food links; develop an educational model to promote these food links; and test an interpersonal approach to encourage new links between local food producers and organizational buyers. Over 250 phone interviews were conducted with food buyers and producers to identify barriers and motivators associated with direct marketing of local foods. An educational video and pamphlets were produced. Meetings were organized between farmers and food buyers. II. Regulation of Prostaglandin Synthesis in Domestic Farm Animals - The sub-project improved understanding of how oxytocin regulates endometrial secretions of prostaglandin and investigated whether feeding fish meal to beef cows during the first 21 days of breeding season increased conception rates. Experiments conducted showed that fishmeal supplementation increased fertility by 15% in postpartum first-calf beef cows. It was hypothesized that the omega-3 fatty acids found in fishmeal become incorporated in uterine endometrium and attenuate PGF2a synthesis during maternal recognition of pregnancy and result in enhanced fertility. Several experiments to determine the molecular and cellular mechanisms that regulate uterine PGF2a synthesis were conducted. III. Improved heat and drought tolerance of Colorado wheat - This sub-project analyzed long-term weather data for eastern Colorado to estimate the frequency and severity of heat and drought stress in relation to wheat developmental stage. Twelve common Colorado wheat cultivars were compared under irrigated and dryland conditions for 2 years and were evaluated for biochemical/physiological characters associated with stress tolerance. Genes were located for Russian Wheat aphid resistance and for yield components under stressed and nonstressed conditions in a recombinant inbred line population. IV. Interdisciplinary program in Johne's disease research - The objectives were to study the epidemiology, molecular genetics, and immunology of paratuberculosis. Seroprevalence studies of Johne's disease were conducted repeatedly on more than 10,000 cows at 16 dairies, and data were compared with other features of the herds. DNA was sequenced and genetic probes were developed. A survey was administered to Crohn's disease patients and to control groups to examine the potential relationship between the agent of Johne's disease and the occurrence of Crohn's disease in humans. V. Building soil landscape models for soil inventories and precision farming - This subproject developed and refined the program of spatial statistics, functions and GIS techniques used towards extracting detailed geographic information from the landscape; developed protocols for the utilization of remote sensing data and progressive soil surveys; and assessed and evaluated satellite data for each set of protocols. Landsat TM imagery was utilized to construct a base map for Costilla county and individual maps of Landsat spectral bands. Vegetation and 3-D terrain maps were developed. Maps, software, and other materials were customized to meet the needs of field scientists.
IMPACT: 1999/03 TO 2004/06
Creating local food links - The research suggests that there are opportunities for direct links between producers and restaurant buyers, but success will likely require co-ops of farmers. Regulation of Prostaglandin Synthesis in Domestic Farm Animals - Results of this project indicate that extracellular signal regulatory kinase (ERK) plays a critical role in regulation of uterine PGF2a synthesis. Improved heat and drought tolerance of Colorado wheat - It was found that a chromosome arm translocation from rye contributes to heat tolerance in wheat. It was found that April rainfall is the most important weather variable for predicting wheat yield in Colorado.
PUBLICATIONS (not previously reported): 1999/03 TO 2004/06
No publications reported this period
PROJECT CONTACT:
Name: Sommers, L. E.
Phone: 970-491-5371
Fax: 970-491-7396
Email: lsommers@lamar.colostate.edu

 
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ACCESSION NO: 0196328 SUBFILE: CRIS
PROJ NO: COLV-BELISLE AGENCY: CSREES COLV
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2003-35204-13685 PROPOSAL NO: 2003-02501
START: 15 SEP 2003 TERM: 14 SEP 2006 FY: 2006 GRANT YR: 2003
GRANT AMT: $200,000
INVESTIGATOR: Belisle, J. T.; Orme, I. M.
PERFORMING INSTITUTION:
Microbiology
Colorado State University
Fort Collins , Colorado 80523
PROTEOMIC DEFINITION OF T CELL ANTIGENS OF JOHNE'S DISEASE.
NON-TECHNICAL SUMMARY: Johne's disease has a major economic impact on the cattle and dairy industry. The inability to control this disease is due to the lack of an effective early diagnosis or effective vaccine. This project will use modern biochemical and immunological approaches to identify the immunodomnant T cell antigens produced by the bacterial agent that causes Johne's disease, Mycobacterium paratuberculosis. These antigens will represent potential vaccine and early diagnostic targets.
OBJECTIVES: The objective of this project are to identify the immunodominant T cell antigens associated with the early stages of M. paratuberculosis infections in cattle. The specific sub-objectives are: 1) Use proteomic methodologies to identify the immunodominant T cell antigens of M. paratuberculosis. 2) Amplify, clone, and express the genes encoding the proteins identified via the proteomic identification of T cell antigens. 3) Use the recombinant products to confirm that the proteins identified through our proteomic approach truly elicit an antigen specific T cell response in M. paratuberculosis infected cattle.
APPROACH: The identification of T cell antigens will be acheived by 2D-liquid phase electrophoresis (LPE) separation of M. paratuberculosis subcellular fractions, followed by testing of the 2D-LPE fractions against whole blood of cattle uninfected and infected with M. paratuberculosis. The read out for immune T cell activation will be IFN-gamma production. The proteins of the fractions that stimulate the most dominant IFN-gamma response will be identified by electrospray mass spectrometry. The genes encoding the proteins defined as immunodominant in the above assays will be cloned into an E. coli or M. smegmatis expression vector and the recombinant proteins produced as His-tagged fusion products. These recombinant proteins will subsequently be used to confirm T cell antigenicity using the whole blood IFN-gamma assay.
PROGRESS: 2003/09 TO 2006/09
During the course of the program strain K10 was adopted as the standard strain due to the availability of the genome sequence. Initially, three subcellular fractions were prepared from M paratuberculosis (Map) namely cell wall, cell membrane and cytosol. We originally proposed to fractionate culture filtrate, however we were unable to culture Map in a liquid media compatible with generating CFP free of media components. Growth on solid media provided the most effective method of culturing limited but sufficient quantities of Map for fractionation. Each subcellular fraction was further fractionated by ammonium sulfate (AS) precipitation generating 4 additional fractions per starting sample. Dependent on the protein amount present in each sample, the fractions were further subjected to chromatafocussing and whole gel elution. This resulted in a total of 93 native protein fractions. Additionally, 22 recombinant proteins were generated based on their unique presence in Map as determined by subtractive hybridization experiments and bioinformatics against M.avium. Following experimental infection of 10 animals, with 5 uninfected controls, PBMCs were harvested at 30, 60, 120 and 271 days post infection and stored under liquid nitrogen until all native protein fractions were generated. PBMCs were tested for reactivity against Johnin, M.avium and M.bovis PPDs using a bovine interferon gamma ELISA. While the T-cells from all animals at all time points were capable of producing interferon gamma in response to non-specific stimulation, none of the cattle demonstrated reactivity to any of the subcellular fractions or PPDs, suggesting that the animals were not infected to a point where a strong cellular immune response developed. Thus, a major objective of defining T cell antigens could not be achieved. During Year 1 of the program we expanded our approach to investigate the humoral response of naturally infected cattle, due to the published efficacy of this approach in identifying proteins of M. tuberculosis that were then shown to be dominant T-cell antigens. Protein arrays were generated using 90 native protein fractions and 22 recombinant proteins printed in triplicate, with E.coli and M.phlei lysates serving as controls. The arrays were then probed with sera from naturally infected cattle, 20 positive and 20 negative sera preabsorbed with M.phlei lysates. In comparison to the control mean, 3 of the cell wall AS fractions and 1 of the cytosol AS fractions were recognized by 6 positive cattle sera. Each positive fraction was subjected to two-dimensional electrophoresis and western immunoblotting with the reactive protein spots identified by mass spectrometry. This analysis identified 6 reactive proteins, 2 of which demonstrated limited homology to proteins in M.tuberculosis (MAP2120c and MAP0494). One of the recombinant proteins, MAP3744, was recognized by 4 of the 20 sera with a further 6 recombinant proteins being recognized by 3 positive sera. We also investigated the relative abundance of LAM in each of the native protein fractions using a polyclonal LAM antibody. A positive correlation between seroreactivity and the presence of LAM was observed.
IMPACT: 2003/09 TO 2006/09
Johnes disease causes significant economic losses for principally the dairy but also beef industries in the US. Control of the disease has been hampered by the lack of robust diagnostics capable of distinguishing M. paratuberculosis infection from other mycobacterial infections. These studies have identified 7 proteins that are strongly recognized by sera from naturally infected cattle. Indeed, 3 of the identified proteins do not appear to have homologues in M. tuberculosis. We have also shown that M. paratuberculosis LAM is strongly recognized by sera from infected cattle. A thorough characterization of the proteins identified as B- and potentially T- cell antigens may lead to the identification of novel protein candidates for the development of improved diagnostics.
PUBLICATIONS (not previously reported): 2003/09 TO 2006/09
No publications reported this period
PROJECT CONTACT:
Name: Belisle, J. T.
Phone: 970-491-6549
Fax: 970-491-1815
Email: jbelisle@colostate.edu

 
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ACCESSION NO: 0196720 SUBFILE: CRIS
PROJ NO: COLV-SALMAN AGENCY: CSREES COLV
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2003-34405-13795 PROPOSAL NO: 2003-06088
START: 15 AUG 2003 TERM: 14 AUG 2004 FY: 2004 GRANT YR: 2003
GRANT AMT: $696,539
INVESTIGATOR: Salman, M.
PERFORMING INSTITUTION:
Environmental Health Research
Colorado State University
Fort Collins , Colorado 80523
ENHANCEMENT OF THE PROGRAM FOR ECONOMICALLY IMPORTANT INFECTIOUS ANIMAL DISEASES.
NON-TECHNICAL SUMMARY: A multidisciplinary research center is needed to study animal diseases of economic importance. Integration of studies covering broad spectrum of disciplines is needed to prevent duplication of existing efforts and programs. A comprehensive study approach is expected to lead to improved disease surveillance, risk assessment, management, and control/prevention strategies. Furthermore, these studies will lead to the generation of fundamental knowledge concerning disease transmission, diagnosis, pathogenesis, and virulence of economically important animal diseases.
OBJECTIVES: A multidisciplinary research center at CSU will be established to study animal diseases of economic importance. The Center will work collaboratively with universities, and state and federal agencies in order to produce results covering a broad spectrum of disciplines without duplication of existing efforts and programs. Expected results are: 1)The development of improved surveillance, risk assessment, management, and control/prevention strategies; 2)Generation of fundamental knowledge concerning transmission, diagnosis, pathogenesis, and virulence of economically important infectious animal diseases.
APPROACH: The Center will include three major sections which will be simultaneously integrated into the research approach: biology of infectious diseases, epidemiology of animal diseases, and risk analysis/assessment. An advisory group will be composed of scientists from all involved disciplines, commodity representatives, state departments of agriculture, and consumer advocates.
PROGRESS: 2003/08 TO 2004/08
TSEs 6 commercial screening assays were evaluated for deer/elk. A project was initiated which could result in diagnostic tests and prevention methods for prionic infections. Personnel were trained on the western blot test for the presence of CNS tissue in food products. PEIIAD hosted a conference TSE in Animal Populations: Facts & Fiction to address research and policy issues. Participation in international organizations in risk assessment and classification of countries for BSE status: Dr. Salman was re-appointed to the scientific working group of the GBR. West Nile Virus (WNV) Epidemiology: A survey with the goal of determining the long-term outcome of WNV-affected horses was initiated. A study has been initiated to compare antibody titers of WNV in vaccinated horses to those recovering from natural infection. APHI personnel also investigated the possibility of development of an ELISA test for detection of IgG to WNV. Vesicular Stomatitis (VS) Participation in the field validation of the newly developed real-time PCR for VSV detection Test development: A one-step single tube multiplex reverse-transcriptase PCR test for detection of the VSV in biological samples and insects was developed and validated. Molecular epidemiology: Molecular fingerprinting is being used in conjunction with GIS analysis to understand the spread of the VSV. Serological data: The team has demonstrated serological evidence of VSV during non-outbreak years. Equine Infectious Diseases Equine clostridiosis Toxoid development: Development of a toxoid against equine clostridiosis for use in broodmares prior to foaling has been investigated. Test development and validation: A test to identify clostridial beta 1 and beta 2 toxins in clinical samples was developed and is now being validated. Treatment: The use of metranidazone and the subsequent development of resistance were investigated. Mycobacterial work M bovis and M tuberculosis Serological testing PCR development and testing Non-domestic species Molecular epidemiology M avium ssp paratuberculosis (Johnes disease) Diagnostic strategies PCR development and testing Assessment of the presence of M avium ssp paratb in selected lymph nodes & other tissues Food Safety and Risk Analysis E. coli O157 testing Fecal sampling protocols Analytical methods Modeling prevalence in clusters Modeling low prevalence Modeling test dependence Modeling transmission Global Vet Epidemiology Researchers have continued to collaborate with several animal health researchers and regulators in the design & implementation of projects related to surveillance and risk analysis. Other Topics Researchers have secured funding to gather corresponding antimicrobial resistance data from intensive livestock raising units in South America to compare to data from the USA New RB51 brucellosis vaccine are being developed and tested for use in bison and wild ungulates. A nationally-recognized biosecurity program for livestock operations, including teaching hospitals, was developed and initiated. PEIIAD personnel provide teaching expertise for the delivery of epidemiology training to USDA VMOs. A new initiative involves the creation of on-line course.
IMPACT: 2003/08 TO 2004/08
The PEIIAD will continue to support detection and prevention of animal diseases that have impact on movement and trade of animals and animal products.
PUBLICATIONS (not previously reported): 2003/08 TO 2004/08
1. Paul S. Morley, Josie L. Traub-Dargatz, et. al. Availability of Antimicrobial Drugs for Use in Animals Without a Prescription. College of Veterinary Medicine and Biomedical Sciences, Colorado State University: Fort Collins, Colorado. Poster presentation, Phi Zeta, Research Day, 2004.
2. Young S, Dunowska M, Hyatt DR, Morley PS. Evaluation of the environmental cleanliness in a veterinary teaching hospital. Poster presented at the Workshop on Nosocomial Infections and Biosecurity for Equine Hospitals. Dorothy Russell Havemeyer Foundation, Lexington, KY, 2003.
3. Serena Young, Magda Dunowska, Doreene R. Hyatt, Paul S. Morley. Evaluation of the Environmental Cleanliness in a Veterinary Teaching Hospital. College of Veterinary Medicine and Biomedical Sciences, Colorado State University: Fort Collins, Colorado. Poster presentation, Phi Zeta Research Day, 2004.
4. Antimicrobial use and resistance in enteric bacteria. Invited presentation presented at the 2003 FDA/CSFSAN-CVM Research Meeting. Baltimore, MD, 2003.
5. Morley PS, Hyatt DR, Dunoswka M. The Effect of Virkon Fogging on Survival of Salmonella enterica on Surfaces in a Veterinary Teaching Hospital. Presentation at the CSU, Phi Zeta research day, January 2004.
6. Evaluation of the Efficacy of Disinfectant Footbaths. Nanea Morris, Paul S. Morley, Doreene R. Hyatt. College of Veterinary Medicine and Biomedical Sciences, Colorado State University: Fort Collins, Colorado. Poster Presentation, Phi Zeta Research Day, 2003.
7. Antognoli, M.C., Hirst H.L, Goodell G., and Salman M.D. Evaluation of Cell Mediated Immunity-based tests for detection of Paratuberculosis in young cattle. Tenth International Symposium of Veterinary Epidemiology and Economics. Abstract325. Vina del Mar, Chile, November 17-21, 2003
8. Antognoli, M.C., Hirst H.L, Goodell G, and Salman M.D. Evaluation of three methods for direct diagnosis of Paratuberculosis in dairy cattle. Tenth International Symposium of Veterinary Epidemiology and Economics. Abstract # 326. Vina del Mar, Chile, November 17-21, 2003
9. Morley PS. Infectious Disease Monitoring and Surveillance at the James L. Voss Veterinary Teaching Hospital. Invited presentation at the Workshop on Nosocomial Infections and Biosecurity for Equine Hospitals. Dorothy Russell Havemeyer Foundation, Lexington, KY, 2003.
10. Dunowska M, Morley PS. Surveillance for Salmonella shedding in large animal patients. Poster presented at the Workshop on Nosocomial Infections and Biosecurity for Equine Hospitals. Dorothy Russell Havemeyer Foundation, Lexington, KY, 2003.
11. Surveillance for Salmonella Shedding in Large Animal Patients. Magda Dunowska and Paul S. Morley, College of Veterinary Medicine and Biomedical Sciences, Colorado State University: Fort Collins, Colorado. Poster presentation, Phi Zeta Research Day, 2003.
12. Burgess BA, Morley PS, Hyatt DR. 2004 Environmental Surveillance for Salmonella in a Veterinary Teaching Hospital. J Am Vet Med Assoc [Submitted].
13. Burgess BA, Morley PS, Hyatt DR. Environmental surveillance for Salmonella in a veterinary teaching hospital. Poster presented at the Workshop on Nosocomial Infections and Biosecurity for Equine Hospitals. Dorothy Russell Havemeyer Foundation, Lexington, KY, 2003.
14 . Burgess BA, Morley PS, Hyatt DR. Salmonella surveillance in a large veterinary teaching hospital. Poster presented at the 4th Annual Phi Zeta Research Day, CSU College of Veterinary Medicine and Biomedical Science, Fort Collins, CO, 2003.
15. Salazer P, Traub-Dargatz JL, Morley PS, et al. Characterization of the 2002 West Nile Virus Epidemic in Nebraska and Colorado equids. J Am Vet Med Assoc [In Press].
16. Geiser S, Seitzinger A, Salazar P, et al. Economic Impact of West Nile Virus on the Colorado and Nebraska Equine Industries: 2002. [Government Report]. USDA:APHIS:VS, Centers for Epidemilogy and Animal Health. Fort Collins, CO, 2003. [available at
http://www.aphis.usda.gov/vs/ceah/cahm/Equine/wnv-info-sheet.pdf] #N394.0403. 4 pp.
17. Traub-Dargatz JL, Salazer P, Morley PS, et al. Vaccination status and outcome of cases of equine west nile virus cases in Colorado and Nebraska in 2002. Proceedings of the 3rd International Veterinary Vaccines and Diagnostics Conference, Guelph, Ontario, 2003, p 68.
18 . Salazer P, Traub-Dargatz JL, Morley PS, et al. Characterization of the 2002 West Nile Virus Epidemic in Nebraska and Colorado equids. Scientific Proceedings of the 4th Annual Phi Zeta Research Day of the Theta Chapter, CSU College of Veterinary Medicine and Biomedical Science, Fort Collins, CO, 2003, p 34.
19. Traub-Dargatz JL, Morley PS, Salazer P, et al. Characterization of the 2002 West Nile Virus Epidemic in Nebraska and Colorado equids. Presented at the 10th International Society for Veterinary Epidemiology and Economics Symposium, Vina del Mar, Chile, 2003.
20. Geiser S, Seitzinger A, Salazar P, Traub-Dargatz J, Morley P, Salman M, Wilmot D, Steffen D, Cunningham W. Economic Impact of West Nile Virus on the Colorado and Nebraska Equine Industries: 2002. Presented at the 10th International Society for Veterinary Epidemiology and Economics Symposium, Vina del Mar, Chile, 2003.
21. International Society for Veterinary Epidemiology and Economics Abstract and Oral Presentation by Dr. Elizabeth Mumford, November 2003 and Abstract and Oral Presentation at Phi Zeta Research Day January 2003.
22 . Hoover, EA. 2003 Chronic wasting disease: Rocky Mountain Virology Conference. November.
23. Mathiason, CK, Sigurdson, CJ, Foos, T, Eliason, G, and Hoover, EA. 2003. Expression of cervid PrPc in Tissues of Deer. Keystone Conference TSEs. Beaver Creek, CO, April.
24 . Sigurdson, CJ, Mathiason, CK, Perrott, MR, Eliason, GA, and Hoover, EA: 2003 Transmission of Chronic Wasting Disease in the Ferret. I
PROJECT CONTACT:
Name: Turney, L.
Phone: 970-491-6229
Email: lturney@cvmbs.colostate.edu
URL: http://www.colostate.edu/CVEADSS

 
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ACCESSION NO: 0097775 SUBFILE: CRIS
PROJ NO: COLV05420 AGENCY: CSREES COLV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 1998 TERM: 30 SEP 2004 FY: 2004
INVESTIGATOR: Niswender, G.
PERFORMING INSTITUTION:
College Administration
Colorado State University
Fort Collins , Colorado 80523
BACTERIAL DISEASES AND THE IMMUNE SYSTEM.
OBJECTIVES: 1) To continue development of new procedures for the rapid and reliable detection of the causative agents of bacterial diseases; 2) To develop new, more efficacious methods for the prevention of diseases caused by bacterial agents and 3) to study the pathogenic processes and epidemiology of bacterial diseases.
APPROACH: Improved diagnostic procedures will be developed for the detection of several important pathogenic bacterial (for example, Brucella ovis; Mycobacterium ovis; Mycobacterium paratuberculosis and clostridium perfringens). Tests will include improved enzyme immunoassays, polymerase chain reaction to detect bacterial DNA and/or RNA, and use of fluorescently labeled recombinant DNA probes and antibodies to specific coat proteins. These reagents will be used to study the disease process and the epidemiology of individual infections. Finally, in some cases more effacacious recombinant vaccines will be developed to prevent the disease.
PROGRESS: 1998/10 TO 2004/09
Mice of the CBA inbred strain background expressing the well characterized mutation designated xid in the cytoplasmic signalling enzyme Bruton's protein kinase have been previously noted to illustrate shifts in T helper type 1 (Th1)/Th2 immunity which is underlined by an apparent failure to produce the regulatory cytokine interleukin-10. This study examined if this extended to infection with Mycobacterium tuberculosis, which also depends on Th1 immunity. Contrary to expectations, xid mice showed evidence of a transient early susceptibility to pulmonary infection, changes in macrophage morphology, and decreased activation of lung natural killer cells, while showing evidence of substantial IL-10 production and accumulation in lung lesions macrophages, but paradoxically this did not influence the course of the chronic disease. In addition, macrophages from the lungs of xid mice also expressed high levels of CD14. These observations suggest that the xid mutation in cellular signalling has much wider effects on the immune system than previously thought. In a separate study, major histocompatibility complex class I tetramer reagent was used to track antigen-specific CD8 T cells in the lungs of mice immunized with the tuberculosis vaccine candidate Mtb72F. The results show that CD8 T cells recognizing an immunodominant Mtb32-specific epitope could be detected in significant numbers over the course of infection in mice exposed to low-dose aerosol challenge with Mycobacterium tuberculosis and that prior vaccination substantially increased the numbers of these cells early in the lungs. The effector phenotype of the cells was shown by the demonstration that many secreted gamma interferon, but very few contained granzyme B. As the course of the infection progressed, many activated CD8 T cells down-regulated expression of CD45RB and upregulated expression of the interleukin-7 receptor alpha chain, indicating a transition of these cells to a state of memory. These data support the hypothesis that M. tuberculosis-specific CD8 T cells can be targeted by vaccination with the Mtb72F polyprotein.
IMPACT: 1998/10 TO 2004/09
Mycobacterial infection in dairy cattle remains a significant economic loss to producers. How the immune system responds to these infections is poorly understood. This limitation has severely hampered the development of a vaccine effective for eliminating Mycobaterial infections (Johne's Disease)in dairy cattle. Some of these studies show that specific bacterial proteins can be used to specifically target components of the immune system, a mechanism that may lead to an effective vaccine for prevention of the multi-billion loss to the dairy industry caused by Johne's disease.
PUBLICATIONS (not previously reported): 1998/10 TO 2004/09
1. Junqueira-Kipnis AP, Kipnis A, Henao Tamayo M, Harton M, Gonzalez Juarrero M, Basaraba RJ, Orme IM. 2005. Interleukin-10 production by lung macrophages in CBA xid mutant mice infected with Mycobacterium tuberculosis. Immunology. 115:246-52.
2. Irwin SM, Izzo AA, Dow SW, Skeiky YA, Reed SG, Alderson MR, Orme IM. 2005. Tracking antigen-specific CD8 T lymphocytes in the lungs of mice vaccinated with the Mtb72F polyprotein. Infect Immun. 73:5809-16.
3. Perry JA, Olver CS, Burnett RC, Avery AC. 2005. Cutting edge: the acquisition of TLR tolerance during malaria infection impacts T cell activation. J Immunol. 174:5921-5.

 
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ACCESSION NO: 0210372 SUBFILE: CRIS
PROJ NO: DCR-2007-01772 AGENCY: CSREES DC.R
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2007-35212-18059 PROPOSAL NO: 2007-01772
START: 01 JUN 2007 TERM: 31 MAY 2008 GRANT YR: 2007
GRANT AMT : $15,000
INVESTIGATOR: Nacy, C. A.
PERFORMING INSTITUTION:
American Society for Microbiology
1752 N Street, NW
Washington , District of Columbia 20036
MYCOBACTERIUM AVIUM PARATUBERCULOSIS : INCIDENTAL HUMAN PATHOGEN OR PUBLIC HEALTH THREAT?
NON-TECHNICAL SUMMARY: There is mounting evidence that supports a role for MAP as the etiologic agent of Crohn's Disease (CD), a chronic relapsing inflammatory human disease of the gut. The possibility that MAP is involved in both JD and CD suggests that MAP could be transferred from cattle to humans. While the direct transfer has not been demonstrated, there is increasing evidence that some percentage of the milk supply is contaminated with MAP from the dairy cattle. We believe that MAP may be an underappreciated and emerging potential public health threat and deserves close examination of its role in animal and human disease and an evaluation of the events underlying MAP transmission from animals to humans. This colloquium and its subsequent report will concentrate on the epidemiology of the MAP and will help to answer some of the questions about whether MAP is a food safety issue. The experts will meet for 2.5 days of deliberations, which will form the foundation of formal report. The report will include a succinct description of the issues, graphical representations, where appropriate, and recommendations for future action.
OBJECTIVES:Mycobacterium avium subspecies paratuberculosis (MAP) is a soil microorganism and the etiologic agent of Johnes Disease (JD), a chronic and progressive enteric infection considered to be one of the most serious diseases affecting cattle and other domestic and wild animals, including sheep, goats, elk, and primates. Clinical progression of the disease includes severe diarrhea and weight loss, and the affected animals eventually either die or are killed. JD is prevalent in domestic animals worldwide; its economic impact in the U.S. alone is stunning, with an estimated loss of $1.5 billion every year. One study estimated the prevalence of MAP in U.S. cattle to be 1.6%, with a significantly higher prevalence in the dairy cattle subset. Studies in more localized herds of dairy cattle have produced even higher estimates; one estimated the prevalence of MAP in cattle in California to be 9.4%. The problem is even more serious in other countries: a study in Denmark estimated the prevalence of MAP in the dairy cattle to be 47%. Mounting evidence supports a role for MAP as an etiologic agent (although it may be one of several) of Crohns Disease (CD), a chronic relapsing inflammatory human disease of the gut. The possibility that MAP is involved in both JD and CD suggests that MAP could be transferred from cattle to humans. While the direct transfer has not been demonstrated, there is increasing evidence that some percentage of the milk supply is contaminated with MAP from the dairy cattle. We believe that MAP may be an underappreciated and emerging potential public health threat and deserves close examination of its role in the epidemiology of animal and human disease, determination of any food safety issues, and an evaluation of the events underlying MAP transmission from animals to humans. It is important to identify the gaps in our knowledge, and to focus new research to resolve these questions.
APPROACH: The American Academy of Microbiology (AAM) will convene a colloquium of 30-40 experts in animal health, agriculture, and human medicine in Salem, Massachusetts, June 15-17, 2007. Attendance at the colloquium is by invitation only, and participants are selected to ensure the greatest scientific balance and diversity. This inter-disciplinary approach will concentrate on the epidemiology of the MAP and will help to answer some of the questions about whether MAP is a food safety issue. The experts will meet for 2.5 days of deliberations, which will form the foundation of formal report. The report will include a succinct description of the issues, graphical representations, where appropriate, and recommendations for future action. The steering committee has composed a set of preliminary questions, intended to be the focus of the colloquium. Each group will discuss questions in the following areas--environmental/zoonotic sources of MAP and control measures; human MAP infection; potential role for MAP in CD; and gap analysis. The bulk of participants time will be spent in small working groups addressing these questions. There will be two general sessions that will bring all colloquium participants together. They will meet at the beginning of the colloquium and at the end to share their working group conclusions and recommendations and discuss any issues raised. Following the colloquium, a science writer (who will attend the colloquium), working closely with the steering committee chair, will develop a draft report for review by colloquium participants. The predicted contributions to the enhancement and improvement of science are: objective analysis of what is known about M. aviumparatuberculosis as a public health problem; and recommendations for future research to clarify the gaps.
PROJECT CONTACT:
Name: Carol Colgan
Phone: 202-942-9227
Fax: 202-942-9353
Email: ccolgan@asmusa.org

 
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ACCESSION NO: 0212066 SUBFILE: CRIS
PROJ NO: FLA-ANS-004680 AGENCY: CSREES FLA
PROJ TYPE: HATCH PROJ STATUS: NEW MULTISTATE PROJ NO: NCERA-199
START: 01 OCT 2006 TERM: 30 SEP 2011
INVESTIGATOR: Elzo, M. A.
PERFORMING INSTITUTION:
Animal Sciences
University of Florida
Gainesville , Florida 32610
IMPLEMENTATION AND STRATEGIES FOR NATIONAL BEEF CATTLE GENETIC EVALUATION.
NON-TECHNICAL SUMMARY: Coordination among researchers and breed associations can maximize adoption of innovations by beef cattle producers and minimize costly duplication of effort among researchers. This project allows people to exchange information about many disconnected research activities that support the national cattle evaluation. The purpose of this project is to develop new ways to share genetic research, including genetic marker information, with breed associations, beef cattle producers and organizations such as the National Cattlemen's Beef Association and Beef Improvement Federation.
OBJECTIVES: 1. Provide a forum for discussion and exchange of information for the many disconnected and diverse research activities--biological, statistical, computational, and economical--that support National Cattle Evaluation (NCE). 2. Develop through this exchange new tools for delivery and use of beef cattle genetic research, including genomic information, to beef breed associations and beef cattle producers. 3. Share research findings with the beef cattle industry in appropriate forums (i.e., Train the Trainer Series). 4. Collaborate with the NBCEC on research and educational outreach activities. 5. Research and develop decision support tool to improve the annual economic value of beef cattle genetic improvement.
APPROACH: 1. An annual meeting with agenda focused on NCE will allow shared ideas and techniques to be rapidly disseminated throughout the entire NCE system. Thus, the NCE will have the latest developments incorporated wherever the actual evaluations are computed or by whom they are distributed. 2. Industry and extension representatives will ensure that the research community is aware of NCE research priorities of the beef cattle industry. Their participation will augment the out reach programs of breed associations as well as those of individual university members. 3. Members of the committee are, and will continue to be, leaders and ubiquitous speakers at the annual meeting and research symposium of the Beef Improvement Federation. The committee will co-sponsor BIF genetic prediction workshops. 4. The Website will be a resource for breed organizations, producers, researchers and extension specialists and other segments of the beef industry.
PROGRESS: 2006/10 TO 2007/09
OUTPUTS: Activities included: 1) Collection of phenotypic reproduction, growth, carcass, management, health, and feed efficiency data from beef cattle populations, 2) Maintenance of databases, 3) Devising and testing new and more diverse multibreed models for genetic evaluation in multibreed populations, 4) Obtaining economic, social, educational information for genetic-economic models in cattle, 5) Creating a DNA repository for the multibreed herd of the University of Florida, 6) Training graduate students. Products were: Ten publications, seven presentations, training of four graduate students, publication of a sire summary, and development of national and international scientific networks. Efforts overlapped with those of project FLA-ANS-04263. PARTICIPANTS: Mauricio A. Elzo Department of Animal Sciences University of Florida D. D. Johnson Department of Animal Sciences University of Florida D. O. Rae Large Animal Clinical Sciences University of Florida Skorn Koonawootrittriron Department of Animal Science Kasetsart University Thailand Arcadio de los Reyes Department of Animal Sciences Federal University of Goias Brazil Gary Hansen North Florida Research and Education Center University of Florida Jeffrey A. Rhone Department of Animal Sciences University of Florida TARGET AUDIENCES: Scientists Graduate Students Producers Industry
IMPACT: 2006/10 TO 2007/09
Outcomes: 1) Database for growth, feed efficiency, reproduction, production, health, survival, and culling data for the multibreed herd of the University of Florida (UF), 2) DNA repository for the multibreed herd of UF, maintained at New Mexico State University (NMSU), 3) Sire summary for a multibreed population, 4) Evidence for negative effects of subclinical paratuberculosis in production and economic terms, 5) Evidence for higher feed efficiency of Brahman than Angus and Angus x Brahman crossbred animals. Impacts: Feed efficiency results suggest further research using genomics and functional genomics tools. Subclinical paratuberculosis results warrants research with national datasets because of the large potential economic impact of this disease.National and international scientific networks increase collaboration levels, efficiency of resource utilization, and promotion of new ideas. Improved training of graduate students.
PUBLICATIONS (not previously reported): 2006/10 TO 2007/09
1. Elzo, M. A. 2006. Genetic evaluation of animals in multibreed cattle populations using linear models. Latin-Amer. Arch. Anim. Prod. 14:154-160.
2. Elzo, M. A., G. R. Hansen, J. G. Wasdin, J. D. Driver, and J. L. Jones. 2007. Evaluation of post-weaning phenotypic residual feed intake in an Angus-Brahman multibreed herd of beef cattle. J. Anim. Sci.85(Suppl.1):475.
3. Elzo, M. A. 2007. Genetic evaluation in multibreed populations. Rev. Col. Cienc. Pec. 20:504-507.
4. Reyes, A. de los, M. A. Elzo, A. C. Sanches, R. B. Lobo, and L. A. F. Becerra. 2006. Effect of sire x maternal grandsire interactions on (co)variance estimates and genetic trends for pre-weaning growth traits in Brazilian Nellore cattle. Brazilian Anim. Sci. 7:365-371.
5. Reyes, A. de los, M. A. Elzo, V. M. Roso, R. Carvalheiro, L. A. Fries, and J. L. Ferreira. 2007. Animal model analyses of additive and non-additive genetic effects for 205-day weight in a Nellore x Hereford multibreed population in Brazil. J. Anim. Sci.85(Suppl.1):475.
6. Koonawootrittriron, S., M. A. Elzo, and T. Tongprapi. 2007. Genetic trends for dairy traits in the Holstein x Other multibreed dairy cattle population in tropical central Thailand. J. Anim. Sci.85(Suppl.1):19.
7. Koonawootrittriron, S., M. A. Elzo, and T. Suwanasopee. 2007. Characterization of a negative halothane gene comercial multibreed swine population for growth and conformation traits in tropical western Thailand. J. Anim. Sci.85(Suppl.1):57.
8. Rhone, J. A., S. Koonawootrittriron, and M. A. Elzo. 2007. Factors affecting milk yield, milk fat, bacterial score, and bulk tank somatic cell count of dairy farms in the central region of Thailand. Trop. Anim. Health Prod. DOI: 10.1007/s11250-007-9074-5.
9. Rhone, J. A., S. Koonawootrittriron, and M. A. Elzo. 2007. Factors affecting bacterial score and bulk tank somatic cell count of dairy farms in the central region of Thailand. J. Anim. Sci.85(Suppl.1):639.
10. Rhone, J. A., S. Koonawootrittriron, and M. A. Elzo. 2007. Factors affecting milk yield and milk fat of dairy farms located in the central region of Thailand. J. Anim. Sci.85(Suppl.2):33.
PROJECT CONTACT:
Name: Elzo, M. A.
Phone: 352-392-7564
Fax: 352-392-7652
Email: elzo@animal.ufl.edu
URL: http://lgu.umd.edu/lgu_v2/homepages/outline.cfm?trackID=7836
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ACCESSION NO: 0184682 SUBFILE: CRIS
PROJ NO: FLA-ANS-03818 AGENCY: CSREES FLA
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 18 FEB 2000 TERM: 30 SEP 2005 FY: 2005
INVESTIGATOR: Elzo, M. A.; Johnson, D. D.; Kunkle, W. E.
PERFORMING INSTITUTION:
Animal Sciences
University of Florida
Gainesville , Florida 32610
IMPROVEMENT OF BEEF CATTLE IN MULTIBREED POPULATIONS: PHASE III.
NON-TECHNICAL SUMMARY: Prediction of genetic values of purebred and crossbred animals in multibreed populations. Estimation of genetic variation and combining ability of animals in multibreed populations. Devise new and more precise genetic-statistical models to predict the genetic values of animals and their combining ability in populations of animals composed of purebred and crossbred animals.
OBJECTIVES: Development of models and procedures to improve the predictive ability and the accuracy of genetic evaluation, selection and mating strategies of straightbred and crossbred animals in national and international multibreed populations for individual traits and for functions of traits under various environmental conditions.
APPROACH: New genetic-statistical models and procedures will be devised, followed by the generation of computer programs to be tested and validated using simulated, experimental, and multibreed field data sets. New computing algorithms will be used and(or) devised as needed. National and international experimental and field data sets will be used. National and international researchers will collaborate in specific parts of this project.
PROGRESS: 2000/02 TO 2005/09
This is the final report of Phase III of this multibreed project in beef cattle. The most important accomplishments of Phase III were: 1) establishment of long-term research collaborations with researchers from Brazil, Chile, and Thailand, and continuation of research collaboration with researchers from Colombia, 2) establishment of a long-term research collaboration with researchers from the College of Veterinary Medicine of UF on the effects of paratuberculosis on production, reproduction, and carcass traits in multibreed beef cattle, 3) development of dedicated software for editing data, constructing connected datasets and pedigree files, and evaluating animals for additive and non-additive genetic effects of growth and dairy traits in multibreed populations in Chile, Thailand, and the US, 4) development of multibreed genetic evaluation models, estimation of additive and nonadditive variance and covariance components, and prediction of genetic values of purebred and crossbred animals from Bos taurus x Bos taurus and Bos taurus x Bos indicus multibreed populations for dairy, growth, and carcass traits, 4) results from the Thai Holstein x Bos indicus dairy cattle population indicated that 5/8 to 3/4 Bos taurus cows would produce greater amounts of milk than animals with higher Bos taurus fractions, that non-additive genetic variation was smaller than additive genetic variation (moderate heritabilities and low non-additive ratios in all breed groups), and that additive, non-additive, and total genetic predictions were highly correlated under Thai tropical conditions, 5) results from the Chilean Holstein x Bos taurus dairy cattle population showed that Holstein and 3/4 Holstein produced the largest amounts of milk, that heritabilities for milk yield were moderate regardless of the Holstein fraction of the breed group, that non-additive variance ratios were negligible but that heterosis was similar to other crossbred dairy populations under temperate climatic conditions, 6) results from the research on non-additive genetic effects in a Brazilian Nellore cattle subpopulation indicated that the variation due to sire x maternal grandsire interaction effects was 1/3 of the additive genetic variation for weight at 120 days and 240 days (weaning), suggesting that sire x maternal grandsire interactions will need to be tested before deciding on a national genetic evaluation model for this breed in Brazil, 7) results from the UF Angus-Brahman multibreed herd showed that sire direct genetic prediction for growth traits tended to increase, but maternal ones tended to decrease from Angus to Brahman, that sire direct genetic predictions for hot carcass weight, yield grade, and shear force tended to increase, and marbling and tenderness tended to decrease from Angus to Brahman, and that there was no trend from Angus to Brahman for sire nonadditive genetic predictions, and 8) the paratuberculosis study found that Brahman fraction of dam and days in lactation were positively associated, and dam weight change, birth weight of calf, and calf preweaning gain were negatively associated with ELISA scores.
IMPACT: 2000/02 TO 2005/09
Phase III of this research had both national and international impact. It influenced the widespread adoption of multibreed genetic evaluation procedures for the evaluation animals in beef and dairy cattle populations both nationally and internationally. Research and development collaborations with scientists from Brazil, Colombia, and Thailand yielded information useful for the implementation of genetic evaluation of animals in multibreed populations in countries with tropical and subtropical environments. Collaboration with Brazilian researchers indicated the need to implement multibreed genetic evaluation procedures in various multibreed subpopulations. Researchers in Thailand are currently using multibreed procedures developed during Phase III to publish national genetic evaluations. Research with the Chilean multibreed population showed the feasibility of using multibreed procedures composed of closely related breeds. Paratuberculosis research in beef cattle at UF found that high ELISA scores were associated with reduced cow weights, small calf weights, and low preweaning gains, possibly due to negative effects of subclinical paratuberculosis.
PUBLICATIONS (not previously reported): 2000/02 TO 2005/09
1. Elzo, M. A., D. O. Rae, S. E. Lanhart, J. G. Wasdin, W. P. Dixon, and J. L. Jones. 2005. Factors associated with ELISA scores for paratuberculosis in an Angus-Brahman multibreed herd of beef cattle. J. Anim. Sci. (In Press).
2. Elzo, M. A., D. O. Rae, S. Lanhart, J. Wasdin, P. Dixon, and J. Jones. 2005. Factors Associated with ELISA Sample/Positive Ratio Scores for Paratuberculosis in an Angus-Brahman Multibreed Herd of Beef Cattle. Page 123 in Proc. 38th Annu. Conf. Am. Assoc. Bov. Pract., Salt Lake City, UT.
3. Elzo, M., D. Rae, S. Lanhart, J. Wasdin, P. Dixon, and J. Jones. 2005. Factors associated with ELISA likelihood s/p ratio scores for paratuberculosis in an Angus-Brahman multibreed herd of beef cattle. J. Anim. Sci. 83 (Suppl. 1):139 (Abstr.)
4. Elzo, M. A., and A. de los Reyes Borjas. 2004. Perspectives for multibreed genetic evaluation of cattle in Brazil. Brazilian Anim. Sci. 5:171-185. Online. Available: http://www.vet.ufg.br/cab5 4 01.pdf. Accessed January 3, 2005.
5. Koonawootrittriron, S., M. A. Elzo, S. Tumwasorn, and T. Tongprapi. 2005. Age at first calving of dairy cattle in a multibreed population of Thailand. Proc. 44th Kasetsart University Conference, February 1-5, 2005, Bangkok. (In press)
6. Koonawootrittriron, S., M. Elzo, P. Sopanarat, S. Prasanpanich, J. Teingthum, C. Chaimongkol, T. Sainui, K. Nithichai, T. Tongprapi, and T. Ralukmun. 2005. D.P.O. Sire & Dam Summary 2004. Dairy Promotion Organization, Ministry of Agriculture and Cooperatives of Thailand, Bangkok. p 1-32.
7. Reyes, A. de los, M. A. Elzo, A. C. Sanches, R. B. Lobo, and L. A. F. Becerra. 2005. Effect of sire x maternal grandsire interactions on (co)variance estimates and genetic trends for pre-weaning growth traits in Brazilian Nellore cattle. Brazilian Anim. Sci. 6 (In Press).
8. Reyes, A. de los, M. A. Elzo, A. C. Sanches, R. B. Lobo, and L. A. F. Becerra. 2005. Inclusion of sire x maternal grandsire interaction in models for the estimation of (co)variances and the prediction of genetic values for pre-weaning growth traits in Brazilian Nellore cattle. Proc. 42nd Annu. Meet. Brazilian Soc. Zootech., Goiania, Goias, Brazil. p 1-4.
9. Reyes, A. de los, M. A. Elzo, R. Lobo, and L. Becerra. 2005. Sire x maternal grandsire interaction for pre-weaning growth traits in Brazilian Nellore cattle. J. Anim. Sci. 83 (Suppl. 1):14 (Abstr.)
PROJECT CONTACT:
Name: Elzo, M. A.
Phone: 352-392-7564
Fax: 352-392-7652
Email: elzo@animal.ufl.edu

 
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ACCESSION NO: 0205856 SUBFILE: CRIS
PROJ NO: FLA-ANS-04263 AGENCY: CSREES FLA
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 2005 TERM: 30 SEP 2010 FY: 2006
INVESTIGATOR: Elzo, M. A.; Johnson, D. D.; Rae, D. O.
PERFORMING INSTITUTION:
Animal Sciences
University of Florida
Gainesville , Florida 32610
IMPROVEMENT OF BEEF CATTLE IN MULTIBREED POPULATIONS: PHASE IV.
NON-TECHNICAL SUMMARY: Accurate prediction of genetic values for economically important traits of purebred and crossbred animals is essential to devise appropriate mating and selection strategies in multibreed populations. This project seeks to develop genetic-economic models and procedures to improve mating and selection strategies in national and international multibreed populations under a variety of environmental conditions.
OBJECTIVES: Development of models and procedures to improve the ability and accuracy of genetic prediction, selection, and mating strategies of straightbred and crossbred animals in national and international multibreed populations for functions of economically important traits under various environmental conditions. Development of applied genetic-economic indicators to assess the worth of straightbred and crossbred animals from national and international multibreed populations for functions of traits under a variety of environmental conditions.
APPROACH: New genetic-statistical models will be devised and subsequently tested and validated using simulated, experimental, and field national and international multibreed datasets of various degrees of unbalancedness. New computing algorithms will be incorporated and(or) devised as needed. National and international researchers will collaborate in various stages of the research.
PROGRESS: 2006/10 TO 2007/09
OUTPUTS: Activities covered: 1) acquisition, collection, and editing of national and international experimental, survey, and field datasets in multibreed populations of beef cattle, dairy cattle, and swine; 2) single-breed and multibreed analysis of datasets from Brazil, Thailand, and USA; 3) collaboration with Brazilian researchers on the development of more detailed and accurate models for multibreed genetic evaluation and estimation of genetic parameters in Bos indicus x Bos taurus multibreed beef cattle populations; 4) collaboration with Bolivian researchers on the development of a genetic evaluation for Criollo cattle; 5) Collaboration with Colombian researchers on indicators of productivity for Bos taurus x Bos indicus crosses in Colombia; 6) collaboration with Thai researchers on regional and national genetic evaluations and genetic trends in Bos indicus x Bos taurus multibreed dairy cattle and swine populations, and training of graduate students; 7) collaboration with researchers from Large Animal Clinical Sciences (LACS) on the impact of subclinical paratuberculosis on reproduction and production traits in multibreed beef cattle populations; 8) collaboration with researchers from NFREC Marianna and ARS-USDA Brooksville on a feed efficiency study using purebred and crossbred beef cattle from the Angus x Brahman multibreed herd of UF, Marianna, and Brooksville. Growth, feed consumption, temperament, and carcass ultrasound measurements were taken for the 2006 calf crop at the NFREC GrowSafe facility in Mariana. Data from the second calf crop will be taken from October to December of 2007. The experiment will run for at least four years; 9) Collaboration with researchers from New Mexico State University (NMSU) on genetic evaluation of purebred and crossbred beef cattle for efficiency of forage utilization using phenotypic, genomics, and functional genomics information; Blood samples were collected for all 2007 calves, sires, and dams from the Angus-Brahman multibreed herd; 10) Collaboration with researchers from Physiology and LACS on physiological genomics aspects of paratuberculosis in beef cattle. Blood samples were taken from all 2007 cows in the Angus-Brahman multibreed herd. Products of these national and international collaborations were: 1) National and international scientific networks, 2) Beef cattle, dairy cattle, and swine experimental, regional, and national databases, 3) Creation of a DNA database for the Angus-Brahman multibreed herd at NMSU (calves, cows, sires) and at UF (cows), 4) Dairy farm surveys in Central Thailand, 5) More detailed and accurate models for genetic evaluation in multibreed field cattle and swine populations, 6) Annual dairy genetic evaluation summary (DPO, Thailand), 7) Training of four MS and PhD students, 8) Twelve publications, 9) Seven presentations in scientific meetings. PARTICIPANTS: Mauricio A. Elzo (PI) Department of Animal Sciences University of Florida D. D. Johnson (Co-PI) Department of Animal Sciences University of Florida D. O. Rae Large Animal Clinical Sciences University of Florida Skorn Koonawootrittriron Department of Animal Science Kasetsart University Thailand Arcadio de los Reyes Department of Animal Sciences Federal University of Goias Brazil Rommy Pena Tropical Agricultural Research Center Bolivia TARGET AUDIENCES: Scientists Graduate Students Producers
IMPACT: 2006/10 TO 2007/09
Outcomes of international collaborations: 1) GrowSafe feed efficiency analyses indicated that bulls were more efficient than steers which were more efficient than heifers, Brahman calves were more efficient than Angus, Brahngus, and Angus x Brahman crossbred calves, low residual feed intake (RFI) calves were more efficient and grew faster than medium and high RFI calves; 2) intralocus and interlocus genetic interactions may need to be included in the model for genetic evaluations for growth in some Bos indicus x Bos taurus multibreed populations; 3) redesigned and simplified surveys to collect information on production, management, health, sociological, and economic aspects of dairy farms in Central Thailand; 4) analysis dairy farms in Central Thailand indicated that small farms had higher milk and fat yields, lower bacterial scores, and received higher prices per kg of milk than medium and large farms; 5) increased number of animals and farms that provided data for dairy genetic evaluation, and conducted the 2007 dairy cattle multibreed genetic evaluation for the Data Promotion Organization (DPO) of Thailand; 6) genetic trends for milk yield, fat yield, and fat percent in the DPO population were near zero suggesting that producers are using a variety of criteria to choose sires in addition to EPD, and that high percent Holstein cows failed to reach their production potential under the management, nutrition, and hot and humid climatic conditions of Thailand; 7) selection for growth and carcass traits in a negative halothane gene commercial swine multibreed population (Pietrain sows, Pietrain, Large White, and Landrace boars) reared in open barns in Thailand resulted in lower ages at first estrous and larger hip widths; other traits (birth and weaning weights, shoulder width, body length) remained unchanged; 8) Estimates of differences between cows with non-zero and zero ELISA scores were associated with longer days open, lower ability of cows to maintain weight, and lower calf birth and weaning weights. Potential losses of income due to subclinical paratuberculosis were estimated to be $62.4 for cows with positive ELISA score. Results from this research have had national and international impact. National impacts: The feed efficiency study found that Brahman calves were more efficient than Angus and Angus-Brahman crossbreds suggesting that further research was warranted (genomics, functional genomics). Results from the paratuberculosis study reinforced previous results indicating that subclinical paratuberculosis had measurable negative effects on traits of economic importance in beef cattle, thus research at regional and national levels seems advisable given the potentially large economic impact of this disease. International impact: Development of effective collaboration networks linking researchers across countries. Development of more effective surveys for data collection, larger population samples, and more complete datasets for genetic evaluation in Thailand. Increased involvement in graduate training of international graduate students. Contributed to the maintenance of Criollo cattle as economically viable cattle populations.
PUBLICATIONS (not previously reported): 2006/10 TO 2007/09
1. Elzo, M. A. 2006. Genetic evaluation of animals in multibreed cattle populations using linear models. Latin-Amer. Arch. Anim. Prod. 14:154-160.
2. Elzo, M. A., G. R. Hansen, J. G. Wasdin, J. D. Driver, and J. L. Jones. 2007. Evaluation of post-weaning phenotypic residual feed intake in an Angus-Brahman multibreed herd of beef cattle. J. Anim. Sci.85(Suppl.1):475.
3. Elzo, M. A., D. O. Rae, S. E. Lanhart, J. G. Wasdin, W. P. Dixon, D. J. Driver, and J. L. Jones. 2007. Relationship Between Cow Reproduction and Calf Preweaning Growth Traits and Cow ELISA Scores for Paratuberculosis in Angus-Brahman Cattle. 2007 Florida Beef Report, IFAS, U. Florida. http://www.animal.ufl.edu/extension/beef/beef cattle report/2007/Management1.pdf.
4. Koonawootrittriron, S., M. Elzo, P. Sopanarat, S. Prasanpanich, J. Teingthum, C. Chaimongkol, T. Sainui, K. Nithichai, T. Tongprapi, and T. Ralukmun. 2007. D.P.O. Sire & Dam Summary 2006. Dairy Promotion Organization, Ministry of Agriculture and Cooperatives of Thailand, Bangkok.
5. Koonawootrittriron, S., M. A. Elzo, and T. Suwanasopee. 2007. Characterization of a negative halothane gene commercial multibreed swine population for growth and conformation traits in tropical western Thailand. J. Anim. Sci.85(Suppl.1):57.
6. Koonawootrittriron, S., M. A. Elzo, and T. Tongprapi. 2007. Genetic trends for dairy traits in the Holstein x Other multibreed dairy cattle population in tropical central Thailand. J. Anim. Sci.85(Suppl.1):19.
7. Koonawootrittriron, S., Elzo, M. A., Tumwasorn, S. , and Tongprapi, T. 2007. Age at first calving of dairy cattle in a multibreed population of Thailand. Proc. 44th Kasetsart University Conference, February 1- 5, 2006, Bangkok.
8. Reyes, A. de los, M. A. Elzo, V. M. Roso, R. Carvalheiro, L. A. Fries, and J. L. Ferreira. 2007. Animal model analyses of additive and non-additive genetic effects for 205-day weight in a Nellore x Hereford multibreed population in Brazil. J. Anim. Sci.85(Suppl.1):475.
9. Reyes, A. de los, M. A. Elzo, A. C. Sanches, R. B. Lobo, and L. A. F. Becerra. 2006. Effect of sire x maternal grandsire interactions on (co)variance estimates and genetic trends for pre-weaning growth traits in Brazilian Nellore cattle. Brazilian Anim. Sci. 7:365-371.
10. Rhone, J. A., S. Koonawootrittriron, and M. A. Elzo. 2007. Factors affecting bacterial score and bulk tank somatic cell count of dairy farms in the central region of Thailand. J. Anim. Sci.85(Suppl.1):639.
11. Rhone, J. A., S. Koonawootrittriron, and M. A. Elzo. 2007. Factors affecting milk yield and milk fat of dairy farms located in the central region of Thailand. J. Anim. Sci.85(Suppl.2):33.
12. Rhone, J. A., S. Koonawootrittriron, and M. A. Elzo. 2007. Factors affecting milk yield, milk fat, bacterial score, and bulk tank somatic cell count of dairy farms in the central region of Thailand. Trop. Anim. Health Prod. DOI: 10.1007/s11250-007-9074-5.
PROJECT CONTACT:
Name: Elzo, M. A.
Phone: 352-392-7564
Fax: 352-392-7652
Email: elzo@animal.ufl.edu

 
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ACCESSION NO: 0185634 SUBFILE: CRIS
PROJ NO: FLA-VME-03878 AGENCY: CSREES FLA
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 JUL 2000 TERM: 30 JUN 2006 FY: 2006
INVESTIGATOR: Rae, D. O.
PERFORMING INSTITUTION:
College of Veterinary Medicine
University of Florida
Gainesville , Florida 32610
RAISING THE CALF CROP IN FLORIDA BEEF CATTLE.
NON-TECHNICAL SUMMARY: Florida, despite enjoying a subtropical climate and nearly year-round grazing, has one of the lowest average calf crops in the continental U.S.; being approximately 76% which is more than 10% below the national average. Contributing factors include the stress of a hot, humid environment and the presence of large numbers of Bos indicus derived cattle. This project aims to identify and characterize a number of female-related factors which contribute to reproductive efficiency in beef cattle.
OBJECTIVES: Objectives: To evaluate methods of assessing reproductive performance in beef cattle in Florida, focusing on factors such as age, phenotype, nutrition, production measures and environmental stressors. To ascertain the best combination(s) of production traits for optimal female selection and management in Florida. To identify biostimulatory effects on female reproduction traits (such as age at puberty and postpartum return to estrus) in Florida beef cattle. To identify and quantify the effects of infectious disease processes on Florida calf crop, commencing with the development of an epidemiological model for Ureaplasmosis and Trichomonosis.
APPROACH: Assessing Reproductive Performance. Examination of the relationships between pregnancy rate, calving interval, body condition score prebreeding, body condition score at pregnancy examination, change in body condition score (spring to fall) and lactational state in four beef breeds in a subtropical environment will be assessed, focusing on the primiparous cow. Data was collected over 5 years from approximately 450 beef cows per year. Data will be analyzed. The statistical model will include: pregnancy rate, calving interval, breed, body condition-spring and fall, change in body condition score and lactational state. Ascertain traits optimal for female selection and management in Florida. Heifer reproductive traits will be obtained at cooperating sites. Data will include age, frame score, body condition score and weight at breeding, and pregnancy rate per cycle. Additional data will include reproductive tract scores, blood progesterone values at breeding, pelvic measures, calf weight and vigor. Bull reproductive data will include breeding soundness parameters and performance traits. Data will be analyzed for age and weight variations. Identify biostimulatory effects on female reproduction traits. Biostimulation is the stimulatory effect generated by the presence of the male on the sexual status of the female. The objective of this study is to assess the effect of biostimulation on reproductive efficiency in beef cows. The study will be conducted prior to and during the breeding season. Within a week of parturition, cows will be allocated to 3 groups and placed on separate pastures. Cows in Group A (n=30) will be placed with an epidydectomized bull (teaser); cows in Group B (n=30) will be placed with another teaser bull; and, cows in Group C (n=30) will serve as controls (no bull). The effect of biostimulation on uterine involution will be assessed by palpation of the uterus per rectum and ultrasonography. The effect of biostimulation on resumption of ovarian activity and cyclicity will be assessed by ovarian ultrasonography, blood progesterone and estradiol concentrations. Pregnancy will be diagnosed using blood progesterone, ultrasonography, and rectal palpation. The effect, of biostimulation on pregnancy will be calculated. Epidemiological Models of Infectious Disease Effects. Infectious disease processes can have dramatic effects on the beef cattle production. Assessment of the effects of disease, and its economic impact, requires the development of a suitable epidemiological models to describe observations in the field. Few models are available or suitable for modeling the extensively managed beef herds in areas such as Florida. Thus, this component of the study will attempt to develop such a model, using Tritrichomonas fetus as the infectious agent. The finding of high prevalence of infection on a major Florida beef ranch and several herd outbreaks in the past several years has provided data and the catalyst to commence the epidemiological model of this disease.
PROGRESS: 2000/07 TO 2006/06
Chlortetracycline in an ad libitum trace mineral salt mix prior to bull exposure was found to be associated with an increased proportion of females pregnant and a reduced time to conception. Other factors which were found to be associated with proportion pregnant were change in body condition score, average daily gain and reproductive tract score. Prevalence of and factors associated with Tritrichomonas fetus in bull populations in the state of Florida was reported. Tritrichomonas fetus infection continues to be prevalent within the natural service beef herds of Florida. The likelihood of disease is greatest in larger herds in more extensive management settings, such as South Florida. T. fetus infection in natural service beef herds in Florida was associated with bull (age, breed), herd and herd management practices. Sero-prevalence of Mycobacterium paratuberculosis (MAP) in Florida beef and dairy cattle was reported to have a true prevalence estimate of 11.2%. Although prevalence was lower than previously reported, cattle sero-prevalence appears to be wide spread. As many as 168,000 cattle in the State of Florida may be infected. In a subsequent study, Dam and calf genetic and environmental factors were evaluated for their association with enzyme-linked immunosorbent assay (ELISA) s/p ratio scores for paratuberculosis in a multibreed beef cattle population. Dams with high ELISA s/p ratio scores produced smaller calves, gained less weight (or lost more weight) during preweaning, and produced less milk, which in turn may have been the cause of calves with smaller preweaning gains. Factors identified here as associated with ELISA s/p ratio scores could help cattle producers with culling decisions related to paratuberculosis control and eradication efforts in beef cattle. In other studies, we have evaluated beef cattle postpartum management (i.e., effect of biostimulation on uterine involution, early ovarian activity and first postparum estrous cycle) and estrus synchronization protocols (i.e., synchronization of Bos indicus x Bos taurus cows for timed AI using GnRH plus PGF2a in combination with melengestrol acetate) to improve reproductive efficiency.
IMPACT: 2000/07 TO 2006/06
Effect of Chlortetracycline. There was an improved pregnancy percentage in groups treated with CTC trace mineral 30-d prior to breeding, and a possible positive effect from feeding during the second 30-d period. Prevalence of T. fetus. Evaluation of herd and bull population prevalence indicated a significant level of trichomonosis in Florida beef bulls. Large herds of breeding-age females were at significant risk of disease, especially in South Florida. Prevalence of Mycobacterium avium subspecies paratuberculosis (MAP). We found MAP prevalent in Florida beef and dairy cattle populations. We found that a high MAP ELISA s/p ratio scores are associated with lower cow weights (and perhaps lower milk production); these were evidenced by lower calf birth weights and lower preweaning gains. Thus, although ELISA tests have low sensitivity, there is evidence of a negative impact of a positive test (i.e., a higher likelihood of subclinical paratuberculosis) on production traits of dams and calves. Factors identified as associated with ELISA s/p ratio scores could help cattle producers with culling decisions related to paratuberculosis control and eradication efforts in beef cattle.
PUBLICATIONS (not previously reported): 2000/07 TO 2006/06
No publications reported this period
PROJECT CONTACT:
Name: Rae, D. O.
Phone: 352-392-4700
Fax: 352-392-7551
Email: owen@rams.vetmed.ufl.edu

 
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ACCESSION NO: 0210324 SUBFILE: CRIS
PROJ NO: FLA-VME-04625 AGENCY: CSREES FLA
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 JAN 2007 TERM: 01 JAN 2012
INVESTIGATOR: Rae, D. O.; Buergelt, C.; Elzo, M. A.
PERFORMING INSTITUTION:
College of Veterinary Medicine
University of Florida
Gainesville , Florida 32610
MYCOBACTERIUM AVIUM PARATUBERCULOSIS (MAP, JOHNES) DISEASE IN FLORIDA BEEF CATTLE.
NON-TECHNICAL SUMMARY: An important disease agent impacting the performance of cattle state-wide is Mycobacterium avium subsp. paratuberculosis (MAP). It is a bacterium that infects ruminants worldwide. It causes chronic, thickening of the gut, Johne's disease. The disease is characterized by chronic diarrhea and weight loss. There is no known cure for the disease and it is eventually fatal. The organism can be isolated from the cow's colostrum and milk and is transmitted primarily by these or a fecal-oral route to their calves early in life. Animals tend to become more resistant as age advances. There is a long incubation period. An animal rarely shows clinical signs until two years of age or more. Control of the disease is difficult. Currently there are no reliable tests for detecting early infection. Detection in older animals is problematic due to low test sensitivity. Using available diagnostic testing modalities and herd measures of performance within the cow-calf herd, different aspects of this project will be assessed. This project aims to identify and characterize a number of factors associated with Mycobacterium aviumparatuberculosis (MAP) disease. We will look at how it impacts cow performance and how management influences disease within the herd. We will focus on the affect on performance of beef cattle in the state of Florida, including performance and morbidity monitoring, genetic selection and genetic marker identification for disease resistance, and application of biosecurity principles in a commercial cattle setting.
OBJECTIVES: To evaluate the influence of Mycobacterium avium paratuberculosis (MAP) associated infection on the innate response of affected cattle populations and their performance among cohorts of [beef] cattle in the state of Florida. 1. To assess cow performance as measured by parameters such as, nutritional status measured by body condition, pregnancy, and calf weight gains, retrospectively as it is associated with likelihood of MAP infection. 2. To assess birth cohorts of calves (by growth, morbidity and mortality factors) born to dams with a greater or lesser likelihood of MAP infection. 3. To evaluate the influence of implemented biosecurity practices on clinical and subclinical cases of MAP, cow performance and environmental contamination. 4. To evaluate the presence/absence of genes associated susceptibility/resistance in multiple breeds of cattle with an aim to identify genetic selection tools for management of MAP. 5. To assess and quantify the effects of the MAP infectious disease process on the Florida calf crop by development of epidemiological models.
APPROACH: Objective 1. To assess cow performance Dam and calf genetic and environmental factors have been evaluated for their association with enzyme-linked immunosorbent assay (ELISA) s/p ratio scores for paratuberculosis (MAP) in a multibreed beef cattle population. The analysis used 359 ELISA s/p ratio scores from 340 dams. Dams with high ELISA s/p ratio scores produced smaller calves, gained less weight (or lost weight) during the preweaning season, and produced less milk, which in turn may have caused calves to have smaller preweaning gains. Factors identified here as associated with ELISA s/p ratio scores could help cattle producers with culling decisions related to paratuberculosis control and eradication efforts in beef cattle. We will continue this evaluation process. Objective 2. To assess birth cohorts of calves. Genetic evaluation for production traits assume that records come from healthy animals. Records from animals suffering from chronic diseases with long subclinical incubation periods may be difficult to identify, thus likely to be included in genetic evaluations, and disease effects not accounted for; this is applicable to MAP infection. A common diagnostic test for paratuberculosis is ELISA. Regression estimates of four cow and two calf traits on ELISA scores were obtained. Cows with greater ELISA scores tended to stay open longer, have larger weight losses, and have calves with lower birth and weaning weights than cows with lesser ELISA scores. We will continue this evaluation process. Objective 3. To evaluate the influence of implemented biosecurity practices on clinical cases of MAP. Biosecurity practices are essential for the successful eradication of MAP in a herd of cattle. Management plus testing is expected to produce a more rapid reduction in prevalence of disease and environmental burden of microbes. Changes in risk of disease, by use of a risk assessment tool, will assess the relative value of biosecurity practices. Objective 4. To evaluate the presence/absence of genes associated susceptibility/resistance in multiple breeds of cattle. There is evidence that resistance to infectious disease in animals has a genetic basis and that genetic variation exists among animals in their response to various infectious challenges. Johnes disease has been demonstrated to have a genetic component. This will be a candidate gene case control association study. The project consists in determining the alleles present in one candidate gene in a population of infected cows (cases) and of non-infected controls to find a resistance allele that could be useful in selection. Objective 5. To assess and quantify the effects of the MAP infectious disease process on the Florida calf crop. Infectious disease processes can have dramatic effects on beef cattle production. Assessment of the effects of disease, and its economic impact, can be estimated by the development of suitable epidemiological models. A disease model will be developed for MAP infection.
PROGRESS: 2007/04 TO 2007/12
Association among serum ELISA, fecal culture, and nested PCR on milk, blood, and feces for the detection of paratuberculosis infection in dairy cows was evaluated. The objective was to analyze the association among a serum ELISA, fecal culture, and nested PCR tests on milk, blood, and feces for Mycobacterium avium subsp. paratuberculosis detection in Holstein cows. Feces, blood and milk samples were collected from 328 lactating dairy cows in four dairy herds to detect paratuberculosis infection. Association between two polymorphisms in the bovine CARD15/NOD2 gene and paratuberculosis infection in Florida dairy and beef cattle was studied. Caspase recruitment domain 15 (CARD15/NOD2) is a gene codifying for a cytosolic protein implicated in bacterial recognition by cells of the innate immune system. The objective of this candidate gene case-control study was to characterize the distribution of two polymorphisms in the bovine CARD15/NOD2 gene and test their association with paratuberculosis infection. The study population consisted of 432 adult cows in four herds. Infection status was determined using five diagnostic tests (serum ELISA, milk/blood/fecal nested PCR, and fecal culture); a parallel interpretation of results was used. Two single nucleotide polymorphisms in the gene were determined for study animals by genotyping assay. It was hypothesized that alleles in our candidate gene would be present in higher frequency in controls compared to cases, suggesting a role in resistance to infection. Association between cow reproduction and calf preweaning growth traits and ELISA scores for paratuberculosis in a multibreed herd of beef cattle was assessed. The objective of this study was to assess the association between 4 cow reproductive and weight traits, and 3 preweaning calf traits and ELISA scores for paratuberculosis in a multibreed herd of cows ranging from 100% Angus to 100% Brahman. Cow data included gestation length, time open, calving interval, and weight change for 502 cows. Calf data consisted of birth, weaning, and adjusted weaning weights for 956 calves. Presentations: 9th International Colloquium for Paratuberculosis, Tsukuba, Japan, Oct 29-Nov 2, 2007, 6A-O2, Association between Two Polymorphisms in the Bovine CARD15/NOD2 Gene and Paratuberculosis Infection in Florida Dairy and Beef Cattle. PJ Pinedo, CD Buergelt, R Wu, GA Donovan, JE Williams, PG Melendez, L Morel, and DO Rae, Poster and Oral. Research Summary, 2007 (20 Sep) American Association of Bovine Practitioners, Annual Conference, Vancouver, BC, Genetic Resistance to Johne's Disease in Four Cattle Breeds: A Candidate Gene Case Control Study, Preliminary Results, Poster and Oral. Emerging Pathogens Institute, Fall Research Retreat, University of Florida, Dec 13, 2007, Poster. Association between CARD15/NOD2 Gene polymorphisms and paratuberculosis infection in Florida Cattle, P Pinedo, C Buergelt, A Donovan, P Melendez, DO Rae, R Wu. 2nd Bi-annual Research Emphasis Day, College of Veterinary Medicine, University of Florida, May 11, 2007, Diagnostic development in bovine paratuberculosis, P Pinedo, DO Rae.
IMPACT: 2007/04 TO 2007/12
The study of association among serum ELISA, fecal culture, and nested PCR on milk, blood, and feces for the detection of paratuberculosis infection in dairy cows analyzed results to establish the association and the level of agreement between pairs of tests. A total of 61 animals (18.6%) tested positive when all the tests were interpreted in parallel. The agreement between results in different pairs of tests was poor, slight and fair. Fecal culture vs. fecal PCR resulted in the highest kappa coefficient (0.39; fair agreement), with the lowest agreement being for serum ELISA vs. PCR on blood (-0.036; poor agreement). Statistically significant association was found between the following test pairs; ELISA fecal culture; ELISA:fecal PCR; PCR on milk:fecal PCR, PCR on blood:fecal PCR and fecal culture:fecal PCR. The complementary sensitivity values obtained in this study suggests the potential use of different tests combinations to increase the overall sensitivity for the diagnosis of paratuberculosis infection. A study of association between two polymorphisms in the bovine CARD15/NOD2 Gene and paratuberculosis infection in Florida dairy and beef cattle resulted in significant differences in allelic frequencies between cases and controls for SNP1 indicating a significant association between infection and mutant allele. Significant association was found between SNP1 and infection status. A significant association between allele combinations and infection status was found when both SNPs (1 and 2) were considered in the genotype. The low representation of the variant allele for SNP1 in Holstein and Jersey breeds raises the prospect of a potential confounding role of breed for its connection with infection. However, a significant association between SNP1 and infection was confirmed when tested within the Brahman-Angus sub-population. Preliminary results suggest a role for CARD15/NOD2 gene in the susceptibility of cattle to paratuberculosis infection. Amino acid substitution C733R (SNP1) appears to be associated to paratuberculosis infection in Florida cattle. These results could be the basis for further research to create a rapid method to select for more resistant individuals, genetically contributing to the control of Johne's disease. Association between cow reproduction and calf preweaning growth traits and ELISA scores for paratuberculosis in a multibreed herd of beef cattle found that estimates of differences between cows with non-zero and zero ELISA scores were associated with lower cow fertility (longer TO), lower ability of cows to maintain weight (negative WC), lower calf BWT, and lower calf weaning weights (WWT and WW205). Considering TO, WC, and WWT, and using average market prices of cows and calves, potential losses of income due to subclinical paratuberculosis were estimated to be $62.4 for cows with positive ELISA score. Further research on the effects of subclinical paratuberculosis in beef cattle at regional and national levels seems advisable considering the large potential economic cost of this disease.
PUBLICATIONS (not previously reported): 2007/04 TO 2007/12
No publications reported this period
PROJECT CONTACT:
Name: Rae, D. O.
Phone: 352-392-4700
Fax: 352-392-7551
Email: raeo@mail.vetmed.ufl.edu

 
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ACCESSION NO: 0199993 SUBFILE: CRIS
PROJ NO: GEOV-0475 AGENCY: CSVM GEOV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 AUG 2003 TERM: 31 JUL 2004 FY: 2004
INVESTIGATOR: Corn, J. L.; Davidson, W. R.; Fischer, J. R.
PERFORMING INSTITUTION:
College of Vet Medicine
University of Georgia
110 Riverbend Road
Athens , Georgia 30602
MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN FREE-RANGING BIRDS AND MAMMALS ON LIVESTOCK PREMISES.
NON-TECHNICAL SUMMARY: Ruminant and non-ruminant wildlife may become exposed to Mptb via feeding on contaminated grain, forage in pastures, feces, or on infected prey. The purpose of this study is to determine if wildlife associated with livestock premises are infected by Mptb.
OBJECTIVES: (a) To determine if infection by Mycobacterium avium subspecies paratuberculosis occurs in free-ranging mammals and birds on infected livestock premesis; (b) To determine if the prevalence of infection by Mycobacterium avium suspecies paratuberculosis is higher in free-ranging mammals and birds on infected livestock premesis versus non-infected premesis; (c) To determine habitat and spatial associations of infected wildlife with livestock on livestock premesis.
APPROACH: Intensive sampling of wildlife will be conducted in the immediate vicinity of livestock premises in the Midwest United States ( Wisconsin) and the Southeast ( Georgia). In each state, specimens will be collected from three premises that contain livestock infected with Mptb, and from one negative control site. Wildlife sampled will be representative of those species with the highest potential for exposure to contaminated materials, and /or that pose the highest risk for contamination of livestock feed or forage. Specimens of ileum, liver, intestinal lymph node, and feces will be harvested from each mammal collected, and ileum, liver, and feces will be collected from birds. A portion of all specimens will be chilled for culture; additional specimens of ileum and intestinal lymph nodes will be fixed in 10% buffered formalin for future histopathological evaluation if deemed necessary. This study will identify which, if any, species of mammals and birds found at infected livestock premises in Georgia and Wisconsin are infected by Mptb, and provide data on the prevalence of infection in wildlife from infected livestock premises as well as the geographic and habitat association of infected animals with livestock. These data will be used to develop further epidemiological studies for use in determining control measures for Mptb in livestock.
PROGRESS: 2003/08 TO 2004/07
The detection of Map infections in a wide range of wildlife species in Wisconsin and Georgia is the first report of Map in non-ruminant wildlife in North America, and is consistent with recent survey results in Scotland(Beard et al.,1999,2001a).In each of these studies both mammals and birds were infected, and infections were detected on numerous premises. In Scotland, the infection prevalence was high in rabbits(up to 67%)(Greig et al.,1997,1999), foxes (85%), stoats (46%), and crows(60%)(Beard et al.,2001a). In our surveys, multiple isolates of Map came from raccoons, armadillos, feral cats, and European starlings, while single isolates were made from several other species. It is evident that a wide range of wildlife can be infected by Map, but the role of wildlife in the maintenance and transmission of this disease agent to livestock or other wildlife is not yet clear. The isolation of Map from both tissue and fecal specimens demonstrates that these were cases of true infection, not simply contamination by recently ingested material. The lack of histological lesions in tissues from our survey leaves questions as to the pathologic capacity of Map in these wildlife species. In Scotland, histological lesions were seen in rabbits (Greig et al., 1997, 1999), foxes, weasels, a stoat, wood mouse, and crow (Beard et al., 2001a). The lack of histological lesions in our specimens may be related to the phase of infection in the small number of culture-positive animals examined, or indicative of a lack of pathology associated with Map-infection in these species. Shedding of Map may result in wildlife to wildlife, wildlife to livestock, and/or livestock to wildlife transmission, and infection pressure via these routes needs additional study. Fecal contamination of the environment on Minnesota dairy farms by Map-infected cows is extensive(Raizman et al.,2004),and given the density of cows on farms, and the volume of contaminated feces produced by infected cows, farm contamination by cows probably is high when compared to contamination produced by infected wildlife. However, Daniels et al. (2001,2003a,2003c) found that rabbits excrete up to 4x106 colony forming units per gram of feces, and as such, postulated that livestock could become infected by ingesting forage contaminated by rabbits. Daniels et al.(2003a) suggest that rabbits are likely to present the greatest risk to livestock due to the high prevalence of infection, high level of fecal contamination of pastures, and the lack of avoidance of rabbit feces on pastures by cattle. Daniels et al.(2003b) developed a model that suggests that fecal contamination of stored feed by wildlife could serve as a source of Map infection for livestock in Scotland. We did not measure environmental contamination by infected wildlife over time, but 5 of the 26 culture-positive animals sampled in our survey, or 5 of the total 674 animals sampled on Map-infected farms, were shedding when sampled. Because specimens from birds were collected as liver and gastrointestinal tract only, it was not possible to determine if shedding occurred in birds in our survey.
IMPACT: 2003/08 TO 2004/07
Individuals of some species of wildlife may live for several years and can have home ranges that cover areas large enough to include more than one farm. Raccoon home ranges average 40-100 ha, and movements may be made to areas outside the home range to visit temporary food sources (Kaufmann, 1982). Infected wildlife may shed Map over a period of time, and this could include shedding on the farm where the infection originated, as well as on nearby farms, depending on movement patterns of the individual animals. Wildlife may not be important to the maintenance of Map on farms when infected livestock are present due to the relatively large amount of Map-contamination by livestock. However, wildlife may be an epidemiologically significant factor for Map control on farms that have eliminated all Map-infected livestock from the premises, and on Map-free farms in the same geographic area as infected farms. Whittington et al. (2003) found that recovery of Map from environmental samples on sheep and goat farms in New Zealand was very low five months after the infected stock were removed, but infected wildlife with longer life spans may continue shedding beyond this period of time.
PUBLICATIONS (not previously reported): 2003/08 TO 2004/07
No publications reported this period
PROJECT CONTACT:
Name: Corn, J. L.
Phone: 706-542-1741
Fax: 706-542-5865
Email: jcorn@vet.uga.edu

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ACCESSION NO: 0201802 SUBFILE: CRIS
PROJ NO: GEOV-0484 AGENCY: CSREES GEOV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 SEP 2004 TERM: 31 AUG 2005 FY: 2006
INVESTIGATOR: Vandenplas, M. L.; Okinaga, T.; Hurley, D. J.
PERFORMING INSTITUTION:
College of Vet Medicine
University of Georgia
110 Riverbend Road
Athens , Georgia 30602
CORRELATING MAP INDUCED NF-KB ACTIVATION WITH NOD2 MUTATIONS IN JOHNE'S DISEASE.
NON-TECHNICAL SUMMARY: Infection with Mycobacterium avium ssp paratuberculosis
(Map) and Johne's disease are a significant economic problem in rearing cattle. It has been recognized for 30 years that resistant and sensitive individuals can be found within the population of cattle. At present, there are no direct methods to identify resistant individuals without challenging them with the organism. This project will utilize a readily measurable physiological response of white blood cells to infection with Map to sort cattle into two groups, and then allow us to look for differences in the genes that control that response as a starting point in a biological definition of resistant and susceptible animals. The results of this project will provide candidate genetic markers for the development of Johne's resistant herds.
OBJECTIVES:Mycobacterium avium subspecies paratuberculosis (Map) is associated with the development of inflammatory gastrointestinal diseases in humans and cattle. In the human manifestation of the disease, known as Crohn's disease, mutations in an inflammatory regulation control gene, NOD2, have been identified in >80% of individuals with disease symptoms, and are a critical factor in susceptibility to Crohn's disease. The mutations impair activation of NF-kB, a pleotropic transcription factor involved in regulation of a wide variety of inflammatory mediators. Mutations in Toll-like receptor-(TLR) 2 and TLR4 genes have also been associated with increased susceptibility to bacterial infections in other species, and are potential candidates for the genes responsible for the susceptibility of some cattle to Johne's disease. Our hypothesis is that activation of NF-kB by viable Map organisms will be significantly less in mononuclear leukocytes obtained from cattle having mutations in one or more of the inflammatory regulation genes (NOD2, TLR2 and TLR4) than in mononuclear leukocytes obtained from cattle lacking mutations in these genes. To test this hypothesis, experiments will be conducted to pursue two goals. The first goal is to demonstrate that cattle can be sorted into two distinct populations based on differences in translocation of NF-kB in their mononuclear leukocytes induced by Map organisms. The second goal is to sequence mRNA for the genes that regulate inflammatory responses to bacteria, specifically NOD2, TLR4, and TLR2. We expect that mutations in NOD2, TLR2 and/or TLR4 will segregate with a reduced ability of Map to activate NF-kB in mononuclear leukocytes. The objectives of this project are: 1) to collect mononuclear leukocytes from 20 cattle, 10 from a herd lacking evidence of Johne's disease for at least 2 years, and 10 from the Johne's disease demonstration herds in Georgia, each of which contains many Johne's disease positive animals. Mononuclear leukocytes in culture will be infected with a Multiplicity of Infection of 10 Map organisms per leukocyte for 12-16 hours; E. coli lipopolysaccharide, and S. aureus muramyl dipeptide will be used as positive TLR4 and TLR 2 controls, respectively. The leukocytes will be collected and NF-kB electrophoretic mobility shift assays performed to assess the level of activation of the transcription factor in cells from each animal; 2) To clone and sequence a full-length cDNA transcript for bovine NOD2 and use the known bovine TLR2 and TLR4 sequences, to design the mRNA sequencing primers needed to establish genetic probes for evaluating NOD2, TLR2 and TLR4 genes, and 3) To sequence the mRNA for NOD2, TLR2 and TLR4 from each of the cattle evaluated in Objective 1 and then to identify single nucleotide polymorphisms that segregate with an altered ability to induce activation of NF-kB.
APPROACH: Animals from two herds will be utilized. Blood samples will be collected from 10 cattle housed at a facility that has gone through two years of screening under the Johne's control program and has no evidence of Johne's disease on the facility. Blood samples from 10 cattle in the Georgia Johne's demonstration herd will also be collected. This herd has active Johne's disease; cattle not showing symptoms of Johne's disease animals will be selected as donors. Blood will be collected and transported to the lab within 6 h of collection. Buffy coat will be collected for each animal, and the buffy, layered over Histopaque 1083 to generate a population of mononuclear cells. PBMCs will be suspended in RPMI + 10% FCS and incubated at 37C with the bacterial products (1 hours) or Map organisms (14 hr) . At the end of the incubation time, a nuclear extract will be prepared, the released nuclear protein will be collected by centrifugation and the protein quantified. The electrophoretic mobility shift assays for each nuclear protein extract will be incubated at room temperature for 20 minutes with 32P-labeled NF-kB oligonucleotide and the complexes resolved on polyacrylamide gels. Each gel will also include nuclear extract from LPS-stimulated human Mono Mac 6 cells as an inter-assay control. The gels will be dried and visualized on a phosphoimager. Relative change in complex intensity will be determined by densitometry and expressed as a percentage of Mono Mac 6 cell nuclear extract. The NOD2 clone will be obtained from BACPAC Resource Center. The insert of the clone will be fully sequenced to obtain 3' half of bovine NOD2. The 5'-end sequence of bovine NOD2 will be obtained by 5'-RACE PCR. Purified mRNA from bovine peripheral blood mononuclear cells, will serve as template in the 5'-RACE PCR reaction using an internal primer designed from the bovine NOD2 sequence. The insert of 5 PCR-derived clones will be fully sequenced for both strands. This will allow us to obtain full-length sequence of bovine NOD2. Using the known bovine TLR2 and TLR4 sequence together with our bovine NOD2, PCR primers will be designed to produce overlapping PCR products that span the entire sequence of mRNA transcripts. Primer pairs will be used in a single step RT-PCR with RNA isolated from each animal as template. After PCR amplification, the products will be purified, size confirmed by agarose gel electrophoresis and then quantified. The PCR product will be sequenced by high throughput sequencing methodology. Sequences of the PCR products generated the RNA sample then will be compared to identify single nucleotide polymorphisms, and the results compared with the NF-kB phenotypic response data.
PROGRESS: 2004/09 TO 2005/08
Johne's disease, a chronic inflammatory bowel disease of cattle caused by Mycobacterium avium subsepecies paratuberculosis (Map), is a bovine disease with high economic impact on the cattle industry. Currently these is no means of identifying genetic susceptibility in cattle herds. Mutations in the NOD2 gene that regulates inflammatory responses to toxic components of bacterial cell walls have been identified in humans has been shown to segregate with the presence of Crohn's disease, which has been associated with Map infection. These NOD2 mutations alter the inflammatory responses of macrophages from these people, resulting in a decreased ability to activate the transcription factor NF-&#954;B. We examined therefore activation of NF-&#954;B in bovine leukocytes in vitro by Map organisms as a preliminary phenotypic screen to identify Map sensitive and resistant animals. While Toll-like receptor ligands, lipopolysaccharide (LPS) and muramyl dipeptide, both induced clear and consistent NF-&#954;B nuclear translocation in bovineleukocytes, treatment of cultured bovine leukocytes with isolated Map at different MOI's and for an extended period did not consistently yield clear NF-&#954;B nuclear translocation. These NF-&#954;B nuclear translocation assays are essential for identifying animals with a potential phenotypic predisposition for the development of Johne's disease. Currently we are attempting to develop new techniques of bovine leukocyte isolation, culture and stimulation with Map organisms that will allow us to reliably monitor Map activation of NF-&#954;B.
IMPACT: 2004/09 TO 2005/08
Johne's disease, a chronic inflammatory bowel disease of cattle caused by Mycobacterium avium subsepecies paratuberculosis (Map), costs the cattle industry in the United States millions of dollars annually. Although it has been known for more than 40 years that some cattle are resistant to mycobacterial infections, including Johne's disease, there has been no clear direct indicator of susceptibility to these organisms. Consequently, there is no way to detect/eliminate susceptible individuals or to select resistant animals for breeding purposes. Furthermore, there are no rapid and reliable methods for identifying animals with subclinical infections, thus making it virtually impossible to eliminate the disease. Development of novel assays to identify animals with genetic predisposition to Map infection and the potential for development of Johne's disease would have a major impact on the cattle industry.
PUBLICATIONS (not previously reported): 2004/09 TO 2005/08
No publications reported this period
PROJECT CONTACT:
Name: Hurley, D. J.
Phone: 706-542-6371
Fax: 706-542-8833
Email: dhurley@vet.uga.edu

 
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ACCESSION NO: 0201882 SUBFILE: CRIS
PROJ NO: GEOV-0485 AGENCY: CSREES GEOV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 JUL 2004 TERM: 30 SEP 2005 FY: 2005
INVESTIGATOR: Quinn, F. D.
PERFORMING INSTITUTION:
College of Vet Medicine
University of Georgia
110 Riverbend Road
Athens , Georgia 30602
CROHNS DISEASE: POTENTIAL ACQUISITION THROUGH PASTEURIZED MILK.
NON-TECHNICAL SUMMARY:Mycobacterium paratuberculosis causes Johnes disease in cattle and other ruminants and may cause Crohns disease in humans. We propose to expose a strain of mouse routinely used in inflammatory bowel disease studies to M. paratuberculosis antigens combined with homogenized and pasteurized milk fat to determine if this exposure is associated with the time of onset and nature of observed Crohns-like illness.
OBJECTIVES:Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) causes Johnes disease, a chronic granulomatous enteritis in cattle and other ruminants. The organism is primarily shed in feces, but has also been isolated in milk from cows with clinical disease, subclinical infection and occasionally from bulk milk. A possible link between M. paratuberculosis and Crohns disease in humans has been postulated with milk as a potential source of infection. Pasteurization of milk is a critical control point in reducing the risk of human consumption of viable M. paratuberculosis bacilli. In a paper tabled at the UK Advisory Committee on the Microbiological Safety of Food it was announced that M. paratuberculosis had been isolated from commercially pasteurized milk. Methods for improving the pasteurization process to completely eliminate viable M. paratuberculosis bacilli have been proposed and are under consideration. However, before the expense and effort are put forth to potentially change industry processes and standards, we suggest ruling out another possibility, that heat-killed M. paratuberculosis bacilli serve as an adjuvant to enhance immune response to antigens in the intestinal lumen. Homogenized milk is an oil-in-water emulsion, and the addition of heat-killed M. paratuberculosis finishes the recipe for Complete Freunds Adjuvant, a powerful stimulant of immune response. While it is unlikely that the form of lipid in milk and the concentrations of lipid and mycobacterial components are optimal for adjuvant activity, they may be sufficient to push susceptible consumers into disease. Much observational and experimental data available support this as a possible mechanism. We propose to expose a strain of mouse routinely used in inflammatory bowel disease studies to M. paratuberculosis antigens combined with homogenized and pasteurized milk fat to determine if this exposure is associated with the time of onset and nature of observed Crohns-like illness. If pasteurization, one of the most successful public health measures ever devised and the basis of our milk safety program is an essential component of this pathogenic mechanism, the dairy industry and regulatory agencies will face a dilemma.
APPROACH: If our hypothesis is correct, persons likely to become ill from foodborne and other exposures to M. paratuberculosis (MAP) will either be immune compromised and develop invasive multi-system disease that may or may not involve the gastrointestinal tract, or have a poorly regulated Th1 response to enteric microflora, perhaps including MAP. The proposed experiments will explore the latter by using a rodent model of spontaneous enteritis that has a predictable onset of clinical disease associated with inflammatory cytokine imbalance. In addition, because of the influence of lipid vehicles on Th1/Th2 response to antigens, MAP delivered in a lipid food matrix is more likely to stimulate a Th1 dominated response. Therefore, this proposal will use high-fat vehicles derived from foods that may be naturally contaminated with MAP. Several animal models of spontaneous and induced intestinal inflammation have been developed to investigate the etiology and pathogenesis of inflammatory bowel disease (Crohns disease and ulcerative colitis). One model, the SAMP1/Yit mouse, develops a syndrome that closely resembles Crohns disease in that it spontaneously develops and most intensely involves the ileum with segmental, transmural granulomatous enteritis. Onset of illness in the mice begins around 20 weeks of age, with nearly 100% affected by 30 weeks of age and continuation of lesions and illness at least through 80 weeks of age. Disease appears to be mediated by an abnormal TNF-alpha &#61472;and IL-12 response. To test the hypothesis that early exposure to MAP antigens may be a triggering event in the pathogenesis of Crohns disease, we propose to expose SAMP1/Yit mice to MAP antigens before their anticipated onset of enteritis to determine if this exposure is associated with modifications in the time of onset and nature of illness. The presence of heat-killed Mycobacterium species in milk gives it adjuvant-like qualities that induce a helper T cell type 1 (Th1)-dominated immune response to bacterial antigens present in the guts of persons with a certain genetic background. The inciting antigens are likely to be normal enteric flora but may be mycobacterial antigens. Twenty-eight SAMP1/Yit mice will be acquired at 4 weeks and 28 mice at 8 weeks from Harlen, U.K. The mice in each age group will be divided into 4 groups of 7 in micro-isolator boxes. The mice will be exposed to cream or cream with live/killed MAP daily for 5 days by gavage at either 5 weeks or 10 weeks of age. The specific concentrations of bacteria will be 0.0 mg bacteria (diluent only), 0.1 mg heat-killed sonicated bacterial, 1.0 mg heat-killed sonicated bacteria, or 10 logarithmic phase viable
MAP bacilli. The mice will be observed daily for clinical signs of illness, especially enteritis. At 20 weeks of age (either 10 or 15 weeks post exposure), blood will drawn from 3 mice from each box and serum obtained. These mice will then be sacrificed and gross necropsy and histopathologic examinations performed. Cytokine Luminex ELISA assays will be performed on the isolated sera. At 30 weeks of age, the remaining mice will be sacrificed and similar analyses performed.
PROGRESS: 2004/07 TO 2005/09
Specific Aims: Mycobacterium paratuberculosis causes a chronic granulomatous enteritis known as Johnes disease in cattle and other ruminants, and is the primary suspect in human Crohns disease; a debilitating disease primarily in children that is increasing in incidence at an alarming rate. These mycobacteria can routinely be detected by PCR from the intestinal biopsies of Crohns disease patients while viable bacilli are only occasionally isolated. A hypothesis to explain this is that heat-killed M. paratuberculosis bacilli are sufficient to induce Crohns disease. Tuberculous mycobacterial cell walls are the key components that make Complete Freunds adjuvant, a powerful stimulant of the immune response. We proposed to test this hypothesis in a mouse model of inflammatory bowel disease (IBD; a Crohns-like illness). 1. Determine if viable and non-viable M. paratuberculosis bacilli can produce Crohns disease-like symptoms in the IBD mouse model 2. Collect and examine mouse serum to determine if levels of key secreted cytokines mimic those detected in Crohns disease. 3. Examine mouse intestinal tissue for early onset of IBD after ingestion of heat-killed M. paratuberculosis bacilli emulsified in pasteurized cream. B. Studies and Results: Several experiments were performed using transgenic IL-10 knock-out mice (IBD model strain). The inocula consisted of sterilized heavy cream, cream with 105 live Mycobacterium paratuberculosis bacilli or cream with 105 killed bacilli (65o C for 2 hours) given to the mice via gavage. Aim 1: Experiment 1: A loss of body mass was observed in mice infected with the live or heat-killed strains but not when the cream control was used. Experiment 2: Serum amyloid A protein levels in infected and control mice were assayed. This protein is a marker of inflammation or infection. By 14-weeks after infection, both live and heat killed bacteria cause an enhanced production of amyloid A in mice while the cream control did not. Aim 2 Experiment 1: Sera, mesenteric lymph nodes (MLN), Peyers patches (PP) or spleen tissues were examined for the presence of IL-2, TNF-alpha, INF-gamma and IL-4 from IL-10 K/O mice infected with live bacilli in cream, cream alone or heat-killed bacilli in cream. Th1 cytokines IL-2, TNF-alpha and INF-gamma&#61472;were selected because they have been shown to be secreted in significant amounts in the blood and diseased intestinal tissues of Crohns patients compared to normal controls. IL-4 is a Th2 cytokine control and is not routinely elevated in association with Crohns disease. Results showed elevated production of IL-2, TNF-alpha and INF-gamma&#61472; but not IL-4 in all tissues and sera after infection with the bacilli (live or heat-killed) relative to levels produced with the cream alone. This indicated a Th1 immune response. Aim 3, experiment 1: Histopathological examination of intestinal tissue from mice given cream or cream with heat-killed bacteria. This work is ongoing and results are not yet available.
IMPACT: 2004/07 TO 2005/09
Significance: Though not yet completed, this research thus far supports our hypothesis that the combination of M. paratuberculosis bacilli (live or dead) and cream may act as an adjuvant to stimulate a cell mediated inflammatory response as defined by the production of amyloid protein and Th1 specific cytokines. Plans: Current and future studies are focusing on the associated histopathology, identifying the specific bacterial antigens responsible for the observed inflammation and eventually performing these same experiments in the more relevant goat model for Johnes disease. The goat model appears to mimic more closely human Crohns disease than any of the currently available mouse models. If confirmed in the goat model, we may need to discuss options and reassess permitted levels of M. paratuberculosis in cow milk.
PUBLICATIONS (not previously reported): 2004/07 TO 2005/09
Singh UP, Singh S, Singh R, Karls RK, Quinn FD, Potter ME and Lillard JW Jr. 2006. Influence of Mycobacterium avium paratuberculosis on T cell response during Crohns disease and murine colitis. Submitted.
PROJECT CONTACT:
Name: Quinn, F. D.
Phone: 706-542-5790
Fax: 706-542-5771
Email: fquinn@vet.uga.edu

 
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ACCESSION NO: 0206455 SUBFILE: CRIS
PROJ NO: GEOV-0496 AGENCY: CSREES GEOV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 15 DEC 2005 TERM: 14 DEC 2007 FY: 2006
INVESTIGATOR: Karls, R.; Pence, M.
PERFORMING INSTITUTION:
College of Vet Medicine
University of Georgia
110 Riverbend Road
Athens , Georgia 30602
ISOLATION OF MYCOBACTERIOPHAGE THAT TARGET M. AVIUM SSP. PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: Antibiotic treatment of cattle infected with Johne's disease is not practical. This purpose of this project is to isolate and develop mycobacteriophages that may serve as a cost-effective intervention strategy to prevent disease transmission.
OBJECTIVES: This project seeks to isolate mycobacteriophage that can target Mycobacterium avium ssp. paratuberculosis that may be developed into a therapeutic intervention strategy to prevent transmission of Johne's disease. The goal is to obtain multiple mycobacteriophage that enter the host via different receptors to minimize the likelihood of resistance to infection.
APPROACH: Soil and manure samples from farms with known Johne's Disease will be screened for the presence of mycobacteriophages using a plaque assay. Once mycobacteriophages have been identified, the mycobacterial host range of each will be determined using both saprophytic and pathogenic mycobacterial species. Those that can infect M. avium ssp. paratuberculosis (MAP) will be characterized further. They will be assayed on a variety of MAP strains to identify whether any target a host receptor that is absent on some clinical MAP strains. Phage plaque morphology will be examined for the presence of a lysogenic growth stage (turbid plaque phenotype). Genetic analysis of phage that target MAP will be performed. If necessary, the repressor of lysogeny will be disrupted to produce phage that grow only in a lytic mode.
PROGRESS: 2005/12 TO 2007/12
OUTPUTS: Contacts were made with Georgia farmers to enable sampling of soil, manure, and pooled liquids from pastures and feedlots. The goals of the project (to isolate phages that infect the bacterial cause of Johne's disease, Mycobacterium paratuberculosis) were discussed with the farmers prior to obtaining samples. Procedures were established to enable stable transport of samples back to the laboratory. Parameters were established to effectively screen samples for mycobacteriophages on various mycobacteria hosts. Results of this research were disseminated to members of the research community within the University of Georgia College of Veterinary Medicine in the forms of laboratory research progress presentations and in discussions with faculty. It is anticipated that the results of this research will provide sufficient data to effectively compete for a grant from USDA or NIAID to continue this research. PARTICIPANTS: A number of people worked on this project. The project was a collaborative effort of two University of Georgia investigators: Dr. Mel Pence, a veterinarian based at the Tifton campus, and Dr. Russell Karls, a research scientist at the Athens campus. Dr. Pence was largely responsible for collecting samples from Georgia farms with cattle positive for Johne's disease. Dr. Karls led the screen for mycobacteriophages and their subsequent analyses. Dr. Karls trained several University of Georgia personnel who participated in this study. Alyson Weber and William Barrow were undergraduates who helped with the initial screening of the soil samples and host range determinations. Rotating graduate students, Jon Gabbard and Alaina Jones, and a volunteer researcher, Leena Malayil, participated in the isolation and examination of DNA from these mycobacteriophages. TARGET AUDIENCES: The hypothetical nature of this project principally targets the scientific community interested in finding alternate strategies to halt the spread of Johne's disease. Presentations were in the form of scientific presentations and one on one discussions withmembers of the scientific community.
IMPACT: 2005/12 TO 2007/12
The initial screenings for mycobacteriophages employed use of a mycobacterium species that replicates quickly. The rapid growth of this bacterium in soft agar overlays made it possible to detect infection of the bacteria by mycobacteriophages in the filtered samples in the form of plaques or clearing zones that appeared within the turbid background formed by bacterial growth. Using this technique, we were able to isolate two distinct mycobacteriophages. One formed small plaques, while the other formed much larger plaques. Upon amplifying the mycobacteriophages in this bacterium and testing for plaque formation on slow growing mycobacteria species, the initial results were negative. However, we discovered that plaques could sometimes be detected with some mycobacteria hosts if the bacteria were allowed to grow on the plates in the soft agar for longer periods of time prior to adding the mycobacteriophages. In some cases this required incubations for several days. Both phages were able to infect the fast growing species Mycobacterium smegmatis and a somewhat slower growing species Mycobacterium avium. Only one of the phages appears to infect Mycobacterium paratuberculosis. Additional mycobacteriophages that can infect this bacterium are necessary to continue to develop an effective intervention strategy to prevent transmission of this bacterium in cattle. Through the resources provided and the activities of those involved, future searches for phages that infect Mycobacterium paratuberculosis are likely to be more fruitful. The results of this study enabled the design of a screen to directly identify mycobacteriophages that infect Mycobacterium paratuberculosis. This may allow for detection of mycobacteriophages that may have been missed by initially screening on Mycobacterium smegmatis.
PUBLICATIONS (not previously reported): 2005/12 TO 2007/12
No publications reported this period
PROJECT CONTACT:
Name: Karls, R.
Phone: 706-542-2584
Fax: 542-5771
Email: rkarls@vet.uga.edu

 
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ACCESSION NO: 0194018 SUBFILE: CRIS
PROJ NO: IND073082 AGENCY: CSREES IND
PROJ TYPE: HATCH PROJ STATUS: REVISED
START: 01 OCT 2005 TERM: 30 SEP 2008 FY: 2007
INVESTIGATOR: Vemulapalli, R.; Wu, C. C.; Lin, T. L.
PERFORMING INSTITUTION:
Veterinary Pathobiology
Purdue University
West Lafayette , Indiana 47907
IMMUNOLOGICAL CHARACTERIZATION OF POTENTIAL PROTECTIVE PROTEINS OF MYCOBACTERIUM PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: No suitable vaccines are presently available for prevention and control of bovine paratuberculosis. Using a mouse model, this project examines the proteins of M. paratuberculosis to identify a protective protein that can used to develop recombinant vaccine against paratuberculosis. The proposed research will aid in the development of a recombinant vaccine against bovine paratuberculosis.
OBJECTIVES: Johne's disease, also known as Paratuberculosis, is a significant health and economic problem to cattle industry worldwide (1,2,3). The causative agent of this diseases is Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis), a slow growing, facultative intracellular bacterium that is very closely related to Mycobacterium avium subspecies avium (M. avium). Several antigens are common to both M. avium and M. paratuberculosis and they contain high percentage of identity at amino acid level. Presently, no effective vaccine is available for use in cattle against this disease. Our current knowledge about M. paratuberculosis antigens involved in eliciting host protective immune responses to the infection is very minimal (3). Researchers' ability to conduct in-depth studies is severely restricted by the lack of a single, suitable laboratory animal model as well as the very slow multiplication rate and prolonged incubation period of M. paratuberculosis (>2 years). In comparison, M. avium multiplies faster and readily infect in-bred strains of mice, and following parenteral inoculation, the bacteria disperse to various organs and replicate to high numbers in spleen, liver and lung. The overall objective of this revised research project is to characterize the immunological properties of several M. paratuberculosis proteins by generating DNA vaccines and characterizing the elicited immune responses in mouse models. Protective potential of the immunogenic proteins will be determined by challenging the immunized mice with M. avium. Objective 1. Generation of DNA vaccines with selected M. paratuberculosis proteins. In Johne's disease, the onset of clinical signs coincident with a switch in the host immune response from Th1 to Th2 type. Therefore, development of a robust Th1 type immune response is required for resistance against clinical disease in M. paratuberculosis infection. DNA vaccines are well known to induce a strong Th1-biased immune responses. We will select several potential protective proteins of M. paratuberculosis and construct their DNA vaccines using commercially available mammalian expression vectors. Objective 2. Immunological and protection studies with the DNA vaccines in BALB/c and/or C57BL/6 strains of mice. We will first immunize BALB/c mice with the DNA vaccines and study their immune responses, particularly phenotypic characterization of antigen-specific T cells, antigen specific production of interferon-g (INF-g) and other cytokines, antigen-specific cytotoxic T cells, and isotypes of antibodies induced, if any.Based on our previous experience, BALB/c mice may not develop specific immune responses against certain M. paratuberculosis proteins. In such cases, we will conduct the immunological studies in C57BL/6 mice. Information thus generated will be used to screen and identify potentially protective proteins of M. paratuberculosis. We will then carryout challenge experiments in both BALB/c and C57BL/6 mice with M. avium to determine their cross protection potential.
APPROACH: Construction of DNA vaccines: DNA vaccines will be constructed using pSecTag2 mammalian expression plasmid (Invitrogen, Inc.). The genomic DNA of a virulent, cattle isolate of M. paratuberculosis will be extracted (4) and the coding sequences of the selected proteins of M. paratuberculosis will be amplified via PCR using the specifically designed primer-pairs and the genomic DNA as template. The amplified fragments will be cloned into pSecTag2 and then sequenced to confirm the integrity of the gene sequence. Confirmation of expression of the protein by DNA vaccine construct. The ability of the DNA vaccine constructs to express the cloned gene products will be confirmed in vitro. For this, COS-7 cells will be transfected with each one of the DNA vaccine plasmids and the cells will be assayed for the expressed products by Western blot analysis using mouse and cattle antisera to M. paratuberculosis. Vaccine preparation. E. coli cells harboring the plasmids containing the DNA vaccine constructs will be grown in large volumes of liquid broth. The plasmids will be extracted from the E. coli cultures using EndoFree Mega Prep kits (Qiagen, Inc.). The extracted plasmids will be dissolved in phosphate buffered saline (PBS) solution at a concentration of 1 mg/ml and stored at -20oC until use. Mice immunization and testing of selected immune responses. Female mice of 6-8 week old will be used. Group of 5 mice will be injected intramuscularly with 100 ug of DNA vaccine. Mice receiving PBS will serve as controls. At 2 and 4 weeks after initial immunization, same dose of vaccine will be given as boosters. All the mice will be bled retro-orbitally to collect serum samples before immunization and at 4 and 6 weeks post-immunization. At week 6, mice will be euthanatized and their splenocytes will be used for characterization of T cell responses. For some vaccines, it may be necessary to give additional booster(s) to induce antigen-specific immune responses. The specific antibody response will be determined using immunoblot (Western) analysis and ELISA. The T cell responses will be analyzed by detection of cytotoxic T lymphocytes and T cells secreting IL-4, IL-5 and INF-g when co-cultured with M. paratuberculois-infected macrophages and upon stimulation with the specific antigens. Protection studies. Groups of 5 mice will be immunized with the selected DNA vaccines as described above. A group of 5 mice will be injected with PBS and serve as unvaccinated control. Two weeks after the last booster immunization, mice will be challenged with 100,000 colony forming units (CFU) of M. avium. Four after the challenge, the mice will be euthanatized and their spleens, livers and lungs will be harvested. The bacterial burden in the collected organs will be determined by homogenizing the tissues and then plating the 10-fold dilutions of the homogenates on Middlebrook 7H9 agar plates supplemented with OADC enrichment. The decrease in the number of bacteria in each organ of the vaccinated mice will be compared to that of unvaccinated mice and used as a measurement to calculate the protective efficacy.
PROGRESS: 2006/10 TO 2007/09
OUTPUTS: During the previous 1 year-reporting period, four DNA vaccines encoding four different proteins of M. paratuberculosis [fibronectin attachment protein (FAP), antigen 85-B, major membrane protein (MMP), and Mn-superoxide dismutase (SOD)] were generated and the potential of the 85B and SOD DNA vaccines to induce a Th1 type immune response was demonstrated. During the past 1 year, we conducted mouse experiments to determine the protective potential of the 85B and SOD DNA vaccines and to characterize the immune responses induced by the FAP and MMP DNA vaccines. To determine the protective ability of the induced 85B- and SOD-specific immune responses, the vaccinated mice were challenged with virulent M. avium, bacteria that are antigenically very closely related to M. paratuberculosis. Groups of 6-weeks old, female BALB/c mice were immunized by intramuscular inoculation of 100 ug/mouse of endotoxin-free recombinant pSecTag plasmid expressing either SOD or 85B. One group each was inoculated with saline and pSecTag alone as controls. All the mice were given two booster immunizations (100 ug/mouse) with the respective recombinant pSecTag plasmid at 2 week-intervals. Two weeks after the last booster immunization, all the mice were challenged with 10000 CFU of M. avium intraperitoneally. Four weeks after the challenge, the mice were euthanized and the number of M. avium in their lungs, spleens and livers were determined. Based on the tissue bacterial burden, mice in the vaccinated groups, in comparison with the unvaccinated control group, did not show significant level of resistance against the challenge. To characterize the antigen-specific immune responses induced by the FAP and MMP DNA vaccines, mouse experiments were conducted as described above. Serum samples were collected after 2, 4 and 6 weeks after initial vaccination for detection of antibody response by ELISA. Mice were euthanised after 6 weeks and their spleens were collected for splenocyte culture. Using the pooled splenocytes from mice of each experimental group, lymphocytes were stimulated according to previously described procedures. Recombinant FAP and MMP purified from over-expressing E. coli DH5alpha were used for stimulating the splenocytes. Cells with no stimulation and cells stimulated with ConA(1 ug) were used as controls. After 72 hour, the cell culture supernatants were collected and used for quantification of interferon-gamma and IL-5 by a sandwich ELISA using recombinant mouse interferon-gamma or IL-5 as a standard. Both the FAP and MMP DNA vaccines were able to induce antigen-specific antibody response. The serum antibodies were predominantly of IgG2a, but not IgG1, isotype. In vitro stimulation of splenocytes from the MMP-immunized mice with MMP protein resulted in interferon-gamma, but not IL-5, production. No detectable levels of interferon-gamma or IL-5 were detected in FAP stimulated culture of splenocytes from the FAP-immunized mice. Splenocytes of all groups secreted similar levels of interferon-gamma and IL-5 upon stimulation with ConA. TARGET AUDIENCES: Veterinarians who are developing vaccines
IMPACT: 2006/10 TO 2007/09
The results of our studies further our understanding of the role of antigen-specific immune responses in mediating protection against mycobacterial infections. Specifically, our studies suggest that the immune responses induced by the 85B and SOD DNA vaccines are insufficient to control M. avium infection. In addition, the FAP and MMP DNA vaccines can induce a Th1 type immune response, but the FAP DNA vaccine fails to induce antigen-specific T cells capable of secreting interferon-gamma, a key effector cytokine required for protection against any mycobacterial infection. Experiments are underway to determine the protective ability of the FAP- and MMP-specific immune responses induced by the DNA vaccines.
PUBLICATIONS (not previously reported): 2006/10 TO 2007/09
No publications reported this period
PROJECT CONTACT:
Name: Vemulapalli, R.
Phone: 765-494-7560
Fax: 765-494-9830
Email: rvemulap@purdue.edu

 
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ACCESSION NO: 0202580 SUBFILE: CRIS
PROJ NO: IND073088AH AGENCY: CSREES IND
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 OCT 2004 TERM: 30 SEP 2009 FY: 2007
INVESTIGATOR: Davis, J. K.
PERFORMING INSTITUTION:
Veterinary Pathobiology
Purdue University
West Lafayette , Indiana 47907
JOHNE'S DISEASE: MOUSE MODEL AND HOST GENE EXPRESSION IN RESISTANT AND SUSCEPTIBLE MICE.
NON-TECHNICAL SUMMARY:Mycobacterium avium ssp. paratuberculosis (MAP) causes Johne's disease, a chronic wasting disease of cattle and sheep. MAP is also one of the organisms that have been implicated in Crohn's disease of humans. Recent studies show that routine pasteurization does not kill the organism. The purpose of this project is to determine which host defense genes are expressed differently in mice that are resistant to MAP vs. those expressed in susceptible mice. The differences in gene expression between the two mouse strains will highlight which host defenses are preventing development of Johne's disease in mice. This information can then be used to guide studies in cattle to determine if similar host responses occur and ultimately to guide the development of vaccines to prevent the disease in ruminants.
OBJECTIVES: Our central hypothesis is that both nonspecific antibacterial defenses and adaptive immune responses operative in the digestive tract comprise an integrated system of cellular and noncellular components whose interactions with Mycobacterium avium ssp. paratuberculosis (MAP) determine disease expression in Johne's disease. Furthermore, this network can be directly analyzed by global gene expression, and disease resistance can be categorically differentiated from disease susceptibility. Effective vaccines will induce alterations in gene expression that simulate patterns seen in animals that are resistant to disease. The best way to test this hypothesis is to compare global gene expression in a well-defined model system where parameters of disease expression can be tightly controlled, yet experimentally manipulated. Thus, our long range objectives are to (i) Characterize and standardize MAP infections of resistant (C3H/HeN) and susceptible (C57BL/6) mice; (ii) Contrast constitutive expression of immune response genes in the gastrointestinal tracts of C3H/HeN and C57BL/6 mice; and (iii) Contrast global expression of immune response genes between resistant and susceptible mice following infection with MAP.
APPROACH: OBJECTIVE I. Standardization of murine model. C3H/HeN mice are resistant, and C57BL/6 mice are susceptible to MAP infection but differences between the two strains have not been quantified. These experiments will characterize the relationships between organism dose and disease progression. Mice will be given MAP by oral gavage in a dose response study. Six mice from each experimental group will be killed at various times PI and sections of the GI tracts, mesenteric lymph nodes, spleen, and liver will be processed for histological assessment of lesions and demonstration of organisms by acid fast stains. Lesions for each organ will be scored by pathologists. Additional portions of these tissues, plus fecal pellets, will be used to quantify the numbers of MAP. The infectious dose 50%, gross lesion dose 50% and histologic lesion dose 50% will be calculated. OBJECTIVE II. Contrast Constitutive Expression of Immune Response Genes Between Mouse Strains. These experiments will identify the constitutive differences in the expression of immune response genes in the GI tracts of susceptible and resistant strains of mice. Definition of these differences in naive animals provides the background database upon which the subsequent experiments are built. Naive mice of both genotypes will be examined at 2 months, 8, 11, 14, and 17 months of age. Serum, jejunum, ileum, cecum, and colon will be collected and processed for isolation of mRNA for use with Affymetrix GeneChip mouse microarrays. This experiment will also determine how gene expression profiles differ among anatomical sites and at different ages. OBJECTIVE III. Contrast global expression of immune response genes between resistant and susceptible mice during infection with MAP. The experimental design concentrates on early events in infection. Animals of both strains will be infected with MAP and 6 will be killed at various times PI. Three animals from each group will be used for microarray analyses and three will be used for enumeration of MAP numbers in tissues. PROCEDURES. Mycobacteria. An oral vaccine candidate developed by Infectious Diseases Inc. (IDI) will be contrasted with a clinical MAP isolate. Quantitative cultures and real time quantitative PCR of MAP from tissues will be done on tissues from the GI tract. Tissues will be collected and processed for histology and stained using standard methods. Each will be transected longitudinally, one half will be used for histology, the other for cultures, quantitative PCR, and microarray analyses. Samples will be processed as recommended for Affymetrix GeneChip mouse microarrays. Data will be analyzed using Affymetrix GeneChip Software. STATISTICS AND DATA INTERPRETATION. Nonparametric data will be analyzed by the Kruskal-Wallis test. All parameteric data will be analyzed by ANOVA. Microarray analyses will focus on testing the hypothesis that global gene expression in the GI tracts of different strains of mice will be perturbed by MAP infection, and that the expression of immune response genes will be related to strain differences in susceptibility.
PROGRESS: 2006/10 TO 2007/09
OUTPUTS: The specific aims of this project were to: 1) define a model of MAP infection in specific pathogen free (SPF), genetically susceptible, mice so that virulence of MAP isolates can be tested, and 2) develop reconstitution models in which vaccine candidates can be tested and the effectiveness of the adaptive immune responses in cattle with natural disease can be evaluated. The latter was to be done by engraftment of naive systemic and/or local bovine lymphocytes or transfer of bovine memory lymphocytes into immunodeficient mice. Because of the limitation in the amount of funds awarded, we concentrated on the second specific aim of the original proposal; i.e., development of reconstitution models. Two immunodeficient mouse models, Severe Combined Immunodeficiency- beige (SCID-bg) and C57Bl6 Rag2-Pfp- (Rag2-Pfp-), were compared for their ability to be engrafted with fetal bovine lymphocytes injected intraperitoneally (IP) with a mixture of lymphocytes purified from the thymus, spleen, liver, mesenteric lymph nodes, and terminal ileum Peyer's patches of a near-term fetal calf. Each mouse received 2 x 107 lymphocytes of which approximately 28% were CD2+CD4+ (T helper cells), and 33% were CD2+CD8+(T cytotoxic cells). In addition, a small percentage of the cells carried markers for mature B cells
(4%), T cells (3%) and macrophage/dendritic cells (5%). TARGET AUDIENCES: Research Veterinarians
IMPACT: 2006/10 TO 2007/09
At 21 days post engraftment (PE), blood smears from the engrafted SCID-bg and Rag2-Pfp- mice showed increases in circulating lymphocytes; however, they appeared more numerous and mature in the Rag2-Pfp- mice. However, the SCID-bg mouse model requires sublethal irradiation, and most of these mice died within 2 months of engraftment with symptoms suggestive of bleeding disorders due to destruction of megakaryocytes. The calculated dose of gamma irradiation given to these mice was less than the normal sublethal dose, but these mice may have been more radiosensitive that expected or the gamma irradiator used may not have been adequately calibrated. Most of the Rag2-Pfp- mice survived and eight were inoculated with irradiated Brucella abortus strain RB51 (antigen made from the vaccine strain) at 42 days PE by intranasal (IN) instillation, given an IP booster injection at 56 days PE, and then given an additional IN booster installation at 84 days PE. Six engrafted mice were not immunized and 3 mice served as non-engrafted controls. Animals were sacrificed at 98 days PE, and blood smears made, serum collected, and tissues taken for histology and immunohistochemistry. In contrast to 21 days PE, very few lymphocytes could be found on blood smears from engrafted animals. However, 2 of 6 mice in the engrafted, non-immunized group and one animal from the engrafted, immunized group had significant amounts of circulating bovine IgM thus proving that engraftment occurred in these three animals. None had bovine IgG suggesting that immunoglobulin class switching did not occur. Flow cytometry analysis suggested that bovine lymphocytes were present, but the amount of non-specific staining was high obscuring our ability to adequately enumerate subtypes of lymphocytes. We are currently examining the spleen, thymus, and lymph nodes by histology to see if engraftment occurred in additional mice. We hypothesize that in the Rag2-Pfp- mice the initial suspension of bovine lymphocytes contained both mature and immature lymphocytes. In the absence of antigen stimulation the mature lymphocytes gradually underwent a normal decline so that at 96 days PE they were almost all gone. Since the number of bovine accessory cells was also low, the same could have happened to bovine lymphocytes in the vaccinated animals as antigen stimulation in the absence of appropriate accessory cells can increase apoptosis. Although the immature lymphocytes survived in some animals, they did not increase in numbers and adequately repopulate the mouse immune system due lack of a suitable microenvironment.
PUBLICATIONS (not previously reported): 2006/10 TO 2007/09
No publications reported this period
PROJECT CONTACT:
Name: Davis, J. K.
Phone: 765-494-1234
Fax: 765-496-7989
Email: davis39@purdue.edu

 
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ACCESSION NO: 0205694 SUBFILE: CRIS
PROJ NO: IND073094 AGENCY: CSREES IND
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 JUL 2006 TERM: 30 JUN 2009 FY: 2007
INVESTIGATOR: Raizman, E. A.; Wu, C. C.; Glickman, L.; Long, T.; Kenyon, S.
PERFORMING INSTITUTION:
Veterinary Pathobiology
Purdue University
West Lafayette , Indiana 47907
EPIDEMIOLOGIC STUDIES OF JOHNES DISEASE IN DAIRY AND BEEF CATTLE.
NON-TECHNICAL SUMMARY: A- There is lack of knowledge about the distribution of Map in the environment of beef cattle. B- Environmental sampling has been used on dairy farms to determine herd infection status, however, it is necessary to improve this method on targeted sampling in cow alleyway and manure storage A. The project will examine the effectiveness of environmental sampling to determine herd infection status on beef operations, especially mother-calf systems. B. The project will try to improve the current environmental sampling
method using cow alley and manure storage.
OBJECTIVES: 1) To determine the prevalence and the distribution of Map in the environment of beef cattle. The hypothesis for this objective is that there is an association between Map herd prevalence and Map prevalence in the environment. 2) Improve predictive value of environmental sampling for assessment of the prevalence of Map on dairy farms using samples from cow alleyway and manure storage. The hypothesis for this objective is that the Map prevalence (bacteria load) and therefore the predictive value of the environmental sampling, will vary among the different lactation groups in the herd (i.e. early, mid, and late lactation).
APPROACH: Objective 1:A cross-sectional study design will be conducted using approximately 40 beef herds in Indiana with a previous history of JD (ELISA or fecal culture). Herds must have at least 50 adult cows and at least 3 years on the current premise. Eligible herds will be randomly selected from the Indian Board of Animal Health data base. Herd owners will be contacted by a letter for voluntary participation. Sample size calculation (i.e. number of herds and number of sampled cows in each herd) assumed fecal culture prevalence 3% with 85% power and 95% confidence. Fecal samples will be tested using the liquid fecal culture method. In each herd, fecal samples will be obtained from up to 100 cows using disposable sleeves and will be ordered randomly into pools of five cows each. In each herd, environmental samples will be collected using the methodology described by Raizman et al (2004). Two environmental samples of manure with or without dirt will be obtained on each farm from different areas where manure accumulation occurs (such as calving area, pasture area, crowding area, etc). The association between herd infection status based on fecal pool prevalence and Map in the environment will be evaluated using appropriate statistical methods. Objective 2: Approximately 20 dairy herds will be in a longitudinal study design. Herds will have at least 120 milking cows and use a free-stall housing system, where cows are divided into several lactation groups (i.e. early, mid, and late lactation). Eligible herds will be randomly selected from the Johnes Disease Control Program data base of Indiana Board of Animal Health. Herd owners will be contacted by a letter for voluntary participation. The Sample size calculation assumed fecal culture prevalence 10% with 85% power and 95% confidence. Two samples will be obtained from different lactation groups pens and up to 4 samples from manure storage areas as described by Raizman et al (2004). In addition, from each lactation pen, up to 20 individual fecal samples will be collected to estimate the pen prevalence. In order to characterize the distribution of Map through the year, in both cow alleyways and manure storage sampling will be performed every 3 months for 3 years.
PROGRESS: 2006/10 TO 2007/09
OUTPUTS: This project has been extended also to study the survival of Mycobacterium paratuberculosis (Map) on crop fields spread with cow manure. An USDA/NRI proposal was submitted last year (with different co-investigators except Dr. Wu) and was not founded. I am planning to resubmit this proposal again for 2008 USDA/NRI. Meanwhile we try to obtain some preliminary results from an experimental study which aims to assess the bacteria leaching ability in sand column under laboratory conditions. PARTICIPANTS: The only person from the original proposal that is currently working with Dr. Raizman on this project is Dr. Wu. Dr.Ron Turco from Purdue is a collaborator as well. TARGET AUDIENCES: Public health EPA authorities should be very intrested in this study PROJECT MODIFICATIONS: Currently, the main goal is to understand the surveillance of Map in the environment of crop fields. We found that this issue is more important and attractive also because of the public health aspects due to increasing reports of Map and Crohn's disease association.
IMPACT: 2006/10 TO 2007/09
Our understanding of the survival of Map in the environment will improve Johne's disease control programs to limit the spread of the bacteria within and among dairy farms.
PUBLICATIONS (not previously reported): 2006/10 TO 2007/09
No publications reported this period
PROJECT CONTACT:
Name: Raizman, E. A.
Phone: 765-494-7727
Fax: 765-494-9830
Email: eraizman@purdue.edu

 
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ACCESSION NO: 0204030 SUBFILE: CRIS
PROJ NO: IND076048AH AGENCY: CSREES IND
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 15 JUN 2005 TERM: 30 SEP 2009 FY: 2007
INVESTIGATOR: Lelievre, S. A.
PERFORMING INSTITUTION:
Veterinary Basic Medical Science
Purdue University
West Lafayette , Indiana 47907
TARGETING APICAL POLARITY TO CONTROL TISSUE DIFFERENTIATION AND BACTERIAL INFECTION.
NON-TECHNICAL SUMMARY: Alterations in apical polarity, a cellular organization typical of differentiated organs, are involved in the development of skin, renal, pulmonary, and bowel pathologies. Apical polarity also influences infection and/or spreading by many viruses and bacteria. Our goal is to identify the genes that specifically control apical polarity and could be used as therapeutic targets.
OBJECTIVES: Baso-apical polarity is a hallmark of differentiated epithelial tissues and refers to the asymmetrical internal organization of epithelial cells within these tissues. Loss of apical polarity is an early event in skin, renal, pulmonary and bowel diseases. In addition, apical polarity plays a critical role in the spreading of a number of devastating viral and bacterial infections. Thus, the control of apical polarity appears critical for the management of many diseases that affect farm animals. Unfortunately, the genomic determinants involved in the proper location of apical polarity markers, and thus functional apical polarity, are not yet identified. When differentiating mammary epithelial cells are induced to demethylate their DNA upon treatment with 5-Aza-deoxycytidine, the formation of apical polarity is specifically prevented. These data demonstrate that genes regulated by methylation play a critical role in the establishment of apical polarity, and provide us with a working model to identify the genes, and subsequently the proteins involved in the control of apical polarity. We will examine tissue cultures of models of polarized and non-polarized cells and analyze gene expression in these cells using microchip technology and multi-pronge statistical analysis to discover the genomic characteristics (the genes and their methylation status) of apical polarity. This project is critical for future development of two major areas of research: (1) the discovery of genes involved in the control of apical polarity (called CAP genes) could be used to design a microchip tailor-made for the identification of methylation-linked alterations in the expression of these genes in a broad range of diseases; (2) the manipulation of methylation has been proposed as a new treatment strategy, therefore pharmacological tools will be sought to selectively modify the expression of CAP genes by manipulating methylation-mediated gene silencing and thus help revert or prevent disease progression. The objective of this five-year project will be achieved by pursuing five goals: Goal (1) To identify potential genes, called CAP genes, involved in the control of tissue polarity, by comparing microarrays of differentiated mammary cells possessing full polarity and mammary cells treated to induce the lack apical polarity upon alteration of the DNA methylation pathway. Goal (2) To analyze the expression of CAP genes in cell culture conditions that mimic pathological losses of polarity, including inverted polarity. Goal (3) To investigate the methylation status of potential CAP genes in polarized and non-polarized tissue structures described in Goals 1 and 2, using bisulfite modification of DNA, followed by methylation sensitive Poly Chain Reaction (PCR) to specifically identify the genes influenced by methylation. Goal (4) To modify the expression of potential CAP genes in fully polarized tissue structures to verify their involvement in the establishment and/or maintenance of apical polarity. Goal (5) To modify the expression of CAP genes in cell cultures used to study bacterial infection and identify key regulators of infection spreading for specific infectious agents.
APPROACH: Goal 1. Microarrays will be performed on polarized (control) and non-polarized (hypomethylated by 5Aza treatment) mammary cells cultured in Matrigel using the Affymetrix GeneChip Instrument System. Statistical analysis will be performed in collaboration with Rebecca Doerge. Significant alterations in gene expression will be confirmed by reverse transcription-PCR. It is expected that genes that are controlled by DNA methylation will be upregulated (hypomethylated) in 5Aza-treated cells. Goal 2. Inverted polarity will be induced by culturing mammary cells within a collagen I-based extracellular matrix (condition 1). In addition, the specific lack of apical polarity will be reproduced by culturing a subline of mammary cells (MCF10A-AP) (condition 2) that cannot form properly organized apical polarity in Matrigel. Microarray analysis will be performed using three different linear model-based statistical analyses that will accommodate the examination of differential expression of each gene for each control/condition comparison, as well as a comparison between the different conditions relative to the control situation. This set of experiments will enable us to determine whether alterations in the expression of potential polarity (CAP) genes identified in Goal 1 accompany polarity disorders. Goal 3. DNA obtained from the different cell samples studied in Goals 1 and 2 will be treated with bisulfite to induce the conversion of non-methylated cytosines (DNA bases) into uracyl, while methylated cytosines will remain unmodified. Primers will be designed to selectively amplify non-methylated or methylated DNA. The presence of a PCR product, following the use of methylated or non-methylated primers, will indicate the methylation status of the gene of interest. This step will be indispensable to confirm that alterations in the expression of CAP genes observed in Goals 1 and 2 are linked to changes in their methylation status. Goal 4. Mammary epithelial cells transfected with each potential CAP gene will be cultured in Matrigel to induce acinar differentiation. The effect on differentiation will be investigated by looking at the location of the different polarity markers. Genes whose overexpression only affects apical polarity will be classified as CAP genes. Similar experiments will be performed on canine kidney MDCK cells induced to polarize on permeable filter supports and long-term culture of colon Caco-2 cells. These cell types are often used to investigate bacterial infection and will be a good control for the preparation of Goal 5. Goal 5. Dr. Ching-Ching Wu has been studying the mechanisms of infection of Mycobacterium paratuberculosis in polarized epithelial cells. In collaboration with Dr. Wu, we will investigate the infection of this bacterium in Caco-2 or MDCK cells expressing the different CAP genes selected upon completion of Goal 4. Infection efficiency will be measured as the ability and degree of adherence and invasion of the bacteria to non-polarized and polarized cells. We expect that specific alterations in apical polarity induced by the expression CAP genes will affect the potency of, or even abrogate, bacterial infection.
PROGRESS: 2006/10 TO 2007/09
OUTPUTS: Polarity is a hallmark of differentiated epithelial tissues and refers to the asymmetrical internal organization of epithelial cells within these tissues. Loss of apical polarity is an early event in differentiation disorders including cancer, as well as diseases, like skin, renal, pulmonary and bowel diseases that impair the well-being of farm animals. In addition, apical polarity plays a critical role in the spreading of a number of devastating viral and bacterial infections. The goal of our project is to develop a novel approach to the study of genomic influence over tissue polarity, and thus discover the genes that specifically control apical polarity (CAP genes), by combining biochemical manipulation of DNA, 3D models of tissue culture, and microarray analysis. In last year's report we had presented the preliminary results obtained from the microarray analyses that compared breast epithelial cells induced to form tissue-like structures with basoapical polarity (control) and cells induced to form tissue-like structures with altered apical polarity upon (1) treatment with GAP junction blocker AGA and (2) treatment with demethylating agent 5-Aza. Using the conservative Holm method we had found that 29 genes were commonly and significantly down-regulated in cells that had lost apical polarity compared to control. During the past year, from this list of 29 genes we have focused on a couple of genes, coding for sprouty-2 and per-1, that seemed particularly promising with regard to their potential influence on proliferation control. Real time PCR analysis has confirmed changes in their expression in several models of apical polarity loss. We are currently pursuing the study of these genes to define if they are a cause or a consequence of apical polarity loss. In addition, we have identified two genes (RhoGAP8 and PDK1) that are significantly up-regulated upon 5-Aza treatment and could be controlling apical polarity as a consequence of their methylation-dependent regulation. We have developed a simplified method compared to last year to assess apical polarity on tissue sections. With this method we can calculate an apical polarity score (APS) for different epithelial units using a combination of markers including connexin 43, Par-3, pals-1 and ZO-1. This APS will be used for the functional analysis of the potential CAP genes. We have also implemented a technique of RNA extraction from paraffinated tissue sections so that we can measure CAP gene expression levels in epithelia that present different APS. Finally, we have discovered a potential link between prostaglandin-linked inflammatory pathways and the control of apical polarity (more experiments are ongoing). In addition, we have recently obtained a grant from the Susan G. Komen Breast Cancer Foundation to study the impact of certain nutritional factors on apical polarity. Results from this work have been disseminated via lectures, oral and poster presentations, as well as abstracts submitted to local, national and international meetings. PARTICIPANTS: Sophie Lelievre (associate Professor) directed this project. Students in training while participating in this project during the past year included graduate students Lesley Chaboub and Hibret Adissu, and undergraduate students Elizabeth Retseck, Jennifer Stanley, and Daniel Stegelman. Collaborators were Alexander Lipka (graduate student from the Biostatistics Department), Rebecca Doerge (Professor, Biostatistics), and Sunil Badve (Pathologist, IU cancer Center). Specific grants came from the NIH/National Cancer Institute, the Oncological Sciences Center ( Purdue Discovery Park) and the Susan G. Komen Breast Cancer Foundation. TARGET AUDIENCES: Undergraduate and graduate students via internships and classroom talks, and scientists via oral and poster presentations.
IMPACT: 2006/10 TO 2007/09
All functional epithelial tissues in the body are asymmetrically organized, as shown by the segregation of different types of proteins between the basal and apical poles of cells. Based on our studies so far, several genes are of interest as potential controllers of apical polarity. We have data to show that inflammatory pathways might participate in the control of apical polarity. We have establish an apical polarity scoring system that could be used as a readout for functional tests needed in order to discover the genes that control apical polarity and to screens for apical polarity-linked disorders. Ultimately, our approach should lead to novel treatment strategies aiming at altering apical polarity in infected cells or revert the loss of tissue polarity in differentiation disorders. Such strategies would help stop the progression of these diseases and minimize tissue destruction and mortality, consequently suppressing the financial burden linked to the development of a number of devastating diseases.
PUBLICATIONS (not previously reported): 2006/10 TO 2007/09
G Chandramouly, PC Abad, DW Knowles and SA Lelievre. The control of tissue architecture over nuclear organization is critical for epithelial cell fate. J. Cell Sci. 120:1596-606, 2007.
PROJECT CONTACT:
Name: Lelievre, S. A.
Phone: 765-496-7793
Fax: 765-494-7881
Email: lelievre@purdue.edu


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ACCESSION NO: 0405016 SUBFILE: CRIS
PROJ NO: 3625-32000-062-00D AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 07 NOV 2001 TERM: 06 NOV 2006 FY: 2006
INVESTIGATOR: Stabel J R; Paustian M; Bannantine J P; Robbe Austerman S
PERFORMING INSTITUTION:
Agricultural Research Service
Ames , Iowa 50010
UNDERSTANDING HOST-PATHOGEN INTERACTIONS FOR THE DIAGNOSIS & CONTROL OF PARATUBERCULOSIS (JOHNE'S).
OBJECTIVES: Sequence the complete M. paratuberculosis (M. ptb) genome. Use functional genomic approaches to identify and characterize unique bacterial genes and proteins that can be used to develop highly sensitive and specific diagnostic tests and vaccines for use in cattle and sheep. Characterize host immune responses during the different stages of disease. Determine shedding of M. ptb in milk of naturally infected cows and evaluate survival of M. ptb in milk after heat treatment. Identify immunogens of M. ptb by random and directed expression library immunization.
APPROACH: Unique genes identified by sequencing the genome of M. paratuberculosis will be evaluated as reagents and vaccine candidates. Studies will be conducted to evaluate therapies to alleviate periparturient immunosuppression in cows with Johne's Disease. Temporal evaluation of current diagnostic tools in an infected herd will be performed for 3 years. Milk from naturally infected cows will be cultured to determine time of shedding. Heat treatment studies will be conducted with a laboratory scale pasteurizer and a slug-flow pasteurizer to evaluate current pasteurization standards for milk, cheese and ice cream. Unique genes identified by sequencing the genome of M. paratuberculosis will be evaluated as reagents and vaccine candidates. Studies
will be conducted to evaluate therapies to alleviate periparturient immunosuppression in cows with Johne's Disease. Temporal evaluation of current diagnostic tools in an infected herd will be performed for 3 years. Milk from naturally infected cows will be cultured to determine time of shedding. Heat treatment studies will be conducted with a laboratory scale pasteurizer and a slug-flow pasteurizer to evaluate current pasteurization standards for milk, cheese and ice cream. BSL-2; Recertified October 15, 2006 (IBC-0092). BSL-Exempt; Recertified October 15, 2006 (IBC-0238). BSL-Exempt (IBC-#0117) canceled 10/7/04. BSL-Exempt 8/3/2006 (IBC-#0261) BSL-2/BSL-3: Recertified 6/21/2006 (IBC-#0280)
PROGRESS: 2005/10 TO 2006/09
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The Johne's Disease Research Project is aligned with NP 103 Animal Health to conduct innovative cutting-edge research, which delivers effective and practical solutions to agricultural problems of high national priority. Paratuberculosis (Johne's disease) is a chronic, progressive enteric disease of ruminants caused by infection with Mycobacterium avium subsp. paratuberculosis. Economic losses are estimated to be $200/infected cow/year and are the result of animal culling, reduced milk production, poor reproductive performance, and reduced carcass value. Johne's disease has become a high priority disease in the cattle industry. Herd prevalence of Johne's disease is estimated to be 22-40% as determined by a recent National Animal Health Monitoring Survey of dairy cattle. There are no adequate estimates of herd prevalence in beef cattle in the U.S. The economic impact of this disease on the dairy industry was estimated to be over $200 million per year in 1996 and is growing each year with the continued spread of this disease. In addition, M. paratuberculosis has been implicated as a causative factor in Crohn's disease, a chronic inflammatory bowel disease of human beings, which has served as a further impetus to control this disease in our national cattle industry. The dairy industry must be assured that they are providing a clean, safe product for consumers. Research in improved diagnostic tests for detection of this disease will provide tools for reducing contamination of the environment with M. paratuberculosis and the spread of infection in a herd. Research on the pathogenesis and immunology of M. paratuberculosis infections of cattle is conducted to allow design of more rational diagnostic and control procedures. Annotation of the genome sequence of M. paratuberculosis will lead to identification of specific antigens for M. paratuberculosis and characterization of these antigens will result in better diagnostic tests to allow early detection of subclinically infected animals and reduce the incidence of disease in herds. Livestock producers, herd veterinarians, diagnostic laboratories, and regulatory agencies will all benefit from improved management tools for paratuberculosis. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY2002): Fill in gaps in sequencing M. paratuberculosis genome, use M. avium sequence to compare. Expression of M. paratuberculosis-specific proteins will be determined by in vitro experiments using macrophage cell lines infected with M. paratuberculosis. Complete host immunity study/calf infection model study in beef and bison calves. Collect data on Pennsylvannia herd study to evaluate diagnostic test efficacy in herd management of disease. Further work to identify protective gene sequences in a DNA immunization format in a mouse model will be conducted. Work on laboratory-scale pasteurization will be completed using milk artificially inoculated with M. paratuberculosis. Year 2 (FY2003): Identify M. paratuberculosis specific genes (annotation) from sequencing data. Perform in vitro infection studies and screen for expression of M. paratuberculosis specific genes. Determine mechanism of Th1-Th2 shift in different stages of disease (host immunity) by studying cytokine expression. Analyze data from Pennsylvannia herd study and evaluate efficacy of diagnostic tools in identifying subclinically infected animals. Perform host immunity studies during the periparturient period with Johne's cows by evaluating various immune function tests and correlating these with infection of cows. Effects of dietary energy will be studied. Results from laboratory-scale experiments on pasteurization of milk inoculated with M. paratuberculosis will be extrapolated to a pilot-scale pasteurizer unit in collaboration with other researchers. Year 3 (FY2004): Annotation of the genome will be completed and function of specific proteins/peptides from the sequencing effort will be determined. Evaluate genes as potential antigens for use in diagnostic tests by testing in ELISA and IFN-gamma assays. Further identification of protective gene pools from M. paratuberculosis using a mouse challenge model will be performed. Protective gene sequences, resulting in reduced colonization of tissues, will be identified and aligned with known sequence from other mycobacteria, including M. avium. Further experiments to determine host immune responses upon vaccination with protective gene sequences will be conducted. Efficacy of destruction of M. paratuberculosis in colostrum using a commercial on-farm pasteurization unit will be completed. Further experiments evaluating host immunity to M. paratuberculosis infection during subclinical and clinical stages of disease will be conducted. Specifically, immune cell function and cytokine expression during the two phases will be determined. Year 4 (FY2005): Expression of M. paratuberculosis-specific proteins will be determined by in vitro experiments using macrophage cell lines infected with M. paratuberculosis. Function of these proteins as part of the microbial defense system will be assessed. Effects of environmental stressors such as iron levels and pH on gene expression will be assessed using M. paratuberculosis gene microarrays. Evaluate protective genes (gene pools) identified in the mouse model in a calf infection model as potential vaccine candidates. Use of specific gene sequences (alone or pooled) as vaccine candidates will be determined in challenge experiments. Further studies on periparturient immune function in M. paratuberculosis- infected dairy cows will be conducted. Diets of experimental animals will be manipulated to reduce immunosuppression during the periparturient period and correlated with fecal and milk shedding of M. paratuberculosis. Year 5 (FY 2006): Gene pools that have successfully protected calves against infection with M. paratuberculosis will be further evaluated in combination with adjunctive cytokine gene therapy to further reduce the incidence of infection in cattle. New diagnostic tests for cattle and sheep will be developed using proteins/peptides identified by genomic sequencing of M. paratuberculosis. Field studies to evaluate methods to distinguish cattle and sheep strains of M. paratuberculosis will be initiated. M. paratuberculosis proteins will be identified for use as immunogens in cell-mediated immune assays such as the skin test and interferon-gamma test for detection of infection in cattle and sheep. Testing of specific antigens will also be conducted using the mouse challenge model. Therapeutic regimes to reduce the severity of periparturient immunosuppression in cows infected with M. paratuberculosis will be evaluated based upon our earlier work. 4a List the single most significant research accomplishment during FY 2006. These accomplishments align with the microbial genomics component of National Program 103, Animal Heath Action Plan. Comparative genomic analysis of M. paratuberculosis isolates from multiple host species Genomic DNA from a total of 35 M. paratuberculosis isolates was isolated and compared to the sequenced M. paratuberculosis K10 isolate by competitive hybridization to DNA microarrays. Isolates were obtained from a variety of host species, including cattle, goats, sheep, bison, starlings, and humans. Our analyses revealed that M. paratuberculosis isolates from sheep can be characterized by a series of inserted and deleted gene clusters, while the isolates obtained from other host species are highly similar to the sequenced bovine isolate M. paratuberculosis K10. These findings have allowed us to develop a more comprehensive understanding of the genetic diversity present among M. paratuberculosis isolates and may answer questions of transmission of M. paratuberculosis from one species to another, along with environmental and host pressures on the evolution of the microorganism. 4b List other significant research accomplishment(s), if any. These accomplishments align with the pathogen detection and disease control strategy components of National Program 103, Animal Health. Two cell-mediated immune assays, the IFN-gamma test and intradermal skin test, were evaluated in calves to determine their efficacy for the early detection of M. paratuberculosis infection. Testing in naturally infected calves (n = 17) demonstrated that paratuberculosis could be deteted in 16/17 calves by IFN-gamma and in 12/17 calves by skin test as early as 6 months of age. This information is critical to producers for control and management of disease within their flocks/herds. A systematic program of antigen discovery from the M. paratuberculosis genome has been continued. In the past year, over 250 M. paratuberculosis genes have been cloned and expressed. These genes will be characterized for immunogenicity and function with the goal of incorporating these findings into immunological diagnostic tests for Johne's disease. 4c List significant activities that support special target populations. Method development, optimization, and evaluation for sheep diagnostic tests for M. paratuberculosis was conducted. The sheep producers in the US specifically requested that ARS perform work in this area. 5. Describe the major accomplishments to date and their predicted or actual impact. These accomplishments align with pathogen detection, microbial genomics, host-pathogen interaction, and disease control strategy components of National Program 103, Animal Health Action Plan. Significant progress has been made in the development and/or optimization of new diagnostic tools for the detection of Johne's disease. Fecal culture methodology has been improved and diagnostic laboratories throughout the U.S. are using this technique. Optimization of the IFN-g test for detection of subclinically infected animals has been achieved through incorporation of our antigen preparations in the test, allowing for detection of infected animals less than 6 months of age. The ability to accurately identify animals in the early stages of disease is a critical control point for producers who must manage the spread of this disease within their herd. Development of a simple, rapid DNA extraction method for M. paratuberculosis from fecal samples may yield more timely and sensitive detection of infected animals that are shedding the bacteria in their feces. This extraction method will be used by diagnostic laboratories conducting testing for Johne's disease and can be applied to multiple species of animals. A new PCR assay that can detect M. paratuberculosis in fecal, milk, and tissue samples has been developed and evaluated. This assay is highly sensitive and specific. Little information is available on host immune responses to M. paratuberculosis infection in cattle and other species of ruminants. Host immune responses in the subclinical and clinical stages of disease have been evaluated using cattle, bison, and a mouse model. Our work has shown that secretion of proteins (cytokines, IFN-g, IL-10, TGF-b) by immune cells and activation of immune cells is affected by the different stages of infection. This information is helpful in developing diagnostic tools for Johne's disease as well as treatment strategies. IFN-g is currently utilized in a test format to detect infection in subclinically infected animals (early infection) but it is not known if treatment with recombinant IFN-g would help resolve disease in clinically infected cattle. M. paratuberculosis has been implicated as a causative factor in Crohn's disease, an inflammatory bowel disease, in humans. A proposed method of infection is via contaminated dairy products from cows with Johne's disease. Our work has demonstrated that pasteurization of milk effectively destroys M. paratuberculosis. This work is critical in assuring a safe, clean product for U.S. consumers. Improvement of diagnostic tools and development of vaccines is dependent upon the identification of specific antigens for M. paratuberculosis. A project for sequencing the M. paratuberculosis genome was initiated and completed, resulting in the identification of several clones specific to M. paratuberculosis. One clone, HspX, has been characterized in our laboratory and has been patented. This gene was recently incorporated in a commercial PCR test kit for M. paratuberculosis (TetraCore). Additional clones have been cloned and expressed and are being evaluated in diagnostic tests. Further characterization of additional clones and expression of proteins/peptides from them will aid in the development of more sensitive diagnostic tests. Gene microarrays of M. paratuberculosis have been developed and have been useful in generating information the expression of genes during different culture conditions. In addition, these arrays have been useful in delineating differences between sheep and cattle isolates of M. paratuberculosis. This information will play an important role in understanding how M. paratuberculosis survives within the host and will allow us to develop intervention strategies to prevent infection. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Polyclonal antibodies to preparations of M. paratuberculosis and monoclonal antibodies to specific M. paratuberculosis proteins/peptides developed in our laboratory and sent to collaborators. Multiple proteins/peptides from M. paratuberculosis have been cloned and expressed and transferred to companies and other researchers (Cooperative Research and Development Agreements or Material Transfer Agreements) in collaborative efforts to develop new diagnostic tools for Johne's disease. Commercial availability of the new diagnostic tools is dependent upon the company involved and successful use of these tools. Diagnostic tests such as the fecal culture method and IFN-g test for the detection of paratuberculosis in cattle have been transferred successfully to APHIS and diagnostic laboratories. Culture methods for milk have also been transferred to other laboratories for use in their research program. The Bovine Macrophage (BoMac) cell line developed in our laboratory has been transferred to over 75 institutions worldwide for research purposes. This cell line is an immortalized bovine macrophage that is useful for in vitro experiments. Unique M. paratuberculosis proteins have been transferred to collaborators at universities to evaluate novel diagnostic tools or host immunity during active infection. Gene microarrays containing more than 4000 open reading frames of M. paratuberculosis have been shared with collaborators. These microarrays are critical to understanding key roles of regulatory genes during M. paratuberculosis infection. Invitations to speak about effects of pasteurization on destruction of M. paratuberculosis have resulted in the transfer of this information to the dairy industry and producers. Invitations to give talks both nationally and internationally about current research in Johne's disease, including development of diagnostic tools and the sequencing of the genome have resulted due to high interest of the cattle industry. Constraints in the utilization of technology for new diagnostic tools are caused by the unique genetic and physical characteristics of the bacteria. The genetic homology between M. paratuberculosis and M. avium, a cross-reactive mycobacterium that is found in field situations, is greater than 98%. This causes significant problems in identifying antigens for diagnostic tools that are highly specific for M. paratuberculosis. Also, problematic is the host immune response to infection with M. paratuberculosis. Host immunity to infection is characterized by cell-mediated immunity in the early stages of disease and humoral immunity in the later stages. This precludes use of one immunologic diagnostic tool for the detection of infected animals. This problem is exacerbated by the lack of knowledge of the immunology and pathogenesis of this disease. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Invited talks: J. R. Stabel - Nov, 2005; Scientific Advisory committee presentation; Johnes committee for USAHA, Hershey, PA. J. R. Stabel - Oct, 2005; UJNR (US-Japan Collaborations), Fuji, Japan. J. R. Stabel - Sept , 2005; MOST (US-China Collaborations), Beijing, China. J. R. Stabel - Nov, 2005; Conference for Research Workers in Animal Disease, St. Louis, MO. J. R. Stabel - April, 2006; Scientific Advisory committee presentation; National Institute for Animal Agriculture, Louisville, KY. J. R. Stabel - July, 2006; American Dairy Science Assoc, Minneapolis, MN. J. R. Stabel - Jan, 2006, Johnes Disease Integrated Program, Davis, CA. S. Robbe-Austerman Nov, 2005; Vaccine Symposium on Johne's Disease; USAHA, Hershey, PA. J. P. Bannantine - August, 2005; Keynote speaker; 8th International Colloquium on Paratuberculosis, Copenhagen, Denmark. J. P. Bannantine - Jan, 2006, Johne's Disease Integrated Program, Davis, CA. J. P. Bannantine - March, 2006; McGill University, Montreal, Canada. J. P. Bannantine - May, 2006; The Veterinary Laboratory Agency, Weybridge, United Kingdom. Articles written about our work: UT develops test for Johne's disease. Tennessee Farm Bureau News- May 2006. New test may improve Johne's diagnosis. Hoard's Dairyman. March 25, 2006. Johnes research seeks answers. Hoard's Dairyman. January 10, 2006. Working through Johne's Disease. Dairy Herd Management. March 1, 2006.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
1. Marri, P.R., Bannantine, J.P., Paustian, M., Golding, G.B. 2006. Lateral gene transfer in Mycobacterium avium subspecies paratuberculosis. Canadian Journal of Microbiology. 52:560-569.
2. Bannantine, J.P., Paustian, M. 2006. Identification of diagnostic proteins in Mycobacterium avium subspecies paratuberculosis by whole genome analysis approach. In: O'Connor, L., editor. Methods im Molecular Biology: Diagnostic Bacteriology Protocols. 2nd edition. Totowa, NJ: Humana Press. p. 185-196.
3. Patel, D., Danelishvili, L., Yamazaki, T., Marta, A., Paustian, M., Bannantine, J.P., Meunier-Goddik, L., Bermudez, L.E. 2006. The ability of Mycobacterium avium subsp. paratuberculosis to enter bovine epithelial cells is influenced by preexposure to a hyperosmolar environment and intracellular passage in bovine mammary epithelial cells. Infection and Immunity. 74(5):2849-2855.
4. Marsh, I.B., Bannantine, J.P., Paustian, M., Tizard, M.L., Kapur, V., Whittington, R.J. 2006. Genomic Comparison of Mycobacterium avium subsp. paratuberculosis Sheep and Cattle Strains by Microarray Hybridization. Journal of Bacteriology. 188(6): 2290-2293.
5. Speer, C.A., Scott, M.C., Bannantine, J.P., Waters, W.R., Mori, Y., Whitlock, R.H., Eda, S. 2006. A Novel Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycobacterium avium subsp. paratuberculosis Infections (Johne's Disease) in Cattle. Clinical and Vaccine Immunology. 13(5):535- 540.
6. Robbe Austerman, S., Krull, A.C., Stabel, J.R., Palmquist, D.E. 2006. Time Delay, Temperature Effects and Assessment of Positive Controls on Whole Blood for the Gamma Interferon ELISA to Detect Paratuberculosis. American Journal of Veterinary Research. 53(5):213-217.
7. Robbe Austerman, S., Stabel, J.R., Palmer, M.V. 2006. Evaluation of the gamma interferon ELISA in sheep subclinically infected with Mycobacterium avium subspecies paratuberculosis using a whole cell sonicate or a johnin purified-protein derivative. Journal of Veterinary Diagnostic Investigation. 18(2):189-194.
8. McMartin, S., Godden, S., Metzger, L., Feirtag, J., Bey, R., Stabel, J.R., Goyal, S., Fetrow, J., Wells, S., Chester-Jones, H. 2006. Heat Treatment of Bovine Colostrum. I: Effects of Temperature on Viscosity and Immunoglobulin G Level. Journal of Dairy Science. 89(6)2110-2118
9. Coussens, P.M., Pudrith, C.B., Skovgaard, K., Xiaoning, R., Suchyta, S.P., Stabel, J.R., Heegaard, P.M. 2005. Johne's disease in cattle is associated with enhanced expression of genes encoding IL-5, GATA-3, tissue inhibitors of matrix metalloproteinases 1 and 2, and factors promoting apoptosis in peripheral blood mononuclear cells. Veterinary Immunology and Immunopathology. 105(3-4)221-234.
10. Stabel, J.R. 2006. Paratuberculosis: an update. Proceedings of 24th World Buiatrics Congress. October 15-19, 2006, Nice, France. p. 1-8.
11. Huntley, J.F., Stabel, J.R., Paustian, M., Reinhardt, T.A., Bannantine, J. P. 2005. Expression Library Immunization Confers Protection against Mycobacterium avium subsp. paratuberculosis Infection. Infection and Immunity. 73(10):6877-6884.
12. Griffin, J.F., Spittle, E., Rodgers, C.R., Liggett, S., Cooper, M., Bakker, D., Bannantine, J.P. 2005. An IgG1 ELISA for Diagnosis of Johne's Disease in Red Deer (Cervus elaphus). Clinical and Diagnostic Laboratory Immunology. p. 1401-1409.
13. Stabel, J.R., Bannantine, J.P. 2005. Development of a Nested PCR Method Targeting a Unique Multicopy Element, ISMap02, for Detection of Mycobacterium avium subsp. paratuberculosis in Fecal Samples. Journal of Clinical Microbiology. 43(9):4744-4750.
14. Bannantine, J.P., Sreevatsan, S., Tizard, M., Michalski, V., Berger, S., Griffin, F., Paustian, M. 2005. Production and Characterization of Monoclonal Antibodies, Aptamers and Single Chain Antibodies to Mycobacterium avium subsp. paratuberculosis. In: Proceedings of the 8th International Colloquium on Paratuberculosis, August 13-17, 2005, Copenhagen, Denmark. p. 358-365.
15. Paustian, M., Zhu, Z., Robbe Austerman, S., Sreevatsan, S., Kapur, V., Bannantine, J.P. 2006. Comparative genomic analysis of Mycobacterium avium subspecies paratuberculosis isolates obtained from multiple host species. In: Proceedings of the 8th International Colloquium on Paratuberculosis, August 13-17, 2005, Copenhagen, Denmark. p. 393-400.
16. Stabel, J.R., Kimura, K., Robbe Austerman, S. 2006. Sensitization with johnin purified protein derivative augments secreted and intracellular interferon-gamma in cows infected with Mycobacterium avium subsp. paratuberculosis. In: Proceedings of the 8th International Colloquium on Paratuberculosis, August 13-17, 2005, Copenhagen, Denmark. p. 135-145.


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ACCESSION NO: 0403720 SUBFILE: CRIS
PROJ NO: 3625-32000-062-01T AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 01 MAY 2000 TERM: 31 DEC 2003 FY: 2004
INVESTIGATOR: Stabel J R
PERFORMING INSTITUTION:
Agricultural Research Service
Ames , Iowa 50010
STUDY OF THERMAL INACTIVATION OF MYCOBACTERIUM PARATUBERCULOSIS IN FLUID MILK.
OBJECTIVES : 1. Use a slug-flow device to determine survivability of M. paratuberculosis in UHT milk at 8 time/temperature combinations. 2. Determine D and Z values for M. paratuberculosis in milk using the slow-flow device. 3. Use a laboratory-scale pasteurizer unit to corroborate results of experiments in objective 1. 4. Use a pilot-scale HTST pasteurization system to determine survivability of M. paratuberculosis in raw milk based upon results from objectives 1 and 3.
APPROACH : UHT milk inoculated with 3 different bovine isolates of M. paratuberculosis will be heated using both a slug-flow heat exchanger and a laboratoryscale pasteurizer unit at 8 different time/temperature combinations ranging from 62.7 C for 30 min to 97.4 C for 25 sec. Treatments will be replicated 3 times with 2 different inoculum levels. After heat treatment, milk will be centrifuged and the pellet resuspended in PBS. Samples are then diluted and then inoculated onto solid medium (HEYM) and in liquid medium (BACTEC). Solid agar slants are inoculated at 37 C up to 6 months. Colonies are confirmed as M. paratuberculosis by acid-fast stain and DNA extraction and PCR analysis using IS900 primers for a target product of 229 bp. FUNDED BY INTERNATIONAL DAIRY FOODS ASSOCIATION
PROGRESS: 2000/05 TO 2003/12
4. What were the most significant accomplishments this past year? D. Progress Report. This report serves to document research conducted under a trust agreement between ARS and the Internationl Dairy Foods Association. Additional details of research can be found in the report for the parent project 3625-32000-062-00D Understanding Host Pathogen Interactions for the Diagnosis and Control of Johne's Disease (Paratuberculosis). Experiments to evaluate 7 proposed time/temperature combinations for heat treatment of milk inoculated with M. paratuberculosis have been completed. These experiments have been completed on both the slug flow and the HTST units, all three bacterial strains at 2 inoculum levels have been tested. Culture work with both solid and liquid mediums has been completed as well as verification of all suspect positives by PCR, subculture and bacterial staining. Results suggest that US pasteurization standards effectively destroy up to 100,000,000 M. paratuberculosis organisms. The exception is the pasteurization of milk for cheese production that was only effective in destroying 100,000 organisms. This work is important to the dairy industry and consumers so they can be assured that pasteurization provides a safe, clean product.
PUBLICATIONS (not previously reported): 2000/05 TO 2003/12
No publications reported this period.

 
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ACCESSION NO: 0406002 SUBFILE: CRIS
PROJ NO: 3625-32000-062-03T AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 01 SEP 2002 TERM: 31 AUG 2005 FY: 2005
INVESTIGATOR: Bannantine J P; Kapur V; Wells S J; Barletta R G
PERFORMING INSTITUTION:
Agricultural Research Service
Ames , Iowa 50010
FUNCTIONAL GENOMIC ANALYSIS OF MYCOBACTERIUM PARATUBERCULOSIS.
OBJECTIVES: Identify all DNA sequences specific to Mycobacterium paratuberculosis. Confirm specificity of these DNA sequences by PCR amplification with several different species of mycobacteria. Produce and purify recombinant proteins from these specific sequences to evaluate utility in a serological-based test or other immunological assay. Validate resulting developed tests for detection of cattle infected with M. paratuberculosis in a whole herd setting.
APPROACH: Computerized DNA sequence alignments will be performed will the complete genome sequences of M. paratuberculosis and the genetically similar M. avium genomes. From these alignments, we will have a list of candidate sequences present in M. paratuberculosis and not M. avium. These sequences will then be checked, again by computerized alignment, against the complete genomes of M. leprae and M. tuberculosis. Sequences specific to only M. paratuberculosis will be further confirmed by PCR amplification and DNA hybridization against genomic DNA from several mycobacterial species. Those sequences that have passed these specificity tests will be cloned and expressed in E. coli. The resulting recombinant proteins will be evaluated for use in a variety of immunologic-based tests aimed at detecting M. paratuberculosis within infected cattle. Developed test(s) will be validated using blood and fecal samples from six well-characterized cattle herds in Minnesota.
PROGRESS : 2002/09 TO 2005/08
4d Progress report. This report serves to document research conducted under a trust agreement between ARS and USDA-CSREES-National Research Initiative-Competitive Grants Program (NRI-CGP). Additional details of research can be found in the report for the parent project 3625-32000-062-00D Understanding Host Pathogen Interactions for the Diagnosis and Control of Paratuberculosis (Johne's). This trust agreement between ARS (NADC) and the NRI-CGP encompasses measures to ensure animal health through the control and management of the disease by improving detection methods for Johne's disease. This project has the objective of analyzing the genome of M. paratuberculosis for identification and analysis of unique genetic sequences that can be used to develop diagnostic tools for the detection of M. paratuberculosis infection. From this analysis, over 39 genes specific to M. paratuberculosis were identified. Fully 35 of these 39 genes have now been cloned and expressed in Escherichia coli. The resulting E. coli expressed protein(s) were purified and analyzed as a potential diagnostic antigen. These proteins have been arrayed on a filter and analyzed with sera from several control and Johnes disease cattle. Unpublished data suggests that at least 12 of these are antigenic with as many as four antigens showing stronger immunogenicity when compare to known M. paratuberculosis antigens. Although we have had trouble deciding which antigens to use in a platform diagnostic (due to many factors) we still plan validation studies in Minnesota dairy herds in 2006.
PUBLICATIONS (not previously reported): 2002/09 TO 2005/08
1. Paustian, M., Amonsin, A., Kapur, V., Bannantine, J.P. 2004. Charatetrization of novel coding sequences specific to mycobacterium avium subsp. paratuberculosis: implications for diagnosis of johne's disease. Journal of Clinical Microbiology. 42:2675-2681.
2. Bannantine, J.P., Hansen, J.K., Paustian, M., Amonsin, A., Li, L.L., Stabel, J.R., Kapur, V. 2004. Expressionand immunogenicity of proteins encoded by sequences specific to mycobacterium avium subsp. paratuberculosis. Journal of Clinical Microbiology. 42:106-114.

 
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ACCESSION NO: 0408777 SUBFILE: CRIS
PROJ NO: 3625-32000-062-06S AGENCY: ARS 3625
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 16 SEP 2004 TERM: 31 JUL 2007 FY: 2004
INVESTIGATOR: Stabel J R; Beiz D
PERFORMING INSTITUTION:
Animal Science
Iowa State University
Ames , Iowa 50011
CELLULAR ACTIVATION IN DIFFERENT STAGES OF INFECTION WITH MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN CATTLE.
OBJECTIVES : The objective of this cooperative research project is to elucidate host immune response mechanisms for cattle during infection with Mycobacterium avium subsp. paratuberculosis.
APPROACH: Peripheral blood mononuclear cells will be isolated from noninfected and naturally infected cattle and analyzed by flow cytometry for activation markers that may be involved in host immune responses. Because monoclonal antibodies for some cell activation markers may not be available for bovine, this project will encompass sequence identification of conserved regions of the gene, peptide synthesis, and monoclonal antibody production to the peptide of interest. Concurrent analyses will be performed to evaluate intracellular expression of cytokines that may be involved in host immune responses to M. paratuberculosis.
PROGRESS: 2005/10 TO 2006/09
Progress Report 4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and Iowa State University. Additional details of research can be found in the report for the parent CRIS 3625- 32000-062-00D Understanding Host-Pathogen Interactions for the Diagnosis and Control of Paratuberculosis (Johne's). This collaborative research project between ARS (NADC) and the Department of Animal Science, Iowa State University, encompasses measures to ensure animal health through the control and management of the disease by improving knowledge of host immunity during infection. The project has the specific objective of elucidating host immune response mechanisms for cattle in different stages of infection (subclinical and clinical) during the periparturient period, a particularly stressful period in the cows production. At the present time, 30 control non-infected and infected cows have been sampled during the 3 weeks pre- and post-partum, their white blood cells isolated, and immune function tests performed in two separate studies. Examples of assays that have been performed on the cells are measures of cytokine expression and secretion. These data will enable us to determine which areas of host immunity are impacted the greatest by the stressors of infection and parturition.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
No publications reported this period.

 
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ACCESSION NO: 0408473 SUBFILE: CRIS
PROJ NO: 3625-32000-062-07R AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 15 APR 2004 TERM: 14 APR 2007 FY: 2006
INVESTIGATOR: Bannantine J P; Paustian M; Robbe Austerman S; Stabel J R
PERFORMING INSTITUTION:
Agricultural Research Service
Ames , Iowa 50010
JOHNE'S DISEASE INTEGRATED PROGRAM IN RESEARCH, EDUCATION, AND EXTENSION.
OBJECTIVES: The Johne's Disease Integrated Program (JDIP) is a multi-institutional USDA program that has the major objective of combating Johne's Disease. This will be done through focused research on several key areas in Johne's Disease. The program is designed such that there are four main research projects and four scientific cores. The principle investigators receiving subcontract funds (John Bannantine and Judy Stabel) are directing two of the scientific cores entitled, Genomics, Antibodies & Proteomics (GAP) and Animal Models & Facilities (AMF). The primary goal of the GAP core is to develop and distribute tools, reagents, and protocols that leverage recent technological advances in genomics and proteomics for members of the JDIP community and stakeholders. The primary goal of the AMF core is to develop and validate animal models and to provide uniform care and maintenance of animals utilized as experimental models for investigations conducted within JDIP.
APPROACH : The GAP core will develop an oligonucleotide array representing all genes in M. paratuberculosis (Map). This core will also produce a clone set of the coding sequences in Map. It will also make available transposon mutants for JD research. The AMF core will perform two specific tasks. (1) Assemble and maintain a current listing of available BL-2 certified animal facilities at various JDIP sponsored institutions and (2) establish calf-challenge models for JD research including study of the immune response to virulent Map and candidate vaccines. Biosafety certification pending.
PROGRESS : 2005/10 TO 2006/09
Progress Report 4d Progress report. This report serves to document research conducted under a reimbursable agreement. Additional details of research can be found in the report for the parent CRIS 3625-32000-062-00D Understanding Host-Pathogen Interactions for the Diagnosis and Control of Paratuberculosis (Johne's). This project (funded by USDA-CSREES) is being done in collaboration with the University of Minnesota, and is designed to facilitate analysis of genomic diversity and gene expression in M. avium subspecies paratuberculosis (MAP). During this fiscal year, an oligonucleotide microarray was been used in genomic diversity experiments at NADC. These experiments showed distinct differences between the cattle isolates and sheep isolates. The genomic differences result mostly from insertions and deletions. Furthermore, the array has been distributed to several collaborators, both within the US and outside the US. One of these collaborators has used the array to show gene transcription differences within MAC-T cells. There are now 1200 transposon mutants of M. paratuberculosis that have been characterized by DNA sequencing. Another task is to clone most of the important genes in the MAP genome and express them to form recombinant proteins. There are currently 267 purified MAP proteins available for in depth studies. These proteins will be used in functional and immunological assays. Finally, we have hosted four investigators in our laboratory for a total of 1.5-months to train them in the use of these valuable resources.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
No publications reported this period.

 
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ACCESSION NO: 0192662 SUBFILE: CRIS
PROJ NO: IOWR-2002-02228 AGENCY: CSREES IOWR
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2002-35204-12321 PROPOSAL NO: 2002-02228
START: 01 SEP 2002 TERM: 31 AUG 2005 FY: 2005 GRANT YR: 2002
GRANT AMT: $285,000
INVESTIGATOR: Bannantine, J. P.; Kapur, V.; Wells, S. J.; Barletta, R. G.; Stabel, J. R.
PERFORMING INSTITUTION:
National Animal Disease Ctr
Ames , Iowa 50010
FUNCTIONAL GENOMIC ANALYSIS OF MYCOBACTERIUM PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: Johne's disease is caused by the bacterium Mycobacterium paratuberculosis and is responsible for the annual loss of more than $220 million dollars to animal production in the United States. A major obstacle in control of Johne's disease is obtaining an accurate diagnosis of M. paratuberculosis-infected animals. Therefore, the goal of this project is to identify DNA sequences from the M. paratuberculosis genome that are useful in diagnosis of Johne's disease and can be applied to testing entire cattle herds. To accomplish this goal, the following objectives are proposed. (1) Identify M. paratuberculosis DNA sequences not present in any other mycobacteria. We will address the need for diagnostic genes or DNA sequences through the use of comparative genomics to identify M. paratuberculosis DNA sequences that are not present in other mycobacteria. (2) Evaluation of specific antigens or proteins for detecting cattle infected with M. paratuberculosis is the second objective. Immunological assays will be used to determine if sera from cattle with Johne's disease detect proteins produced from these unique genes or sequences. (3) The final objective involves validation of developed tests for detection of cattle with Johne's disease. Diagnostic tests developed in the proposed studies will then be used to test well-characterized whole cattle herds in Minnesota.
OBJECTIVES: Identify all DNA sequences specific to Mycobacterium paratuberculosis. Confirm specificity of these DNA sequences by PCR amplification with several different species of mycobacteria. Produce and purify recombinant proteins from these specific sequences to evaluate utility in a serological-based test or other immunological assay. Validate resulting developed tests for detection of cattle infected with M. paratuberculosis in a whole herd setting.
APPROACH: Computerized DNA sequence alignments will be performed will the complete genome sequences of M. paratuberculosis and the genetically similar M. avium genomes. From these alignments, we will have a list of candidate sequences present in M. paratuberculosis and not M. avium. These sequences will then be checked, again by computerized alignment, against the complete genomes of M. leprae and M. tuberculosis. Sequences specific to only M. paratuberculosis will be further confirmed by PCR amplification and DNA hybridization against genomic DNA from several mycobacterial species. Those sequences that have passed these specificity tests will be cloned and expressed in E. coli. The resulting recombinant proteins will be evaluated for use in a variety of immunologic-based tests aimed at detecting M. paratuberculosis within infected cattle. Developed test(s) will be validated using blood and fecal samples from six well-characterized cattle herds in Minnesota.
PROGRESS: 2002/09 TO 2005/08
The goal of this proposal, entitled, Functional genomic analysis of Mycobacterium paratuberculosis, is to identify coding sequences (DNA that encodes for proteins) from the M. paratuberculosis genome that are useful in diagnosis of Johne's disease. By combining a comparative genomics approach with PCR amplification and microarray analysis, we have identified a total of 39 genes that are present uniquely in M. paratuberculosis. Those gene sequences that were successfully cloned and expressed in E. coli were evaluated for immunogenicity using sera from clinical cattle. Five to six solid candidate antigens have been identified as a result of these studies. Novel antigens have already been incorporated into a new diagnostic assay (involving flow cytometry) and the validation in dairy cattle herds in Minnesota and Japan is nearing completion. In summary, all of the objectives of this project have been successfully completed.
IMPACT: 2002/09 TO 2005/08
Because of the current difficulties in diagnosis of Johne's disease dairy cattle, this new flow cytometry assay (and subsequent ELISA based test-manuscript in preparation) is expected to make a large impact on identifying infected animals faster and thus breaking the insidious transmission cycle that perpetuates Johne's disease throughout herds. This means greater control of the disease and a lessening of the economic costs attributed to the disease. Cost reductions could be as large as 10-fold!
PUBLICATIONS (not previously reported): 2002/09 TO 2005/08
1. Eda, S., B. Elliott, M. C. Scott, W. R. Waters, J. P. Bannantine, R. H. Whitlock, and C. A. Speer. 2005. New method of serological testing for Mycobacterium avium subsp. paratuberculosis (Johne's Disease) by flow cytometry. Foodborne Pathogens and Disease 2:250-262.
2. Paustian, M. L., V. Kapur, and J. P. Bannantine. 2005. Comparative genomic hybridizations reveal genetic regions within the Mycobacterium avium complex that are divergent from Mycobacterium avium subsp. paratuberculosis isolates. Journal of Bacteriology 187:2406-2415.
3. Li, L., J. P. Bannantine, Q. Zhang, A. Amonsin, B. J. May, D. Alt, N. Banerji, S. Kanjilal, and V. Kapur. 2005. The complete genome sequence of Mycobacterium avium subspecies paratuberculosis. Proceedings of the National Academy of Sciences 102:12344-12349.
4. Huntley, J. F. J., J. R. Stabel, and J. P. Bannantine. 2005. Immunoreactivity of the Mycobacterium avium subsp. paratuberculosis 19-kDa lipoprotein. BMC Microbiology 5:3.
5. Paustian, M. L., A. Amonsin, V. Kapur, and J. P. Bannantine. 2004. Characterization of novel coding sequences specific to Mycobacterium avium subsp. paratuberculosis: implications for diagnosis of Johne's disease. Journal of Clinical Microbiology 42:2675-2681.
6. Bannantine, J. P., J. K. Hansen, M. L. Paustian, A. Amonsin, L. Li, J. R. Stabel, and V. Kapur. 2004. Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp. paratuberculosis. Journal of Clinical Microbiology 42:106-114.
7. Waters, W. R., B. J. Nonnecke, M. V. Palmer, S. Robbe-Austermann, J. P. Bannantine, J. R. Stabel, D. L. Whipple, J. B. Payeur, D. M. Estes, J. E. Pitzer, and F. C. Minion. 2004. Use of recombinant ESAT-6:CFP-10 fusion protein for differentiation of infections of cattle by Mycobacterium bovis and by M. avium subsp. avium and M. avium subsp. paratuberculosis. Clinical and Diagnostic Laboratory Immunology 11:729-735.
8. Bannantine, J. P., E. Baechler, Q. Zhang, L. Li, and V. Kapur. 2002. Genome scale comparison of Mycobacterium avium subspecies paratuberculosis with Mycobacterium avium subspecies avium reveals potential diagnostic sequences. Journal of Clinical Microbiology 40:1303-1310.
PROJECT CONTACT:
Name: Bannantine, J. P.
Phone: 515-663-7340
Fax: 515-663-7458
Email: jbannant@nadc.ars.usda.gov

 
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ACCESSION NO: 0202769 SUBFILE: CRIS
PROJ NO: IOWV-109-05-03 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JUL 2003 TERM: 30 JUN 2006 FY: 2005
INVESTIGATOR: Hostetter, J.; Jones, D.; Wannemuehler, M.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
S. And 16th Elwood
Ames , Iowa 50011
COMPARISON OF BOVINE DENDRITIC CELL STIMULATION OF T CELLS ISOLATED FROM MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS (M. A. PTB).
NON-TECHNICAL SUMMARY: Johne's disease is an economically significant enteric disease of ruminants. A major problems in the control of Johne's disease is the significant number of clinically healthy, but infected animals, which escape detection through current diagnostic methods. It is the pupose of this study to test the hypothesis that a dendritic cell induced proliferation assay of peripheral blood T cells can distinguish M. a. ptb infected from non-infected cattle.
OBJECTIVES : The objectives include purification of dendritic cells (DC).Bovine dendritic cells will be generated from bovine peripheral blood mononuclear cells (PBMC). The animals used for blood collection will be positive for M. a. ptb infection by fecal culture; however; will not be demonstrating clinical signs (weight loss, diarrhea). Dendritic cells will be identified by morphologic features, cell surface marker expression, and potential for endocytosis. Dendritic cells once characterized will be used to identify M. a. ptb infected animals by initiating strong recall responses in autologous T cells. Such a recall response will be negative in animals not infected with M. a. ptb.
APPROACH: Blood samples will be collected from M. a. ptb infected cattle. From these blood samples we will generate dendritic cells from peripheral blood mononuclear cells via culture with GM-CSF and IL-4 cytokines for 7 days. In addition, autologous T-lymphocytes will be isolated. Dendritic cells will be characterized by morphology, phenotype, and by functional assays. Dendritic cells will be pulsed with M. a. ptb antigen, then incubated with autologous T lymphocytes (isolated from the same blood sample). We will then evaluate the T-lymphocyte responses (proliferation, production of proinflammatory cytokines). We will use dendritic cells and T lymphocytes from non infected cattle as a negative control. We expect to identify proliferation and cytokine production by the T-lymphocytes isolated from peripheral blood of the M. a. ptb infected animals, but not in T lymphocytes from M. a. ptb negative animals.
PROJECT CONTACT:
Name: Hostetter, J.
Phone: 515-294-0953
Email: jesseh@iastate.edu

 
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ACCESSION NO: 0194202 SUBFILE: CRIS
PROJ NO: IOWV-109-05-49 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JUL 2002 TERM: 30 JUN 2004 FY: 2005
INVESTIGATOR: O'Connor, A.; Robbe, S.; Evans, R.; Ensley, D.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
S. And 16th Elwood
Ames , Iowa 50011
ESTIMATING THE SENSITIVITY AND SPECIFICITY OF THE THREE COMMERCIAL ELISA'S, THE SKIN TEST AND FECAL CULTURE IN BEEF CATTLE.
NON-TECHNICAL SUMMARY: Once a producer's herd becomes infected with Johne's disease, it is very difficult to develop an eradication program that is timely, reasonable in cost, and effective. Recently, new statistical methods have been developed to evaluate the performance of diagnostic tests using several populations of animals with different prevalence of disease. This technique allows us to evaluate newer and more sensitive tests in real productions systems at a reasonable cost.
OBJECTIVES: Johne's disease is a chronic bacterial disease of ruminants caused by Mycobacterium avium subspecies paratuberculosis. Once a producer's herd becomes infected with Johne's disease, it is very difficult to develop an eradication program that is timely, reasonable in cost, and effective. Therefore developing and using prevention programs is critical for producers. The majority of producer's herds become infected by purchasing infected subclinical animals. The diagnostic tests commercially available are not capable of accurately identifying exposed or infected subclinical animals. New and developmental tests that detect cell-mediated immune responses have the potential of improving our ability to detect subclinically infected animals. However, one of the difficult challenges researchers face when attempting to evaluate a new diagnostic test is evaluating that test against a current gold standard. Recently, new statistical methods have been developed to evaluate the performance of diagnostic tests using several populations of animals with different prevalence of disease. This technique allows us to evaluate newer and more sensitive tests in real productions systems. Although these methods will never replace experiments, they do allow us to evaluate diagnostic tests, especially false positive rates within the context of the production system and at a reasonable cost. The majority of the research concerning Johne's disease has been done in dairy cattle, not in beef cattle. Consequently we have extrapolated the performance of diagnostic tests from dairy to beef and automatically have assumed the specificity and sensitivity of the diagnostic tests are the same in extensive beef cattle operations and intensive dairy operations. This may be a reasonable assumption, but the population dynamics and stress levels are very different between beef cattle and dairy cattle. For example, the average age of a beef cow is usually between 7-8 years of age, the average age of a dairy cow is 3-4 years. Consequently there is a critical need to evaluate these tests in beef cattle. Our objective is to evaluate the following tests in beef cattle: 1. Three commercial ELISA's: HerdChek by IDEXX, ParaChek by Biocor, and SERELISA by Synbiotics; 2) The skin test with the new NVSL Johnin antigen, and compare the results of the skin test against the gamma interferon; 3) The fecal culture on HEY media. Once we collect the results of all the diagnostic tests, the data will be entered into a Hui and Walter model. Sensitivities, specificities, and prevalence of the populations will be estimated.
APPROACH: 1.) Identify 3 positive herds and 3 negative herds. These herds must have 100+ animals and have had confirmed Johne's' disease. 2.) Draw blood using the tail vein, collect feces, and inject 0.1 ml of Johnin in the caudal fold of the tail. 3.) Age of the animals and body condition scores will also be collected. These samples will all be collected in the summer and fall of 2002. 4.) The skin test injection site will be read in 72 hours. 5.) These results will be evaluated using Bayesian statistical methods and the Hui and Walter model.
PROGRESS: 2002/01 TO 2002/12
New project: no progress report at this time.
PUBLICATIONS (not previously reported): 2002/01 TO 2002/12
No publications reported this period
PROJECT CONTACT:
Name: O'Connor, A.
Phone: 515-294-5012
Fax: 515-294-1072
Email: oconnor@iastate.edu


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ACCESSION NO: 0195167 SUBFILE: CRIS
PROJ NO: IOWV-400-23-03 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 SEP 2002 TERM: 31 AUG 2004 FY: 2005
INVESTIGATOR : Thoen, C. O.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
S. And 16th Elwood
Ames , Iowa 50011
APPLICATION OF A SKIN TEST USING MYCOBACTERIUM AVIUM SS PARATUBERCULOSIS PPD FOR DETECTION OF EARLY STAGES OF JOHNE'S DISEASE IN CATT. . . (Note: Title exceeded the title field character limit.)
NON-TECHNICAL SUMMARY: Johne's disease causes major economic losses to the dairy and beef industry. Available information indicates Johne's disease is present in 40 percent of the dairy herds in Iowa and Minnesota. The purpose of this work is to develop a reliable, practical test for detecting Johne's disease exposure to control and eradicate the disease.
OBJECTIVES: Efforts to control Johne's disease in cattle has been limited by the lack of reliable diagnostic tests for identifying all M. avium paratuberculosis-infected animals. Several serologic tests have been developed for detecting antibodies in sera of cattle experimentally or naturally exposed to M. avium paratuberculosis. However, a high percentage of the cattle infected with Ma ptb fail to have detectable mycobacterial antibodies in sera in the early stages of disease. Cell-mediated immune responses (CMI) occur before detectable antibody in mycobacterial infections, therefore the use of an improved PPD (antigen) in skin test to monitor the CMI would provide for the detection of cattle in the early stages of disease. Specific objectives are: 1) evaluate the sensitivity of a Ma ptb PPD in skin test for use in the diagnosisof Johne's disease in cattle; 2) determine the specificity of the PPD skin test in cattle in Johne's negative herds; 3) evaluate a gamma interferon assay in animals that respond on skin test.
APPROACH: Skin tests will be conducted using M. avium ss ptb PPD on 400 cattle, 10-14 months of age and 400 cattle 20-24 months of age in 10 or more herds in which Johne's disease has been diagnosed by mycobacteriologic examination and on 800 cattle of the same age in 10 herds in whch Johne's disease has not been diagnosed for 5 years. Skin thickness will measured before injection of Maptb PPD in skin of the mid-cervical region and at 72 hours following injection. The difference in sin thickness will be determined, recorded, and anlayzed. Gamma interferon assays will be conducted on blood of animals that are positive on the skin test.
PROGRESS: 2003/01 TO 2003/12
Skin tests using the new PPD produced from ATCC 19698 (Neotype Strain of Mycobacterium avium ss paratuberculosis) have been conducted in cattle in 26 herds. Positive responses were observed in 15 of 494 cattle, 10 to 26 months of age originating 17 herds in which Johne's disease had not been diagnosed. Based on these results the specificity of the new PPD skin test is 97%. Skin tests were also conducted on 757 cattle in 9 herds in which Johne's disease had been diagnosed previously. Positive responses were observed in 121 (16%) of the 757 animals tested. Most important, positive responses were observed in 10 to 26 month-old cattle in 8 of 9 herds in which Johne's disease had been diagnosed. ELISA was positive in only 6 cattle of the same age group in 2 of 9 herds.
IMPACT: 2003/01 TO 2003/12
Information indicates Johne's disease is present in 40% of the dairy herds in Iowa. Moreover, diagnostic tests are not suitable for monitoring young replacement cattle for the presence of infection or for early detection of infection in cattle in which disease persists. In order to control and eradicate the disease it will be essential to have tests with improved sensitivity and specificity for detecting early stages of Ma ptb infection.
PUBLICATIONS (not previously reported): 2003/01 TO 2003/12
No publications reported this period
PROJECT CONTACT:
Name: Thoen, C. O.
Phone: 515-294-7608
Fax: 515-294-8500
Email: cthoen@iastate.edu

 
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ACCESSION NO: 0083779 SUBFILE: CRIS
PROJ NO: IOWV-400-23-09 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 AUG 1980 TERM: 17 SEP 2007 FY: 2007
INVESTIGATOR: Thoen, C. O.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
Ames , Iowa 50011
MYCOBACTERIAL INFECTIONS IN ANIMALS.
OBJECTIVES: Characterization of mycobacterial isolates from domestic and exotic animals.
APPROACH: In vitro investigations will include biochemical and drug susceptibility tests; ELISA will be conducted to select humoral antibodies in experimentally or naturally infected animals.
PROGRESS: 2003/01 TO 2003/12
Mycobacteriologic examinations were conducted on tissues and fecal specimens submitted from 497 animals. Mycobacterium avium ss paratuberculosis was isolated from 35 (7%) of the specimens. M. avium ss avium was isolated from 8 specimens. Rapidly growing nonphotochromogenic mycobacteria were isolated from 11 specimens. M. tuberculosis was isolated from 2 specimens.
IMPACT: 2003/01 TO 2003/12
The isolation of M. avium ss paratuberculosis is in support of research to develop improved diagnostic tests for Johne's disease in cattle. The disease is widespread in dairy herds in Iowa and causes significant economic losses to producers.
PUBLICATIONS (not previously reported): 2003/01 TO 2003/12
No publications reported this period
PROJECT CONTACT:
Name: Thoen, C. O.
Phone: 515-294-7608
Fax: 515-294-8500
Email: cthoen@iastate.edu

 
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ACCESSION NO: 0192185 SUBFILE: CRIS
PROJ NO: IOWV-411-23-0021 AGENCY: CSREES IOWV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2001 TERM: 30 SEP 2004 FY: 2004
INVESTIGATOR: Jones, D. E.; Steadham, E.; Hostetter, J.; Waters, R.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
S. And 16th Elwood
Ames , Iowa 50011
BOVINE MACROPHAGE AND T CELL RESPONSES TO MYCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS INFECTION.
NON-TECHNICAL SUMMARY: Currently there is no vaccine available to prevent or treat the cause of Johne's disease. Development of a vaccine, or treatment, is likely to be impeded without an understanding of the mechanisms that allow Map to survive in vivo. The purpose of this project is to understand those mechanisms.
OBJECTIVES: Johne's disease (Mycobacterium avium subspecies paratuberculosis or Map) has a significant impact on the dairy industry nationwide. Milk production can be reduced from 2 to 19 percent per cow when compared to uninfected herdmates. The impact of Map on beef cattle is not as readily apparent, but loss of body weight and inefficient feed conversion, as well as premature culling, are consequences of Map infection that can adversely effect cattle revenues. Currently there is no vaccine available to prevent or treat the cause of Johne's disease. The ability of Map to produce disease is related to its ability to survive and replicate within the host macrophage. Paradoxically, macrophages typically play an essential role in host defense of Map by ingesting and killing invading bacilli as part of a cell-mediated immune response that is driven by IFN-g from antigen-specific CD4+ T cells (Th1 cells). The mechanisms by which Map survives within macrophages are not known, nor are the mechanisms that subvert the Th1 cell response and the induction of protective immunity. Development of a vaccine, or treatment, is likely to be impeded without an understanding of the mechanisms that allow Map to survive in vivo. The objectives of this proposal are based on our understanding that persistent infection develops because bovine macrophages infected with Map fail to kill the bacteria. We hypothesize that there is a failure in the ability of Map-specific CD4+ a/b T cells to maintain Th1 phenotype and effectively activate macrophages to kill bacteria. This project has two objectives towards understanding the immunopathology associated with Johne's disease in cattle and will critically examine one of the four scenarios, outlined above, that may impact the expression of the Th1 CD4+ T cell phenotype during this chronic disease. Objective 1: Determine the effects of CD4+ T cell secreted or membrane associated products on macrophage activation in vitro and their influence on Map killing and intracellular trafficking. Objective 2: Test the hypothesis that there is a failure of CD4+ a/b T cells to remain committed to a Th1 cell phenotype.
APPROACH: We will test the hypothesis by assaying bacterial killing by macrophages when stimulated with committed Th1 cells or cell products. Th1 cells will be produced in vitro by culturing CD4+ T cells from naive animals with anti-CD3 and IL-12. Infected macrophages will be cultured with the Th1 cells and/or their supernatants. At various time points after infection the bacteria will be recovered from the macrophages and their viability will be determined by staining and CFU assay. We will also evaluate several other aspects of macrophage activation under these conditions, specifically, the production of reactive nitrogen and oxygen intermediates and phagosome maturation. We will also determine the commitment of CD4+ cells to the Th1 phenotype by repeatedly stimulating cells in vitro and assaying for proliferation and the production of IFN-g. IL-12 receptor b1 and b2 levels will be assayed as an additional indicator of Th1 phenotype. Receptor expression will be determined by amplifying the mRNA from Th1 cells by PCR. We will also determine the Th1 cell phenotype of Map-specific CD4+ T cells obtained from in vivo Map infections. Their commitment to a Th1 phenotype will be tested by repeated stimulation in vitro. These Th1 cells from Map infected animals will be compared to the equivalent cell type from M. bovis-infected animals. M. bovis infection of cattle results in a persistent Th1 type immune response and are used as a positive control. Evaluation of these aspects of the immune response to Map will facilitate our understanding of the immune response of cattle to Map infection and help in the evaluation of vaccine candidates
PROGRESS: 2002/01 TO 2002/12
A method of experimentally infecting calves with Mycobacterium avium subspecies paratuberculosis (Map) was developed. This has allowed us to investigate the development of the infection through the first eight months of exposure. Five months after intra-tonsilar inoculation with the bacteria, calves developed an antibody and lymphocyte response to Map antigens. The cells proliferating upon exposure to Map antigens, in vitro, were evaluated by flow cytometry and found to be CD4+ T lymphocytes. In addition, these cells were found to be the primary cell type producing IFN-gamma. We have also titrated naive primary bovine monocyte-derived macrophages for their response to IFN-gamma in vitro. We have developed assays for evaluating reactive nitrogen intermediates, reactive oxygen intermediates, and changes in the cellular morphology of macrophages in response to IFN-gamma. This was a necessary first step in evaluating the ability of Map antigen-specific T cells to activate Map-infected bovine macrophages by the IFN-gamma secreted into their medium in vitro.
IMPACT: 2002/01 TO 2002/12
The inability to experimentally infect calves with Map has retarded progress in studying Johne's disease. For the first time we are able to describe the length of time required for an antibody response, as well as a cell-mediated response, to develop. In addition, we will have a reproducible and consistent source of antigen responsive T cells to use in determining their ability to activate Map infected macrophages.
PUBLICATIONS (not previously reported): 2002/01 TO 2002/12
No publications reported this period
PROJECT CONTACT:
Name: Jones, D. E.
Phone: 515-294-4682
Fax: 515-294-5423
Email: jonesdou@iastate.edu


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ACCESSION NO: 0194210 SUBFILE: CRIS
PROJ NO: IOWV-430-23-15 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JUL 2002 TERM: 31 MAR 2005 FY: 2005
INVESTIGATOR : Simutis, F. J.; Jones, D. E.; Cheville, N. F.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
S. And 16th Elwood
Ames , Iowa 50011
GAMMA-DELTA T CELL FUNCTION IN MYCOBACTERIAL INFECTIONS.
NON-TECHNICAL SUMMARY : In search for more effective vaccines against pathogenic mycobacteria, the roles of the different lymphocyte subsets must be better understood in order to develop stimulatory components which lead to the most effective response by requisite subsets and, if necessary the downregulation of subsets which are ineffective or counterproductive. The purpose of this research is to seek answers to the fundamental question of whether or not gamma-delta T cells respond to mycobacterial infection by activating infected macrophages.
OBJECTIVES: The functional role of gamma-delta T cells in controlling mycobacterial infection is presently unclear. Recent work with Mycobacterium avium and M. avium subsp. paratuberculosis (Map) using gamma-delta T cell receptor knockout mice suggests a role for gamma-delta T cells in recruiting lymphocytes and neutrophils to the site of infection and in granuloma formation and organization. While studies in knockout mice are essential for determining the contribution of gamma-delta T cells to the overall immune response to mycobacterial disease, studies evaluating the in vivo and in vitro functions of gamma-delta T cells in mycobacterial infection could best be accomplished using an animal that normally has large numbers of gamma-delta T cells present and is a natural host for the disease. The bovine calf is an exceptional model for examining the responses of gamma-delta T cells as neonatal ruminants have large numbers of ciruculating gamma-delta T cells, comprising as much as 75 percent of the circulating T cell pool. Preliminary data from a bovine model suggests a substantial role for gamma-delta T cells in macrophage activation and bactericidal activity during early infection of bovine calves with Map. We have developed a bovine model of localized mycobacterial infection which permits the functional analysis of large numbers of antigen-responsive gamma-delta T cells during early mycobacterial infection. In this model, neonatal calves are inoculated subcutaneously with a virulent strain of Map, the causative agent of ruminant paratuberculosis (Johne's disease) and a significant enteric pathogen of ruminant species worldwide. Following experimental infection, the draining lymph node is surgically removed, and lymph node-derived mononuclear cells are isolated for proliferation and functional studies. Specific aims of the project are: 1) Demonstrate that gamma-delta T cells are a predominant T cell subset that proliferates and produces IFN-gamma in early M. avium subsp. paratuberculosis infection; and 2) Test the hypothesis that antigen-specific gamma-delta T cells are capable of activating macrophages during early infections with M. avium subsp. paratuberculosis.
APPROACH: For specific aim 1: Preliminary data from our laboratoary regarding the immune response to subcutaneous inoculation with Map suggests substantial gamma-delta T cell proliferation with concomitant IFN-gamma production within the draining lymph node at post-inoculation day (PID) 60. Although not ordinarily prominent within lymph nodes, gamma-delta T cells from the lymph node draining the site of local infection in half of the animals in our experiements proliferated more extensively in vitro than either CD4+ or CD8+ alpha-beta T cells from the same lymph node. In the experiments we will examine antigen-specific gamma-delta T cell proliferation and IFN-gamma production in vitro using three-color flow cytometry, and we will use 5-bromo-2'-deoxyuridine (BrdU) incorporation as a marker of cellular proliferation for immunohistochemical studies in order to correlate our in vitro data with in vivo lymphoid changes within the draining lymph node. For specific aim 2: Although there are reports of gamma-delta T cell-mediated increases in macrophage TNF-alpha production during exposure to lipopolysaccharide (LPS) and gamma-delta T cell regulation of nitric oxide production in experimental candidiasis, there are no published reports of macrophage activation by gamma-delta T cells during mycobacterial infection. The function of gamma-delt T cells in the modulation of protective immunity against mycobacteria has not been established as beneficial, redundant, or deleterious. In our model, bacteria are cleared from inoculation sites and draining lymph nodes before PID 60, which suggests that proliferating gamma-delta T cells activate macrophages to eliminate Map during early infection. To test the hypothesis that antigen-specific gamma-delta T cells activate macrophages to upregulate mycobactericidal activity, we will compare the in vitro ability of gamma-delta T cells from Map-sensitized calves to activate autologous infected macrophages with the macrophage activation capability of thawed, cryopreserved autologous gamma-delta T cells obtained prior to experimental infection.
PROGRESS: 2003/01 TO 2003/12
We used a series of assays to test the hypothesis that gamma-delta T cells activate infected macrophages to kill intracellular Mycobacterium avium subsp. paratuberculosis (Map). For these experiments, purified gamma-delta T cells were incubated with autologous Map-infected macrophages, bacterial killing was assessed, and macrophage activation was determined by measuring the production of bactericidal substances (nitric oxide and reactive oxygen species). In addition, IFN-gamma (a mediator of macrophage activation produced by gamma-delta T cells) was quantified. (1) Incubation of Map-infected macrophages with gamma-delta T cells reduces bacterial viability. Bacterial viability was assessed by plating dilutions of lysed macrophage:gamma-delta T cell co-cultures onto 7H10 agar for mycobacteria. Cultures of Map-infected macrophages incubated with unstimulated, autologous gamma-delta T cells or antigen-stimulated autologous gamma-delta T cells for 24 hours yielded 53.1 +/- 3.9% (mean +/- SE) and 51.5 +/- 11.8% fewer viable bacteria, respectively, compared to infected macrophages without added gamma-delta T cells. These decreases in bacterial viability were statistically significant. In contrast, treatment of infected macrophages with IFN-gamma and lipopolysaccharide, a combination known to activate macrophages, was ineffective at reducing bacterial viability. (2) Nitrite concentrations within culture supernatants are not increased at 24 hours in Map-infected cultures containing gamma-delta T cells. To indirectly test for nitric oxide production, we measured nitrite levels within supernatants of cultures of infected macrophages with and without added gamma-delta T cells. 24 hours after gamma-delta T cell transfer there were no significant increases in nitrites in infected cultures containing added gamma-delta T cells compared to infected macrophages alone (despite marked bacterial killing). This finding was true of cultures from calves which had been previously exposed to Map and from control (uninfected) calves. (3) Production of reactive oxygen species (ROS) by Map-infected macrophages is not induced by gamma-delta T cells. Changes in nitroblue tetrazolium (NBT) reduction were followed by assessing the absorbance at 550 nm immediately after addition of NBT and again 18-24 hours later. We measured the fold increases in A550 for macrophage cultures from four animals, and in all cases, there was no significant increase in NBT reduction by infected macrophage cultures containing added gamma-delta T cells compared to Map-infected macrophage cultures without added T cells. (4) gamma-delta T cells upregulate IFN-gamma production when cultured with Map-infected macrophages. At 48 hours post-T cell transfer, infected macrophage cultures containing unstimulated gamma-delta T cells produced significantly greater IFN-gamma compared to infected macrophage cultures containing no added T cells. Co-cultures containing antigen-stimulated gamma-delta T cells also produced increased IFN-gamma, but this increase was not statistically significant. This production of IFN-gamma was true of calves which had been previously exposed to Map and control (uninfected) calves.
IMPACT: 2003/01 TO 2003/12
Purified gamma-delta T cells, whether rested for 7 days or stimulated with Map protein for 7 days, and from control or Map-inoculated calves, markedly reduced the viability of Map in macrophage cultures. This finding is consistent with recent studies in which gamma-delta T cells have been shown to be bactericidal to mycobacteria in other experimental systems. Killing of Map by gamma-delta T cells appears to be independent of nitric oxide and reactive oxygen species, known products of macrophage activation.
PUBLICATIONS (not previously reported): 2003/01 TO 2003/12
No publications reported this period
PROJECT CONTACT:
Name: Simutis, F. J.
Phone: 515-294-3282
Fax: 515-294-5423
Email: fsimutis@iastate.edu

 
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ACCESSION NO: 0206074 SUBFILE: CRIS
PROJ NO: IOWV-HOST-412-23-21 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 30 SEP 2005 TERM: 29 SEP 2006 FY: 2006
INVESTIGATOR: Hostetter, J. M.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
S. And 16th Elwood
Ames , Iowa 50011
DEVELOPMENT OF FLOWMETRICS ASSAY.
NON-TECHNICAL SUMMARY: Situation: Currently our ablility to accurately diagnose Johne's disease in cattle is limited. Effective implementation of Johnes disease control programs will rely on more accurate diagnostic tools. Purpose: This project will use a novel serologic assay for detection of M. paratuberculosis infected cattle at different stages of disease. This assay would allow differentiation of cattle infection with M. paratuberculosis from other Mycobacterial species.
OBJECTIVES: The overall purpose of this project is to develop a sensitive, reproducible, and rapid assay for the detection of M. paratuberculosis infection. The objective of this proposal is to begin testing of a novel serological assay currently under development in our laboratory. The assay we are developing utilizes recombinant proteins cloned from either M. bovis or M. aviumparatuberculosis. Significant advantages of this approach include the ability to multiplex the assay, the small volume of sample required, and the ability to evaluate large numbers of samples in a short period of time. One of the critical aspects in controlling the impact of Johnes disease in a herd is the ability to effectively detect infected animals and to accurately differentiate affected animals from those exposed to other mycobacterial species. The potential of this assay to incorporate a panel of proteins unique for M. paratuberculosis as well as other mycobacterial pathogens, including M. bovis and M. avium, provides the opportunity to use serum antibodies to effectively screen cattle for exposure to these agents. Recently, serum antibodies to M. paratuberculosis antigens have been detected as early as two weeks post infection. These findings demonstrate that generation of serum antibodies to M. paratuberculosis is not limited to late in the disease course, and may have broad diagnostic potential (25). We have two desired results from this proposal. First, that we will be able to successfully detect M. paratuberculosis infection in the experimentally infected animals despite failure of commercial serum ELISA kits to detect circulating M. paratuberculosis antibody. Second, that we will able to discriminate M. paratuberculosis infected cattle from M. bovis infected cattle. The outcome of this project would be the basis for a novel diagnostic approach with increased sensitivity and specificity. The utility of this fluorescent-based technology will allow for the simultaneous detection of as many as 50 different antigens in a single reaction tube by use of the Luminex 100 system. The assay can be adapted to test for the presence of the agent or host antibody to specific antigens. Because the assay can be performed in a microtiter plate format, hundreds to thousands of samples could be performed in a single day. We expect that such an assay would greatly facilitate implementation of the VBJDCP through high versatility, increased confidence of test results, and ease of testing.
APPROACH: These proposed studies will use experimental M. avium subspecies paratuberculosis infection in cattle to evaluate the specificity and sensitivity of a novel flowmetrics-based assay as a method diagnostic tool. Development of this assay is currently underway in our laboratory with support from the Iowa Livestock Health Advisory Council. The approach is built upon advances in genomic analysis of mycobacterial pathogens including M. paratuberculosis and M. bovis and the availability of a more sensitive fluorescent-based technology that facilitates the ability to multiplex the detection method. Recent genomic analysis of each organism has identified a series of proteins that are unique to each mycobacterial species and have been shown to be reactive with serum antibodies from infected cattle. Proteins can be chosen that identify infected animals during the early stages of infection as well as the chronic stages of the disease. The conjugation of specific protein antigens to fluorescent beads will permit the development of a multiplexed assay that has enhanced sensitivity and is differential, rapid, and cost effective for the serological diagnosis of Johnes disease.
PROJECT CONTACT:
Name: Hostetter, J. M.
Phone: 515-294-3282
Fax: 515-294-5423
Email: jesseh@iastate.edu

 
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ACCESSION NO: 0207581 SUBFILE: CRIS
PROJ NO: IOWV-HOSTE-109-05-03 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: NEW
START: 01 JUL 2006 TERM: 30 JUN 2007 FY: 2007
INVESTIGATOR: Hostetter, J. M.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
S. and 16th Elwood
Ames , Iowa 50011
ANTIGEN DISCOVERY: SCREENING FOR IMMUNOGENIC POTENCIAL OF RECOMBINANT PROTEINS DERIVED FROM MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCUL[OSIS.]
NON-TECHNICAL SUMMARY: One of the problems in the prevention of Johne's disease is that until a vaccine is designed that leads to reliable protection without compromising the specificity of tuberculosis and paratuberculosis skin testing; the use of immunization on a broad scale will likely remain impractical. The currently approved vaccine is available only on a limited basis and has the potential to interfere with tuberculosis and paratuberculosis skin testing, thereby hindering its widespread application. Implementation of an effective immunization strategy against Mycobacterium avium subspecies paratuberculosis (M. a. ptb) in combination with reliable diagnostic techniques would greatly enhance our ability to control and ultimately eradicate Johne's disease.
OBJECTIVES: Establish the immunogenicity of M. a. ptb proteins in vitro.
APPROACH: Dendritic cells are one of the initial cell types to interact with antigens at vaccination sites and their function is essential to development of protective immune responses. Therefore, in this objective we will screen for immunogenic recombinant M. a. ptb proteins by identifying those that induce Th-1 polarizing DC function. Our key approach will be to compare the ability of recombinant M. a. ptb protein pools with a whole bacterial sonicate to induce DC maturation and antigen presentation. Initially two overlapping protein pools each consisting of 100 known proteins will be tested. The first pool is composed of proteins derived from M. a. ptb surface membrane and the second composed of proteins that are unique to M. a. ptb. Protein uptake will be confirmed by conjugating proteins with FITC and measuring FTIC positive DC by flow cytometry. We will determine DC maturation phenotype by measuring expression of MHCI and MHCII byimmunofluorescent labeling and flow cytometry. We will measure gene expression of the co-stimulatory molecules CD40 and CD80 by Q-RT-PCR. To determine cytokine profiles we will measure IL-10, IL-12 and TNF-a production in DC culture supernatants. Using a mixed lymphocyte reaction (MLR),we will measure DC allostimulatory activity (T cell proliferation and IFN-. and/or IL-4 production) as a measure of antigen presentation ability. we will measure DC allostimulatory activity (T cell proliferation and IFN-. and/or IL. We will define Th-1 polarizing activity as DC maturation (high CD40, CD80, MHCI, and MCHII), production of IL-12 and TNF-a, and induction of T cell proliferation and IFN-. production. With detection of immunogenicity, individual proteins will be serially subtracted from the pool. In this fashion it will be possible to identify individual or groups of proteins that are strongly immunogenic. Data evaluation and controls: For each parameter comparisons will be made for M. a. ptb recombinant protein pools with the whole bacterial sonicate. Uptake of FITC labeled proteins and surface marker expression (MHCI, MHCII) will be expressed as mean fluorescent intensity of DC within gated regions. Relative mRNA quantification (CD40, CD80) will be determined by calculating a ratio between target mRNA in DC to medium treated DC (calibrator). The internal reference will be beta-actin mRNA. The following calculation will be used to determine mRNA values: Fold change = Efficiency target .Ct (calibrator - target) / Efficiency reference.Ct (calibrator - target). All samples will be run in triplicate. Cytokine production (IL-10, IL-12, TNF-a, IFN-.) will be expressed as ug/ml culture supernatant. T cell proliferation will be expressed as a proliferation index as determined by Modfit software. Negative controls will consist of DC incubated in medium alone. Positive controls will consist of DC infected with live M. a. ptb then activated with LPS. It is our experience that this combination strongly induces DC maturation and antigen presentation. Statistical analysis: Normality and variance of the data will be assessed.
PROJECT CONTACT:
Name: Hostetter, J. M.
Phone: 515-294-0953
Fax: 515-294-5423
Email: jesseh@iastate.edu


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ACCESSION NO: 0207582 SUBFILE: CRIS
PROJ NO: IOWV-THOEN-109-05-27 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: NEW
START: 01 JUL 2006 TERM: 30 JUN 2007 FY: 2007
INVESTIGATOR: Thoen, C. O.
PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
S. and 16th Elwood
Ames , Iowa 50011
EVALUATION OF REDUCED DOSEGE [DOSAGE?] OF MYCOPAR(MYCOBACTERIUM PARATUBERCULOSIS-HEAT KILLED CELLS IN OIL)FOR USE TO VACCINATE CALVES IN JOHN. . . (Note: Title is incomplete due to title character limits for the database.)
NON-TECHNICAL SUMMARY: Johne's disease is widespread in dairy and beef herds in the United States (9, 12). Efforts to eradicate Johne's disease from cattle in herds in which MAP-infection persist using available diagnostic tests (ELISA and/or culture of feces) and slaughter have not often been successful. Therefore, there has been an increased interest in the use of Mycopar to vaccinate calves in the control of Johne's disease (1). Available information shows that use of Mycopar as a vaccine usually eliminates the occurrence of clinical cases and markedly reduces or eliminates shedding of MAP in feces (2, 3, 4, 5). Mycopar is effective in limiting the economic losses due to Johne's disease; however, a few animals may develop a softball size or larger swelling at the injection site in the brisket. Usually only golf ball or baseball size swellings are observe at the injection site. Although these swellings do not affect the market value of the animals, some producers and veterinarians are concerned about the appearance of larger swellings which may persist as long as the animal remains in the herd. The large swellings that occur in cattle are believed to be related to the dosage of Mycopar vaccine used; therefore, there is a need to explore the use of reduced dosage of Mycopar to minimize or eliminate swellings at the injection site.
OBJECTIVES: The objective is to determine the occurrence of injection site swellings in calves following the use of Mycopar at routine dosage (1 ml.) and in calves following the use of a reduced dosage (1/2 dose) of Mycopar in herds in which Johne's disease has been diagnosed by an organism based test (PCR or Culture). In Phase I the objective is to determine if swellings induced by the use of Mycopar, (heat killed Mycobacterium paratuberculosis in oil), in calves can be minimized or eliminated by using a reduced dosage of Mycopar. Injection sites will be observed and measured In Phase II the objective is to determine the shedding of MAP in feces of cattle in each of the three groups at 36, 48, and 60 months post vaccination and the occurrence of clinical disease during each year.
APPROACH: Twelve-hundred heifer calves 1 to 35 days of age in two or more herds negative on tuberculin skin test in which the incidence of MAP-infection in cows is estimated to be 20% or greater will be used in the investigation. The calves will be divided into three groups of 400 calves per group. A 20 gauge needle will be used in making injections and calves in each of the three groups will be inoculated within a 30 day time period. Group I calves will be inoculated subcutaneously in the brisket with 1 ml of Mycopar. This is the dose routinely used and approved for calves. Group II calves will be inoculated with half of the dose of Mycopar in l ml at the same site. Group III calves will be non-vaccinated controls and will be injected subcutaneously with 1 ml of mineral oil at the same site. The injection site will be measured (cm)before administration of Mycopar or oil alone and at 4, 12, and 20 months post-injection. Producers will be requested to note any swellings observed at other time intervals and to notify the Veterinary Practitioner at the time of herd health checks. Fecal specimens will be collected at 20 months post-exposure for mycobacteriologic examination and processed as described in Laboratory Methods in Veterinary Mycobacteriology, NVSL, USDA.
PROJECT CONTACT:
Name: Thoen, C. O.
Phone: 515-294-7608
Fax: 515-294-8500
Email: cthoen@iastate.edu

 
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ACCESSION NO: 0407353 SUBFILE: CRIS
PROJ NO: 6445-12630-001-00D AGENCY: ARS 6445
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 11 JUN 2003 TERM: 08 NOV 2005 FY: 2006
INVESTIGATOR: Sistani K R; Tewolde H; Bolster C H; Cook K L; Loughrin J H
PERFORMING INSTITUTION:
Agricultural Research Service
4660 Nashville Road
Bowling Green , Kentucky 42104
ANIMAL WASTE MANAGEMENT RESEARCH.
OBJECTIVES: Research objectives are to investigate, develop, and evaluate management practices and treatment technologies that protect water quality, reduce air emissions, and control pathogens at animal production facilities, manure storage areas, and field application sites. This research is curative, exploratory, and protective. Conduct solution-oriented research that aid farmers in solving problems associated with animal waste in an environmentally sound manner considering the unique problems associated with karst topography in Kentucky and elsewhere. Specifically, quantify the environmentally important nitrogen (N), phosphorus (P), and other elements fluxes and transformation in soil, water, and air as a result of long-term land application of animal manure. Quantify changes taking place in soil chemical, physical, and biological characteristics resulting from long-term application of animal manure. In the animal facilities, gas losses of nutrients which impact air quality and fertilizer value are modified through management and assessed for newer animal production systems. The potential for water pollution is measured for row crops and forage management, soil types and animal manure fertilization in watersheds for karst topography.
APPROACH: Research approach will focus on the major manure management issues such as: excess nutrient of soil and water; atmospheric emissions of ammonia, particulates, volatile organic compounds (odor), hydrogen sulfide, and greenhouse gases; and pathogenic microorganisms and pharmaceutically active compounds. Specifically, determine the impacts of animal manure nutrient quality, quantity of application, and timing of application on row crops or forage productivity, on rates of nutrient removal in grain or harvested hay, on accumulation of excess nutrients in soil, and on nutrient mobility in the soil. Determine soil quality characteristics, nutrient speciation, mineralization rates, and soil nutrient balances and imbalances with long term fertilization with animal manure. Determine emissions from animal production operations; develop and test management practices for emissions reduction; develop process models to predict emissions, and develop tools to predict dispersion of emissions. Determine the water quality of runoff and subsurface water with animal manure applications to watersheds and pasture botanical changes with and without animal grazing on three soil types. With regard to pathogens research, detect pathogens in complex matrices; assess pathogen survival under a range of conditions; predict and control transport and dissemination; develop cost-effective treatment technologies; and assess risks associated with manure pathogens.
PROGRESS : 2003/06 TO 2005/11
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Animal manure generated annually in the US contains more than 8.5 million tons of nitrogen and 3.5 million tons of phosphorus and many other plant nutrients. However, manure in general is underutilized as a nutrient source for row crops such as corn and cotton production. Significant environmental impacts can also occur if manure is improperly managed at the production site and when applied to land. Animal agriculture has also been the focus of much attention as a potential source of pathogenic microorganisms associated with live animal, food- and water-borne diseases. The problem of malodorous compound emissions from farms and rearing facilities are two-fold: reliable quantification of malodorous compounds from these facilities is needed, as are practices and techniques for odor abatement. Therefore, animal manure if not properly managed becomes a liability rather than a sustainable on-farm resource. There is now a critical need for development of the rational bases for land application of animal manure that results in sustained crops and pastures production and alleviates contamination of water, air, and soil resources from excessive nutrients, atmospheric emission, and pathogens. Nutrient management planning is a tool to address these issues. Maintaining an environmentally sound and sustainable livestock production in regard to hazards from manure nutrients, atmospheric emissions, and pathogens is critical to the economic viability of agriculture in Kentucky, the Southeast, and the nation. This challenge creates an opportunity to develop new, biological, physical, and chemical treatments via a systems approach to solving problems facing livestock and poultry industries. The research under this newly established research project, 6445-12630- 001-00D, in Bowling Green, Kentucky, will address several components of the NP 206, Manure and Byproduct Utilization Action Plan, specifically, Problem Areas 3 (products 1, 2, and 5) and 4 (products 1, 2, and 3) (Nutrient); Problem Areas 1, 2, and 3 (Emission); and Problem Areas 2a and 2b (Pathogen). This research also contributes to research activities related to NP 201, Water Quality and Management and NP 202, Soil Resource Management. 2. List the milestones (indicators of progress) from your Project Plan. This is a newly established Location with new research project 6445-12630- 001-00D. The first project plan is still being reviewed by OSQR, however, research related to all components of NP 206 (Nutrients, Emission, and Pathogen) in relation to our project plan are ongoing. Many peer-reviewed publications and scientific presentations listed in this report are the indicators of progress for the past year. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. New research project will start in September 2005. Milestone Not Met Other 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 2006: 1. Establish field plots, collect background soil samples, begin manure application (objective 1). 2. Collect soil samples to 120 cm depth; complete analyses of all soil and plant tissue samples collected in the previous season. Continue research on banding experiment (objective 1). 3. Locate sampling sites; install and test sampling equipment; conduct dye tracing experiments at Cave Spring Caverns to determine groundwater catchments (objective 2). 4. Develop and optimize QRT-PCR reactions for target groups (objective 3). 5. Identify adsorbents that have superior performance for the collection of malodorous compounds from air samples for use in passive dosimeters. Also, determine the effects of added NO3 to agricultural wastewater in regards to the levels of malodorous compounds. Complete studies on solid- phase extraction techniques for the quantification of malodors in water (objective 4). 6. Characterize community; develop primer/probes for QRT-PCR (objective 5) . 2007: 1. Harvest yield, collect soil for incubation and column studies, conduct chemical analyses of soil, plant, and manure samples (objective 1). Repeat activities of year 1; data evaluation, presentation, and publications (objective 1). 2. Continue dye tracing experiments at Cave Spring Caverns; collect base flow water samples at Logsdon River (objective 2). Field studies, quantification and correlation to biochemical emissions (objective 3). 3. Evaluate the effects of added iron to remediation of odors in agricultural wastewater and start field studies on the quantification of odors in air using passive dosimeters (objective 4). 4. Bench scale and field tests to examine survival (objective 5). 2008: 1. Finish soil characterization and incubation studies, continue column experiment, present results, publication (objective 1). 2. write manuscripts from banding and incorporation studies; test subsurface banding on a farm-scale in cooperation with state extension service (objective 1). 3. Continue base flow sampling at Logsdon, Collect storm event data at Logsdon River, and begin sampling waterfalls at Cave Springs Caverns (objective 2). 4. Continue field studies, scientific publications/presentations (objective 3). 5. Complete studies on the effects of added nitrate and iron on the remediation of odors in agricultural wastewater and determine if humates and other quinone containing substances can enhance utilization of iron by waste bacteria (objective 4). 6. Scientific publications, presentations on incidence/survival of Campylobacter (objective 5). 4b List other significant accomplishments, if any. Completion of the new temporary facilities to accommodate Labs. and Office spaces for the Unit. Hiring 3 SY's and other support staff. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Information related to the use of animal manure particularly poultry litter and best management practices were outlined and disseminated during the local field days. Information on the animal manure utilization for forages (bermudagrass and ryegrass) and row crops (cotton, corn, and soybeans) and in the area of emission, odor, and water quality have been published in scientific outlets, also available for incorporation into states nutrient management guidelines (USDA-NRCS codes 590 and 633), and utilization by the county extension agents, and USDA-NRCS district conservationists. In the current climate of awareness of air and water quality issues, the technology has unlimited durability. The greatest constraint to adaptation is the lack of adequate site specific and on- farm data for the sound scientific recommendation of a particular management practice. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Read, J.J., Sistani, K.R., Brink, G.E., Rowe, D.E. 2004. Forage yield and plant reflectance in annual ryegrass fertilized with poultry litter. Agron. Abstract. Sistani, K.R., Rowe, D.E., Johnson, J.R., Tewolde, H. 2004. Supplemental nitrogen effect on broiler-litter fertilized cotton. Agron. Abstract. Tewolde, H., Sistani, K. R., Rowe, D.E., Adeli, A. Lack of Incorporation Reduces Benefits of Poultry Litter Applied to No-Till Cotton. Proceedings of the Beltwide Cotton Conferences. January 4-7. NewOrleans, LA. 2005. Shalamar, Tewolde, H., Way, T, Sistani, K. R., Rowe, D.E. Cotton. Proceedings of the Beltwide Cotton Conferences. January 4-7. New Orleans, LA. 2005. Loughrin, J.H., Szogi, A.A., Vanotti, M.B. Reduction of Malodorous Compounds in a Swine Waste Treatment System Without Lagoon. ASA/SSSA/CSSA Annual Meetings. Oct 31-Nov 4, 2004. Loughrin, J.H., Szogi, A.A., Vanotti, M.B. Evaluation of an Advanced Waste Treatment System for Reduction of Malodorous Compounds from Swine Waste. Thirty-first Mississippi Water ResourcesConference. Jackson, MS. April 26-27, 2005. Cook, K.L., Britt, J.A., Pike, A. 2005. Evaluation of Mycobacterium avium subsp. paratuberculosis survival in the environment. Poster presented at the 2005 Conference on Gastrointestinal Function. April 12, 2005. Chicago, IL. Participated in a field day organized by Kentucky University, Department of Agriculture on July 29, 2005 in Princeton KY. The benefit of animal manure application and management on pasture and row crops were discussed with farmers, and extension agents.
PUBLICATIONS (not previously reported): 2003/06 TO 2005/11
1. Sistani, K.R., Brink, G.E., Adeli, A., Tewolde, H., Rowe, D.E. 2004. Year- round soil nutrients dynamics from broiler litter application to bermudagrass cultivars. Agronomy Journal. 96:525-530.
2. Adeli, A., Sistani, K.R., Varco, J.J., Rowe, D.E. 2005. Effects of swine lagoon effluent relative to commercial fertilizer applications on warm- season forage nutritive value. Agronomy Journal. 97:408-417.
3. Tewolde, H., Sistani, K.R., Rowe, D.E., Adeli, A., Tsegaye, T. 2005. Estimating cotton leaf area index nondestructively with a light sensor. Agronomy Journal. 97:1158-1163.
4. Brink, G.E., Sistani, K.R., Rowe, D.E. 2004. Yield and nutrient uptake of bermudagrass cultivars fertilized with broiler litter. Agronomy Journal. 96:1509-1515.
5. Adeli, A., Sistani, K.R., Rowe, D.E., Tewolde, H. 2005. Effect of broiler litter on soybean production and soil nitrogen and phosphorus concentrations. Agronomy Journal. 97:314-321.
6. McLaughlin, M.R., Sistani, K.R., Fairbrother, T.E., Rowe, D.E. 2005. Effects of overseeding cool-season annuals on hay yield and nitrogen and phosphorus uptake by Tifton 44 bermudagrass fertilized with swine effluent. Agronomy Journal. 97:479-486.
7. McLaughlin, M.R., Sistani, K.R., Fairbrother, T.E., Rowe, D.E. 2005. Overseeding common bermudagrass with cool-season annuals to increase hay yield and nitrogen and phosphorus uptake in a hay field fertilized with swine effluent. Agronomy Journal. 97:487-493.
8. Bromley, J., Bolster, C., Jones, S. 2005. Effect of nutrient and doc concentrations on recovery of chlorine - exposed Escherichia coli in estuarine microcosms. Enviro. Sci. Technol. Vol. 39, 3083-3089
9. Tewolde, H., Sistani, K.R., Rowe, D.E. 2005. Broiler litter as the sole nutrient source for cotton: N, P, K, Ca, and Mg concentrations in different plant parts. Journal of Plant Nutrition. 28(4):605-619.
10. Adeli, A., Sistani, K.R., Bal'a, M.F., Rowe, D.E. 2005. Phosphorus dynamics in broiler litter-amended soils. Communications in Soil Science and Plant Analysis. 36:1099-1115.

 
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ACCESSION NO: 0408828 SUBFILE: CRIS
PROJ NO: 6445-12630-003-00D AGENCY: ARS 6445 k
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 09 NOV 2005 TERM: 31 OCT 2010 FY: 2006
INVESTIGATOR: Sistani K R; Lovanh N C; Bolster C H; Cook K L; Tewolde H; Loughrin J H
PERFORMING INSTITUTION:
Agricultural Research Service
4660 Nashville Road
Bowling Green , Kentucky 42104
EFFICIENT MANAGEMENT AND USE OF ANIMAL MANURE TO PROTECT HUMAN HEALTH AND ENVIRONMENTAL QUALITY.
OBJECTIVES: Increase the current effort to develop and evaluate management practices and treatment technologies that reduce air emissions of ammonia and odor causing compounds from animal production operations, manure storage areas, and field application sites. The overall goal of the research project formulated in a real partnership between ARS and Western Kentucky University (WKU) is to conduct cost effective and problem solving research associated with animal waste management. The research will evaluate management practices and treatment strategies that protect water quality, reduce atmospheric emissions, and control pathogens at the animal production facilities, manure storage areas, and field application sites, particularly for the unique karst topography. This Project is a unique situation in the sense that non-ARS scientists from a university are included in a research project to conduct research under the same National Program. Hence, to achieve the ultimate goal of this project, the integration and coordination of scientific expertise of the scientists from ARS and WKU are required within and across all objectives. The objectives and related specific sub-objectives are organized according to the three major components (Nutrient, Emission, and Pathogen) of the National Program 206, which mostly apply to this project. The specific objectives for the next 5 years are: Nutrient Component Objective 1: Develop management practices and decision tools for long-term use of animal manure as an alternative source of fertilizer for forages and row crops with regard to the following factors: Impacts on crop yield, nutrient loading, availability and uptake, application rate and timing, tillage, methods of application, and soil quality. Objective 2: Determine if nutrient loading from agricultural watersheds in karst terrain is a function of physical watershed characteristics. Emission Component Objective 3: Reduce odiferous emissions by developing innovative molecular-based methods to identify and quantify microorganisms and biological activities responsible for production of odorous compounds in livestock wastes. Objective 4: Develop new analytical approaches to quantify gases (e.g. methane, H2S), volatile odor compounds (e.g. p-cresol, skatole, and other VOCs) and evaluate treatment technologies for odor abatement at animal production facilities and manure-applied fields. Pathogen Component Objective 5: Employ molecular-based methods to improve detection, quantification, and evaluation of transport, and survival of pathogens from animal manure. Also, compare survival of these pathogens with indicator organisms through a series of laboratory and watershed studies.
APPROACH: This research project was conceived as a cooperative/partnership and comprehensive research program between USDA-ARS Animal Waste Management Research Unit (AWMRU) and Western Kentucky University (WKU). The research is designed to utilize the scientific expertise and facilities of both institutions to conduct problem-solving research related to the animal waste management in Kentucky and the Southeastern US. The research effort will be multi-disciplinary and multifaceted in support of decision making and systems development. Research focuses will be on all three components (Nutrient, Atmospheric Emission, and Pathogens) of the National Program 206. State-of-the-art laboratories and equipments exist at both AWMRU and WKU, which can be accessed by the scientists. Main instruments include: ICP, GC-MS, Lachat, C/N Analyzer, Real time PCR, etc.
PROGRESS: 2005/10 TO 2006/09
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Animal manure generated annually in the US (estimated to exceed 335 million tons) contains more than 10 million tons of nitrogen (N) and 6 million tons of phosphorus (P) and many other plant nutrients. However, manure in general is underutilized as a nutrient source for row crops and forages. For example, cotton and corn farmlands have the potential to assimilate a substantial amount of the excessive poultry litter generated in the southeastern US. Significant environmental impact can occur if manure is improperly managed at the production site and when applied to land. Animal agriculture has also been the focus of much attention as potential source of pathogenic microorganisms associated with live animal, food- and water-borne diseases. The problem of malodorous compound emissions from farms and rearing facilities are two-fold: reliable quantification of malodorous compounds from these facilities is needed, as are practices and techniques for odor abatement. The objectives of this research are to investigate the environmental problems related to the improper use of animal manure such as nutrients, pathogens, trace elements, greenhouse gasses, odor-causing volatile organic compounds (VOCs), dust and sediment associated with animal production facilities and manure application that can degrade soil, water and air quality, and pose a threat to human and animal health. Objectives are (1) develop management practices and decision tools for long-term use of animal manure as an alternative source of fertilizer for forages and row crops with regard to the following factors: impacts on crop yield, nutrient loading, availability and uptake, manure application rate and timing, tillage, methods of application, and soil quality; (2) determine if nutrient loading from agricultural watersheds in karst terrain is a function of physical watershed characteristics; (3) reduce odiferous emissions by developing innovative molecular-based methods to identify and quantify microorganisms and biological activities responsible for production of odorous compounds in livestock wastes; (4) develop new analytical approaches to quantify gases (e.g. methane, H2S), volatile odor compounds (e.g. p-cresol, skatole, and other VOCs) and evaluate treatment technologies for odor abatement at animal production facilities and manure-applied fields; and (5) employ molecular-based methods to improve detection, quantification, and evaluation of transport, and survival of pathogens from animal manure. Also, compare survival of these pathogens with indicator organisms through a series of laboratory and watershed studies. The research under this newly established research project 6445-12630- 003-00D in Bowling Green, Kentucky, will address several components of the NP 206 Manure and Byproduct Utilization Action Plan, specifically, Problem Areas 2, 3, and 4 (Nutrient); Problem Areas 1, 3, and 4 (Emission) ; and Problem Areas 1, 2a and 2b (Pathogen). This research also contributes to research activities related to NP 201 Water Quality and Management and NP 202 Soil Resource Management. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2006): Establish field plots, collect background soil samples, begin manure application. Collect background soil samples; establish bermudagrass and tall fescue forages; begin manure banding application; start collecting runoff water. Collect soil samples to 120 cm depth; complete analyses of all soil and plant tissue samples collected in the previous season. Continue research on banding experiment. Initiate long-term residual effect study on commercial farms; complete rotation research; analyze data collected from litter vs fertilizer research. Locate sampling sites; install and test sampling equipment; conduct dye tracing experiments at Cave Spring Caverns to determine groundwater catchments. Develop and optimize Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR) reactions for target groups. Develop protocols for improved capture of volatile organic compounds (VOCs) by evaluating various phases and adsorbents. Perform comparisons of solid-phase extraction techniques on liquid wastes. Evaluate effect of added NO3 to wastes. Characterize community; develop primer/probes for QRT-PCR. Development of laboratory protocols for column experiments; selection of field sites for collection of water for survival studies. Year 2 (FY 2007): Harvest yield, collect soil for incubation and column studies, conduct chemical analyses of soil, plant, and manure samples. Harvest forage yield, conduct chemical analysis of soil, plant, and manure samples. Repeat activities of year 1, data evaluation; presentation; publications. Adjust treatments, continue research; develop manuscripts on litter vs fertilizer. Continue dye tracing experiments at Cave Spring Caverns; collect base flow water samples at Logsdon River. Field studies, quantification and correlation to biochemical emissions. Field studies started to determine fluxes and concentrations of VOCs. Evaluate effect of added NO3 to wastes. Bench scale and field tests to examine survival. Conduct column experiments; conduct survival studies. Year 3 (FY 2008): Review of research results at South East Poultry Litter Research Group meeting; Finish soil characterization and incubation studies, continue column experiment, present results, write publication. Repeat the field and lab activities done in Yr 2; make presentation, write publications. Writing manuscripts from banding and incorporation studies; test subsurface banding on a farm-scale in cooperation with state extension service. Continue long-term residual study. Continue base flow sampling at Logsdon; collect storm event data at Logsdon River; begin sampling waterfalls at Cave Springs Caverns. Continue field studies, prepare scientific publications/presentations. Continue field studies on fluxes of VOCs; determine transport potential of VOCs. Evaluate effects of added quinones and humic substances to wastes containing insoluble iron. Prepare scientific publications, presentations on incidence/survival of Campylobacter. Continue survival studies. Year 4 (FY 2009): Review of research results at South East Poultry Litter Research Group meeting; finish soil characterization and incubation studies, continue column experiment, present results, write publication. Make treatment adjustment based on 3-year results, present results, and write publication. Write manuscripts from banding and incorporation studies; test subsurface banding on a farm-scale in cooperation with state extension service. Test selected BMPs on a farm-scale in cooperation with state extension service. Continue base flow and storm event sampling at Logsdon River; continue sampling of waterfalls at Cave Springs Caverns. Scientific information regarding the microbial populations involved in emissions and the correlation to biochemical analyses. Continue field studies on fluxes of VOCs; determine transport potential of VOCs. Evaluate effects of added quinones and humic substances to wastes containing insoluble iron. Prepare scientific publications, presentations on incidence/survival of Campylobacter. Prepare scientific publications and presentations on transport and survival behavior of Campylobacter. Year 5 (FY 2010): Develop guidelines/products; technology transfer; publications, outcome. Develop guidelines; BMPs, disseminate products/ technology transfer by presenting at scientific meetings, local/regional field days, extension agents; prepare publications. Prepare guideline of poultry litter BMPs suitable for end-users in the form of experiment station bulletins or similar outlets; make presentations and publications. Conclude long-term residual effect research; prepare BMP guidelines based on research results in the form of experiment station bulletin. Prepare publications and presentations. Scientific information regarding the microbial populations involved in emissions and the correlation to biochemical analyses. Development of transport models that are capable of predicting transport and fate of VOCs in complex terrains. Prepare presentations/publications on the effects of electron acceptor manipulations. Prepare data sets on inactivation rates for Campylobacter agricultural wastes. Compilation of transport parameters and inactivation rates for Campylobacter; attempt to incorporate data into predictive models. 4a List the single most significant research accomplishment during FY 2006. 1) Completed a study to evaluate the survival of C. jejuni and E. coli in groundwater microcosms that resulted in 1 submitted publication and 4 abstracts and talks. Results of the study suggest that E. coli does not serve as an adequate indicator of C. jejuni survival and that survival is significantly influenced by the nutrient makeup of the water source. 2) Developed an equilibrium sampler for the measurement of malodorous compounds in water. This allows measurement of these compounds on site in waste lagoons, anaerobic pits and other sites on animal production facilities. 3) Established four experiments to study broiler litter seasonal and method of applications. 4b List other significant research accomplishment(s), if any. 1) Completed all the milestones related to objectives 1.1 to 1.4. 2) Methodology has been developed for quantifying malodorous sulfides (hydrogen sulfide, methyl mercaptan, etc.) from waste slurries and microbial cultures. This methodology is being used to determine if probes developed for the quantification of sulfate reducing bacteria can be used to predict sulfide emissions from lagoons, anaerobic pits and other waste storage areas. 3) Completed development of QRT-PCR assays to target Mycobacterium avium subsp paratuberculosis,C. jejuni and E. coli in environmental samples. 4) Conducted dye and nutrient injection experiment in Logsdon River. Located additional site for conducting injection experiments. 4c List significant activities that support special target populations. 1) Presented the Unit goal, mission, and research accomplishments at the Field Days and other local and regional meetings in Kentucky and Alabama. 2) Presented Unit goal, mission and research interests at the Stakeholders Technical Committee meeting which will likely become a yearly event. 3) Invited talk entitled "Emerging Trends in Pasture Forage Fertilization" at the University of Florida 2006 Extension Symposium meeting. 4) Presented information about the ARS to middle school students at the annual Science Day event held in Decatur, AL, by one of the Unit scientist. 5. Describe the major accomplishments to date and their predicted or actual impact. Research results showed litter application in bands -- a new practice that buries the litter about 2 inches under the soil in narrow bands for forages and row crops is better than the conventional broadcast application. For example, the yield of cotton fertilized by banding 3 ton/ac litter was about the same as the yield of cotton fertilized with 5 ton/ac applied by the customary broadcast application. This suggests applying litter in bands under the soil surface conserves litter-derived nutrients vulnerable to loss by volatilization and possibly in runoff water. Optimization of a QRT-PCR assay to detect Mycobacterium avium paratuberculosis (MAP) in environmental samples is complete. Using this assay, MAP was monitored in weekly samples were taken from the pen of a cow that had clinical Johnes disease (caused by MAP). The duration of survival of MAP in the environment after removal of the animal was determined. An enrichment study was conducted to enrich for and characterize microorganisms associated with skatole-production. Samples were extracted and compared using molecular-based methods (DGGE and clonal library sequence analysis). Data were used for two poster presentations, and paper is in preparation. A proceedings paper was prepared using data from preliminary enrichment studies. An equilibrium sampler was developed that allows for the sampling of malodors in situ on animal production facilities. This sampler is being used to quantify malodors at various depths in swine waste lagoons and will serve in quantifying odor sources for flux measurements of odor from the lagoons. Equipment is being developed to quantify malodorous compounds near the air-water interface for these measurements and preliminary experiments have been conducted. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Information related to best management practices (BMPs) of animal manure particularly poultry litter were outlined and disseminated to farmers/producers during the regional field days and during Kentucky Farm Bureau different commodity meetings. Information on the animal manure utilization for forages (bermudagrass and ryegrass) and row crops (cotton and corn), also in the area of emission, odor, and water quality have been published in scientific outlets. Scientific research results are available for incorporation into states nutrient management guidelines (e.g. USDA-NRCS codes 590 and 633) to be utilize by the county extension agents, and USDA-NRCS district conservationists. At the request of our cooperating farmers, prepared and provided FACT SHEETS of poultry litter use on cotton based on research results. Similar research results were also communicated with other farmers at a meeting of Producer Advisory Council in North Mississippi and Alabama. Information related to the movement of nutrients through karst soils was outlined and disseminated during an invited talk to the Workshop on the Nature, Study, and Protection of Karst Resources presented to scientists and land managers from The Nature Conservancy along with state conservation officials from Kentucky and Tennessee. In the current climate of public awareness of the environmental quality issues, the technology have unlimited durability. The greatest constraint to adaptation is the lack of adequate site specific and on-farm data for the sound scientific recommendation of a particular management practice. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Participated in a field day organized by Auburn University, Extension Services on June 29, 2006 in Crossville, AL. The benefit of animal manure application and management on pasture and row crops were discussed with farmers, and extension agents.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
1. Sistani, K.R., Tewolde, H., Adeli, A., Rowe, D.E. 2005. Substituting chemical fertilizers with poultry manure to reduce environmental impact. International Conference on Environmental Management. pp 305-309
2. Sistani, K.R., Adeli, A., Tewolde, H., Brink, G.E., Read, J.J., Owens, P. 2005. Mineralization of broiler litter nitrogen: laboratory incubation and field validation. Agronomy Society of America, Crop Science Society of America, Soil Science Society of America Meeting. Agronomy Abstracts, CD Nov 2005.
3. Read, J.J., Sistani, K.R., Brink, G.E., Rowe, D.E., Oldham, J.L. 2005. Effects of overseeding bermudagrass with annual ryegrass on removal of excess soil nutrients from broiler litter applications [abstract]. Agronomy Abstracts. 2005 CDROM.
4. Adeli, A., Rowe, D.E., Sistani, K.R. 2006. Soil chemistry after fifteen years intensive applications of swine lagoon effluent [abstract]. In: Proceedings of World Congress of Soil Science, Frontiers of Soil Science, July 9-15, 2006, Philadelphia, Pennsylvania. p. 458.
5. Tewolde, H., Sistani, K.R., Rowe, D.E., Adeli, A. Time of application affects fertilizer potency of poultry litter. Agronomy Society of America, Crop Science Society of America, Soil Science Society of America Meeting. Agronomy Abstracts, CD Nov 2005.
6. Cook, K.L., Loughrin, J.H. 2006. Characterization of Skatole-Producing Microbial Populations in Enriched Swine Lagoon Slurry. Proceedings of the Workshop on Agricultural Air Quality: State of the Science. pp 547-551
7. Britt, J., Pike, A., Cook, K.L. The Western Kentucky University Johnes Eradication Project. pgs 25-30
8. Loughrin, J.H. Comparison of solid phase micro-extraction techniques for the quantification of malodorous compounds in wastewater. Agronomy Society of America, Crop Science Society of America, Soil Science Society of America Meeting. Agronomy Abstracts CD
9. Cook, K.L., Britt, J., Pike, A. Evaluation of Mycobacterium avium subsp. paratuberculosis survival in the environment. Conference on Gastrointestinal Function. Abstract CD-ROM
10. Cook, K.L. 2005. Detection of Mycobacterium avium subsp. paratuberculosis (map) in the environment. American Society for Microbiology Branch Meeting.
11. Bolster, C.H., Cook, K.L. 2006. Is Escherichia coli a good indicator of the transport of Campylobacter jejuni in ground water environments. ASABE Annual International Meeting.
12 . Cook, K.L., Bolster, C.H. 2006. Survival of Campylobacter jejuni and Escherichia coli in groundwater during prolonged exposure to low temperatures. American Society for Microbiology. (ISBN 1-55581-389-5)
13. Cook, K.L. 2006. Targeting Mycobacterium avium subsp. paratuberculosis in the environment. American Society for Microbiology.(ISBN 1-55581-389-5)
14. Brink, G.E., Sistani, K.R., Oldham, J.L., Pederson, G.A. 2006. Maturity effects on mineral concentration and uptake in annual ryegrass. Journal of Plant Nutrition. 29:1143-1155.
15. Rowe, D.E., Fairbrother, T.E., Sistani, K.R. 2006. Winter cover crop and management effects on summer and annual nutrient yields. Agronomy Journal. 98:946-950.
16. Loughrin, J.H., Szogi, A.A., Vanotti, M.B. Reduction of malodorous compounds from a treated swine anaerobic lagoon. Journal of Environmental Quality. 35:194-199.
17. Loughrin, J.H., Szogi, A.A. Free fatty acids and sterols in swine manure. Journal of Environmental Science and Health part B, 41:31-42, 2006
18. Bolster, C.H., Walker, S.L., Cook, K.L. 2006. Comparison of the transport behavior of Escerichia coli and Camplobacter jejuni in saturated porous media. Journal of Environmental Quality. 35:1018-1025 (2006)
19. Tewolde, H., Sistani, K.R., Rowe, D.E. Nutrient accumulation in cotton fertilized with poultry litter. National Cotton Council Beltwide Cotton Conference. National Cotton Council, Memphis, TN. PP. 2056-2060.
20. Sistani, K.R., Mclaughlin, M.R. Soil nutrient dynamics from swine effluent application to common bermudagrass overseeded with cool-season annuals. Journal of Sustainable Agriculture. 28:101-116.
21. Janaki, L., Cook, K.L., Berk, S.G. Interactions between Mycobacterium avium subsp. paratuberculosis and protozoa isolated from a watering trough of a cow with Johne's disease. American Society for Microbiology. CDROM
22 . Adeli, A., Sistani, K.R., Tewolde, H., Rowe, D.E. 2005. Broiler litter effects on selected soil chemical properties under two contrasting management systems [abstract]. Agronomy Abstracts. 2005 CD-ROM.
23. Pote, D.H., Way, T.R., Kingery, W.L., Aiken, G.E., Sistani, K.R., Han, F.X., Moore Jr, P.A. 2006. [CD-ROM] Incorporating poultry litter into perennial grassland to improve water quality. Proceedings of the Arkansas Water Research Center Conference.

 
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ACCESSION NO: 0404260 SUBFILE: CRIS
PROJ NO: 6445-12630-003-01S AGENCY: ARS 6445
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 04 APR 2001 TERM: 30 MAR 2006 FY: 2005
INVESTIGATOR: Sistani K R; Ferrell B
PERFORMING INSTITUTION:
Biology
Western Kentucky Univ
Bowling Green , Kentucky 42101
EFFICIENT, SAFE UTILIZATION OF POULTRY LITTER PHOSPHORUS.
OBJECTIVES: Determine poultry litter nutrient effects and fate on Kentucky soils and adapted forages. Determine phosphate transporter and phosphatase gene effects on transformed plants. Determine polyphosphate accumulating bacteria for litter and its solubility.
APPROACH: Apply poultry litter to grasses (fescue and sudan) at different rates and then monitor herbage yield and nutrient concentrations, fate of nutrient forms, and soil quality. Introduce phosphate transporter and phosphatase gene to transform plants. Determine P concentrations in plant and plant parts over time in contrast to untransformed plants in low and high P soils. Isolate polyphosphate accumulating bacteria adapted to poultry litter environment and test for P availability in the litter when field applied. Measure P in soil leaching tests, runoff from micro watersheds, and in incubations with soil to which litter is commonly applied.
PROGRESS: 2004/10 TO 2005/09
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Number of broilers produced in Kentucky increased 142% from 1997 to 2002 (Kentucky Agricultural Statistics, 2002). Since the majority of production is occurring in the western third of the state, this presents challenges with respect to environmentally responsible management of litter in a restricted geographical region. These challenges are important to the poultry and other livestock producers, community stakeholders, and regulatory and conservation agencies. Utilization of poultry litter to provide optimum crop quality and yield while reducing potential soil phosphorous, copper, and zinc accumulations (accumulations relating to greater probability of water and soil pollution). Problems related to excess phosphorus (P) in soils are being addressed using a plant-based bioremediation (phytoremediation) technology. The problem is remediation of phosphates in poultry waste. Research also was conducted on the possibility of using normal poultry litter microflora to absorb and maintain high intracellular levels of phosphate thereby reducing the amount of phosphate available for immediate run-off into local watersheds. Produce useful products from poultry litter by studying the combustion behavior of co-firing poultry litter with coal, and investigating multi- utilization and treatment technology of poultry litter for production of energy. New research projects are ongoing related to the animal waste management such as; production of useful products from poultry litter by studying the combustion behavior of co-firing poultry litter with coal, and also investigating multi-utilization and treatment technology of poultry litter for production of energy such as utilizing poultry litter to generate activated carbon, which could be used as sorbents to capture the mercury produced in the coal combustion process, by testing several thermal chemical processes. In another experiment, research is providing new information on the environmental contamination of cattle housing areas with Mycobacterium avium paratuberculosis (MAP) an organism that is shed in the feces and causes chronic untreatable diarrhea in cattle and other species. There is speculation that there is a relationship between MAP and Crohns disease in humans. These problems directly relate to Manure and Byproduct Utilization (NP 206). 2. List the milestones (indicators of progress) from your Project Plan. a) Applied fertility treatments to plots; collected and analyzed soil and forage samples; presented findings at national/regional meetings; preparing paper for submission to Agronomy Journal; and beginning to quantify best management practices; b) Identified plants suitable for high P accumulation; characterized P accumulator plants under different conditions, such as pH, mode of planting, etc.; prepared and analyzed a cDNA library and a mRNA subtraction library representing the phosphorus sufficient and deficient gulf ryegrass; isolated a partial clone of a high affinity phosphate transporter induced under phosphate deficiency in gulf ryegrass; c) Co-firing poultry litter with coal in a lab-scale fluidized bed combustion and a bench-scale circulating fluidized bed combustion system to identify the optimal combustion conditions. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Completed litter rate studies in tall fescue and corn (grain). Milestone Substantially Met 2. Training and education of several agricultural undergraduate students in field research using poultry litter application to forage crops. Milestone Substantially Met 3. A lab-scale fluidized bed combustor (FBC) was set up for testing co- firing of poultry litter and coal. Milestone Substantially Met 4. Compared an ELISA test against a new FCT test for diagnosis of MAP using bovine serum. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? 2006: a) Collect background soil samples, establish research plots, apply broiler litter and inorganic fertilizer; b) Finish all experiments on the lab-scale fluidized bed combustor, and start to test on the bench-scale circulating fluidized bed combustor; complete the test on two screw reactors; establish the fluidized bed reactor for char activation, optimize the fluidized bed for activated carbon and RDF gasification; c) Continue screening plants for higher P accumulation, establishment of tissue cultures and development of a repeatable regeneration protocol for alfalfa Isolate, analyze and catalog sets of phosphate-induced genes from gulf ryegrass libraries; d) Find the most productive methods of sampling the environment for MAP organisms, do more environmental sampling of pasture, harvested forage and manure storage areas; e) Determine what environmental conditions influence expression of skatole synthesis. Skatole is a major odorant in manures, monitor indole producing organisms in in vitro experiments to determine which bacterial groups are most important in this process. 2007: a) Gather soil and forage samples, analyze soil and forage samples, present findings at national/regional meetings; b) Complete all experiments on the bench-scale circulating fluidized bed combustor, and develop a mobile apparatus for disposing poultry litter and test in the lab first. Integrate two screw reactors with fluidized bed reactor to set up the whole system for poultry litter multi-utilization, make the economic analysis and environmental impact for this technology; c) Clone some of the phosphate-induced genes (e.g. phosphate transporters, phosphatase, phytase etc.) in to the binary vector containing AtPT2 promoter, infection and co-cultivation of plant parts/callus with Agrobacterium strains containing plasmids, regeneration of plantlets, optimize transformation of Alfalfa using both reporter genes and target genes identified above; d) evaluate the effect of anaerobic digestion of manure on the viability of the MAP organism; e) Isolation of skatole cultures from various manures and feces. This will allow, in part, for the determination of the microbial diversity responsible for this odor. 2008: a) Publication of peer-reviewed articles, field days/tours for target populations/stakeholders, develop a producer tour, begin to quantify best management practices; b) Test this mobile apparatus on site, perfect this unit, and transfer the technology to the poultry industry, put the technology into the practice in poultry industry; c) Standardization of high frequency regeneration of transformed plantlets, southern blot, Northern blot and Western blot analyses of transformed alfalfa tissues/plantlets, transgenic plantlets will be assayed for phytase activity in rhizosphere, transgenics will be tested for use of phytate as a sole source of P in growth media. Based on the outcome of the above analyses in gulf ryegrass, we will choose 3 to 4 genes for further analysis to evaluate their roles in phosphate sequestration by plants. Analyze the expression of sets of phosphate starvation induced genes in phosphate efficient Gulf and Marshall rye grasses and in phosphate inefficient relatives of rye grasses; d) possibly look at vaccination as a control procedure for MAP; e) Isolation of the genes responsible for skatole synthesis. This will allow for the design of molecular probes to monitor this odorant. 4a What was the single most significant accomplishment this past year? a) Completed litter rate studies in tall fescue and corn(grain). Findings indicate no agronomic advantage to applying greater than 2 T/A litter on tall fescue or 4 T/A litter on corn; b) co-combustion of poultry litter and coal, set up the lab-scale fluidized combustions for studying the co-combustion of poultry litter and coal, understand mechanisms the dry/pyrolysis/activation of chicken waste; c) Characterization of P accumulations in annual ryegrass. Also, generation and analysis of molecular tools such cDNA and mRNA subtraction libraries; d) Demonstration that the phosphate binding capabilities of endogenous poultry litter microorganisms is increased by nutrient deprivation. 4b List other significant accomplishments, if any. Completed original orchardgrass and sorghum-sudangrass studies. Both of these studies are being modified for the coming year to better determine agronomically sustainable ways of removing excess soil nutrients; Construction of a phylogeny of tryptophanase genes which will allow for construction of molecular probes to monitor the potential for indole production. 4c List any significant activities that support special target populations. Training and education of several Agricultural Undergraduate Students in field research using poultry litter application to forage crops. 4d Progress report. a) All milestones have been fully or substantially met. We are in the process of preparing a manuscript for peer-reviewed publication; b) Studies on physiological and growth parameters have confirmed the ability of Gulf and Marshall Grasses to sequester high levels of phosphorus in above ground parts. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. a) There were 2 field days for target populations. Another major accomplishment was finding that applying litter based on crop P requirements, and supplementing with inorganic N, produced forage yield and quality similar to what is produced with inorganic fertilizers or litter applied based on crop N requirements; b) Co-firing of poultry litter with coal could bring the following benefits: First, SO2 emission could be reduced due to very low sulfur content in poultry litter. Second, co-firing poultry litter with coal could abate NOx emission level effectively due to NH3 released from poultry litter. Finally, mercury content in poultry litter is very low, but chlorine content in poultry litter is much higher than that in coal. These characteristics could be helpful to capture mercury emission from the coal combustion process; c) Generation of transgenic plants efficient in sequestering phosphate in above ground parts should also aid in phytoremediation of excess phosphate in soils. Isolation and analysis of genes induced during phosphate deficiency in Gulf rye grass may lead to identification of molecular targets for improving phosphate uptake and efficiency in plants. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? a) Lower litter rates and supplement with inorganic N. There is no agronomic advantage to applying more than 2 T/A litter on tall fescue or 4 T/A litter on corn. These findings have been passed on to producers and other researchers; b) A phytoremediation strategy to deal with the excess soil P problem will be the outcome of this research after a few years; c) A presentation at the Kentucky Dairymans conference provided information to over 100 dairy industry persons about MAP and how we are dealing with the disease and its eradication. We have also made several farm visits to discuss with farmers their manure management issues. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Gilfillen, R.A., Willian, W.T, Henderson, H. 2004. Soil Nutrient Accumulations in Corn Soil as Influenced by Poultry Litter Application Rate (4241). Proc. American Society of Agronomy Annual Meeting. Nov. 6-10. Seattle, WA. Henderson, H., Gilfillen, R.A., Sleugh, B.B., Willian, W.T. 2004. Available N and P in Orchardgrass-Alfalfa soils following poultry litter application. Proc. American Society of Agronomy Annual Meeting. Nov. 6-10. Seattle, WA. Sleugh, B.B., Gilfillen, R.A., Willian, W.T., Henderson, H., Embrey, D., Simmons, J. 2004. Nutrient Uptake and Forage Quality of SorghumSudangrass Under Different Poultry Litter Fertility Programs. Proc. American Society of Agronomy Annual Meeting. November 6-10. Seattle, WA. Willian, W.T., Gilfillen, R.A., Sleugh, B.B, Sistani, K.R., Henderson, H. D. 2004. Soil nutrient accumulation and field corn yield as influenced by poultry litter application rate. Proc. American Society of Agronomy Annual Meeting. Nov. 6-10. Seattle, WA. Sleugh, B.B., Gilfillen, R.A., Willian, W.T., Henderson, H.D. 2005. Poultry Litter Rate Study in Tall Fescue. Proc. American Forage and Grassland Council Annual Meeting. June 11-15. Bloomington, IL. Whitely, N. R., Ozao, R., Wu, C.H., Chen, D., Pan, W.P. Solving the Chicken Litter Problem: Development of Novel Practices for Chicken Litter Management and Disposal, Proceedings, 35th Mississippi Water Resources Conference and 2nd Symposium on Safe Management and Utilization of Animal Waste. Jackson, MS. April 26-27, 2005. Whitely, N.R., Ozao, R., Wu, C.H., Chen, D., Pan, W.P. Combustion Behavior of Chicken-litter Coal Blends, March 15-17. Prepr. Pap.-Am. Chem. Soc. Div. Fuel Chem. 2005. 50(1):249. Sharma, N. C., Sahi, S.V. Strategy for phosphate phytoremediation. Proceedings of the 4th International Phosphorus Workshop (Eds. Chardon, W. J. and Koopmans, G.F. Wageningen. The Netherlands. 2004. Britt J, Pike A, Cook K. The Western Kentucky University Johnes Eradication Program, Proceedings Kentucky Dairymans Conference. March 2005. P 25-32. Mason, B. P., Vadari, Y., Doerner, K.C. 2005. Characterization of Phosphate Hyper-Accumulating Staphylococcus sp. Isolated from Poultry Litter. In Abstracts of the 105th General Meeting of the American Society for Microbiology. American Society for Microbiology. Washington D.C. Groves, C, Bolster, C., Meiman, J. Spatial and Temporal Variations in Epikarst Storage and Flow in South Central Kentuckys Pennyroyal Plateau Sinkhole Plain. USGS Karts Interest Group Conference Proceedings. 2005. Accepted 7/2005. Ozao, R., Okabe, T., Arii, T., Nishimoto, Y., Cao, Y., Whitely, N., Pan, W.P. TGA/DTA/GC-MS Study of Odorless Woodceramics from Chicken Wastes. Journal of Thermal Analysis and Colorimetry. 2005. 80:489-493. Sharma, N. C., Sahi, S.V. Characterization of phosphate accumulation in Lolium multiflorum for remediation of phosphorus-enriched soils. Environmental Science and Technology. 39:5475-5480. 2005. Doerner, K.C., Mason, B.P. 2005. Nutritional Stress Increases Intracellular Phosphate and Polyphosphate in Poultry Litter Microflora. [Accepted for publication 25 June 2005] Lett. Appl. Microbiol. (LAM 2005- 0506.R1)
PUBLICATIONS (not previously reported): 2004/10 TO 2005/09
No publications reported this period.

 
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ACCESSION NO: 0189284 SUBFILE: CRIS
PROJ NO: LAB93527 AGENCY: CSREES LA.B
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 MAY 2001 TERM: 30 OCT 2006 FY: 2007
INVESTIGATOR: Elzer, P. H.
PERFORMING INSTITUTION:
Veterinary Science
Louisiana State University
Baton Rouge , Louisiana 70893
EVALUATION OF B. ABORTUS RB51 AS A MULTIVALENT VACCINE TO GENERATE IMMUNE RESPONSES AGAINST BRUCELLOSIS, TUBERCULOSIS AND JOHNE'S IN CATTLE.
NON-TECHNICAL SUMMARY: Brucellosis, Tuberculosis and Johne's Disease are three major bacterial diseases which have a negative impact on the cattle industry in the United States. The long term goal of our research program is to develop a recombinant RB51 strain that would function as a highly efficacious live multivalent vaccine against three important chronic intracellular bacterial diseases: brucellosis, tuberculosis (Tb), and paratuberculosis (Johne's).
OBJECTIVES: 1.Determine the localization and number (colony forming units CFU) of Brucella abortusRB51 constructs expressing Mycobacterium bovis and M. aviumparatuberculosis antigens in the lymphoid tissues of Brucella , Mycobacteruim naive, sexually mature, female cattle at 7, 14, 21, 28 and 42 days post inoculation. 2. Determine the localization of selected rough B. abortus mutants expressing the mycobacterial antigens and their pathogenic potential when administered to late gestational cattle.
APPROACH: Sixty beef cattle, which are not vaccinated for brucellosis and tuberculosis negative, will be used in this study and will be divided into 6 groups. 1. Strain RB51 overexpressing SOD and expressing the 85A antigen from M. bovis BCG (strain RB51SOD/85A) 2. Strain RB51 overexpressing SOD and expressing the 35 kDa antigen from M. paratuberculosis (strain RB51SOD/35) 3. Strain RB51 overexpressing SOD and expressing the ESAT6 antigen from wild type M. bovis (strain RB51SOD/ESAT6) 4. Strain RB51 overexpressing SOD and expressing the three antigens, ESAT-6, 85A, and 35 kDa antigen (strain RB51SOD/3xAG) 5. RB51 overexpressing SOD (vector control) 6. Saline (negative control) All animals will receive intramuscularly (im) 1-3 x1010 CFU of RB51 expressing the different antigens. At 7, 14, 21, 28, and 42 days post-vaccination, 2 animals from each group will be sacrificed using a captive bolt. Forty-eight hours prior to necropsy, skin tests will be performed using brucella and Tb antigens. The animals will then be necropsied, and sera and tissues will be removed for the culturing of Brucella and for histology. The skin test site and a small section of each tissue will be removed and placed in 10% buffered formalin for histology. The remaining tissues will be individually homogenized and plated on Brucella-selective media. After 3 to 14 days of incubation at 37C in 5% CO2, the plates will be counted; and selective isolates will be tested using molecular techniques to make sure the mutant strains have not changed. Fixed tissues will be stained using H&E and morphological changes will be recorded compared to control tissues. Prevaccination and necropsy serum samples will be tested using standard brucellosis diagnostic tests (all which should remain negative), Western blot analysis, and ELISA. Thirty sexually mature beef cattle, which are not vaccinated for brucellosis and tuberculosis negative, will be used in this study and will be divided into 6 groups. The animals will be bred, and pregnancies will be time-dated. At 200 days pregnancy, 5 animals from each experimental group will be injected intramuscularly with1-3x1010 cfu of the different RB51 strains and 5 animals will be infected conjunctivally with 1x107 cfu of the virulent field strain 2308.Pregnancies will be monitored; and abortions, premature or live births will be recorded. At the time of birth or abortion, the fetus, fetal membranes, and maternal membranes will be collected. A portion of the fetal and maternal membranes will be taken and placed in 10% buffered formalin and the remainder will be cultured. The following tissues will be taken for histology and Brucella culture from the fetus: lungs, liver, spleen, adrenal glands, thymus, internal iliac ln, and blood. The cows will be sacrificed and necropsied as described in Specific Aim 1. Histological and immunological analyses will also be performed as described above.
PROGRESS: 2001/05 TO 2006/10
Vaccinating animals against brucellosis, specifically cattle and swine, with a vaccine that is safe and efficacious, aids in the protection of domestic and wild animals from this zoonotic or potential agroterrorist pathogen. Rough Brucella abortus vaccine derivatives of strain RB51 were used to express heterologous antigen preparations from Mycobacterium bovis (MB), M. aviumparatuberculosis (MAP) and Pseudorabies virus (PRV). Cattle vaccinated with RB51 expressing MB or MAP antigens generated the appropriate cell mediated and or humoral responses to the antigens. These vaccines provided significant protection against virulent brucellae challenge. When RB51-MB vaccinated cattle were challenged with MB, significant protection was observed with the vaccine strain expressing Esat-6 of MB. These results were also confirmed with histological observations. Swine vaccinated with RB51-PRV or a rough strain of B. suis expressing PRV antigens (VTRS-PRV) generated humoral immune responses to the PRV antigen, and both vaccines provided significant protection against virulent brucellae challenge in swine. When used in pregnant animals, none of the above vaccines induced abortions or any negligible gross pathological lesions. Vaccination with RB51 expressing antigens from other facultative intracellular pathogens provides protective immunity against both homologous and heterologous organisms. A duel purpose vaccine will be of benefit to producers in areas where the above mentioned diseases pose a risk of transmission to traditional livestock populations from feral or wild animals.
IMPACT: 2001/05 TO 2006/10
A disease-free food animal population is imperative to the well-being of all individuals. The regulatory disease addressed in this study deleteriously impacts the economics of cattle and swine producers, directly affecting the market price and interstate and international import/export potential of the animals, which in turn influences all consumers. As zoonotic organisms, Brucella species pose a human health threat, hence a protected animal population benefits the general public. Brucellosis animal vaccine work has a significant impact in protecting the human population since Brucella species are also known as bioterrorist agents or "agents of mass destruction."
PUBLICATIONS (not previously reported): 2001/05 TO 2006/10
1. Nielsen, K, P. Smith, W. Yu, P. Nicoletti, P.H. Elzer, C. Robles, R. Bermudez, T. Renteria, A. Ruiz, C. Massengill, Q. Muenks, G. Jurgersen, T. Tollersrud, L. Samartino, S. Conde, L. Forbes, D.Gall, B. Perez, X. Rojas, and A. Minos (2005). Towards a single screening test for brucelloisis. Res. Sci. Off. Int. Epiz 24(3):1027-1038.
2. Zygmunt, M.S., S.D. Hagius, J.V. Walker, and P.H. Elzer. (2006). Signature tagged mutagenesis identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host. Microbes Infect. 2006 Oct 16; [Epub ahead of print]
3. Roux, C.M., N.J. Booth, B.H. Bellaire, J.M. Gee, R.M. Roop, M.E. Kovach, R.M. Tsolis, P.H. Elzer, and D.G. Ennis. (2006). RecA and RadA proteins of Brucella abortus do not perform overlapping protective DNA repair functions following oxidative burst. J. Bacteriology, 188(14):5187-95.
4. Kahl-McDonagh, M.M., P.H. Elzer, S.D. Hagius, J.V. Walker, Q.L. Perry, C.M. Seabury, R.M. Tsolis, L.G. Adams, D.S. Davis and T. Ficht. Evaluation of novel Brucella melitensis unmarked deletion mutants for safety and efficacy in the goat model of brucellosis. (2006) Vaccine. Jun 12;24(24):5169-77.
PROJECT CONTACT:
Name: Elzer, P. H.
Phone: 225-578-4763
Fax: 225-578-4890
Email: pelzer@agctr.lsu.edu

 
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ACCESSION NO: 0405602 SUBFILE: CRIS
PROJ NO: 1265-32000-074-00D AGENCY: ARS 1265
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 01 JUN 2002 TERM: 07 DEC 2005 FY: 2006
INVESTIGATOR: Karns J S; Van Kessel J S; Higgins J A; Jenkins M C
PERFORMING INSTITUTION:
Beltsville Agr Res Center
Beltsville , Maryland 20705
DAIRY MANAGEMENT PRACTICES AND THE TRANSMISSION OF ZOONOTIC PATHOGENS IN MILK.
OBJECTIVES: To evaluate the presence and distribution of selected bacterial and viral pathogens on the dairy farm and in bulk milk. To further identify the critical microbial ecological relationships on dairy farms that affect maintenance of pathogens in the dairy environment and then to evaluate management practices that affect the occurrence of zoonotic pathogens in the milk supply with emphasis on the contributions of dairy cattle waste. To provide strategies and decision aid tools for dairy producers that minimize or eliminate zoonotic dairy waste pathogens that may enter the environment and milk supply.
APPROACH: The needed molecular techniques will be developed to rapidly and accurately identify the pathogenic forms of E. coli, Salmonella Bacillus and Listeria and to employ them to determine the source of the forms that contaminate raw milk on the dairy farm. Utilizing molecular techniques such as isolates taken from raw milk will be compared to establish phylogenetic relationships and variation in genotype profiles that occur within each bacterial species analyzed both from single farm studies and multi-farm studies. Based on various surveys for microorganisms, on farm practices that may be involved in the maintenance and transmission of the zoonotic pathogens in the dairy production system, with particular regard to their transmission to milk will be identified and evaluated.
PROGRESS: 2002/06 TO 2005/12
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This project is aimed at reducing or eliminating the risk of human pathogen transmission through milk and determining the on-farm microbial ecology and management practices that contribute to contamination of raw milk. Research is designed to develop and evaluate the needed technologies for detecting and identifying bacterial pathogens from various on-site niches and for minimizing or eliminating pathogens of zoonotic importance that enter the food supply as a result of current dairy production practices. The project will also determine the extent of contamination of raw milk with viruses that may potentially pose emerging disease threats to humans. Molecular techniques such as PCR and sequence analysis will provide the data to finally evaluate the relationships of on-farm practices to contamination of raw milk. It is fully expected that the culmination of this research project will be the identification of suitable dairy management practices that will lessen the impact of or eliminate those pathogenic microbes that contaminate the milk supply system. This information will be made available to producers, commodity action groups and regulatory agencies that are concerned with transmission of human disease through consumption of milk and milk products. 2. List the milestones (indicators of progress) from your Project Plan. 1.) Develop molecular techniques to rapidly and accurately identify pathogenic forms of E. coli, Salmonella and Listeria and to determine the sources responsible for milk contamination on the dairy farm. 2.) Compare (using rep-PCR, nucleotide sequencing and other molecular techniques), on-farm isolates with isolates from raw milk to establish phylogenetic relationships and variations in genotype profiles among bacterial isolates from single and multi-farm studies. 3.) Identify and evaluate on-farm practices responsible for the maintenance and transmission of the pathogens in the dairy production system, with particular emphasis on their transmission to milk. 4.) Evaluate the presence and abundance of viruses or virus sequences in raw milk and identify the potential for transmission of viruses from raw milk to humans, with particular bovine enteroviruses as an indicator for contamination. 5.) Identify and evaluate specific farms designated through the RDQMA that will serve as long-term sites for in depth studies of on-farm microbial ecology; evaluate specific management practices that influence the presence and contamination by Mycobacterium avium paratuberculosis and Salmonella spp. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why? 1. Develop molecular techniques to rapidly and accurately identify pathogenic forms of E. coli, Salmonella and Listeria and to determine the sources responsible for milk contamination on the dairy farm. Milestone Substantially Met 2. Compare (using rep-PCR, nucleotide sequencing and other molecular techniques), on-farm isolates with isolates from raw milk to establish phylogenetic relationships and variations in genotype profiles among bacterial isolates from single and multi-farm studies. Milestone Substantially Met 3. Identify and evaluate on-farm practices responsible for the maintenance and transmission of the pathogens in the dairy production system, with particular emphasis on their transmission to milk. Milestone Not Met Other 4. Evaluate the presence and abundance of viruses or virus sequences in raw milk and identify the potential for transmission of viruses from raw milk to humans, with particular bovine enteroviruses as an indicator for contamination. Milestone Substantially Met 5. Identify and evaluate specific farms designated through the RDQMA that will serve as long-term sites for in depth studies of on-farm microbial ecology; evaluate specific management practices that influence the presence and contamination by Mycobacterium avium paratuberculosis and Salmonella spp. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This project will terminate as of 5/31/06. Milestones for FY2006 include: PCR method for the reliable and routine detection of Listeria monocytogenes will either be chosen from those reported in the literature or commercially available or will be developed in-house. Archived samples from the NAHMS 2002 Dairy Survey and the dairy farm characterization studies will be used to validate the assay. Phylogenetic relationships among various on-farm bacterial pathogens will continue to be evaluated. Surveillance for organisms will be carried out in collaboration with surveys established for protozoan parasites on farms (CRIS# 1265-32000-073-00D.)and from on-going longitudinal studies of three dairy farms in the northeast US. The genetic and serological database for these identified pathogens will be built and analyzed over the next three years. Other molecular typing techniques such as multilocus sequence typing, and restriction fragment length polymorphisms will be conducted on the well-characterized isolates in our inventory. It is expected that given additional funding, full-length genomic sequence analysis may be implemented for Listeria isolates from the EMSL archived organisms bank to further understand genetic relationships among the pathogenic forms and the differences in pathogenic and non-pathogenic forms. Longitudinal studies on 3 dairy farms will continue and more farms added as funding permits. Management changes designed to limit the propagation and spread of disease organisms will be proposed, chosen, and implemented and studies continued to assess their effectiveness. 4a What was the single most significant accomplishment this past year? Analysis of milk samples from the NAHMS 2002 Dairy Survey with a commercial PCR kit for the detection of Salmonella indicated that the level of Salmonella contamination of US bulk is much higher than previously determined using standard culture techniques. While culture techniques indicated that 2.6% (22 /861) of the samples were contaminated while PCR indicated that 11.8% (101/854) contained Salmonella. Although the initial levels of Salmonella in the milk were determined to be very low, the presence of this organism represents a risk to consumers of raw milk or products made from raw milk. PCR methods allow for more rapid testing of raw milk, providing results in 24 h as opposed to 48 to 96 h for conventional culture methods. 4b List other significant accomplishments, if any. A real-time PCR method was developed for the analysis of Salmonella in milk, feces, and environmental samples. Use of commercial kits for PCR detection of Salmonella is expensive and availability of the kits is uncertain. A PCR method combining SYBR green dye as the detection agent with a well defined PCR method derived from the literature allows sensitive, reliable, and semi-quantitative detection of Salmonella in samples from dairy farms at reasonable cost. A commercial system for the reliable use of REP-PCR methods for the molecular characterization of microorganisms was used to characterize a large number of isolates of Salmonella and Listeria monocytogenes from dairy farms. Molecular characterization of pathogenic bacteria may be used to distinguish between different strains of the same organism. The results indicated that REP-PCR can be used distinguish different serotypes of these organisms but probably does not have the resolving power to reliably distinguish between isolates within a serotype. Since serotyping with conventional methods can be expensive, time consuming, and uses labile reagents, a molecular typing method that determines serotype can be useful in research labs. A Salmonella outbreak on an operating dairy farm was detected and characterized. The zoonotic foodborne pathogen Salmonella is a frequent contaminant of bulk tank milk from US dairies. Knowledge of the factors involved in the establishment and maintenance of Salmonella infections in dairy cows will help define factor that can be controlled to limit pathogen levels and prevent their transmission to milk. In this case, a large percentage of the cows were excreting Salmonella in their feces even though they showed disease symptoms. Although Salmonella was frequently detected in the milk filter, it was rarely detected in the milk, indicating that the milking hygiene practiced on the farm was effective in limiting the transmission of high numbers of Salmonella to the milk. This project was accomplished through a collaboration between EMSL and the Department of Veterinary Science at The Pennsylvania State University. 4d Progress report. This project has matured substantially since its redirection. The past year has seen the use of sophisticated molecular tracking techniques to identify and characterize farm isolates of Listeria, Salmonella and E. coli. This has helped to provide a national view of pathogens on the US dairy farm. It has also provided a mechanism to track pathogens in their various niches on the farm. With access to three specific farms with our collaboration with the four northeastern Universities and access to scores of other farms through collaboration with the CRIS 1265-32000-073- 00D, the laboratory is in a position to become a leader in characterizing molecular epidemiological relationships among a host of important dairy pathogens. With the loss of an investigator to the CDC in October 2004, the virology portion of this project has ended. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. Following re-direction of this project to detection and characterization of management practices to influence microbial pathogens, the tools to evaluate microbial ecology are now all in place and data directly related to incidence and importance are being gathered. The Dairy 2002 study headed by the National Animal Health Monitoring Systems has been completed and the data are now being published. This data is going to provide the most comprehensive look at on-farm pathogen distribution that has ever been made available to the Dairy industry and the regulatory agencies. A variety of molecular probes and primers for characterizing E. coli 0157:H7, Listeria monocytogenes, and Salmonella spp., and bovine enteroviruses have been developed and characterized and these data are likewise being published. The molecular tools needed for portable microbial quality assessment of bulk tank milk are in place. Collaborations with 4 universities in the northeast US have established access to 3 operating dairy farms for long term studies that will result in data sets that will help identify management practices that influence the establishment and maintenance of zoonotic bacterial pathogens on the farm. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Results have been reported at field days and producer meetings such as the BARC Poster Day, 2005 Cornell University Leadership Program and lectures to students at Johns Hopkins University. Technical presentations were made at several professional meetings and workshops. Summaries of our final research results obtained from the NAHMS Dairy 2002 study were presented to FDA/CFSCAN and to the National Milk Producers Federation. Also these results were presented and discussed as a by- product of our recent In-Depth Laboratory review in which an external panel of experts reviewed EMSL. More results of the development of culturing and molecular detection techniques were passed on to collaborators at APHISs National Animal Health Monitoring System, FSIS, and to collaborating scientists at FDA-CFSAN. Important information on the distribution of zoonotic pathogens on the dairy farm and how they might enter the raw milk collection system was provided to our customers through the reports of the NAHMS Dairy 2002 reports. Data from a variety of assays were provided to researchers in support of our CRADA with a molecular detection company (Idaho Technology) to develop and make available detection kits for a variety of pathogens that might be found in milk. Sample collection techniques and data have been made available to our collaborators within the Regional Dairy Quality Alliance/National Milk producers federation sponsored study that includes investigators from several universities in the northeast US. Transfer presentations made: ARS-FSIS Meeting, January 2005, Shepherdstown, WV: "Pilot Study of Factors Affecting Maintenance of Mycobacterium,Salmonella,E. coli and Listeria on Dairy Farms in the Northeast U.S." Northeast US Animal Health Association Meeting, Groton, CT. The Salmonella story, Chapter 1. Meeting on Molecular Methods in Milk Quality, Quality Milk Program, October 2004, Cornell University, Ithaca, NY. Real-time PCR in Milk: Food Safety in Time of War and Peace 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). ARS Collaborates in Regional Dairy Quality Management Alliance Agricultural Research, April 2005.
PUBLICATIONS (not previously reported): 2002/06 TO 2005/12
1. Van Kessel, J.S., Karns, J.S., Gorski, L.A., McCluskey, B.J., Perdue, M.L. 2004. Prevalence of Salmonellae, Listeria monocytogenes and fecal coliforms in bulk tank milk on U.S. dairies. Journal of Dairy Science. p. 2822-2830.
2. Blas-Machado, U., Boileau, M.J., Saliki, J.T., Caseltine, S.L., Goens, S.D. , Duffy, J.C., Welsh, R.D. 2004. Ulcerative and hemorrhagic typhlocolitis in an angus heifer associated with natural bovine enterovirus type-1 infection [abstract]. American College of Veterinary Pathologists. p 2.
3. Whiteaker, J., Karns, J.S., Fenselau, C., Perdue, M.L. 2004. Analysis of Bacillus anthracis spores in milk using mass spectrometry. Foodborne Pathogens and Disease. l(3):185-194.
4. Karns, J.S., Van Kessel, J.S., McCluskey, B.J., Perdue, M.L. 2005. Prevalence of Salmonella enterica in bulk tank milk from US Dairies as determined by PCR. International Association for Food Protection Proceedings, August 14-17, 2005, Baltimore, MD. p.1
5. Blas-Machado, U., Boileau, M.J., Saliki, J.T., Caseltine, S.L., Goens, S.D. , Duffy, J.C., Welsh, R.D. 2004. Ulcerative and hemorrhagic typhlocolitis in an angus heifer associated with natural bovine enterovirus type-1 infection. [Abstract]. American Association of Veterinary Laboratory Diagnosticians 47th Annual Meeting. p.10.
6. Van Kessel, J.S., Karns, J.S., Gorski, L.A., Perdue, M.L. 2005. Subtyping Listeria monocytogenes from bulk tank milk using automated repetitive element-based PCR. In: International Association for Food Protection Proceedings, August 14-17, 2005, Baltimore, MD. p. 14.

 
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ACCESSION NO: 0409832 SUBFILE: CRIS
PROJ NO: 1265-32000-078-00D AGENCY: ARS 1265
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 08 DEC 2005 TERM: 07 DEC 2010 FY: 2006
INVESTIGATOR: Karns J S; Van Kessel J S; Higgins J A
PERFORMING INSTITUTION:
Beltsville Agr Res Center
Beltsville , Maryland 20705
DAIRY MANAGEMENT PRACTICES AND THE TRANSMISSION OF ZOONOTIC PATHOGENS IN MILK .
OBJECTIVES: Objective 1 - Determine the environmental compartments within dairy farming systems that support the survival of the zoonotic pathogens Salmonella enterica,Escherichia coli, and Listeria monocytogenes and characterize their contribution to the pathogen content of milk. Objective 2 - Characterize the role of management practices in the introduction and maintenance of Salmonella enterica, Escherichia coli, and Listeria monocytogenes on dairy farms and evaluate changes in management practices that might reduce or eliminate pathogens. Objective 3 - Use molecular typing methods to determine the relationship between isolates of Listeria, Salmonella, and pathogenic E. coli from dairy cows, the farm environment, and from bulk tank milk with those known to have caused human disease. Objective 4 - Develop new methods for the rapid and sensitive detection of Bacillus anthracis and Listeria monocytogenes in bulk tank milk and milk products.
APPROACH: Although pasteurization and regulations controlling the processing of any products made with unpasteurized milk have an excellent record of assuring the biological safety of dairy products marketed in the US, there is increasing concern about the presence of zoonotic pathogenic microorganisms in raw milk. For various cultural and economic reasons the consumption of raw milk and desire for products made from raw milk seems to be increasing and outbreaks of food-borne gastrointestinal disease due to contamination of dairy products have been documented. This project focuses on the ecology of the zoonotic bacterial pathogens Salmonella, Listeria monocytogenes, and Escherichia coli on dairy farms in the Northeastern United States, and the relationship of the pathogens found in farm animals and the farm environment with those found in bulk tank milk from those farms. Intensive longitudinal sampling will be performed on three typical farms with collection of milk, milk filters, blood, feces, and various environmental samples. We will analyze samples for the three pathogens by both molecular and culture techniques; collaborators will analyze samples for MAP, Campylobacter, and enterococci. Molecular characterization techniques will be used to equate any pathogens found in bulk tank milk with those found on the farm. Management changes will be suggested to the farmers and the results of those changes will be documented. The relationships between Listeria monocytogenes from the farm and those associated with human disease will be investigated. Methods will be developed for improved detection of bacterial pathogens in milk and environmental samples.
PROGRESS: 2005/10 TO 2006/09
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This project is aimed at reducing or eliminating the risk of human pathogen transmission through milk and determining the on-farm microbial ecology and management practices that contribute to contamination of raw milk. This project is is aligned with the Food Safety (Animal and Plant Products) Program 108. Objectives specifically relate to action plan component 1.1.1 Sampling, isolation, identification and quantification of pathogens in animal fluids and tissues, manure; and the environment, including feed, water, and wild animals, 1.2.1 Ecology and assessment of risk factors of pathogens in food producing animals including those carrying antibiotic resistance, outside of the host animal, and 1.4.1 Develop intervention strategies that reduce colonization and shedding of pathogens in animals used for food. Research is designed to develop and evaluate the needed technologies for detecting and identifying bacterial pathogens from various on-site niches and for minimizing or eliminating pathogens of zoonotic importance that enter the food supply as a result of current dairy production practices. Molecular techniques such as PCR, molecular sub-typing, and sequence analysis will provide the data to finally evaluate the relationships of on-farm practices to contamination of raw milk. It is fully expected that the culmination of this research project will be the identification of suitable dairy management practices that will lessen the impact of or eliminate those pathogenic microbes that contaminate the milk supply system. This information will be made available to producers, commodity action groups and regulatory agencies that are concerned with transmission of human disease through consumption of milk and milk products. 2. List by year the currently approved milestones (indicators of research progress) 1. Analyze samples from dairy farms for the presence of Salmonella, E. coli, and Listeria monocytogenes. 2006-2010 2. Alter dairy management techniques to reduce the incidence of zoonotic pathogens in bulk tank milk. 2007-2010. 3. Report Farm results 2006-2010 4. Refine methods for detection of Salmonella, E. coli, and Listeria monocytogenes in farms samples. 2006-2010. 5. Molecular fingerprinting of Listeria isolates. 2007-2010. 6. Develop methods for detection of Listeria in milk. 2006-2009. 7. Develop methods for detection of Bacillus anthracis in milk.2006- 2009. 4a List the single most significant research accomplishment during FY 2006. A. Single Most Significant Accomplishment for each research project during FY 2006.Mycobacterium avium paratuberculosis (MAP) supershedders: A longitudinal study of Salmonella, Listeriamonocytogenes, pathogenic Escherichia coli, and Mycobacterium avium paratuberculosis (MAP) on dairy farms in the northeast conducted in collaboration with 4 universities in the northeastern US has resulted in the discovery of cows that shed extremely high levels of MAP (the causative agent of Johnes disease in cattle). These supershedders appear to be responsible for high environmental loads of MAP and the anomaly of animals testing positive when in fact an infection had not been established. Removal of the supershedder animals from each of three farms resulted in significant reductions in environmental load and, more significantly, MAP-positive animals. (National Program Component 6 Objective 3.2) 4b List other significant research accomplishment(s), if any. A Salmonella outbreak on an operating dairy farm was detected and characterized. The zoonotic foodborne pathogen Salmonella is a frequent contaminant of bulk tank milk from US dairies. Knowledge of the factors involved in the establishment and maintenance of Salmonella infections in dairy cows will help define factor that can be controlled to limit pathogen levels and prevent their transmission to milk. In this case, a large percentage of the cows were excreting Salmonella in their feces even though they showed no disease symptoms. The milk filter was shown to be an indicator of the level of Salmonella shedding in a herd. Although Salmonella was frequently detected in the milk filter, it was rarely detected in the milk, indicating that the milking hygiene practiced on the farm was effective in limiting the transmission of high numbers of Salmonella to the milk. However, the consistent detection of Salmonella in the milk filter was correlated with a high percentage of animals shedding the organism. This project was accomplished through collaboration between EMSL and the Department of Veterinary Science at The Pennsylvania State University. NP108 component 1.2.1 Water troughs were consistently determined to be contaminated with Salmonella on a farm that has a high prevalence of animals infected with Salmonella. Results of a three month study showed that daily cleaning of the water troughs was not more effective than weekly cleaning for eliminating Salmonella. More aggressive intervention steps are needed. NP108 component 1.4.1 Twenty-eight isolates of E. coli were recovered from the feces and internal organs of cattle from both RDQMA and BARC herds, and subjected to genotyping via a triplex PCR reaction. The predominant genotype was B1 (27%) followed by genotype A and genotype B2. Genotype D, which includes E. coli O157:H7, was present at < 4 %. E. coli isolates from other animals associated with the dairy farm environment, such as birds, goats, and houseflies, also had comparatively few genotype D isolates. These results suggest that genotype D E. coli (and by extension E. coli O157:H7) , are not major representatives of the normal flora of dairy cattle participating in these studies. NP108 component 1.1.1 and 1.2.1 4d Progress report. The past year has seen the use of sophisticated molecular tracking techniques to identify and characterize farm isolates of Listeria,Salmonella and E. coli. This has helped to provide a national view of pathogens on the U.S. dairy farm. It has also provided a mechanism to track pathogens in their various niches on the farm. With access to three specific farms with our collaboration with the four northeastern Universities, the laboratory is in a position to become a leader in characterizing molecular epidemiological relationships among a host of important dairy pathogens. Significant progress has been made towards milestone one with all scheduled sampling and analysis done on the study farms. Sampling rates on one participant farm were increased dramatically to document an outbreak of Salmonella. Several management practice alterations have been implemented on a participant farm (milestone 2) in order to measure the effect on the maintenance of Salmonella in the herd. Results of the monitoring of a Salmonella outbreak on a participant dairy farm were presented at the Annual meeting of the Dairy Science Society of America (milestone 3). The ability of REP-PCR to distinguish between isolates of Listeria monocytogenes was tested (milestone 3). Work was done with several collaborators to test various methods for improved detection of pathogenic agents in milk and other matrices and a national survey of Salmonella in bulk-tank milk by PCR was completed and published. 5. Describe the major accomplishments to date and their predicted or actual impact. Analysis of virulence factors associated with pathogenic forms of E. coli in bulk tank milk samples from the Dairy 2002 study headed by the National Animal Health Monitoring Systems has been completed and the data are now being published. This data is going to provide the most comprehensive look at on-farm pathogenic E. coli distribution that has ever been made available to the Dairy industry and the regulatory agencies (NP108 components 1.1.1 and 1.2.1). A variety of molecular probes and primers for characterizing E. coli 0157:H7 and Salmonella spp. have been developed and characterized and these data are likewise being published (NP 108 component 1.1.1). Collaborations with 4 universities in the northeast US have established access to 3 operating dairy farms for long term studies that will result in data sets that will help identify management practices that influence the establishment and maintenance of zoonotic bacterial pathogens on the farm (NP108 components 1.1.1, 1.2.1 and 1.4.1). 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Results have been reported at field days and producer meetings such as the 2006 Cornell University Leadership Program and lectures to students at Johns Hopkins University. Technical presentations were made at several professional meetings and workshops. Summaries of our final research results obtained by analysis of samples from the NAHMS Dairy 2002 study were presented to FDA/CFSCAN and to the National Milk Producers Federation. More results of the development of culturing and molecular detection techniques were passed on to collaborators at APHISs National Animal Health Monitoring System, FSIS, and to collaborating scientists at FDA-CFSAN. Important information on the distribution of zoonotic pathogens on the dairy farm and how they might enter the raw milk collection system was provided to our customers through the reports of the NAHMS Dairy 2002 reports. Data from a variety of assays were provided to researchers in support of our CRADA with a molecular detection company (Idaho Technology) to develop and make available detection kits for a variety of pathogens that might be found in milk. Sample collection techniques and data have been made available to our collaborators within the Regional Dairy Quality Alliance/National Milk producers federation sponsored study that includes investigators from several universities in the northeast U.S. Transfer presentations made: ARS-FSIS Meeting, ARS/RDQMA Dairy Project Update, March 8, 2006, Annapolis, MD. Northeast U.S. Animal Health Association Meeting, Dairy Project Update: Salmonella, Listeria and E. coli, March 13, 2006, Atlantic City, NJ. Joint Meeting of ADSA and ASAS, A long-term, sub-clinical outbreak of Salmonella enterica subsp. enterica Cerro in a Pennsylvania dairy herd, July 11, 2006, Minniapolis, MN.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
1. Karns, J.S., Van Kessel, J.S., Mccluskey, B.J., Perdue, M.L. 2005. Prevalanece of Salmonella enterica in bulk tank milk from U.S. Dairies as determined by pcr. Journal of Dairy Science. 88:3475-3479.
2 . Hovingh, E., Whitlock, R.H., Sweeney, R.W., Fyock, T., Wolfgang, D.R., Smith, J., Schukken, Y.H., Van Kessel, J.S. 2006. Identification and implications of map supershedders. Joint Meeting of the ADSA, AMSA, ASAS and PSA, Minneapolis, MN, July 9-13, 2006.
3. Van Kessel, J.S., Karns, J.S., Wolfgang, D.R., Hovingh, E., Schukken, Y. 2006. A long term, sub-clinical, outbreak of Salmonella enterica subsp. enterica Cerro in a Pennsylvania dairy herd. Joint Meeting of the ADSA, AMSA, ASAS and PSA, Minneapolis, MN, July 9-13, 2006.
4. Chapagain, P.P., Van Kessel, J.S., Karns, J.S., Wolfgang, D., Schukken, Y. H., Grohn, Y.T. 2006. Mathematical modeling of the dynamics of Salmonella Cerro infection in a U.S. dairy herd. American Physical Society. p. 1.
5. Van Kessel, J.S. 2005. Salmonella, E. coli and Listeria on dairy farms [abstract]. USDA-MOST Food Safety/Ag Processing Workshop. p. 12.
6. Van Kessel, J.S., Karns, J.S., Gorski, L.A., Perdue, M.L. 2006. Subtyping Listeria monocytogenes from bulk tank milk using automated repetitive element-based PCR. Journal of Food Protection. 6:2707-2712.


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ACCESSION NO: 0210006 SUBFILE: CRIS
PROJ NO: MICK-2007-00054 AGENCY: CSREES MICK
PROJ TYPE: SMALL BUSINESS GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2007-33610-17933 PROPOSAL NO: 2007-00054
START: 15 MAY 2007 TERM: 14 JAN 2009 GRANT YR: 2007
GRANT AMT: $79,941
INVESTIGATOR: Mathew, F. P.
PERFORMING INSTITUTION:
Rapid Biosense, Llc
Dexter , Michigan 48130
LOW-COST CONDUCTIMETRIC BIOSENSOR FOR DETECTION OF MULTIPLE BIOTERRORISM AGENTS.
NON-TECHNICAL SUMMARY: Combating bioterrorism effectively will require diagnostic devices that can detect potential bioterrorism agents quickly with a high level of sensitivity, specificity, and simplicity in any sample matrix. This study will develop a single target proof-of-concept rapid diagnostic device, which will pave the way for development of a novel multi-target biosensor to be used as an "immediate response" device in veterinary hospitals and diagnostic labs for detection of bacteria, viruses, and toxins.
OBJECTIVES: The Phase I research will demonstrate proof-of-concept for a low cost, easy-to-use diagnostic device that can be employed to detect bioterrorism agents in a wide variety of samples and testing environ-ments. The proposed sensor, uses a conductimetric detection technique, combining antibodies with a conductive molecular transducer on a disposable test strip. The proposed research is focused on three specific aims: Aim 1: Fabrication of a single-sample biosensor for M. paratuberculosis: Using improvements to methods from preliminary studies at Michigan State University, biosensors will be fabricated for detection of MAP. Aim 2: Biosensor testing for M. paratuberculosis in fecal samples: Protocols will be developed to enable MAP detection using the biosensor with minimum preprocessing of samples. Biosensor will then be tested for MAP in well characterized fecal samples in collaboration with North Dakota State University (NDSU). Aim 3: Biosensor testing for M. paratuberculosis in milk samples: Protocols will be developed to detect MAP in milk samples. The biosensor will be tested for MAP in raw and pasteurized milk samples in collaboration with NDSU to validate the performance of the proposed biosensor.
APPROACH: Detection of MAP, the etiological agent of Johne s disease (JD) in animals and a potential bioterrorism agent, in milk or fecal samples requires a minimum of 5-16 weeks to complete and is expensive and laborious. Existing rapid tests for fecal samples, housed in research and diagnostic labs, require extensive technical expertise (e.g., PCR) and up to 6 weeks (rapid liquid culture) to obtain results. For animal biosecurity, there is an urgent need to develop highly sensitive and specific, low cost and rapid diagnostic devices for JD that can identify MAP in milk and fecal samples. The central determinants of the sensitivity and specificity of the proposed conductimetric biosensor detection technology to a certain bioterrorism agent are optimization of the biosensor performance as well as optimum preparation of appropriate sample matrices for analysis using the biosensor. This research will address the following questions relevant to establishing the technical feasibility: A) Can this diagnostic technology be adapted for detection of any target agent? Specifically, using the fabrication procedures previously developed, can a biosensor specific to Mycobacterium paratuberculosis (MAP) be fabricated? B) Can this diagnostic technology work with different sample matrices? Specifically, what are the sample preparation steps required for detection of MAP in feces and milk samples? Answering these questions will enable customization of the sensor materials, electronics, and signal processing to deliver optimum biosensor performance for this application. The approach used in this research is to take pre-enrichment procedures used currently by the diagnostic laboratories for M. paratuberculosis isolation and detection in milk and fecal samples and tailor these procedures for a biosensor-based detection so as to minimize the time-to-results and the labor/expertise required.
PROJECT CONTACT:
Name: Mathew, F. P.
Phone: 800-579-4913
Fax: 800-579-4913
Email: finny@rapidbiosense.com

 
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PROJ NO: MICL02071 AGENCY: CSREES MICL
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START : 01 AUG 2003 TERM: 31 DEC 2005 FY: 2006
INVESTIGATOR: Coussens, P. M.; Grooms, D.; Bolin, C.; Bolin, S.; Kuipel, M.; Barletta, R.
PERFORMING INSTITUTION:
Animal Science
Michigan State Univ
East Lansing , Michigan 48824
JOHNE'S DISEASE PATHOGENESIS AND HOST RESPONSE TO MYCOBACTERIUM PARATUBERCULOSIS .
NON-TECHNICAL SUMMARY: Johne's disease is a major concern in the US dairy industry. There are currently no viable vaccines for Johne's disease and often diagnosis is highly problematic. This project will benefit efforts to create vaccines and diagnostics for Johne's disease as well as provide a background of basic information on the bovine immune system. It is anticipated that results from this project will also benefit studies on prevention and control of bovine tuberculosis and other infectious diseases.
OBJECTIVES: Our long-term objective relevant to the current project is to elucidate the complex interplay of immune cells and modulators that lead to peripheral and local immune responses in cattle infected with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle. Based on existing literature and preliminary results, one of our primary hypotheses is that MAP-specific and general immune suppression observed in clinically infected animals is at least partly due to specific suppressor cell populations that arise during the long subclinical phase of infection. Specific Objectives (1) Correlate MAP stimulated changes in immune cell gene expression with immune cell population profiles using peripheral blood mononuclear cells (PBMCs) from cattle representing subclinical and clinical stages of MAP infection. (2) Test the hypothesis that specific immune cell types are responsible for repressed gene expression in PBMCs from clinical Johne's disease cows in response to MAP. (3) Correlate and contrast observations from peripheral immune cells with infected intestinal tissue and mesenteric lymph nodes from the same animals used in Objectives 1 and 2. (4) Investigate interactions between MAP and bovine macrophages that lead to presistence of MAP in a cell designed to kill invading pathogens.
APPROACH: One hypothesis to explain gene expression differences between immune cells from Johnes disease positive and control cows is that immune cell populations in infected and control cows are initially different. A second, equally valid hypothesis is that MAP induces apoptosis in specific immune cell subsets thus leading to lower gene expression relative to controls. To test and distinguish between these hypotheses, we will conduct a series of experiments comparing responses of PBMCs from infected and control cows to MAP, in which gene expression profiles are linked to cell distribution, measures of apoptosis, and immune cell proliferation. We will also address the hypothesis that a particular immune cell type is responsible for quenching a rapid and vigorous response to MAP. To test this hypothesis, we will use immunomagnetic separation with cell-type specific antibodies, removing specific cell types from PBMCs. We will focus on the use of quantitative reverse transcriptase real-time polymerase chain reaction (Q-RT-PCR). we will also examine differences between intestinal tissues and mesenteric lymph nodes from Johne's disease positive cows and similar tissues from control uninfected cows. One specific hypothesis is that overexpression of IL-1 and TRAF 1 in clinical Johnes disease has allowed cells in the intestinal lumen and mesenteric lymph nodes to adopt a survival phenotype by preventing apoptosis. A second hypothesis to be tested is that, despite an apparent Th 2-like peripheral response in clinical cows, local sites and nodes from infected tissues retain a strong pro-inflammatory pattern of gene expression. Data needed to test our specific hypotheses will be acquired using both Q-RT-PCR and cDNA microarray analysis. We will extend our preliminary results to a larger pool of Johnes disease positive cows and compare expression of key genes between control, subclinical, and clinical infected animals. In addition, we will add a comparison of expression in mesenteric lymph nodes, not included in our original analysis. Inhibition of phagosome-lysosome fusion by Mycobacteria appears to have two main components. These components are: 1) preventing the phagosome from maturing into a phagolysosome through interruption of protein expression and/or trafficking and 2) avoidance of normal signaling through the Toll-like receptor (TLR) pathway. Specific Objective 4 will address the first component by directly examining phagosomes in macrophages following phagocytosis of latex beads and E. coli and two Mycobacteria species. Resting macrophages are used as a control in these studies, allowing us to also identify changes in gene expression and protein trafficking during the general processes of phagocytosis and activation.
PROGRESS: 2003/08 TO 2005/12
Our work during the past 12 months has focused on examining the effects of M. paratuberculosis on cultured bovine macrophages. These studies were initiated because of prior results that showed M. paratuberculosis infected macrophages at sites of infection have dramatically enhanced levels of IL-1alpha and TRAF1 protein expression. TRAF1 is a member of a protein family that is intricately involved in intracellular signaling, including activation of macrophages following contact with cytokines or activated T cells. One hypothesis is that high levels of TRAF1 would inhibit signaling through TNF receptor superfamily members, including TNFR1, Fas, and TNFR2. We have now demonstrated that in cultured macrophages, infection of cells with M. paratuberculosis indeed upregulates IL-1alpha and TRAF1 expression and that TRAF1 expression is dependent upon continued release of IL-1alpha. These results suggest that drives high expression of TRAF1 in lesions from cattle with Johnes disease is likely driven by IL-1alpha. Importantly, uninfected macrophages newly recruited to sites of infection would be exposed to high levels of IL-1alpha and may thus be deficient in activation and signaling of T cells. These studies will be continued under a recently warded USDA-NRI grant and designated as project number MICLO8369.
IMPACT: 2003/08 TO 2005/12
Results from this project suggested that M. paratuberculosis has profound effects on gene expression and possibly on intracellular signaling in infected macrophages. In addition, our results provided a foundation for understanding how M. paratuberculosis might interfere with the ability of macrophages to interact with T cells and to become activated following these interactons.
PUBLICATIONS ( not previously reported): 2003/08 TO 2005/12
1. Aho, A.D., McNulty, A.M., and P.M. Coussens. (2003). \ Enhanced Expression of IL-1? and TRAF 1 in Ileal Tissues of Cattle Infected with Mycobacterium paratuberculosis. Infection and Immunity, 71(11), 6479-6486.
2. Coussens, P.M., N. Verman, M.A. Coussens, M.D. Elftman, and A.M. McNulty. (2004). Cytokine Gene Expression in Peripheral Blood Mononuclear Cells and Tissues of Cattle Infected with Mycobacterium avium subspecies paratuberculosis: Evidence for an Inherent Pro-inflammatory Gene Expression Pattern. Infection and Immunity, 72(3):1409-1422.
3. Tooker, B.C. and P.M. Coussens. (2004). Phagocytosis of M. paratuberculosis fails to Activate Expression of NADH Dehydrogenase and Nucleolin-Related Protein in Bovine Macrophages. FEMS Immunology Letters. 93: 137-142.
4. Coussens, P.M. 2004. A Model for Immune Responses to Mycobacterium paratuberculosis. Infection and Immunity (Minireviews), 72: 3089-3096.
PROJECT CONTACT:
Name: Coussens, P. M.
Phone: 517-353-3158
Fax: 517-353-1699
Email: coussens@msu.edu

 
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ACCESSION NO: 0180740 SUBFILE: CRIS
PROJ NO: MICL03381 AGENCY: SAES MICL
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 DEC 1998 TERM: 30 NOV 2003 FY: 2005
INVESTIGATOR: Lloyd, J.; Peterson, C.; Harsh, S.
PERFORMING INSTITUTION:
Agricultural Economics
Michigan State Univ
East Lansing , Michigan 48824
STRATEGIC MANAGEMENT IN AGRIBUSINESS USING KEY PERFORMANCE INDICATORS.
OBJECTIVES : The objectives of this research are: 1) to define the awareness of, attitudes toward, and levels of adoption for formal strategic management in both the Michigan dairy industry and the veterinary profession, and 2) to identify the key performance indicators (KPI's) that are most useful for managing dairy farms and veterinary practices in a strategic management framework, based on firm-specific goals and objectives.
APPROACH: Preliminary data on both strategic management and KPIs will be collected through individual focus group meetings for dairy producers and practicing veterinarians. Case studies of individual dairy farms and veterinary practices will also be employed to provide descriptive information on how to implement various strategic managment practices in real time. Subsequently, surveys will be conducted to quantify the findings from the focus groups for both dairy producers and practicing veterinarians. Collaborations will be developed with ongoing studies of both the dairy industry and the veternary profession to determine the strength of any potential correlation between strategic management and firm performance. Beyond these econometric studies, computer models will be developed for both dairy farms and veterinary practices to evaluate potential changes in tactics for their possible impact on the KPIs of individual agribusinesses.
PROGRESS: 1998/12 TO 2003/11
The objectives of this research project were: 1) to define the awareness of attitudes toward, and levels of adoption for formal strategic management in both the Michigan dairy industry and the veterinary profession, and 2) to identify the key performance indicators (KPIs) that are most useful for managing dairy farms and veterinary practices in a strategic management framework, based on firm-specific goals and objectives. As this project evolved over the course of its lifetime, the scope of work changed somewhat from the original intent. During early consideration of strategic management in both the Michigan dairy industry and the veterinary profession, a strong need was encountered for a broader set of personal attributes to achieve effective leadership and management in the veterinary profession. Subsequent research in this arena revealed a wide diversity of training programs across the Association of American Veterinary Colleges without any real consensus on educational content or approach. This finding clearly signaled the need for development of standardized curriculum. Results of this project also indicate that these non-technical skills, knowledge, aptitudes, and attitudes (SKAs) are not always effectively modeled in the veterinary teaching hospitals,, and admission processes may, in effect, be screening out some of the desired characteristics. A broad-based need for leadership skills has also been identified for the veterinary profession. With regard to KPIs, project results have provided insight into strategic aspects of paratuberculosis, drug management, herd depopulation, culling and replacement, BST use, and management intensive grazing in the dairy industry. Similarly, the strategic importance of starting salaries and debt loads of veterinary graduates, customer satisfaction, and pricing decisions have been addressed for the veterinary profession.
IMPACT: 1998/12 TO 2003/11
As a result of our work, Michigan dairy producers have a clearer picture of: the impact of paratuberculosis on production, reproduction and economic performance; dairy herd reproduction with BST use; determinants of drug management practices; reliability of on-farm, residue detection assays; risk factors for violative drug residues in milk; the potential impact of management intensive grazing on profitability and economic efficiencies; and the financial aspects of herd depopulation, culling and replacement selection, and BST use. To the extent that these findings are being used, dairy production should be more efficient, profitable, and sustainable as a result. In addition, the impacts of the veterinary medical profession on the State of Michigan are more fully understood, as are both the KPIs used in managing veterinary practices and the teaching and research needs related to non-clinical skills, knowledge, aptitudes, and attitudes (SKAs) in the veterinary profession. Our demonstration projects and applied research have sensitized the veterinary profession to the applicability and utility of conducting applied market research in a strategic management framework. Further, this project has made it apparent that enhanced veterinary teaching hospital management, veterinary school admissions processes, and veterinary leadership development programs are needed. Across the veterinary profession, curricula and research priorities related to SKAs, teaching hospital management, admissions, and leadership are currently being scrutinized based, in large part, on our work.
PUBLICATIONS (not previously reported): 1998/12 TO 2003/11
No publications reported this period

 
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ACCESSION NO : 0187072 SUBFILE: CRIS
PROJ NO: MICL03402 AGENCY: SAES MICL
PROJ TYPE: STATE PROJ STATUS: REVISED
START: 01 OCT 2005 TERM: 30 SEP 2010 FY: 2007
INVESTIGATOR: Coussens, P. M.; Kuipel, M.
PERFORMING INSTITUTION:
Animal Science
Michigan State Univ
East Lansing , Michigan 48824
IMMUNE MODULATION AND GENE EXPRESSION SIGNATURES ASSOCIATED WITH INFECTION OF CATTLE WITH M. PARTUBERCULOSIS.
NON-TECHNICAL SUMMARY: A. Johne's disease, caused by M. paratuberculosis, is one of the most costly infectious disease problems in the US dairy herd. A. This project will enhance knowledge on how M. paratuberculosis survives in macrophage cells and how infected cells interact with other components of the immune system. B. This project will also be the first demonstration of using gene expression profiling to diagnose an infection in domestic species.
OBJECTIVES: I. We will conduct In vitro work on the effect of M. paratuberculosis (MAP) on macrophage signaling, activation, and T cell interactions. These studies include: 1) Examination of the relationship between MAP infection and expression of IL-1alpha, TRAF1, and TRAF2 in cultured monocyte derived macrophages (MDM). 2) Determination of the effect of MAP infection and IL-1alpha treatment on CD40-CD154 and TNFalpha signaling in MDM. 3) We will determine the effect of MAP infection on interactions between MDM and autologous activated T cells. 4) We will Investigate the effect of MAP infection on susceptibility/resistance to apoptosis in MDM. II. We will work to Develop an infection signature for diagnosis of MAP and to aid understanding of MAP effects on the peripheral immune system. To accomplish this, we will: 1) Complete Q-RT-PCR mediated validation of 45 potential MAP infection signature genes on a cohort of 150 infected cattle and 50 controls. 2) We will complete statistical analysis of the Q-RT-PCR data and look for correlations between gene expression differences and infection parameters assessed by various standard diagnostic tests. We will also attempt to correlate immune tests (serum ELISA, IFN? test results, and fecal culture) with gene expression events. 3) Finally, we will extend our gene expression signature work to other pathogens, including Brucella abortus, M. bovis, BVDV and BLV.
APPROACH: Specific Objective 1: Ontology of TRAF1 and IL-1alpha mRNA and protein expression in MDM infected with MAP. monocyte-derived macrophages (MDM) prepared from 4 different cows will be infected with MAP in vitro. Uninfected MDM will serve as negative controls. Infected and uninfected MDM will be collected at various times post-infection. Cultures will be harvested for RNA and protein at each time point. TRAF1, TRAF2, and IL-1alpha mRNA expression at each time point will be assessed by Q-RT-PCR. Relationship between IL-1alpha expression and TRAF1 upregulation. Uninfected MDM from 4 cows will be treated with rIL-1alpha in the presence and absence of an excess of IL-1 receptor antagonist (IL-1Ra) for 24 hours. Cells will be harvested for mRNA and protein analysis. TRAF1 and TRAF2 gene expression and protein content will be analyzed by Q-RT-PCR and western blotting. MDM from 4 cows will be infected with MAP in the presence and absence of IL-1Ra. Lysates will be collected at 0, 0.5, 1, 3, 6, 12, 24, 48, and 72 hours following exposure to MAP. Expression of TRAF1 mRNA and protein will again be monitored. Establish the relationship between TRAF1 and TRAF2 in MAP infected MDM. Protein extracts from MAP infected and IL-1alpha treated MDM will be used to examine the composition of TRAF1/TRAF2 complexes by immunoprecipitation pull-down coupled with western blotting. Specific Objective 2: Determine the effect of MAP infection and TRAF1 overexpression on CD40 signaling in MDM. First, CD154-CD40 mediated activation of NFkB in MDM will be assayed. Second, we will directly examine activation of IKK, a regulator of NFkB. Third, expression of IL-10, IL-12p35, IL-12p40, TNFalpha, MIP-1alpha and production of nitric oxide will be monitored by Q-RT-PCR in control and MAP infected MDM. Specific Objective 3: Interaction of autologous T cells with MAP infected macrophages. Cultures of MDM from all cows will be infected with MAP at a MOI of 5 to 10 and allowed to settle. Fresh PBMCs will be prepared from each cow and stimulated with MAP antigens in vitro, with general mitogens, or without antigen stimulation for 24 hours. Following stimulation, autologous T cells will be isolated using magnetic cell sorting. Isolated T cells will be fixed and applied to autologous infected or control MDM and allowed to interact for 4 to 24 hours. Macrophage activation parameters to be assayed include production of IFN?, IL-10, IL-12p35, IL-12p40, TNFalpha, MIP-1alpha, and IL-1alpha mRNA by Q-RT-PCR. Specific Objective 1: Evaluate and validate a previously determined subset of 45 host immune response genes by Q-RT-PCR for diagnosis of M. paratuberculosis infection in populations of 150 subclinical infected and 50 control cattle. Preliminary data, clearly indicates that inherent gene expression profiles in immune cells from M. paratuberculosis infected cows is different than profiles in cells from control cows. We have now selected 45 of the most significantly differentially expressed genes for validation and testing in a large group of infected and control cows using high-throughput Q-RT-PCR.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Johnes disease in cattle has a significant economic impact on the global dairy industry, with losses in the US estimated at over 1 billion dollars annually. There are no effective vaccines available to prevent infection with Mycobacterium paratuberculosis (MAP), the causative agent of Johnes disease. A hallmark of infection with MAP, as with other mycobacteria, is survival of the organism in host macrophage cells where immune destruction is difficult. Why infected macrophages do not destroy invading MAP organisms is a central question in MAP immunobiology and in study of other mycobacteria, such as M. bovis. Our work is focused on discovering mechanisms employed by MAP to suppress macrophage functions in the hopes of finding new methods to combat Johnes disease. Our main hypothesis is that MAP reduces the ability of infected macrophages to react to normal T cell signaling, failing to activate and destroy MAP, and failing to properly signal T cells to respond. Infected macrophages are also induced to resist apoptosis, prolonging survival of intracellular MAP. This project has four main objectives. These are to 1) examine the relationship between infection and expression of interleukin (IL)-1alpha, TRAF1, and TRAF2 in cultured monocyte derived macrophages (MDM). 2) Determine the effect of MAP infection and IL-1alpha treatment on CD40-CD154 and TNF&#945; signaling in MDM. 3) Determine the effect of MAP infection on interactions between MDM and autologous activated T cells. 4) Investigate the effect of MAP infection on susceptibility/resistance to apoptosis in MDM. In related studies, we have begun to examine how development of specific regulatory T cells might impact development of clinical Johnes disease and affect macrophage-T cell interactions. Previously we had shown the IL-1alpha and TRAF1 were expressed at haig concentrations in MAP infected tissues and cells. We have explored this relationship further, using in vitro model systems. This work led us to examine signaling mechanisms within MAP infected macrophages. We discovered that MAP infected macrophages were defective in some aspects of signaling through CD40 receptors, leading to under expression of the critical cytokine IL-12 and the inducible nitric oxide synthetase (iNOS). We have also examined possible mechanisms for this defect, examining the roles of various mitogen-activated protein kinases. In the regulatory T cell area, we have definitively demonstrated that these cells do exist in cattle, that MAP-reactive regulatory T cells develop in the course of natural infections with MAP, and that they respond to MAP antigens by production of IL-10, suppressing production of interferon gamma, a cytokine required to clear infections with MAP. Our results have been presented at several major international meetings, including the Conference for Research workers in Animal Disease, Dec. 3-5, Chicago IL; the International Vet. Immunology Symposium, Ouro Preto, Brazil, August 2007; The Johnes Disease Integrated Project Meeting, College Station Texas, and the 9th Intl.Colloquium on Paratuberculosis. Tsukuba, Japan, October 2007. PARTICIPANTS: P.M. Coussens, Principal Invvestigator, Professor, Department of Animal Science, Michigan State University. C.J. Colvin, Research Technician II, Department of Animal Science, Michigan State University. D. Almeida, Postdoctoral fellow, Department of Animal Science, Michigan State University. S. Sommer, Postdoctoral fellow, Department of Animal Science, Michigan State University. S.K. Chiang, Visiting Sceintist, Department of Animal Science, Michigan State University. E. Kabara, Graduate Research Assistant, Department of Animal Science, Michigan State University. Collaborators W. Davis, Professor, Washington State University. S. Sreevatsan, Associate Professor, University of Minnesota. TARGET AUDIENCES: The primary target audiences for our work are other scientists working in Johnes disease and on other species of mycobacteria. Additional target audiences are scientists working in the animal health industry, particularly those associated with projects to develop vaccines against Mycobacterium paratuberculosis (MAP) and diagnostics to detect MAP
infection in cattle.
IMPACT: 2007/01 TO 2007/12
Work completed to date on this project has demonstrated that MAP infection of macrophages enhances expression of interleukin (IL)-1 leading to activation of the transcription factor NfkappaB and subsequent activation of the TRAF1 gene. Enhanced expression of TRAF1 in cultured macrophages can also be achieved with direct treatment of macrophages with recombinant IL-1, while the reverse is not true. Furthermore, sustained expression of TRAF1 requires continued presence of IL-1. These results demonstrated that MAP may have profound effects on intracellular signaling within infected macrophages and led to the hypothesis that MAP may alter the ability of infected macrophages to activate and to properly signal T cells to respond to infection. These results have led to a series of new investigations, including a search for MAP factors that might be responsible for this effect and a more global functional genomics based investigation of MAP-induced gene expression changes in macrophage cells. CD40 interactions with its ligand on activated T cells is a major mechanism used to activate macrophages, eliciting expression of numerous immune response genes in the target macrophage. Our subsequent work demonstrated that MAP is able to subvert a subset of macrophage responses to CD40 signaling, in particular expression of IL-12 and iNOS. These two factors are critical for macrophages to destroy MAP and direct T cell responses toward a Th1 or proinflammatory activity, which is required for clearance of MAP infections. This result offers a possible explanation for why the Th1 proinflammatory response to MAP is lost during the course of natural infections in cattle that proceed to clinical disease. In addition, our results suggest that promoting IL-12 and perhaps iNOS production may help alleviate development of clinical illness due to MAP and allow clearance of this fastidious pathogen. Furthermore, our results are being incorporated into modified live vaccine development strategies, searching for MAP mutants that do not inhibit iNOS and IL-12 production in macrophages. Regulatory T cell biology and especially development of peripheral Treg cells in response to infection is a relatively new area in immunobiology. Our work with MAP-specific Treg cells has clearly demonstrated that these cells do exist in cattle, a novel finding. These studies have therefore opened a new avenue of research in bovine immunobiology. Development of Treg cells in MAP infection can, in addition to the results discussed above, help explain why immune responses to MAP appear to be limited or re-directed in animals that proceed to clinical disease. Again, these results are being used to examine MLV vaccine candidates, since any effective vaccine should not elicit a Treg response.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
1. Coussens, P.M., C.J. Colvin, and D.E. Almeida. 2008. Antigen-specific regulatory T cells in bovine paratuberculosis. Veterinary immunology and immunopathology, In Press.
2 . Chiang, S., Sommer, S., Aho, A., Kuipel, M., Colvin, C., Tooker, B.R., and P.M. Coussens. 2007. Relationship between IL-1alpha and TRAF1 in primary bovine macrophages infected with Mycobacterium avium subspecies paratuberculosis. VII, In Press: Veterinary Immunol. Immunopath. 116: 131-144.
3. Murphy, J.T., S. Sommer, E.A. Kabara, N.Verman, M.A. Kuelbs, P. Saama, R. Halgren, and P.M. Coussens. 2006. Gene Expression Profiling of Monocyte-Derived Macrophages Following Infection with Mycobacterium avium subspecies avium and Mycobacterium avium subspecies paratuberculosis. Physiological Genomics, 28: 67-75.
4. Janagama, H.K., K.I. Jeong, V. Kapur, P.M. Coussens, and S. Sreevatsan. 2006. Cytokine responses of bovine macrophages following phagocytosis of diverse clinical Mycobacterium avium subspecies paratuberculosis strains. BMC Microbiology 2006, 6:10
5 . Sommer, S., Pudrith, C. B., Colvin, C. J., and Coussens, P. M., (2007). Mycobacterium avium subspecies paratuberculosis suppresses CD40 signaling induced IL-12p40 and iNOS gene expression in bovine monocyte-derived macrophages. Veterinary Immunology and Immunopathology, In Press.
6 . Sommer, S., Romeih, M., and Coussens, P. M., (2007). Advances in the fight against Johnes disease. Michigan Dairy Review, 12(3):10-12.
PROJECT CONTACT:
Name: Coussens, P. M.
Phone: 517-353-3158
Fax: 517-353-1699
Email: coussens@msu.edu

 
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ACCESSION NO: 0207811 SUBFILE: CRIS
PROJ NO: MICL04002 AGENCY: CSREES MICL
PROJ TYPE: HATCH PROJ STATUS: NEW MULTISTATE PROJ NO: NC-1027
START: 01 OCT 2006 TERM: 30 SEP 2011 FY: 2007
INVESTIGATOR: Grooms, D. L.
PERFORMING INSTITUTION:
Large Animal Clinical Sciences
Michigan State Univ
East Lansing , Michigan 48824
AN INTEGRATED APPROACH TO CONTROL OF BOVINE RESPIRATORY DISEASE.
NON-TECHNICAL SUMMARY: Bovine respiratory disease (BRD) continues to be the most costly disease problem facing the cattle industry. The focus of this multistate project is on the nature of factors that put cattle at risk for respiratory disease and on the pathogenesis of single agent and multi-agent respiratory diseases.
OBJECTIVES: 1. To evaluate the prevalence of viral and bacterial agents of respiratory disease by developing, validating and disseminating new state-of-the-art molecular diagnostics for rapid identification of these agents. 2. To develop management and prevention strategies that incorporate new vaccines and treatment protocols to combat bovine respiratory disease and reduce economic loss
APPROACH: Through collaboration with multiple departments at Michigan State University, multipe approaches will be taken to develope novel biosensor technologies for detecting pathogens important in the pathogeneisis of bovine respiratory disease. Pathogens important in the pathogenesis of bovine respiratory disease will contiue to be monitored for through submissions to the Diagnsotic Center for Population and Animal Health. Strategies for controlling Mycoplasma bovis will be evaluated, including vaccination and antibiotic intervention.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Outputs from this project in 2007 have included further refinement of the conductometric biosensor used to detect BVDV and the exploration of new biosensor technology aimed at detecting pathogens important to animal and human health. This project has and continues to serve as the basis for training of students at various academic levels including undergraduate, graduate and post-graduate. It is also the basis of active collaboration between researchers that are expanding the knowledge learned in this project the to detection of other important animal and human pathogens. These inclulde Mycobacterium paratuberculosis in cattle, and important food pathogens including E. coli and Salmonella. Finally, this project has resulted in the application for a new patent on processes related to manufacturing of the biosensor. New to the project this year is the launching of the Michigan Upper Peninsula BVDV Eradication Project. The goal of this project is to eradicate BVDV form a region of the US and study the impacts on cattle health, including bovine respiratory disease. Launched in the fall of 2007, initial outreach activities included a series of nine (9) meetings across the Upper Peninsula of Michigan focusing on educating producers and industry partners about BVDV. Over 150 people attended these meetings. Included in these meetings were educational handouts describing BVDV and the program in general. PARTICIPANTS: Vangie Alocilja, Michigan State University, Department of Agriculture and Biosystems Engineering. Collaborative partner on the conductometric biosensor project. Dr. Steve Bolin, Michigan State University, College of Veterinary Medicine. Collaborative partner on the BVDV Eradication Project. Dr. Roger Maes, Michigan State University, College of Veterinary Medcine. Collaborative partner on the BVDV Eradication Project. Dr. Ben Bartlett, Michigan State University Extension, Collaborative partner on the BVDV Eradication Project. Michigan Department of Agriculture and the United States Department of Agriculture, APHIS, VS. Collaborative partner on the BVDV Eradication Project. Pfizer Animal Health. Collaborative partner on the BVDV Eradication Project. TARGET AUDIENCES: The primary target audience for this project is the cattle industry where bovine respiratory disease is of major concern. The cattle industry includes producers, veterinarians and other livestock industry support personnel (nutritionists, feed sales, pharmaceutical sales, marketing personnel, etc).
IMPACT: 2007/01 TO 2007/12
A rapid detection of BVDV in cell culture media using a biosensor has been demonstrated. Detection of cattle persistently infected with BVDV using ear notches was shown to be most consistent when compared to serum and nasal swabs Further development may make this a useful tool for the rapid and economical identification of BVDV PI cattle. The Michigan regional BVDV Eradication program has been launched. This program has the potential to significantly impact cattle health and marketability on a regional basis.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12Muhammad-Tahir, Z., Alocilja, E.C., Grooms, D.L. (2007) Indium Tin Oxide-Polyaniline Biosensor: Fabrication and Characterization. Sensors 7:1123-1140.
PROJECT CONTACT:
Name: Grooms, D. L.
Phone: 517-432-1494
Fax: 517-432-1042
Email: groomsd@cvm.msu.edu

 
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ACCESSION NO: 0187832 SUBFILE: CRIS
PROJ NO: MICL06901 AGENCY: CSREES MICL
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2000 TERM: 30 SEP 2005 FY: 2006
INVESTIGATOR: Coussens, P.; Burton, J.
PERFORMING INSTITUTION:
Animal Science
Michigan State Univ
East Lansing , Michigan 48824
DYNAMICS OF IMMUNE RESPONSES TO JOHNE'S DISEASE IN INFECTED AND VACCINATED CALVES.
NON-TECHNICAL SUMMARY: Johne's disease is a chronic infection in cattle that is untreatable with conventional antibiotics and ultimately leads to death. The immune response to M. paratuberculosis, the causative agent of Johne's disease, is poorly understood, though recent progress has begun to define parameters that might affect the susceptability of various animals to disease. This project will help to further define the response of cattle to infection with M. paratuberculosis. This infromation is critical to control programs and development of successful vaccines.
OBJECTIVES: The Molecular Pathogenesis Laboratory, Department of Animal Science at MSU is dedicated to understanding the relationship between host immune response and microbial associated pathogenesis in animals. Mycobacterial infections are responsible for major diseases of most animals, including humans. Mycobacteria hide within cells of the immune system (macrophages) and appear to direct host responses away from beneficial cell mediated immunity towards an ineffective production of antibody and a harmful chronic inflammatory response. The long-term objective addressed by work presented in this project is to help unravel some of the host immune events in infection of cattle with M. paratuberculosis, the causative agent of Johne's disease. Our specific objectives under this project are to: 1) Establish groups of Holstein calves that have been vaccinated with killed M. paratuberculosis, experimentally infected with live M. paratuberculosis, and control animals free of contact with M. paratuberculosis. 2) Monitor the response of various lymphocyte subsets to M. paratuberculosis antigens at weekly or biweekly intervals following vaccination or infection and 3) Determine the Th-1 and Th-2 cytokine profiles of lymphocytes and lymphocyte subsets following vaccination or infection.
APPROACH: Holstein calves will be vaccinated or experimentally infected with M. paratuberculosis (5 per group). Two additional calves will be held under similar conditions in a separate room to serve as controls. Following a 4 day acclimation period, initial blood samples will be drawn as a baseline for cell population, proliferation, and serology studies. Five calves will be infected with 2 gm wet weight of live M. paratuberculosis using gastric tubing. Five additional calves, housed separately, will be vaccinated with 400mg wet weight of killed M. paratuberculosis suspended in 1 ml of mineral oil. At 14 days post initial vaccination, animals will be given booster injections of 200mg sonicated M. paratuberculosis in the brisket and 200mg intradermally in the neck. Intragastric dosing of the infection group will also be repeated at this time. Blood samples from all animals will be collected at least once weekly following infection or vaccination and analyzed as described below. Fecal samples will be collected on a weekly basis from each animal and used to assay for presence of M. paratuberculosis by polymerase chain reaction (PCR) as well as by direct culture. Animals will be maintained for at least 10 months following treatment. Our proposed experimental design will allow a direct comparison of vaccination and experimental infection. This is necessary since M. paratuberculosis lives in, and can have profound effects on, bovine macrophages. Macrophages are an essential component of the immune system, expressing a number of cytokines that can affect and even direct the resulting immune response. Vaccination would not necessarily have the same effect on this important cell population as would infection with viable M. paratuberculosis. Cell subsets will be monitored by fluorescence activated flow cytometric analysis using commercially available antibodies directed against subset specific cell-surface markers. These makers are: CD4 (GC50A1); CD8 (CACT80C); IgM (B-cells); CD2 (all T-cells); and the gd-T cell marker N2 (BAQ4A). Cell subsets will be visualized using FITC conjugated secondary antibodies appropriate to the primary antibody type. To monitor cells proliferating in response to M. paratuberculosis antigens, we will employ a non-radioactive balstogenesis assay system (Chemicon International) based on cleavage of tetrazolium salt WST-1 (TZ-WST-1) to formazan by mitochondrial enzymes. Cytokine profiles of cells in response to M. paratuberculosis antigens will be analyzed from cells prepared as described above and incubated with M. paratuberculosis antigens for 24 hours under similar conditions. Cytokine profiles will be examined using quantitative competitive RT-PCR. Specific cytokines to be monitored include: Th-1 cytokines (IL-1, IL-2, IL-6, IFN-gamma, and IL-12); and Th-2 cytokines (IL-4, IL-5, and IL-10).
PROGRESS: 2000/10 TO 2005/09
This study began in an effort to identify host immune cell genes that respond to M. paratuberculosis antigens in naturally infected cows. In addition, we were interested in examining the gene expression patterns in intestinal tissues and mesenteric lymph nodes from infected cows versus patterns observed in uninfected controls. During the course of these studies, we completed a number of cDNA microarray studies that compared gene expression patterns in peripheral blood mononuclear cells (PBMCs) from M. paratuberculosis infected cows (Johnes disease positive) following stimulation with antigens from M. paratuberculosis. Gene expression patterns in intestinal tissues and lymph nodes were also examined by cDNA microarray. In addition, we completed a large-scale analysis of cytokine gene expression in PBMCs, gut lymph tissue, and lesions from cows with Johnes disease. For the microarray studies, we investigated the effect of antigen stimulation over time and compared the effect of antigen stimulation between cells from infected cows and healthy controls. These studies led to the surprising conclusion that stimulation with antigen was not necessary to detect gene expression differences in PBMCs from Johnes disease positive cows and healthy controls. These studies have also led to many novel observations
regarding the immune response to M. paratuberculosis and have indicated that novel genes, such as CIDE-A, MMP9, and MMP23 are important in immune responses to M. paratuberculosis. In addition, our results confirmed earlier observations that suggested that the cytokine IL-10 was important in immune responses to M. paratuberculosis. In fact, IL-10 was one of the few cytokine genes to be consistently activated by antigen stimulation of PBMCs when monitored by quantitative real time PCR (Q-RT-PCR). Our results in these areas were recently summarized and published as a review that established a new model for understanding immune responses to M. paratuberculosis.
IMPACT: 2000/10 TO 2005/09
Our gene expression profiling work has led to identification of numerous new targets for diagnosis of M. paratuberculosis infection in cattle. Continuation of this work proceeds using high-throughput Q-RT-PCR under funding from USDA-APHIS. In addition, our work has also identified IL-10 as one of the important cytokines produced by immune cells in response to M. paratuberculosis stimulation and has led to the hypothesis that regulatory T cells develop over the course of infection. This discovery is likely to impact current diagnostics, including IFN-gamma test and has implications for vaccine development.
PUBLICATIONS (not previously reported): 2000/10 TO 2005/09
1. Coussens, P. M., (2001). Interactions Between Mycobacterium paratuberculosis and the Bovine Immune System, Animal Health Research Reviews 2, 141-161.
2 . Yao, J., Burton, J. L., Saama, P., Sipkovsky, S., and Coussens, P. M., (2001). Generation of EST and cDNA Microarray Resources for the Study of Bovine Immunolobiology, Acta Vet Skandivica 42, 391-406.
3. Coussens, P. M., and Nobis, W., (2002). Bioinformatics and High-Throughput Approach to Create Genomic Resources for Study of Bovine Immunobiology, Veterinary Immunology and Immunopathology 86: 229-244
4. Coussens, P.M., C.J. Colvin, K. Wiersma, A. Abouzied, and S. Sipkovsky (2002). Gene Expression Profiling of Peripheral Blood Mononuclear Cells from Cattle Infected with M. paratuberculosis. Infection and Immunity 70:5494-5502.
5. Coussens, P.M, Colvin, C.H., Rosa, G., Perez Laspuir, J., and M. Elftman. (2003). Evidence for a Novel Gene Expression Program in Peripheral Blood Mononuclear Cells from M. paratuberculosis Infected Cattle. Infection and Immunity. 71(11), 6487-6498.
6. Aho, A.D., McNulty, A.M., and P.M. Coussens. (2003). Enhanced Expression of IL-1alpha and TRAF 1 in Ileal Tissues of Cattle Infected with Mycobacterium paratuberculosis. Infection and Immunity, 71(11), 6479-6486.
7. Coussens, P.M., A. Jeffers, and C.J. Colvin. (2003). Rapid and transient activation of gene expression in peripheral blood mononuclear cells of Johnes disease positive cows exposed to Mycobacterium paratuberculosis in vitro. Microbial Pathogenesis. 36(2):93-108.
8. Coussens, P.M., N. Verman, M.A. Coussens, M.D. Elftman, and A.M. McNulty. (2004). Cytokine Gene Expression in Peripheral Blood Mononuclear Cells and Tissues of Cattle Infected with Mycobacterium avium subspecies paratuberculosis: Evidence for an Inherent Pro-inflammatory Gene Expression Pattern. Infection and Immunity, 72(3):1409-1422.
9. Coussens, P.M. 2004. A Model for Immune Responses to Mycobacterium paratuberculosis. Infection and Immunity (Minireviews), 72: 3089-3096.
10. Coussens, P.M., C. B. Pudrith, X. Ren, S. P. Suchyta, J. R. Stabel, K. Skovgaard, and P. M.H. Heegaard. 2005. Johnes disease in cattle is associated with enhanced expression of genes encoding IL-5, GATA-3, tissue inhibitors of matrix metalloproteinases 1 and 2, and factors promoting apoptosis in peripheral blood mononuclear cells. Veterinary Immunology and Immunopathology. 105: 221-234.
PROJECT CONTACT:
Name: Coussens, P. M.
Phone: 517-353-3158
Fax: 517-353-1699
Email: coussens@msu.edu

 
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ACCESSION NO: 0188199 SUBFILE: CRIS
PROJ NO: MICL07664 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2001-34427-10444 PROPOSAL NO: 2001-03570
START: 01 JUL 2001 TERM: 30 JUN 2004 FY: 2004 GRANT YR: 2001
GRANT AMT : $303,339
INVESTIGATOR: Kaneene, J. B.; Fitzgerald, S. D.; Bartlett, P. C.; Griffore, F.; Bolin, S.; Bolin, C.
PERFORMING INSTITUTION:
Population Medicine Center
Michigan State Univ
East Lansing , Michigan 48824
EPIDEMIOLOGY AND RISK ANALYSIS OF MYCOBACTERIUM BOVIS IN WILD AND DOMESTIC ANIMALS IN MICHIGAN.
NON-TECHNICAL SUMMARY: Bovine tuberculosis (TB) in Michigan is being recognized as more prevalent in wildlife and livestock than originally thought. We have seen bovine TB in beef and dairy cattle and in a number of wildlife species. The current combination of active and passive surveillance programs, as well as control and eradication efforts, have shown that more research is needed to provide information needed to deal with the problem. The first focus of these projects is to identify factors that will influence whether a cattle herd will develop TB. Next, these projects will look at the effectiveness of tests used to detect TB in cattle. Finally, the projects will look at the impact of bovine TB on farm families and communities in the TB-affected area.
OBJECTIVES: Specific objectives are to: 1) conduct epidemiological studies to determine major risk factors influencing TB transmission in livestock and deer, 2) refine and validate preliminary risk analysis models, 3) evaluate the effect of M. paratuberculosis status on the reliability of the CFT in cattle, 4) use new risk analysis approaches to estimate the rate of false positives on the CFT and CCT, 5) determine whether wild rodents are possible TB reservoirs, and 6) determine the social impact of bovine TB on farm families and farm communities.
APPROACH: Objective 1: A retrospective epidemiological study will examine the association between M. bovis infection and physical landscape factors. Environmental samples will be taken from livestock operations with confirmed M. bovis infections and processed for bacterial isolation, identification, and typing. A cross-sectional study will identify different wildlife species with bovine TB, determine the most likely routes of infection for each species, and estimate the potential of each species to be hosts for M. bovis by describing pathogenicity and assessing the possibility of shedding through different routes. A retrospective epidemiological analysis will be conducted to examine the association between the occurrence of M. bovis on farms with M. bovis in deer, deer-related supplemental feeding, and physical landscape factors. Objective 2: A stochastic simulation model, using cattle herd factors, deer factors, deer feeding factors, and land use factors, will be developed to estimate the risk of a dairy or beef cattle herd developing TB infection in a year. Objective 3: From dairy herds in the Michigan Johne's Control Program, three herds will be selected: one high prevalence herd (> 15%); one low prevalence herd (< 2%); and one Johne's-free herd. Each animal will receive a bovine TB caudal fold test, comparative cervical test, and gamma-interferon tests, and TB test results will be compared by Johne's disease status. Objective 4: A database and data entry template for infected Michigan cattle herds will be developed for CFT and CCT data. Risk analysis software will be used to generate frequency distributions of tuberculosis prevalence in different sub-populations of domestic ruminants. Risk analysis software will be used to generate frequency distributions of rates of false positives and negatives, and true positives and negatives, from the Michigan TB testing program for livestock. Objective 5: M. bovis of deer-origin from Michigan will be used to inoculate prairie voles by oral gavage and intranasal installation. Groups of oral-inoculated, nasal-inoculated, and control rodents will be euthanized at days 30 and 60 post-inoculation. Necropsies, histopathology, acid-fast staining and mycobacterial isolation will be evaluated for rodent susceptibility to infection with M. bovis and ability to shed the organism. Objective 6: Follow up interviews with farm families affected by bovine TB in northeastern Michigan will be conducted to examine patterns of adaptive behavior and long-term impact of bovine TB on farm families. Families with new TB herds, and farmers in the area who have not yet been directly affected by TB will be interviewed. Interviews have been conducted with members of various stakeholder groups, and a subset of these interviewees will be contacted to aid in clarification of the results. A public opinion survey will collect attitudes about the Michigan TB situation from key personnel in state-level agricultural and natural resource agencies across the US. Data collected by the questionnaire will be evaluated to assess respondents' reactions to the TB situation and areas where additional information or education are needed.
PROGRESS: 2001/07 TO 2004/06
The first aim of this project was to determine the spatial relationships of bovine TB (bTB) in white-tailed deer, relating to factors in the physical landscape and location-specific human activity. Spatial clusters of TB were detected in areas that encourage deer to congregate for long periods of time. One paper has been submitted for publication. To identify factors that may influence whether a cattle herd will develop TB, a matched case-control study of herds was conducted to identify herd management factors and environmental conditions associated with TB (Kaneene et al., 2002). A stochastic risk assessment model for herd TB status was developed, based on results of this study, and has been integrated with economic data to create a management tool to develop recommendations to reduce the TB risk for individual cattle farms. The on-farm program is undergoing field testing. The study on the effect of Johnes disease (JD) on the caudal fold tuberculin test (CFT) in herds without TB was completed and submitted for publication. Fecal culture and antibody ELISA for M. avium ssp. paratuberculosis, were performed on cattle from 10 herds. Blood samples were taken and subjected to gamma-interferon (GI) tests for M. bovis and JD. Cattle positive for JD by fecal culture, ELISA, or GI appear to be more likely to be false + on CFT than were negative cattle, and no associations were found between + fecal culture or ELISA with GI for M. bovis. To determine the effectiveness of current TB testing in Michigan cattle, cattle from TB-infected herds were examined by gross necropsy, histopathologic exam, mycobacterial culture, and PCR. Bayesian inference was used to estimate the sensitivity and specificity of the CFT and comparative cervical tuberculin test (CCT) using two-population-two-tests latent-class models. Bayesian estimates of the sensitivity and specificity of the CFT and CCT were 85 and 94%, and 76 and 99%, respectively, which agrees with reports from other studies in the U.S. Two papers have been submitted for publication. To evaluate the role of rodents as possible reservoirs of M. bovis, an experimental study was conducted to study the relative susceptibility of 'wild-type' rodents to inoculation with M. bovis by the oral and intranasal routes. M. bovis was cultured from the feces of 9 oral inoculates and 8 intranasal inoculates on day 1 post-inoculation, and from fecal samples of 3 intranasal inoculates at day 30 post-inoculation, and also from pooled tissue samples. Results of this study are being prepared for publication. In-person interviews were conducted with farm families to measure the social impacts of bTB, and study findings indicate that families can adapt to the changes imposed by the presence of TB on their farms, but they experienced problems in receiving information in a timely fashion, and inconsistency and inequity in the application of government policies and procedures. To lessen negative impacts, families should be accorded more attention and consideration when policies are made, and should have a more substantial role in decision-making as it relates to their own farms. Two manuscripts are being submitted for publication.
IMPACT: 2001/07 TO 2004/06
Control and eradication of TB from the Michigan livestock industry requires an understanding of the disease and how it spreads, efficient disease detection methods, and the development of tools for control of the disease at the farm level. Risk assessment models can be used to develop sound, cost-effective disease control programs. The reliability of the caudal fold skin test, used for TB testing in livestock, may be affected by an animal's disease or vaccination status. These factors should be taken into consideration when interpreting TB skin test results, or designing a TB surveillance program. Current TB tests require great time, effort and expense, and false results are common with existing skin and blood tests. Even with TB lesions, many times associated with acid-fast bacilli, bacteria cannot be cultured because of poor samples, freeze-thaw, or lack of available fresh tissues. New DNA-based testing methods (cDNA microarray analysis of gene expression and laser-capture microscopy for isolating DNA for PCR) have the potential to become rapid, sensitive methods to identify TB in tissue samples in an efficient and reliable way. With a better understanding of the stresses and social impacts of bovine TB on Michigan farm families, programs can be designed to reduce the negative impact of TB control and surveillance programs on families' lives.
PUBLICATIONS (not previously reported): 2001/07 TO 2004/06
1. Dunn, J.R., Kaneene, J.B., Grooms, D.L., Bolin, S.R., Bolin, C.A., Bruning-Fann, C.S. Testing positive for Mycobacterium avium subsp. paratuberculosis and the lack of significant effect on the caudal fold tuberculin (CFT) and gamma interferon tests for bovine tuberculosis. Am J Vet Res, accepted for publication 2004.
2. Fitzgerald, S.D., Boland, K.G., Clarke, K.R., Wismer, A., Kaneene, J.B., Berry, D.E., Church, S.V., Hattey, J.A., C.A. Bolin. Experimental Inoculation of Mallard Ducks (Anas platyrhynchos) with Mycobacterium bovis. Avian Dis. submitted 2004.
3. Griffore, R., Phenice, L. The Impact of Bovine TB on the Farm Family Ecosystem. In preparation for submission to Family Relations, 2004.
4. Griffore, R., Phenice, L; Kaneene, J.B. Veterinarians: A Complex Role of Mediation Between the State and Farm Families. In preparation for submission to Journal of the American Veterinary Medical Association, 2004.
5. Miller, R., Kaneene, J.B., Schmitt, S.M., Lusch, D.P., Fitzgerald, S.D. Geographic distribution and spatial analysis of Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus) in Michigan. Prev Vet Med. accepted 2004.
6. Norby, B., Bartlett, P.C., Fitzgerald, S.D., Granger, L., Bruning-Fann, C., Whipple, D.L., J.B. Payeur. The Sensitivity of Gross Necropsy, Caudal Fold and Comparative Cervical Tests for the Diagnosis of Bovine Tuberculosis. J Vet Diagn Invest. 16:126-131, 2004.
7. Norby, B., Bartlett, P.C., Grooms, D.L., Kaneene, J.B., Bruning-Fann, C.S. Herd-level sensitivity, specificity, and predictive values of bovine tuberculosis skin tests in Michigan. Am J Vet Res. submitted 2004.
8. Norby, B. Tempelman, R.J., Hansonc, T.E., Kaneene, J.B., Bartlett, P.C. Estimation of sensitivity and specificity of bovine tuberculosis skin tests in Michigan when a perfect reference test is not available. Prev Vet Med. submitted 2004.
9. O'Brien, D.J., Schmitt, S.M., Berry, D.E., Fitzgerald, S.D., Vanneste, J.R., Lyon, T.J., Church, S.V., Fierke, J.S., Schooley, A.M., Cooley, T.M., Magsig, D., Zwick, L., and B.V. Thomsen: Estimating the True Prevalence of M. bovis in Hunter-harvested White-tailed Deer in Michigan. J Wildl Dis. 40:42-52, 2004.
PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-353-5941
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu

 
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ACCESSION NO: 0191695 SUBFILE: CRIS
PROJ NO: MICL07676 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2002-34427-11829 PROPOSAL NO: 2002-06051
START: 01 SEP 2002 TERM: 31 AUG 2004 FY: 2006 GRANT YR: 2002
GRANT AMT: $297,445
INVESTIGATOR: Kaneene, J. B.; Fitzgerald, D. D.; Bolin, S. R.; Bartlett, P. C.; Bolin, C. A.
PERFORMING INSTITUTION:
Population Medicine Center
Michigan State Univ
East Lansing , Michigan 48824
BOVINE TUBERCULOSIS: EPIDEMIOLOGY, DIAGNOSIS, AND PATHOGENESIS.
NON-TECHNICAL SUMMARY: Since bovine TB control programs for livestock have significant costs, there is a need to improve the performance of these programs. The proposed research will evaluate the costs of current TB control programs, the reliability of TB tests being used, and factors that influence the effectiveness of these programs. In addition, the study will try to identify new, more efficient methods of identifying bovine TB in cattle.
OBJECTIVES: There are six projects with specific objectives: Project 1 Objective: to develop a predictive risk assessment model, combining epidemiological and economic risk factors, for the effects of bovine TB infection on the Michigan cattle industry; Project 2 Objectives: 1) determine the sensitivity, specificity and predictive values and reliability of the CFT and the gamma-interferon blood test in Johne's-positive cattle herds, using the comparative cervical skin test (CCT) and mycobacterial culture for validation; 2) compare results of the CFT from Johne's-positive cattle with results from Johne's-negative cattle, using the CCT and mycobacterial culture for validation; and 3) to determine if cattle with advanced Johne's Disease are immunologically competent to respond appropriately to M. bovis antigens; Project 3 Objectives: 1) study the relative susceptibility of wild-type rodents to oral inoculation with M. bovis; 2) attempt to isolate M. bovis from inoculated rodents through fecal culture at multiple times post-inoculation, and from various organs at necropsy; 3) evaluate various tissues microscopically at multiple times post-inoculation to understand the pathogenesis of M. bovis in these rodents; and 4) evaluate rodents for the threat they pose in re-introduction of M. bovis to cattle farms, and their potential as sentinel species for infection on farms; Project 4 Objectives: 1) construct probability distributions of the prevalence of bovine TB in different sub-populations of Michigan domestic ruminants; 2) construct probability distributions of the false and true positives and false and true negatives resulting from the current serial testing (CFT/CCT) program in Michigan; and 3) estimate the rates of human injuries and deaths suffered during the bovine TB testing program in Michigan; Project 5 Objectives: 1)
identify altered gene expression for bovine cytokines in cattle sensitized to M. bovis, using lymphocytes stimulated in vitro with purified protein derivative (PPD) from M. bovis or M. avium; and 2) identify additional genes from bovine lymphocytes that show altered expression attributable to exposure with M. bovis, using a cDNA microarray made from bovine lymphocytes and mRNA from lymphocytes stimulated in vitro with PPD from M. bovis or M. avium.
APPROACH: Project 1: Herd-based risk assessment models and models of TB in wild white-tailed deer in northeastern Michigan will be used to develop industry-level predictive models of levels of TB and its economic consequences to the state's cattle industry, so that the impact of herd-level or industry-level TB control measures, and their attendant costs, can be projected over periods of time and be used to determine which measures would be most efficient and cost-effective for the industry. Project 2: Objectives 1 & 2: To test effect of Johne's Disease (JD) on the caudal fold and comparative cervical skin tests and the gamma-interferon test for Mycobacterium bovis, cattle from dairy herds with no/low/high levels of JD will have TB skin tests administered. Blood and fecal samples will be collected to compare animal JD status (by fecal culture, ELISA and gamma-interferon for M. paratuberculosis) with results of TB skin tests and gamma-interferon testing. Objective 3: TB-negative cattle with and without JD will be injected with killed M. bovis antigen in a dose response study to compare the immune response of cattle with and without JD to respond to the M. bovis antigen. Project 3: Norway rats and wild rodents will be orally inoculated with high and low doses of M. bovis, with some sham-inoculated controls. Fecal cultures and body weights will be collected throughout the study. Groups will be euthanatized at different intervals, and results of gross necropsy, histopathology, acid-fast staining and bacterial culture will be assessed. Project 4: Objectives 1 & 2: Probabilistic risk assessment models will be developed to assess the prevalence of bovine TB on cattle operations in Michigan. Risk analysis software will be used to generate frequency distributions of rates of false-positives and -negatives from the Michigan TB livestock testing program. Objective 3: A survey on worker injuries will be administered to a random sample of the veterinarians involved with TB testing. Project 5: Objective 1: Whole blood will be collected from calves sensitized to M. bovis, and stimulated by incubation with PPD made from M. bovis or M. avium. Lymphocytes will be harvested and total cellular RNA will be obtained. RT-PCR and a real-time PCR detection system will be used to monitor up- and down-regulation, and altered product-ratios for bovine cytokines. Results from sensitized cattle will be compared to results from animals with natural infection. Objective2: Available cDNA microarrays made from bovine lymphocytes will be used to identify genes that are up-regulated or down-regulated after in vitro exposure of lymphocytes with either M. bovis or proteins derived from M. bovis. Total cellular RNA will be harvested from lymphocytes after stimulation with antigen, and reverse transcribed using an oligo (dT)15 primer to incorporate aminoallyl-modified dUTP into the single strand product. The fluorescent-labeled probe cDNAs will be hybridized to microarrays, the microarrays will be washed several times, dried, and scanned to create reports of spot intensity ratios to identify genes that have altered levels of expression.
PROGRESS: 2002/09 TO 2004/08
Project 1: A stochastic risk assessment model for herd TB status was developed, based on results from a case-control study to identify herd management factors and environmental conditions associated with TB status. A method to examine trade-offs between expected benefits and expected costs of biosecurity management practices and investments was developed. The model is being updated with additional data from over 10 new TB-positive herds, and is being implemented in a form for on-farm use. Project 2: A prospective study was designed to determine if cattle infected with Mycobacterium. avium subsp. paratuberculosis (JD) have a higher proportion of false positive caudal fold tuberculin test (CFT) results for TB when compared to uninfected cattle. Blood and fecal samples from 1043 cattle were subjected to M. bovis and M. avium gamma-interferon (INF-gamma) and JD antibody ELISA testing, and fecal culture. The overall false positive rate on the CFT was 17%. The high (>15%) and low (<15%) prevalence herds averaged 21 and 14% positive on the CFT, respectively, and 32 and 19% of JD positive (+) cows from high and low prevalence herds, respectively, were CFT positive. These results indicate an association between JD disease and false positive CFT. Project 3: The experimental infection study of wild house mice was completed, and mice were found to be highly susceptible to M. bovis and may pose a real threat to infected farms that are depopulated and later repopulated. The final analysis, combining results of several studies, shows that voles were the most susceptible to infection, mice were highly susceptible, and rats being highly resistant to both infection and shedding. A manuscript for publication comparing and summarizing these findings is in preparation. Project 4: The sensitivities of the CFT, CFT and comparative cervical tests (CCT) in series, and gross necropsy were 93, 88, and 86%, respectively. Sensitivities of skin tests were slightly higher when at least 2 lesions were found at gross necropsy. If 1 TB+ animal is enough to declare a herd TB+ and the tests used to classify a single animal as TB+ has a specificity of 1.0, the herd level performance of the TB skin tests in Michigan is very good. When prevalence is low, herd level sensitivity is correlated with TB prevalence and the size of the tested herd. Herd negative predictive value is very high and decreases slightly when herd size decreases. These results show that attention should be paid to smaller herds to meet the goal of TB eradication. Project 5: Whole blood from 5 false-positive CCT reactors, 1 lesion+ animal, 1 INF-gamma reactor, and 1 cow sensitized with sensitinogen was stimulated with bovine PPD before harvest of total cellular RNA for determination of levels of cytokine gene expression compared with levels of 2 housekeeping genes. There was an increase in gene expression for 6 cytokines and INF-gamma, and decreased expression of IL-4 in the CCT reactors. The lesion+ animal showed an increase in expression of TNF-alpha, and IL-10 or INF-gamma. Additional RNA has been collected for further study, from a TB positive herd and a negative herd located outside of the TB endemic area.
IMPACT: 2002/09 TO 2004/08
Control and eradication of TB from the Michigan livestock industry requires an understanding of the disease and how it spreads, efficient disease detection methods, and the development of tools for control of the disease at the farm level. Risk assessment models can be used to develop sound, cost-effective disease control programs. The reliability of the caudal fold skin test, used for TB testing in livestock, may be affected by an animal's disease or vaccination status. These factors should be taken into consideration when interpreting TB skin test results, or designing a TB surveillance program. Current TB tests require great time, effort and expense, and false results are common with existing skin and blood tests. Even with TB lesions, many times associated with acid-fast bacilli, bacteria cannot be cultured because of poor samples, freeze-thaw, or lack of available fresh tissues. New DNA-based testing methods (cDNA microarray analysis of gene expression and laser-capture microscopy for isolating DNA for PCR) have the potential to become rapid, sensitive methods to identify TB in tissue samples in an efficient and reliable way. Determining what wildlife species can serve as reservoirs of M. bovis is fundamental to understanding the epidemiology of TB, which is necessary to develop effective disease eradication programs
PUBLICATIONS (not previously reported): 2002/09 TO 2004/08
No publications reported this period
PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-355-2269
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu

 
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ACCESSION NO: 0195202 SUBFILE: CRIS
PROJ NO: MICL07681 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2003-34427-13292 PROPOSAL NO: 2003-06030
START: 01 JUL 2003 TERM: 30 JUN 2006 FY: 2007 GRANT YR: 2003
GRANT AMT: $323,193
INVESTIGATOR: Kaneene, J. B.; Fitzgerald, S. D.; Grooms, D.; Bolin, S. R.; Bolin, C. A.; Wolf, C. A.; Fine, A.
PERFORMING INSTITUTION:
Large Animal Clinical Sciences
Michigan State Univ
East Lansing , Michigan 48824
BOVINE TUBERCULOSIS: EPIDEMIOLOGY, DIAGNOSIS, AND PATHOGENESIS.
NON-TECHNICAL SUMMARY: Since bovine TB control programs for livestock have significant costs, there is a need to improve the performance of these programs. The proposed research will evaluate the costs of current TB control programs, the reliability of TB tests being used, and factors that influence the effectiveness of these programs. In addition, the study will try to identify new, more efficient methods of identifying bovine TB in cattle.
OBJECTIVES: There are four projects with specific objectives. Project 1 Objectives: 1) to validate the epidemiological and economic risk assessment models for a cattle herd becoming infected with bovine tuberculosis; and 2) develop a user-friendly software package to predict a herd's risk for the bovine tuberculosis, and estimate the costs associated with changing management practices identified as contributing to the herd risk for bovine tuberculosis. Project 2 Objectives: 1) determine the level of association between false positive rates in the caudal fold skin tests and levels of M. paratuberculosis infection in the cattle herd; 2) determine the incidence of M. paratuberculosis infection in cattle that are classified as suspects or reactors by the comparative cervical tuberculin test; 3) determine whether the administration of a vaccine containing a modified-live bovine viral diarrhea virus changes the response to the caudal fold tuberculin test or a-interferon test for bovine tuberculosis; and 4) determine whether the administration of a new L. borgpetersenii serovar Hardjo bacterin changes the response to the caudal fold tuberculin test or a-interferon test for bovine tuberculosis. Project 3 Objectives: 1) compare patterns of gene expression in WBC RNA from comparative cervical suspects or reactors from tuberculosis free herds, and cattle naturally infected with M. bovis; 2) optimize the DNA extraction process and the PCR reaction conditions to increase the sensitivity for detection of M. bovis in lesions that lack observable acid fast organisms; 3) develop and standardize DNA extraction and PCR techniques for detection of M. bovis in known positive animal tissues; 4) compare the sensitivity and specificity of this new technique to existing PCR on formalin-fixed tissue techniques; and 5) determine the utility of this new technique for wildlife and domestic animal tuberculosis surveillance, as well as experimental inoculation study application. Project 4 Objectives: 1) use bacteriologic culture and DNA-based testing techniques to identify M. bovis in environmental substrates from TB-affected cattle farms and areas with identified clusters of TB-infected white-tailed deer; 2) assess the effect of environmental conditions (humidity, temperature, and light) on the probability and duration of M. bovis survival in the environment; 3) study the relative susceptibility of mallard ducks to oral and intra-tracheal inoculation with M. bovis and attempt to isolate M. bovis from inoculated birds through fecal culture at multiple times post-inoculation, and from various organs at necrospy; 4) evaluate various tissues microscopically at multiple times post-inoculation to understand the pathogenesis of M. bovis in these birds; and 5) evaluate wild bird species for the threat they pose in re-introduction of M. bovis to cattle farms.
APPROACH: Project 1: Herd-based risk assessment models and economic models of TB in cattle herds in northeastern Michigan will be used to develop predictive models for a herd's risk for TB and its economic consequences, so that the impact and costs of herd-level TB control measures can be projected over periods of time and be used to determine which measures would be most efficient and cost-effective. Project 2: Objectives 1-2: Cattle from dairy herds with no/low/high levels of Johne's Disease (JD) will receive caudal fold skin tests (CFT). Blood and feces will be collected to compare animal JD status with results of CFT and a-interferon testing. Objective 3: Calves free of BVDV will be sensitized to M. bovis, vaccinated with a modified-live virus vaccine containing attenuated BVDV, bovine herpesvirus-1, bovine respiratory syncytial virus, and parainfluenza-3. The CFT will be given after vaccination, and comparisons made between vaccinated and unvaccinated calves. Objective 4: Calves free of Leptospira will be sensitized to M. bovis, vaccinated with a monovalent serovar Hardjo vaccine, and given CFTs after vaccination. Comparisons will be made between CFT results from sensitized and unsensitized calves. Project 3: Objectives 1-2: Whole blood collected from calves sensitized to M. bovis will be stimulated with M. bovis or M. avium PPD to obtain total cellular RNA. RT-PCR and real-time PCR will monitor up- and down-regulation, and altered product-ratios for bovine cytokines. Available cDNA microarrays made from bovine lymphocytes will be used to identify genes that are up- or down-regulated after in vitro exposure of lymphocytes with M. bovis or proteins derived from M. bovis. Objectives 3-5: Extraction of DNA and PCR will be used on sections of formalin fixed, paraffin embedded tissue with microscopic lesions consistent with TB. A laser capture microsdissection system will be used to dissect out microscopic granulomatous lesions for capture using HS Caps, DNA will be extracted, and real-time PCR will be conducted. Project 4: Objective 1: Soil, hay, and water samples will be inoculated with M. bovis. Processed samples will be analyzed for the presence of M. bovis by culture and PCR, and the most efficient method for processing environmental samples will be determined. Objectives 2-3: Locations on TB-infected cattle farms will be identified and samples of feces, feeds, open water, and pasture grass will be taken. Similar samples will be taken from sites in areas with high TB-prevalence in wildlife. Analysis for M. bovis will be done by culture, DNA probe, and IS6110 primer-based PCR. Objectives 3-5: Mallard ducks will be orally inoculated with high and low doses of M. bovis, with some sham-inoculated controls. Fecal cultures and body weights will be collected throughout the study. Groups will be euthanatized at different intervals, and results of gross necropsy, histopathology, acid-fast staining and bacterial culture will be evaluated.
PROGRESS: 2003/07 TO 2006/06
Project 1: The test version of a user-friendly software package to predict herd risk for TB, identify conditions on the farm associated with increasing TB risk, and estimate the economic costs associated with changing management practices identified as contributing to the herd risk for TB for on-farm use is undergoing field-testing and refinement, and the predictive risk assessment model for a herd becoming reinfected with TB after depopulation is being completed. Project 2: Four groups of 9 cattle each were sensitized with antigen from M. bovis to stimulate an immune response that would mimic natural infection when the cattle were tested for tuberculosis, using currently approved testing methods. One group was vaccinated with a modified live virus commonly used to control respiratory pathogens, another group was vaccinated with a commonly used inactivated bacterin for Leptospirosis, and 2 groups served as nonvaccinated controls. Neither vaccine affected the caudal fold test (CFT), and only the respiratory vaccine negatively affected the whole blood gamma interferon assay for tuberculosis. Project 3: Blood was collected 2 TB positive cattle, 5 skin test positive/lesion negative cattle, and 1 cow experimentally sensitized with inactivate antigen from M. bovis to determine if using cDNA microarrys to analyze gene regulation in response to stimulation of white blood cells with antigens derived from M. bovis would identify gene targets for secondary testing for TB. Altered gene expression patterns among cattle were detected using a cDNA microarry created from differentially expressed genes in bovine lymphocytes, but no clear targets were found. To determine if M. avium ssp paratuberculosis (MAP) infection affects currently used tests for TB, formalin fixed, paraffin embedded tissues from 300 cattle were tested for DNA from MAP. The cattle were lesion negative for TB on post mortem examination and tested as TB suspects on the CFT or were CFT negative. MAP was not associated with a positive reaction on the CFT. Project 4: Methods for processing environmental samples capable of detecting small numbers of M. bovis were established. Based on a cross-sectional study of environmental substrates collected on TB-affected cattle farms, M. bovis was not isolated from any samples (soil, water, feed) collected from 13 TB-affected cattle farms and 5 wildlife areas with known TB. The study to determine the effect of substrate (water, soil, hay, grain) and environmental conditions (humidity, temperature, sunlight) on the persistence of viable M. bovis in the environment found that M. bovis can persist for 6-10 weeks in cooler seasons. These results are being prepared in 3 papers for publication in scientific journals. Mallard ducks are highly resistant to oral infection with TB, and do not shed the organism in feces. Results of this study were published in 2005.
IMPACT: 2003/07 TO 2006/06
Control and eradication of TB from the Michigan livestock industry requires an understanding of the disease and how it spreads, efficient disease detection methods, and the development of tools for control of the disease at the farm level. Risk assessment models can be used to develop sound, cost-effective disease control programs. The reliability of the caudal fold skin test, used for TB testing in livestock, may be affected by animal disease or vaccination status. These factors should be taken into consideration when interpreting TB skin test results, or designing a TB surveillance program. Current TB tests require great time, effort and expense, and false results are common with existing skin and blood tests. Even with TB lesions, many times associated with acid-fast bacilli, bacteria cannot be cultured because of poor samples, freeze-thaw, or lack of available fresh tissues. New DNA-based testing methods (cDNA microarray analysis of gene expression and laser-capture microscopy for isolating DNA for PCR) have the potential to become rapid, sensitive methods to identify TB in tissue samples in an efficient and reliable way. Determining where in the environment and ecosystem M. bovis exists, and the length of time it survives and remains infective, is fundamental to understanding the epidemiology of TB, which is necessary to develop effective disease eradication programs. Experiments have shown that some wild bird species may contribution to the maintenance and spread of TB in wildlife and livestock.
PUBLICATIONS (not previously reported): 2003/07 TO 2006/06
Fine, A.E. The role of indirect transmission in the epidemiology of bovine tuberculosis in cattle and white-tailed deer in Michigan. Ph.D. Thesis, Michigan State University, East Lansing, Michigan, 2006.
PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-353-5941
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu


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ACCESSION NO: 0199414 SUBFILE: CRIS
PROJ NO: MICL07692 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO : 2004-34427-14585 PROPOSAL NO: 2004-06033
START: 01 SEP 2004 TERM: 31 AUG 2007 FY: 2007 GRANT YR: 2004
GRANT AMT: $288,804
INVESTIGATOR: Kaneene, J. B.; Fitzgerald, S. D.; Bolin, S. R.; Grooms, D. L.; Bolin, C. A.
PERFORMING INSTITUTION:
Large Animal Clinical Sciences
Michigan State Univ
East Lansing , Michigan 48824
BOVINE TUBERCULOSIS: EPIDEMIOLOGY, DIAGNOSIS, AND PATHOGENESIS.
NON-TECHNICAL SUMMARY: Since bovine TB control and prevention programs for livestock have significant costs, there is a need to improve the performance of these programs. The proposed research will evaluate current TB control and prevention programs, the reliability of TB tests being used, factors that influence the effectiveness of these tests and programs, and the efficacy of two TB vaccines for animals.
OBJECTIVES: There are four projects with specific objectives. Project 1 Objectives: 1) Identify risk factors associated with reacquiring TB after repopulation, including factors associated with herd biosecurity, cattle movement to and from affected herds, cattle feeding practices, cattle housing, and wildlife access to livestock and livestock feed; 2) Evaluate the spatial relationship between risk for reacquiring TB and proximity to other herds affected by TB, deer habitat, surface water features, and other ecological features in the region; and 3) Determine association between herd TB status and herd-related management factors, geographic location of environmental conditions, proximity to TB positive herds, levels of TB in deer. Project 2 Objectives: 1) Determine the level of association between false positive rates in the caudal fold skin tests (CFT) and levels of M. paratuberculosis infection in the cattle herd; and 2) Determine the prevalence of M. paratuberculosis infection in cattle that are classified as suspects or reactors by the comparative cervical tuberculin test (CCT); 3) determine whether the administration of a vaccine containing a modified-live bovine viral diarrhea virus changes the response to the caudal fold tuberculin test or gamma interferon test for bovine tuberculosis; 4) determine whether the administration
of a new L. borgpetersenii serovar Hardjo bacterin changes the response to the caudal fold tuberculin test or gamma-interferon test for bovine tuberculosis; 5) Retrospectively apply PCR to formalin-fixed, paraffin-embedded sections of ileum and determine the prevalence of M. paratuberculosis infected cattle that falsely test as suspects or reactors by 1) the CFT, 2) the CCT, and 3) cattle that are skin test negative; and 6) Compare resulting prevalences of false reactors by the two skin tests with the negative test population as a negative control to determine effect of Johnes infection on test results. Project 3 Objectives: 1) Identify targets of altered gene expression after 0, 1, 2, 4 or 20 hours of antigen stimulation to determine optimal time for whole blood stimulation; and 2) compare patterns of gene expression in WBC RNA from comparative cervical suspects or reactors from tuberculosis free herds, and cattle naturally infected with M. bovis. Project 4 Objectives: 1) Evaluate BALB/c mice vaccinated with a RB51 vector vaccine or a BCG vaccine over several months after challenge with deer-origin M. bovis for fecal shedding of the organism, clinical signs, and terminal necropsy for histologic evaluation and mycobacterial isolation and titration; and 2) Compare results with unvaccinated, M. bovis-challenged mice to evaluate vaccine efficacy in increasing disease resistance, decreasing lesion development and mycobacterial organism replication, and controlling shedding of M. bovis.
APPROACH: Project 1, Part A: A case-control study will compare risk factors (farm location, ecological data, herd management practices before and after restocking) between herds diagnosed with TB that were depopulated and repopulated (controls) with repopulated herds that have become reinfected with TB (cases). Project I, Part B: A retrospective case-control study will compare spatial risk factors between herds diagnosed with TB since the beginning of the current disease outbreak (cases) and herds that have not been diagnosed with TB (controls) from the five county area. Case herds will be those. Risk factors include livestock housing location data (located by Global Positioning Satellite (GPS) systems), specific ecological conditions in cattle housing areas, and herd management practices. Project 2, Part A: The effect of infection with M. paratuberculosis on results of the bovine TB caudal fold test (CFT) test and gamma interferon assay will be assessed by comparing the false negative rate for these two assays in Johnes disease high prevalence dairy herds and herds known to be free of Johnes disease. Project 2, Part B: The effect of use of select veterinary vaccines on the reliability of the CFT for TB in cattle will be tested by comparing CFT test results and M. bovis gamma interferon production between groups of calves sensitized to M. bovis that are unvaccinated or vaccinated with a commercially available vaccine containing modified-live bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis (IBR), PI-3 and bovine respiratory syncital virus (BRSV) (Pyramid, Ft. Dodge Animal Health, Fort Dodge, IA). Project 2, Part C: A retrospective study of the role of M. paratuberculosis in false reactor response on the CFT and the comparative cervical test (CCT) will compare levels of M. paratuberculosis between CFT positive/M. bovis culture negative cattle and CFT negative cattle by PCR for M. paratuberculosis from sections of ileum and ileal-cecal lymph nodes. Project 3, Part A: The optimal time of exposure of whole blood to bovine ppd for detection of diagnostic gene targets in caudal fold suspect cattle will be determined by comparing levels of altered expression of 10 genes after 0, 1, 2, 4 or 20 hours of antigen stimulation, between cattle that have been sensitized to M. bovis and cattle that have not been sensitized. Project 3, Part B: Optimal gene targets for detection of bovine tuberculosis will be identified by stimulating blood with phosphate-buffered saline (PBS) (negative control), M. avium purified protein derivative (PPD), and M. bovis PPD, and harvesting total cellular RNA to generate cDNA for use in real-time quantitative polymerase chain reaction (RT-QPCR). Project 4: The efficacy of BCG vaccine and a recombinant vector vaccine (RB-51) for M. bovis in laboratory mice will be tested by comparing lesion development and mycobacterial isolation results from vaccinated and unvaccinated mice challenged with deer-origin M. bovis.
PROGRESS: 2004/09 TO 2007/08
Project 1: The predictive risk assessment model for reacquiring TB and proximity to other herds affected by TB, deer habitat, surface water features, and other ecological features in the region has been finalized. Updated information on data from newly infected cattle herds (biosecurity, cattle movement to and from affected herds, cattle feeding practices, cattle housing, and wildlife access to livestock and livestock feed) and additional retrospective data collection was used to refine the existing predictive model, which is undergoing testing. Project 2: Dairy cattle with Johne's disease (JD, infection with M. avium ssp. paratuberculosis [n=11]) and age-matched cattle without JD (n=8) were sensitized with antigen from M. bovis to stimulate an immune response that would cause the cattle to test positive for TB using currently approved testing methods. JD did not affect the result of the caudal fold test (CFT), as all sensitized cattle showed a positive reaction, but did adversely affect the results of the whole blood gamma interferon test in some cattle. Project 3: To identify altered gene expression patterns in cattle that are false positive on current TB tests as potential diagnostic targets for TB detection, whole blood was collected from 60 lesion negative cattle suspect for TB on the CFT and the comparative cervical test (CCT) or the whole blood gamma interferon test, and 19 microarray analyses have been completed for RNA samples from blood pre-stimulated with M. bovis antigen for 4 hrs (n=15; 7 to CFT reactors, 4 double reactors, 4 TB positive) and 0-hr (n=4; all double reactors). Altered gene expression of 5-fold or greater was seen in each group: 10 up- and 2 down-regulated genes from CFT reactors, 223 up- and 5 down-regulated genes from double reactors, and 12 up- and 5 down-regulated genes from TB positives. No genes demonstrating altered expression levels were shared among or between these groups of cattle. The 0-hr microarrays showed only 4 genes with altered expression and none showed altered levels that were greater than 5-fold above or below control values. Project 4: BALBc mice were vaccinated twice with standard BCG or a new recombinant vaccine, then challenged intranasally with live M. bovis. 12 unvaccinated controls and 12 recombinant vaccine mice lost weight, became moribund, and were sacrificed within 4 weeks of challenge. BCG vaccinates maintained activity and body weight, and only 2 of 12 mice were sacrificed 7 weeks post challenge. The recombinant vaccine showed marked reduction in mortality, gross and microscopic lesions compared to controls, which shows promise for development of a vaccine that will not interfere with current tests. A second trial was performed using a modified subunit vaccine:weight loss, lesions development, and mortality were reduced compared to controls, but slightly higher compared to BCG vaccinates. Final summarization of the study is pending on final mycobacterial isolation and titration. A study was conducted on Michigan strains of M. bovis isolates from 1999 and 2004, to look for evidence of antimicrobial resistance, and no evidence of antimicrobial resistance development was seen.
IMPACT: 2004/09 TO 2007/08
Infection of cattle with Johne's disease does not appear to have major influence on the rate of false positive skin TB tests in Michigan, but may cause cattle to test negative on some secondary laboratory diagnostic assays. Results of gene expression profiling showed that there are promising gene targets that may be used for developing diagnostic tools to detect TB in live cattle. The intranasal challenge in BALBc mice is an efficient system for testing efficacy of tuberculosis vaccines. BCG vaccine demonstrated good protection to challenge, and the redesigned recombinant vaccine was much more effective that earlier recombinant vaccines. Lack of antimicrobial resistance in M. bovis from Michigan is important for the treatment of TB if cases in humans occur.
PUBLICATIONS (not previously reported): 2004/09 TO 2007/08
1. Clarke, K.R. 2005. Effects of Mycobacterium bovis inoculation in select potential reservoir or spillover wildlife host species. Ph.D. Thesis, Michigan State University, East Lansing, Michigan, 2005.
2. Clarke, K.R., Fitzgerald, S.D., Zwick, L.S., Church, S.V., Kaneene, J.B., Wismer, A.R., Bolin, C.A., Hattey, J.A., Yuzbasiyan-Gurkan, V. Experimental inoculation of meadow voles (Microtus pennsylvanicus), and Norway rats (Rattus norvegicus) with Mycobacterium bovis. J. Wildl. Dis., 43(3): 353-365, 2007.
3. Daly, M., Diegel, K.L., Fitzgerald, S.D., Schooley, A., Berry, D.E., Kaneene, J.B., Patterns of antimicrobial susceptibility in Michigan wildlife and bovine isolates of Mycobacterium bovis . J Vet Diagn Invest, Vol 18: 401-404, 2006.
4. Miller, R. and Kaneene, J.B. Evaluation of historical factors influencing the occurrence and distribution of Mycobacterium bovis infection among wildlife in Michigan. AJVR, Vol. 67 (4): 604-615, 2006.
5. Miller, R., Kaneene, J.B., Schmitt, S.M., Lusch, D.P., Fitzgerald, S.D. Spatial analysis of Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus) in Michigan, USA. Prev. Vet. Med., doi:10.1016/j.prevetmed.2007.05.011, 2007.
6. Norby, B., Bartlett, P.C., Grooms, D.L., Kaneene, J.B., Bruning-Fann, C.S. Use of simulation modeling to estimate herd-level sensitivity, specificity, and predictive values of diagnostic tests for detection of tuberculosis in cattle. Am. J. Veterinary Research, 66(7): 1285-1291, 2005.
PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-355-2269
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu

 
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ACCESSION NO: 0204959 SUBFILE: CRIS
PROJ NO: MICL08369 AGENCY: CSREES MICL
PROJ TYPE : NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2005-35204-16474 PROPOSAL NO: 2005-01809
START: 01 SEP 2005 TERM: 31 AUG 2008 FY: 2007 GRANT YR: 2005
GRANT AMT : $350,000
INVESTIGATOR: Coussens, P. M.; Kiupel, M.; Davis, W.
PERFORMING INSTITUTION:
Animal Science
Michigan State Univ
East Lansing , Michigan 48824
IMMUNE MODUATION IN BOVINE PARATUBERCULOSIS: MACROPHAGE-T CELL INTERACTIONS.
NON-TECHNICAL SUMMARY: Johnes disease is caused by M. paratuberculosis and is a devastating problem in the US dairy industry. There are no effective vaccines against M. paratuberculosis and it appears that the immune system of infected cows is affected by the organism such that this response becomes ineffective over time. This project makes use of an in vitro system to determine how M. paratuberculosis affects cells of the bovine immune system and prevents proper interactions between various immune cells, thus escaping control.
OBJECTIVES: 1) We will examine the relationship between Mycobacterium paratuberculosis (MAP) infection and expression of IL-1alpha, TRAF1, and TRAF2 in cultured monocyte derived macrophages (MDM). These studies follow up a previous observation that MAP infected intestinal tissues have enhanced levels of IL-1alpha and TRAF 1. 2) The effect of Mycobacterium paratuberculosis (MAP) infection and IL-1alpha treatment on CD40-CD154 and TNFalpha signaling in MDM will be investigated. These studies are designed to determine if MAP or IL-1alpha interfere with normal activation of macrophages. 3) The effect of autologous activated T cells will be examined to determine if MAP interfres with normal macrophage-T cell signaling. 4) Investigate the effect of MAP infection on susceptibility/resistance to apoptosis in MDM. These studies will determine if MAP promotes cell death or cell survival in infected macrophages.
APPROACH: Monocyte derived macrophages (MDM) will be used to investigate the effects of infection with M. paratuberculosis (MAP) on macrophage activation, signaling, interactions with T cells, and apoptosis. 1) MAP infected and uninfected MDM will be collected at various times post-infection, including: 0 (no MAP treatment), 1, 3 , 6 , 12 , 24 , and 48 hours. Cultures will be harvested for DNA, RNA, and protein at each time point. DNA will be used to confirm infection and survival of MAP by quantitative IS900 PCR. TRAF1, TRAF2, and IL-alpha mRNA expression at each time point will be assessed by Q-RT-PCR. TRAF1 and TRAF2 protein expression will be assayed by western blot analysis. Relative amounts of TRAF1 and TRAF2 will be determined. IL-1alpha protein in culture supernatants will be measured by ELISA. We will thus identify optimal times for TRAF1 and IL-1alpha mRNA and protein upregulation in MAP infected MDM. We also expect to confirm that TRAF1 and IL-1alpha mRNA and protein upregulation occurs coincidentally. 2) TRAF1 is overexpressed in intestinal lesions of MAP infected cows, relative to tissues from healthy cows. Signaling through CD40, TNFR1, and TNFR2 depends upon the balance of TRAF1 and TRAF2 cells. We will test the hypothesis that enhanced TRAF1 expression increases non-signaling TRAF1 homotrimers and (TRAF1)2-(TRAF2)1 heterotrimeric complexes within MDM, using a combination of immune precipitation and western blotting. 3) The effect of enhanced TRAF1 expression on CD40 signaling will be assayed by measuring activation of NFkappaB in MDM by electrophoretic mobility shift assay (EMSA). The ratio of phosphorylated (activated) to non-phosphorylated (inactive) NFkappaB p65 subunit will also be determined. Finally, activation of IkappaKinase in treated cultures will be measured. We will monitor mRNA levels for expression of IL-10, IL-12p35, IL-12p40, TNFalpha, MIP-1alpha and production of nitric oxide in MAP infected and IL-1alpha treated MDM in response to soluble CD154. 4) Engagement of the T cell receptor (TCR) with MHC II + antigen on macrophages and binding of other co-stimulatory factors are of paramount importance in macrophage T cell interactions. To monitor these interactions, fresh PBMCs will be prepared and stimulated with MAP antigens in vitro, for 24 hours. Following stimulation, autologous T cells corresponding to each MDM culture will be isolated using magnetic cell sorting. Isolated T cells will be fixed and applied to infected or control MDM and allowed to interact for 4 to 24 hours. Macrophage activation parameters to be assayed include production of IFNgamma, IL-10, IL-12p35, IL-12p40, TNFalpha, MIP-1alpha, and IL-1alpha mRNA by Q-RT-PCR. 5) The large numbers of macrophages found at sites of MAP infection suggest that infected macrophages have become resistant to normal clearance mechanisms, including apoptosis. The ability of two common apoptosis-inducing ligands to signal apoptosis in MAP infected and control uninfected MDM will be investigated. Apoptosis will be measured flow-cytometrically using annexin V staining and propidium iodide (PI).
PROGRESS: 2006/09 TO 2007/08
OUTPUTS: Johnes disease in cattle has a significant economic impact on the global dairy industry, with losses in the US estimated at over 1 billion dollars annually. There are no effective vaccines available to prevent infection with Mycobacterium paratuberculosis (MAP), the causative agent of Johnes disease. A hallmark of infection with MAP, as with other mycobacteria, is survival of the organism in host macrophage cells where immune destruction is difficult. Why infected macrophages do not destroy invading MAP organisms is a central question in MAP immunobiology and in study of other mycobacteria, such as M. bovis. Our work is focused on discovering mechanisms employed by MAP to suppress macrophage functions in the hopes of finding new methods to combat Johnes disease. Our main hypothesis is that MAP reduces the ability of infected macrophages to react to normal T cell signaling, failing to activate and destroy MAP, and failing to properly signal T cells to respond. Infected macrophages are also induced to resist apoptosis, prolonging survival of intracellular MAP. This project has four main objectives. These are to 1) examine the relationship between infection and expression of interleukin (IL)-1alpha, TRAF1, and TRAF2 in cultured monocyte derived macrophages (MDM). 2) Determine the effect of MAP infection and IL-1alpha treatment on CD40-CD154 and TNF&#945; signaling in MDM. 3) Determine the effect of MAP infection on interactions between MDM and autologous activated T cells. 4) Investigate the effect of MAP infection on susceptibility/resistance to apoptosis in MDM. In related studies, we have begun to examine how development of specific regulatory T cells might impact development of clinical Johnes disease and affect macrophage-T cell interactions. Previously we had shown the IL-1alpha and TRAF1 were expressed at high concentrations in MAP infected tissues and cells. We have explored this relationship further, using in vitro model systems. This work led us to examine signaling mechanisms within MAP infected macrophages. We discovered that MAP infected macrophages were defective in some aspects of signaling through CD40 receptors, leading to under expression of the critical cytokine IL-12 and the inducible nitric oxide synthetase (iNOS). We have also examined possible mechanisms for this defect, examining the roles of various mitogen-activated protein kinases. In the regulatory T cell area, we have definitively demonstrated that these cells do exist in cattle, that MAP-reactive regulatory T cells develop in the course of natural infections with MAP, and that they respond to MAP antigens by production of IL-10, suppressing production of interferon gamma, a cytokine required to clear infections with MAP. Our results have been presented at several major international meetings, including the Conference for Research workers in Animal Disease, Dec. 3-5, 2007, Chicago IL; the International Veterinary Immunology Symposium, Oro Puerto, Brazil, August 2007; The Johnes Disease Integrated Project Meeting in College Station Texas, 2007 and the 9th International Colloquium on Paratuberculosis. Tsukuba, Japan, October 2007 PARTICIPANTS: P.M. Coussens, Principal Investigator, Professor, Department of Animal Science, Michigan State University. S. Sommer, Postdoctoral fellow, Department of Animal Science, Michigan State University. D. Almeida, Postdoctoral fellow, Department of Animal Science, Michigan State University. E. Kabara, Graduate Student, Department of Animal Science, Michigan State University. C.J. Colvin, Research Technician II, Department of Animal Science, Michigan State University. Collaborators: M. Kiupel, Professor, Department of Pathobiology, Michigan State University W. Davis, Professor, Washington State University. S. Sreevatsan, Associate Professor, University of Minnesota. TARGET AUDIENCES: Primary target audience of our work are other scientists working in the Mycobacterium paratuberculosis field, as well as those working on other mycobacteria. Additional target audiences include scientists working on development of diagnostics and vaccines for Johnes disease in cattle and other species.
IMPACT: 2006/09 TO 2007/08
Work completed to date on this project has demonstrated that MAP infection of macrophages enhances expression of interleukin (IL)-1 leading to activation of the transcription factor NfkappaB and subsequent activation of the TRAF1 gene. Enhanced expression of TRAF1 in cultured macrophages can also be achieved with direct treatment of macrophages with recombinant IL-1, while the reverse is not true. Furthermore, sustained expression of TRAF1 requires continued presence of IL-1. These results demonstrated that MAP may have profound effects on intracellular signaling within infected macrophages and led to the hypothesis that MAP may alter the ability of infected macrophages to activate and to properly signal T cells to respond to infection. These results have led to a series of new investigations, including a search for MAP factors that might be responsible for this effect and a more global functional genomics based investigation of MAP-induced gene expression changes in macrophage cells. CD40 interactions with its ligand on activated T cells is a major mechanism used to activate macrophages, eliciting expression of numerous immune response genes in the target macrophage. Our subsequent work demonstrated that MAP is able to subvert a subset of macrophage responses to CD40 signaling, in particular expression of IL-12 and iNOS. These two factors are critical for macrophages to destroy MAP and direct T cell responses toward a Th1 or proinflammatory activity, which is required for clearance of MAP infections. This result offers a possible explanation for why the Th1 proinflammatory response to MAP is lost during the course of natural infections in cattle that proceed to clinical disease. In addition, our results suggest that promoting IL-12 and perhaps iNOS production may help alleviate development of clinical illness due to MAP and allow clearance of this fastidious pathogen. Furthermore, our results are being incorporated into modified live vaccine development strategies, searching for MAP mutants that do not inhibit iNOS and IL-12 production in macrophages. Regulatory T cell biology and especially development of peripheral Treg cells in response to infection is a relatively new area in immunobiology. Our work with MAP-specific Treg cells has clearly demonstrated that these cells do exist in cattle, a novel finding. These studies have therefore opened a new avenue of research in bovine immunobiology. Development of Treg cells in MAP infection can, in addition to the results discussed above, help explain why immune responses to MAP appear to be limited or re-directed in animals that proceed to clinical disease. Again, these results are being used to examine MLV vaccine candidates, since any effective vaccine should not elicit a Treg response.
PUBLICATIONS (not previously reported): 2006/09 TO 2007/08
1. Coussens, P.M., C.J. Colvin, and D.E. Almeida. 2008. Antigen-specific regulatory T cells in bovine paratuberculosis. Veterinary immunology and immunopathology, In Press.
2. Sommer, S., C.B. Pudrith, C.J. Colvin and Paul M. Coussens. 2008. Mycobacterium avium subspecies paratuberculosis suppresses CD40 signaling induced IL-12p40 and iNOS gene expression in bovine monocyte-derived macrophages. Veterinary Immunology and Immunopathology, In Press.
PROJECT CONTACT:
Name: Coussens, P.
Phone: 517-353-3158
Fax: 517-353-1699
Email: coussens@msu.edu
URL: www.cafg.msu.edu

 
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ACCESSION NO: 0403699 SUBFILE: CRIS
PROJ NO: 3625-32000-062-02S AGENCY: ARS 3625
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 58-3625-0-137
START: 07 SEP 2000 TERM: 28 FEB 2004 FY: 2002 GRANT YR: 2000
GRANT AMT: $146,000
INVESTIGATOR: Bannantine J P; Stabel J R; Kapur V
PERFORMING INSTITUTION:
Veterinary Pathobiology
Univ of Minnesota
St Paul , Minnesota 55108
GENOME SEQUENCING OF MYCOBACTERIUM PARATUBERCULOSIS.
OBJECTIVES: The objective of this cooperative research project is to analyze the genome of Mycobacterium paratuberculosis to identify targets for the development of diagnostic tests and control measures for Johne's Diseases in cattle.
APPROACH: The genome sequence of one isolate of M. paratuberculosis will be analyzed to find sequences which differentiate between M. paratuberculosis and other closely related mycobacteria. The genome will also be searched to detect sequences coding for potential virulence factors and factors critical for survival of the bacterium within the host. Gene expression inside macrophages will be determined using DNA microarrays.
PROGRESS: 2000/09 TO 2004/02
4. What were the most significant accomplishments this past year? D. Progress Report. This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Minnesota. Additional details of research can be found in the report for the parent project 3625-32000-062-00D Understanding Host-Pathogen Interactions for the Diagnosis and Control of Paratuberculosis (Johne's). This agreement between ARS(NADC) and the University of Minnesota encompasses measures to ensure animal health through an increased understanding of the biology of Mycobacterium paratuberculosis, the bacterial agent that causes Johne's disease. This project has the objective of sequencing the entire genome of M. paratuberculosis. Sequencing of this genome has now been completed in our laboratory in collaboration with researchers at the University of Minnesota. The manuscript describing the genome is now in preparation as well as a searchable database of genome information. Preliminary results from the genome sequence include identification of potential virulence genes and vaccine candidate sequences. We have constructed a complete microarray representing all of the genes present in M. paratuberculosis is being constructed.
PUBLICATIONS (not previously reported): 2000/09 TO 2004/02
No publications reported this period.

 
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ACCESSION NO: 0406864 SUBFILE: CRIS
PROJ NO: 3625-32000-062-04S AGENCY: ARS 3625
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: TERMINATED
START: 04 JUN 2003 TERM: 31 AUG 2005 FY: 2003
INVESTIGATOR: Bannantine J P; Kapur V; Wells S
PERFORMING INSTITUTION:
Univ of Minn
2642 Univ. Ave
St Paul , Minnesota 55114
FUNCTIONAL GENOMIC ANALYSIS OF MYCOBACTERIUM PARATUBERCULOSIS ( UNIV. OF MN).
OBJECTIVES: The development of diagnostic tests and control measures for Johne's Disease in cattle as outlined in the USDA-NRI grant #2002-02228. The specific objectives of this cooperative research project are to identify unique antigens from the genome of Mycobacterium paratuberculosis, incorporate these antigens into a diagnostic test, and evaluate this test in dairy cattle herds.
APPROACH: The genome sequence of a bovine isolate of M. paratuberculosis will be analyzed to find DNA sequences which differentiate between M. paratuberculosis and other closely related mycobacteria. Efforts will then be made to clone, express and purify proteins from these novel M. paratuberculosis sequences. These purified proteins will be evaluated in a variety of immunological assays to determine which ones are best for diagnostic test development. The test will be optimized and used in whole-herd field tests of known and unknown infection status.
PROGRESS: 2003/06 TO 2005/08
4d Progress report. This report serves to a document research conducted under a specific cooperative agreement between ARS and the University of Minnesota. This specific cooperative agreement represents a subcontract of USDA-CSREES funds to the University of Minnesota. Additional details of research can be found in the report for the parent project 3625-32000-062-00D Understanding Host-Pathogen Interactions for the Diagnosis and Control of Paratuberculosis (Johne's). This specific cooperative agreement between ARS (NADC) and the University of Minnesota encompasses measures to ensure animal health through the control and management of disease by improving detection methods for Johne's disease. This project has the objective of identifying any remaining sequence that are specific to Mycobacterium paratuberculosis and are not present in closely related mycobacteria. To date, we have recently identified and confirmed a number of genes that brings the total of M. paratuberculosis-specific sequences up to 39. We also have Minnesota dairy herds online and ready for validation studies.
PUBLICATIONS (not previously reported): 2003/06 TO 2005/08
No publications reported this period.

 
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ACCESSION NO: 0210343 SUBFILE: CRIS
PROJ NO: MIN-62-025 AGENCY: SAES MIN
PROJ TYPE: STATE PROJ STATUS: NEW
START: 01 JUN 2007 TERM: 30 JUN 2009
INVESTIGATOR: Sreevatsan, S.; Singer, R.; Wells, S.; Isaacson, R.
PERFORMING INSTITUTION:
Veterinary Population Medicine
Univ of Minnesota
St Paul , Minnesota 55108
MODULATION OF INTESTINAL MICROBIAL COMMUNITY STRUCTURE AND FUNCTIONING BY MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS INFECTION .
NON-TECHNICAL SUMMARY: The understanding of microbial population structure in a pathogen induced environment would enable more rigorous risk analysis for food pathogen carriage. These studies will also provide critical missing information on the specific microbial populations that may be depleted as a consequence of Johne's disease and provide clues on how probiotic approaches may help in controlling this spread of Mycobacteria.
OBJECTIVES: (1)Determine alterations in microbial community structure that occur as a result of infection by MAP in the intestines of MAP-shedding and MAP-exposed and non shedding (healthy) animals using well-established terminal RFLP method; (2) Determine the prevalence of Shiga toxin-producing E. coli (STEC)and Salmonella enterica serovar Typhimurium carriage in MAP-infected and shedding, and MAP-exposed and not shedding dairy animals using well-established microbiological procedures and pathogen-specific primers and probes coupled to quantitative real time PCR analyses. At the conclusion of these studies, we expect to be able to document the magnitude of public health threat of MAP infection in animal populations. These studies will establish a baseline for future analysis and quantitation of the impact of JD on food safety and public health.
APPROACH: We propose to study intestinal microbial community structure and function in JD infected and control animals in a prospective study design. Animals culture positive and negative for MAP residing on infected herds and status negative herds will be sampled for fecal culture and microbial community profiling monthly over a one year period. All samples will be analyzed by terminal-restriction fragment length polymorphism analysis for microbial population profiles and cultured for MAP, Salmonella and E. coli O157. Shifts in microbial structure will be correlated with MAP status and carriage of Salmonella and E. coli O157. In addition, when animals are culled from the herd, they will be purchased and euthanized for a detailed histopathology of the intestines and mucosal microbial profiles using fluorescence in situ hybridization methods.
PROGRESS: 2007/07 TO 2007/12
OUTPUTS: The project was initiated in July of 2007 and work is underway. No outputs have been recorded for this period yet. PARTICIPANTS: Dr. Michael Paustian; National Animal Disease Center, Ames, IA Dr. Gary Andersen; Lawrence Berkeley National Laboratories, CA PROJECT MODIFICATIONS: We have identified collaborations with Dr. Andersen at the Lawrence Berkeley national laboratories to perform our analyses using more robust, unambiguous, and quantitative Phylochips. Phylochips are 16 rDNA based high density microarrays encompassing both eubacteria and archaea. The arrays are expected to provide quantitative microbial identities within each community which would not have been available with the proposed TRFLP protocols. Thus, we propose to continue the work with Phylochips.
IMPACT: 2007/07 TO 2007/12
Since initiation of this project, we have identified a Johne's disease infected herd. Forty fecal samples from culture positive (moderate to heavy shedders) and 10 uninfected animals have been collected. Total microbial community DNA has also been extracted from these samples. Of these, twenty fecal DNA samples have been amplified for universal 16s rDNA encompassing eubacterial and archaeal communities in a gradient PCR. All gradient PCR generated amplicons will be poled by sample prior to analysis with a Phylochip.
PUBLICATIONS (not previously reported): 2007/07 TO 2007/12
No publications reported this period
PROJECT CONTACT:
Name: Sreevatsan, S.
Phone: 612-625-3769
Fax: 612-625-6241
Email: sreev001@umn.edu

 
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ACCESSION NO: 0208096 SUBFILE: CRIS
PROJ NO: MINV-62-027 AGENCY: CSREES MINV
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2007-35204-17798 PROPOSAL NO: 2006-01731
START: 01 MAR 2007 TERM: 28 FEB 2010 FY: 2007 GRANT YR: 2007
GRANT AMT: $364,443
INVESTIGATOR: Godden, S. M.
PERFORMING INSTITUTION:
Veterinary Population Medicine
Univ of Minnesota
St Paul , Minnesota 55108
EFFECT OF FEEDING HEAT-TREATED COLOSTRUM ON PREWEANING HEALTH, ECONOMICS AND TRANSMISSION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS IN . . . (Note: Title exceeded the title field character limit.)
NON-TECHNICAL SUMMARY : Johne's disease is one of the most significant and costly infectious diseases affecting animal performance, animal welfare, profitability of the dairy herd enterprise and, quite possibly, public health. Disease in preweaned dairy calves is another important source of economic loss to dairy producers. But what do Johne's disease and calf hood disease have in common? One of the earliest potential exposures of dairy calves to infectious agents arrives with the first colostrum feeding. We need to develop colostrum management practices that could help to prevent pathogen transmission to newborn calves. The goal of this study is to perform a prospective controlled field study in commercial dairy herds to evaluate the effect of feeding heat-treated colostrum on preweaning health, economics, and Johne's disease transmission in dairy calves. The first objective of this study is to describe the efficacy and cost-benefit of feeding heat-treated bovine colostrum as a control point for reducing disease in preweaned dairy calves (Phase I). The second (long-term) objective of this study will be to describe the efficacy and cost-benefit of feeding heat-treated bovine colostrum as a critical control point for reducing transmission of Johne's to calves (Phase II). The long-term outcomes of this research will include the development of more scientifically and economically sound comprehensive Johne's control programs, improved animal health and well being, improved profitability and sustainability of commercial dairy farms, and improved food safety in the United States.
OBJECTIVES: The goal of this study is to complete a controlled on-farm field study in Johne's-infected commercial dairy herds to evaluate the effect of a novel management tool, feeding heat-treated bovine colostrum, on preweaning health, economics, and transmission of Mycobacterium avium subspecies paratuberculosis (Map) in dairy calves. This goal will be achieved through the two major objectives in two separate phases of the project. Objective 1 (Phase I): Evaluate the efficacy and cost-benefit of feeding heat-treated bovine colostrum as a critical control point for reducing morbidity and mortality in preweaned calves. Note: Only objective 1 will be addressed in the current proposal. Objective 2 (Phase II): Evaluate the efficacy and cost-benefit of feeding heat-treated bovine colostrum as a critical control point for reducing transmission of Map to newborn dairy calves. Note that this objective is not considered in the current proposal or budget (will come under a separate submission to be completed in the future).
APPROACH: Study Farms. This study will be completed in 8 to 10 commercial Holstein dairy herds in Minnesota and Wisconsin. Herds must be on a regular DHIA test program, have a history of clinical Johne's disease, and have had at least one fecal culture positive animal confirmed in the previous 3 years. One-time testing of 80 adult cows in their second or older lactation will be performed, using a pooled fecal culture method, to estimate the prevalence of Johne's infection in the herd. Colostrum Preparation. Unique batches of fresh colostrum (> 8 L per batch) will be well mixed and then split into two equal 4L duplicate aliquots. One of the 4 L aliquots will be selected for heat-treatment at 60 degrees C x 60 minutes in a commercial on-farm batch pasteurizer. For each batch, duplicate 50 ml samples of fresh (raw) and of heat-treated colostrum will be collected and frozen. Immediately after processing, fresh (3.8 L) and heat-treated (3.8 L) colostrum will be transferred into sanitized covered plastic feeding bottles, labeled, and refrigerated. Calf-Enrollment. Singleton newborn heifer calves will be removed from the dam by one hour of age and before suckling. Date of birth, dam ID, calf ID, gender and birth weight will be recorded. The calf will be systematically assigned to be fed 3.8 L of either fresh or heat-treated colostrum within 1-2 hours of birth. The colostrum bottle will be removed from the refrigerator, warmed to 95-105 degrees F in a warm water bath that is < 125 degrees F, mixed well, and then fed to the calf using the standard feeding method used by that farm. The colostrum batch number, date and treatment group will be recorded. After the first colostrum feeding, all calves will be housed separately and fed the standard milk feeding program that is in use on that farm. 1200 heifer calves (600/group) will be enrolled. Follow-up and Sampling During the Preweaning Period. Calves will be observed daily for illness and records will be kept of the calf ID, illness date, on-farm diagnosis (or veterinary diagnosis if available), treatments provided, and number of days treated. Death events will be recorded as calf ID, date, and on-farm diagnosis (or veterinary diagnosis if available). Body weight (and date) will be recorded at time of weaning. A study technician will visit the farm once per week to collect a 10 ml blood sample from the jugular vein for each study calf between 24 hrs to 8 days of age. Laboratory Testing. Serum from blood samples will be split into equal aliquots, frozen, and then submitted for analysis of serum total protein (gm/dl) and serum immunoglobulin G (mg/ml) concentrations. Frozen duplicate paired (pre- and post-heat treatment) colostrum samples will be analyzed for colostrum IgG concentration (mg/ml) and bacteria counts. 20 ml of one colostrum sample from each batch will be tested for Mycobacterium avium subsp. paratuberculosis using a nested real-time PCR assay with a specific probe allowing for quantification.
PROJECT CONTACT:
Name: Godden, S.
Phone: 612-625-8177
Fax: 612-625-6241
Email: godde002@umn.edu

 
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ACCESSION NO: 0199163 SUBFILE: CRIS
PROJ NO: MINV-62-052 AGENCY: CSREES MINV
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 2004-35605-14243 PROPOSAL NO: 2006-00794
START: 15 APR 2004 TERM: 14 APR 2009 FY: 2007 GRANT YR: 2005
GRANT AMT: $149,666
INVESTIGATOR: Kapur, V.; Wells, S.
PERFORMING INSTITUTION:
College of Veterinary Medicine
Univ of Minnesota
St Paul , Minnesota 55108
JDIP: JOHNE'S DISEASE INTEGRATED PROGRAM IN RESEARCH, EDUCATION, AND EXTENSION.
NON-TECHNICAL SUMMARY: Johne's disease is a disease which results in lost productivity in dairy herds and potential mobidity/mortality. There is also some concern that the bacterium itself is related to the onset of Crohn's disease in humans. The JDIP program aims to control, and potentially eliminate, Johne's through a combination of research projects on how to manage the disease, and potentially eliminate it. This is being done through a broad collaboration of research universities and USDA groups, with the collaboration and support of the impacted private sector.
OBJECTIVES: To establish an integrated program in Johne's disease research with a focus on developing a strong translational pipeline of new diagnostic tests, vaccine candidates and strategies to manage, prevent and control the disease.
APPROACH: The above noted objectives will be accomplished via thematic research units on: Johne's disease epidemiology; diagnostic test development including strain differentiation; M. paratuberculosis basic biology and pathogenesis; and strategies for enchancing T and B cell immune response to M. paratuberculosis. These research units will be supported by scientific support cores in: Biostatics and Epidemiology; Diagnostics and Strain Differentiation; Gene and Protein Expression; and Animal Experimentation. In addition, an Administrative Core will provide support for both research units and the scientific cores while a Communications and Extension Core will facilitate communication within the overall JDIP as well as translating the outcomes from the research into educational information for the broadly affected Johne's community. An external advisory board, chosen from public and private stakeholder, will provide guidance to the JDIP team in pursuit of the overall objectives.
PROGRESS: 2006/01 TO 2006/12
We are well on our way towards achieving each of our strategic objectives as outlined in the original Johne's Disease Integrated Program (JDIP) proposal and have made documented progress through the creation and maintenance of functional and highly productive integrated research teams and core facilities that are interdisciplinary and multi-institutional. In brief, the CAP program has enabled JD research, education, and extension to move rapidly forward in a manner that would not be possible through traditional funding mechanisms from the USDA. In particular, the founding and continued support of JDIP has enabled the community to, for the first time since JD was described more than a century ago, develop an integrated and coordinated program with a focus on developing a strong translational pipeline of new diagnostic tests, vaccine candidates, strategies to manage, prevent and control the disease, and the formulation of an outstanding education and training program. In the brief period since the founding of the program, JDIP investigators have conducted path-breaking research and development that has resulted in: 1. A better understanding of Map on-farm transmission dynamics that is helping identify critical control points in the transmission chain; 2. The development of alternative sampling and testing strategies for detection of infected animals and herds that are being adopted by the national voluntary control program for JD; 3. The optimization and standardization of laboratory protocols for Map culture and PCR for reducing timelines for rapid detection of infected animals; 4. Characterization of genetic differences between isolates of Map for molecular epidemiologic analyses and tracking of strains in infected animals and the environment; 5. Development of standards for animal challenge models with Map for the evaluation of vaccine efficacy; 6. Identification of key genes, proteins and lipids in Map for development of the next generation of diagnostic tests and vaccines; 7. Development and widespread use of an on-line JD veterinary certification program; and, 8. Development of community resources including Map isolates, serum samples and other clinical material for the development and validation of diagnostic tests, genomic microarrays, recombinant proteins, and mutant strain banks of Map for identification of potential vaccine candidates; In addition to our research accomplishments, we have developed a strong communications and extension plan that includes workshops, newsletters, regular conference calls, and an annual conference of JD researchers. Hence, JDIP has brought together scientists and stakeholders with a shared vision and well-defined plan to support and facilitate research, extension and education activities and enhance animal health through biosecurity by addressing well-documented and emerging needs in JD and is well on its way to meeting and exceeding all of the project objectives as per plan.
IMPACT: 2006/01 TO 2006/12
Johne's disease (JD) is a serious economic and animal health problem in domesticated ruminants (primarily dairy and beef cattle, sheep, and goats) throughout the world. JD in wildlife species is also of great concern since it may limit opportunities to control or eradicate JD from domesticated animals. JDIP, through its projects and cores, develops science-based solutions to enhance animal biosecurity to limit the spread of JD within herds and prevent its introduction into previously uninfected herds. We have assembled a strong scientific team with an outstanding record of accomplishments who are working together under the auspices of JDIP to help develop and implement a comprehensive plan to enhance animal biosecurity through the targeted development of rational methods for the diagnosis, prevention, and control of Johne's disease.
PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
1. Amonsin, A., L. L. Li, Q. Zhang, J. P. Bannantine, A. S. Motiwala, S. Sreevatsan, and V. Kapur. 2004. Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains. J Clin Microbiol 42:1694-702.
2. Bannantine, J. P., R. G. Barletta, J. R. Stabel, M. L. Paustian, and V. Kapur. 2004. Application of the genome sequence to address concerns that Mycobacterium avium subspecies paratuberculosis might be a foodborne pathogen. Foodborne Pathog Dis 1:3-15.
3. Bannantine, J. P., J. K. Hansen, M. L. Paustian, A. Amonsin, L. L. Li, J. R. Stabel, and V. Kapur. 2004. Expression and immunogenicity of proteins encoded by sequences specific to Mycobacterium avium subsp. paratuberculosis. J Clin Microbiol 42:106-14.
4. Bannantine, J. P., and M. L. Paustian. 2006. Identification of diagnostic proteins in Mycobacterium avium subspecies paratuberculosis by a whole genome analysis approach. Methods Mol Biol 345:185-96.
5 . Benedictus, A., R. H. Whitlock, J. M. Widmann, R. Sweeney, T. Fyock, M. Linde, R. M. Mitchell, and Y. H. Schukken. 2005. Calculation of parameters on the transmission of Mycobacterium avium subspecies paratuberculosis in a dairy herd going through a control program. 8th International Colloquium on Paratuberculosis, Copenhagen, Denmark.
6. Benedictus, A., R. H. Whitlock, J. M. Widmann, R. Sweeney, T. Fyock, M. Linde, R. M. Mitchell, and Y. H. Schukken. 2006. Calculation of transmission parameters of Mycobacterium avium subspecies paratuberculosis infections in a dairy herd going through a control program. Pre Vet Med (in press).
7. Berger, S., D. Hinz, J. P. Bannantine, and J. F. Griffin. 2006. Isolation of high-affinity single-chain antibodies against Mycobacterium avium subsp. paratuberculosis surface proteins from sheep with Johne's disease. Clin Vaccine Immunol 13:1022-9.
8. Chapagain, P., R. M. Mitchell, S. M. Stehman, Y. H. Schukken, and Y. T. Grohn. 2006. Characteristics of a mathematical model of Mycobacterium avium subsp. paratuberculosis (MAP) transmission. Symposium on Veterinary Epidemiology and Economics, Cairns, Australia.
9 . Choi, Y. K., I. A. Gardner, W. O. Johnson, and M. T. Collins. 2005. Bayesian modeling of ROC curves without a gold standard. Presented at 85th Conference of Research Workers in Animal Disease, Chicago, USA.
10. Choi, Y. K., W. O. Johnson, M. T. Collins, and I. A. Gardner. 2006. Bayesian inferences for receiver operating characteristic curves in the absence of a gold standard. Journal of Agricultural Biological and Environmental Statistics 11:210-229.
11 . Davis, W. C., H. C. Koo, Y. H. Park, M. J. Hamilton, A. J. Allen, G. M. Barrington, K. T. Park, J. B. Kim, J. L. Dahl, W. R. Waters, and A. K. Storset. 2006. Flow cytometric analysis of the immune response to M. avium subsp. paratuberculosis in experimentally infected calves. 8th International Colloquim on Paratuberculosis. Proceedings, International Association for Paratuberculosis.Inc., Publication Department:98-107.
12. Eckstein, T. M., S. Chandrasekaran, S. Mahapatra, M. R. McNeil, D. Chatterjee, C. D. Rithner, P. W. Ryan, J. T. Belisle, and J. M. Inamine. 2006. A major cell wall lipopeptide of Mycobacterium avium subspecies paratuberculosis. J Biol Chem 281:5209-15.
13. Eda, S., J. P. Bannantine, W. R. Waters, Y. Mori, R. H. Whitlock, M. C. Scott, and C. A. Speer. 2006. A highly sensitive and subspecies-specific surface antigen enzyme- linked immunosorbent assay for diagnosis of Johne's disease. Clin Vaccine Immunol 13:837-44.
14 . Gardner, I. A., W. O. Johnson, A. J. Branscum, and M. Georgiadis. 2005. Sample sizes for evaluation of diagnostic tests for bovine paratuberculosis in the absence of a gold standard. 8th International Colloquium on Paratuberculosis, Copenhagen, Denmark.
15. Gollnick, N., R. M. Mitchell, S. Klaessig, and Y. H. Schukken. 2005. Macrophage killing capacity in Mycobacterium A. paratuberculosis infected cows compared to uninfected controls, 8th International Colloquium on Paratuberculosis, Copenhagen, Denmark.
16. Grewal, S. K., S. Rajeev, S. Sreevatsan, and F. C. Michel, Jr. 2006. Persistence of Mycobacterium avium subsp. paratuberculosis and other zoonotic pathogens during simulated composting, manure packing, and liquid storage of dairy manure. Appl Environ Microbiol 72:565-74.
17. Harris, N. B., J. B. Payeur, V. Kapur, and S. Sreevatsan. 2006. Short-sequence-repeat analysis of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium subsp. avium isolates collected from animals throughout the United States reveals both stability of loci and extensive diversity. J Clin Microbiol 44:2970-3.
18. Hines, M. E., S. Stiver, D. Giri, L. Whittington, C. Watson, J. Johnson, J. Musgrove, M. Pence, D. Hurley, C. Baldwin, I. A. Gardner, and S. Aly. 2006. Efficacy of spheroplastic and cell wall competent vaccines for Mycobacterium avium subsp. paratuberculosis in experimentally-challenged baby goats. Vaccine (accepted).
19. Janagama, H. K., K. I. Jeong, V. Kapur, P. Coussens, and S. Sreevatsan. 2006. Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains. BMC Microbiol 6:10.
20. Johansen, K. A., E. E. Hugen, and J. B. Payeur. 2006. Growth of Mycobacterium avium subsp. paratuberculosis in the presence of hexadecylpyridinium chloride, natamycin, and vancomycin. J Food Prot 69:878-83.
21 . Keun, S. S., S. U. Lee, Y. H. Park, W. C. Davis, L. K. Fox, and G. A. Bohach. 2006. Long-term enterotoxin exposure induces soluble factor mediated immunosuppression by bovine CD4 and CD8 T cells. Infection and Immunity (accepted for publication)
22. Koo, H. C., Y. H. Park, J. Ahn, W. R. Waters, M. J. Hamilton, G. Barrington, A. A. Mosaad, M. V. Palmer, S. Shin, and W. C. Davis. 2004. New latex bead agglutination assay for differential diagnosis of cattle infected with Mycobacterium bovis and Mycobacterium avium subsp. paratuberculosis. Clin Diagn Lab Immunol 11:1070-4.
23 . Koo, H. C., Y. H. Park, M. J. Hamilton, G. M. Barrington, C. J. Davies, J. B. Kim, J. L. Dahl, W. R. Waters, and W. C. Davis. 2004. Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves. Infect Immun 72:6870-83.
24. Marri, P. R., J. P. Bannantine, M. L. Paustian, and G. B. Golding. 2006. Lateral gene transfer in Mycobacterium avium subspecies paratuberculosis. Can J Microbiol 52:560-9.
25. Marsh, I. B., J. P. Bannantine, M. L. Paustian, M. L. Tizard, V. Kapur, and R. J. Whittington. 2006. Genomic comparison of Mycobacterium avium subsp. paratuberculosis sheep and cattle strains by microarray hybridization. J Bacteriol 188:2290-3.
26. McDonald, J., and E. A. Horn. 2005. An Online Program for All Regions: Thinking Globally-Implementing Locally, Proceedings of the 21st Conference on Distance Teaching and Learning. Continuing and Vocational Education, UW-Madison, Madison, WI, USA
27. Mitchell, R. M., S. M. Stehman, R. H. Whitlock, A. Benedictus, and Y. H. Schukken. 2005. A deterministic mathematical model of Mycobacterium avium subsp. paratuberculosis (MAP) transmission on commercial US dairy farms. 8th International Colloquium on Paratuberculosis, Copenhagen, Denmark.
28. Mitchell, R. M., R. H. Whitlock, S. M. Stehman, A. Benedictus, P. Chapagain, Y. H. Grohn, and Y. T. Schukken. 2006. Mathematical modeling of Mycobacterium avium subsp. paratuberculosis (MAP) on commercial US dairy farms. Pre Vet Med (in press).
29. Motiwala, A. S., A. Amonsin, M. Strother, E. J. Manning, V. Kapur, and S. Sreevatsan. 2004. Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species. J Clin Microbiol 42:1703-12.
30. Motiwala, A. S., L. Li, V. Kapur, and S. Sreevatsan. 2006. Current understanding of the genetic diversity of Mycobacterium avium subsp. paratuberculosis. Microbes Infect 8:1406-18.
31 . O'Shea, B., S. Khare, K. Bliss, P. Klein, T. A. Ficht, L. G. Adams, and A. C. Rice-Ficht. 2004. Amplified fragment length polymorphism reveals genomic variability among Mycobacterium avium subsp. paratuberculosis isolates. J Clin Microbiol 42:3600-6.
32. Patel, D., L. Danelishvili, Y. Yamazaki, M. Alonso, M. L. Paustian, J. P. Bannantine, L. Meunier-Goddik, and L. E. Bermudez. 2006. The ability of Mycobacterium avium subsp. paratuberculosis to enter bovine epithelial cells is influenced by preexposure to a hyperosmolar environment and intracellular passage in bovine mammary epithelial cells. Infect Immun 74:2849-55.
33. Paustian, M. L., A. Amonsin, V. Kapur, and J. P. Bannantine. 2004. Characterization of novel coding sequences specific to Mycobacterium avium subsp. paratuberculosis: implications for diagnosis of Johne's Disease. J Clin Microbiol 42:2675-81.
34. Robbe-Austerman, S., I. A. Gardner, B. V. Thomsen, D. G. Morrical, B. M. Martin, M. V. Palmer, C. O. Thoen, and C. Ewing. 2006. Sensitivity and specificity of the agar-gel-immunodiffusion test, ELISA and the skin test for detection of Johne's disease in United States midwest sheep populations. Vet Res (in press).
35. Seo, K. S., L. S. U., P. Y. H., D. W. C., L. K. Fox, and G. A. Bohach. 2006. Long-term enterotoxin exposure induces soluble factor mediated immunosuppression by bovine CD4 and CD8 T cells. Infection and Immunity (In press).
36. Speer, C. A., M. C. Scott, J. P. Bannantine, W. R. Waters, Y. Mori, R. H. Whitlock, and S. Eda. 2006. A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle. Clin Vaccine Immunol 13:535-40.
37. Stratmann, J., B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull. 2004. A 38-kilobase pathogenicity island specific for Mycobacterium avium subsp. paratuberculosis encodes cell surface proteins expressed in the host. Infect Immun 72:1265-74.
38. Wang, C., B. Turnbull, Y. T. Grohn, and S. S. Nielsen. 2006. Bayesian nonparametric estimation of ROC curves when the true disease state is unknown. J. Agric. Biolog. Environ. Statist (in press).
39. Wang, C., B. W. Turnbull, Y. T. Grohn, and S. S. Nielsen. 2006. Estimating receiver operating characteristic curves with covariates when there is no perfect reference test for diagnosis of Johne's disease. J Dairy Sci 89:3038-46.
PROJECT CONTACT:
Name: Kapur, V.
Phone: 612-625-7712
Fax: 612-626-0623
Email: vkapur@umn.edu

 
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ACCESSION NO: 0200698 SUBFILE: CRIS
PROJ NO: MINV-62-054 AGENCY: CSREES MINV
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2004-35204-14756 PROPOSAL NO: 2004-01413
START: 01 AUG 2004 TERM: 31 JUL 2008 FY: 2007 GRANT YR: 2004
GRANT AMT: $454,506
INVESTIGATOR: Godden, S.
PERFORMING INSTITUTION:
Veterinary Population Medicine
Univ of Minnesota
St Paul , Minnesota 55108
EVALUATION OF CRITICAL CONTROL POINTS IN YOUNGSTOCK AND ADULT DAIRY COW MANAGEMENT TO REDUCE TRANSMISSION OF MYCOBACTERIUM AVIUM SUBSPECIES. . . . (Note: Title exceeded the title field character limit.)
NON-TECHNICAL SUMMARY: The modes of transmission of Mycobacterium paratuberculosis (Map) are inadequately understood, and the efficacy and cost-effectiveness of current recommended control programs have not been formally evaluated. This project will critically evaluate the efficacy, magnitude of impact, and cost-effectiveness of several commonly recommended practices surrounding young stock management, for reducing the transmission of Map in infected dairy herds.
OBJECTIVES: The long-term goal of this proposal is to complete a series of prospective controlled field studies designed to critically evaluate the efficacy, magnitude of impact, and cost-effectiveness of several commonly recommended practices surrounding the management of young stock and adult dairy cattle, for reducing the transmission of in infected dairy herds. This goal will be achieved through the following five objectives: 1. Evaluate the Effect of Maternity Pen Management on Transmission Mycobacterium avium subspecies paratuberculosis in Newborn Calves. 2. Evaluate the Effect of Off-Site Heifer Rearing on Transmission of Mycobacterium avium subspecies paratuberculosis in Youngstock. 3. Evaluate the Effect of Feeding of Bovine Colostrum (vs. a Commercial Colostrum Substitute) on the Risk for Transmission of Mycobacterium avium subspecies paratuberculosis in Newborn Calves. 4. Evaluate the Effect of Feeding Pasteurized Waste Milk (vs. Commercial Milk Replacer) to Control Transmission of Mycobacterium avium subsp. paratuberculosis in Dairy Calves. 5. Evaluate the Effect of Delaying Exposure to Mycobacterium avium subspecies paratuberculosis until Adulthood on the Development of New Infections in Adult Dairy Cows.
APPROACH: The first objective is to evaluate the effect of maternity pen management on M. paratuberculosis (Map) transmission. This will be evaluated in 4 large dairy herds with a high seroprevalence of Johnes disease (at least 10 percent of cows ELISA-positive). Each herd will randomly allocate 50 percent of cows to calve in individual pens (treatment) that are cleaned between uses. The remainder of cows will calve in the usual location (e.g. multiple cow loose housing maternity areas). This allocation will continue for up to 12 mos. After removal from calving areas, all replacement heifers will be raised segregated from adult cattle and their feces until 20 mos. of age. The second objective is to evaluate the effect of off-site heifer rearing on Map transmission. This will be conducted in 2 herds of greater than 1000 cows that currently have a high seroprevalence (greater than 10 percent ELISA positive cows). In each herd, 300 heifer calves will be randomly allocated to each treatment group (off-site or on-site rearing). Enrollment of calves will take approximately 12 mos. The third objective will continue a field study designed to evaluate the effect of feeding of bovine colostrum on Map transmission. The enrollment phase for this study was completed between July through Sept., 2003, wherein approximately 500 newborn heifer calves from 12 Midwest herds were removed from the dam within 30 to 60 minutes of calving and then systematically assigned to be fed either 4 L of fresh bovine colostrum or 2 L of a commercial colostrum substitute (Secure, A.P.C. Inc., Ames, IA). The fourth objective is to evaluate the effect of feeding pasteurized waste milk to control Map transmission. The enrollment phase of this study included was completed between Dec. 2001 and Oct. 2003 and involved one large commercial dairy farm with a 12-15 percent seroprevalence for Johnes infection. A total of 439 heifer and bull calves delivered to these cows were transported to an off-site heifer grower at 1-2 d of age, where they were systematically assigned to be fed either batch pasteurized waste milk (n equals 222) or a conventional 20:20 milk replacer (n equals 217). The fifth objective is to evaluate the effect of delaying exposure to Map until adulthood on the development of new MAP infection in adult cows. This will be evaluated by comparing rates of subclinical and clinical Johnes disease in cattle raised in an environment presumed free of Johnes disease to those of cattle raised in an environment presumed to be infected. Through collaboration with the MN Board of Animal Health, we have identified unclassified or high Johnes disease incidence herds that have purchased replacement cattle from uninfected herds. A total of 100 case cattle (exposed equals raised in low risk Johnes disease infected herds and introduced into Johnes disease infected herds as springing heifers) will be identified, along with 3 homebred control cows per case (non-exposed) matched within herd and by lactation number and stage of lactation. For all objectives, follow-up fecal culture and serum ELISA testing of study animals will performed on an annual basis at approx. 24 and 36 months of age.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Research activities on this project continued throughout 2007. Results from one early objective of the study was published in the peer-reviewed veterinary literature (J. American Veterinary Medical Assoc.). Preliminary results from other objectives were also presented at the Annual Meeting of the Minnesota Dairy Health Management Conference (+ published in proceedings), and at the 40th Midwestern sectional meeting of the American Society of Animal Science and the American Dairy Science Association. DesMoines, IA. Mar 19-21, 2007. PARTICIPANTS: Sandra Godden, University of Minnesota, Principal Investigator. Scott Wells, University of Minnesota, Co Principal investigator. Ian Gardner, University of California, Davis, Collaborator. Judy Stabel, USDA, Ames, IA, Collaborator. John Fetrow, University of Minnesota, Collaborator. Heather Swan, University of Minnesota, MPH graduate student. Patrick Pithua, University of Minnesota, PhD graduate student. Russ Bey, University of Minnesota, Collaborator. Hugh Chester-Jones, University of Minnesota, Collaborator. TARGET AUDIENCES: Dairy kProducers. Dairy Veterinarians, Dairy Extension Educators, Dairy Professionals (nutritionists, pharmaceutical companies, other), Professional Veterinary Students, Animal Science Students.
PROJECT MODIFICATIONS: None.
IMPACT: 2007/01 TO 2007/12
One major finding of an early objective in this study was that dairy calves fed pasteurized non-saleable milk have a significantly higher growth rate and lower morbidity and mortality rates than do calves fed conventional milk replacer, and that feeding pasteurized non-saleable milk could be an economically viable strategy for dairy calf producers. This work has been published and has had an impact on the U.S. dairy industry, which is seeing increasing adoption of on-farm commercial pasteurization systems for feeding non-saleable milk to calves. This practice should result in improved economic efficiencies for producers and improved calf health and welfare, where it is adopted.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
1. Swan, H., S. Godden, R. Bey, S. Wells, J. Fetrow, H. Chester-Jones. 2007. Passive transfer of immunoglobulin G and preweaning health in Holstein calves fed a commercial colostrum replacer. J Dairy Sci. 90:3857-3866.
2. Pithua, P., S.J. Wells, S.M. Godden. 2007. Effect of maternity pen management on neonatal calf health during the first 90 days of life. Proceedings of the Minnesota Dairy Health Conference. Pp 105
3. Pithua, P., S.M. Godden, S.J. Wells. 2007. Effect of feeding a commercial colostrum substitute on risk for transmission of Mycobacterium avium subsp. paratuberculosis in newborn calves descriptive preliminary data. Proceedings. of the 40th Midwestern sectional meeting of the American Society of Animal Science and the American Dairy Science Association. DesMoines, IA. Mar 19-21, 2007. Pp 23.
PROJECT CONTACT:
Name: Godden, S.
Phone: 612-625-8177
Fax: 612-625-6241
Email: godde002@umn.edu

 
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ACCESSION NO: 0204095 SUBFILE: CRIS
PROJ NO: MINV-62-055 AGENCY: CSREES MINV
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2005-35204-16106 PROPOSAL NO: 2005-01426
START: 01 AUG 2005 TERM: 31 JUL 2008 FY: 2007 GRANT YR: 2005
GRANT AMT: $350,000
INVESTIGATOR: Sreevatsan, S.
PERFORMING INSTITUTION:
Veterinary Population Medicine
Univ of Minnesota
St Paul , Minnesota 55108
STUDIES ON GENOTYPE-SPECIFIC HOST-PATHOGEN INTERACTIONS IN MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY : The proposed studies attempt to improve animal health and prevent disease caused by MAP through a program of fundamental research that will enable the development of superior diagnostic reagents, vaccines and antimicrobial agents.
OBJECTIVES: Johne's disease (JD), a chronic inflammatory disease caused by infection with Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most prevalent and costly diseases of cattle and sheep worldwide. Our long-term objective is to provide a comprehensive understanding of bacterial population genetics and host-pathogen interactions in MAP infections. Our preliminary studies have revealed substantial differences in survival among genotypically distinct MAP strains in bovine macrophages and strain-specific variation in transcript profiles. Thus, we hypothesize that genetically distinct subtypes of MAP differ in their effect on host macrophage and dendritic cells and these differences play a major role in determining the character of infection. In the current application, we propose to study the differential gene expression by the host and MAP strains in a peripheral blood monocyte derived macrophage and dendritic cell models. Toward this end, the following objectives are proposed: (1) Establish baseline, early and late post macrophage and dendritic cell-exposure gene expression profiles in genetically distinct and identical MAP strains isolated in the United States from a variety of host species. We propose to use a well-established enrichment approach termed, Selective Capture of Transcribed Sequences (SCOTS) to compare and contrast the differentially expressed genes from the genotypically distinct strains of MAP and (2) Establish baseline and post MAP-exposure cytokine and gene expression profiles in bovine macrophages and dendritic cells at the transcript and protein levels using well-established bovine total leukocyte microarrays and ELISAs, respectively. The proposed studies attempt to improve animal health and prevent disease caused by MAP through a program of fundamental research that will enable the development of superior diagnostic reagents, vaccines and antimicrobial agents.
APPROACH: We propose to use a well-established enrichment approach termed, Selective Capture of Transcribed Sequences (SCOTS) to compare and contrast the differentially expressed genes from the genotypically distinct strains of MAP and well-established bovine total leukocyte microarrays to interrogate host gene expression profiles.
PROGRESS: 2006/08 TO 2007/07
OUTPUTS: In the proposed research we hypothesized that genetically distinct subtypes of MAP differ in their gene expression as well as in their effect on host macrophage and dendritic cells and these differences play a major role in determining the character of infection. The following objectives were proposed: (1) Establish baseline, early and late post macrophage and dendritic cell-exposure gene expression profiles in genetically distinct and identical MAP strains isolated in the United States from a variety of host species and(2) Establish the host cell-responses to the same subset of isolates using a microarray-based transcriptional profiling. APPROACH: We used a well-established enrichment approach termed, Selective Capture of Transcribed Sequences (SCOTS) to compare and contrast the differentially expressed genes from the genotypically distinct strains of MAP and studied baseline and post MAP-exposure gene expression profiles in bovine macrophages at the transcript and protein levels using well-established bovine total leukocyte microarrays and ELISAs, respectively. PARTICIPANTS: Dr. Xiaochun Zhu. Univerity of Minnesota. (MS recipient) Dr. Harish Janagama. University of Minnesota (PhD Student) Dr. Paul Coussens. Michigan State University, East Lansing, MI Mr. Ed Kabaara. Michigan State University, East Lansing, MI (PhD Student) Dr. Zhu was awarded an MS in Veterinary medicine for his work on pathogen gene expression profiling in macrophages, by the University of Minnesota.
IMPACT: 2006/08 TO 2007/07
Progress toward Objective 1: In this study we asked if genotypic variation in MAP is associated with common themes in macrophage-induced gene expression. We applied selective capture of transcribed sequences (SCOTS) on three genotypically diverse MAP isolates from cattle, human, and sheep. Six cDNA libraries were created from the three MAP isolates exposed to primary bovine monocyte derived macrophages for 48 h and 120 h and sequenced. Sequence annotation against mycobacterial and sixteen actinobacteriaceae genomes revealed that at 48-h and 120-h post-infection (PI), the cattle isolate up-regulated 27 and 241 genes; the human isolate up-regulated 22 and 53 genes, and the sheep isolate up-regulated 35 and 358 genes, respectively. Thirteen to 33% of the genes identified did not have any annotated function and were classified as "unknown". In general, the sheep isolate showed higher transcriptional activity and up-regulated higher numbers of genes associated with oxidative stress and fatty acid biosynthesis. The human isolate appeared to enter and survive the intracellular environment without much transcriptional activity when compared with the animal isolates. Despite several variations in the genes identified, the patterns of expression fell into overlapping cellular functions as inferred by pathway analysis. For example, about 10-12% of the genes expressed by all three strains at each time point were associated with cell-wall biosynthesis. All three strains of MAP studied appeared to use pathways to combat oxidative stress, metabolic and nutritional starvation, and up-regulated genes involved in cell survival. Taken together, this comparative transcriptional analysis suggests that diverse MAP genotypes may use similar modus operandi for survival in the host. Progress toward Objective 2: In this study we hypothesized that MAP alters the transcription of a core group of genes for its intracellular survival. To test this hypothesis, we investigated host gene expression by infecting MDM from five uninfected Holstein cattle with 10 different strains of MAP strains from various host backgrounds. Differential gene expression was assessed using a bovine immune-specific microarray (BOTL5). The microarray data was statistically analyzed, resulting in the detection of 99 host genes with altered, either up- or down-regulated, expression. Of these 99 genes, 82 had significant annotations with grouping into several distinct functional categories. Using the DAVID analysis software, up-regulated and down-regulated gene sets were analyzed to measure enrichment in functional groups. Up-regulated genes were over-represented in DNA-binding functional groups while down-regulated genes showed enrichment in proteolytic functional groups. Cluster analysis was preformed using the Cluster 3.0 program to analyze similarity between host gene expression patterns of the 10 different MAP strains. The cluster analysis showed a possible link between bacterial phenotype and host gene alteration. Validation on a select number of the identified host genes is being performed by Q-RT-PCR.
PUBLICATIONS (not previously reported): 2006/08 TO 2007/07
1. Motiwala, A. S., H. Janagama, M. L. Paustian, X. Zhu, J. Bannantine, V. Kapur, and S. Sreevatsan. 2006. Microarray analysis of human macrophage transcriptional responses to different strains of Mycobacterium avium subspecies paratuberculosis. Infect Immun. 74(11):6046-56.
2. Janagama, H. K., K. I. Jeong, V. Kapur, P. Coussens, and S. Sreevatsan. 2006. Cytokine responses of bovine macrophages to diverse clinical Mycobacterium avium subspecies paratuberculosis strains. BMC Microbiol 6:10.
3. Motiwala, A. S., L. Li, V. Kapur, and S. Sreevatsan. 2006. Current understanding of the genetic diversity of Mycobacterium avium subsp. paratuberculosis. Microbes Infect 8:1406-1418.
4. Golnick, N. S., R. M. Mitchell, M. Baumgart, H. K. Janagama, S. Sreevatsan, and Y. H. Schukken. 2007. Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages is not affected by host infection status but depends on infecting bacterial genotype. Vet. Immunol. Immunopathol. Aug 2.
5 . Weiss, D., C. D Souza, O. Evanson, S. Sreevatsan. 2007. Cell membrane receptors on bovine mononuclear phagocytes involved in phagocytosis of Mycobacterium avium subsp. paratuberculosis. Am. J. Vet Res. 68(9):975-80.
6. Zhu, X., Z. Tu, P. M. Coussens, V. Kapur, H. Janagama, and S. Sreevatsan. 2007. Diverse strains of Mycobacterium avium subspecies paratuberculosis show Unifying Themes of Gene Expression in Primary Bovine Monocyte Derived Macrophages. Manuscript in preparation.
7. Zhu, X. 2007. Diverse strains of Mycobacterium avium subspecies paratuberculosis show Unifying Themes of Gene Expression in Primary Bovine Monocyte Derived Macrophages. Masters's Degree Thesis submitted to University of Minnesota.
PROJECT CONTACT:
Name: Sreevatsan, S.
Phone: 612-625-3769
Fax: 612-624-4906
Email: sreev001@umn.edu

 
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ACCESSION NO: 0204556 SUBFILE: CRIS
PROJ NO: MINV-63-028 AGENCY: CSREES MINV
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2005-35204-16198 PROPOSAL NO: 2005-01447
START: 01 AUG 2005 TERM: 31 JUL 2008 FY: 2007 GRANT YR: 2005
GRANT AMT: $350,000
INVESTIGATOR: Rutherford, M.
PERFORMING INSTITUTION:
Veterinary Biomedical Sciences
Univ of Minnesota
St Paul , Minnesota 55108
BOVINE MUCOSAL IMMUNE RESPONSE TO MYCOBATERIAL INFECTION: ROLE OF INTERLEUKIN-10 AND CD10 AND CD25 T-CELLS.
NON-TECHNICAL SUMMARY: Results of recent studies have established that Johne's disease is one of the most prevalent and costly diseases of ruminants worldwide. It has been estimated that 35% of U.S. dairy herds are infected with M. a. ptb. Beef herds are also infected. Johne's disease is also a threat to wild ruminant populations, including deer, elk, and moose. Most investigators agree that eradication of Johne's disease will be difficult or impossible because M. a. ptb has a long subclinical period, survives for long periods in the environment, is highly resistant to disinfectants, and is widely spread throughout wild animal and bird populations. Although current vaccines have some capacity to reduce the occurrence of clinical disease, they have not been effective in preventing infection or eradicating the disease. Therefore, we propose that it is essential to understand host-organism interactions that lead to chronic infection. Because the infection is localized in the intestinal tract, it is particularly important to study innate and adaptive immune responses occurring in the lamina propria. To address this, we propose to undertake in vitro studies, using bovine mononuclear leukocytes and in vivo studies of mucosal immune responses of calves experimentally infected with M. a. ptb.
OBJECTIVES: Specific Aim #1: To determine the role of MAPK-p38 in regulating expression of IL-10 in bovine mononuclear phagocytes ingesting M. a. ptb organisms. The working hypothesis for this research aim is that survival of M. a. ptb within macrophages is largely due to the capacity of the organism to induce phosphorylation of MAPK-p38 with subsequent expression of IL-10. To address this hypothesis, we will evaluate the effect of specific inhibitors (chemical and interfering RNA) of MAPK-p38 on IL-10 production and transcription factor binding to the IL-10 promoter. Specific Aim 2: To determine the biological significance of MAPK-p38 signaling in the interaction of mononuclear phagocytes with M. a. ptb organisms. The working hypothesis for this research aim is that MAPK-p38 enhances IL-10 production and downregulates IL-12 production, thereby attenuating the capacity of macrophages to mount an antimicrobial response. To address this hypothesis, we will incubate bovine mononuclear phagocytes with M. a. ptb organisms with and without addition of MAPK-p38 siRNA and determine the capacity of macrophages to ingest and kill the organisms, express IL-10 and IL-12 mRNA, and acidify phagosomes. To define novel effects of MAPK-p38 inhibition on macrophage-M. a. ptb interactions, we will conduct microarray studies. Specific Aim 3: To determine the role of IL-10 and regulatory T cells in attenuating mucosal immune responses in calves experimentally infected with M. a. ptb. The working hypothesis for this research aim is that overproduction of IL-10 attenuates innate and adaptive immune responses in the mucosa. To address this hypothesis, we will evaluate the mucosal immune response of calves at multiple time points after oral inoculation of M. a. ptb organisms.
APPROACH: We will use both in vitro and in vivo approaches to study the pathogenesis of Johne's disease. In the invitro studies, we will evaluate cell signaling pathways that are involved in attenuating inflammatory and immune responses when macrophages ingest M. a. ptb organisms. Our preliminary studies have identified interleukin-10 (IL-10) as a critical factor in attenuating inflammatory and immune responses and indicate that M. a. ptb-induced IL-10 production is primarily mediated by the p38 mitogen-activated protein kinase pathway. In the in vivo studies, we will infect calves with M. a. ptb organisms and investigate the role of IL-10 and CD25+ regulatory T cells in preventing an effective immune response to the organism.
PROGRESS: 2005/08 TO 2006/07
We have investigated the interaction of Mycobacterium avium subsp. paratuberculosis (MAP), the causative organism in Johne's disease, with bovine monocytes to better understand how the organism enters and survives within the host cell. We specifically investigated host cell membrane receptors involved in MAP adhesion, phagocytosis, and initiation of cell signaling responses. We further investigated monocyte cell signaling initiated by MAP that lead to survival within monocytes. The following results were obtained. 1. MAP phagocytosis is controlled primarily by integrin receptors (CD11CD18) on the monocyte surface and to a lesser extent by mannose receptors. 2. Incubation of MAP with monocytes results in rapid activation of p38, JNK, and ERK1/2mitogen-activated protein kinases (MAPK) in monocytes. 3. Blocking experiments indicate that the MAPK-p38 pathway induces IL-10 expresses and suppresses IL-12 expression in MAP-infected monocytes. 4. Blocking the MAPK-p38 pathway increased phagosome acidification and increased the capacity of monocytes to kill MAP organisms. 5. Blocking experiments indicate that the MAPK-JNK pathway stimulates tumor necrosis factor-alpha expression but suppress interleukin-1, interleukin-12, and interleukin-18 expression in MAP-infected monocytes. 6. Blocking experiments indicate that the MAPK-ERK pathway stimulates tumor necrosis factor alpha expression in MAP-infected monocytes.
IMPACT: 2005/08 TO 2006/07
In this study, we are trying to determine the mechanisms Mycobacterium avium subsp. paratuberculosis (MAP) organisms use to survive within macrophages. Once we know this, we expect to be able to design novel vaccines to prevent this disease. We have shown that one of the major ways that MAP avoids being destroyed is by inducing the macrophage to produce large amounts of interleukin-10 which is a major inhibitor of inflammatory and immune responses. In this study, we have shown that interleukin-10 expression is activated through the p38 mitogen-activated protein kinase pathway.
PUBLICATIONS (not previously reported): 2005/08 TO 2006/07
No publications reported this period
PROJECT CONTACT:
Name: Rutherford, M.
Phone: 612-625-4281
Fax: 612-625-5203
Email: ruthe003@umn.edu

 
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ACCESSION NO: 0098315 SUBFILE: CRIS
PROJ NO: MINV-65-006 AGENCY: CSVM MINV
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 OCT 1983 TERM: 30 JUN 2008 FY: 2007
INVESTIGATOR: Collins, J. E.; Goyal, S.
PERFORMING INSTITUTION:
Veterinary Diagnostic Medicine
Univ of Minnesota
St Paul , Minnesota 55108
ANIMAL DISEASE DIAGNOSTIC LABORATORY.
OBJECTIVES: Provide laboratory diagnostic service to poultry and livestock producers and veterinary practitioners for State of Minnesota, conduct preliminary investigations and initiate minor research on new animal disease problems, develop improved diagnostic procedures for selected disease problems.
APPROACH: Case histories and diagnostic laboratory data will be recorded for each case presented. Appropriate laboratory procedures will be employed for each case or specimen. Field diagnostic investigations will be made for animal disease problems as necessary. Efforts will be made to improve existing diagnostic laboratory procedures through minor research studies and new diagnostic procedures will be utilized when developed.
PROGRESS: 2006/01 TO 2006/12
The number of cases submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) increased from 66,801 in 2005 to 69,123 in 2006. The number of procedures also increased from 1,364,618 in 2005 to 1,474,680 in 2006. Paratuberculosis (Johne's disease), bovine viral diarrhea, and enteritis by rotavirus, coronavirus, and Salmonella were the major problems. Tuberculosis was also detected in five Minnesota beef herds and one wild white-tailed deer. In pigs the major problems were caused by porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus, and swine influenza virus. Avian pneumovirus continues to be a problem for the turkey industry. Fewer Minnesota deer were tested for chronic wasting disease this year because no cases of this disease were found in the last 4 years of testing.
IMPACT: 2006/01 TO 2006/12
The Veterinary Diagnostic Laboratory plays an important role in protecting the health of animals as well as to protect the public from food borne and zoonotic diseases.
PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
1. Goyal, S.M. (Ed.). 2006. Viruses in Foods. Springer, New York, NY, 345 pp.
2. Tiwari, A., Patnayak, D.P., and Goyal, S.M. 2006. Attempts to improve on a challenge model for subtype C avian pneumovirus. Avian Pathol. 35:117-121.
3. Patnayak, D.P., and Goyal, S.M. 2006. Duration of immunity engendered by a single dose of cold adapted strain of avian pneumovirus. Can. J. Vet. Res. 70:65-67.
4. Allwood, P.B., Malik, Y.S., Maherchandani, S., Hedberg, C.W., and Goyal, S.M. 2006. Effect of temperature on the survival of F-specific RNA coliphage, feline calicivirus, and Escherichia coli in chlorinated water. Int. J. Environ. Res. Publ. Hlth. 2:442-446.
5. Clay, S., Maherchandani, S., Malik, Y.S., and Goyal, S.M. 2006. Survival on uncommon fomites of feline calicivirus, a surrogate of noroviruses. Am. J. Infect. Control. 34:41-43.
6. Malik, Y.S., Allwood, P.B., and Goyal, S.M. 2006. Disinfection of fabrics and carpets artificially contaminated with calicivirus: relevance in institutional and health care centers. J. Hosp. Infect. 63:205-210.
7. Malik, Y.S., and Goyal, S.M. 2006. Virucidal efficacy of sodium carbonate against FCV, a Norovirus surrogate. Int. J. Food Microbiol. 109:160-163.
8. Chander, Y., Goyal, S. M. and Gupta, S. C. 2006. Antimicrobial resistance of Providencia sp. isolated from animal manure. Vet. J. 172:188-191.
9. McMartin, S., Godden, S., Metzger, L., Feirtag, J., Bey, R., Stabel, J., Goyal, S., Fetrow, J.,Wells, S., and Chester-Jones, H. 2006. Heat-Treatment of Bovine Colostrum. I: Effects of temperature on viscosity and immunoglobulin G level. J. Dairy Sci. 89:2110-2118.
10 . Tiwari, A., Patanayak, D.P. and Goyal, S.M. 2006. Survival of two avian respiratory viruses on porous and nonporous surfaces. Avian Dis. 50:284-287.
11. Farnsworth, J.E., Goyal, S.M., Kim, S.W., Kuehn, T.H., Raynor, P.C., Ramakrishnan, M.A., Anantharaman, S., and Tang, W. 2006. Development of a method for bacteria and virus recovery from Heating, Ventilation, and Air Conditioning (HVAC) filters. J. Environ. Monit. 8:1006-1013.
12. Godden, S., McMartin, S., Feirtag, J., Stabel, J., Bey, R., Goyal, S., Metzger, L., Fetrow, J., Wells, S., and Chester-Jones, H. 2006. Heat-Treatment of Bovine Colostrum II: Effects of Heating Duration on Pathogen Viability and Immunoglobulin G. J. Dairy Sci. 89:3476-3483.
13. Vincent AL, Lager KM, Ma W, Lekcharoensuk P, Gramer MR, Loiacono C, Richt JA. 2006. Evaluation of hemagglutinin subtype 1 swine influenza viruses from the United States. Vet Microbiol. 118:212-22.
14. Ma W, Gramer M, Rossow K, Yoon KJ. 2006. Isolation and genetic characterization of new reassortant H3N1 swine influenza virus from pigs in the midwestern United States. J Virol. 80:5092-6.
15. Chou J, Wunschmann A, Hodzic E, Borjesson DL. 2006. Detection of Borrelia burgdorferi DNA in tissues from dogs with presumptive Lyme borreliosis. J Am Vet Med Assoc. 229:1260-5.
16. Wunschmann A, Ziegler A. 2006. West Nile virus-associated mortality events in domestic Chukar partridges (Alectoris chukar) and domestic Impeyan pheasants (Lophophorus impeyanus). Avian Dis. 50:456-9.
17. Finno CJ, Valberg SJ, Wunschmann A, Murphy MJ. 2006. Seasonal pasture myopathy in horses in the midwestern United States: 14 cases (1998-2005). J Am Vet Med Assoc. 229:1134-41.
18. Dean J, Latimer KS, Oaks JL, Schrenzel M, Redig PT, Wunschmann A. 2006. Falcon adenovirus infection in breeding Taita falcons (Falco fasciinucha). J Vet Diagn Invest. 18:282-6.
19. Fano E, Jiang Y, Faaberg K, Murtaugh MP, Guedes A, Collins JE, Joo HS. 2006. The impact of animal age, bacterial coinfection, and isolate pathogenicity on the shedding of porcine reproductive and respiratory syndrome virus in aerosols from experimentally infected pigs. Can J Vet Res. 70:297-301.
20. Wells SJ, Collins MT, Faaberg KS, Wees C, Tavornpanich S, Petrini KR, Collins JE, Cernicchiaro N, Whitlock RH. 2006. Evaluation of a rapid fecal PCR test for detection of Mycobacterium avium subsp. paratuberculosis in dairy cattle. Clin Vaccine Immunol. 13:1125-30.
21. Cho JG, Dee SA, Deen J, Guedes A, Trincado C, Fano E, Jiang Y, Faaberg K, Collins JE, Murtaugh MP, Joo HS. 2006. Evaluation of the effects of animal age, concurrent bacterial infection, and pathogenicity of porcine reproductive and respiratory syndrome virus on virus concentration in pigs. Am J Vet Res. 67:489-93.


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ACCESSION NO: 0205414 SUBFILE: CRIS
PROJ NO: MOV-2-FF39 AGENCY: CSREES MO.V
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: EXTENDED
START: 01 OCT 2005 TERM: 30 SEP 2007 FY: 2007
INVESTIGATOR: Schultz, L.; Nagy, D. W.
PERFORMING INSTITUTION:
Veterinary Medicine & Surgery
University of Missouri
Columbia , Missouri 65211
SPECIFICITY OF SEROLOGY IN THE DIAGNOSIS OF JOHNE'S DISEASE IN ENDEMIC HERDS.
NON-TECHNICAL SUMMARY: Reported specificity of the Johne's ELISA is 95 to 100 percent. The documented performance of this test is much higher than we perceive clinically. A lower specificity is supported by our preliminary data set. The proposed research will provide a more accurate assessment of the specificity of serologic tests for Mycobacterium aviumparatuberculosis.
OBJECTIVES: The scientific objectives of the project are to: 1) characterize the specificity of the ELISA test in herds with infected cattle, and 2) evaluate the utility of expanded testing protocols in serologically positive dairy cows.
APPROACH: Over a 2-year period, 70 cattle will be drawn from dairy herds that are currently enrolled in the Missouri State Johne's Control Program. Herds with an apparent prevalence of plus-minus 10 percent will be eligible for participation in the study. Sampling will be restricted to cattle with positive ELISA results associated with annual whole herd serologic testing. Client-owned animals with one positive serologic test for Mycobacterium avium subspecies paratuberculosis will be eligible for inclusion. Inducements for participation will be explained. Enrolled cattle will either be transported to the Univ of Missouri Food Animal Clinic or a sampling team will travel to the farm for collection. True positive status will be defined as a cow with a positive isoltion of MAP from either biopsied tissue or feces. Cattle with initial negative culture results later identified as positive by follow up of fecal cultures will be classified as true positives as well. The specificity of the ELISA after initial sampling and after follow up fecal cultures on cows with initial negative cultures will be calculated. After completion of the data collection, an economic analysis of the intensive sampling protocol will be performed using decision tree methodology. The models used will permit all possible permutations of testing strategies.
PROGRESS: 2006/01 TO 2006/12
This proposal was originally prepared as a two year proposal. Over the past year complete samples were obtained from 5 cows. While these samples have added to our preliminary data set, it is far short of the projected 35 for the first year. Despite identification of possible candidates, enrollment has been challenging.
IMPACT: 2006/01 TO 2006/12
We hope to generate important data on the predictive value and performance of various testing modalities for Johnes disease. This information will provide a more sound basis for testing strategies in current control programs.
PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
No publications reported this period
PROJECT CONTACT:
Name: Dusty Nagy
Phone: 573-882-6857
Fax: 573-884-5444
Email: NagyD@missouri.edu

 
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ACCESSION NO: 0056879 SUBFILE: CRIS
PROJ NO: MONB00022 AGENCY: SAES MONB
PROJ TYPE: STATE PROJ STATUS: REVISED
START: 01 OCT 2006 TERM: 30 SEP 2011 FY: 2006
INVESTIGATOR: Quinn, M. T.
PERFORMING INSTITUTION:
Veterinary Molecular Biology
Montana State University
Bozeman , Montana 59717
MISCELLANEOUS INVESTIGATIONS IN VETERINARY MOLECULAR BIOLOGY.
NON-TECHNICAL SUMMARY: Infectious disease causes considerable loss for livestock producers by reducing production of animal units and by reduced sales because of food safety concerns. Veterinary Molecular Biology (VMB) is the only research unit in Montana focused on animal health, particularly on the study of infectious diseases of cattle. New faculty members joining VMB are required to initiate new research projects. In addition, other faculty not on Montana Agricultural Experiment Station (MAES) funding may be hired to develop new short-term projects. These projects are in support of the respective missions of MAES and VMB. This departmental project is to be used by these scientists prior to their approved MAES project. Support is also provided to maintain and operate departmental research facilities.
OBJECTIVES: The short and long term objectives of research in VMB are the development of new drugs, vaccines, and diagnostic tools for fighting infectious diseases of livestock. This includes scholarly discovery and dissemination of science and technology related to diseases affecting livestock and wildlife, as well as zoonotic diseases that can be transmitted to humans.
APPROACH: VMB scientists utilize state-of-the-art molecular approaches to address basic and applied problems in infectious disease research. These research programs require laboratories, large and small animal facilities, clinics, and modern research equipment, such as flow cytometers, DNA sequencers, and genomics analysis facilities. Specific methods and procedures utilized are dependent upon program type and necessary protocols. New or existing faculty members must develop an understanding of Montana and regional issues and a more in-depth understanding of existing research programs, prior to developing their own MAES project proposals.
PROGRESS: 2007/01 TO 2007/12
Funds were used to support seed projects for three junior faculty members in VMB. Dr. Jovanka Voyich is pursuing the strain specific identification of Staphylococcus aureus isolates causing mastitis in Montana Dairy Farms. Dr. Jay Radke is studying gene expression in bovine monocytes infected with Mycobacterium avium ssp. paratuberculosis. Dr. Robert Cramer is investigating the role of aspergillus infection in death of honey bee colonies.
IMPACT: 2007/01 TO 2007/12
All of these seed projects focus on infectious diseases important to Montana and the Nation. The impact is high, and it is hoped that these projects will lead to a better understanding of these diseases.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
No publications reported this period
PROJECT CONTACT:
Name: Quinn, M. T.
Phone: 406-994-4705
Fax: 406-994-4303
Email: mquinn@montana.edu

 
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ACCESSION NO: 0208406 SUBFILE: CRIS
PROJ NO: MONB00588 AGENCY: CSREES MONB
PROJ TYPE : ANIMAL HEALTH PROJ STATUS: EXTENDED
START: 01 OCT 2006 TERM: 30 SEP 2008 FY: 2006
INVESTIGATOR: Radke, J. R.
PERFORMING INSTITUTION:
Veterinary Molecular Biology
Montana State University
Bozeman , Montana 59717
ANALYSES OF GENE EXPRESSION IN BOVINE MONOCYTES INFECTED WITH MYCOBACTERIUM AVIUM SSP. PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY:Mycobacterium avium ssp. paratuberculosis infects an estimated 22 percent of commercial beef and dairy cattle herds in the US and results in production losses that approach $250 million annually. These experiments will identify host cell mRNAs altered in response to M. paratuberculosis infection.
OBJECTIVES: We will use Affymetrix genechip technology and the Bovine Genome Chip to compare gene expression in bovine monocytes infected with Mycobacterium avium ssp. paratuberculosis and Mycobacterium avium ssp. avium.
APPROACH: Johne's disease continues to have a major economic impact on the beef and dairy cattle industry in the US [1, 2]. The inability to control this disease is based on lack of early detection [37], effective therapeutic intervention once detected [39] and the absence of a fully efficacious vaccine [42, 43]. Unlike, M. paratuberculosis,M. avium does not cause disease in ruminants and can be cleared effectively by the host response. We believe the molecular elements underlying the differential fate of these two closely related Mycobacteria will identify specific bovine mRNAs pertinent to host-pathogen interactions underlying persistence and disease. We will use the Affymetrix genechip technology and the Bovine Genome Array containing > 24,000 sequences to complete a functional genomic analysis of expressed mRNAs in infected bovine monocytes. Gene expression profiles at 2 h post-infection compared with those 24 h post-infection will distinguish between early and late (er) populations which may uncover alterations relevant to acidification of the phagosome, induction of apoptotic pathways and influences on host cell signaling that may direct pathogenesis.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: We have conducted a preliminary analyses of altered gene expression in monocyte-derived bovine marophages infected with Mycobacterium avium ssp. avium (MAV) and Mycobacterium paratuberculosis ssp. paratuberculosis (MAP) using the Affymetrix Bovine Chip Array. We have assessed mRNA levels at 2 and 24 h post-infection and quantified changes when comparing infection with avirulent MAV and virulent MAP isolates. Comparison of mRNA levels in primary bovine macrophages infected with MAP identified 156 mRNAs altered 5-fold when compared with uninfected controls and 172 are altered by infection with MAV. Subsequent analyses identified 108 mRNAs commonly altered by both MAP and MAV infection; and 48 mRNA altered by MAP infection alone, compared with 64 after infection with MAV (Fig. 1A and Table 1). Direct comparison of host mRNAs altered by MAV and MAP identified 135 differentially altered after infection. MAV infection altered the levels of 101 mRNAs over those seen by infection with MAP compared with 34 mRNAs altered by MAP PARTICIPANTS: Jay Radke, PhD Veterinary Molecular Biology Montana State University Bozeman, Montana 59717 Philip D. Fox Veterinary Molecular Biology Montana State University Bozeman, Montana 59717 Amy Eibs, Veterinary Molecular Biology Montana State University Bozeman, Montana 59717
IMPACT: 2007/01 TO 2007/12
Analyses of gene expression results are in progress.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
No publications reported this period
PROJECT CONTACT:
Name: Radke, J. R.
Phone: 406-994-3799
Fax: 406-994-4303
Email: jradke@montana.edu

 
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ACCESSION NO: 0406865 SUBFILE: CRIS
PROJ NO: 3625-32000-062-05S AGENCY: ARS 3625
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: TERMINATED
START: 28 FEB 2003 TERM: 01 JAN 2005
INVESTIGATOR: Bannantine J P; Barletta R
PERFORMING INSTITUTION:
University of Nebraska
Lincoln , Nebraska 68583
FUNCTIONAL GENOMIC ANALYSIS OF MYCOBACTERIUM PARATUBERCULOSIS ( UNIV. OF NE).
OBJECTIVES: The overall objective of this agreement is the development of diagnostic tests and control measures for Johne's Disease in cattle as outlined in the USDA-NRI grant #2002-02228. The specific objectives of this cooperative research project are to identify unique antigens from the genome of Mycobacterium paratuberculosis, incorporate these antigens into a diagnostic test, and evaluate this test in dairy cattle herds.
APPROACH: The genome sequence of a bovine isolate of M. paratuberculosis will be analyzed to find DNA sequences which differentiate between M. paratuberculosis and other closely related mycobacteria. Efforts will then be made to clone, express and purify proteins from these novel M. paratuberculosis sequences. Because of the potential for 50 or more genes to be evaluated by these labor intensive methods, purification of proteins encoded by unique genes must be split between both parties. The resulting purified proteins will be evaluated in a variety of immunological assays to determine which ones are best for diagnostic test development. The test will be optimized and used in whole-herd field tests of known and unknown infection status.
PROGRESS: 2003/02 TO 2005/01
4d Progress report. This report serves to document research conducted under a specific cooperative agreement between ARS and the University of Nebraska. This specific cooperative agreement represents a subcontract of USDA-CSREES funds to the University of Nebraska. Additional details of research in the report for the parent project 3625-32000-062-00D Understanding Host- Pathogen Interactions for the Diagnosis and Control of Paratuberculosis (Johne's). This collaborative research project between ARS (NADC) and the University of Nebraska encompasses measures to ensure animal health through an increased understanding of the biology of Mycobacterium paratuberculosis, the bacterial agent that causes Johne's disease. This project has the objective of purifying recombinant M. paratuberculosis proteins. No new progress has been made over this past year as proteins have been purified in previous years.
PUBLICATIONS (not previously reported): 2003/02 TO 2005/01
No publications reported this period.

 
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ACCESSION NO: 0411458 SUBFILE: CRIS
PROJ NO: 5438-32000-026-01T AGENCY: ARS 5438
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 01 NOV 2006 TERM: 31 MAY 2007
INVESTIGATOR: Wells J; Shackelford S D; Wheeler T L; Harhay D M; Guerini M N; Kalchayanand N
PERFORMING INSTITUTION:
Agricultural Research Service
Clay Center, Nebraska 68933
PREVALENCE OF MYCOBACTERIUM AVIUM PARATUBERCULOSIS IN LYMPH NODES & ON HIDES & CARCASSES FROM CULL COW & FED CATTLE SLAUGHTER PLANTS.
OBJECTIVES: Determine the prevalence of Mycobacterium avium subspecies paratuberculosis in cattle ileocecal lymph nodes, on cattle hides, and on pre-evisceration and post-intervention carcasses in cull cow and fed cattle.
APPROACH: Cull cow and fed cattle slaughter plants will be sampled in different regions of the U.S. with multi-member crews. Samples for hide, pre-evisceration carcass, post-intervention carcass and lymph nodes will be obtained. Bacterial DNA will be extracted from bacterial pellets collected from each sample. Extracted bacterial DNA will be subjected to PCR analysis using primers targeting MAP-specific IS900 repeat genome sequences and analyzed using gel electrophoresis. Bacterial pellets from carcass swabs will be decontaminated and enriched using standard techniques. MAP enrichments will be sampled, and bacterial DNA will be extracted and analyzed using PCR.

 
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ACCESSION NO: 0153376 SUBFILE: CRIS
PROJ NO: NEB-14-059 AGENCY: SAES NEB
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 13 DEC 1990 TERM: 31 DEC 2020 FY: 2006
INVESTIGATOR: Schmitz, J. A.; Doster, A. R.; Johnson, J. L.
PERFORMING INSTITUTION:
Veterinary and Biomedical Sciences
University of Nebraska
Lincoln , Nebraska 68583
VET DIAGNOSTIC LAB SYSTEM: DIAGNOSTIC SURVEILLANCE & DISEASE INVESTIGATION IN NE LIVESTOCK & POULTRY.
NON-TECHNICAL SUMMARY : Diagnosis of animal diseases requires sophisticated technology, equipment and trained personnel. This project provides personnel expertise equipment and facilities for disease investigation and diagnosis.
OBJECTIVES: 1. Esta,+k!a management structure & operating program for a unified state- wide veterinary diagnostic laboratory system. 2. Provide timely & accurate disease diagnosis & surveillance for the Nebraska livestock industry. 3. Provide information & data for research projects currently conducted in the Department of Veterinary Science. Focus attention on emerging disease syndromes which need additional investigation. 4. Provide continuing education for students, clients, and veterinary producers through formal class session, off-campus continuing education programs, interaction with clients on case materials, and contact with departmental extension veterinarians.
APPROACH: 1. Establish a single revolving account for all three laboratories for expenditures of funds. 2. Discontinue inter-laboratory charging. 3. Establish base budgets from appropriated funds for each laboratory & seek funding on the basis of a unified front. 4. Establish uniform quality control practices to insure a high degree of accuracy. 5. Develop a computer system to manage laboratory records at all three laboratories. 6. Prepare monthly summaries of laboratory activities, disease trends, seasonal patterns, etc.
PROGRESS: 2006/10 TO 2007/09
The lab received over 15,000 requests for diagnostic assistance from producers. Foreign animal diseases are included in the differentials and excluded based on laboratory examination or clinical data. We assist state health officials with monitoring programs for M paratuberculosis, avian influenza, Newcastle disease, classical swine fever, CWD and West Nile virus. Testing for BVDV PI status was performed on over 200,000 calves. Positive animals are removed from production to prevent spread of virus. The BVDV testing was compared to PCR pooling strategies in side by side comparisons of ELISA, IHC and pooled PCR. On over 6,000 samples. It was determined that accuracy of PCR was satisfactory on pools of 10 samples and perhaps up to twenty, but was reduced beyond that. Robust testing. A poster was presented at the AAVLD meeting and a publication is in preparation. CWD prevalence studies in Nebraska are ongoing and the disease appears to be stable and endemic in the western aspects of the state. We continued to support Johnes disease control programs and evaluation of different testing strategies and PCR sensitivity issues. Dwarfism investigations continued and DNA samples shared with ISU for genetic analysis resulted in identification of a marker for the disease. A commercial test has been licensed. Investigations into deaths of wildlife and zoo animals led to recognition of a novel herpes virus in Hyrax. The isolate was sequenced and a poster presented and manuscript is being presented. The full host range is still undetermined. We also provided Immunohistochemical analysis on alpaca samples for characterization of BVDV persistent infection in alpacas and for determination of the prevalence of acute and persistent infection of aplaca's with BVDV. A poster was presented and papers are in progress.
IMPACT: 2006/10 TO 2007/09
BVDV infections rate at 1% means over 1,500 persistently infected calves, the reservoir for virus were eliminated from production facilities. We determined that some widely used BVDV pooled testing approaches were insensitive and unfit for some uses and made the sceintific community aware of these issues. We determined the scope of the BVDV problem for the alpaca industry and characterized acute and persistent disease in that species. Routine surveillance testing supports free movement of livestock products across state and national boundaries and identifies endemic diseases providing useful data for management and treatment of diseases that affect livestock profitability. The CWD and Johnes surveys will provide base line statistically valid prevalence data for the state so that effectiveness of intervention can be measured. Identification of and publications describing, emerging diseases (hyrax herpes, alpaca BVDV, dwarfism) of domestic and wild animals aids those responsible for animal health in humane management of those resources.
PUBLICATIONS (not previously reported): 2006/10 TO 2007/09
No publications reported this period
PROJECT CONTACT:
Name: Schmitz, J. A.
Phone: 402-472-2952
Fax: 402-472-9690
Email: JSchmitz@unl.edu

 
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ACCESSION NO: 0181124 SUBFILE: CRIS
PROJ NO: NEB-14-103 AGENCY: CSREES NEB
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 NOV 2003 TERM: 31 OCT 2008 FY: 2006
INVESTIGATOR: Cirillo, J. D.
PERFORMING INSTITUTION:
Veterinary and Biomedical Sciences
University of Nebraska
Lincoln , Nebraska 68583
PATHOGENIC MECHANISMS OF BACTERIAL RESPIRATORY PATHOGENS .
NON-TECHNICAL SUMMARY : Respiratory problems are the number one cause of death in most livestock. This project is designed to develop novel methods for the prevention and treatment of respiratory infections.
OBJECTIVES: Respiratory infections are the number one cause of morbidity and mortality in cattle, swine and humans. Similarities have been observed between the mechanisms of pathogenesis of a number of bacterial lung pathogens. The identification of genes that are common to pathogens that infect specific organs may lead to effective methods for vaccination and antibiotic therapy. To whether the presence of similar pathogenic mechanisms in different bacterial pathogens indicates that they play an important role in the ability to cause disease we will: 1) Determine the mechanisms of macrophage infection by mycobacteria, 2) Evaluate the mechanisms of macrophage infection by Legionella and 3) develope molecular genetic systems for Francisella.
APPROACH: Through the use of molecular, cellular and genetic techniques we will examine the role of a number of genes involved in the pathogenesis of bacteria that cause lung infections. Through the analysis of the function, regulation and effect upon virulence we hope to develop a better understanding of the genes that are critical to the ability to cause lung diseases. This strategy is expected to result in novel methods for prevention and treatment of lung infections through the development of improved vaccines and antimicrobial agents.
PROGRESS: 2003/11 TO 2008/10
Respiratory pathogens are the number one cause of death in both domesticated animals and humans throughout the world. Respiratory problems, including bacterial infections, are the number one cause of mortality in cattle and calves leading to greater than 478 million dollars in economic loss in the U.S. Respiratory problems are also the number one cause of nursery deaths in swine. We have begun investigation of the causes of bacterial respiratory pathogens and the common molecular bases for pathogenesis. At present, we have identified more than 55 genes that are involved in virulence of respiratory pathogens and are in the process of constructing mutations in them. Careful characterization of the effects of different mutations on virulence has led to a better understanding of the mechanisms involved. In Legionella we recently used microarray analyses to identify a potential receptor for colonization that appears to be associated with smoking. This could be a link between smoking and this potentially lethal pneumonia. We have also identified more than 31 genes involved in the mechanism of macrophage infection by mycobacteria that are present in virulent mycobacterial species and is likely to be a key component of athogenesis in M. bovis and M. paratuberculosis. We have completed sequence and complementation analysis of more than 15 of these genes. Further studies are necessary to determine the host receptors and we have made significant progress toward understanding the signal transduction pathways involved. In addition, we have developed a novel model system for the study of the virulence mechanisms of fish pathogens. These studies are likely to have a significant impact on our understanding of respiratory pathogens as well as important pathogens in aquaculture. Thus, we have made significant progress in our characterization of virulence determinants in respiratory pathogens and have been able to demonstrate that the determinants we have isolated play an important role in pathogenesis.
IMPACT: 2003/11 TO 2008/10
Respiratory Infections in the cattle and swine industry lead to greater than 478 million dollars in economic loss in the U.S. Our laboratory has developed methods that are likely to be useful in the prevention and treatment of these infections. In addition, we have made significant progress in our characterization of virulence determinants in respiratory pathogens and have been able to demonstrate that these factors play an important role in respiratory diseases in animals.
PUBLICATIONS (not previously reported): 2003/11 TO 2008/10
1. R. Bartzatt, S.L.G. Cirillo, J.D. Cirillo. 2004. Molecular Properties and Antibacterial Activity of the Methyl and Ethyl Ester Derivatives of Ampicillin. Physiol. Chem. Phys. Med. NMR. 36:85-94
2. E. Miltner, K. Daroogheh, P.K. Mehta, S.L.G. Cirillo, J.D. Cirillo, L.E. Bermudez. 2005. Identification of Mycobacterium avium Genes That Affect Invasion of the Intestinal Epithelium. Infect. Immun. 73:4214-4221
3. S.L.G. Cirillo, P.K. Mehta, A.K. Pandey, S. Subbian, B. Park, M. Khounlotham, S.H. El-Etr, M.M. Samrakandi and J. D. Cirillo. 2005. Identification of Mycobacterial Genes That Affect Interactions With Macrophages. US- Japan Coop. Med. Sci. Prog., submitted: 40:77-81
PROJECT CONTACT:
Name: Cirillo, J. D.
Phone: 402-472-8587
Fax: 402-472-9690
Email: jcirillo1@unl.edu

 
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ACCESSION NO: 0184662 SUBFILE: CRIS
PROJ NO: NEB-14-108 AGENCY: CSREES NEB
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 1999 TERM: 30 SEP 2005 FY: 2005
INVESTIGATOR: Barletta, R. G.
PERFORMING INSTITUTION:
Veterinary and Biomedical Sciences
University of Nebraska
Lincoln , Nebraska 68583
MOLECULAR GENETIC ANALYSIS OF MYCOBACTERIUM PARATUBERCULOSIS AND RELATED MYCOBACTERIAL PATHOGENS.
NON-TECHNICAL SUMMARY:Mycobacterium paratuberculosis is the etiologic agent of Johne's disease, an infectious disease affecting all ruminants, characterized by diarrhea, weight loss, and ultimately death. The purpose of this study is to learn more about the mechanisms of pathogenesis with the goals of developing more effective disease control strategies.
OBJECTIVES: 1) Develop a selection strategy to identify M. paratuberculosis transposon mutants with reduced replication in bovine macrophages; 2) characterize candidate attenuated mutants at the molecular level and test their virulence in animal models; 3) identify, clone, overproduce, and characterize immunogenic M. paratuberculosis secreted and cellular proteins; and 4) identify drugs or drug combinations effective against M. paratuberculosis.
APPROACH: Herein is proposed a novel methodology to select for mutants with reduced ability to survive and multiply in bovine macrophages from a transposon mutant bank. A fluoroquinolone will be used to kill intracellularly growing bacteria and select for mutants unable to replicate in bovine macrophages. For mutants that demonstrate an attenuated phenotype in their interaction with bovine macrophages, the patterns of gene expression in intracellular and extracellular conditions will be compared and the virulence of candidate attenuated mutants will be tested in beige mice. To test the role of superoxide dismutase and alkylhydroperoxidase, the corresponding genes will be subcloned into E. coli expression vectors and purified in large amounts. The purified antigens will be used in assays for humoral and cellular immunity. To identify drugs or drug combinations effective against M. paratuberculosis, a variety of M. paratuberculosis strains isolated from cattle and Crohn's disease patients will be tested for drug susceptibility. M. paratuberculosis strains will be grown and transformed with plasmids carrying either the firefly luciferase or the green fluorescent protein reporter genes. The minimal inhibitory concentrations of individual drugs or pair-wise drug combinations will be determined in broth culture or M. paratuberculosis infected macrophages. For drug combination experiments, isobolograms will be constructed to determine if the drug interaction is indifferent, synergistic, or antagonistic.
PROGRESS: 1999/10 TO 2005/09
Johnes disease is a chronic enteritis with worldwide distribution and a significant impact on the world economy. In the United States, losses to the cattle industry have been estimated at $1.5 billion per year. Koch postulates have been applied to demonstrate that Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent. The objectives of this project were to: 1) develop a selection strategy to identify MAP transposon mutants with reduced replication in bovine macrophages; 2) characterize candidate attenuated mutants at the molecular level and test their virulence in animal models; 3) identify, clone, overproduce, and characterize immunogenic MAP secreted and cellular proteins; and 4) identify drugs or drug combinations effective against MAP. The following results were obtained during the lifetime of the project. In objective 1, a transposon mutant bank of MAP K-10 was created using the mycobacterial transposon Tn5367 carrying a kanamycin resistance marker. A total of 13,536 individual mutants were collected and evaluated for replication in a bovine macrophage cell line. Evaluations of bacillary ingestion and intracellular growth were performed by microscopic examination of acid fast-stained monolayers on glass coverslips. Results indicated that the fraction of macrophages infected with wild-type MAP K-10 is maintained over four days of incubation. During this period, a two-fold increase in the number of bacilli per infected macrophage was observed, demonstrating that MAP multiplies in tissue culture. These data were similar for the mutant strains tested. Additional experiments used the fluoroquinolone Bay y 3118 to selectively enrich pools of mutants for those unable to grow in minimal media lacking amino acids of interest. These mutants are not killed in the presence of the drug since they are not actively replicating. Using this positive selection, 107 auxotrophic mutants were isolated. In objective 2, nine colony morphology mutants were identified by visual screen of the complete mutant bank. One mutant (4H2) was shown to be replication deficient and thus attenuated in bovine macrophages. In objective 3, analysis of the MAP superoxide distmutase gene (sodA) indicated that the 207 amino acid encoded protein was secreted into culture supernatant fluids. The immunogenicity of antigen 85 and its potential as a subunit vaccine were also demonstrated. Furthermore, the immunodominant determinants of MAP protein P34 were analyzed and found to have diagnostic significance. In objective 4, the mutant bank was also screened to identify mutants susceptible to the peptidoglycan synthesis inhibitor D-cycloserine. We identified 63 mutants that were unable to grow at 20 microgram per ml of drug, while wild-type and most of the mutant strains were able to grow unimpaired under this condition. In addition, this project was instrumental in securing additional extramural funds from USDA competitive programs (USDA BARD IS-2564-95, USDA BARD US-3413-03, USDA-NRI 99-35204-7789, USDA-NRI 2004-35204-14231 and USDA-NRI 2004-35605-14243) necessary to expand our research.
IMPACT: 1999/10 TO 2005/09
Paratuberculosis and related mycobacterioses cause an estimated one billion dollars in annual losses to U.S. agriculture alone. Molecular genetic studies of MAP and related pathogens may aid in the development of a vaccine to control JD and bovine tuberculosis.
PUBLICATIONS (not previously reported): 1999/10 TO 2005/09
Thoen, C.O., and R. G. Barletta. 2005. Chapter 4: Pathogenesis, In Press. In C.O. Thoen, J.H. Steele and M.J. Gilsdorf (eds.) Mycobacterium bovis Infection in Animals and Humans, Second Edition. Blackwell Publishing
PROJECT CONTACT:
Name: Barletta, R. G.
Phone: 402-472-8543
Fax: 402-472-9690
Email: RBarletta@unl.edu

 
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ACCESSION NO: 0199138 SUBFILE: CRIS
PROJ NO: NEB-14-129 AGENCY: CSREES NEB
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO : 2004-35204-14231 PROPOSAL NO: 2003-02206
START: 01 FEB 2004 TERM: 31 OCT 2007 FY: 2006 GRANT YR: 2004
GRANT AMT: $270,000
INVESTIGATOR: Barletta, R. G.; Czuprynski, C. J.
PERFORMING INSTITUTION:
Veterinary and Biomedical Sciences
University of Nebraska
Lincoln , Nebraska 68583
MOLECULAR ANALYSIS OF A MYCOBACTERIUM PARATUBERCULOSIS COLONY-MORPHOLOGY ATTENUATED MUTANT.
NON-TECHNICAL SUMMARY : Mycobacterium paratuberculosis is the etiologic agent of Johne's disease, a chronic infectious disease of ruminants, characterized by diarrhea, weight loss, and ultimately death. The purpose of this study is to further characterize at the cellular, genetic and biochemical levels a colony-morphology mutant strain of the infectious agent shown to be attenuated by in vitro assays.
OBJECTIVES: Objective 1. Determine the molecular basis of the colony morphology and attenuated phenotype of Mycobacterium paratuberculosis mutant strain 4H2. The experimental design for this objective includes the use of both genetic and molecular-biochemical approaches: complementation analysis, gene expression studies and biochemical characterization. Objective 2. Determine the intracellular fate of mutant 4H2 within bovine macrophages. This objective will analyze the survival of the mutant strain 4H2 in stimulated and non-stimulated bovine macrophages, determine the localization of this M. paratuberculosis mutant strain within bovine macrophages and establish the in vitro susceptibility of the mutant strain to superoxides and nitric oxide.
APPROACH: In the first objective, PCR and Southern blotting analyses will be used to confirm the location of the transposition insertion within the suspected DNA sequence, located adjacent to a gene encoding one of the proline-rich proteins of M. paratuberculosis. The wild-type allele of the candidate ppe gene will be introduced back into the mutant strain. Bovine macrophage survival assays will be used to confirm the restoration of the virulent wild-type phenotype. Wild type and mutant strains will be grown on both low and high iron media, as well as intracellularly within macrophages. Gene expression under these conditions will be monitored at the transcriptional level by Northern and primer extension analyses, RT-PCR and transcriptional fusions to the firefly luciferase gene. Further biochemical characterization will involve the over-expression and purification of the wild-type gene product from recombinant E. coli. Using the purified product, monospecific polyclonal antibodies will be produced. In the second objective, a recombinant version of the mutant strain expressing the green-fluorescence protein will be used to assess their ability to survive and co-localize within lysosomes. To determine survival, both unstimulated and interferon-gamma stimulated macrophages will be infected with the wild-type and mutant strains of M. paratuberculosis and monitored for both its ability to infect macrophages and persist intracellularly over time. Confocal microscopy will be used for co-localization studies of bacteria-containing phagosomes with lysosomes.
PROGRESS: 2004/02 TO 2007/10
The main findings for the entire period were: (1) primary bovine monocytes exhibited greater ability to phagocytose Mycobacterium avium paratuberculosis (MAP) as compared with the BOMAC cell line, (2) phagocytosis of MAP by monocytes, but not the cell line, was enhanced by the addition of autologous serum, (3) the number of viable MAP cells in monocytes increased within the first 4 days and then declined, while this number remained unchanged in the cell line during the incubation period, (4) the number of microscopically visible acid-fast bacilli increased with time in monocytes, but not in the cell line, (5) Southern blot and PCR analysis are consistent with a transposon insertion site upstream of the gene MAP1152, (6) transcription of MAP1152 in broth cultures is not affected in the mutant strain and (7) complementation analysis demonstrated that the MAP1156 wild type gene restored the altered colony morphology of the mutant strain to wild type. Complementation analysis was done in the current period. Shuttle plasmids carrying either MAP1152 or MAP1156 genes were introduced into the MAP by transformation. Transformants of the wild type strain with recombinant plasmids (over-production controls) did not result in any colony alteration. Likewise, transformants of the mutant strain with the plasmid carrying MAP1152 yielded similar results. However, transformants of the mutant with the plasmid carrying MAP1156 restored the altered colony morphology of the mutant strain to wild type. In addition, the attenuated phenotype of the mutant strain was confirmed with primary bovine macrophages. In other studies, it was shown that the addition of serum from infected or uninfected cattle increased ingestion of MAP without affecting the overall pattern of bacilli survival. Differential live/dead staining of bacilli from infected monocytes showed that the percentage of viable bacilli decreased from 70% at day 0 to 25% at day 8. Regarding the role of reactive intermediates (RI), bovine monocytes did not produce increased amounts of oxygen- or nitrogen-RI after infection with MAP. Monocytes infected with live MAP exhibited 30%, while monocytes that ingested heat killed MAP showed about 94% phagosome-lysosome fusion at 24 hours after infection. Addition of 5 mM ATP to MAP-infected bovine monocytes resulted in 50% cytotoxicity of bovine monocytes at 24 h. Addition of a longer-lived ATP homologue and a purinergic receptor agonist significantly increased membrane pore activation. Neither ATP nor its homologue reduced the survival of MAP in bovine mononuclear phagocytes. Bovine monocytes constitutively secreted ATP during an 8 day incubation period in vitro; however, MAP infection did not enhance ATP release. Removal of extracellular ATP by the addition of apyrase increased the viability of infected monocytes, but surprisingly decreased the number of viable intracellular bacilli. In contrast to previous reports, addition of extracellular ATP (1 mM) increased intracellular survival of MAP in bovine monocytes. Neither apyrase nor ATP altered production of oxygen- or nitrogen-RI by bovine monocytes.
IMPACT: 2004/02 TO 2007/10
A definite linkage was established between the altered colony morphology mutant and the role of the gene encoded by MAP1156. The results suggest that the mutant strain displays an altered expression of MAP1156 and that the wild type gene provided in trans is capable of complementing this defect. Another aspect studied was the ability of MAP to survive and multiply in bovine mononuclear phagocytes. These studies demonstrated that concomitant bacillary growth and killing can occur in bovine monocytes. Evidence was also obtained that phagosome-lysosome fusion is inhibited by viable MAP. Thus, bovine monocytes are able to both support the survival or and kill intracellular MAP. However, the relatively poor induction of mycobactericidal mechanisms by infected bovine monocytes might explain in part the intracellular survival of MAP. Some factors
were also ruled out as significant contributors to MAP survival: extracellular ATP did not induce the killing of intracellular MAP in bovine mononuclear phagocytes. Likewise, ATP release from MAP infected bovine monocytes improved rather than decreased the intracellular survival of MAP.
PUBLICATIONS (not previously reported): 2004/02 TO 2007/10
1. Woo, S.-R., J.A. Heintz, R. Albrecht, R.G. Barletta and C.J. Czuprynski. 2007. Life and death in bovine monocytes: the fate of Mycobacterium avium subsp. paratuberculosis. Microbial Pathogenesis, 43: 106-113.
2. Woo, S.-R., R.G. Barletta and C.J. Czuprynski. 2007. Extracellular ATP Is Cytotoxic to Mononuclear Phagocytes but Does Not Induce Killing of Intracellular Mycobacterium avium subsp. paratuberculosis. Clinical and Vaccine Immunololgy,
14: 1078-83.
3. (Submitted) Woo S.R., R.G. Barletta and C.J. Czuprynski. 2007. ATP release by infected bovine monocytes increases the intracellular survival of Mycobacterium paratuberculosis. Comparative Immunology, Microbiology and Infectious Diseases.
PROJECT CONTACT:
Name: Barletta, R. G.
Phone: 402-472-8543
Fax: 402-472-9690
Email: rbarletta@unl.edu

 
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ACCESSION NO: 0205570 SUBFILE: CRIS
PROJ NO: NEB-14-141 AGENCY: CSREES NEB
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 OCT 2005 TERM: 30 SEP 2010 FY: 2006
INVESTIGATOR: Barletta, R. G.
PERFORMING INSTITUTION:
Veterinary and Biomedical Sciences
University of Nebraska
Lincoln , Nebraska 68583
MOLECULAR GENETIC ANALYSIS OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS (MAP) AND RELATED MYCOBACTERIAL PATHOGENS.
NON-TECHNICAL SUMMARY : MAP is the etiologic agent of Johne's disease, an infectious disease affecting all ruminants, characterized by diarrhea, weight loss, and ultimately death. The purpose of this study is to use a genetic approach to study MAP virulence determinants and related pathogens. We expect to identify and characterize attenuated MAP mutants that could serve as candidate vaccine strains.
OBJECTIVES: The long term objective of this project is to understand, at the molecular level, the mechanisms of MAP pathogenesis and related mycobacterial pathogens, and to develop molecular tools to control and diagnose mycobacterioses in human and animal populations. In this context, the following specific objectives are proposed: Objective 1: To identify and characterize genes involved in entry of MAP into bovine epithelial cells. Objective 2: To identify and characterize genes involved in replication and survival of MAP in bovine macrophages. Objective 3: Determine the intracellular trafficking of MAP mutant and wild-type strains within bovine macrophages. Objective 4: Characterize molecular targets of MAP and related pathogens for drug therapy.
APPROACH: Objective 1) We propose to screen a MAP signature tagged transposon bank that will be used to infect Madin-Darby bovine kidney (MDBK) cells in vitro to identify genes or virulence determinants in MAP encoding for proteins (or enzymes involved in the synthesis of lipids or carbohydrates) that participate in the process of binding and invasion of the intestinal mucosa. Once a deficiency in invasion is established, the clones will be sequenced and searched against the MAP genome. Methods will include screening MDBK epithelial cells for MAP invasion and complementation analysis to confirm that a phenotype observed is due to a specific gene inactivation. Objective 2) DNA microarrays will be used to identify genes exclusively expressed within macrophages and defined targeted MAP mutants will be constructed and evaluated for replication and survival in bovine macrophages. We will use whole MAP genome oligonucleotide DNA microarrays to profile the transcription level of each encoded gene. Next, MAP isogenic mutants will be constructed to inactivate the gene of interest. Finally, we will test the virulence of selected MAP mutants to survive and replicate in bovine macrophages. Objective 3) We will use peripheral blood monocytes and the BoMac cell line to test whether MAP mutants have a diminished ability to prevent the fusion of phagosomal compartments. This will include confocal microscope studies with recombinant GFP-expressing mutant strains ability to survive and co-localize within lysosomes. In addition, we will test the ability of MAP mutant and wild-type strains ability to survive in stimulated and nonstimulated bovine macrophages. Objective 4) We will focus on identifying and characterizing drug targets in the D-alanine branch of peptidoglycan biosynthesis to develop novel drugs for the therapy of M. tuberculosis and M. avium subsp. avium infections in humans, Johne's disease or bovine tuberculosis in animals, and Crohn's disease in humans. We will need to determine the essentiality of a given target gene by constructing merodiploid strains using the putative wild-type gene in an integrating vector. Next we will overproduce and purify the recombinant target proteins. D-alanine racemase activity will be assayed in the direction of the conversion of L-alanine into D-alanine by a modification of the coupled-spectrophotometric method while D-alanine:D-alanine ligase assays will be carried out by a modified thin layer chromatography based method. Lastly, methods will be developed to screen a large number of chemical compounds for inhibitors of the purified enzymes.
PROGRESS: 2007/01 TO 2007/11
The focus of this reporting period was on Objective 2. One new attenuated mutant of Mycobacterium avium subsp. paratuberculosis (MAP) strain K-10 was characterized, the transposon insertion site was mapped within the open reading frame designated MAP1566 in the genome sequencing project. This ORF seems to be contained within an operon that also encodes ABC transporter components (mod genes). A homologous gene was found in M. avium subsp avium strain 104. Both strains K-10 and 104 display the same gene organization in this region. Homologues were also found in Mycobacterium smegmatis and Mycobacterium tuberculosis, but in these cases the corresponding homologues were not linked to ABC transporter component genes. In addition, collaborative studies were carried out. With the Kris Huygen Laboratory at the Institute Pasteur in Brussels, a luminescent MAP strain of bovine origin expressing the luxAB genes of Vibrio harveyi were used to evaluate the effect of the Slc11a1 (formerly Nramp1) polymorphism on susceptibility against MAP. A series of inbred mouse strains were infected intravenously with luminescent MAP S-23 and monitored for bacterial replication in spleen, liver and lungs for 12 weeks. In BALB/c, congenic BALB.B10 (BALB/c background, H-2b), C57BL/6 and mutant C57BL/6bg/bg mice (all Slc11a1s) bacterial numbers in spleen and liver remained unchangedduring the first 4 weeks of infection, whereas in DBA/2 and congenic C.D2 mice (both Slc11a1r) and in (C57BL/6xDBA/2)F1 (Slc11a1s/r) the bacterial number had decreased more than 30 fold at 4 weeks post-infection in both male and female mice. At later time points, additional differences in bacterial replication were observed between the susceptible mouse strains, particularly in the liver. Whereas bacterial numbers in the liver gradually decreased more than one-hundred fold in C57BL/6 mice between week 4 and week 12, bacterial numbers remained more or less constant in liver from BALB/c and mutant C57BL/6bg/bg mice. Mycobacteria-specific IFN-&#947; responses developed earlier and to a higher magnitude in C57BL/6 mice than in BALB/c mice and were lowest in resistant C.D2 mice. In collaboration with the William Davis Laboratory at Washington State University, an efficient allelic exchange method was developed to generate directed mutations within preselected genes of MAP. Using this method, we obtained a 78 to 100% allelic exchange frequency and a 9.5 x 10-8 to 1.6 x 10-7 transduction frequency. Three genes were selected to demonstrate the utility of the method: pknG and relA are genes known to be important virulence factors in mycobacteria and lsr2 is a gene that regulates lipid biosynthesis. Mutants were successfully generated in the MAP K-10 strain and a variant of K-10 containing the green fluorescent protein gene.
IMPACT: 2007/01 TO 2007/11
MAP is the causative pathogen of paratuberculosis or Johne's disease, a chronic inflammatory wasting disease of the intestine in cattle. Paratuberculosis and related mycobacterioses cause an estimated $1 billion in annual losses to U.S. agriculture. The disease has been difficult to control because of the lack of an effective vaccine. The analysis of mutant strains for replication and survival in bovine macrophages may aid in the development of a vaccine to control Johne's disease and bovine tuberculosis. Mice studies indicate that, as for MAV, innate resistance to infection is genetically controlled by Slc11a1. These results support the hypothesis that MAP may play a role as an etiologic or opportunistic pathogen of Crohn's disease (CD) in humans. In this context, CD is also associated with this type of genetic control. The improved efficiency of disrupting selected genes in MAP should accelerate development of mutants to test as vaccines.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/11
1. Roupie, V., V. Rosseels, V. Piersoel, D.K. Zinniel, R.G. Barletta and K. Huygen. 2007. Innate resistance of mice to M. avium subsp. paratuberculosis is controlled by Slc11a1. Proceedings of the 9th International Colloquium on Paratuberculosis.
2. Park, K.T., J. Dahl, J.P. Bannantine, R. Barletta, J. Ahn, A.J. Allen, M.J. Hamilton, W.C. Davis. 2007. Construction of &#916;pknG, &#916;relA and &#916;lsr2 in Mycobacterium avium subsp. paratuberculosis by allelic exchange mutagenesis. Proceedings of the 9th International Colloquium on Paratuberculosis.
PROJECT CONTACT:
Name: Barletta, R. G.
Phone: 402-472-8543
Fax: 402-472-9690
Email: rbarletta1@unl.edu


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ACCESSION NO: 0407651 SUBFILE: CRIS
PROJ NO: 1265-32000-078-01S AGENCY: ARS 1265
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 01 SEP 2003 TERM: 31 AUG 2008
INVESTIGATOR: Karns J S; Schukken Y H; Van Kessel J S
PERFORMING INSTITUTION:
Veterinary Preventive Medicine
Cornell University
Ithaca , New York 14853
PILOT STUDY OF FACTORS AFFECTING MAINTENANCE OF MYCOBACTERIUM, SALMONELLA, E. COLI, AND LISTERIA ON DAIRY FARMS.
OBJECTIVES: The objective of this research is to set up a Pilot Program consisting of two dairy herds to focus on the protocol development, laboratory set-up, program logistics, and database and bio-bank development needed to evaluate the impact of intervention strategies on Johne's disease dynamics, milk and beef quality (particularly with respect to zoonotic bacterial pathogens), economics and sustainability through intensive longitudinal follow up of selected research/demonstration dairy farms. Long term goals are to validate intervention strategies to support BMP's and to optimize intervention and monitoring strategies given the constraints on time, labor and financial resources in modern dairy herds. In addition, a national resource bank (data and biological specimens on well-characterized animals) will be created for current and future monitoring and research on dairy cattle diseases. Emphasis will be on longitudinal data collection on endemic infectious diseases of public health and animal health concern in dairy herds.
APPROACH: Pathogens are of increasing concern on dairy farms and in dairy products. The production of safe and wholesome food from US farms requires control of the production process on the farm. Specific focus areas in this process are biosecurity, food safety and animal health. To be able to scientifically support regional process control programs there is a need for longitudinal research on commercial dairy farms throughout the United States. In this collaboration, Cornell University, The Pennsylvania State University, University of Vermont and the University of Pennsylvania, all participants in the Regional Dairy Quality Management Alliance (RDQMA), and the USDA's Environmental Microbial Safety Laboratory (EMSL) will investigate the sites on operating dairy farms that act as reservoirs for pathogenic microorganisms that affect animal health and that decrease product quality because of their zoonotic nature. Several farms will be identified as candidates for this investigation and 3 will be chosen for extensive sampling based on the previous occurrence of Johne's disease and/or Salmonella infection. Serum, feces, bulk tank milk, and environmental samples (water, bird and rodent feces, feed, etc.) will be taken on each farm. In addition, tissue samples will be obtained from carcasses of culled animals. Samples will be distributed among the university and ARS researchers for analysis to determine the presence of Mycobacterium avium paratuberculosis (the causative agent of Johne's disease in cattle) and for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes (human food-borne pathogens of concern in dairy products). This research will be the first to attempt a comprehensive analysis of both Johne's disease and food-borne pathogens on working dairy farms. It will allow us to determine a baseline for these organisms on two farms and set the stage for investigation of the effect of interventions, in the form of best management practices (BMPs), on animal health and product quality.
PROGRESS: 2005/10 TO 2006/09
Progress Report 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and Cornell University. Additional information can be found in the reports for the SCAs with the other universities involved in the project (1265-32000-078-02S, 03S, and 04S) and in the report for the parent project 1265-32000-078-00D "DAIRY MANAGEMENT PRACTICES AND THE TRANSMISSION OF ZOONOTIC PATHOGENS IN MILK". This cooperative agreement is part of a larger project involving 4 universities, the University of Pennsylvania, The Pennsylvania State University, University of Vermont, and Cornell University, and formulated in consultation with the Northeastern Regional Dairy Quality Alliance (RDQMA) and the National Milk Producers Federation. Currently, this project provides longitudinal access to three dairy herds totaling 575 milk cows (NY herd-340 milk cows; PA herd-110 milk cows and VT herd-125 cows). Fecal samples from adult cows on the NY farm are collected twice a year and sent to collaborating labs to be tested for the presence of Mycobacterium avium Paratuberculosis (MAP, the causative agent of Johne's disease), and the zoonotic foodborne bacterial pathogens Salmonella, Escherichia coli, Listeria monocytogenes, Campylobacter, and Enterococcus. Blood serum is collected quarterly to test for the presence of antibodies to the Johnes Disease (JD) organism. Environmental samples of manure, feed, water, vermin, birds, flies, etc are taken 4 times a year for analysis by collaborators. Samples of bulk tank milk and milk filters are taken weekly. The purpose of the project from the perspective of our lab is to characterize the microbial populations on the farm with particular interest in Salmonella, Escherichia coli, and Listeria monocytogenes. Initial findings from the NY farm have shown the occasional presence of Salmonella in fecal samples and the presence of Listeria monocytogenes in fecal and environmental samples as well as in the milk filter and bulk tank milk. In addition, MAP has been found in fecal and environmental samples. A database of the data from all farms is maintained so that analysis of the influence of management practices can be conducted.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
No publications reported this period.

 
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ACCESSION NO: 0193449 SUBFILE: CRIS
PROJ NO: NYC-127461 AGENCY: CSREES NY.C
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 OCT 2002 TERM: 30 SEP 2005 FY: 2007
INVESTIGATOR: Thonney, M. L.; Smith, M. C.; Stehman, S. M.
PERFORMING INSTITUTION:
Animal Science
Cornell University
Ithaca , New York 14853
EFFECT OF VACCINATION AGAINST PARATUBERCULOSIS (JOHNE'S DISEASE) IN SHEEP.
NON-TECHNICAL SUMMARY: Paratuberculosis (Johne's Disease) causes early death and poor reproductive performance in sheep and dairy flocks. This project tests the efficacy of vaccination to help control paratuberculosis in sheep.
OBJECTIVES: The overall objective of this proposal is to evaluate the effectiveness of vaccination against Johne's Disease in sheep. Correlated objectives are to improve the control of Johne's disease in sheep flocks to increase production efficiency and to eradicate the disease from the Cornell Dorset flock, which is one of the only flocks in the world intensely selected for out of season breeding. A successful vaccination program would make the Cornell a seasonal genotype available to New York farmers which would enable them to meet the year-round demand from ethnic consumers for lamb in the Northeast.
APPROACH : One-hundred control ewes and 100 ewes vaccinated as lambs with Mycopar are in an experiment started in 1999. The presence and size of granulomas was monitored in the vaccinated lambs. The ewes in the vaccination trial are managed with the other ewes in our flock. At 2, 3, 4, and 5 years of age, about one-fourth of the control ewes and one-fourth of the vaccinated ewes are being slaughtered so that the intestinal tracts can be examined by histopathology to determine if they are infected. The reason for the serial slaughter is to determine if there is a difference between the control and vaccinated groups in severity of pathology with age if some ewes in the vaccinated group become infected. The ewes are randomly sampled as they finish a lambing/lactation period. Prior to slaughter, samples of blood and feces are obtained for development of fecal culture methodology and correlation of AGID results with histopathology data. At slaughter, the intestinal tracts are retrieved and returned on ice to the College of Veterinary Medicine where sample tissues from the ileocecal junction, the ileocecal node and the mesenteric node are collected and stored in formalin. Those tissues are then processed and stained for histological examination by a veterinary pathologist. The histopathological results are the gold standard by which the effectiveness of vaccination will be judged and to which results from AGID and fecal culture will be compared. Statistical analysis will test the null hypothesis that there is no difference between vaccination groups in the incidence of paratuberculosis. Incidence of paratuberculosis will be analyzed in a 2 (ewe vaccination) 4 (year) contingency table using 2.
PROGRESS: 2002/10 TO 2005/09
Finnsheep x Dorset ewes managed under the Cornell STAR accelerated lambing system were used to test vaccination as a method to help control Johne's Disease (Mycobacterium avium subsp. paratuberculosis) in sheep. A total of 135 ewe lambs vaccinated against Johne's Disease using Mycopar(R) (a licensed cattle vaccine used with permission) and 125 contemporaneous control ewe lambs started the project across four birth groups (March 1999, June 1999, January 2000, March 2000). Fifty control and 60 vaccinated ewes died of natural causes, 73 control and 72 vaccinated ewes were culled, with 66 control and 64 vaccinated ewes old enough at culling for sampling for histopathology, and 2 control and 3 vaccinated ewes were missing (lost eartags) by the end of the experiment in August 2005. Granuloma areas in the vaccinated ewes ranged from 0 to 7 cm2 and appeared to reach maximum size by 10 to 15 days post-vaccination. An overall 18 percent death loss to two years of age was higher than expected. More (P = 0.073) vaccinated than control ewes died at young ages. Within birth groups, however, the effect was significant only for the ewes born in the January 2000 lambing season. There was no significant difference in natural death rates by the end of the experiment. Of those ewes that lived long enough that they could have lambed, 6 of 113 (5.3 percent) control ewes and 12 of 107 (11.2 percent) vaccinated ewes had no records of lambing. These 18 ewes were kept in the flock to ensure that sufficient animals were available to sample at older ages. Of the ewes that were available for lambing, 51.8 percent had lambed by the end of the first 73-day period and 77.7 percent had lambed by the end of the second 73-day period, but the cumulative 146-day-total represented 70.1 percent of vaccinated ewes compared to 85.0 percent of control ewes (P = 0.008). Although there was no difference between vaccination groups in lambs delivered, born alive, or weaned per lambing, control ewes lambed more frequently (P = 0.049). Among ewes with available histological samples from the ileum, cecum, and mesenteric lymph node, 13 of 92 control ewes and 3 of 86 vaccinated ewes had one or more pathological lesions consistent with Johne's Disease. Thus, only 14.1 percent of control animals that died or were culled purposefully for sampling had evidence of infection by histopathology. Even with this moderate control-animal infection rate, vaccination reduced (P = 0.013) infection. Among animals that were purposely culled for sampling, the infection rates were 15.1 and 3.1 percent for control and vaccinated animals (P = 0.018), respectively. Thus, vaccination against Johne's Disease using Mycopar(R) will reduce the incidence of infection and help to control the disease in sheep flocks.
IMPACT: 2002/10 TO 2005/09
Vaccination against Johne's Disease (Mycobacterium avium subsp. paratuberculosis) using Mycopar(R) will reduce the incidence of infection and help to control the disease in sheep flocks. This will reduce mortality in breeding flocks, improving efficiency of production to make sheep farming more profitable and encourage the expansion of environmentally-sustainable sheep farms.
PUBLICATIONS (not previously reported): 2002/10 TO 2005/09
No publications reported this period
PROJECT CONTACT:
Name: Decker, D. J.
Phone: 607-255-2224
Fax: 607-255-9499
Email: cuaes@cornell.edu

 
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ACCESSION NO: 0197984 SUBFILE: CRIS
PROJ NO: NYC-127468 AGENCY: CSREES NY.C
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 OCT 2003 TERM: 30 SEP 2006 FY: 2006
INVESTIGATOR: Gavalchin, J.; Thonney, M. L.; Stehman, S. M.; Smith, M. C.
PERFORMING INSTITUTION:
Animal Science
Cornell University
Ithaca , New York 14853
IDENTIFICATION OF PROTECTIVE IMMUNE RESPONSES IN SHEEP AFTER VACCINATION WITH THE MYCOPAR VACCINE FOR PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY : Johne's Disease is widely distributed in cattle, sheep and goats. The pathogenesis of Johne's is not understood, and its diagnosis is difficult in live animals. No vaccine for Johne's is approved for use in sheep in the US. This study will identify the immune responses of sheep after infection with MAP, and after vaccination with the cattle vaccine, Mycopar. Our goal is to understand the pathogenesis of Johne's, and find immune biomarkers that can be used for its diagnosis.
OBJECTIVES: Johne's Disease, caused by Mycobacterium avium paratuberculosis (MAP), is widely distributed in cattle, sheep and goats. In the US, estimates of 20 to 40 percent of dairy herds are infected with MAP, leading to losses of over $220 million/year. Many of the sheep flocks in NY are infected, with weight loss, poor production, increased parasitism, and faster turnover rate. This has limited sales, which is a particular problem for the Cornell Dorset breeding flock, since it is one of the only flocks in the world selected for out of season breeding. Because the only time that Johne's disease can be conclusively diagnosed is during its late stages, when many organisms are shed in the feces, or when organisms can be identified in tissues of infected animals upon necropsy, it is hard to determine the exact number of infected flocks in the United States. This situation would improve if we were able to define the immunological events in natural infection with MAP, so that infected animals could be culled before the onset of clinical disease. Vaccination against MAP also may be effective; however it only modulates clinical signs of disease and does not prevent an animal from becoming a carrier of MAP. Further, the mechanisms of protection afforded by vaccination are not well understood. Ultimately, a successful vaccination program will help to eradicate the disease in all sheep flocks and increase the availability of a seasonal Cornell sheep to NY farmers. In addition, the transfer of results to cattle could also have a significant economic impact on the New York dairy cattle industry. Using sheep that are part of an ongoing trial to test the effectiveness of the cattle vaccine, Mycopar on MAP infection, we propose to investigate whether modulation of immune responses of sheep after infection with MAP, or after vaccination with Mycopar, will lead to immune responses that are effective in limiting or preventing Johne's, according to the following objectives. Objective 1: We assume that all of the animals in the Cornell University Mycopar vaccine trial have been exposed to M. paratuberculosis, but only some will be infected. Since the onset of disease may take anywhere from 2 weeks to years, we will first define the stages of disease in the flock by assaying sera for levels of interferon gamma, which is a biomarker for early disease, and MAP-specific antibodies, which are characteristic of late disease. Cell-mediated responses will be measured by skin testing with the Johnin antigen. The results of these tests, as well as fecal culture and PCR detection of organism in feces, will be used to characterize each animal with regard to MAP infection/exposure. Objective 2: We will define the immune cell phenotypes and functions that are modulated in MAP infection and Mycopar vaccination by in vitro methods. These studies will also identify additional biomarkers for immune responses to MAP.
APPROACH: There are 184 sheep remaining in the original Mycopar vaccination trial; 88 were vaccinated with Mycopar at weaning either in July 1999, September 1999, May 2000, and July 2000, and there are 96 age-matched unvaccinated controls. In each of the 3 years of this grant, the immune responses to MAP will be determined, in order to correlate specific immune responses with disease kinetics and clinical stages. Objective 1: We will first define the stages of infection in the flock by assaying sera for levels of interferon gamma, which is a biomarker for early disease, and MAP-specific antibodies which appear in late disease. Skin testing with Johnin will also be done. The results of these tests, and fecal organism load, will be used to characterize each animal with regard to MAP exposure/infection. Approach: Ten mls of blood will be collected from each ewe. Serum will be obtained by centrifugation. Fecal samples will be collected for determination of organism load by PCR. DTH to Johnin and TB will be conducted by an accredited veterinarian (Mary Smith, DVM). Gamma interferon levels will be determined by the Bovigam assay. The Cornell Veterinary Diagnostic Lab will perform the AGID assay for antibodies to Johnin. Johnin-specific antibodies will also be quantitated using the Parachek test (Biocor). DNA will be isolated from feces using the QIAamp DNA Stool Mini Kit. Enzymatic amplification of MAP DNA will be performed using PCR primers MP3 (5'-CTGGCTACCAAACTCCCGA-3') and MP4 (5'-GAACTCAGCGCCCAGGAT-3'). The results of the individual assays will be analyzed by Student's T-test. The data analysis program SPSS will be used to perform logistic regressions, correlations, cluster centers, and cross tabulations to identify correlated immune responses and stages of infection. Objective 2: We will further define the immune cell phenotypes and functions that are modulated in MAP infection and Mycopar vaccination by in vitro methods. The results of these studies will identify additional biomarkers for immune responses to MAP. Approach: Peripheral blood mononuclear cells (PBMC) will be isolated and analyzed by flow cytometry for the proportion of T lymphocytes (CD4, CD8), B lymphocytes (anti-ovine IgG), monocytes (CD14), and neutrophils, as well as expression of activation markers. Cells will be cultured in vitro with Con A to determine the overall proliferative responses, and with Johnin, to determine the MAP-specific responses. Immunoglobulin production will be measured by ELISA. Johnin-specific cytokine production will be evaluated by culture of PBMC with either Con A or Johnin, and will include interleukin (IL)-2 and interferon- gamma to measure the Th-1-type response and IL-4 and IL-10 to measure to the Th2-type response. IL-1 and TNF alpha levels will also be measured. Data will be analyzed by Student's T-test.
PROGRESS: 2003/10 TO 2006/09
Mycobacteruim avium subsp. paratuberculosis (Map) causes a wasting disease, Johnes disease, that results in granulomatous lesions of the lymph nodes of the small intestine in ruminants. According to the Johnes Information Center, it is estimated that seven percent of beef herds and twenty-two percent of dairy herds in the U.S. are infected with Map. Animals apparently are infected when young but, while shedding the organism via feces, these animals may not show clinical symptoms for several years. Because the organism resides in macrophages in the intestinal mucosa and associated lymph nodes, infected animals may not have obvious clinical signs of disease. At the present time, there is no treatment or vaccination program in the United States that effectively prevents Johnes disease. Current control methods are based upon minimizing exposure to feces from infected animals in dairy herds, requiring early identification and culling of infected animals. Vaccines against Map may cause animals to react positively when tested for Mycobacterium bovis (Bovis). Therefore, in the present work, the effect of vaccination on MAP Ab and skin-test responses to MAP and Bovis were compared in 25 ewes vaccinated against MAP with 24 control ewes in an infected flock three years post-vaccination. MAP-specific Ab levels were seven times higher (P = 0.001) in vaccinated ewes than in control ewes. All increases in skinfold-thickness from 0 to 48 h were greater (P = 0.0001) than zero while increases in skinfold-thickness from 48 to 72 h were greater (P = 0.05) than zero for Johnin but not for Bovis. The Vaccine x Ag x Time interaction for skinfold-thickness was significant (P = 0.001) with greater increases to Johnin than to Bovis, but with much greater increases in vaccinated ewes. These data confirm that vaccines against MAP developed from whole organisms increase the likelihood that animals will test positive to Bovis, but they also show that vaccination initiates both humoral and cell-mediated MAP-specific responses. We are continuing the studies to determine whether immune responses to Johnin can be used to identify infection by Map or vaccine-induced protection. These responses include T cell proliferation, and Johnin-specific interferon gamma and immunoglobulin production. Unfortunately this has proven to be difficult as the assay that we have been using to identify infected sheep, AGID, has given us false positive results, with culled AGID-positive sheep found to be negative by histopathology. This has caused us to turn our attention to the development of a more sensitive and specific diagnostic assay. We are currently working to develop a low-cost, easy quantitative lateral flow (immuno-chromatographic) Map antibody assay suitable for field use. Our approach will be to use proprietary 'avidity' constructs capable of capturing all Map antibodies as they flow across a nitrocellulose membrane. The avidity constructs will be applied to the nitrocellulose membrane using ink-jet deposition to construct a quantitative inexpensive dipstick-based Map antibody assay that can be used under non-laboratory conditions.
IMPACT: 2003/10 TO 2006/09
Johnes Disease, caused by Mycobacterium avium paratuberculosis (MAP), is widely distributed in cattle, sheep and goats. In the US, estimates of 20 to 40 percent of dairy herds are infected with MAP, leading to losses of over $220 million/year. Many of the sheep flocks in NY are infected, with weight loss, poor production, increased parasitism, and faster turnover rate. The pathogenesis of Johne's is not understood, and its diagnosis is difficult in live animals. No vaccine for Johne's is approved for use in sheep in the US. This study, to identify the immune responses of sheep after infection with MAP, and after vaccination with the cattle vaccine, Mycopar, will provide an understanding of the pathogenesis of Johne's, and identify immune biomarkers that can be used for its diagnosis.
PUBLICATIONS (not previously reported): 2003/10 TO 2006/09
Nedrow, A.J., Gavalchin, J., Smith, M.C., Stehman, S.M., Maul, J.K., McDonough, S.P, and M. L. Thonney. 2007. Antibody and skin-test responses of sheep vaccinated against Johnes disease. Vet. Immunol. Immunopathol. (in press)
PROJECT CONTACT:
Name: Decker, D. J.
Phone: 607-255-2224
Fax: 607-255-9499
Email: cuaes@cornell.edu


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ACCESSION NO: 0212151 SUBFILE: CRIS
PROJ NO: NYC-127471 AGENCY: CSREES NY.C
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 2007 TERM: 30 SEP 2010
INVESTIGATOR: Gavalchin, J.; Thonney, M. L.; Smith, M.; Stehman, S.; Chang, Y. F.; Lane, M. J.
PERFORMING INSTITUTION:
Animal Science
Cornell University
Ithaca , New York 14853
DEVELOPMENT OF A SENSITIVE, LOW-COST POINT-OF-USE LATERAL FLOW ASSAY FOR THE DIAGNOSIS OF JOHNE'S DISEASE.
NON-TECHNICAL SUMMARY: Johne's Disease, caused by M. paratuberculosis (Map), affects cattle, sheep and goats; 20 to 40 percent of dairy herds in the US may be infected, leading to losses of over $220 million/yr. The objective of an effective management program to control Map infection is to make the diagnosis early, in order to cull infected animals. However, while current diagnostic assays for Johne's are specific, with few false-positives, sensitivity is often less than 50 percent, so that infection may be undetected. Further, all of the tests require trained personnel, and the time required to obtain test results may be quite long. The cost of the test may also prohibit routine testing. We propose to develop a low-cost, more sensitive lateral flow-based Map antibody assay to detect early Map infection, suitable for field use. We will construct and optimize the assay for Map antibody binding in a dipstick format, to obtain maximum assay specificity and sensitivity. Next, we will test the Map antibody-specific dipstick assay for detection of anti-MAP antibody levels using sera from known Map-positive and negative cattle. We will also format the assay for detection of anti-MAP antibody levels in sheep. Finally, we will determine the performance characteristics of the MAP antibody lateral flow dipstick assay, including its specificity, sensitivity, reproducibility and stability.
OBJECTIVES: Johne's Disease, caused by M. paratuberculosis (Map), is widely distributed in cattle, with losses in the US of over $220 million/yr. Control methods require identification and culling of infected animals; however, sick animals, infected when young, may not show clinical symptoms for years. While current tests for Johne's are specific, with few false-positives, sensitivity is often less than 50 percent. The tests also require trained personnel, a long time to result and are cost prohibitive for routine screening. We propose to develop a low-cost, more sensitive lateral flow-based Map antibody assay to detect early Map infection, suitable for field use. In aim 1, we will construct and optimize the Map antigen avidity constructs for Map antibody binding in a dipstick format, to obtain maximum assay specificity and sensitivity. In aim 2, we will evaluate the Map antibody-specific dipstick assay for detection of anti-MAP antibody levels using sera from known Map-positive and negative cattle. In aim 3, we evaluate the efficacy of the MAP antibody lateral flow dipstick assay for detection of anti-MAP antibody levels in known MAP-positive and negative animals in other species. In aim 4, we will determine the performance characteristics of the MAP antibody lateral flow dipstick assay, including its specificity and sensitivity for detecting MAP antibody levels.
APPROACH: In aim 1, we will construct and optimize the assay for anti-Map antibodies (Ig). In practice, a BD microtainer capillary blood collection device will be used to collect 0.10 mL of whole blood from the ear of an animal, which is deposited into a vial containing wash buffer. The vial will be shaken and the dipstick (nitrocellulose membrane) printed with capture lines composed of conjugates of purified Map proteins/DNA that bind anti-Map Ig will be inserted into the vial. The blood will flow up the membrane, then the dipstick will be placed in blocking and wash solution. The dipstick is then reacted with anti-bovine Ig conjugated to alkaline phosphatase directly or the signal will be amplified several thousand-fold by non-enzymatic means. Detection of bound Ig is by BCIP/NBT substrate. The titer of anti-Map Ig will be determined by counting the number of lines that have reacted with substrate. Total time to completion should be 20 minutes. In aim 2, we will evaluate the Map antibody lateral flow dipstick assay for detection of anti-Map antibody levels in known Map-positive and Map-negative cattle. Repository samples from the JDIP Diagnostic Core (n=25) and other infected (low, moderate, and high shedders) and control samples (n=120), will be used to determine of the sensitivity and specificity of the assay. In aim 3, we will format and evaluate the Map antibody lateral flow dipstick assay for detection of anti-Map antibody levels in sheep using samples from Map histopathology-positive (n =13) and negative ewes (n=15). In aim 4, we will determine the performance characteristics of the Map antibody lateral flow dipstick assay, including specificity and sensitivity. The dynamic range of the assay will be determined by using the assay strips to analyze antibody levels in a set of sera containing calibrated or known titers of anti-Map antibody. The assay will also be evaluated for accuracy, reproducibility and stability.
PROJECT CONTACT:
Name: Hoffmann, M. P.
Phone: 607-255-2224
Fax: 607-255-9499
Email: cuaes@cornell.edu

 
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ACCESSION NO: 0186568 SUBFILE: CRIS
PROJ NO: NYC-143808 AGENCY: CSREES NY.C
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 OCT 2000 TERM: 30 SEP 2003 FY: 2004
INVESTIGATOR: Wiedmann, M.; Scarlett, J.; Worobo, R.
PERFORMING INSTITUTION:
Food Science
Cornell University
Ithaca , New York 14853
IMPROVED TOOLS FOR PREDICTING THE PATHOGENIC POTENTIAL OF FOODBORNE MICROORGANISMS.
NON-TECHNICAL SUMMARY: Foodborne diseases cause an estimated 76 million illnesses and 5000 deaths annually in the US. To protect consumers from these diseases it is important to understand exactly which bacteria can cause human disease. This project examines bacteria from humans and animals with bacterial diseases and from contaminated foods by DNA fingerprinting methods to define which bacterial types cause human foodborne disease.
OBJECTIVES: We propose a multidisciplinary approach for developing science-based microbial food safety criteria. Current regulations for foodborne pathogens are commonly based on historical taxonomic identifications that may not correlate to the ability of an organism to cause human disease. This project will identify distinct genetic criteria that define the specific human pathogenic potentials of clonal bacterial groups focusing on E. coli O157:H7, Streptococcus agalactiae, and Mycobacterium paratuberculosis. Objective 1: Assemble a collection of animal, human, and environmental (where applicable) bacterial isolates to be characterized by molecular subtyping methods. Objective 2: Epidemiologically define the relative human pathogenic potentials of different clonal groups of these pathogens. Objective 3: Explore the functional and genetic basis of differences in the abilities of clonal subgroups to cause human disease using tissue culture and animal models.
APPROACH: Objective 1: Human, food, and animal isolates of E. coli O157:H7, St. agalactiae, and M. paratuberculosis will be collected in collaboration with the NYS Veterinary Diagnostic Laboratory, the Dept. of Agriculture and Markets, and the Dept. of Health. All isolates will be characterized by DNA subtyping methods including Southern blotting, ribotyping and sequencing of selected virulence genes. Objective 2: Bacterial subtype distribution among human, food, and animal isolates will be statistically analyzed to identify specific subgroups linked to human infections. Objective 3: Selected subgroups differing in human and animal host specificities will be used to infect appropriate tissue culture cell lines to determine differences in their pathogenic potentials.
PROGRESS: 2000/10 TO 2003/09
Most food safety regulations regarding bacterial foodborne pathogens specify action limits for the presence of specific bacterial species. These classical taxonomic definitions of bacterial species often do not correlate to the ability of a group of bacteria to cause human disease. Rather, somewhat related bacteria that may differ in their abilities to cause human and/or animal disease may be grouped together into the same species. This project is designed to develop a phylogenetic and population genetics framework to allow the specific definition of bacterial clonal groups with human pathogenic potential. Our work specifically focused on Streptococcus agalactiae, which is not only a major cause of mastitis in cattle, but is also responsible for severe invasive disease in adult and neonate humans, as well as asymptomatic infections in women. To probe the population genetics and the interspecies transmission potential of this species, we used molecular subtyping and phenotypic methods to characterize temporally matched bovine milk and clinical human invasive isolates (52 each) collected in New York State over 18 months. EcoRI ribotyping differentiated 17 ribotypes and DNA sequencing of the housekeeping gene sodA and of a 950 bp fragment of hylB, encoding hyaluronidase, differentiated 17 and 7 allelic types, respectively. Human and bovine isolates were not randomly distributed between ribotypes and hylB and sodA allelic types. Combined analysis of all subtyping methods allowed differentiation of 39 clonal groups, 26 groups contained only bovine isolates. Only two clonal groups contained both human and bovine isolates. EcoRI ribotyping also showed a significantly higher genetic diversity among bovine isolates (SID = 0.90 +/- 0.05) as compared to human isolates (0.42 +/- 0.15), providing further evidence that human and bovine isolates represent distinct populations. Eight human but no bovine isolates showed an IS1548 transposon insertion in hylB. Hyaluronidase activities determined for 43 representative isolates showed that human isolates on average showed lower activities than bovine isolates. Isolates with the IS1548 insertion in hylB showed no hyaluronidase activity. In addition, tissue culture invasion assays showed that human and bovine isolated did not differ in their ability to invade human epithelial cells. Our data show that (i) EcoRI ribotyping and hylB and sodA sequencing provide discriminatory subtype analysis of S. agalactiae, (ii) human invasive and bovine S. agalactiae represent distinct populations with limited interspecies transmission, and (iii) hyaluronidase activity is not required for all human infections.
IMPACT: 2000/10 TO 2003/09
Most current food safety and public health regulations use classical taxonomic species definitions to identify microorganisms that represent a public health hazard. Bacteria within a given species may vary tremendously in their ability to cause human disease though. This work clearly shows that bacteria within a given species, even if associated with animal and human disease, are not necessarily transmitted between these hosts. A better understanding of bacterial host specificity for zoonotic and foodborne pathogens will contribute to the development of science-based regulations and intervention strategies targeting those bacterial subtypes that have the potential to cause human disease.
PUBLICATIONS (not previously reported): 2000/10 TO 2003/09
No publications reported this period
PROJECT CONTACT:
Name: Coffman, W. R.
Phone: 607-255-2224
Fax: 607-255-9499
Email: ded7@cornell.edu

 
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ACCESSION NO: 0201752 SUBFILE: CRIS
PROJ NO: NYC-478462 AGENCY: CSREES NY.C
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 OCT 2004 TERM: 30 SEP 2007 FY: 2007
INVESTIGATOR: Chang, Y. F.; Baeummer, A. J.; Shin, S. J.; Stehman, S.; Nydam, D. V.
PERFORMING INSTITUTION:
Vet Population Medicine & Diagnostic Science
Cornell University
Ithaca , New York 14853
BIOSENSOR FOR RAPID DETECTION OF MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: Paratuberculosis (Johne's disease) occurs worldwide as a chronic granulomatous enteritis of domestic and wild animals. A sensitive and rapid test is urgently needed. The purpose of this study is: 1) improve the sensitivity of the PCR test directly from fecal samples, milk, and colostrum or liquid media culture system by using a novel laser-induced cell lysis technique to prepare the DNA extracts and (2) develop a rapid, inexpensive, and sensitive biosensor for detection of MAP based on RNA detection.
OBJECTIVES: Molecular techniques for the diagnosis of Johne's disease based on nucleic acid amplification (regular PCR and/or real-time PCR) have been widely used in animal health diagnostic laboratories. However, the sensitivity of the PCR-based test directly from fecal samples, especially from the low shedders, is still very low. Furthermore, PCR detection in milk and colostrum cannot distinguish between viable and non-viable organisms. Thus, in order to have sufficient material, very long cell culture incubation times are required until a definite answer can be found using PCR. Therefore, we propose to improve the PCR based test by using a novel cell lysis technique, the laser-lysis technology, to improve the yield of DNA extract preparation. The immensely thick lipid layer of MAP is the main obstacle for current lysis methods. Since the laser-lysis technique is based on the local and specific increase of intracellular water molecules, we postulate that it will not be hindered by the presence of the thick lipid layer but will successfully lyse cells without any damage to DNA, RNA and intracellular protein molecules. Our central hypothesis is that the use of the laser lysis technique will increase the template DNA recovery, and therefore, it will increase the sensitivity of PCR test directly from fecal samples, which results in a significantly shorter cell incubation time as required currently. At the same time, biosensor technology will enable us to simplify the detection system and make it inexpensive and even more rapid while keeping its high specificity for the detection of Johne's disease.
APPROACH: 1. The use of laser lysis technique will increase the DNA template recovery from fecal sample preparation; thus, it will increase the sensitivity of PCR tests from either fecal sample directly and/or from Trek culture system. If this technique is successful, then, it may be applied to fecal samples directly and/or early detection of Trek liquid culture system. 2. The RNA biosensor based on liposome technology is a rapid, inexpensive and sensitive system to detect the MAP directly from fecal samples and/or Trek culture system. The antibody biosensor is also a rapid, inexpensive and sensitive system to detect the Mycobacterium avium subsp. paratuberculosis (MAP) directly from fecal samples and/or Trek culture system. To accomplish the objective of this study, we will apply two approaches: (1) Increase the PCR sensitivity by the improvement of cell lysis and thus DNA extraction using laser-induced cell lysis. (2) Apply RNA Biosensor for MAP diagnosis.
PROGRESS: 2004/10 TO 2007/09
OUTPUTS: Mycobacterium avium subsp.paratuberculosis (MAP) is the causative agent of Johne's disease (JD), a chronic intestinal granulamatous infection affecting domestic ruminants such as cattle, sheep and goats. Apart from domestic ruminants, wild ruminants like rabbits, deer, bison, and antelopes are also affected by JD. The primary route of infection is through ingestion of contaminated feed and water, colostrum and milk. Although cattle are usually infected early in life, clinical signs do not develop until 2 to 4 years of age which makes early diagnosis of this infection a difficult task. JD is considered as an economically important disease and accounts for an annual loss of $220 million to the US dairy industry due to premature culling and production losses. The need for an effective JD control program has increased as the costs of this infection become more apparent. The proposed, but poorly defined association of MAP with Crohn's disease in human beings is also of concern. Effective control of JD is hampered by diagnostic techniques that can not consistently detect infected animals. Currently, JD diagnosis is typically based on either detecting the etiological agent or detecting the host's immune responses to the infection. The former method employs culturing of MAP organisms and detecting gene specific sequences from clinical samples. Though, isolation and identification of MAP is the most definitive test for diagnosis, it is time consuming and labor intensive, requiring 8-12 weeks. Contamination is an added problem when MAP is cultured from fecal samples. Although, PCR for IS900 sequences is of diagnostic value, it may lead to false positive amplification due to the presence of environmental bacteria with similar sequences. Novel sequences identified recently in the genome of MAP appear specific and may also be used in nucleic-acid based diagnostic tests. Alternatively, PCR can be coupled with southern blotting to get a definite diagnosis, but again this method too takes more time. Serological tests like agar gel immunodiffusion test, complement fixation test and enzyme linked immunoassay (ELISA) are relatively easy to perform but may lack sensitivity. Serum samples from animals exposed to environmental mycobacteria may also cross react with MAP antigen resulting in false positive reaction. Most diagnostic laboratories continue to use traditional culture methods for identification of MAP, while few laboratories employ molecular methods along with culture methods. Current diagnostic tests have been found to be more useful on a herd basis rather than in testing individual animals. In this scenario, the general feeling is the need for improved, rapid diagnostic techniques for JD. Therefore, we develop a bioanalytical system like biosensors coupled with a reverse transcriptase PCR to achieve low limits of detection for a rapid and accurate detection of MAP. This manuscript has submitted to Journal for publication. We will disseminate our results through publication. TARGET AUDIENCES: The target audiences are diagnosticians of Johne's disease
IMPACT: 2004/10 TO 2007/09
A simple, membrane-strip-based lateral-flow (LF) biosensor assay and a high-throughput microtiter plate assay have been combined with reverse transcriptase polymerase chain reaction (RT-PCR) for rapid detection of a small number (ten) of viable Mycobacterium avium subsp. paratuberculosis (MAP) cells from fecal samples. The assays are based on the identification of the RNA of the IS900 element of MAP. For the assay, RNA was extracted from fecal samples spiked with known quantity of (101 to 106) MAP cells and amplified using RT-PCR and rapidly identified by the LF biosensor and the microtiter plate assay. While the LF biosensor assay requires only 30 min assay time, the overall process took 10 h for the detection of 10 viable cells. The assays are based on an oligonucleotide sandwich hybridization assay format and use either a membrane flow through system with an immobilized DNA probe that hybridizes with the target sequence or a microtiter plate well. Signal amplification is provided when the target sequence hybridizes to a second DNA probe that has been coupled to liposomes encapsulating the dye sulforhodamine B. The dye in the liposomes provides a signal that can be read visually, quantified with a hand-held reflectometer or with a fluorescence reader. Specificity analysis of the assays revealed no cross reactivity with other mycobacteria such as M.avium complex, M. ulcerans, M.marium, M.kansasii, M.abscessus, M.asiaticum, M.phlei, M.fortutitum, M.scrofalaceum, M.intracellular, M.smegmatis and M.bovis. The overall assay for the detection of MAP in live organisms is less expensive and extremely rapid, especially in comparison to standard MAP detection using culture method requiring 6 - 8 weeks of incubation time, and is significantly less expensive than real-time PCR.
PUBLICATIONS (not previously reported): 2004/10 TO 2007/09
1. Chen, L. H., K. Kathaperumal, C. J. Huang, S. P. McDonough, S. Stehman, B. Akey, J. Huntley, C.F. Chang and Y. F. Chang. 2007. Immune responses in mice to Mycobacterium avium subsp. paratuberculosis following vaccination with a novel 74F recombinant polyprotien. Vaccine, (in Press)
2. Kathaperumal, K., S. U. Park, S. P. McDonough, S. Stehman, B. Akey, J. Huntley, S. Wong, L. H. Chen, C.-F. Chang and Y. F. Chang. 2007. Vaccination with recombinant Mycobacterium avium subsp.paratuberculosis proteins induces differential immune responses and protects calves against infection by oral challenge. Vaccine (submitted).
3. Kumanan, V. S. R. Nugen, A. J. Baeumner, and Y. F. Chang. 2007. A rapid biosensor assay for the detection Mycobacterium avium subsp. paratuberculosis from fecal samples. Anal. Bioanal. Chem. (Submitted).
PROJECT CONTACT:
Name: Decker, D. J.
Phone: 607-255-2224
Fax: 607-255-9499
Email: cuaes@cornell.edu

 
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ACCESSION NO: 0202603 SUBFILE: CRIS
PROJ NO: NYC-478475 AGENCY: CSREES NY.C
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 OCT 2004 TERM: 30 SEP 2007 FY: 2007
INVESTIGATOR : Schukken, Y. H.; Simpson, K. W.; Gilbert, R. O.
PERFORMING INSTITUTION:
Vet Population Medicine & Diagnostic Science
Cornell University
Ithaca , New York 14853
BOVINE SPECIFIC CYTOKINE ASSAYS TO EVALUATE TH1/TH2 POLARIZATION OF THE IMMUNE RESPONSE.
NON-TECHNICAL SUMMARY: Dairy cows are often affected by chronic bacterial infections. The immune response to these infections is not well understood. We will contrast the immunological response of the cow in transient versus chronic infections. We intend to develop a set of quantitative assays for bovine cytokines. Using these assays we expect to gain a greater understanding of the early immunological response of the dairy cow to intramammary and intrauterine infections.
OBJECTIVES: The first objective of this project is to develop species-specific quantitative cytokine assays. Of specific interest is the development of assays for cytokines that represent either classical T-helper cell 1 immune response (Th1), or the T-helper cell-2 immune response (Th2). Th1 cells produce IFN-gamma, IL-2 and IL-12 whereas Th2 cells produce IL-4, IL-10 and IL-13. Cytokines will be cloned and expressed to facilitate development of assays to measure cytokine concentrations in blood, milk and tissues from cattle. The second objective is to determine which cytokines are involved in polarization of the cellular immune response, and how cytokines are influenced by transient versus chronic infections. We will quantify cytokine kinetics during the early immune response of cattle to infectious diseases of the mammary gland and of the uterus. In both organ systems transient and chronic infections are present. A better understanding of the dynamics of the early immune response is necessary to be able to develop effective intervention options such as treatment or vaccination for these infections. The specific goals to reach the two objectives of this project are defined as: 1. Development of assays to quantitate bovine cytokine mRNA. Reverse transcription reactions will be carried out in a RT-PCR system. 2. Application of quantitative PCR for cytokine mRNA to bovine cells in vitro. PCR primers and probes for cytokines such as bovine interferon gamma (IFN-gamma), IL-2, IL-6, IL-8, IL-10, IL-12 will be synthesized and used to amplify their respective cDNA. An in-vitro infection experiment comparing chronic versus transient infection strains will be performed. 3. Develop assays to quantify the aforementioned bovine cytokines. These cytokines will be targeted for cloning, expression and development of quantitative assays.
APPROACH: The experimental procedures necessary to reach these four goals are described in more detail below. Development of quantitative bovine cytokine mRNA assays. Composite milk samples will be collected from cows. Each milk sample will be centrifuged at 700 g for 20 min and the pellet is washed twice in 50 ml phosphate-buffered saline. Cells (5 X 10E6) are then lysed with 350 l lysis buffer according to the manufacturer's recommendations (RNeasy mini kit, Qiagen, Valencia, CA, USA). Similarly, endometrial biopsies will be processed and kept at -80C until RNA extraction and complementary DNA (cDNA) synthesis. Total RNA (tRNA) is extracted from lysed milk cells using RNeasy mini kit (RNeasy mini kit; Qiagen). The cDNA will be synthesized in 20 l RT mix. Ad 2 Application of quantitative PCR for cytokine mRNA in bovine cells. PCR primers and probes for bovine interferon gamma (IFN-g: Accession M29867), TNF-a (Z14137) and interleukin IL-2 (M12791), IL-6 (X62501), IL-8 (S74436), IL-12p40 (U14416) have been described for cattle and would be synthesized by Applied Biosystems and used to amplify theirrespective cDNA (TABLE 1). Primers and probes against (IL) 1-a (M37210), IL-1b (M35589), IL-4 (M77120), TGF B and IL-10 (U00799) would be designed with Primer Express software (Applied Biosystems) using sequences present in the NCBI database (see accession numbers listed above). Where possible primers would be designed to be exon spanning and their ability to distinguish GDNA from cDNA confirmed by PCR and gel electrophoresis. We have successfully employed this approach to quantitate canine and feline mucosal cytokines. A fragment from the bovine GAPDH (AF022183) gene and a universal 18S rRNA detection kit (Applied Biosystems) would be used to monitor the amount of RNA present in the reaction. Develop assays to quantify bovine cytokines. PCR will be performed using primers that target full length bovine cytokine genes. The amplified bands corresponding to cytokine cDNAs will be excised from the gel and purified using the Geneclean kit (Bio 101, USA). The purified DNA fragments will be ligated into the pGEM-T easy expression vector ( Promega, USA), and transformed into a competent E. coli DH-5. Purified recombinant cytokines will be used to make monoclonal antibodies. These antibodies will form the basis of sandwich ELISAs (capture and detection) for bovine cytokines. Evaluation of cytokine dynamics during transient and chronic in vivo infections. In-vivo challenge experiments will be conducted with transient and chronic E.coli strains. These experiments are currently in the planning phase and will be funded from other sources.
PROGRESS: 2004/10 TO 2007/09
OUTPUTS: The most important outputs of this project have been the developed assays for bovine cytokines. We now have a series of cytokine assays available that were not available before the start of this project. This project was mostly a developmental project where new assays were developed for use in a range of applications. The project was directed by three collaborating scientists who each have produced outputs with the assays developed. The assays were developed by the principle investigators, supported by their technical staff, PhD students and post-doctoral candidates. The assays were developed in the laboratories of the PI's. The assays were validated and used for applications in bovine mastitis and reproduction. Both in-vivo and in-vitro inflammatory responses were studied. Outputs in terms of completed standard operating procedures for the cytokine assays were shared with scientists in New York, throughout the US and with international collaborators. The National Mastitis Council has adopted the assays developed under this project as the standard for future use in mastitis challenge studies. Results of the studies performed with the developed assays have been reported to New York dairy farms and veterinarians at several events in the State. Both Drs Gilbert and Schukken presented studies that were conducted with the aid of the assays developed under this project. This included presentations to the Fall Dairy Conference in both 2006 and 2007, the annual meeting of the National Mastitis Council and several local producer meetings. At the winter dairy management meetings (21 meetings in total throughout New York), approximately 1000 dairy producers were informed about the patters of inflammatory responses after bovine intra-mammary infections. PARTICIPANTS: Y.H. Schukken, P.I. K.W. Simpson, P.I. R.O. Gilbert, P.I. S. Klaessig, laboratory technician. N.Gollnick, graduate student. R.M. Mitchell, graduate student. B. Dogan, post-doctoral associate R. Quesnell, post-doctoral associate. TARGET AUDIENCES: There are two major audiences: 1. fellow scientists who will use the developed assays in this project. This includes scientists in veterinary colleges and animal science departments throughout the United States and the world. 2. dairy farmers, who will use the information gained from the experiments PROJECT MODIFICATIONS: The assays that we developed were not available to the scientific community before the start of this project. Our understanding of TH1/TH2 immune switches during the dry period has been altered dramatically due to the application of the assays developed during this project.
IMPACT: 2004/10 TO 2007/09
The outcomes of this project are the availability of several bovine cytokine ELISA assays available to dairy scientists. These assays were developed and validated through this project. The standard operating procedures (SOP's) from these assays have been adapted by the National Mastitis Council in early 2008 as the industry standards. Several studies were completed using the assays that were developed and these studies have contributed importantly to our understanding of the immune system of the dairy cow. Two studies will be highlighted here: First, a study by graduate student Nicole Gollnick and other (including technician Suzanne Klaessig) reported on the ability of different Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains to survive in bovine monocyte-derived macrophages (MDMs) of cows naturally infected with M. paratuberculosis and control cows not infected with this organism. We tested the hypotheses that survival of M. paratuberculosis in macrophages is dependent on the strain. Following differentiation, MDMs where challenged in vitro with 4 M. paratuberculosis strains of different host specificity (cattle, sheep). Two hours and 2, 4, and 7 days after infection, ingestion and intracellular survival and cytokine patterns of M. paratuberculosis strains were determined by fluorescence microscopy and cytokine ELISA assays. Important strain differences were observed. These findings suggest that some M. paratuberculosis strains interfere more successfully than others with the ability of macrophages to kill intracellular pathogens which will make it important to include strain typing when designing control programs for Johne's disease on dairy farms. Second, a number of intramammary challenge experiments were conducted by Post doctoral associate Rebecca Quesnell in collaboration with Suzanne Klaessig. For these studies four cows were challenged with approximately 30 cfu of E.coli at approximately 14 days before the expected calving day (hence, in the dry period). Cows were sampled before challenge, intensively (every 3 hours) immediately after challenge and after calving and weekly thereafter. Cytokine patterns were determined in all samples. When cows developed a case of clinical mastitis another sequence of intensive sampling ensued. All samples were evaluated for cytokine patterns using the developed cytokine ELISA assays for bovine IL-1, IL-6, IL-8, IL-10, TNF-alpha and Gamma-interferon. The results of these studies showed a very clear TH1-TH2 switch at the time of calving in the dairy cow. Challenge infections in the dry period resulted in a full blown TH2 response with strong IL-10 response and virtually no pro-inflammatory cytokine response. Post-partum inflammatory response was a full blown TH1 response with an early pro-inflammatory cytokine response and a delayed IL-10 response. This is the first time that the TH1-TH2 switch in dairy cows has been reported. The developed ELISA assays were essential to show this. The results of this study will be very important for our understanding of the bovine immune system and for further development of preventative programs for bovine mastitis.
PUBLICATIONS (not previously reported): 2004/10 TO 2007/09
1. Baumgart M., Dogan B., Rishniw M., Weitzman G., Bosworth B., Yantiss R., Orsi RH., Wiedmann M., McDonough P., Kim S.G., Berg D., Schukken Y., Scherl E., Simpson K.W. 2007. Culture independent analysis of ileal mucosa reveals a selective increase in invasive Escherichia coli of novel phylogeny relative to depletion of Clostridiales in Crohn's disease involving the ileum. ISME J. 1:403-418.
2. Gollnick N.S., Mitchell R.M., Baumgart M., Janagama H.K., Sreevatsan S., Schukken Y.H. 2007. Survival of Mycobacterium avium subsp. paratuberculosis in bovine monocyte-derived macrophages is not affected by host infection status but depends on the infecting bacterial genotype. Vet Immunol Immunopathol. 120:93-105.
3. Wilson D.J., Mallard B.A., Burton J.L., Schukken Y.H., Grohn Y.T. 2007. Milk and serum J5-specific antibody responses, milk production change, and clinical effects following intramammary Escherichia coli challenge for J5 vaccinate and control cows. Clin Vaccine Immunol. 14:693-699.
4. Kummer S., Hayes G.R., Gilbert R.O., Beach D.H., Lucas J.J., Singh B.N. 2008. Induction of human host cell apoptosis by Trichomonas vaginalis cysteine proteases is modulated by parasite exposure to iron. Microb Pathog.44:197-203.
5. Herath S., Williams E.J., Lilly S.T., Gilbert R.O., Dobson H., Bryant C.E., Sheldon I.M. 2007. Ovarian follicular cells have innate immune capabilities that modulate their endocrine function. Reproduction. 134:683-693.
6. Janeczko S., Atwater D., Bogel E., Greiter-Wilke A., Gerold A., Baumgart M., Bender H., McDonough P.L., McDonough S.P., Goldstein R.E., Simpson K.W. 2008. The relationship of mucosal bacteria to duodenal histopathology, cytokine mRNA, and clinical disease activity in cats with inflammatory bowel disease. Vet Microbiol. 128:178-193.
7. Schukken Y.H., Gonzalez, R.N., Bennett, G.J., Schulte, H.F., Welcome, F.L., Tikofsky, L.L., Coffin, L.M., Santisteban, C.G., Zadoks, R.N., and Zurakowski, M.J. 2007. Quality Milk Production Services: 60 years of milk quality improvement in New York State. Proceedings National Mastitis Council 2007 pp 159-173.
PROJECT CONTACT:
Name: Hoffmann, M. P.
Phone: 607-255-2224
Fax: 607-255-9499
Email: cuaes@cornell.edu

 
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ACCESSION NO: 0136980 SUBFILE: CRIS
PROJ NO: NYCV-478306 AGENCY: CSVM NYCV
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 OCT 1988 TERM: 30 SEP 1998 FY: 2007
INVESTIGATOR: Lein, D. H.; Shin, S. J.; Jacobson, R. H.
PERFORMING INSTITUTION:
Diagnostic Laboratory
Cornell University
Ithaca , New York 14853
DEVELOPMENT OF IMPROVED DIAGNOSTICS FOR PARATUBERCULOSIS.
OBJECTIVES: Develop improved diagnostics for Paratuberculosis via culturing techniques and serodiagnosis using specific antigens in automated kinetics ELISA (KELA) systems.
APPROACH: Improve selective media for Mycobacterium paratuberculosis to reduce contamination by various environmental bacteria and fungi. In methodology various gemination procedures for spores will be explored as well as various antibiotic combinations. Specific antigens are being employed in KELA serodiagnostic assays for more accurate and timely detection of cattle infected with paratuberculosis. Epidemiological surveys will be conducted using a validated test.

 
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ACCESSION NO: 0197918 SUBFILE: CRIS
PROJ NO: ND04401 AGENCY: SAES ND.
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 JUL 2003 TERM: 30 SEP 2008 FY: 2007
INVESTIGATOR: Freeman, D.; Dyer, N.; Ringwall, K.; Faller, T.
PERFORMING INSTITUTION:
Veterinary & Microbiological Sciences
North Dakota State Univ
Fargo , North Dakota 58105
BIOSECURITY: DISEASE SURVEILLANCE AND FOOD SAFETY.
NON-TECHNICAL SUMMARY: Introduction of pathogenic organisms into the ND agricultural sector may have devastating effects on animal health and the agricultural industry. This project addresses methods to identify, diagnose and contain a disease outbreak as rapidly as possible.
OBJECTIVES: 1. Enhance diagnostic capabilities at NDSU Veterinary Diagnostic Laboratory (VDL). 2. Expand participation in state and national public health and veterinary diagnostic networks. 3. Develop methods for rapid communication between the field, diagnostic lab and state agencies. 4. Develop continuing education for target groups to enhance preparedness. 5. Develop a rapid response team for animal handling and management in a disease outbreak. 6. Enhance the rapid diagnostic methods and disease prevention / vaccination protocols available for selected pathogens.
APPROACH: 1. Augment NDSU VDL facilities from Biosafety Level 2 to 3, and hire a Clinical Diagnostic Microbiologist with molecular experience. 2. Strengthen surveillance coordination the Department of Health Lab through creation of overflow capacity or supporting diagnostics. Work with national and regional labs to participate in the National Animal Health Laboratory Network. 3. Establish a communications center in western ND (DSU) that will allow remote, wireless teleconferencing and data transmission from the field to NDSU and the ND State Veterinarian. 4. Work with NDSU Extension and national experts to develop continuing education programs for "first observer" groups in a disease outbreak (such as border guards, practicing veterinarians, producers). Create programs that can be delivered potentially on-line or via CD, as well as live. 5. Equip and train a group of animal specialists from the DREC and HERC to use GPS technology and locate/muster/evaluate all stock in any geographic area. This will include trucks, mobile lab, animal handling/restraint, communications and initial diagnostic equipment. 6. Develop a series of projects that model novel vaccine types (novel antigens and delivery systems); and expand research into virulence factors of selected pathogens that can be exploited for disease prevention and diagnosis.
PROGRESS: 2005/10 TO 2006/09
Ongoing diagnostic work continues in the Veterinary Diagnostic Lab, including the development of rapid diagnostics utilizing PCR for several pathogens, including anthrax. The VDL is performing liquid culture and subsequent Real-time PCR on suspect fecal samples for Mycobacerium paratuberculosis (Johne's Disease); from January to May 2006, the lab ran over 400 fecal samples in liquid culture and PCR, and reported out 15 positive results. Gas chromatography, HPLC and mass spectrophotometry are being utilized to analyze fatty acids from the isolates, as these are suspected to be involved in the immunopathology. Faculty and graduate students in Veterinary and Microbiological Sciences were supported in a variety of current research projects related to models of disease pathogenesis, diagnosis and disease surveillance. In addition to the development of new models and enhanced understanding of microbial disease, these efforts provide a source of training for the next generation of scientists and health professionals. High frequency RFID tags have been tested extensively in laboratory or warehouse conditions. In addition, we concentrated on testing whether passive HF RFID could work in field conditions. Some of these activities include Doorway Gate Design, Alley Gate Design, and development of short life and medium life RFID ear tags. In the past year, we have developed several techniques to measure the 'quality' of an RFID tag. This is a necessary activity to design and optimize our own RFID tag in the size and shape of a cattle tag. We are now able mathematically model and predict the performance of an RFID antenna design using ANSOFT providing the antenna is at least 1.5m away from the RFID tag. We are now designing and building our own RFID antennas. Based on trace back studies, current methods of cattle traceability work (greater than 99%) including branding, provided cattle are not co-mingled and resorted. Once co-mingled and resorted cattle were not traceable (0%) by any current method primarily due to cut tags, 79% within backgrounders and 13% within feeders, and simple logistics of re-working larger groups of calves. Cattle must maintain original electronic ear tags recorded in the USDA-APHIS-VS network of approved databases. The Biosurveillance Working Group (BWG) has continued to meet statewide and coordinate activities, including the state and federal veterinarians.
IMPACT: 2005/10 TO 2006/09
The animal RFID and trace back research is generating novel data that should serve the national need in animal id for producers and regulatory agencies. Studies in disease pathogenesis, epidemiology, engineering and communication continue and provide critical new data in addition to opportunities for student education and training. This project has continued to improve our ability to respond to a disease outbreak, conduct related research, communicate critical information and educate our clientele.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
1. Horne, S.M., and B.M. Pruess. 2006. Global gene regulation in Yersinia enterocolitica: effect of FliA on the expression levels of flagellar and plasmid-encoded virulence genes. Arch. Microbiol. 185:115-126.
2. Pruess, B.M., C. Besemann, Denton, A. and Wolfe, A.J. 2006. A complex transcription network controls the early stages of biofilm formation. J. Bacteriol. 188:3731-3739.
3. Khaitsa, ML, M. L. Bauer, G. P. Lardy, D. Doetkott, R. B. Kegode and P. S. Gibbs, 2006. Fecal shedding of Escherichia coli O157:H7 in North Dakota feedlot cattle in the fall and spring. J Food Prot. 69(5)1154-1158.
4. Khaitsa, ML, R. Barigye, N.W. Dyer, D. Doetkott, JR Foster. 2006. Serologic and other Diagnostic Evidence of Neospora caninum presence in North Dakota Beef Herds. The Bovine Practitioner 40(1) 51-56.
5. Khaitsa, ML, R. Kegode, M. L. Bauer, P. S. Gibbs, G. P. Lardy and D. Doetkott. 2006. A longitudinal study of Salmonella shedding and antimicrobial susceptibility patterns in North Dakota feedlot cattle (J. Food Protection, In press).
6. Khaitsa, M.L., R. Barigye, N.W. Dyer, D. Doetkott, J.R. Foster. 2006. Extension Publication: Evidence of Neospora caninum presence in North Dakota Beef Herds. Unified Beef Cattle and Range Research Report: Agricultural Experiment Station, Dept. Animal and Range Sciences, North Dakota State University.
7. Theis, M., M.L. Khaitsa, D. Doetkott, N. Dyer, P. Gibbs. 2005. Salmonella Occurrence in North Dakota Grass Fed Beef Cattle: Prevalence, Characterization and Comparison with reports from the Veterinary Diagnostic Laboratory and North Dakota Department of Health. In: Proceedings of the 86th Annual Meeting of the Conference of Research Workers in Animal Diseases. St Louis, MO, Dec.3 -6, 2005. Abstract No. 36.
PROJECT CONTACT:
Name: Freeman, D. A.
Phone: 701-231-8504
Fax: 701-231-7514
Email: douglas.freeman@ndsu.nodak.edu

 
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ACCESSION NO: 0206890 SUBFILE: CRIS
PROJ NO: ND05348 AGENCY: CSREES ND.
PROJ TYPE: SPECIAL GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2006-34475-17127 PROPOSAL NO: 2006-06092
START: 01 AUG 2006 TERM: 31 JUL 2008 FY: 2007 GRANT YR: 2006
GRANT AMT: $1,317,014
INVESTIGATOR: Freeman, D.; Logue, C.; Sellnow, T.; Hinsz, V.; Nganje, W.; Khaitsa, M.; Gibbs, P.; Panigrahi, S.; Hall, C.; Glower, J.
PERFORMING INSTITUTION:
Veterinary & Microbiological Sciences
North Dakota State Univ
Fargo , North Dakota 58105
FOOD SAFETY RISK ASSESSMENT, ND.
NON-TECHNICAL SUMMARY: Annually, millions of people in the US suffer from food borne illness, resulting in thousands of deaths. The purpose of these projects are to develop and test models designed to predict risk of pathogen contamination, and to develop improved quality sensors, with the ultimate goal of enhancing food safety.
OBJECTIVES: A. Risk Assessment Objective 1 Evaluate Salmonella contamination associated with farm production and carryover; evaluate consumer exposure in domestic homes and the effect of portion size on consumer exposure to Salmonella. Objective 2 Food Safety Risk and Economic Optimality in Ready to Eat Turkey Products Objective 3 Evaluate strategies for communication comprehension, efficiency, and compliance related to risk in food processing consumption. Objective 4 Evaluate how psychological and emotional factors contribute to food safety risk. Objective 5 Risk Assessment of Salmonella contamination of different turkey meat products including organic, fresh, frozen, and ready to eat products from US Midwestern retail outlets, and different turkey meat products consumed by specific risk groups. Objective 6 Merging and Data Analysis (Drs. Logue, Nganje, Sellnow, Hinsz and Khaitsa) data collected from the five objectives will be merged and included in the risk model to address issues of data gaps and new research which will enhance the project. B. Risk Assessment for Pathogens of Food Safety Concern in Milk and Powdered Milk Products. Objective 1 Assess the potential for raw milk as a source of Enterobacter sakazakii in powdered infant formula, and then examine isolates for relatedness to outbreak isolates that have been documented in the United States. Objective 2 Assess the potential for milk and milk products as a source of other members of the Enterobacteriaceae, and as a source of antimicrobial resistance mechanisms. Objective 3 Assess the potential for milk and milk products as a source of Mycobacterium avium subspecies paratuberculosis. C. Intelligent Quality Sensors for Food Safety Objective 1: Identification of liquid metabolites associated with meat contaminated with Salmonella. Objective 2: Characterization of liquid metabolites using Raman Spectroscopy. Objective 3: Fabrication and evaluation of different sensing materials for detecting specific indicator gaseous compounds associated with meat contamination due to Salmonella. Objective 4: Investigate on developing pattern recognition techniques for enhancing the performances of our electronic nose modules. Objective 5: Evaluate the performance of integrated electronic nose module in field-conditions.
APPROACH: 1) Studies will investigate Salmonella in turkeys from entry into rearing barns to slaughter. Molecular analysis will further define the pathotype of the organism and the transfer between flock rotations. Domestic exposure of consumers to Salmonella in homes will be evaluated. 2) The empirical model will include optimal intervention strategy at the retail (consumer) level. The associated risks, costs and benefits of alternative mitigation strategies (e.g., generic and augmented HACCP) for Salmonella are evaluated jointly. 3) Studies will test potential risk communication messages that contain varying strategies related to culture, learning styles, public outrage, and message source in order to determine message receptivity. The messages will be tested with diverse publics, including general American macro-culture and underserved populations. We will test differences in message receptivity, and develop strategies for adapting risk messages to meet the needs of various audiences. 4) Studies will test whether the activation of the disgust emotion heightens perceptions and judgments of risk and diminishes willingness to approach and interact with foods appearing to have properties associated with contamination. Food handlers and preparers reports of key practices will be assessed. 5) Different turkey meat products including organic, fresh, frozen, and ready to eat products from retail outlets will be randomly sampled and tested for the presence of Salmonella. 6) Data and information from all studies will be merged and reported in a range of media. B. Risk Assessment for Pathogens of Food Safety Concern in Milk and Powdered Milk Products. Initial Approach to All Objectives: Assess the potential for milk and milk products as a source of Enterobacter sakazakii, other members of the Enterobacteriaceae, and M. avium subsp. Paratuberculosis on raw milk samples. Culturing procedures for E. sakazakii and M. avium subsp. paratuberculosis have been optimized with newly developed and commercially available media. An improved method for isolation of M. avium subsp. paratuberculosis from bulk tank milk samples will be implemented. C. Intelligent Quality Sensors 1) Representative meat samples will be analyzed using HPLC technique to determine their constituents and identify indicator metabolites. 2) The identified metabolites (compounds) will be characterized using SERS (surface enhanced) Raman spectroscopy. 3) Detectors will be fabricated based on their novel sensing characteristics to provide additional sensitivity to the indicator compounds. 4) We will use the responses of different electronic nose modules to determine probability density functions (pdf), which will be used to generate additional numbers of simulated data sets which will be used to develop and evaluate classification or prediction models using neural networks techniques. 5) The framework for integrated prototype electronic nose will be developed. The integrated nose modules will then be further evaluated for discriminating Salmonella contaminated meat samples.
PROGRESS: 2006/08 TO 2007/07
OUTPUTS: The microbiologists have explored Salmonella and Campylobacter in poultry production and assessing risks using rapid methods for the detection of Salmonella in turkey and comparative genetics for analysis of Campylobacter. Papers and presentations have been generated. Our psychologists have explored the role that psychological factors have on perceptions and judgments of risk as well as serve as a motivation to prevent unsafe activities with food. Presentations have been generated. Our risk communications experts evaluated individual responses to risk communication messages about food contamination or adulteration (unintentional and unintentional) by testing risk communication messages to determine the degree to which the messages are efficient, comprehendible, and promote compliance. Papers and presentations were generated. Our economists have developed stochastic optimization and stochastic dominance models to evaluate food safety risk mitigation strategies at retail facilities using real option models to evaluate where investments will reduce intentional contamination risks in the food supply chain. From this they have developed models to test the existence of off-setting behavior in food safety policy/information delivery and evaluated incentives and efficient policy design to mitigate foodborne pathogen outbreaks. Presentations and papers were generated. New projects have focused Enterobacter sakazakii, Enterobacteriaceae, and M. avium subsp. paratuberculosis in milk and milk products from supermarket and raw milk samples of North Dakota dairies. Our epidemiologists have studied the prevalence of E. coli O157:H7 and Salmonella shedding at the time of slaughter in cattle in North Dakota. They are measuring the efficacy and cost effectiveness of an intervention strategy to reduce shedding of E. coli O157:H7 by cattle pre-harvest and the prevalence of E. coli O157:H7 and Salmonella in raw ground beef. Papers and presentations were generated. Our agricultural engineers have investigated different sensing materials for indicator compounds associated with Salmonella contamination of packaged meat. A configuration for an integrated electronic nose system has been developed. Experiments have been conducted to characterize liquid metabolites in Salmonella contaminated meat using LC-MS. Parallel work has investigated strategies for small datasets associated with a typical biological study including mega-trend diffusion (MTD) and functional virtual population (FVP) techniques for data domain expansion and synthetic sample generation. Artificial neural networks were used for classifications. A presentation was generated. PARTICIPANTS: Catherine M. Logue PI Associate Professor of Microbiology; Chantal W. Nde - Graduate Researcher; Mohamed K. Fakhr Post Doctoral Researcher; Margaret L. Khaitsa - Assistant Professor of Epidemiology; Redempta Kegode - Graduate Researcher; James Oloya - Post Doctoral Researcher; William E. Nganje - Associate Professor of Agribusiness and Applied Economics; Simeon Kaitibie - Post Doctoral Researcher; Alexandre Sorin - Graduate Researcher; Linda Lehrke - Graduate Researcher; Agnes Lyonga - Graduate Researcher; Timothy L. Sellnow - Professor of Communication; Steven Venette - Post Doctoral Researcher; Julie Novak - Graduate Researcher; Verlin Hinsz - Professor of Psychology; Gary Nickell - Professor of Psychology - Research Associate TARGET AUDIENCES: Policy makers and Regulators - eg. USDA FSIS, FDA; State and Federal Regulators; Researchers in Microbiology; Researchers in Epidemiology; Researchers in Food Safety and Risk; Researchers in Psychology; Researchers in Communication; Researchers in Engineering; Researchers in Economics and Agribusiness; Producers and Processors; Consumer groups; Food safety advocates;
IMPACT: 2006/08 TO 2007/07
Microbiologists assessed rapid methods for the detection of Salmonella in turkey and comparative genetics for analysis of Campylobacter, and examined the prevalence of virulence and resistance genes in Salmonella associated with poultry to understand their distribution in the food chain. The psychologists examined experimental activation of motives among participants and its influence on judgments of risk for food-borne contamination and found motives influenced judgments of contamination. The study examined how psychological traits motivate individuals to care more about food safety and act to avoid contamination. The trait of conscientiousness of food handlers and preparers was assessed; those with higher levels of conscientiousness had more positive opinions about following food safety practices and being more likely to engage in such behaviors. The communications team surveyed diverse public groups including general American macro-culture and underserved populations to explore the impact of culture, learning styles, public outrage, and message source on message receptivity. The economists developed real option models (Tomato Garden Models) to: a) evaluate where investments reduce intentional contamination risks in the food supply chain, b) test the existence of off-setting behavior in food safety policy/information delivery and c)examine the effect of incentives and efficient policy design to mitigate foodborne pathogen outbreaks. Enterobacter sakazakii has not been detected in milk to date. Antimicrobial susceptibilities of fecal coliforms (E. coli, Klebsiella,Enterobacter spp. non-sakazakii), indicate many possess therapeutic level resistance to tetracycline, cephalothin, ampicillin, and nalidixic acid. The epidemiologists investigated E. coli O157:H7 and Salmonella shedding at time of slaughter in North Dakota cattle, and their antimicrobial susceptibility profiles. They are measuring the efficacy and cost effectiveness of an intervention strategy to reduce shedding of E. coli O157:H7 by cattle pre-harvest and the prevalence of E. coli O157:H7 and Salmonella in raw ground beef. The agricultural engineers investigated sensing materials for indicator compounds associated with Salmonella in packaged meat. Composite polymers were made using conducting polymers and carbon black particles and deposited on interdigitated gold electrodes to develop sensor heads. The heads were used for sensing ethanol and acetic acid. Polyvinylphenol and Polyethylenimine showed best sensitivity. Ongoing work is validating sensitivities at low ppm levels. A configuration for an integrated electronic nose system has been developed; design of the sensing chamber, sampling devices is complete. Experiments characterizing liquid metabolites of Salmonella contaminated meat using LC-MS found liquid samples are complex; investigations are determining non-volatile constituents. Parallel work investigating strategies for small dataset analysis using mega-trend diffusion (MTD) and functional virtual population (FVP) techniques for data domain expansion and synthetic sample generation is ongoing.
PUBLICATIONS (not previously reported): 2006/08 TO 2007/07
1. Fakhr, M.K. and Logue, C.M. 2007. Sequence variation in the outer membrane protein encoding gene cmeC, conferring multidrug resistance among Campylobacter jejuni and Campylobacter coli strains isolated from different hosts. Journal of Clinical Microbiology 45:3381-3383.
2. Khaitsa, ML, R. Kegode, M. L. Bauer, P. S. Gibbs, G. P. Lardy and D. Doetkott. 2007. A longitudinal study of Salmonella shedding and antimicrobial susceptibility patterns in North Dakota feedlot cattle. J Food Prot. 70:2:476-481.
3. Nganje, William, Simeon Kaitibie, and Alexandre Sorin. 2007. HACCP Implementation and Economic Optimality in Turkey Processing. Agribusiness, An International Journal Volume 23, No. 2.
4. Nganje, William and Linda Lehrke. 2007. Evaluating Cost-Effective Food Safety Risk Reduction Strategies, FSRRN Journal, Vol II,: 3:1-5, 06.
5. Agnes Lyonga, William Nganje, Timothy Sellnow, Simeon Kaitibie and Steven Vennette. 2007. Human Factor Risk in Turkey Processing and High Reliability Culture. Food Protection Trends 26(7):593-600.
6. Sellnow, T.L., Ulmer, R.R., Seeger, M.W., and Veil, S.R. 2007. Terrorism as chaos: A chaos model for managing random acts of terror. In D.H. O Hair, and R.L. Heath (Eds.). Community preparedness and response to terrorism: Communication and the media. Praeger: Westport, CT. Book chapter.
7. Seeger, M. W., Sellnow, T. L., Ulmer, R. R., Novak, J. M. 2007. Applied Communication Ethics: A Summary and Critique of the Research Literature. In L. Frey and K. Cissna (Eds.). Handbook of applied communication research. Book chapter.
8. Blume, M.P., Hinsz, V.B., and Nickell, G.S. 2007. Dispositional disgust as a motivating emotion in food safety. Abstr. Society for Personality and Social Psychology, Memphis, TN.
9. Lawrence, D.M., Hinsz, V.B., and Nickell, G.S. 2007. Optimistic bias and food safety: Getting sick from home cooking or the street fair. Abstr. Association for Psychological Science, Washington, D.C.
PROJECT CONTACT:
Name: Freeman, D. A.
Phone: 701-231-8504
Fax: 701-231-9692
Email: douglas.freeman@ndsu.edu

 
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ACCESSION NO: 0059542 SUBFILE: CRIS
PROJ NO: OHO00416 AGENCY: CSREES OHO
PROJ TYPE: HATCH PROJ STATUS: TERMINATED MULTISTATE PROJ NO: NC-107
START: 01 OCT 2001 TERM: 30 SEP 2006 FY: 2004
INVESTIGATOR: Sreevatsan, S.
PERFORMING INSTITUTION:
Food Animal Health Research Program
Ohio State University
1680 Madison Avenue
Wooster , Ohio 44691
EVOLVING PATHOGENS, TARGETED SEQUENCES, AND STRATEGIES FOR CONTROL OF BOVINE RESPIRATORY DISEASE.
NON-TECHNICAL SUMMARY: Chronic respiratory disease is one of the most important disease problems in cattle. The project addresses emerging and reemerging agents, pathogenesis, and intervention strategies of chronic respiratory diseases of cattle.
OBJECTIVES: Our research will focus on the following three objectives: 1) Identify emerging and re-emerging agents and develop diagnostic methods for bovine respiratory disease (BRD); 2) Characterize mechanisms and intervention targets in pathogenesis of BRD at the molecular, cellular and host level; 3) Develop intervention strategies for critical control points to reduce impact of BRD.
APPROACH: The new project will generate data on pathogen patterns and better diagnostic methods that can be used for better treatment and control measures. Understanding the mechanisms used by the various agents of BRD to persist on the host, and produce inflammatory and immune responses are needed for the characterization of novel intervention targets that can minimize the use of antibiotics.
PROGRESS: 2001/10 TO 2006/09
This project was terminated.
IMPACT: 2001/10 TO 2006/09
This project was terminated.
PUBLICATIONS (not previously reported): 2001/10 TO 2006/09
No publications reported this period
PROJECT CONTACT:
Name: Sreevatsan, S.
Phone: 330-263-3745
Fax: 330-263-3677
Email: sreevatsan.1@osu.edu
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ACCESSION NO: 0211331 SUBFILE: CRIS
PROJ NO: ORE00885 AGENCY: CSREES ORE
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 15 APR 2007 TERM: 30 SEP 2011 FY: 2007
INVESTIGATOR: Bermudez, L. E.; Gustafson, S. B.; Hase, C.; Jin, L.; Pastey, M. K.; Rockey, D. D.; Sarker, M. R.
PERFORMING INSTITUTION:
College of Veterinary Medicine
Oregon State University
Corvallis , Oregon 97331
ANIMAL HEALTH AND DISEASE.
NON-TECHNICAL SUMMARY : The consequences of disease have severe implications, both on the economics of agriculture as well as on public health. The identification of disease mechanisms, animal models, vaccines, and antibiotic resistance can bring much needed development to agriculture. This project will focus on understanding the genetic makeup of pathogens and infection processes in animals, developing recombinant vaccines, controling hemorrhages, and developing diagnostic tools and models.
OBJECTIVES: To mitigate the impact of animal diseases on the economics of agriculture and on public health. Program objectives are: 1. To develop a recombinant vaccine using alpha-herpes virus as a vector for type A influenza virus to allow large production of flu virus antigen in a short timeframe (Jin); 2. To determine the genetic difference between alpaca BVDV and bovine BVDV (Jin); 3. To determine the efficacy and biocompatibility of various forms of chitosans in controlling hemorrhages (Gustafson); 4. To develop conditions under which the Chlamydia suis genomic island that carries a tetracycline resistance gene can be used as a tool for transformation of chlamydia (Rockey); 5. To identify the various proteins of Vibrio parahaemolyticus that are expressed during association of the bacteria with oysters as part of an effort to develop novel targeted intervention strategies to increase seafood safety and reduce occurrence of seafood-borne illnesses (Hase); 6. To replicate highly pathogenic avian influenza H5N1 viruses in pigs in a long-term effort to study the molecular bases of the ability of a virus to spread among a range of hosts and the pathogenicity of influenza viruses (Pastey); 7. To define the role of a sensorhistidine kinase, Spo0A in sporulation-regulated Clostridium perfringens enterotoxin (CPE) synthesis (Sarker); 8. To identify bacterial genes involved in early stages of infection, or entry of the intestinal mucosa, by Mycobacterium paratuberculosis (Bermudez).
APPROACH: Researchers will investigate different pathways for how viruses and bacteria operate or distribute in animals and shellfish, identify genetic transformation systems, find molecular mechanisms underlying Vibrio bacterial-shellfish interaction and CPE synthesis, and identify how disease interacts with internal organs and cellular wall. Experiments will be conducted to determine persistence of infection. Diagnostic tools for detection and control plus mew generation recombinant vaccines and chitosan bandages will be developed. IBC and IACUC approval will be sought when experiments and procedures are finalized.
PROJECT CONTACT:
Name: Bermudex, L. E.
Phone: 541-737-6538
Email: luiz.bermudez@oregonstate.edu

 
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ACCESSION NO: 0407659 SUBFILE: CRIS
PROJ NO: 1265-32000-078-02S AGENCY: ARS 1265
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 01 SEP 2003 TERM: 31 AUG 2008 FY: 2005
INVESTIGATOR: Karns J S; Whitlock R H; Van Kessel J S
PERFORMING INSTITUTION:
Veterinary Preventive Medicine
Univ of Pennsylvania
Philadelphia , Pennsylvania 19104
PILOT STUDY OF FACTORS AFFECTING MAINTENANCE OF MYCOBACTERIUM, SALMONELLA, E. COLI, AND LISTERIA ON DAIRY FARMS.
OBJECTIVES: The objective of this research is to set up a Pilot Program consisting of two dairy herds to focus on the protocol development, laboratory set-up, program logistics, and database and bio-bank development needed to evaluate the impact of intervention strategies on Johne's disease dynamics, milk and beef quality (particularly with respect to zoonotic bacterial pathogens), economics and sustainability through intensive longitudinal follow up of selected research/demonstration dairy farms. Long term goals are to validate intervention strategies to support BMP's and to optimize intervention and monitoring strategies given the constraints on time, labor and financial resources in modern dairy herds. In addition, a national resource bank (data and biological specimens on well-characterized animals) will be created for current and future monitoring and research on dairy cattle diseases. Emphasis will be on longitudinal data collection on endemic infectious diseases of public health and animal health concern in dairy herds.
APPROACH: Pathogens are of increasing concern on dairy farms and in dairy products. The production of safe and wholesome food from US farms requires control of the production process on the farm. Specific focus areas in this process are biosecurity, food safety and animal health. To be able to scientifically support regional process control programs there is a need for longitudinal research on commercial dairy farms throughout the United States. In this collaboration, Cornell University, The Pennsylvania State University, University of Vermont, and the University of Pennsylvania, all participants in the Regional Dairy Quality Management Alliance (RDQMA), and the USDA's Environmental Microbial Safety Laboratory (EMSL) will investigate the sites on operating dairy farms that act as reservoirs for pathogenic microorganisms that affect animal health and that decrease product quality because of their zoonotic nature. Several farms will be identified as candidates for this investigation and 3 will be chosen for extensive sampling based on the previous occurrence of Johne's disease and/or Salmonella infection. Serum, feces, bulk tank milk, and environmental samples (water, bird and rodent feces, feed, etc.) will be taken on each farm. In addition, tissue samples will be obtained from carcasses of culled animals. Samples will be distributed among the university and ARS researchers for analysis to determine the presence of Mycobacterium avium paratuberculosis (the causative agent of Johne's disease in cattle) and for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes (human food-borne pathogens of concern in dairy products). This research will be the first to attempt a comprehensive analysis of both Johne's disease and food-borne pathogens on working dairy farms. It will allow us to determine a baseline for these organisms on two farms and set the stage for investigation of the effect of interventions, in the form of best management practices (BMPs), on animal health and product quality.
PROGRESS: 2005/10 TO 2006/09
Progress Report 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and the University of Pennsylvania. Additional information can be found in the reports for the SCAs with the other universities involved in the project (1265-32000-078-01S, 03S, and 04S) and in the report for the parent project 1265-32000-078-00D "DAIRY MANAGEMENT PRACTICES AND THE TRANSMISSION OF ZOONOTIC PATHOGENS IN MILK". This cooperative agreement is part of a larger project involving 4 universities, the University of Pennsylvania, The Pennsylvania State University, University of Vermont, and Cornell University, and formulated in consultation with the Northeastern Regional Dairy Quality Alliance (RDQMA) and the National Milk Producers Federation. Currently, this project provides longitudinal access to three dairy herds totaling 575 milk cows (NY herd-340 milk cows; PA herd-110 milk cows and VT herd-125 cows). The feces of each adult cow is culture tested twice a year for the presence of Mycobacterium avium Paratuberculosis (MAP, the causative agent of Johne's disease) and blood serum is tested quarterly with an ELISA for the presence of antibodies to the Johne's Disease (JD) organism. Frequency of fecal sampling is increased to quarterly for any cow previously identified as culture positive or ELISA positive. As adult milk cows are culled from the herd, attempts are made to retrieve fecal samples and tissues at slaughter for culture to provide further evidence of JD. The initial prevalence in each herd was as follows. Herd A: 2.6 to 2.8%; Herd B: 1.8% to 6.8%; and Herd C: 13.6%, for a total number of 41 culture positive out of 575 or 5.9% apparent prevalence across the study. New techniques for quantitation of MAP were developed and four cows were identified as likely "super-shedders" and one cow progressed from a low-moderate shedder to a "super-shedder" over an 8 month time period. Based on the numerical assessment of Johne's bacteria in colony forming units per gram of manure, a single "super-shedder" could contaminate the environment with more bacteria than 160 heavy shedders, more than 6,000 moderate shedders and more than 60,000 low shedders. Each of the three herds in the study had a super-shedder cow at their farm. Not only do these super-shedders contribute to environmental contamination and risk of disease transmission on the farm, they may also cause false-positive fecal culture results in uninfected cows because of gastrointestinal "pass through" of Johne's bacteria. The importance of "super-shedders" for the transmission of JD in these dairy herds was studied by working with the producers to encourage culling of supershedders. After culling the incidence of MAP positive animals dropped drastically. Based on these results, management practices can be devised to further reduce the prevalence of JD in these and other dairy herds.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
No publications reported this period.

 
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ACCESSION NO: 0407665 SUBFILE: CRIS
PROJ NO: 1265-32000-078-03S AGENCY: ARS 1265
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 01 SEP 2003 TERM: 31 AUG 2008
INVESTIGATOR: Karns J S; Jayarao B M; Van Kessel J S
PERFORMING INSTITUTION:
Veterinary Science
Pennsylvania State University
Pennsylvania , Pennsylvania 16202
PILOT STUDY OF FACTORS AFFECTING MAINTENANCE OF MYCOBACTERIUM, SALMONELLA, E. COLI AND LISTERIA ON DAIRY FARMS.
OBJECTIVES: The objective of this research is to set up a Pilot Program consisting of two dairy herds to focus on the protocol development, laboratory set-up, program logistics, and database and bio-bank development needed to evaluate the impact of intervention strategies on Johne's disease dynamics, milk and beef quality (particularly with respect to zoonotic bacterial pathogens), economics and sustainability through intensive longitudinal follow up of selected research/demonstration dairy farms. Long term goals are to validate intervention strategies to support BMP's and to optimize intervention and monitoring strategies given the constraints on time, labor and financial resources in modern dairy herds. In addition, a national resource bank (data and biological specimens on well-characterized animals) will be created for current and future monitoring and research on dairy cattle diseases. Emphasis will be on longitudinal data collection on endemic infectious diseases of public health and animal health concern in dairy herds.
APPROACH: Pathogens are of increasing concern on dairy farms and in dairy products. The production of safe and wholesome food from US farms requires control of the production process on the farm. Specific focus areas in this process are biosecurity, food safety and animal health. To be able to scientifically support regional process control programs there is a need for longitudinal research on commercial dairy farms throughout the United States. In this collaboration, Cornell University, The Pennsylvania State University, University of Vermont and the University of Pennsylvania, all participants in the Regional Dairy Quality Management Alliance (RDQMA), and the USDA's Environmental Microbial Safety Laboratory (EMSL) will investigate the sites on operating dairy farms that act as reservoirs for pathogenic microorganisms that affect animal health and that decrease product quality because of their zoonotic nature. Several farms will be identified as candidates for this investigation and 3 will be chosen for extensive sampling based on the previous occurrence of Johne's disease and/or Salmonella infection. Serum, feces, bulk tank milk, and environmental samples (water, bird and rodent feces, feed, etc.) will be taken on each farm. In addition, tissue samples will be obtained from carcasses of culled animals. Samples will be distributed among the university and ARS researchers for analysis to determine the presence of Mycobacterium avium paratuberculosis (the causative agent of Johne's disease in cattle) and for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes (human food-borne pathogens of concern in dairy products). This research will be the first to attempt a comprehensive analysis of both Johne's disease and food-borne pathogens on working dairy farms. It will allow us to determine a baseline for these organisms on two farms and set the stage for investigation of the effect of interventions, in the form of best management practices (BMPs), on animal health and product quality.
PROGRESS: 2005/10 TO 2006/09
Progress Report 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and The Pennsylvania State University. Additional information can be found in the reports for the SCAs with the other universities involved in the project (1265-32000-078-01S, 02S, and 04S) and in the report for the parent project 1265-32000-078-00D "DAIRY MANAGEMENT PRACTICES AND THE TRANSMISSION OF ZOONOTIC PATHOGENS IN MILK". This cooperative agreement is part of a larger project involving 4 universities, the University of Pennsylvania, The Pennsylvania State University, University of Vermont, and Cornell University, and formulated in consultation with the Northeastern Regional Dairy Quality Alliance (RDQMA) and the National Milk Producers Federation. Currently, this project provides longitudinal access to three dairy herds totaling 575 milk cows (NY herd-340 milk cows; PA herd-110 milk cows and VT herd-125 cows). Fecal samples from adult cows on the PA farm are collected twice a year and sent to collaborating labs to be tested for the presence of Mycobacterium avium Paratuberculosis (MAP, the causative agent of Johne's disease), and the zoonotic foodborne bacterial pathogens Salmonella, Escherichia coli, Listeria monocytogenes, Campylobacter, and Enterococcus. Blood serum is collected quarterly to test for the presence of antibodies to the Johne's Disease (JD) organism. Environmental samples of manure, feed, water, vermin, birds, flies, etc are taken 4 times a year for analysis by collaborators. Samples of bulk tank milk and milk filters are taken weekly. The purpose of the project from the perspective of our lab is to characterize the microbial populations on the farm with particular interest in Salmonella, Escherichia coli, and Listeria monocytogenes. Initial findings from the PA farm demonstrated an outbreak of Salmonella and this organism was frequently detected in the milk filter and occasionally in bulk tank milk. The rate of fecal and environmental sampling was increased to fully document the outbreak. Sampling continues at the increased rate. Several serotypes of Salmonella have been detected. The milk filter was shown to be an indicator of the rate of Salmonella shedding within the herd. In addition, MAP has been found in fecal and environmental samples. Milk samples from all three farms have been analyzed for general quality indicators.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
No publications reported this period.

 
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ACCESSION NO: 0180178 SUBFILE: CRIS; HNRIMS
PROJ NO: PEN03677 AGENCY: CSREES PEN
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 JAN 1999 TERM: 31 DEC 2003 FY: 2004
INVESTIGATOR: Correll, P. H.
PERFORMING INSTITUTION:
Veterinary Science
Pennsylvania State University
University Park , Pennsylvania 16802
ROLE OF THE STK RECEPTOR IN MACROPHAGE ACTIVATION AND INFLAMMATION.
OBJECTIVES: Examine how the absence of the STK signaling pathway in IFN-gamma-activated macrophages affects the production of Nitric Oxide, the biochemical method by which this occurs, and the effect of STK on Mycobacterium paratuberculosis infection.
APPROACH: Utilize STK-deficient mice to determine whether STK is acting through other known regulators of NO production. Utilize the RAW264.7 macrophage cell line to determine the biochemical mechanisms by which STK suppresses NO. Infect STK-deficient mice with Mycobacterium paratuberculosis to determine the effect of STK on the infection process and its potential role in Johne's and Crohn's disease.
PROGRESS: 1999/01 TO 2003/12
Macrophages are a diverse and dynamic population of cells that play a key role in host defense as well as in the maintenance of normal tissue structure and function. However, inappropriate activation of macrophages can promote inflammation and tissue damage through the production of inflammatory mediators, such as nitric oxide, and the support of Th1 differentiation and the production of IFN-(gamma) which feeds back positively to further activate pro-inflammatory macrophage activities. It has been suggested that changes in the threshold of signal required for classical macrophage activation may promote the progression of autoimmune disease by enhancing Th1 differentiation through the upregulation of IL-12 and MHC Class II, thus generating a positive feedback loop for further tissue destruction. We propose that the threshold for classical macrophageactivation is set, in part, by the expression of receptor tyrosine kinases (RTKs) on these cells and that RTKs may represent a new class of therapeutic targets for regulating the tissue-damaging effects of autoimmunity. We have shown previously that the STK receptor tyrosine kinase inhibits the production of nitric oxide through the downregulation of inducible nitric oxide synthase (iNOS) expression while, at the same time enhancing expression of arginase, an enzyme that competes with iNOS for the substrate, L-arginine, resulting in cell proliferation and matrix synthesis. Thus we concluded that the expression of STK on tissue-resident macrophages tips the balance of these cells from cytotoxic mediators to cells with more reparative functions. More recently, we have shown that STK inhibits the production of IL-12 and the expression of MHC Class II by macrophages, resulting in a reduction in the ability of these cells to support Th1 differentiation in vitro. Subsequently, mice with a targeted deletion in STK are more susceptible to endotoxic shock and exhibit more inflammation in a delayed-type hypersensitivity response. In the past year, we have set out to determine whether the expression of the STK receptor tyrosine kinase on tissue-resident macrophages regulates the progression of autoimmunity. Our preliminary data suggests that the progression of autoimmune disease is dramatically enhanced in the absence of STK.
IMPACT: 1999/01 TO 2003/12
Regulation of macrophage activation is critical in order to maintain a balance between an effective immune response and inflammatory-mediated tissue damage and cell death. This work is an important step toward understanding the regulatory mechanisms that govern macrophage activation and may eventually lead to the identification of targets for regulating chronic inflammation.
PUBLICATIONS (not previously reported): 1999/01 TO 2003/12
1. Forgie A, Wyatt S, Correll PH and Davies AM. 2003. Macrophage stimulating protein (MSP) is a novel target-derived neurotrophic factor for developing sensory and sympathetic neurons. Development. 130 (5): 995-1002.
2. Teal HE, Craici A, Paulson RF and Correll PH. 2003. MSP potentiates erythropoietin receptor signaling in primary erythroid progenitors. J Hem Stem Cell Res 12, 165-177.
3. Lutz MA, Liu Q-P and Correll PH. 2003. Activation of complement-mediated phagocytosis by MSP requires the RON receptor, tyrosine kinase activity, phosphatidylinositol 3-kinase and protein kinase C-(geta). J Leuko Biol 73 (6), 802-814

 
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ACCESSION NO: 0204163 SUBFILE: CRIS
PROJ NO: PEN04087 AGENCY: CSREES PEN
PROJ TYPE: SPECIAL GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2005-34163-16014 PROPOSAL NO: 2005-06119
START: 01 AUG 2005 TERM: 31 JUL 2007 FY: 2007 GRANT YR: 2005
GRANT AMT: $656,525
INVESTIGATOR: Jayarao, B. M.; Perdew, G.; Key, D.; Love, B. C.; DebRoy, C.; Narasimha, H. V.; Heinrichs, A.; Wolfgang, D. R.
PERFORMING INSTITUTION:
Veterinary Science
Pennsylvania State University
208 Mueller Laboratory
University Park , Pennsylvania 16802
BIOREPORTER AND MOLECULAR BEACON-BASED TECHNOLOGIES FOR DETECTION OF ORGANIC TOXICANTS AND BACTERIAL PATHOGENS IN MILK AND MILK PRODUCTS.
NON-TECHNICAL SUMMARY: Develop state of the art diagnostics for detection of organic toxicants and foodborne pathogens in milk and milk products This project will focus on developing reliable and highly sensitive assays for detection of organic toxicants and foodborne pathogens in milk and milk products that can be used for monitoring and safeguarding milk supply and milk products.
OBJECTIVES: PROJECT 1: Bioreporter-based technology for detection of organic toxicants directly from milk and milk products Objective 1.1 Develop promoter-reporter gene constructs for detection of BETTX (benzene, ethylbenzene, toluene, trichloroethylene, and xylene) and PCBs. Objective 1.2 Standardize bioreporter assays for detection of BETTX (benzene, ethylbenzene, toluene, trichloroethylene, and xylene) and PCBs in milk and milk products. Objective 1.3 Survey milk and milk products for BETTX (benzene, ethylbenzene, toluene, trichloroethylene, and xylene) and PCBs along the food chain continuum. PROJECT 2: Molecular beacon-based technology for detection of bacterial pathogens directly from milk and milk products Objective 2.1 Develop and standardize molecular-beacon based real-time PCR assays for , Campylobacter jejuni, Escherichia coli, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus, Bacillus anthracis and Mycobacterium avium subsp. paratuberculosis. Objective 2.2 Survey milk and milk products for bacterial pathogens along the food chain continuum using molecular beacon-based real-time PCR assays.
APPROACH: PROJECT 1: The toluene degradation operon (tod) that degrades benzene, ethylbenzene, toluene, tricloroethylene, and xylene (BETTX) will be amplified from the genomic DNA of the Pseudomonas putida (ATCC53902). The primers for the PCR amplification for the tod promoter and regulator elements will be designed using the tod sequence reported by Mosqueda and Ramos (2000). The biphenyl dioxygenase gene (bphA1) has been shown to degrade PCBs. The gene will be amplified from genomic DNA of the Burkholderia cepacia (ATCC 55487). The primers for PCR amplification for the bphA1gene will be designed using the bphA1gene sequence (AJ251217) in the NCBI nucleotide database. The PCR amplified product will be cloned in to pGlow-TOPO vector (Invitrogen, Carlsbad, CA) containing green fluorescent protein (GFP) as reporter. This individual plasmid constructs will then be introduced in to E. coli TOP10 (Invitrogen) by electroporation (BioRAD micropluser, Hercules, CA) for use as a bioreporter. The transformed E. coli strains will be tested rigorously by exposing the strains to specific toxicants (concentration ranging from 10 microMol to 1 mMol) suspended in buffered peptone water. Expression of GFP proteins by bioreporter E. coli strains will be measured by reading the intensity of fluorescence in a spectrofluorimeter at intervals of 90-, 120-, 180-, 210-, and 240 minutes of incubation. PROJECT 2: Molecular beacons, PCR primers and PCR conditions that will be used to develop the assays will be from techniques reported in literature for C. jejuni (Sails et al., 2003), E. coli (Fortin et al, 2001), L. monocytogenes (Nogva et al 2000), Salmonella (Chen et al., 2000), B. anthracis (Verma-Basil et al., 2004), and M. avium subsp. paratuberculosis (Rodrigeuz-Lazaro et al., 2004). The assays will be standardized using control molecular beacons, bovine cellular DNA and common milk bacterial DNA as internal standards. The molecular beacon-based PCR assays will be developed and standardized individually for all seven organisms. ASSAY APPLICATIONS: Milk and milk products, milk filters and colostrum will be collected and analyzed for organic toxicants and C. jejuni, E. coli, L. monocytogenes, Salmonella, S. aureus, B. anthracis and M. avium subsp. paratuberculosis. Raw milk from 10 dairy producers that supply milk to the processing plant will be traced along the food continuum (farm---milk hauler---milk silo---pasteurized milk in retail markets). Samples will be collected over a period of 6 months from the farm, milk trucks, milk from the processing plant, and pasteurized milk sold by the processing plant in the retail markets. In addition, cheese (domestic and imported), evaporated and condensed milk, non fat dry milk from retail stores will also be examined for bacterial pathogens. A total of 10 dairy herds will be selected (milk collected by the same milk hauler and on the same milk route) and samples (milk filters, bulk tank milk, colostrum) will be collected once every 2-3 weeks by a trained technician.
PROGRESS: 2006/08 TO 2007/07
OUTPUTS: Project 1: Bioreporter-based technology for detection of organic toxicants directly from milk and milk products. The toluene degradation operon (tod) that degrades benzene, ethylbenzene, toluene, tricloroethylene, and xylene (BETTX) was isolated using the method called substrate induced gene expression screening (SIGEX). For detection of PCBs similar approach was used but the genomic DNA from Burkholderia cepacia (ATCC 55487) was used. Following exposure of the genomic libraries to BTEX and PCBs (2mM concentration), clones expressing GFP were cell sorted using a flow cytometer. Clones that showed the highest GFP expression were identified. These clones (BETTX-1 and BETTX-3) were successfully tested and standardized using buffered peptone water and raw milk contaminated with different concentrations of BTEX and PCBs and validated by GC-MS technique. A total of 394 raw bulk tank milk samples from 41 counties in PA, 60 samples each of cottage cheese, cheddar, mozzarella, ricotta, parmesan cheese, 2% and 3.5% pasteurized milk from retail stores in State College were examined for BETTEX and PCBs using respective bioreporter organisms, and GC-MS technique. The bioreporter assay identified 3 (0.76%) raw bulk tank milk, 1 (1.6%) sample of 3.5% pasteurized milk, and 2 (3.2%) samples of parmesan cheese were positive for BETTEX. While the GC-MS technique revealed that none of the samples positive by bioreporter assay were positive for BETTEX. The results of the study showed that the bioreporter assay gave false positive results. Further testing of the bioreporter clones showed that the clones gave positive results in the presence of other organic organic compounds in milk. It was concluded that bioreporter assay although promising has its limitations. Project 2. Molecular beacon-based technology for detection of bacterial pathogens directly from milk and milk products. A total of 394 raw bulk tank milk samples from 41 counties in PA were examined for Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella, Staphylococcus aureus, and bioterrorism agent Bacillus anthracis using molecular beacon-based technology and conventional microbiological techniques as described by BAM. It was observed that 2 (0.50%), 11 (2.79%),3 (0.76%), 48 (12.1%), and 10 (2.53%) bulk tank milk samples were positive for L. monocytogenes, Salmonella, E. coli O157:H7, S. aureus, and C. jejuni, respectively by both molecular beacon based analysis and conventional microbiology. PARTICIPANTS: Bhushan Jayarao, Principal Investigator; Coordinate project and report results. Dr.David Wolfgang, Co-Investigator; Oversee sample collection and processing Dr. Key, Co-Investigator; Advice on developing molecular beacon PCR assay Dr. Perdew, Co-Investigatgor; Advice on PCB analysis Dr. Brenda Love, Co-Investigator; Advice on bacteriological analysis of foodborne pathogens Dr. DebRoy, Co-Investigator; Advice on molecular beacon PCR assays Dr. Heinrichs, Co-Investigator; Identify and solicit participation of dairy herds Dr. Narsimha Hegde, Post doctoral research associate; Develop, test and standardize bioreporter assays Ms. Beth Houser, Research Technician; oversee day to day operations of project Ms. Sarah Donaldson, Research Technician; conduct molecular beacon assays. TARGET AUDIENCES: Animal Diagnostic Laboratories Food Testing Laboratories Federal, State and Local government agencies with focus on food safety Food Safety Experts
IMPACT: 2006/08 TO 2007/07
A bioreporter-based diagnostic tests for detection of organic toxicants such as benzene, toluene, ethylbenzene, trichloroethylene, and xylene and PCBs directly from milk and milk products was developed. The assay was rapid, and had a very high throughput. However the assay gave a considerably high level false positive results, the threshold of detection was considerably lower that that of GC-MS technique. Further refinements are needed before a bioreporter based technology can be made available for diagnostic use. The molecular beacon based real time PCR assays for detection of foodborne pathogens including Campylobacter jejuni, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella, Staphylococcusaureus, and bioterrorism agent Bacillus anthracis were developed. The molecular beacon based real time PCR assays provided the test result in about 4 hours, as compared to coventional microbiological technqiues which required 3-5 days. The cost of molecular beacon assay is about $9 per assay which is quite economical especially when it comes to saving time and reagents. The molecular beacon assay can be easily incorporated into routine testing in most diagonstic laboratories.
PUBLICATIONS (not previously reported): 2006/08 TO 2007/07
No publications reported this period
PROJECT CONTACT:
Name: Jayarao, B. M.
Phone: 814-863-5939
Fax: 814-863-6140
Email: bmj3@psu.edu

 
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ACCESSION NO: 0207998 SUBFILE: CRIS
PROJ NO: PEN04147 AGENCY: CSREES PEN
PROJ TYPE: SPECIAL GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2006-34163-17583 PROPOSAL NO: 2006-06147
START: 15 SEP 2006 TERM: 14 SEP 2008 FY: 2007 GRANT YR: 2006
GRANT AMT: $728,285
INVESTIGATOR: Knabel, S. J.; Cutter, C. N.; Debroy, C.; Jayarao, B. M.
PERFORMING INSTITUTION:
Food Science
Pennsylvania State University
208 Mueller Laboratory
University Park , Pennsylvania 16802
TRACKING AND CONTROLLING ZOONOTIC PATHOGENS THROUGHOUT THE DAIRY FOOD SYSTEM.
NON-TECHNICAL SUMMARY: Zoonotic pathogens, those that are transmitted from animals to humans, are the main caused of foodborne disease due to consumption of raw and pasteurized dairy products. Most illnesses are caused by a subset of highly virulence strains, known as epidemic clones. In order to control these zoonotic pathogens we must first be able to track them throughout the dairy system. The purpose of this study is to enhance the safety of raw and pasteurized dairy products by developing methods for tracking and controlling epidemic clones of zoonotic foodborne pathogens throughout the dairy system.
OBJECTIVES: The overall objective of this project is to determine the prevalence, reservoirs and routes of transmission of zoonotic pathogens throughout the dairy food system and to use this information to develop effective intervention strategies that enhance the safety of raw and pasteurized dairy products. Specific objectives include: 1) To determine the sero-prevalence for Mycobacterium avium subspecies paratuberculosis (MAP) in humans exposed to dairy environments; 2) To determine the prevalence of Shigatoxin-producing E. coli (STEC) in humans that consume raw milk; 3) To detect and track MAP, STEC and Listeria monocytogenes (LM) throughout the dairy food system, which will then allow development of effective intervention strategies for control; and 4) To determine the factors affecting biofilm formation by LM in dairy production control.
APPROACH: Objective 1. Serum and fecal samples from Chrohn's [Crohn’s?], colitis and control patients will be subjected to serological and bacteriological analyses. Data will be analyzed using various statistical procedures and CDC's Epi Info. Objective 2. DNA from human fecal samples from humans that work on dairy farms will be extracted from suspect colonies of STEC and subjected to PCR. Two sets of multiplex PCR will be conducted, one for detecting the genes stx1 and stx2 and eae and a second multiplex for detecting the genes rfbE of O157 and fliC for H7. Positive samples will be identified based on the presence of bands of the expected sizes compared to results with positive control strain E. coli ATCC 43895. Objective 3. Milk samples and milk filters from organic and non-organic dairy farms will be collected and screened for the presence of MAP, STEC and LM. MAP will be recovered and identified as previously described by Jayarao et al., 2004. STEC will be detected using the methods described in part 3b. Epidemic clones of LM will be detected using the muliplex PCR method developed in 3c and confirmed using the SNP method in 3d. Objective 4. Initial and attachment phases of biofilm formation will be assayed using dye binding and microscropy as previously described. Various environmental conditions will be manipulated to determine their effect on biofilm formation. To determine if the outbreak strains co-habitate with other strains LM cells will be tagged with a fluorescent marker and incubated with various other bacteria strains that are common in foods. Epifluorence microscopy will be used to examine the location and movement of LM cells with the multi-species biofilm. Genome sequences of biofilm-forming and non-biofilm-forming LM will be compared to determine if the biofilm-forming strains have unique sequences that may be involved in biofilm formation. Knock-out mutants will then be created to confirm whether these genes are indeed required for biofilm formation.
PROGRESS: 2006/09 TO 2007/09
OUTPUTS: Dr. Cutter co-directed and taught in the PSU Sanitation Shortcourse (SSC) in October, 2007. She introduced the concept of biofilms to 65 participants of the SSC. Participants were food processors from all over the U.S. Dr. Cutter began collaborating with Dr. Gylen Uhlech from USDA-ARS Eastern Regional Research Center on biofilm research. Dr. Uhlech has provided information from a similar project with E. coli O157:H7 that will be incorporated into the existing Listeria monocytogenes biofilm project. Dr. Knabel began working with Dr. Peter Evans (USDA-FSIS) and Dr. Todd Ward (USDA-ARS) on a project involving analysis of USDA-FSIS Listeria monocytogenes isolated from meat plants in the Northeastern U.S. that appear to be epidemic clone II by PFGE analysis. Dr. Knabel's laboratory confirmed that they were epidemic clone II and found that they were the 1998-1999 outbreak clone, using multi-virulence-locus sequence typing and new a prophage PCR method that was recently developed in his laboratory as part of this grant proposal. Dr. Knabel is now collaborating with Dr. Edward Dudley in the Food Science Department to apply these new DNA-sequence-based subtyping technologies to track E. coli O157:H7 in dairy processing plants. Dr. Jayarao solicited the participation of the Crohn's and Colitis Foundation Chapter in Philadelphia to seek individuals of dairy farm origin with Crohn's disease, irritable bowel syndrome, ulcerative colitis and other gastrointestinal conditions. Dr. Jayarao combined a part of this study with an ongoing statewide study on zoonotic diseases in dairy farm operations. Dairy producers who had indicated in their response to survey that could be contacted, were solicited to participate in this study. A total of 112 adult dairy producers consented to participate in the study. The 42 dairy producers comprised of individuals with (cases; n=19) and without (controls; n=23) chronic gastrointestinal conditions. The cases and controls were matched based on geographical location of the farm, sex and age of the participants. PARTICIPANTS: Dr. Stephen J. Knabel, PI, is responsible for Objective 3 and the development of DNA-sequence-based molecular subtyping methods for tracking zoonotic pathogens. Dr. Bhushan Jayarao, PI, is responsible for Objective 1, to determine the sero-prevalence for Mycobacterium avium subspecies paratuberculosis (MAP) in humans exposed to dairy environemnts. Dr. Chirita Debroy, PI, is responsible for Objective 2, and the charactization of all zoonotic agents that are isolated as part of this project. Dr. Catherine Cutter, PI, is responsible for Objective 4, to determine the factors affecting biofilm formation by Listeria monocytogenes (LM) in dairy production and processing environments and to develop intervention strategies for their control. Dr. Edward Dudley will serve as a co-advisor to a M.S. student working on developing a DNA-sequence-based molecular subtyping strategy for tracking E. coli O157:H7. TARGET AUDIENCES: Target audiences include graduate students working on this project and undergraduate and graduate students taking courses on food microbiology, including Food Microbiology, Fundamentals of Food Science, Epidemiology and Molecular Epidemiology. Two visiting scientists from Italy are working on this project and one technician working on this project in our laboratories is an African-American. Target audiences also include participants in short courses sponsored by the Department of Food Science, including the Penn State Sanitation Short Course and the Food Microbiology Short Course. Target audiences also include food industry associations such as the Eastern Meat Packers Association, the Pennsylvania Association for Food Protection, and the Institute of Food Technologists. Target audiences also include government associations such as the Central Atlantic States Association of Food and Drug Officials and the National Advisory Committee for the Microbiological Criteria for Foods (NACMCF). Farm families in Pennsylvania were also served by the Mycobacterium avium subspecies paratuberculosis part of this project. Given their high risk for zoonotic foodborne illness, this project specifically targets infants, the elderly and those individuals with compromised immune systems, such as patients with AIDs and cancer and those on immunosuppressive drug therapy.
IMPACT: 2006/09 TO 2007/09
Dr. Cutter identified a doctoral student and assigned this student to the biofilm project. The student spent three months researching the biofilm literature and researched methods for biofilm detection. The student conducted a series of experiments that indicated that some strains of Listeria monocytogenes from foodborne outbreaks are better biofilm formers than others. This information may be used as another means of characterizing the pathogen and may provide insight into how to control the pathogen in food processing establishments. Dr. Knabel identified a Ph.D. student and a M.S. student to work on this project. The M.S. student is co-advised by Dr. Dudley and will work on developing molecular subtyping methods for tracking E. coli O157:H7. The M.S. student will continue our work on developing DNA-sequence-based methods for tracking Listeria monocytogenes. Both students completed their literature reviews this semester and will start research on their projects next semester. Dr. Jayarao conducted a detailed analysis (60 survey questions ranging from dietary habits to work practices) between cases and controls which did not reveal any significant association between dietary habits (raw milk consumption, consumption of meat/vension, source of water) and work practices (manure handling, history of Johne's disease etc). Bulk tank and milk filter samples will be collected starting December 14, 2007, once a week for 6 weeks from all 38 participating dairy herds. All the samples will be examined for the presence of Salmonella, Escherichia coli, Listeria monocytogenes, Campylobacter jejuni, Mycobacterial spp. including Mycobacterium avium subsp. paratuberculosis using real time PCR assays developed in Dr. Jayarao's laboratory. Samples positive on real time PCR assay will be cultured for the respective pathogens and provided to other collaborators including Dr. DebRoy (Escherichia coli,Salmonella and Campylobacter) and Dr. Knabel (Listeria monocytogenes and Escherichia coli) for further characterization. E. coli strains will be further characterized in Dr. DebRoy's laboratory. These will be serotyped to determine if they belong to O157:H7 or any other shiga-toxin producing serogroups. The presence of virulence genes such as shiga toxins (Stx), heat-stable toxins (Sta), heat labile toxins (LT) and other virulence attributes such as adherence and effacing gene (eae) and bundle forming pili (bfp) will be determined by PCR. Salmonella strains will be tested for the presence of gene encoding for invasive gene (inv) known to be a virulence factor. Campylobacter isolates will be tested for cytotoxic distending toxin (cdt). Presence or absence of these pathogens in the stool of cases and control will be matched to that in bulk tank milk and milk filters to determine if there is a causal relationship between gastroenteric disease and the occurrence of over and excess of a pathogen
PUBLICATIONS (not previously reported): 2006/09 TO 2007/09
No publications reported this period
PROJECT CONTACT:
Name: Unger, R. L.
Phone: 814-865-3136
Fax: 814-863-6152
Email: rlh12@psu.edu

 
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ACCESSION NO: 0212120 SUBFILE: CRIS
PROJ NO: PEN04213 AGENCY: CSREES PEN
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2008-55620-18710 PROPOSAL NO: 2007-01019
START: 15 DEC 2007 TERM: 14 DEC 2008 GRANT YR: 2008
GRANT AMT: $1,200,000
INVESTIGATOR: Kapur, V.
PERFORMING INSTITUTION:
Veterinary Biomedical Sciences
Pennsylvania State University
208 Mueller Laboratory
University Park , Pennsylvania 16802
JDIP: JOHNE'S DISEASE INTEGRATED PROGRAM IN RESEARCH, EDUCATION AND EXTENSION.
NON-TECHNICAL SUMMARY: Johne's disease is a disease which results in lost productivity in dairy herds and potential morbidity/mortality. There is also some concern that the bacterium itself is related to the onset of Crohn's disease in humans. The JDIP program aims to control, and potentially eliminate, Johne's through a combination of research projects on how to manage the disease and potentially eliminate it. This is being done through a broad collaboration of research universities and USDA groups with the collaboration and support of the impacted private sector.
OBJECTIVES: Continuation of an integrated program in Johne's disease research with a focus on developing a strong translational pipeline of new diagnostic tests, vaccine candidates, and strategies to manage, prevent, and control the disease.
APPROACH: The objectives will be accomplished via thematic research units on: Johne's disease epidemiology; diagnostic test development including strain differentiation; M. paratuberculosis basic biology and pathogenesis; and strategies for enhancing T and B cell immune response to M. paratuberculosis. These research units will be supported by scientific support cores in: biostatistics and epidemiology; diagnostics and strain differentiation; gene and protein expression; and animal experimentation. In addition, an administrative core will provide support for both research units and the overall JDIP as well as translating the outcomes from the research into educational information for the broadly affected Johne's community. An external advisory board, chosen from public and private stakeholders, will provide guidance to the JDIP team in pursuit of the overall objectives.
PROJECT CONTACT:
Name: Unger, R. L.
Phone: 814-865-3136
Fax: 814-863-6152
Email: runger@psu.edu
URL: http://www.jdip.org
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ACCESSION NO: 0209669 SUBFILE: CRIS
PROJ NO: PENV2007-5033 AGENCY: CSREES PENV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 OCT 2006 TERM: 30 SEP 2007
INVESTIGATOR: Fecteau, M. E.; Whitlock, R.; Sweeney, R.
PERFORMING INSTITUTION:
School of Veterinary Medicine
Univ of Pennsylvania
Philadelphia , Pennsylvania 19104
DETERMINATION OF THE EXTENT OF MAP TISSUE INFECTION OF YOUNG DAIRY CATTLE FOLLOWING GRAZING OF PASTURES USING LEADER-FOLLOWER GRAZING SYS IN HER . . . (Note: Title exceeded the title field character limit.)
NON-TECHNICAL SUMMARY: Johne's Disease is a costly infectious intestinal disease of cattle. To determine whether exposure to MAP at 12 months of age resulted ONLY in pass through (i.e. passive shedding), or whether the yearlings developed permanent infection.
OBJECTIVES: To determine if cattle exposed to MAP for the first time at one year of age by following the adult herd on grazing pasture are pass-through positive only or if they become permanently infected with MAP. To determine the extent of MAP infection in the tissues of these yearling cattle. To determine the extent of MAP in manure of yearling cattle in a dairy herd using the leader-follower system.
APPROACH: The experimental approach will be to perform extensive cultures for MAP of intestinal and mesenteric lymphoid tissue obtained at the time of slaughter in May 2007. The intestinal tract and associated lymph nodes will be obtained at the processing plant and transported to our laboratory. Thirty separate samples of intestinal tissue and associated lymph node, collected along the length of the GI tract will be harvested for culture from each animal. These samples will be processed for MAP culturing using standard methods employed extensively in our laboratory9-12. Additionally, fecal samples obtained from the distal colon will be subjected to MAP culture.
PROJECT CONTACT:
Name: Salsbury, P.
Phone: 610-925-6320
Fax: 610-925-8100
Email: salsbury@vet.upenn.edu

 
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ACCESSION NO: 0213331 SUBFILE: CRIS
PROJ NO: PENV2008-5033 AGENCY: CSREES PENV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 JAN 2008 TERM: 30 SEP 2008
INVESTIGATOR: Fecteau, M.
PERFORMING INSTITUTION:
School of Veterinary Medicine
Univ of Pennsylvania
Philadelphia , Pennsylvania 19104
ANTIMICROBIAL ACTIVITY OF GALLIUM NITRATE AND MONENSIN SODIUM AGAINST MYCOBACTERIUM AVIUM SUBSP. PARATUBERCULOSIS (MAP) IN VITRO.
NON-TECHNICAL SUMMARY: Paratuberculosis (Johne's disease), caused by Mycobacterium avium subsp. paratuberculosis (MAP) is a costly infectious intestinal disease of cattle and other ruminants, characterized by progressive weight loss and chronic diarrhea. Infections with MAP are difficult to control because of long incubation periods (1-10 years), the absence of clinical signs until advanced stages of the disease, and the lack of completely reliable diagnostic methods in the preclinical stages of the disease. It is general knowledge that most calves become infected very early in life. Therefore, control programs are based on preventing transmission from adult cattle shedding the MAP organisms in feces to young replacement stock on the farm. Control programs specifically focus on calving-pen management, milk- and colostrum-feeding practices and rearing of young stock. There are currently no drugs approved for the prevention or treatment of Johne's disease in cattle. Vaccination, available on a limited basis in the United States, may reduce the incidence of clinical disease, and to a lesser extent the prevalence of infection, but vaccinates are not fully protected from infection. The purpose of this study is to test the in vitro susceptibility of various virulent bovine strains of MAP to gallium nitrate and monensin sodium. We hypothesized that both gallium nitrate, and monensin sodium have antimicrobial efficacy against MAP, and thus could be used as an aid in the treatment and/or control of paratuberculosis in cattle.
OBJECTIVES: 1. To evaluate the in vitro susceptibility of various virulent strains of MAP to gallium nitrate. 2. To evaluate the in vitro susceptibility of various virulent strains of MAP to monensin sodium. 3. To investigate the potential use of gallium and monensin as prophylactic and therapeutic agents for the control of MAP infections in cattle.
APPROACH: In this study, the in vitro susceptibility of several virulent strains of MAP to gallium nitrate and monensin sodium will be tested using broth culture with detection of MAP growth by a non-radiometric automated detection methodology (BACTEC MGIT 960, BD Diagnostics, Sparkes MS), which employs fluorometric detection of oxygen consumption.
PROJECT CONTACT:
Name: Salsbury, P.
Phone: 610-925-6320
Fax: 610-925-6767
Email: salsbury@vet.upenn.edu

 
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ACCESSION NO: 0204725 SUBFILE: CRIS
PROJ NO: PENV528607 AGENCY: CSVM PENV
PROJ TYPE: STATE PROJ STATUS: NEW
START: 01 JAN 2005 TERM: 31 DEC 2006 FY: 2006
INVESTIGATOR: Sweeney, R.
PERFORMING INSTITUTION:
School of Veterinary Medicine
Univ of Pennsylvania
Philadelphia , Pennsylvania 19104
ORAL VACCINATION FOR JOHNE'S DISEASE IN CATTLE.
NON-TECHNICAL SUMMARY: Johne's disease is widespread, costly and incurable in cattle. An effective oral vaccine will significantly enhance efforts to eliminate Johne's disease from infected herds.
OBJECTIVES: Johne's disease is a widespread, costly incurable, intestinal infection of cattle caused by Mycobacterium paratuberculosis. Currently available subcutaneous vaccination does not prevent infection of vaccinated cattle. The goal of this project is to develop an oral faccination [vaccination?] protocol that will stimulate mucosal immunity and prevent intestinal cononization of cattle with M. paratuberculosis. The influence of vaccine dose, boostering and use of cytokine as a mucosal adjuvant will be measured. Intestinal immune responses will be tested. Once the optimal combination of dose, adjuvant and boostering is determined, the ability of the oral vaccine to protect against intestinal colonization will be tested using a short-term experimental infection model.
APPROACH: Johne's disease is a costly, progressive and ultimately fatal infection of cattle and other ruminants. Currently available parenteral vaccines do not prevent colonization of the intestines with Mycobacterium paratuberculosis. Thus, vaccinated cattle may become infected and shed the organism in feces, representing a continued source of environmental contamination and infection for other susceptible animals. The hypothesis we propose to test follows: oral vaccination, by stimulating a protective immune response at the point of entry of the infection, represents the greatest promise for an effective vaccine against paratuberculosis.
PROGRESS: 2006/01 TO 2006/12
This project was divided into 3 experiments. Experiment 1 was designed to test the hypothesis that liposome/IL-12 would enhance the gastrointestinal immune response to orally administered killed Mycobacterium avium subsp. paratuberculosis (MAP). Experiments 2 and 3 build on the results of the first experiment, testing different dosing schemes, with and without booster doses, eventually using an experimental infection model to show protection against MAP infection. The results of phase 1 reported previously showed a weak response to a single dose of killed vaccine. Experiment 2 was completed during the current reporting period. Calves were vaccinated with 2 doses of killed oral vaccine, and the immune responses compared to unvaccinated controls. Intestinal tissue and peripheral blood were collected to test for antigen-specific production of Interferon-gamma and Interleukin-10, proteins that are produced by lymphocytes that stimulate, and inhibit the immune response respectively. Our results showed a trend for the vaccinated calves to have lower production of IL-10, the protein that would be expected to inhibit resistance to infection. The differences in IFN-gamma were not as pronounced. However, if oral vaccination is able to suppress IL-10, this could result in a better immune response to live challenge with the pathogen. The next experiment will involve challenge with live MAP following a series of two oral vaccinations.
IMPACT: 2006/01 TO 2006/12
If successful, the results of this study will identify a method for vaccinating cattle against Johne's disease, an infectious disease of significant economic concern for the dairy and beef cattle industries in the United States.
PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
No publications reported this period

 
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ACCESSION NO: 0206409 SUBFILE: CRIS
PROJ NO: SC-1700303 AGENCY: CSREES SC.
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 JUL 2006 TERM: 31 OCT 2006 FY: 2007
INVESTIGATOR: Fitts, M. G.
PERFORMING INSTITUTION:
Livestock-Poultry Health
Clemson University
Clemson , South Carolina 29634
METABOLIC PROFILING OF MICROBES OF ECONOMIC AND ANIMAL HEALTH SIGNIFICANCE.
NON-TECHNICAL SUMMARY:Mycobacterium avium subsp. paratuberculosis is the causative agent of Johne's disease and a suspect agent in Crohn's disease. Some estimates have the economic loss to the US cattle industry due to Johne's disease at $1.5 billion annually. Pseudomonas aeruginosa is a problematic microbe capable of causing diseases in plants, animals and humans. It is almost ubiquitous in nature, residing in water and soil. It is also a model organism for biofilm formation, a process that renders it highly resistant to antibiotics (for infections of artificial implants or chronic infection of the lung) or cleaning agents (for fouling of industrial equipment). It is hoped that this work will identify factors vital to the growth and survival of Pseudomonas aeruginosa and Mycobacterium avium subsp. paratuberculosis. These findings may then be exploited in more applied research in an attempt to manage, treat and/or diagnose infections caused by these organisms.
OBJECTIVES: Metabolic profiling is the identification of the small molecule components of a cell and has diverse applications in drug discovery, clinical diagnostics, biomarker discovery and plant and animal biochemistry. For microbes the metabolome is a measure of an organisms response to its environment over time and reflects the overall condition of the culture. I intend to explore the small molecule repertoire of the clinically significant microbes Mycobacterium avium subsp. paratuberculosis (MAP) and Pseudomonas aeruginosa. Using reference strains and clinical isolates from our laboratory, I intend to identify unique and relative changes in small molecule production under a variety of culture conditions. It is hoped that the basic research described in this grant proposal will identify factors vital to the growth and survival of P. aeruginosa and MAP. These findings may then be exploited in more applied research in an attempt to manage, treat and/or diagnose infections caused by these organisms.
APPROACH : In-house and reference strain microbes are cultured on select solid media either alone or co-cultured with another microbe of interest and harvested at different time intervals. Bacterial suspensions are heated and bacterial debris removed by centrifugation. The resulting supernatant is applied to a size exclusion column to collect a fraction containing molecules less than 3,000 daltons. These extracts are applied to a liquid chromatography - mass spectrometer (LC-MS) to identify small molecules of interest. Selected molecules that cannot be matched to any libraries will be submitted to the University of South Carolina NMR facility for structural determinations. Once these molecules are identified by NMR, we will attempt to determine their role in the survival and/or virulence of these strains in addition to determining if they have any diagnostic value.
PROGRESS: 2006/07 TO 2006/10
No research accomplished. Project terminated early (10-31-2006) due to
resignation of principal investigator.
IMPACT: 2006/07 TO 2006/10
none
PUBLICATIONS (not previously reported): 2006/07 TO 2006/10
No publications reported this period
PROJECT CONTACT:
Name: Fitts, M. G.
Phone: 803-788-2260
Fax: 803-699-8910
Email: mfitts@clemson.edu


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ACCESSION NO: 0198049 SUBFILE: CRIS
PROJ NO: TEN00291 AGENCY: CSREES TEN
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 2003 TERM: 30 SEP 2008 FY: 2007
INVESTIGATOR: Speer, C. A.
PERFORMING INSTITUTION:
Forestry Fisheries & Wildlife
University of Tennessee
569 Dabne Hall
Knoxville , Tennessee 37996
DEVELOPMENT OF AN EARLY DIAGNOSTIC TEST FOR JOHNE'S DISEASE IN CATTLE.
NON-TECHNICAL SUMMARY: Johne's disease affects numerous ruminants (including cattle) in which it is characterized as a chronic wasting disease. Losses are estimated at $250 million annually in the U.S. alone. Johne's disease is inordinately difficult to diagnose. The purpose of this project is to develop a specific and sensitive monoclonal antibody-based test for Johne's disease. Project contact: C.A. Speer
OBJECTIVES: 1. Generate monoclonal antibodies against the Ag85a complex of Mycobacterium avium paratuberculosis. 2. Compare the sensitivity and specificity of the Ag85a antibody test with the ELISA and the fecal culture tests for the detection M. a. paratuberculosis. 3. Establish a serum bank of M. a. paratuberculosis-positive and negative cattle
APPROACH: Generate monoclonal antibodies against Ag85a peptides and then test for reactivity and specificity against Ag85a from M. a. paratuberculosis in sera of infected cattle.
PROGRESS : 2006/01 TO 2006/12
There are no effective diagnostic tests, chemotherapies or vaccines against Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP). Recently, we developed an enzyme-linked immunosorbent assay (ELISA) that is subspecies specific (>96%) and highly sensitive (>95%) in detecting pre-patent as well as chronic MAP infections. The ELISA plates for diagnosis of JD were prepared by using surface antigens gently dislodged from the surface of MAP bacilli. The plates can be prepared quickly and safely with a relatively small amount of bacteria and have a shelf-life of at least 7 weeks.
IMPACT: 2006/01 TO 2006/12
Worldwide, JD is one of the most prevalent and economically important diseases of livestock, causing an annual loss of more than $200 million to the dairy industry alone. We have developed a highly sensitive and subspecies-specific surface antigen ELISA for the rapid diagnosis of JD in cattle. The ELISA will be used to control JD in beef and dairy herds.
PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
1. Eda, S., J.P. Bannantine, W.R. Waters, Y. Mori, R.H. Whitlock, M.C. Scott and C.A. Speer. 2006. A highly sensitive and subspecies-specific surface antigen enzyme-linked immunosorbent assay for diagnosis of Johne's disease. Clin. Vaccine Immunol. 13:837-844.
2. Speer, C.A., M.C. Scott, J.P. Bannantine, W.R. Waters, Y. Mori, R.H. Whitlock and S. Eda. 2006. A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's disease) in cattle. Clin. Vaccine Immunol. 13:535-540.

 
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ACCESSION NO: 0211563 SUBFILE: CRIS
PROJ NO: TEN02007-01613 AGENCY: CSREES TEN
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2007-35204-18462 PROPOSAL NO: 2007-01613
START: 15 AUG 2007 TERM: 14 AUG 2009 FY: 2007 GRANT YR: 2007
GRANT AMT: $293,000
INVESTIGATOR : Eda, S.
PERFORMING INSTITUTION:
Forestry Fisheries & Wildlife
University of Tennessee
569 Dabne Hall
Knoxville , Tennessee 37996
OPTIMIZATION AND EVALUATION OF NOVEL SERUM AND MILK ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR JOHNE'S DISEASE.
NON-TECHNICAL SUMMARY: Johne's disease, caused by a bacterial pathogen (Mycobacterium avium subsp. paratuberculosis), affects many ruminants and, after several years of infection, a chronic wasting disease develops which may ultimately result in death. Johne's disease occurs in domestic and wild animals worldwide and, in the United States, it causes an estimated annual loss of $220 million to the agricultural economy. Despite its heavy economic burden on the agricultural industry, there are still no practical chemotherapeutic agents or vaccination programs against Johne's disease. Further, current diagnostic tests have limitations related to low sensitivity and long turn-around time. We developed recently a novel diagnostic test for Johne's disease. The diagnostic test can be completed in 3-4 hours, has a higher level of accuracy compared to currently available tests, and is capable of diagnosing early stages of the disease. The purposes of this work are to improve the performance of the test and to evaluate the accuracy of the test by using a large number of serum and milk samples from diary cattle. The national and international impact of this work is far-reaching because early diagnosis of Johne's disease will help guide veterinary authorities in targeting or re-directing control and eradication resources and efforts, ultimately leading to the potential for improvements in surveillance of Johne's disease in the United States.
OBJECTIVES: Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), has a significant economic impact on the US dairy cattle industry. Currently, removal of MAP infected cattle from herds is a common intervention used to promote the control of the disease. However, most widely used procedures for diagnosing Johne's disease, such as fecal culture and enzyme-linked immunosorbent assay (ELISA) tests, have major limitations due to low sensitivity, high labor intensity, and long turn-around time. We developed a novel ELISA, called EVELISA, for detection of MAP and demonstrated that the test was highly sensitive; in a previous study the EVELISA identified 98% of fecal-culture positive cattle compared to a current ELISA that identified 50%. The long-term objectives are to (a) contribute to efforts for early and rapid diagnosis of Johne's disease by developing and evaluating a new ELISA with high sensitivity, (b) quantify the performance accuracy of the absorbed EVELISA relative to other available tests, and (c) promote control and eradication of Johne's disease through improved surveillance. The objectives of this project are to optimize further the conditions of the EVELISA and to evaluate performance characteristics of the optimized EVELISA for testing of serum and milk samples for Johne's disease in cattle.
APPROACH: Our previous data showed that species-specific antigens expressed on various mycobacteria could be extracted specifically using 80% ethanol. An ELISA test developed using ethanol-extracted surface antigens of MAP, named EVELISA, showed the level of diagnostic sensitivity higher than 95% in detecting bovine MAP infections, whereas that of current ELISAs for MAP infections was shown to be lower than 30%. In this project, conditions of the EVELISA will be optimized by using different strains of mycobacteria for serum pre-absorption and different reagents for antibody reaction. The reproducibility, sensitivity, and other performance measures of the optimized EVELISA will be evaluated by statistical methods including Bayesian model.
PROJECT CONTACT:
Name: Shigetoshi, E.
Phone: 865-974-5008
Fax: 865-974-4714
Email: seda@utk.edu

 
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ACCESSION NO: 0173019 SUBFILE: CRIS
PROJ NO: TEX08409 AGENCY: CSREES TEX
PROJ TYPE: HATCH PROJ STATUS: REVISED
START: 15 JUL 2004 TERM: 14 JUL 2009 FY: 2006
INVESTIGATOR: Adams, L. G.; Ficht, T. A.; Ficht, A. R.; Womack, J. E.; Thomas, T. L.; Baumler, A. J.; Tsolis, R. M.; Zhu, G.; Clarke, N. P.; Walker, D. H.; Peters, C. J.; Yilma, T.
PERFORMING INSTITUTION:
Veterinary Pathobiology
Texas A&M Univ
College Station , Texas 77843
MOLECULAR PATHOGENESIS OF EMERGING & ZOONOTIC INTRACELLULAR PATHOGENS OF RUMINANTS AS THE BASIS FOR IMPROVED DIAGNOSIS AND VACCINATION.
NON-TECHNICAL SUMMARY: This will provide deterrents to biological warfare through innate resistant cattle, biosignatures for diagnostics, and improved vaccines. The outcomes from these projects have application for improving public health, producing safer foods and minimizing agricultural and human bioterrorism.
OBJECTIVES: The specific objectives of our research program project are to: 1. Establish the in vitro and in vivo patterns of temporal gene expression by massively parallel signature sequencing and macro- and microarray analysis of Salmonella entericaTyphimurium, Brucella abortus, Cryptosporidium parvum, and Mycobacterium avium subspecies paratuberculosis infected target tissue culture cells and intact bovine ileum. 2. Apply bioinformatics and computational biology to compare the temporal gene profiles for common and unique expression patterns for Salmonella entericaTyphimurium, Brucella abortus, Cryptosporidium parvum, and Mycobacterium avium subspecies paratuberculosis. 3. Confirm selected common and uniquely expressed genes by real-time PCR, Northern analysis, RNAse protection and/or RNA interference assays and define the roles of candidate genes in major cell physiological pathways critical to survival of the pathogen or the host. 4. Sequence host genes essential for survival of each pathogen in nuclear families of cattle segregating differential response to pathogens and identify single nucleotide polymorphisms (SNPs).
APPROACH: Because the central theme of all investigators on this team hinges on the availability of specimens from the host:pathogen interface, DNA microarray analysis, and bovine genomic analysis, we will further strengthen shared core laboratories to meet the requirements of the individual research projects as described below. Ruminant Ileal Loop Core Laboratory: For the in vivo genomic comparative analysis of ruminant host responses to the selected intracellular pathogens, the initial focal point of all investigators is at the level of the host:pathogen entry interface and subsequent interactions in Peyers patches. Samples for bacteriologic culture, histopathology and ultrastructural studies are collected as required for the specific pathogens, for example at 5, 15, and 30 minutes and 1, 2, 3, 4, 5, 6, 8, 10, and 12 hours. For gene expression analysis, total RNA is extracted immediately after dissection of the mucosa from samples obtained at specified time intervals post-infection. Microarray & Informatics Core Laboratory: We will use a commercial bovine microarray to study the differential expression of bovine genes in response to the microbial infections, bacterial and protozoal pathogens will be inoculated into bovine ligated ileal loops or appropriate cell lines. Total RNA will be isolated from infected and control bovine tissues or cells using appropriate RNA isolation kits and mRNA may be further purified from the total RNA to increase the ratio of under-representative transcripts that include many regulatory or defensive genes critical to the host-pathogen interaction. Hybridization is detected using cDNA probes synthesized with one of two color fluorescent deoxyribonucleotide precursors. The intensities of hybridization in microarrays will be quantified and analyzed using a GeneSight software. Using microarray techniques and ratio analysis, we expect to identify bovine genes whose expression levels are strongly regulated by the invasion of microbial pathogens for future confirmation by real-time PCR. Analysis of Genes in Radiation Hybrid Cell Lines: The development of a 5000 rad radiation hybrid (RH) panel for cattle genome mapping ushered in high-resolution comparative mapping of the cattle genome relative to the genomes of human and mice. Two-point linkage and multipoint map construction will be carried out with the RHMAPPER package. The most likely order will be subjected to a multipoint maximum likelihood algorithm to determine breakage frequency and cR distances.
PROGRESS: 2007/01 TO 2007/12
Brucellosis is a zoonotic disease with a worldwide distribution that can be transmitted via intentional or accidental aerosol exposure. Unmarked deletion mutants BADeltaasp24 and BMDeltaasp24 consistently conferred superior protection to mice against homologous and heterologous aerosol challenge infection, thus were considered viable candidates as vaccine strains against brucellosis. The virB operon, encoding a Type IV secretion system (T4SS), is essential for intracellular survival and persistent infection by Brucella spp. Infection studies using type I interferon receptor knockout mice found that a lack of type I interferon signalling did not affect Brucella replication during the first 4 weeks of infection. Thus, induction of type I interferons does not appear to be an essential mechanism by which the T4SS promotes persistent infection by Brucella. Mycobacterium tuberculosis complex species survive and replicate in phagosomes of the host cell. Cell death (CD) has been highlighted as one of the probable outcomes in this host-pathogen interaction. Our data suggest that M. bovis-induced CD in bovine macrophages is caspase independent with apoptosis-inducing factor participation. The viaB locus contains genes for the biosynthesis and export of the Vi capsular antigen of Salmonella enterica serotype Typhi. Introduction of the cloned viaB region into serotype Typhimurium resulted in a significant reduction of GROalpha and IL-17 expression and in reduced fluid secretion. Our data support the idea that the viaB region plays a role in reducing intestinal inflammation in vivo. The presence of a new fatty acyl coenzyme A (acyl-CoA) elongation system was confirmed in Cryptosporidium and the functional characterization of the key enzyme, a single long-chain fatty acid elongase (LCE), in this parasite. CpLCE1 gene transcripts are present at all life cycle stages, but the levels are highest in free sporozoites and in stages at 36 h and 60 h postinfection that typically contain free merozoites. The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. Data from experiments ultimately validated the function of the CpPKS1-AL-ACP unit, and making it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.
IMPACT: 2007/01 TO 2007/12
Data derived from a series of in vitro and in vio experiments involving Brucella spp. Mycobacterium spp., Salmonella spp. and Cryptosporidium parvum interactions with their respective hosts revealed insightful new information that may have application to the design of new detection methods, protective immunity or possibly novel drug targets.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
1. Raffatellu, M., Santos, R. L., Chessa, D., Wilson, R. P., Winter, S. E., Rossetti, C. A., Lawhon, S. D., Chu, H., Lau, T., Bevins, C. L., Adams, L. G., and Baumler, A. J. 2007. The capsule encoding the viaB locus reduces interleukin-17 expression and mucosal innate responses in the bovine intestinal mucosa during infection with Salmonella enterica serotype Typhi. Infection & Immunity 75:4342-50.
2. Roux, C. M., Rolan, H. G., Santos, R. L., Beremand, P. D., Thomas, T. L., Adams, L. G., and Tsolis, R. M. 2007. Brucella requires a functional Type IV secretion system to elicit innate immune responses in mice. Cellular Microbiology 9:1851-69.
3. Kahl-McDonagh, M. M., Arenas-Gamboa, A. M., and Ficht, T. A. 2007. Aerosol infection of BALB/c mice with Brucella melitensis and Brucella abortus and protective efficacy against aerosol challenge. Infection & Immunity 75:4923-32.
4. Fritzler, J. M., Millership, J. J., and Zhu, G. 2007. Cryptosporidium parvum long-chain fatty acid elongase. Eukaryot Cell 6:2018-28.
5. Fritzler, J. M. and Zhu, G. 2007. Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase. Int J Parasitol 37:307-16.
PROJECT CONTACT:
Name: Adams, L. G.
Phone: 979-845-9816
Fax: 979-862-1088
Email: gadams@cvm.tamu.edu
URL: http://www.cvm.tamu.edu/vtpb

 
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ACCESSION NO: 0178985 SUBFILE: CRIS
PROJ NO: TEX08637 AGENCY: SAES TEX
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 06 AUG 1998 TERM: 05 AUG 2004 FY: 2005
INVESTIGATOR: Brumbaugh, G. W.
PERFORMING INSTITUTION:
Veterinary Physiology & Pharmacology
Texas A&M Univ
College Station , Texas 77843
PHYSIOLOGIC, PATHOPHYSIOLOGIC, AND PHARMACOLOGIC MANAGEMENT OF ANIMAL HEALTH.
NON-TECHNICAL SUMMARY: Management practices to enhance or sustain health of animals become outdated with the introduction of new technology and research findings. This project will critically and rationally evaluate published information about continued or new animal health issues and application of techniques in order to develop rational management practices or to eliminate irrational practices.
OBJECTIVES: To enhance or sustain health of animals by targeted management based on understanding, physiologic or pathophysiologic processes associated with disease and pharmacologic mechanisms of treatments.
APPROACH: Critical and rational evaluation of published information about continued or new animal health issues and application of techniques to evaluate processes involved in order to develop rational management practices or to eliminate irrational practices.
PROGRESS: 1998/08 TO 2004/08
The susceptibility in vitro of Mycobacterium avium sbsp. paratuberculosis to monensin sodium and to tilmicosin phosphate were evaluated. Subsequent to exposure to those compounds, the infectivitiy of the organism in vivo was evaluated using a murine model. This project contributed to the knowledge base about the organism's response to two chemical agents that may be evaluated as part of the control of cattle with bovine paratuberculosis, globally.
IMPACT: 1998/08 TO 2004/08
The two compounds evaluated are commonly used in cattle around the world for other indications. Bovine paratuberculosis is a globally recognized disease for which there is no treatment and for which control measures are customized. With results of this study, studies with diseased cattle should be conducted next to evaluate the role of these compounds for the control of this disease.
PUBLICATIONS (not previously reported): 1998/08 TO 2004/08
Brumbaugh GW. Simpson RB, Edwards JF, Anders DR, Thomson TD. 2003. Susceptibility of Mycobacterium avium sbsp. paratuberculosis to monensin sodium or tilmicosin phosphate in vitro and resultant infectivity in a murine model. (Accepted for publication, 2004, Canadian Journal of Veterinary Research)
PROJECT CONTACT:
Name: Brumbaugh, G. W.
Phone: 979-845-7257
Fax: 979-845-6544
Email: gbrumbaugh@cvm.tamu.edu

 
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ACCESSION NO: 0203969 SUBFILE: CRIS
PROJ NO: TEX09114 AGENCY: CSREES TEX
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2005-35204-16087 PROPOSAL NO: 2005-01639
START: 01 SEP 2005 TERM: 31 AUG 2008 FY: 2006 GRANT YR: 2005
GRANT AMT: $314,984
INVESTIGATOR: Fosgate, G. T.; Norby, B.; Ward, M. P.; Gayle, L.; Ellis, D.
PERFORMING INSTITUTION:
Veterinary Integrative Biosciences
Texas A&M Univ
College Station , Texas 77843
FOREIGN ANIMAL DISEASE SURVEILLANCE SYSTEMS FOR THE FUTURE: EVALUATION OF METHODS FOR DATA ACQUISITION AND ANALYSIS.
NON-TECHNICAL SUMMARY: The intentional or accidental introduction of a foreign animal disease poses a great risk to the animal agriculture industry of the United States. The purpose of this project is to determine the most effective surveillance system for foreign animal diseases of cattle by creating a network of veterinary practitioners and its comparison to other survey techniques.
OBJECTIVES: 1. Create a veterinary practitioner network dedicated to the surveillance of foreign animal diseases. The working hypothesis for this research objective is that private veterinary practitioners are the first line of defense to prevent the incursion of an FAD from becoming an uncontrolled epidemic. The skills and expertise of practicing veterinarians are not being exploited for active surveillance of FADs. A skilled network of practicing veterinarians will improve our ability to detect FADs and protect U.S. agriculture. 2. Determine the effectiveness of three active surveillance techniques for detection of foreign animal diseases by using currently endemic cattle pathogens that mimic the epidemiology, pathogenicity, and virulence of FADs. The working hypothesis for this research objective is that active surveillance strategies using specimens that are currently collected for other disease programs are an equally sensitive method for detecting low prevalence, low morbidity diseases as more comprehensive, and expensive, survey methods. It is hypothesized that current brucellosis surveillance programs could be modified to include testing for select agents determined to be of high consequence to U.S. agriculture and public health. It is further hypothesized that active surveillance of clinically ill cattle (targeted) is necessary for the rapid detection of diseases that are more virulent with higher morbidity proportions. Using endemic cattle diseases that mimic FADs is necessary for the development of future FAD surveillance programs in the absence of actual outbreaks. 3. Develop a decision support system for the initiation of a foreign animal disease (FAD) investigation based on spatiotemporal clustering of results from an imperfect screening test. The working hypothesis for this research objective is that intensive surveillance programs for FADs using tests with imperfect diagnostic specificity will lead to the unnecessary investigation of false-positive test results unless the appropriate decision tools are developed. It is hypothesized that all screening tests will have some proportion of false-positive results and mechanisms can be developed to account for background levels of spatiotemporal clustering of these results to prevent unnecessary and costly FAD investigations including quarantines and hold orders. Developing these mechanisms is necessary for the design and implementation of future surveillance systems.
APPROACH: A practitioner network has been formed based on the American Veterinary Medicine Association (AVMA) member directory. A brief mailing was performed describing the objectives of the current proposal and included a postcard for the veterinarian to return stating his or her interest level in participating in this type of study. Educational materials will be developed concerning foreign animal disease diagnosis and surveillance and distributed to the veterinarians participating in this network in addition to currently available materials such as the CD version of the Grey Book available from the United States Animal Health Association (USAHA). A monthly newsletter will be created including a disease of the month feature spotlighting pertinent pathological and epidemiological characteristics of different foreign animal diseases. The newsletter will also contain updates concerning the research activities including the number of samples submitted and disease prevalences. Practitioners enrolled as part of the surveillance network will be requested to submit serum samples from cattle with illnesses that cannot be definitively diagnosed based exclusively on physical exam findings. Practitioners will be asked to submit serum and data from 2 cattle per month over a 2-year sample collection period. A random sample of serum specimens submitted to the State-Federal Laboratory in Austin, Texas will be selected for inclusion in the research project. A non-probability sample of Texas cattle will also be evaluated as part of this research project to compare to the other methods of active surveillance. These cattle were sampled in a manner proportional to the overall cattle distribution throughout Texas. The total number of serum samples tested over the 2-year period will be 1000 for each surveillance system. Submitted samples will be tested for select infectious agents including Paratuberculosis, Bovine Leukemia Virus, Bovine Virus Diarrhea, and West Nile Virus. The owner may also choose not to view the results in which case only the researchers directly involved with the study will ever know the disease status of the sampled animals. Statistical simulation models will be developed and used as a decision support system to identify disease clusters caused by the introduction of a foreign animal disease. Spatiotemporal scan statistical methods are often used for these types of problems. These models can also be adjusted for covariates to account for a non-random distribution of cases through time and space (non-uniform baseline risks). The surveillance for FADs will necessitate modeling the baseline risk of a false-positive test result, rather than the baseline level of disease. Statistically significant clusters found after adjusting for the covariate-adjusted risk of false-positives will be identified as clusters. Covariates that may be important for modeling false-positive reactions include cattle type (beef versus diary) and ecological factors such as climate, which may be associated with presence of cross-reacting organisms.
PROGRESS: 2006/09 TO 2007/08
A surveillance network comprised of 58 private veterinary practitioners has been developed. These veterinarians have been asked to submit blood samples to test for bovine virus diarrhea (BVD), Bovine Leukosis Virus (BLV), Johne's disease (JD), and West Nile Virus (WNV) when confronted with a cattle illness of unknown etiology. A total of 79 samples have been submitted. A total of 1084 cattle have been tested from 13 geographically dispersed herds throughout Texas. Texas was categorized based on ecological zones and the number of cattle tested per zone was proportional to the total cattle population in each zone. This component of the surveillance project has been completed. A total of 25 cattle from each of 4 markets corresponding to 4 different areas of Texas have been tested per month. This sampling has also been completed following the12 month period for a total of 1200 tested cattle. Foreign animal disease (FAD) specific posters and newsletters have been created to keep this network informed concerning progress of the research as well as providing information for highlighted FADs. Seven quarterly reports (4 new this reporting period) comprised of FAD posters and research-specific newsletters have been sent to these practitioners. The FAD topics for the first 7 newsletter/poster combinations have been rinderpest, avian influenza, classical swine fever, heartwater, screwworm myiasis, contagious equine metritis, and peste des petits ruminants. Future topics that will be developed include foot-and-mouth disease, African horse sickness, exotic Newcastle disease, Rift Valley fever, and African swine fever.
IMPACT: 2006/09 TO 2007/08
The overall prevalences of the 4 diseases are 1.3% (30/2363), 13.5% (318/2363), 2.3% (55/2363), and 0.3% (8/2363) for BVD, BLV, JD, and WNV respectively. Herd testing has revealed prevalences of these diseases of 0.4% (4/1084), 2.8% (30/1084), 2.6% (28/1084), and 0% (0/1084). Market cattle testing has shown 1.9% (23/1200), 21.5% (258/1200), 1.6% (19/1200), and 0.4% (5/1200). Veterinarian submitted sample results are 3.8% (3/79), 38.0% (30/79), 10.1% (8/79), and 3.8% (3/79). These findings support our hypothesis that different methods of sample collection affect the observed prevalence of disease. The intentional or accidental introduction of a foreign animal disease (FAD) poses a great risk to animal agriculture of the United States. The purpose of this project is to determine the most effective surveillance system for FADs of cattle by creating a network of veterinary practitioners and its comparison to other possible sampling techniques. Development and maintenance of this surveillance network will improve practitioner awareness of FAD conditions and reporting procedures. Dissemination of educational materials related to select FADs will also improve the preparedness of practitioners to recognize and respond to potential outbreaks of these devastating diseases. Comparison of the 3 types of surveillance methods will also help determine if existing surveillance methods for regulated disease, for example brucellosis surveillance, might be effective to routinely screen for select FADs. Statistical models will be developed for the recognition of spatially-associated clusters of a FAD based on the results of an imperfect diagnostic test by using statistical techniques that adjust for the underlying population at risk and the non-random probability of false-positive reactions. These data will provide an estimate of the number of positive results from an imperfect screening test that would be expected in the absence of a FAD introduction.
PUBLICATIONS (not previously reported): 2006/09 TO 2007/08
1. Pearce BH, Fosgate GT, Norby B, Ward MP, Roussel AJ. 2006. Describing the efficiency of three methods of surveillance for detecting Johne's disease in Texas cattle. 87th Conference of Research Workers in Animal Diseases (CRWAD), December 3-5. Epidemiology and Animal Health Economics, Abstract No. 81.
2. Pearce BH, Fosgate GT, Norby B, Ward MP, Roussel AJ. 2007. Using spatial risk maps to evaluate three surveillance methods for detecting Johne's disease in Texas cattle. 3rd Annual Conference, Johne's Disease Integrative Program (JDIP), January 19-21. Paper #5.
PROJECT CONTACT:
Name: Fosgate, G. T.
Phone: 979-845-3203
Fax: 979-847-8981
Email: gfosgate@cvm.tamu.edu

 
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ACCESSION NO: 0211243 SUBFILE: CRIS
PROJ NO: TEX09251 AGENCY: CSREES TEX
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 27 JUN 2007 TERM: 30 SEP 2008
INVESTIGATOR: Khare, S.; Ficht, A. R.; Ficht, T. A.
PERFORMING INSTITUTION:
Veterinary Pathobiology
Texas A&M Univ
College Station , Texas 77843
3-D CELL CULTURE MODEL FOR THE STUDY OF THE HOST INNATE RESPONSE TO INFECTION BY MYCOBACTERIUM AVIUM PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: A. Johne's disease remains a serious agricultural problem with no treatment or prevention available. B. The initial response of cattle to infection is through the innate immune system which may serve to protect from infection. A. This project develops a 3D tissue culture system for the study of innate responses to infection with Johne's agent. B. The purpose of this study is to compare results of 3D tissue culture infection with our established infection model in the bovine small intestine.
OBJECTIVES: The goal of the proposed research is to understand the innate responses of ileal tissue to an intestinal pathogen, Mycobacterium avium ssp. paratuberculosis (MAP). Our central hypothesis is that early interaction of MAP with host ileal mucosa play major role in the progression of the Johne's Disease (JD). The bacteria (MAP) are detected in intestinal macrophages within hours of oral ingestion and persist in subepithelial macrophages. The objective of the proposed research proposal is to identify host and pathogen associated factors involved in the virulence, persistence and progression of the disease. We will test our hypothesis with the following specific aims: Specific aim 1. To develop and characterize 3-dimentional small intestine tissue-like aggregates (TLAs). We propose to develop a physiologically relevant bovine ileal 3-D in-vitro cell culture model that conserves morphological differentiation and cell-specific functions of ileum. Specific aim 2. Study and compare the infection of 3-D TLAs with that of calf ileum using microarray and RT-PCR. We propose to infect the TLAs with Mycobacterium avium ssp. paratuberculosis (MAP), a chronic pathogen. The 3-D model will serve as a tool to understand the innate responses of host against MAP and other intestinal pathogens. Mechanistic investigations will be possible for microbe-cell interactions, innate responses of the host, recognition of pertinent diagnostic biomarker and a means for rapid evaluation of candidate therapeutic agents in the target system tissue.
APPROACH: There are many limitations to the study of the innate response of the host in Johne's Disease. It is widely accepted that failure of the innate immune responses to contain a pathogen lead to infection. Research progress on MAP has been slow due in large part to the extreme difficulty of culturing the organism (3-4 months), and the length of time needed to produce an experimental infection (2 years). Thus, there remains a gap in our understanding of the host innate response to MAP, which is a key barrier for this pathogen to breach in order to ultimately establish chronic disease. The long range goal is to elucidate the pathogenesis of JD. Much is documented regarding the host response of chronically infected individuals, but little is known concerning the events in the host immediately surrounding infection in bovine and human systems. Bovine ligated ileal loop model (BLIL) has been used in our lab to identify the early events of host (cattle)-pathogen (MAP) interaction at the site of infection (gut associated lymphoid tissue). Though BLIL model reflects the very early responses of the host, it has the limitations to study the innate responses of a chronic pathogen like MAP; for which the innate responses are usually defined as the responses of host from few days to few weeks. The establishment of 3-D small intestine TLA culture system will provide a more relevant model to study the responses of host in response to MAP infection.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: ACTIVITIES The first SPECIFIC AIM of the proposal is to Develop and characterize 3-D small intestine TLAs. Following objectives were conducted to fulfill the SPECIFIC AIM 1: OBJECTIVE 1. Development of 3-D small intestine Tissue Like Aggregates (TLAs). In order to develop 3-D small intestine TLAs successfully we have drawn on the expertise and support of M.T. Suderman, President and CSO, Cell Systems-3D, LLC, League City, Texas. Bovine ileum was collected from the Animal Sciences Department. Six to nine centimeter sequential ileum containing both absorptive epithelium (AE) and follicle-associated epithelium (FAE) was removed from a humanely euthanized 3 week old calf. After removal of fat and mesentery, the ileal tissue is placed in ice-cold PBS with 2% (v/v) penicillin-streptomycin, 1% fungizone, 1% D-glucose, 0.2% glucose and 4 mM L-glutamine for primary tissue disassociation and recovery of enterocytes-M cells-fibrocytes. Dissociated cells are in the culture to achieve large numbers. After achieving the appropriate number of cells TLA model will be initialized in the rotatory bioreactor. OBJECTIVE 2. Characterization of bovine intestine and dissociated cells by transmission electron microscopy (TEM): The original sample of the bovine intestine was fixed in the fixative and processed for the TEM. TEM is underway and the sections will be evaluated for cell-cell junctional contacts, apically-orientated villous structures, lumen-orientated enterocytes with microvilli, mucin producing goblet cells, identification of M cells or lymph follicular tissue and fibrocyte-enterocyte junctional contact. This original TEM will be compared with the TEM of TLAs EVENTS Sangeeta Khare (PI) attended and presented a research paper in the Annual meeting of Conference of Research Workers in Animal Disease at Chicago (1-4 December' 2007). Sangeeta Khare participated in a training on "Safe practices in Biosafety laboratory 3" by University of Texas Medical Branch-Galveston-Texas (August 14-17' 2007) SERVICE Sangeeta Khare developed a course on the Laboratory Biosafety to prepare students to safely work in the Biological Laboratory. PRODUCTS The cell cultures are in the process of proliferation. To achieve this, we have drawn the expertise of Mr. Suderman. We expect to build and characterize 3-D small intestine TLAs. We have also established collaborations with Dr. Radhey Shyam Kausik an Assistant Professor at South Dakota State University. Dr. Kaushik has an expertise in the establishment of fetal bovine intestinal epithelial cell cultures. DISSEMINATION The project is in the developmental stage. Expertise of Dr. Suderman and Kaushik will help us to conduct the planned experiments successfully. PARTICIPANTS: Individuals: Sangeeta Khare Research Assistant Professor Department of Veterinary Pathobiology COllege of Veterinary Medicine and Biomedical Sciences College Station-TX-77843 Partner Organizations: Cell Systems-3D, LLC, League City, TX. Collaborators and contacts: M.T. Suderman, President and CSO, Cell Systems-3D, LLC, League City, TX. TARGET AUDIENCES: Target audiences: Sciectific community working in the area of host-pathogen interaction Efforts: Course development in the "Laboratory Biosafety" PROJECT MODIFICATIONS: We proposed to collect bovine ileum from the slaughter house. Due to the limitations of sterile tissue collection process we changed the collection facility and now the bovine ileum are collected from the Meat Sciences laboratory at Texas A&M University. Meat Sciences Laboratory will not harvest cow for another two months. We are looking for the alternative sources to collect more bovine tissues.
IMPACT: 2007/01 TO 2007/12
OUTCOME: CHANGE IN KNOWLEDGE To our knowledge this is the first study to constitute a 3-D cell culture model for the bovine intestine. We are under the developmental process to design the tissue like aggregates that will mimic the bovine intestine. CHANGE IN ACTIONS We proposed to collect bovine ileum from the slaughter house. Due to the limitations of sterile tissue collection process we changed the collection facility and now the bovine ileum are collected from the Meat Sciences laboratory at Texas A&M University. They will not harvest cow for another two months. We are looking for the alternative sources to collect more bovine tissues. CHANGE IN CONDITIONS We had a great success in collection of epithelial cells from bovine intestine. However, we had problems in isolating and culturing the fibrocytes from bovine ileum. We plan to collect more bovine tissue for the isolation of fibrocytes. Meat Sciences laboratory at Texas A&M University will not harvest cow for another two months. We are looking for the alternative sources to collect more bovine tissues.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
No publications reported this period
PROJECT CONTACT:
Name: Khare, S.
Phone: 979-845-9814
Fax: 979-862-1088
Email: skhare@cvm.tamu.edu

 
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ACCESSION NO: 0193040 SUBFILE: CRIS
PROJ NO: TEXR-2002-02212 AGENCY: CSREES TEXR
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 2002-35204-12634 PROPOSAL NO: 2002-02212
START: 01 SEP 2002 TERM: 31 AUG 2005 GRANT YR: 2002
GRANT AMT: $249,000
INVESTIGATOR: Ficht, A. R.; Ficht, T. A.; Adams, G. L.
PERFORMING INSTITUTION:
Texas A&M University System Health Sci Ctr
407 Reynolds Medical Bldg.
College Station , Texas 77843
GENETIC DIVERSITY APPLIED TO THE DIAGNOSIS AND EPIDEMIOLOGY OF MYCOBACTERIUM AVIUM PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: Johne's disease ('paratuberculosis') is a chronic, infectious, wasting disease with significant impact on herd productivity resulting in a corresponding economic loss to U.S. dairy operations. According to the USDA National Animal Health Monitoring System's (NAHMS) 1996 national dairy study, Johne's-positive herds experience an economic loss of almost $100-200 per cow resulting from reduced milk production and increased cow-replacement costs. When averaged across all herds, Johne's disease costs the US dairy industry $200-250 million annually in reduced productivity. The experiments described are designed to enhance early detection of Johne's disease and to correctly identify and track virulent isolates. We have developed a sensitive detection method for Johne's disease organisms which employs magnetic bead recovery of organisms from cattle milk or fecal samples. We are also utilizing a newly developed genetic approach, Amplified Restriction Fragment Polymorphism (AFLP) in combination with bioinformatics to develop new tools for tracking individual and herd isolates of Johne's organisms. Research here will provide new tools for diagnosis and control of subclinical infection and permit the elimination of infected animals before the organism has a chance to spread within herds.
OBJECTIVES: Characterize the extent of genetic diversity among and between M. avium avium (M. avium) and M. avium paratuberculosis (MapTB) isolates using amplified fragment length polymorphism (AFLP). Characterize specific genetic differences by isolation and sequence analysis of AFLP fragments. Utilize diversity data to develop PCR primers for diagnostics and epidemiology.
APPROACH: AFLP, a relatively new experimental approach, will be used to reveal polymorphisms useful in cataloging the genetic diversity of of MapTB clinical isolates from Central and North America. This approach will also be used in subdividing and tracking clinical isolates of MapTB. AFLP will first be used to establish relationships among MapTB isolates and between MapTB and nonpathogenic M. avium. Secondly, AFLP fragments unique to MapTB will be isolated and sequenced and the sequences compared to that of M. avium. PCR primers capable of specifically detecting MapTB isolates will be derived from this data and used in diagnostic and epidemiological analysis.
PROGRESS: 2002/09 TO 2003/08
One hundred ninety two AFLP primer sets have been employed to characterize 20 Mycobacterium avium subspecies paratuberculosis (MparaTb) field isolates, one ATCC MparaTb isolate (ATCC 19698), and two M. avium avium isolates (ATCC 35716 and Mac 104) totaling 4,416 independent PCR reactions. Specifically, 16 MseI primer sets bearing selective dinucleotides at the 3'end were used in combination with 12 PstI primers bearing 3' selective dinucleotides. Diagnostic patterns have been established for the identification of MparaTb isolates and five AFLP fragments unique to the MparaTb genome have been cloned and sequenced. BLAST search results for these five AFLP fragments reveals sequence homology to genes such as the primosomal protein N' (PPN') of Brucella melitensis, P44k protein of Rhodococcus erythropolis, putative polyketide type 1 synthase (PPKS) of Streptomyces, replication protein of Rhodococcus erythropolis, and one region showed no significant similarity to any known protein. Primers were generated that were internal to these regions and when applied to the 20 MparaTb field isolates in this study an appropriate PCR product was obtained in 99 of 100 reactions. The M. avium avium isolates failed to act as templates for PCR amplification in 9 of 10 reactions. This work revealed the presence of polymorphisms in the genomes of MparaTb and M. avium avium. In addition to these PCR reactions we also carried out PCR with 4 out of the 5 MparaTb specific primer sets on an NVSL Johnes test set. This test set consisted of 25 samples, 17 determined to be MparaTb and 8 were determined to be negative for MparaTb genomic DNA through IS900 PCR and verification from NVSL. PCR amplification with diagnostic primer sets for regions 1-4 designed in this study were performed on these 25 NVSL isolates. Region 1 primer set amplified its corresponding region in 15 of 17 MparaTb isolates. Region 2 primer set amplified its corresponding region in 11 of the 17 MparaTb isolates. Region 3 primer set amplified its corresponding region in 12 of the 17 MparaTb isolates. Finally, Region 4 primer set amplified its corresponding region in only 7 of the 17 MparaTb isolates. One of the 17 MparaTb isolates (#15) showed no amplification with any primer set and none of the MparaTb-specific primer sets described here produced amplification products from the negative samples. This data suggests that there is a high degree of heterogeneity among these 17 MparaTb isolates and that these polymorphic regions are not conserved in all MparaTb isolates. Examining one of the MparaTb specific sequences more closely revealed a 5,145bp region with no homolog in the M. avium avium genome. Within this region are genes responsible for integrase/recombinase function. An additional MparaTb specific sequence revealed a 15kb region with no homologue in the M. avium avium genome. Both of these missing regions were verified to be absent from the M. avium avium genome through PCR primer walks. Three additional MparaTb-specific regions have been cloned, revealing a number of house keeping genes; all were evaluated for their diagnostic and epidemiological value.
IMPACT: 2002/09 TO 2003/08
The work we have completed will enable a rapid diagnostic test to be put in place utilizing isolation of organisms from milk or feces followed by PCR. This test will enable a diagnosis within a matter of days rather than the 6 or more weeks normally required for culture growth and identification. As we continue to investigate a spectrum of field isolates and analyze them via AFLP, we will identify more DNA targets and additional PCR diagnostic primer sets that will facilitate not only identification of M. paratuberculosis isolates, but epidemiological tracking of organisms.
PUBLICATIONS (not previously reported): 2002/09 TO 2003/08
1. O'Shea, Brian, Khare, Sangeeta, Bliss, Katherine, Klein, Patricia, Ficht, Thomas A., Adams, L. Garry, Rice-Ficht, Allison C. (2003) Genotyping and Characterization of Mycobacterium avium subsp. paratuberculosis Using Amplified Fragment Length Polymorphism (submitted).
2. O'Shea, Brian (2002) Genotyping and Characterization of Mycobacterium avium paratuberculosis Using Amplified Fragment Length Polymorphism, International Conference on Emerging Infectious Diseases; Atlanta, Georgia; March 24-27, 2002.
3. O'Shea, Brian (2002) Genotyping and Characterization of Mycobacterium avium subsp. paratuberculosis Using Amplified Fragment Length Polymorphism for the Development of a Diagnostic Tool, Conference of Research Workers in Animal Diseases, St. Louis, Missouri, November 10-11, 2002.
PROJECT CONTACT:
Name: Ficht, A. R.
Phone: 979-458-1024
Fax: 979-847-9481
Email: a-ficht@tamu.edu

 
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ACCESSION NO: 0187543 SUBFILE: CRIS
PROJ NO: UTA00468 AGENCY: SAES UTA
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JUL 2000 TERM: 31 DEC 2003 FY: 2004
INVESTIGATOR: Bingham, H. R.; Bagley, C. V.
PERFORMING INSTITUTION:
Animal Dairy & Vet Science
Utah State University
Logan , Utah 84322
SEROPREVALENCE OF MYCOBACTERIUM AVIUM SUBSPECIES PARATUBERCULOSIS IN UTAH CULL DAIRY AND BEEF CATTLE.
NON-TECHNICAL SUMMARY: Dairy and beef cattle infected with the bacteria Mycobacterium paratuberculosis may eventually develop chronic diarrhea (commonly known as Johne's disease) which decreases the market value and productivity of the infected animal and spreads the organism to uninfected cattle. This research will estimate the prevalence of Mycobacterium paratuberculosis infection in Utah cull dairy and beef cattle and provide useful information to help educate Utah veterinarians and cattle producers about Johne's disease.
OBJECTIVES: 1. Estimate the seroprevalence of antibodies specific for Mycobacterium avium subspecies paratuberculosis in Utah cull dairy and beef cattle. 2. Compare the level of agreement between the Johne's ELISA test and the Johne's Tip-Test.
APPROACH: Individual blood samples will be collected from 2000 cull dairy and beef cattle from a single abattoir in northern Utah. One milliliter aliquots of serum will be placed into cryovials and frozen at -18 C (0 F) until all samples are collected and can be submitted as a single lot. Serum samples will be analyzed for Mycobacterium avium subspecies paratuberculosis specific antibodies using both the Johne's ELISA test and the Johne's Tip-Test. Test results will be entered into a spreadsheet (Excel). The auction market of origin will also be recorded for each individual cow. Mycobacterium avium subspecies paratuberculosis seroprevalence rates will be calculated from these data for the overall state and for each of the 8 auction markets in Utah. The level of agreement between the Johne's ELISA test and the Johne's Tip-Test will also be calculated and reported.
PROGRESS : 2000/07 TO 2003/12
The four objectives of this project are as follows:1) Describe the seroepidemiology of Mycobacterium avium subspecies paratuberculosis (MAP) in cull dairy and beef cattle of Utah origin; 2) Compare the agreement between two MAP antibody detection tests (IDEXX ELISA, ImmuCell Tip-Test); 3) Estimate MAP herd prevalence in Northern Utah dairies; 4) Identify unique production and management practices that are associated with MAP infection in Northern Utah dairies. All project objectives have been completed. Objective 1: Serum samples from Utah cull dairy and beef cattle were collected and analyzed for MAP antibodies. The apparent seroprevalence of MAP in Utah cull dairy and beef cattle is 3.84% (42/1096) and 0.09% (1/1086), respectively. County seroprevalence rates are available upon request. Objective 2: Two MAP antibody detection tests were compared. There was a moderate level of agreement (Kappa = 0.48) between the IDEXX ELISA and the ImmuCell Tip-test. However, the two MAP antibody detection tests gave statistically different results (P <0.05). These results suggest that the two tests should not be considered as similar for use in Johnes Disease control programs. Objective 3: Blood and fecal samples were collected from 86 dairy herds in Box Elder, Cache, and Weber counties representing almost 50% of the total cow population in Utah. Blood was collected from 30 randomly selected, 2nd lactation or older cows within each herd and analyzed for MAP antibody using the IDEXX ELISA test. Fecal samples were collected from MAP seropositive cows and cultured for a minimum of 16 weeks. Bacterial colonies were confirmed as MAP using both Zehl-Neelsen stain and PCR. A MAP positive herd was defined as one where MAP was definitively identified (by culture & PCR) from a cow within that herd. The apparent MAP herd prevalence in northern Utah dairies is 19.8% (true prevalence: 23.5-59.9%). Objective 4: A survey evaluating 82 aspects of herd production and management was also completed during on farm sample collection by the herd owner. The relationship between these factors and MAP positive herds was evaluated using multiple regression analysis. Dairy herds that had purchased heifers or cows within the last five years were 40 times more likely to be MAP positive compared to herds that did not purchase heifers or cows or just purchased bulls. Jersey herds were 2.8 times and mixed breed herds were 14.7 times more likely to be MAP positive compared to Holstein herds. Herds that shared a common fence line between 2-6 month old calves were 12.3 times more likely to be MAP positive compared to those which did not share a common fence line. These data suggest that Johnes Disease is common in Utah dairy herds, particularly in herds that purchase heifers/cows and that true MAP prevalence is higher among Jersey and mixed breed herds compared to Holstein herds. The data also suggest buying bulls and showing cattle are not risk factors for MAP in Utah herds.
IMPACT: 2000/07 TO 2003/12
MAP infection in cattle causes a chronic, debilitating, and production limiting condition in cattle referred to as Johnes Disease. The information in this study demonstrates that Johnes Disease is common in Utah dairy herds and much more common in herds with certain characteristics. The results from this study will be used to help educate Utah cattlemen about Johne's Disease and to promote and direct efforts of a Utah state Johnes Disease control program.
PUBLICATIONS (not previously reported): 2000/07 TO 2003/12
No publications reported this period
PROJECT CONTACT:
Name: Bingham, H. R.
Phone: 435-797-7146
Fax: 435-797-3959
Email: hbingham@cc.usu.edu

 
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ACCESSION NO: 0409023 SUBFILE: CRIS
PROJ NO: 1265-32000-078-04S AGENCY: ARS 1265
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 30 SEP 2004 TERM: 29 SEP 2009
INVESTIGATOR: Karns J S; Van Kessel J S; Smith J
PERFORMING INSTITUTION:
University of Vermont
Burlington , Vermont 05405
PILOT STUDY OF FACTORS AFFECTING MAINTENANCE OF MYCOBACTERIUM,SALMONELLA, E. COLI, AND LISTERIA ON DAIRY FARMS.
OBJECTIVES: The objective of this research is to identify and characterize a third dairy herd to be included in a Pilot Program that currently consists of two dairy herds and which focuses on the protocol development, laboratory set-up, program logistics, and database and bio-bank development needed to evaluate such that the impact of intervention strategies on Johne's disease dynamics, milk and beef quality (particularly with respect to zoonotic bacterial pathogens), economics and sustainability can be evaluated through intensive longitudinal follow up of selected research/demonstration dairy farms. Long-term goals are to validate intervention strategies to support best management practices (BMPs) and to optimize intervention and monitoring strategies given the constraints on time, labor and financial resources in modern dairy herds. In addition, a national resource bank (data and biological specimens on well-characterized animals) will be created for current and future monitoring and research on dairy cattle diseases. Emphasis will be on longitudinal data collection on endemic infectious diseases of public health and animal health concern in dairy herds.
APPROACH: Pathogens are of increasing concern on dairy farms and in dairy products. The production of safe and wholesome food from US farms requires control of the production process on the farm. Specific focus areas in this process are biosecurity, food safety and animal health. To be able to scientifically support regional process control programs there is a need for longitudinal research on commercial dairy farms throughout the United States. In the past year, Cornell University, The Pennsylvania State University, and the University of Pennsylvania, which are all participants in the Regional Dairy Quality Management Alliance (RDQMA), have collaborated with the USDA's Environmental Microbial Safety Laboratory to investigate 2 operating dairy farms to determine sites that act as reservoirs for pathogenic microorganisms that affect animal health and that decrease product quality because of their zoonotic nature. Farms in Vermont (also an RDQMA member) will be identified as candidates for inclusion in this investigation and 1 will be chosen for extensive sampling based on the previous occurrence of Johne's disease and/or Salmonella infection. Serum, feces, bulk tank milk, and environmental samples (water, bird and rodent feces, feed, etc.) will be taken on the farm. In addition, tissue samples will be obtained from carcasses of culled animals. Samples will be distributed among the university and ARS researchers for analysis to determine the presence of Mycobacterium avium paratuberculosis (the causative agent of Johne's disease in cattle) and for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes (human food-borne pathogens of concern in dairy products). This research will be the first to attempt a comprehensive analysis of both Johne's disease and food-borne pathogens on working dairy farms. It will allow us to determine a baseline for these organisms on 3 farms and set the stage for investigation of the effect of interventions, in the form of BMPs, on animal health and product quality.
PROGRESS: 2005/10 TO 2006/09
Progress Report 4d Progress report. This report serves to document research conducted under a Specific Cooperative Agreement between ARS and The University of Vermont. Additional information can be found in the reports for the SCAs with the other universities involved in the project (1265-32000-078-01S, 02S, and 03S) and in the report for the parent project 1265-32000-078-00D "DAIRY MANAGEMENT PRACTICES AND THE TRANSMISSION OF ZOONOTIC PATHOGENS IN MILK". This cooperative agreement is part of a larger project involving 4 universities, the University of Pennsylvania, The Pennsylvania State University, University of Vermont, and Cornell University, and formulated in consultation with the Northeastern Regional Dairy Quality Alliance (RDQMA) and the National Milk Producers Federation. Currently, this project provides longitudinal access to three dairy herds totaling 575 milk cows (NY herd-340 milk cows; PA herd-110 milk cows and VT herd-125 cows). Fecal samples from adult cows on the VT farm are collected twice a year and sent to collaborating labs to be tested for the presence of Mycobacterium avium Paratuberculosis (MAP, the causative agent of Johne's disease), and the zoonotic foodborne bacterial pathogens Salmonella, Escherichia coli, Listeria monocytogenes, Campylobacter, and Enterococcus. Blood serum is collected quarterly to test for the presence of antibodies to the Johne's Disease (JD) organism. Environmental samples of manure, feed, water, vermin, birds, flies, etc are taken 4 times a year for analysis by collaborators. Bulk tank milk and the milk filter are collected weekly. The purpose of the project from the perspective of our lab is to characterize the microbial populations on the farm with particular interest in Salmonella, Escherichia coli, and Listeria monocytogenes. Initial findings from the VT farm have shown the occasional presence of Listeria monocytogenes in fecal and environmental samples. In addition, MAP has been found in fecal and environmental samples. A cow that was excreting massive amounts of MAP was identified and culled resulting in a drastic reduction of environmental MAP and MAP positive animals on the farm. This work will lead to changes in the approach for reduction of MAP in dairy production environments.
PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
No publications reported this period.

 
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ACCESSION NO: 0204359 SUBFILE: CRIS
PROJ NO: VA-137194 AGENCY: CSREES VA.
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 JUL 2005 TERM: 30 JUN 2006 FY: 2006
INVESTIGATOR: Boyle, S. M.; Sriranganathan, N.; Gwazdauskas, F. C.
PERFORMING INSTITUTION:
College of Vet Medicine
Virginia Polytechnic Institute
Blacksburg , Virginia 24061
IMMUNE RESPONSE IN CATTLE TO BRUCELLA ABORTUS RB51 OVER-EXPRESSING PROTECTIVE ANTIGENS.
NON-TECHNICAL SUMMARY: Current vaccines against brucellosis do not afford specific protection against other cattle diseases. the development of a multivalent vaccine to protect against brucellosis and other cattle diseases would make vaccination programs more cost effective. This project assesses the ability of a second generation vaccine derived from vaccine strain RB51 to induce antibodies or cellular immune responses consistent with protection
against mycobacterium infections
OBJECTIVES: 4. Objectives: The overall purpose of the objectives outlined is to test the immunogenicity of the new vaccines following subcutaneous administration to cattle: a) of RB51 strains over-expressing either Mycobacterium ESAT6 antigen or B. abortus SOD antigen. By injecting these RB51 platform vaccines, we will produce immunized subjects that will serve as a source of serum and cells to measure immune parameters that can correlate with protection against tuberculosis and/or brucellosis in cattle. b) and testing the humoral and cellular immune responses (adaptive). By measuring the levels and isotypes of the immunoglobulins to specific antigens (Brucella, Mycobacterium and anthrax) as well as the cellular immune responses ( i.e. cytokines), we will learn if the vaccines are inducing immune correlates consistent with protection.
APPROACH: Our ability to raise healthy cattle, free of zoonotic diseases such as Brucella spp., has had a positive impact on both the health and expenditures of producers and consumers of animal foods in Virginia and the USA. Since the implementation of the Brucellosis eradication program in 1950s, it is estimated that the steady reduction over the past 50 years and virtual elimination of brucellosis in cattle herds in 2000 has saved the US millions of dollars annually in terms of cattle and human costs (1). There are still concerns about other zoonotic agents including Mycobacterium bovis, M. paratuberculosis for which no effective vaccines are available for cattle and threaten the health of both cattle and humans. Thus new vaccines that can take advantage of proven vaccines to deliver protective antigens represent an efficient means to immunize susceptible populations. In the proposed work, the USDA approved cattle vaccine, Brucella abortus RB51, will be assessed for its ability to act as a platform to deliver specific antigens to induce immune responses in cattle that correlate with protection against specific pathogens i.e. in addition to Brucella spp. If such protective immune responses (e.g. humoral and cellular) are observed, then additional funding will be sought at the federal level to set up challenge studies in cattle. The development of such a platform-based vaccine will allow for the prevention of several diseases with one one dose of an attenuated vaccine. This type of technology will be cost effective and efficient and minimize side effects seen with multiple vaccination schemes. In addition, a platform based vaccine that can be rapidly formulated and prevent several types of diseases will allow us to respond quickly to biological threats whether they be initiated by natural or terrorist means.
PROGRESS: 2005/07 TO 2006/06
Heifers (4-5 female Holsteins, 8-10 months of age per group) were immunized subcutaneously with either saline, vaccine strains B. abortus RB51, strain RB51/WboA or RB51/SOD/Esat6. 30ml of blood per calf was collected via jugular venipuncture every other week for six weeks. Plasma was harvested from 10mL of whole blood and stored for evaluation of antibody production using antigen specific ELISA. From the remaining 20ml of blood collected, we separated lymphocytes and measured proliferation in response to the vaccine strains as assessed by Alamar blue colorimetric assay as well as the amount of interferon gamma produced as measured by ELISA. At 2, 4 and 6 weeks post immunization relative to the saline treatment, the serum IgG immune responses exhibited a progressive titer increase in all heifers receiving the vaccines: RB51 - 5, 15, 21 mg/dl; RB51/wboA - 6, 8, 14 mg/dl; RB51/SOD/Esat6 - 6, 5, 14 mg/dl respectively. The corresponding interferon-gamma levels relative to saline treatment (measured in supernatants of stimulated lymphocytes) showed a maximum at 2 weeks post-immunization in all groups regardless of the Brucella extract (RB51, RB51/wboA, RB51/SOD/Esat6) used to stimulate the proliferation; the ability to stimulate interferon gamma levels from lymphocytes decreased at 4 and 6 weeks. The extract derived from strain RB51/wboA stimulated the highest level of interferon-gamma (7-9 ng/ml at 2 weeks) production in lymphocytes independent of the Brucella vaccine used toimmunize. In contrast, the extract derived from strain RB51/SOD/Esat6 stimulated the smallest level of interferon-gamma and was significantly less than that stimulated by extract from strain RB51; this suggests an immunosuppressive effect. There were no statistically significant differences in leukocyte differential counts (remained normal) or in serum cortisol levels as a function of the Brucella vaccines at 2, 4 and 6 weeks post-immunization. We conclude that these novel platform based B. abortus RB51 based vaccines (RB51/wboA and RB51/SOD/Esat6) are not detrimental to the calves. It appears that strain RB51/wboA is able to better stimulate interferon gamma production in lymphocytes than either strain RB51 or RB51/SOD/Esat6. It will be necessary to perform challenge studies to determine if strain RB51/wboA is able to more effectively protect cattle that strain RB51 against a virulent Brucella challenge. In addition, because of the low levels of gamma-interferon stimulated relative to strain RB51, it appears that strain RB51/SOD/Esat6 would not be a good candidate to protect cattle against a Mycobacterium bovis infection.
IMPACT: 2005/07 TO 2006/06
These results show that it is possible to modifiy a USDA/APHIS approved vaccine to prevent brucellosis in cattle and improve its ability to protect against brucellosis but not tuberculosis. It will be necessary to perform challenge studies of cattle immunized with the novel RB51 vaccine to show efficacy in the field. If proven to be effective, it may be possible to lower the effective dose of the vaccine and pass this savings on to the livestock producers.
PUBLICATIONS (not previously reported): 2005/07 TO 2006/06
Amanda Hurt, Immune Response in Cattle to Brucella abortus RB51 Over-expressing Homologous and Heterologous Protective Antigens, M.Sc. Thesis, Virginia Tech, submitted June 2007.
PROJECT CONTACT:
Name: Boyle, S. M.
Phone: 540-231-4641
Fax: 540-231-3426
URL: smboyle@vt.edu


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ACCESSION NO: 0198916 SUBFILE: CRIS
PROJ NO: WNP00568 AGENCY: CSREES WN.P
PROJ TYPE: HATCH PROJ STATUS: NEW MULTISTATE PROJ NO: NRSP-8
START: 01 OCT 2003 TERM: 30 SEP 2008 FY: 2007
INVESTIGATOR: Jiang, Z.; Neibergs, H.
PERFORMING INSTITUTION:
Animal Science
Washington State University
Pullman , Washington 99164
NATIONAL ANIMAL GENOME RESEARCH PROGRAM
NON-TECHNICAL SUMMARY: Livestock genome analysis has advanced significantly in recent years, yet its progress lags behind the human, mouse and rat. The purpose of this study is to develop the cutting-edge comparative strategies and approaches to facilitate genome mapping in animals by mainly taking advantage of and building on the information available through the mammalian genome programs.
OBJECTIVES: 1.Enhance and integrate genetic and physical maps of agriculturally important animals for cross species comparisons and sequence annotation. 2.Facilitate integration of genomic, transcriptional, proteomic and metabolomic approaches toward better understanding of biological mechanisms underlying economically important traits.
APPROACH: HAPPY mapping panels will be generated to facilitate genome mapping in livestock species. Orthologous genes will be selected at an interval of 2 Mb based on the human genome sequencing information, while primers will be designed based on livestock gene or EST (expressed sequence tags) sequences. These orthologous genes/markers will be typed on the HAPPY panels and maps will be constructed using RHMAP (version 3) software. The gene products will be amplified using pooled DNA samples of Chinese and Western pigs and used for direct sequencing in order to reveal single nucleotide polymorphisms in the livestock genomes. A Livestock Orthologous Gene (LOG) database will be developed to gather information on gene sequencing, mapping, expression profiling and functional analysis in livestock species.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Corticotropin releasing hormone (CRH) is a promising candidate gene for correlation beef marbling score (BMS) and subcutaneous fat depth (SFD) in beef cattle. In the present study, a total of five single nucleotide polymorphisms(SNPs) were identified in the bovine CRH gene and genotyped on ~250 F2 progeny; four were selected as tagging SNPs for association analysis. Statistical analysis showed g.9657C>T, c.10718G>C and c.10936G>C as well as their haplotypes had significant effects on SFD, but only c.10936G>C was significantly associated with BMS. Overall, our results provide further evidence that CRH is a promising candidate gene for a concordant QTL related to lipid metabolism in mammals. A novel type of sequence variation - multiple nucleotide length polymorphisms (MNLPs)was discovered in the bovine genome. In the present study, we discovered a novel type of sequence variation, i.e., multiple nucleotide length polymorphisms (MNLPs) in bovine UCN3 and its receptor CRHR2 genes. Both MNLPs featured involvement of multiple nucleotides length polymorphisms (5 - 18 bases), low sequence identity and 1.7 - 11 fold changes in promoter activity between two alleles. Therefore, this novel genetic complexity would contribute significantly to the evolutionary, functional and phenotypic complexity of genomes within or among species. Development of a novel model for mapping cryptorchidism in sheep and initial evidence for association of INSL3 with the defect. In the present study, we developed a four-generation cryptorchid family in sheep, which consists of 99 individuals. The ovine INSL3 gene (GenBank accession no: DQ473569) was cloned and three SNPs were identified. The family based association tests revealed that g.2489C>T, but not c.2714C>A, was significantly associated with cryptorchidism in the family (P=0.000465). The Wagyu resources at Washington State University (WSU). The Wagyu cattle breed in the United States resulted from importation of just a few hundred animals. The objective of this study was to characterize the Wagyu in comparison with other breeds in the degree of relatedness, by evaluating the nucleotide divergence of sequences used for animal identification or parentage. Blood or semen samples of twenty-four Wagyu animals of diverse heritage were obtained and the DNA extracted. Sequencing has been initiated with 16 genomic regions in 12 animals completed. Disease gene mapping. Cattle show significant variation in their responses to disease challenges. Some of this variation is attributable to genetics. The objective of this research is to identify loci that increase the resistance or tolerance of cattle to Mycobacterium avium paratuberculosis (MAP). MAP is the bacterial pathogen that is responsible for Paratuberculosis, also known as Johne's disease. Dairy cattle from 5 herds have been sequentially tested for MAP and tissues collected following the culling of the animal (regardless of reason). Extraction of DNA from tissues of matched cases and controls animals is proceeding prior to genotyping. PARTICIPANTS: Washington State University: G.A. Williams, J.J. Michal, C.T. Gaskins, R.W. Wright, T.F. Daniels, T. Kunej, Z. Wang, N.S. Magnuson, Q. Xiao, T.A. Wibowo, X.L. Wu, J.J. Reeves, J.R. Busboom, G.H. Thorgaard. Penn State University: T.L. Ott. TARGET AUDIENCES: Livestock producers, product processors, consumers, genome mapping community, health educators.
IMPACT: 2007/01 TO 2007/12
Genome research at Washington State University provides insights into understanding the genome structure, function and evolution, and resources/tools for developing quantitative and molecular genomic approaches for improving animal production, product quality and health. The characterization of highly informative markers in Wagyu compared to other breeds provides the opportunity for individual identification of parentage and traceability of Wagyu and Wagyu crossbred animals. Reducing the prevalence or severity of Paratuberculosis in cattle will economically benefit the US cattle industry by reducing morbidity and mortality of cattle disease, thereby enhancing production. This in turn provides an increase in consumer access to safe and affordable food.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
1. Jiang, Z., Wang, Z., Kunej, T., Williams, G.A., Michal, J.J., Wu, X.L., and Magnuson, N.S. 2007. A novel type of sequence variation - multiple nucleotide length polymorphisms discovered in the bovine genome. Genetics 76:403-407.
2. Williams, G.A., Ott, T.L., Michal, J.J., Gaskins, C.T., Wright, R.W., Daniels, T.F., and Jiang, Z. 2007. Development of a novel model for mapping cryptorchidism in sheep and initial evidence for association of INSL3 with the defect. Animal Genetics 38:189-191.
3. Wibowo, T.A., Michal, J.J., and Jiang, Z. 2007. The corticotropin releasing hormone is a promising candidate gene for marbling and subcutaneous fat depth in beef cattle. Genome 50:939-945.
4. Jiang, Z., and Rothschild, M.F. 2007. Swine genome science comes of age. International Journal of Biological Sciences 3:129-131.
5. Rothschild, M.F., Hu, Z., and Jiang, Z. 2007. Advances in QTL mapping in pigs. International Journal of Biological Sciences 3:192-197.
6. Jiang, Z., Pappu, S.S., and Rothschild, M.F. 2007. Hitting the jackpot twice: identifying and patenting gene tests related to muscle lipid accumulation for meat quality in animals and type 2 diabetes/obesity in humans. Recent Patents on DNA & Gene Sequences 1:100-111.
7. Kunej, T.,Wang, Z., Michal, J.J., Daniels, T.F., Magnuson, N.S., and Jiang, Z. 2007. Functional UQCRC1 polymorphisms affect promoter activity and body lipid accumulation. Obesity 15(12):2896-2901.
8. Chen, J., Yang, X.J., Xia, D., Chen, J.P., Wegner, J., Jiang, Z., and Zhao, R.Q. 2007. Sterol regulatory element binding transcription factor 1 expression and genetic polymorphism significantly affect intramuscular fat deposition in the longissimus muscle of Erhualian and Sutai pigs. J. Anim. Sci. 86:57-63.
PROJECT CONTACT:
Name: Jiang, Z.
Phone: 509-335-8761
Fax: 509-335-1082
Email: jiangz@wsu.edu

 
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ACCESSION NO: 0150147 SUBFILE: CRIS
PROJ NO: WNP00858 AGENCY: CSREES WN.P
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: REVISED
START: 01 NOV 2006 TERM: 31 OCT 2009 FY: 2007
INVESTIGATOR: Fox, L. K.; Hancock, D. D.; Gay, J. M.
PERFORMING INSTITUTION:
Animal Science
Washington State University
Pullman , Washington 99164
INVESTIGATION OF FOOD ANIMAL DISEASE PROBLEMS IN THE STATE OF WASHINGTON.
NON-TECHNICAL SUMMARY: The control animal disease and of zoonotic and food-borne disease agents can be addressed by epidemiological research at the level of the farm. Research that can define the epidemiology of zoonotic pathogens in the livestock industry can lead to HACCP procedures that will reduce their impact on the well being of the public. The program continues to have a significant impact on the on-farm definition and control of animal disease problems. Studies continue to define the on farm epidemiology of zoonotic agents such as E. coli O157:H7, Salmonella spp and Listeria monocytogenes. The program also addresses diseases of importance to livestock in the State on Washington.
OBJECTIVES: 1. To define and resolve important and emerging animal diseases in agricultural production systems. 2. To define and resolve problems of food safety and food quality generated at the farm level by on-farm research in animal agricultural production systems. 3. To define and resolve problems of zoonotic disease in agricultural animals by on-farm research.
APPROACH: The methodology of investigation will vary according to the problem under consideration. In general, it will include the detailed definition of the problem by farm visitation, data recording, and record analysis and the establishment of hypotheses and risk factors for its cause and spread by standard epidemiologic analysis, laboratory analysis, post mortem analysis and response trial analysis. There will be a concentration of effort in the areas of mastitis, and the ecology of zoonotic disease agents - especially Salmonella typhimurium DT 104 and Escherichia coli 0157:H7 and Mycobacterium paratuberculosis; and multi-drug resistant Salmonella sp. Efforts will also be directed at advancement of research in studies of the epidemiology of Neospora caninum. For those diseases where there are specific research thrusts and funded projects study design and analysis is according to the grant proposal. Study herds are identified that have management or other characteristics required for the study. Herds may be selected on demographic characteristics, production characteristics or other parameters for survey studies or in selected cross sectional, case control or cohort studies.
PROGRESS: 2007/01 TO 2007/12
Last reported was completion of the lupine induced beef calf physical abnormality project, (crooked calf disease). Publications appeared in press this year. Studies of antibiotic resistant zoonotic agents continue and a new project (has been initiated) examining the reduction in pathogen transport to surface water and to soils and the reduced risk of herd-herd transmission of pathogens between farms that participate in a community based anaerobic manure digester. On zoonoses, Salmonella Typhimurium circulating in food animal populations and carrying resistance to antimicrobial agents represent a potential human health risk. We evaluated the rise of extended spectrum cephalosporin resistance in a new clone of Salmonella enterica serovar Typhimurium isolated from cattle and humans in the Pacific Northwest. In vitro susceptibility profiles were determined from 115 bovine isolates and 26 human isolates of this clone that were detected from 1999 through 2006. This clone is identified by pulsed-field gel electrophoresis (PFGE) patterns, (WA TYP035/ TYP187), which differ by only a single band. Multiple-locus variable number of tandem repeat analysis (MLVA) was used to discriminate among these isolates. Using ceftazidime as a screening drug for extended spectrum cephalosporin resistance, resistance increased in bovine isolates from zero in 1999 to 73% (27/37) in 2006. In 2006 62% (5/8) human isolates were resistant to ceftazidime. Dendrogram analysis of the MLVA data suggests that extended spectrum cephalosporin resistance (presumably acquisition of blaCMY-2 bearing plasmids) occurred on multiple independent occasions during the expansion of this TYP035/187 clone. This PFGE type may be largely restricted to Washington and the local antimicrobial selection pressure likely in cattle resulted in the development of increased ceftazidime resistance in this clone. A unique aspect of the anaerobic digester (AD) project is that we are measuring water quality and pathogen levels in soils and livestock of individual farms before and after the adoption of the community AD. A community AD of a continuous flow design with liquid-solid separation and solids composting will be the central technology to achieve pathogen reduction and will be tested on 7 dairies, 3000 cows. Post-digestion manure liquids and solids will be returned to participating dairies for use as a nutrient source for crop growth. Weekly sampling of manure entering the community AD, and at each point of handling post AD (liquid, solid, and compost) will be used to demonstrate the reduction in pathogens or pathogen surrogates of Salmonella spp., enterococci, generic E. coli, Mycobacterium avium subsp. paratuberculosis (Map or Johnes), Listeria spp., Campylobacter spp., and enterovirus. Microbial source tracking technology will be applied to the generic E. coli isolates to determine origins.
IMPACT: 2007/01 TO 2007/12
Identification of farm agents that can cause human disease potentially has significant impact on human health. This impact is even more significant when the pathogens develop resistance to antibiotics. It is imperative then to study the epidemiology of these agents to determine when, and perhaps, how, they develop resistance. With this knowledge intervention strategies can be developed to control resistance. It is expected that two major outcomes from the AD project utilizing a community based manure anaerobic digester will be: 1) determination of the level of reduction in transport of pathogens to surface water from land applied manure that has been AD treated, and 2) demonstration of the reduction in pathogens that are of significance in inter-herd transmission, therefore reducing the risk (producer concern) of between-farm movement of pathogens. We expect that this project will demonstrate that community based anaerobic digesters can have the primary water quality advantage of improved surface water quality via reduced transport of nutrients and pathogens to surface water. In addition, it is expected that information gained from the individual herds will demonstrate herd-herd transmission of pathogen is of limited risk, therefore eliminating any adoption barriers.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
1. Panter, K.E., Wierenga, T.L., and Pfister, J.A., eds. Poisonous Plants: Global Research and Solutions. CABI Publishing, 2007. ISBN 9781845932732:
2. Panter, K.E., James, L.F., Wierenga, T.L., Gay, C.C., Motteram, E.S., Lee, S.T., Gardner, D.R., Pfister, J.A., Ralphs, M.H., and Stegelmeier, B.L. 2007. Research on lupine-induced 'crooked calf disease' at the poisonous plant research laboratory: past, present and future. Poisonous Plants: Global Research and Solutions, 58-65.
3. Gay, C.C., Tibary, A., Panter, K.E., Mealey, K.L., Gay, J.M., Hjartarson, S.W., Motteram, E., Wierenga, T., and James, L.F. 2007. Lupine-induced crooked calf diseases: plasma disposition of lupine alkaloids following repeated challenge in cattle that have given birth to affected calves or those that have borne normal calves. Poisonous Plants: Global Research and Solutions, 151-155.
4. Gay, C.C., Panter, K.E., Motteram, E., Gay, J.M., Hantz, H., Wierenga, T., and Platt, T. 2007. Risk factors for lupine-induced crooked calf disorder in East-central Washington State. Poisonous Plants: Global Research and Solutions, 156-164.
5. Ralphs, M.H., Panter, K.E., Gay, C., Motteram, E., and Lee, S.T. 2007. Cattle grazing velvet lupine (Lupinus leucophyllus): influence of associated forages, alkaloid levels and population cycles. Poisonous Plants: Global Research and Solutions, 401-406.
6. Pfister, J.A., Lee, S.T., Panter, K.E., Motteram, E.S., and Gay, C.C. 2007. Effects of experience and lactation on lupine consumption by cattle. Poisonous Plants: Global Research and Solutions, 407-410.
7 . Gay, C.C., Panter, K.E., Motteram, E.S., Gay, J.M., Wierenga, T., and Platt, T. 2007. Effect of shade on alkaloid content of Lupinus leucophyllus. Poisonous Plants: Global Research and Solutions, 411-413.
8. Gay, C.C., Motteram, E.S., Panter, K.E., and Wierenga, T. 2007. Year-to-year variation in alkaloid concentration in Lupinus leucophyllus growing on the scablands of Central Washington. Poisonous Plants: Global Research and Solutions, 414-419.
9. Lee, S.T., Cook, D., Panter, K.E., Gardner, D.R., Ralphs, M.H., Motteram, E.S., Pfister, J.A., and Gay, C.C. Lupine Induced "Crooked Calf Disease" in Washington and Oregon: Identification of the Alkaloid Profiles in Lupinus sulfureus,Lupinus leucophyllus, and Lupinus sericeus. J Agric Food Chem. 2007 Dec 26;55(26):10649-55. Epub 2007 Nov 27.
10. Adhikari, B., Hancock, D.D., Besser, T.E., Gay, J.M., Fox, L.K., and Davis, M.A. Emerging third-generation cephalosporin resistance in a new clone of SalmonellaTyphimurium in Northwest cattle and humans. Proceedings of the 88th Annual Meeting of Conference of Research Workers in Animal Diseases (CRWAD), Chicago, 2007, edited by Robert P. Ellis, (2007).
PROJECT CONTACT:
Name: Fox, L. K.
Phone: 509-335-0786
Fax: 509-335-1082
Email: fox@wsu.edu
URL: http://www.vetmed.wsu.edu/depts-vcs/fdiu/

 
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ACCESSION NO: 0191142 SUBFILE: CRIS
PROJ NO: WNV-2001-2282 AGENCY: CSREES WN.V
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2002-35204-11688 PROPOSAL NO: 2001-02282
START: 15 DEC 2001 TERM: 30 DEC 2003 FY: 2004 GRANT YR: 2002
GRANT AMT: $117,000
INVESTIGATOR: Barrington, G. M.; Davis, W. C.; Eriks, I. S.; Gay, J. M.
PERFORMING INSTITUTION:
Animal Health Research Center
Washington State University
Pullman , Washington 99164
CD4+ T-CELL IFN-Y EXPRESSION IN BOVINES INFECTED WITH M. PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY: Paratuberculosis (Johne's disease) is a chronic, debilitating, gastrointestinal disease of cattle, worldwide. Strategies for control will ultimately be based on a thorough understanding of the biology and epidemiology of the disease. Currently, the single largest impediment to improving our knowledge of this disease lies in the absence of a sensitive diagnostic test that identifies infected animals during the early phases of disease (Stage 1 or the 'silent infection' stage). The primary goal of this project is to develop a novel diagnostic test to identify animals infected with Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis, during the early stages of disease. To accomplish this goal, we will develop a test to measure substances produced by specific cells of the immune system in response to infection. Once validated in known-infected animals, the test will be used to identify the earliest time in which infection can be identified in calves infected with Map, from birth until 15 months of age. The test will also help define changes in the immune system of infected animals during progression of disease. This diagnostic test will provide a critical tool to accelerate investigations of many aspects of paratuberculosis, eventually leading to development of vaccines and other preventative strategies that will reduce the prevalence of this infection in cattle. Such knowledge will have a substantial positive benefit for U.S. and international animal industries and animal health.
OBJECTIVES: The objective of this project is to develop a flow cytometric-based assay capable of identifying cattle infected with Mycobacterium avium subsp. paratuberculosis (Map), the causative agent of paratuberculosis, during the earliest stages of disease.
APPROACH: The most promising early-stage diagnostic approach to paratuberculosis
involves recognition of the cell-mediated immune response as it is the first detectable host response to Mycobacteria infections. To date, the most proficient technology to evaluate such a response is multiparameter flow cytometry. In this proposal, we hypothesize that a flow cytometry assay will demonstrate intracellular expression of interferon (IFN) gamma in activated CD4+ T lymphocytes of animals silently infected by Map. Two specific aims are proposed to examine this hypothesis. Specific aim 1: Demonstrate the expression of IFN-gamma in activated CD4+ T lymphocytes of known Map-infected animals using multiparameter flow cytometry. This aim will validate the flow cytometric assay in animals known to be infected with Map. Study animals will be chosen based on their infection status as determined by enzyme linked immunosorbent assay (ELISA), fecal and milk polymerase chain reaction (PCR), and fecal culture results. Samples from non-infected control animals will be obtained from a separate paratuberculosis-free dairy (no paratuberculosis diagnosed for 30+ years). Three groups of cattle will be tested including 5 subclinically infected animals, 5 clinically infected animals and 5 non-infected control animals. A variety of antigens will be used to stimulate cytokine production by memory T-cells including preparations of whole Map organisms, Johnin purified protein derivative (PPD), and a non-specific T-cell mitogen. Flow cytometry will be used to identify T-cell subsets responding to Mycobacteria antigens by recognizing which subsets are producing cytokines. Specific aim 2: Identify the age at which IFN-gamma expression by CD4+ T lymphocytes can be demonstrated in calves experimentally infected with Map. A total of 8 male, castrated calves will be used for this study. The calves will be separated into 2 groups of 5 infected and 3 control calves. Calves will be infected multiple times during the first week of life to assure infection and to mimic exposure in an endemic herd. Infected calves will be obtained from a dairy with endemic paratuberculosis mentioned in aim 1. Control calves will be obtained from the paratuberculosis-free dairy previously described. Sampling of calves will occur bi-monthly until the calves are 15 months of age. This age is the approximate time that heifers are bred and is therefore a critical juncture for management decisions in the dairy industry. Sampling during this period will identify the earliest age at which a cell mediated immune (CMI) response occurs and will facilitate characterizing the dynamics of the CMI response over time. It is possible that calves will develop a CMI response at different ages and some calves may even develop clinical disease within the 15-month period. We believe that data obtained from these calves will be critical for beginning to elucidate the heterogeneity of responses to Map infection. Future studies will be directed at utilizing this assay in calves naturally infected with Map.
PROGRESS: 2002/01 TO 2002/12
Newborn Holstein calves were obtained for experimental infection with M. paratuberculosis (Map). The day after birth, 5 calves were orally infected with 1x107 Map organisms for 7 consecutive days. Three calves have been serving as non-infected controls. Fecal, peripheral blood leukocytes and serum have been serially obtained from all calves on a bi-monthly to monthly basis for approximately the last 7 months. Fecal cultures have been negative from infected calves thus far. The validation of our assay to measure intracellular IFN-gamma in cells isolated from cattle infected with Map is still in progress. Studies are ongoing to characterize leukocyte subsets from the control and Map-infected calves. Thus far, no major differences in leukocyte populations from Map-infected and control calves have been identified. Work is also ongoing to identify cell surface molecule expression in leukocyte populations obtained from Map-infected calves. Of great interest, we have observed differences in memory T-cell surface expression of CD25 (interleukin-2 receptor-alpha, IL2R-alpha) and CD26 (activation molecule 3, ACT-3) between Map-infected and control calves, whereby expression of these molecules is up-regulated in the infected calves.
IMPACT: 2002/01 TO 2002/12
Early and accurate detection of cattle infected with M. paratuberculosis is essential for investigating the immunology, pathobiology, and epidemiology of paratuberculosis. A more thorough understanding of these areas will be critical for implementing successful and appropriate control and prevention strategies.
PUBLICATIONS (not previously reported): 2002/01 TO 2002/12
Barrington GM, Gay JM, Eriks IS, Davis WC, Evermann JF, Emerson C, O?Rourke JL, Hamilton MJ, Bradway DS. 2003. Temporal patterns of diagnostic results in serial samples from cattle with advanced paratuberculosis infections. J Vet Diag Invest
(in press)
PROJECT CONTACT:
Name: Barrington, G. M.
Phone: 509-335-0703
Fax: 509-335-0880
Email: geob@vetmed.wsu.edu

 
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ACCESSION NO: 0208084 SUBFILE: CRIS
PROJ NO: WIS01081 AGENCY: CSREES WIS
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 2006 TERM: 30 SEP 2010 FY: 2007
INVESTIGATOR: Landick, R.
PERFORMING INSTITUTION:
Biochemistry
Univ of Wisconsin
Madison , Wisconsin 53706
RECOMBINANT MYCOBACTERIAL RNA POLYMERASES FOR FUNCTIONAL STUDY AND INHIBITOR SCREENING.
NON-TECHNICAL SUMMARY: RNA polymerase is a good target for developing new antibiotics needed to control agricultural pathogens. This project will develop new methods to produce RNA polymerases from agricultural pathogens and to use them to identify new antibiotic candidates.
OBJECTIVES: This project will generate new methods for study of mycobacterial RNAPs by developing methods to produce them in recombinant form using E. coli (mycobacteria are exceptionally hard to grow in the lab, making direct purification difficult). The recombinant mycobacterial RNAPs will be used to characterize the function of different mycobacterial sigma initiation factors involved in the mycobacterial life cycle, to characterize unique structural features of mycobacterial RNAP, to determine the termination mechanism of mycobacterial RNAPs, which is currently unknown, and to develop a novel method to screen for new inhibitors of mycobacterial RNAPs. The work is directly relevant to major bacterial threats to Wisconsin agriculture, M. bovis and M. paratuberculosis.
APPROACH: We will construct overexpression plasmids for mycobacterial RNAPs and produce purified mycobacterial RNAPs for in vitro study. These will include plasmids to produce M. bovis RNAP and M. paratuberculosis RNAP. We will use recombinant RNAPs to study sigma interactions and intrinsic signal specificity.We will also test use of foreign and native sigmas by mycobacterial RNAPs, including E. coli sigma70, T. aquaticus sigmaA, M. bovis sigmaA, sigmaB, and sigmaF. We will construct a deletion mutant to test the function of a sequence insertion (SI5) in mycobacterial RNAP beta prime subunit. We also will identify mycobacterial transcriptional terminators in vitro. Finally, we will use mycobacterial RNAP expression plasmids to screen for lineage-specific inhibitors of mycobacterial RNAP.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: Results of this work were described in a poster presentation at the Molecular Genetics of Bacteria and Phages meeting in Madison, WI in Aug. 2007. PARTICIPANTS: Principal Investigator: Robert Landick Postdoctoral Associate: Agata Czyz Graduate Research Assistant; Kook Sun Ha TARGET AUDIENCES: Microbial and pharmaceutical scientific research communities with the intent of increasing knowledge.
IMPACT: 2007/01 TO 2007/12
During the past project year, mycobacterial RNA polymerase was successfully expressed in active form in the test orgnanism E. coli. This expression was coupled to expression of a reporter gene encoding green fluorescent protein. This combination of RNA polymerase and reproter expression was thus demonstrated to be a viable assay for identification of new inhibitors of mycobacterial RNA polymerase as mead compounds for antibiotic developement.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
No publications reported this period
PROJECT CONTACT:
Name: Landick, R.
Phone: 608-265-8475
Fax: 608-262-9865
Email: landick@bact.wisc.edu


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ACCESSION NO: 0208080 SUBFILE: CRIS
PROJ NO: WIS01093 AGENCY: CSREES WIS
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 OCT 2006 TERM: 30 SEP 2009 FY: 2007
INVESTIGATOR: Talaat, A.
PERFORMING INSTITUTION:
Animal Health & Biomedical Science
Univ of Wisconsin
Madison , Wisconsin 53706
GENOMIC PLASTICITY OF MYCOBACTERIUM PARATUBERCULOSIS CAUSING JOHNE'S DISEASE.
NON-TECHNICAL SUMMARY: Cattle infection with Mycobacterium paratuberculosis causes Johnes disease. Millions of dollars (from the dairy industry) are lost every year because of the Johnes disease especially in Wisconsin. To better understand infection with M. paratuberculosis and to identify sound control measures.
OBJECTIVES: The main goal of this project is to evaluate the impact of genomic plasticity on the virulence of M. paratuberculosis, especially those circulating in human and animal populations.
APPROACH: We will begin our efforts by assessing the overall genomic rearrangements among a large number of M. avium and M. paratuberculosis isolates. The virulence of representative isolates of each clade of mycobacteria will be examined using the mouse model of infection. Moreover, we will delineate the role of M. paratuberculosis-specific GIs in mycobacterial persistence using homologous recombination techniques. The gained knowledge from this project will lead to the design of novel vaccines and diagnostics that are necessary to combat Johnes disease.
PROGRESS: 2007/01 TO 2007/12
OUTPUTS: The main goal of this project is to evaluate the impact of genomic plasticity on the virulence of mycobacterial strains, especially those pathogenic to humans and animals. So far, we used both PCR and sequencing-based strategies to assess the overall genomic plasticity among more than 70 isolates of M. avium (M.av) and M.ap. Isolates were collected from humans, animals and environmental samples. Interestingly, PCR-based approach did not yield significant difference in the virulence gene contents of all isolates. However, sequencing of 6 virulence genes from all isolates revealed several loci of polymorphism indicating significant changes on the nucleotide level. Currently, we are using J774A.1 cell culture model of infection to test the contribution of the discovered polymorphism on the ability of mycobacterial strains to survive in macrophages. More analysis is currently underway to evaluate the evolution of virulence among M. ap isolates collected from different sources. PARTICIPANTS: Project director: Adel M. Talaat, designed the experiments and performed data analysis.Graduate student: Elizabeth Vue, run experiments and helped with the data analysis. TARGET AUDIENCES: Researchers and veterinary practitioners in the field of Johne's disease.
IMPACT: 2007/01 TO 2007/12
The new knowledge base generated throughout this project will improve our chances to address more questions related to the evolution of pathogenic strains of mycobacteria. We will also be able to design better approaches to control Johne's disease.
PUBLICATIONS (not previously reported): 2007/01 TO 2007/12
Wu, C-W, Schmoller, S.K., Shin, S.J. and Talaat,A.M. Defining the stressome of Mycobacterium avium subspecies paratuberculosis in vitro and in naturally infected cows. J. Bacteriol. 2007. 189: 7877-7886.
PROJECT CONTACT:
Name: Talaat, A.
Phone: 608-262-2861
Fax: 608-262-7420
Email: atalaat@wisc.edu

 
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ACCESSION NO: 0209427 SUBFILE: CRIS
PROJ NO: WIS01155 AGENCY: CSREES WIS
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2007-35205-17884 PROPOSAL NO: 2006-04849
START : 15 JAN 2007 TERM: 14 JAN 2009 FY: 2007 GRANT YR: 2007
GRANT AMT: $449,458
INVESTIGATOR: Shook, G. E.; Kirkpatrick, B. W.; Collins, M. T.
PERFORMING INSTITUTION:
Dairy Science
Univ of Wisconsin
Madison , Wisconsin 53706
IDENTIFICATION OF GENETIC MARKERS FOR SUSCEPTIBILITY TO PARATUBERCULOSIS IN DAIRY CATTLE.
NON-TECHNICAL SUMMARY: Our ultimate goal is to develop a genetic approach to reducing paratuberculosis susceptibility in dairy cattle. Paratuberculosis, commonly called Johnes disease, is a chronic infection of the small intestine that occurs in cattle and other ruminants but is most prevalent in dairy cattle. A national study has found that paratuberculosis occurs in more than 40% of US dairy herds. The disease lacks an effective vaccine, is incurable, and causes annual economic losses to dairy producers that total $200 to 250 million in the US dairy industry. Losses accrue from loss of body weight, decreased milk production, and premature removal of diseased animals from herds. A further concern is that the pathogen for paratuberculosis may be a cause of Crohns disease in humans, although the evidence is not conclusive at this time. Thus, the pathogen is potentially a food safety concern. By study of closely spaced DNA markers on all chromosomes, we anticipate finding a number of reliable markers for genes that have large effects on susceptibility to paratuberculosis. The most significant markers will identify short chromosome segments that harbor genes that cause differences in disease susceptibility. We will then examine individual genes in an attempt to identify those that cause susceptibility differences. Disease incidence in dairy cattle could be reduced by selecting bulls for artificial insemination (which is widely practiced in dairy) that carry markers or gene forms associated with low susceptibility.
OBJECTIVES: Our ultimate goal is to develop a genetic approach to reducing paratuberculosis susceptibility in dairy cattle. This goal can be accomplished by selecting sires for artificial insemination that carry DNA markers associated with low disease susceptibility. The aim of this project is to identify those markers and, ultimately, to identify the causative genes. The specific objectives are: 1) Conduct a genome-wide association test to map quantitative trait loci (QTL) associated with paratuberculosis susceptibility. 2) Validate QTL findings from objective 1 in an independent sample of animals. 3) Examine effects on other economically important traits of DNA markers for paratuberculosis QTL. 4) Identify candidate genes within the QTL regions found in objectives 1 and 2; identify single nucleotide polymorphisms (SNPs) within those genes, and test their association with paratuberculosis susceptibility.
APPROACH: OBJECTIVE 1 (genome-wide screening stage): In previous work, we obtained paratuberculosis test results and DNA from 5,611 cows, mainly daughters of 12 project bulls. Samples were collected from 300 herds located throughout the US. A case-control design will be used to map loci associated with disease susceptibility. Depending on cost-effectiveness, we will use a 10K or 25K SNP chip to genotype 172 case cows and 172 matched control cows. Statistical analysis of the 5,611 disease records will produce an age-adjusted probability of infection for each animal considering sire effects, herd effects, and individual animal test results. Case animals will include those with the highest probabilities of infection, and they will tend to come from herds and sire families with higher infection levels. Each diseased cow will be matched with its half-sib born in the same herd that has the lowest probability of infection. SNP allele frequencies will be contrasted between case and control groups. Loci with large case-control differences and exceed the suggestive linkage threshold (nominal P<0.0001) will be examined in the confirming stage. OBJECTIVE 2 (confirming stage): Significant markers will be examined in an independent population of cows to eliminate false positive markers from stage 1 results. This will involve 5,000 cows in 9 herds that participate in an ongoing National Johnes Demonstration Project. Each cow will be sampled one time. These cows will be genotyped for a set of 144 significant SNPs found in stage 1. Again a case-control design will be used to compare allele frequencies between infected and non-infected cows. Case animals will be those with highest probability of infection and will number about 400. Two control cows with low probability of infection will be matched by herd, age, and family relationship with each case cow. Final determination of QTL significance will be based on a joint analysis of data from both stages. OBJECTIVE 3: Markers that are significant for paratuberculosis will be examined for association with other economic traits that are routinely evaluated in the national dairy genetic evaluation system. This analysis will enable an overall economic evaluation of the paratuberculosis QTL. This study will involve 960 Holstein sires in 25 paternal half-sib families for which we have previously obtained DNA. Regression of sons predicted transmitting ability for each trait on number of copies of the allele at each locus will assess the effect of QTL genotype on each trait. OBJECTIVE 4: Polymorphisms in candidate genes will be examined at loci with significant association in the joint analysis of objective 2. Comparative mapping information will be used to identify regions of the human genome corresponding to the location of bovine SNP markers with validated associations. Gene expression information will be examined to identify genes with expression patterns potentially relevant to Johne's disease susceptibility along with information on gene function and association with disease phenotypes in humans. Association of candidate gene polymorphisms with disease susceptibility will be determined.
PROGRESS: 2007/01 TO 2008/01
OUTPUTS: This project is in the early stages; there are no outputs to report at this time. Significant DNA markers identified in objective 1 and confirmed in objective 2 will enable marker assisted selection to reduce susceptibility to Johne's disease in dairy cattle. The artificial breeding industry will use these markers as one among many criteria to select bulls for their semen markets. Producers will use the marker information in two ways: a) to select among AI bulls for matings in their herds and b) to select cows as dams of young bulls for the artificial breeding industry. PARTICIPANTS: INDIVIDUALS: G. E. Shook - project director, recruit cooperating dairy herds, data base management, statistical analysis, overall coordination. M. T. Collins - conducts disease testing and interpretation of test results. B. W. Kirkpatrick - DNA extraction, genotyping, analysis and interpretation of genotyping results. TRAINEES: Two post-doctoral trainees are conducting the work under objectives 1 and 3; one graduate student is conducting the work on objective 2; five undergraduate students are doing the DNA extractions for objective 2. PARTNER ORGANIZATIONS: Johne's Disease Integrated Program - JDIP supported our group for analysis of association of the CARD15 locus with susceptibility to Johne's disease. JDIP is a collaboration among 21 universities, USDA, and international partners that is funded in part by NRI "to promote development and support of projects designed to enhance knowledge, promote education, develop real-world solutions, and mitigate losses associated with Johne's disease". COLLABORATIONS: ** New Zealand - Informal collaboration with Johne's disease project at AgResearch and LIC. ** Israel - Submitted two proposals to the US-Israel Bi-National Agricultural Research and Development fund to conduct joint marker discovery and replication of the discovered markers. Proposals were not funded. ** Dr. Hasan Khatib, Dairy Science Department, University of Wisconsin-Madison, has used the DNA samples from our project to study association of SNPs in candidate genes (STAT1, STAT5A, OPN, PPARGC1A) with production and fertility traits in dairy cattle. This work has resulted in four publications during the past two years. TARGET AUDIENCES: Producers, Veterinarians, Extension Specialists in animal genetics and animal health, Animal Biotechnology Industry, Animal Breeding Industry, Scientific Community PROJECT MODIFICATIONS: Subsequent to submitting our proposal, a new ELISA antibody assay with improved sensitivity (i. e., higher probability of detecting infected animals) was developed in the laboratory of co-investigator Collins. We have re-tested all serum samples with this new assay. The serum samples had been collected under a previous project, and the results of this new diagnostic test will be used in the current project. Subsequently, we repeated a bivariate quantitative analysis of fecal culture and ELISA antibody disease tests to estimate effects of herd, lactation number, sire, and cow. Results from this new analysis were used to select cows for genotyping.
IMPACT: 2007/01 TO 2008/01
** Objective 1: Disease test results and DNA samples were obtained from 5611 cows in 300 herds during a previous project. This resource will be used for the current project. Disease tests included fecal cultures for the Mycobacterium paratubercuolsis pathogen and serum ELISA assays for antibodies against the pathogen. Recently, a new, more sensitive assay (i. e., higher probability of detecting diseased animals) has been developed in our collaborating laboratory. We have re-assayed all of the original serum samples with the new method. Subsequently, we performed a bivariate quantitative analysis of fecal culture and ELISA antibody disease tests to estimate effects of sire, herd, and lactation number on the test results. The effects were estimated simultaneously to account for the confounding among design factors that is inherent in field data sampling. Using results of the quantitative analysis, a susceptibility index was calculated for each animal that was the sum of the sire effect, herd effect, and individual animal residual term for both disease tests. This effectively adjusted the susceptibility index for lactation number effects that increase with cow age. Animals with the highest susceptibility index (i. e., most highly infected) were chosen for genotyping. The most important term in the index was the individual animal residuals. Among animals with similar residual terms, those from sires and herds with higher disease prevalence were favored for genotyping by this selection process. DNA samples from 250 cows have been submitted for genotyping with a 60K SNP chip. ** Objective 2: Resources consisting of serum ELISA and fecal culture disease tests and DNA samples are being accumulated. Samples will be collected from 3,600 Holstein and 300 Jersey cows in 9 herds over a 15 month period that will end in June 2008. Disease prevalence in these data is 100% at the herd level and 10% among cows.
PUBLICATIONS (not previously reported): 2007/01 TO 2008/01
No publications reported this period
PROJECT CONTACT:
Name: Shook, G. E.
Phone: 608-263-3486
Fax: 608-263-9412
Email: geshook@wisc.edu

 
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ACCESSION NO: 0211031 SUBFILE: CRIS
PROJ NO: WIS01244 AGENCY: CSREES WIS
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2007-35204-18400 PROPOSAL NO: 2007-01577
START: 01 SEP 2007 TERM: 31 AUG 2010 FY: 2007 GRANT YR: 2007
GRANT AMT: $374,119
INVESTIGATOR: Talaat, A.
PERFORMING INSTITUTION:
Pathobiological Sciences
Univ of Wisconsin
Madison , Wisconsin 53706
PERSISTENCE AND VIRULENCE OF MYCOBACTERIUM PARATUBERCULOSIS DURING INFECTION: A FUNCTIONAL GENOMICS APPROACH.
NON-TECHNICAL SUMMARY : A) M. paratuberculosis causes sever economic losses to the dairy industry (Johnes disease) B) No current vaccine is effective enough to control Johne's disease. A) Understand how the organisms cause infection and establish themselves in the host B) Identify new targets for vaccine and drug development against Johnes disease
OBJECTIVES: Infection of cattle with Mycobacterium avium ss paratuberculosis (M. ap) causes severe economic losses to the dairy industry in the USA and world-wide. In this phase of the project we will build on findings gained so far to examine key aspects of the persistence and virulence of M. ap. For example, we will examine the impact of genomic rearrangements on M. ap antigenicity and virulence using proteomic, lipidomic and immunoblot analyses. Additionally, we will define the M. ap genetic responses to key host cells such as macrophage and intestinal epithelial cells using DNA microarrays. Finally, we will take advantage of our library of insertional mutants to examine the role of in vivo-expressed genes in mycobacterial virulence using the murine model of paratuberculosis.
APPROACH: In this phase of the project, we will harness the power of comparative genomics and transcriptome, cellular biology and classical genetics to answer key questions and investigate important stages of M. ap infection. We already have generated a strong base of experimental data and assembled an impressive team of investigators with diverse expertise to: I) Examine the impact of genomic rearrangements on mycobacterial proteome, lipidome as well as mycobacterial antigenicity. II) Identify the mycobacterial response to key host cells implicated in the progression of JD such as macrophages and intestinal epithelial cells. III) Characterize the virulence of mycobacterial strains with deletions in in vivo activated genes or those with variable phylogenomes. The outcomes of the proposed goals will provide novel insights into the pathogenesis of JD on a molecular level. More importantly, our study will also provide new leads for developing novel vaccines and diagnostics desperately needed to control Johnes disease. The goals of this project are strongly supported by the priorities of the Animal Protection and Biosecurity program.
PROJECT CONTACT:
Name: Talaat, A.
Phone: 608-262-2861
Email: atalaat@wisc.edu

 
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ACCESSION NO: 0186799 SUBFILE: CRIS
PROJ NO: WIS04471 AGENCY: CSREES WIS
PROJ TYPE: OTHER GRANTS PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 00-52100-9621 PROPOSAL NO: 2000-04294
START: 15 SEP 2000 TERM: 15 SEP 2005 FY: 2005 GRANT YR: 2000
GRANT AMT: $477,220
INVESTIGATOR: Shook, G. E.; Collins, M. T.; Kirkpatrick, B. W.
PERFORMING INSTITUTION:
Dairy Science
Univ of Wisconsin
Madison , Wisconsin 53706
GENETIC RESISTANCE TO PARATUBERCULOSIS IN DAIRY CATTLE
NON-TECHNICAL SUMMARY: Paratuberculosis, commonly known as Johne's Disease, affects 22 percent of all U. S. dairy herds and nearly 40 percent of herds larger than 300 cows. This incurable disease continues to spread and is estimated to cost the U. S. dairy industry over $200 million annually. This project seeks to discover locations of genes on chromosomes that influence resistance and susceptibility to Johne's Disease. Knowledge of these chromosomal regions will enable the dairy industry to breed cattle that are genetically more resistant to this disease. The project will test a total of 6,000 daughters of 15 prominent U. S. Holstein sires. Diagnostic tests for Johne's Disease will be carried out for these daughters in cooperating commercial herds throughout the U. S. Genotyping will be done for 140 genetic markers spread across all 29 cattle chromosomes [excluding sex chromosomes]. Genetic markers inherited from the sires will be compared between infected and uninfected daughters to locate the genes for susceptibility and resistance. For the 15 sire families involved in this research it will be possible to identify specific sons and daughters that inherited chromosomes with the resistance genes. Breeding for genetic resistance has been a successful strategy for control of many plant diseases; this approach has not been applied in animals. Genetic resistance to infectious disease has been well documented in laboratory animals, poultry, and livestock. This project will be an early practical example of using molecular genetics as an aid to preventing disease in livestock.
OBJECTIVES: The long-term objective of our collaboration is to develop a genetic approach to prevention of paratuberculosis in cattle. The specific objectives for this project are: 1) Establish a resource of DNA samples and paratuberculosis test results for 15 half sib families of Holstein cattle for study of genetic resistance to paratuberculosis; 2) Search for segregating DNA markers throughout the cattle genome that are associated with resistance or susceptibility to paratuberculosis in five sire families. The paratuberculosis test results and DNA samples obtained under objective 1 will later be used to complete a genome wide scan in the remaining ten sire families; 3) Disseminate the results of this research to: a) producers, veterinarians, and dairy extension agents throughout the country, b) sire procurement personnel in the artificial insemination industry, and c) students in our undergraduate, graduate, and veterinary medicine courses.
APPROACH: A genome wide search for DNA markers linked with major genes that influence paratuberculosis resistance or susceptibility will be carried out. This scan will involve 140 heterozygous markers across all 29 autosomal chromosomes. We propose to study a total of 6000 daughters of 15 prominent sires in 400 to 600 commercial dairy herds throughout the country. This project will complete the disease testing for all 15 sire families and genotyping for five sire families. Subsequently, with additional funding, the genome wide search will be completed for the remaining ten sire families. Paratuberculosis will be assessed by the combination of an ELISA test for serum antibody to the pathogen and bacterial culture of the pathogen from fecal samples. In herds with disease prevalence between 5 and 25%, predictive values for both positive and negative test results are greater than 90% for the combined tests. Genotyping will be performed selectively for all daughters that are positive for either of the two diagnostic tests. Advantages for genotyping positive animals include a considerable reduction in the number of animals genotyped and the fact that the diagnostic tests have a low rate of false positive results. Animals with negative test results may have escaped exposure to the disease and the diagnostics have a moderately high rate of false negative results; therefore, these cases would not indicate resistance to disease even though test results are negative. Data analysis will be carried out within sire families: For loci with no effect on disease resistance, affected daughters are expected to have inherited with equal frequency one of the two alleles from their sire. Loci at which the two sire alleles differ in their disease resistance will be revealed by a high frequency of the susceptible allele and low frequency of the resistant allele among affected daughters. We plan a proactive nationwide program of extension activities and website development to make our findings known to producers, veterinarians, the animal genetics industry, and dairy extension agents.
PROGRESS: 2000/09 TO 2005/09
Phase 1 of the project involved collecting blood and fecal samples for disease testing and a second blood sample for DNA extraction. A total of 308 cooperating commercial, pedigreed herds submitted samples. Our final resource population consisted of 4233 daughters of 12 project bulls in a daughter design. The 12 project sires were selected based on large numbers of daughters in production and low genetic relationships among sires. Two disease diagnostic tests for M. paratuberculosis were performed on each cow: a serum antibody ELISA and a fecal culture for the pathogen. A cow was classified as infected if positive to either test. Only daughters from herds with direct evidence of disease were included in these studies. Phase 2 involved quantitative analysis of the disease test results. Predicted apparent prevalence of Johne's disease in bulls' daughter groups ranged from 5.3 to 10.4% and averaged 7.9%. Heritability for disease susceptibility from the combined antibody and fecal culture tests was 10.2%. These results demonstrate that important genetic variation exists among bulls and that selection against disease susceptibility would be effective. Phase 3 involved identification of molecular markers associated with susceptibility to paratuberculosis infection. We have completed a genomewide scan for quantitative trait loci (QTL) with DNA pools in 3 of our 12 half-sib sire families. These families were chosen for their large numbers of daughters (252, 570, and 400) and relatively high predicted future daughter disease prevalence (7.2, 8.0, and 9.0%). The sires were genotyped for a set of 270 microsatellite markers to determine each sire's heterozygosity for each marker. One sire was heterozygous for 176 markers and the other two were heterozygous for 151 markers. Correct sire identification was verified with 8 independent, polymorphic markers for which the sires were heterozygous. Incorrect parentage was found for 15% of the daughters which is similar to the industry average. Parentage verified daughters were genotyped within family by selective DNA pooling. Infected daughters (positive pool) were matched with 2 of their non-infected herdmates (negative pool) in the same lactation to control for herd and age effects. The DNA pools for each sire were genotyped for markers that were heterozygous in the sire. Nine chromosomal regions (BTA 7, 10, 12, 14, 15, 16, 18, 20, and 25) (P < 0.01) were putatively associated with M. paratuberculosis infection in at least one family. Three of these regions (BTA 7, 10, and 12) were significantly associated with infection in 2 families. Interval mapping is in progress to validate significance and refine location of these effects. Also, a linkage disequilibrium analysis is in progress for the remaining nine sire families. For this experiment, DNA pools were formed across families and were genotyped on a commercial10K bovine SNP chip which uses molecular inversion probe technology. Pool genotyping has been completed and data analysis is in progress.
IMPACT: 2000/09 TO 2005/09
The project has assisted more than 300 dairy producers throughout the US to identify the presence or absence of Johne's disease in their herds. This is the first study of its kind in US Holsteins, the predominant US dairy breed. We found that 10% of variation among cows for Johne's disease susceptibility is attributable to genetics. Disease prevalence in the most susceptible sire lines is twice the prevalence in the least susceptible lines. Practically, this knowledge will enable geneticists and producers to select dairy bulls that confer to their offspring decreased susceptibility to Johne's disease. Scientifically, this work will identify chromosomal regions in cattle with genes that have a major effect on Johne's disease resistance/ susceptibility. Chromosomal regions significantly associated with susceptibility to M. paratuberculosis infection with our linkage and linkage disequilibrium analyses are good candidates for future fine-mapping studies. The resource of DNA samples, antibody samples, and disease test results will continue to be exploited for genetic studies of Johne's disease and other diseases.
PUBLICATIONS (not previously reported): 2000/09 TO 2005/09
1. Shook, G. E., M. G. Gonda, B. W. Kirkpatrick, and M. T. Collins. 2004. A search for major genes that cause susceptibility to Johne's disease. Pages 106-111 in Proc. 20th Tech. Conf. Artificial Insemination and Reproduction. Nat'l. Assoc. Anim. Breeders, Columbia, MO.
2. Gonda, M. G., Y. M. Chang, G. E. Shook, M. T. Collins, and B. W. Kirkpatrick. 2005. Effect of Johne's disease status on production, reproduction, and health traits in U. S. Holstein cows. J. Dairy Sci. 88 (Suppl. 1):133. (Abstract).
3. Gonda, M. G., Y. M. Chang, G. E. Shook, M. T. Collins, and B. W. Kirkpatrick. 2005. Genetic variation of Johne's disease susceptibility in U. S. Holstein cattle. J. Dairy Sci. 88 (Suppl. 1):231. (Abstract).
4. Gonda, M.G., G.E. Shook, B.W. Kirkpatrick, and M.T. Collins. 2006. Mapping quantitative trait loci affecting Mycobacterium paratuberculosis infection in cattle. Page 234 in Proceedings Plant and Animal Genome Conference XIV, San Diego, CA. (Abstract).
PROJECT CONTACT:
Name: Shook, G. E.
Phone: 608-263-3486
Fax: 608-263-9412
Email: shook@calshp.cals.wisc.edu

 
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ACCESSION NO: 0198094 SUBFILE: CRIS
PROJ NO: WIS04770 AGENCY: CSREES WIS
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2003 TERM: 30 SEP 2006 FY: 2006
INVESTIGATOR: Czuprynski, C.
PERFORMING INSTITUTION:
School of Veterinary Medicine
Univ of Wisconsin
Madison , Wisconsin 53706
EFFECT OF MACROPHAGE RECEPTORS ON UPTAKE, PHAGOSOMAL FUSION AND INTRACELLULAR FATE OF MYCOBACTERIUM PARATUBERCULOSIS.
NON-TECHNICAL SUMMARY:Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic progressive enteritis of cattle and other ruminant species that causes considerable economic losses to the dairy and beef cattle farmers of Wisconsin and the nation. The overall goal of the proposed research is to investigate how activation of surface receptors on bovine macrophages affects the uptake, compartmentalization and fate of Mycobacterium paratuberculosis.
OBJECTIVES:Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic, progressive enteritis of cattle and other ruminant species. There is no effective treatment; nor is it likely that an economically acceptable treatment will be developed. It has been estimated that up to 34 percent of Wisconsin's dairy herds are infected with M. paratuberculosis. This in turn results in losses due to increased mortality rate, culling and replacement heifer costs, and decreased milk production. One estimate placed the cost of Johne's disease to the U.S. dairy industry at 200 to 250 million dollars annually. Efforts to prevent or control Johne's disease in cattle have been stymied by our limited understanding of the pathogenesis of paratuberculosis. Efforts to improve diagnostic and management techniques are limited by our understanding of how the bacilli enter their preferred niche (i.e. macrophage) and how alterations in this process might influence the course of the infection. The overall goal of the proposed research is to investigate how activation of surface receptors on bovine macrophages affects the uptake, compartmentalization and intracellular fate of M. paratuberculosis. The central hypothesis is, that opsonization of M. paratuberculosis with antibody or complement proteins before entry into bovine macrophages, affects maturation of the resulting phagosome and survival of the bacilli. A second hypothesis is that activation of the P2X7 purinergic receptor enhances apoptotsis in infected macrophages and concomitantly reduces the viability of the bacilli themselves. There are two specific aims: 1) Compare phagosome maturation in bovine macrophages that ingest IgG-opsonized, complement opsonized or unopsonized M. paratuberculosis; and 2) Determine whether macrophage apoptosis and inactivation of ingested bacilli is affected by ATP activation of the P2X7 purinergic receptor on bovine macrophages. The proposed work will provide much needed information about the pathogenesis of paratuberculosis, and how the host response to infection might alter the survival of infected macrophages. Such information could be used to devise new vaccines that elicit an immune response that facilitates ingestion of M. paratuberculosis via a macrophage receptor pathway that enhances anti-mycobacterial activity. Achieving this long-term goal would `promote the general welfare through the improved health and productivity of domestic livestock . which are essential to the Nation's food supply;' . In a more general sense, the proposed work will `promote the general welfare through expanded programs of research and extension to improve animal health.' (Section 1429 Title XIV of Public Law 95-113).
APPROACH: In the proposed work we will determine whether prior opsonization with IgG or complement affects