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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
(27)
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
(28)
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
(29)
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
(30)
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
(31)
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-κB. We examined therefore activation of NF-κB 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-κB 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-κB nuclear translocation. These NF-κB 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-κB.
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 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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 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 activate