USDA and USDA Sponsored Research

 

 

PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 JUN 2005 TERM: 31 May 2008 FY: 2005
INVESTIGATOR: Habtemariam, T.; Yehualaeshet, T.
PERFORMING INSTITUTION: Microbiology, Tuskegee University, Tuskegee, Alabama 36088
SUBJECT: 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 non-culturable 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
PROJECT CONTACT:
     Name: Yehualaeshet, T.  Phone: 334-727-8107.  Fax: 334-724-4277
     Email: teyehual@tuskegee.edu
     URL: http://compepid.tuskegee.edu/

 

PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 01 DEC 1998 TERM: 12 JUN 2002 FY: 2002
INVESTIGATOR: Stanker L H; Ravva S V; Duffy B K
PERFORMING INSTITUTION: Western Regional Research Center. Albany, California 94710
SUBJECT:  Treatment of animal manure to prevent pathogen transmission.
OBJECTIVES:  1) Develop methods to identify Salmonella, Campylobacter, and other bacterial and parasitic pathogens, as they are found in animal waste, and define their survival characteristics; 2) Develop methods to handle and treat animal manure during production in order to preclude transmission of these pathogens to land and/or crops for human food.
APPROACH: A combination of microbiological and biochemical approaches will be employed to characterize the biology of pathogens in animal waste.  Pathogens in manure will be studied by confocal and scanning electron microscopy to determine whether biofilms are present.  New methods for detection and identification of pathogens will be developed. Pathogen reporter strains will be produced.  The survival of pathogens in manure will be studied by microbiology and molecular biology approaches.  New compounds and/or treatment methods for minimizing pathogens will be developed and tested.  This research should be conducted in collaboration with the U.S. Salinity Laboratory in Riverside.  FY99 Prgm Inc.($540,000) for treatment of animal manure to prevent pathogen transmission.  Previously was 5325-42000-023-00D (05/01).
PROGRESS: 1998/12 TO 2002/06.  1. What major problem or issue is being resolved and how are you resolving it?  The increasing number of outbreaks of human disease as a result of microbial contamination of meat, poultry, fruits and vegetables is a major concern to all US citizens.  Manure, irrigation water, and bioaerosols are recognized as potential sources of this contamination.  Basic and applied research to help understand the biology of food-borne pathogens in manure and the environments influenced by manure are required to facilitate development of new strategies for minimizing pathogens in manure, water and air.  We are developing methods for tracking pathogens, identifying mechanisms crucial to pathogen survival in manure, and developing new strategies for minimizing pathogens in manure.  2. How serious is the problem?  Why does it matter?  More than eighty million tons of manure are produced each year in the United States.  Due to the confinement of many food-producing animals, the manure is concentrated in relatively small areas and is difficult to control.  Improperly managed manure can harm the environment and become a serious food safety and public health issue.  Food-borne pathogen outbreaks are very serious.  They are considered by most consumers, food processors, food safety researchers and regulatory agencies to be the most important food safety problem today.  It is estimated that there are 10 million to 100 million cases of food-borne illnesses each year.  In addition, there is concern that the virulence of some pathogens may be increasing (e.g. hemolytic uremic syndrome, Guillain-Barre syndrome, reactive arthritis).  Research that helps to minimize the number and virulence of pathogens in food is important for maintaining confidence in the food supply. 3. How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned?  The project addresses pathogen research components of National Programs 108 on 1) Food Safety and 2) Manure and Byproduct Utilization.  Our program is addressing many of the goals of the National Programs including "the survival, transport and ultimate fate of pathogens from manure and other byproducts" and "the effects of manure management on harmful organisms".  4. What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2001 year: The survival of human pathogenic E.coli O157:H7 marked with rifampin resistance is being monitored in aerated and non-aerated manure lagoon water from a California dairy farm with active manure management practices that include separation of manure solids followed by aeration in lagoons.  Laboratory scale manure water microcosms aerated with miniaturized circulating aerators provided by our MOU partners were used to monitor the survival and proliferation of pathogens.  The populations of E.coli O157:H7 declined rapidly from 105 cells/mL to undetectable levels.  Strain variations were noted.  However, a strain of O157:H7 isolated from a dairy survived longer than the isolates from the culture collection.  These studies on pathogen survival in manure lagoons are critical in designing management strategies and are of much interest to dairymen and the manufacturer of circulating aerators.  B. Other Significant Accomplishments, if any: Detection of Mycobacterium avium paratuberculosis MAP: Real-time quantitative PCR methods to detect MAP the causal agent of Johne's disease at low numbers was developed.  Oligonucleotide primers and probes encoding the amplification of 50 to 65 base pair fragments of the DNA insertion sequence, IS900, unique for MAP were designed and optimized. Methods were developed to detect the presence of amplicons in both isolated DNA and whole cells of MAP.  We anticipate the detection of MAP in environmental samples in less than half-a-day instead of the 2-6 month period by the traditional culture methods.  Pathogen transfer to feed crops: Research examining factors that influence transfer of E. coli O157:H7 and Salmonella enterica Thompson to different cultivars typically grown for use as animal feed that are irrigated and/or fertilized with manure, and if contaminated waste application may be a means of increasing pathogen numbers on crops.  Significant differences in pathogen growth on the roots of the different cultivars was observed, but there was little transfer of pathogens to leaves.  The significance of this work are 1) cultivars may be selected to reduce the potential risk of pathogen re-growth on dairies, 2) evidence of some degree of host specificity for pathogens generally considered to be opportunistic invaders of agroecosystems is provided, and 3) the need to consider host genotype when interpreting work on pathogen: plant interactions is necessary.  C. Significant Accomplishments/Activities that Support Special Target Populations: 5. Describe your major accomplishments over the life of the project, including their predicted or actual impact?  Our group has developed a number of research directions related to understanding the biology of pathogens in food and environments crucial to food production.  These include production of reagents for detection of pathogens, pathogen reporter strains for studies of gene regulation, assays for measuring and tracking pathogens in food, identification of non-toxic anti-microbials, methods for fast and sensitive identification of pathogens, and the role of biofilms in pathogen survival.  It is anticipated that innovative methods for detecting pathogens in manure and other environments will be developed, and new methods for minimizing pathogens in the environment will be produced.  6. What do you expect to accomplish, year by year, over the next 3 years?  Year 1: Develop methods for separation and detection of bacterial pathogens in complex environments related to manure, water and compost.  Year 2: Determine the survival and proliferation of multiple human pathogens in manure and crop environments.  Develop methods for recovering and sampling pathogens in aerosols and fog.  Screen for novel anti- pathogen compounds suitable for use in manure management.  Year 3: Determine the life cycle of human pathogens transported from manure to soils and fruit and vegetable crops.  Determine the survival of pathogens in aerosols and resultant deposition of pathogens on plants.  Develop a prototype strategy for minimizing pathogens in manure and crops treated with manure and compost.  7. What technologies have been transferred and to whom?  When is the technology likely to become available to the end user (industry, farmer other scientist)?  What are the constraints, if known, to the adoption durability of the technology?  7.The project is new and we anticipate developing transferable technologies in the near future.  8. List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below)
1. Ravva, S.V. Presentation to the Environmental Committee of the Almond Board of California on "Environmental factors affecting human pathogen survival and proliferation in manure and almond orchards." 11/16/01. Modesto, CA.
2. Duffy, B.K., Ravva, S.V. Discussed the "Efficacy of manure solids separation in nutrient conservation" to the members of Diamond D, Mellos and Dias Dairies and Structures Plus (a manure solid separation company). Kingsburg, CA.
3. Ravva, S.V. Presentation to the members of the Citrus Board of California on "Foodborne human pathogens in dairy environments." 11/16/01. Modesto, CA.
4. The work on manure solids separation and nutrients in manure has been quoted in "Hoards Dairyman."
5. Duffy, B., Sarreal, C., Stevenson, R., Ravva, S., Stanker, L. Regrowth of pathogenic bacteria in compost teas and risk of transmission to strawberry plants. Proceedings of 2002 International Symposium: Composting and Compost Utilization. 2002. Columbus, OH.
PUBLICATIONS: 1998/12 TO 2002/061.
1. Ravva, S.V., Duffy, B.K., Stanker, L.H., Mandrell, R.E. Foodborne pathogens in dairy environments. 30th United States-Japan Cooperative Program in Natural Resources (UJNR) Protein Resources Panel Meeting. October 15-19, 2001 in Tsukuba, Ibaraki, Japan. p. 64-71.
2. Stanker, L.H., Sheffield, C., Beier, R.C., Andreotti, P., Venkateswaran, K., McBride, M.T., Ravva, S.V., Duffy, B.K., Mandrell, R.E. Bacterial detection using sensitive immunological methods. 30th United States-Japan Cooperative Program in Natural Resources (UJNR) Protein Resources Panel Meeting, October 15-19, 2001 Tsukuba, Ibaraki, Japan. p. 33-40.

 

PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 13 JUN 2002 TERM: 31 MAY 2006 FY: 2005
INVESTIGATOR: Mcgarvey J A; Ravva S V; Hernlem B J
PERFORMING INSTITUTION: Western Regional Research Center, Albany, California 94710
SUBJECT:  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: 2003/10 TO 2004/09  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?  Americans suffer from 76 million cases of food poisoning each year, causing 5,000 deaths, and costing $7 billion in lost productivity.  Most food poisoning is caused by bacteria such as those present at high levels in manure.  These include strains of E. coli, Salmonella, and Campylobacter.  Livestock also suffers from transfer of bacterial pathogens, including Mycobacterium avium paratuberculosis, responsible for Johne's Disease in cattle.  Farm animals and their manure are often present at high density near croplands.  Microbes from manure may be transferred to crops by aerosol, especially in dust-, wind- and fog-prone areas.  In addition, manure is often used as fertilizer on crops for animal feed and fodder, as well as crops intended for human consumption.  This can allow direct contact between manure and food/feed, as well as causing widespread contamination of soil.  Over 80 million tons of animal manure are produced each year in the US.  Proper management of this resource is important to minimize infection of farm animals, contamination of farm facilities and equipment, and transfer to food and feed.  Treatments such as composting of solids can significantly reduce levels of potentially harmful bacteria in manure.  But handling may also create aerosols.  Ideal methods for manure treatment may be relatively expensive and unwieldy, especially for small- to medium- scale commodity farm economics, so a practical approach is necessary.  We are comparing effectiveness and cost of various methods for handling and treatment of manure, specifically to reduce transfer of pathogens from manure to crops.  This research falls under National Program 108, Food Safety.  Specifically this research addresses the following goals in the National Program Action Plan: 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.  1.6.1 Determine how food safety is affected by manure handling and utilization.  2. List the milestones (indicators of progress) from your Project Plan.  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.  3. Milestones: The following milestones were scheduled for FY04 and were fully met: Culture methods for M. a. paratuberculosis.  Factors influencing pathogen regrowth on plants.  The following two milestones are planned for FY05: Aeration treatment of manure lagoons.  A few commercial-scale lagoons have already been studied for population dynamics of E. coli, Salmonella, and Campylobacter with various rates of aeration.  We will study M. a. paratuberculosis in this setting, and also identify microorganisms that reduce odor.  Electroflotation treatment of manure lagoons.  This technology is being added from a food processing water project.  We will determine whether electroflotation is a practical treatment for lowering pathogen levels of M a. paratuberculosis, E. coli O157:H7 and Salmonella pathogens in manure lagoons.  This project will undergo OSQR with new Project Objectives beginning FY06.  Year 5 (FY06) Role of bacteriophage in pathogen survival in manure lagoons.  We will determine whether bacteriophage activity affects survival and proliferation of E. coli.  Sampling microbes from aerosol and fog.  New efficient high-throughput air sampling devices will be used for capture of M. a. paratuberculosis, E. coli O157:H7 and Salmonella microbes.  Special cultures and assays will study the prevalence of pathogens in air at various distances from sites of intensive manure production and handling.  Year 6 (FY07) Determine whether aerosol transported M. a. paratuberculosis, E. coli O157:H7 and Salmonella survive and proliferate in crop environments, especially the rhizosphere.  Examine seasonal variations of pathogens in manure lagoons and crop rhizosphere.  Evaluate a prototype electroflotation device on a dairy farm.  Monitor surrogates (and pathogens, if any) and nutrient levels.  Evaluate solar powered devices to reduce operating costs.  4.  What were the most significant accomplishments this past year?  Determining biological factors for pathogen survival and proliferation in manure lagoons is critical in developing intervention strategies for on- site pathogen control.  Scientists in the FCR, WRRC, Albany, CA. introduced E. coli O157:H7 into samples of manure lagoon water from Prins Dairy (Oakdale, CA) and monitored survival of the introduced pathogen as well as native protozoa.  We found that protozoan populations proliferated, while pathogens declined to undetectable levels during eight days of incubation.  Thus, encouraging the growth of protozoans may lead to improvement in pathogen control in manure.  B. Other significant accomplishment(s), if any. FCR Scientists studying manure lagoons noticed that those with circulators have a distinct pink coloration and noticeably less odor compared to static lagoons.  They took samples back to the lab and characterized the organisms responsible for the color.  Although previously undescribed, these novel bacteria (Thiocapsa and Achromatium spp.) appear related to species known to consume volatile organic compounds (VOC).  We are now investigating these organisms, their metabolism, and their potential role in reduction of odor and VOC.  C. Significant activities that support special target populations.  While making field observations and gathering samples for analysis, we have frequent conversations with dairy farmers and vendors of farm equipment, answering their questions about technology and feasibility.  We often participate in more formal presentations, such as the following: McGarvey J (2004) "Analysis of dairy waste water, a molecular approach", Presented to Natural Aeration Inc., Innovative Approaches to Nutrient Cycling Field Day in Modesto, CA. McGarvey J (2004) "Bacterial community structure of dairy waste water", Presented to The Ohio State University.  D. Progress Report opportunity to submit additional programmatic information to your Area Office and NPS (optional for all in-house ("D") projects and the projects listed in Appendix A; mandatory for all other subordinate projects).  Growing industry, public, and scientific interest in M. a. paratuberculosis is steering our efforts toward this difficult-to-culture pathogen, especially in dairy agriculture.  5.  Describe the major accomplishments over the life of the project, including their predicted or actual impact.  Field studies and inoculation experiments in laboratory models suggest dairy manure lagoons are not conducive for growth of E. coli O157:H7 and Salmonella enterica pathogens.  S. enterica declined faster from aerated manure water, while E. coli O157:H7 populations declined similarly from both aerated and non-aerated waters.  This indicates that regrowth of these organisms may be the major factor in pathogen transfer.  Action Plan components 1.2.1.1, 1.6.1.1, and 1.6.1.2.  Pathogens grow rapidly during the traditional methods of making compost teas supplemented with molasses.  Essential oils and natural products inhibit pathogen re-growth in compost teas.  Action Plan component 1.6.1.4.  Developed new immunochemical and molecular methods for sensitive detection of M a. paratuberculosis.  This technology should also prove useful for concentration of M. a. paratuberculosis in dilute environmental samples, making detection and quantitation faster and cheaper.  Action Plan component 1.1.1.2.  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?  While we are working with several vendors of manure treatment (solids separation and aeration) equipment, we have not transferred any new science/technology.  We are evaluating and refining existing commercialized technologies for their effects on pathogen populations.  Farmers naturally focus on optimization of production of their commodity, rather than on manure.  Furthermore, manure management practices that provide ideal levels of pathogen reduction are relatively expensive and impractical.  We are studying implementations that vary in size and complexity, in order to be able to provide cost-benefit information to stakeholders.  These results will be published and presented in FY05. 7.  List your most important publications in the popular press and presentations to organizations and articles written about your work. Stanker LH, Silva CJ, Onisko BC, McGarvey JA, and Ravva SV 2003 New challenges in food safety: Prions and risk factors associated with prion disease and animal manure as a pathogen, Annual meeting of the United States-Japan Cooperative Program in Natural Resources Panel on Toxic Microorganisms.
PUBLICATIONS: 2003/10 TO 2004/091.
1. Duffy, B., Sarreal, C., Stevenson, R., Ravva, S., Stanker. L. Regrowth of pathogenic bacteria in compost teas and risk of transmission to strawberry plants. F.C. Michel, Jr., R.F. Rynk and H.A.J. Hoitink,editors. The JG Press, Inc., Emmaus, PA. Proceedings of 2002 International Symposium: Composting and Compost Utilization, 2002. p. 1142-1149. Available from http://www.composting.2002.org
2. Stanker, L.H., Ravva, S.V. Survival of E.coli O157:H7 in aerated dairy manure lagoons. In Proceedings of the 31st United States-Japan Cooperative Program in Natural Resources (UJNR) Protein Resources Panel Meeting. December 1-7, 2002. Monterey, CA.
3. Mcgarvey, J.A., Bermudez, L.E. 2004. Differential gene expression in mononuclear phagocytes infected with pathogenic and non-pathogenic mycobacteria. [No Journal name given] 136(6):490-500.

 

PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: NEW
START: 07 MAR 2005 TERM: 31 DEC 2006 FY: 2005
INVESTIGATOR: Mcgarvey J A; Mitloehner F; Zhang R
PERFORMING INSTITUTION:  University of California. Davis, California 95616
SUBJECT:  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.

 

PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 OCT 2001 TERM: 30 SEP 2006 FY: 2005
INVESTIGATOR: Cullor, J. S.
PERFORMING INSTITUTION: Population Health & Reproduction, University of California (Vet-Med) Davis, California 95616.
SUBJECT:  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: 2005/01 TO 2005/12  The primary objective of this project is to determine the limit of detection of a wash culture plate assay coupled with real time PCR on bovine manure spiked with viable cells of Mycobacterium paratuberculosis (MAP) (the Johne's agent).  To date varying concentrations of a laboratory MAP broth culture have been spiked into bovine manure and then concentrated using a modified version of the conventional culture NADC method (Stabel, 1997).  The spiked samples have been spirally plated onto agar plates.  After a plate incubation time of 3 days the surface of the plates have been washed and concentrated into a pellet by centrifugation.  The DNA from the resulting pellet has been extracted and subjected to real time MAP PCR.  Thus far, the limit of detection of this assay is ~10 CFU/g after only 3 days incubation, which compares very favorable to conventional culture assays that may take many months to detect about 100 CFU/g. IMPACT: 2005/01 TO 2005/12.  The preliminary results may indicate that screening culture plates via PCR can substantially increase both the sensitivity of the detection as well as the turn around time for results compared to conventional culture.  This may help facilitate whole herd testing which is important in efforts to control the spread of the disease on the farm.
PUBLICATIONS: 2005/01 TO 2005/12
No publications reported this period.
PROJECT CONTACT:
     Name: Cullor, J. S.  Phone: 559-688-1731.  Fax: 559-686-4231

     Email
: mailto:jscullor@ucdavis.edu

     
URL: http://www.vmtrc.ucdavis.edu/dfsl/dfsl.html

 

PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 98-35204-6535 PROPOSAL NO: 9802523
START: 01 DEC 1998 TERM: 30 NOV 2002 FY: 2003 GRANT YR: 1998
GRANT AMT: $149,906
INVESTIGATOR: Gardner, I. A.
PERFORMING INSTITUTION:  Medicine and Epidemiology.  University of California (Vet-Med).  Davis, California 95616
SUBJECT:  Test dependence affects diagnosis and surveillance of animal disease.
OBJECTIVES: 1. To determine the effects of conditional dependence of test sensitivity when combinations of tests are used for individual and herd diagnosis.  2. To estimate sensitivity and specificity of two or more tests in combination when there are more than two populations and no Gold standard.
APPROACH: To achieve Objective #1, we will use existing data on tests for bovine paratuberculosis, swine brucellosis, swine toxoplasmosis and beta-lactan residues in low milk.  As a model for other diseases, we will determine the optimal combination of tests to determine an individual's and herd's paratuberculosis status, incorporating decision and cost-benefit analyses, where ever appropriate.  For Objective #2, we will evaluate data sets that include results for multiple tests for paratuberculosis and other animal diseases and use the estimation-maximization algorithm and Gibbs sampler to perform calculations.
PROGRESS: 1998/12 TO 2002/11.  We have implemented a Bayesian method for estimation of sensitivity and specificity when there are 2 correlated (dependent) tests and 2 populations with different prevalences and the true disease status is unknown (i.e. there is no gold standard).  All uncertainty in the accuracy of tests and disease prevalence is modeled through the use of probability distributions.  Our previous work identified that a positive correlation between test results lead to an overestimation of the accuracy of both tests using traditional statistical methods that assume that the tests are conditionally independent (uncorrelated).  Correlation typically occurs when the 2 tests measure similar biologic processes e.g. serum antibody responses.  The model has been applied to the evaluation of 2 serologic tests for toxoplasmosis in pigs and we have shown that the model yields unbiased estimates of test sensitivity and specificity.  We have developed a web-based interface using an HTML form for implementation of methods to estimate test sensitivity and specificity in the absence of a gold-standard.  The programs can incorporate data obtained from several populations, results of multiple tests and can account for data from a reference population in which the true status (infected or not infected) or each individual is known exactly.  Two estimation methods are used and both assume test independence conditional on the infection status of individuals and constant test accuracy in each population.  The program is available at www.epi.ucdavis.edu/diagnostictests/
IMPACT: 1998/12 TO 2002/11  The development of a web-based program will allow for more widespread implementation of these methods by scientists involved in the assessment of accuracy of animal diagnostic tests.
PUBLICATIONS: 1998/12 TO 2002/11
1. Gardner IA, Stryhn H, Lind P, Collins MT. Conditional dependence affects the diagnosis and surveillance of animal diseases. Prev Vet Med 2000; 45: 107-122.
2. Hanson TE, Johnson WO, Gardner IA. Log-linear and logistic modeling of dependence among diagnostic tests. Prev Vet Med 2000; 45: 123-137.
3. Enoe C, Georgiadis MP, Johnson WO. Estimation of the sensitivity and specificity of diagnostic tests and disease prevalence when the true disease state is unknown.  Prev Vet Med 2000; 45: 61-81.
4. Pouillot R, Gerbier G, Gardner IA. "TAGS", a program for the evaluation of test accuracy in the absence of a gold standard. Prev Vet Med 2002; 53(1-2):67-81.
5. Hanson TE, Johnson WO, Gardner IA. Hierarchical models for the estimation of disease prevalence and the sensitivity and specificity of dependent tests in the absence of a gold standard. J Agric Biol Environ Stat 2002 (in press).
6. Georgiadis MP, Johnson WO, Singh R, Gardner IA. Correlation-adjusted estimation of sensitivity and specificity of two diagnostic tests. Appl Stat 2002 (in press).

 

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 & Epidemology. University of California (Vet-Med).  Davis, California 95616
SUBJECT:  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 Johne’s Disease Herd Status Program relative to herd certification for paratuberculosis.
PUBLICATIONS: 2001/10 TO 2003/101.
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

 

PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 15 APR 2005 TERM: 14 APR 2009 FY: 2005
INVESTIGATOR: Gardner, I. A.; Adaska, J. M.; Whitlock, R. H.
PERFORMING INSTITUTION:  Medicine & Epidemology.  University of California (Vet-Med).  Davis California   
SUBJECT:  Environmental sampling to assess the bio-burden of California dairies.
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: 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: 2005/01 TO 2005/12.  The specific objectives of the study are to 1) quantify concentrations of Map, as measured by culture on Herrolds egg yolk medium (HEYM) and in liquid culture (Trek ESP II), in environmental samples including recycled lagoon water used for flushing cow alleyways, 2) collect environmental Map data biannually with the goal of correlating changes in current values with changes in cohort specific Map risk when females born in the herd at the time of sampling are in the second or later lactation, and 3) quantify the Map colony counts on HEYM tubes where results are recorded as too numerous to count (TNTC), equivalent to approximately more than 70 colonies per tube.  We started our investigation in a dairy with about 2700 lactating cows (aggregated in 12 strings), 300 dry cows and about 150 pregnant heifers.  Currently, fecal culture prevalence and ELISA seroprevalence of cows (n=976) at dry-off are 9.4% and 4.6%, respectively.  Testing of fecal slurry samples in February 2005 collected from alleyways using a standardized protocol showed that 100% (12/12) were positive on HEYM, including 25% (3/12) alleyways had at least one HEYM tube that had Map colonies that were TNTC.  Repeat testing of the herd in October 2005 using the Trek ESP II system yielded similar findings.  In November 2005, we investigated logistical aspects of detection of TNTC cows in this herd.  All environmental samples were positive by quantitative real-time (qrt) PCR and one sample was positive using the qualitative PCR available through the Minnesota Diagnostic Laboratory.  We are currently doing follow-up testing of 3 strings identified as having different likelihoods (high, moderate and low, respectively) of having cows that are shed 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.  
IMPACT: 2005/01 TO 2005/12.  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.
PUBLICATIONS: 2005/01 TO 2005/12.
Berghaus RD, Farver TB, Anderson RJ, Jaravata CC, Gardner IA.  Environmental sampling for detection of Mycobacterium avium ssp. paratuberculosis on 23 large California dairies. J Dairy Sci 2006; 89: 963-970.
PROJECT CONTACT:     
     Name
: Gardner, I. A.  Phone: 530-752-6992.  Fax: 530-752-0414
     Email
: iagardner@ucdavis.edu

 

PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 2003-35204-13374 PROPOSAL NO: 2003-02398
START: 01 AUG 2003 TERM: 31 JUL 2006 FY: 2005 GRANT YR: 2003
GRANT AMT: $157,000
INVESTIGATOR: Gardner, I. A.; Johnson, W. O.
PERFORMING INSTITUTION:  Medicine & Epidemology.  University of California (Vet-Med)  Davis, California 95616
SUBJECT:  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: 2005/01 TO 2005/12.  Our long-term 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.  As part of our first objective, we developed and applied 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.  We considered 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 bovine paratuberculosis and a second for Salmonella in pig herds, were used to demonstrate application of the methods in field studies (Su et al., 2006).  As part of our second objective, we developed a Bayesian approach to sample size calculations for cross-sectional studies designed to estimate sensitivity and specificity of one or more diagnostic tests.  Sample size calculations can be made for common study designs such as one test in one population, two conditionally independent or dependent tests in at least 2 populations, and three tests at least 2 populations.  We determined a sample size combination that yields high predictive probability, with respect to the future study data, of accurate and precise estimates of sensitivity and specificity.  We also consider hypothesis testing for demonstrating the superiority or equivalence of one diagnostic test relative to another.  The predictive probability can also be computed when the sample size combination is fixed in advance, thereby providing a power-like measure for the future study.  The method is straightforward to implement using the S-Plus/R library emBedBUGS together with WinBUGS (Branscum et al., 2006).
IMPACT: 2005/01 TO 2005/12.  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.  Our research findings allow users greater flexibility in study planning and data analysis than are sometimes available with traditional statistical approaches.  Wherever possible, 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/
PUBLICATIONS: 2005/01 TO 2005/12
1. Branscum AJ, Johnson WO, Gardner IA. Sample size calculations for studies designed to evaluate diagnostic test accuracy. J Agric Biol Environ Stat 2006 (in press).
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 (in press)
3. Su, CL, Gardner IA, Johnson WO. Bayesian estimation of aggregate test accuracy based on different sampling schemes. J Agric Biol Environ Stat 2006 (tentatively accepted)
PROJECT CONTACT:     
     Name: Gardner, I. A.  Phone: 530-752-1363.  Fax: 530-752-0414
     
Email: iagardner@ucdavis.edu

 

PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2000 TERM: 30 SEP 2004 FY: 2003
INVESTIGATOR: Gardner, I. A.
PERFORMING INSTITUTION: Medicine & Epidemology.  University of California (Vet-Med)  Davis, California 95616
SUBJECT:  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: 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

 

PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 APR 2005 TERM: 31 MAY 2007 FY: 2005
INVESTIGATOR: Cullor, J. S.
PERFORMING INSTITUTION:  Population Health and Reproduction.  University of California (Vet-Med)  Davis, California 95616
SUBJECTApplications of high sample throughput PCR diagnostics for animal health, food safety and food security.
NON-TECHNICAL SUMMARY: In order to insure animal health, food safety and security, and supply needed scientific information to develop better management practices and support and enforce regulatory decisions, there is a need for a broad based detection system to rapidly detect potential vectors of animal and foodborne diseases on the dairy farm environment.  This project seeks to develop a rapid and high sample throughput detection system that can be applied to a wide variety of on-farm sample types including milk, cattle feed and water for the control of infectious agents.
OBJECTIVES: The overall goal of this project is to further develop, optimize and characterize the sensitivity, specificity and ease of performance of an automated DNA extraction method coupled with PCR technology on a variety of on-farm sample types, for the identification of infectious agents, or potential vectors of infectious diseases.  These sample types include 1.) mastitic milk, for the direct and rapid detection of various bacterial pathogens including Mycoplama bovis and streptococcus species 2.) cattle feed, for the detection of prohibited ingredients including ruminant DNA to prevent the spread of Mad Cow disease and 3.) agricultural water for the presence of potential waterborne pathogens including E. coli 0157 and Salmonella which can be shed by asymptomatic cattle.
APPROACH: 1.) To evaluate a automated DNA extraction protocol to be used on milk, cattle feed and agriculture water for its ability to abrogate the effects of inhibitory agents and rapidly process numerous samples.  2.) To further develop PCR primers and florescent probes to detect and identify various waterborne infectious agents including E. coli 0157 and Salmonella.  3.) To optimize and characterize each DNA extraction /PCR assay for sample throughput, lower limit of detection, sensitivity, specificity and ease of performance using laboratory spiked samples.
PROGRESS: 2005/01 TO 2005/12.  An automated DNA extraction system based on magnetic bead technology has been purchased, set-up, and its use, including training, offered to other research units within the School of Veterinary Medicine.  The system is capable of extracting up to 32 samples within one hour with minimal hands-on labor required.  The system has also been evaluated in a side by side comparison of a manual DNA extraction method using milk spiked with viable Mycobacterium paratuberculosis cells (MAP).  In brief bulk tank milk samples were spiked with different MAP cell/ml concentrations.  The samples were centrifuged and the DNA from the resulting pellet was extracted by both a manual DNA method, and the automated method.  The resulting DNA was then assayed with a real time MAP PCR assay with the results recorded as either positive or negative for each spiked sample in order to compare the lower limits of detection for each method.  The automated DNA extraction method was able to return essentially the same results as the best used manual DNA extraction method on the samples studied.  These results suggest that the automated DNA extraction system is as capable as the best manual method available in efficiently extracting mycobacterial DNA from milk.
IMPACT: 2005/01 TO 2005/12.  The automated DNA extraction system thus far studied may be utilized for high sample throughput milk quality applications allowing for very sensitive bacterial detection in a labor saving manner.
PUBLICATIONS: 2005/01 TO 2005/12.
No publications reported this period
PROJECT CONTACT:
     Name: Cullor, J. S.  Phone: 559-688-1731.  Fax: 559-686-4231    
     
Email: jscullor@ucdavis.edu

 

PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 APR 2005 TERM: 31 MAY 2010 FY: 2005
INVESTIGATOR: Gardner, I. A.; Adaska, J. M.; Whitlock, R. H.
PERFORMING INSTITUTION:  Medicine and Epidemiology.  University of California (Vet-Med)  Davis, California 95616
SUBJECT:  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: 2005/01 TO 2005/12  The objectives of the study were to evaluate the effectiveness of various testing methods for detection of Map-infected herds at the initial step of herd classification, and 2) to determine the most cost-effective testing strategies for detection of Map-infected herds for various herd demographic factors.  A stochastic simulation model was used to compare the cost-effectiveness of 7 currently-available testing methods for detection of Map-infection in California dairies.  Testing methods were culture of environmental samples (ENV), culture of fecal pools containing 10 individual fecal samples per pool (PFC), culture of individual fecal samples (IFC), a qualitative fecal PCR assay, 2 commercial ELISA assays: ELISA A (IDEXX Laboratories, Inc., Westbrook, ME) and ELISA B (ParaCheck; CSL/Biocor, Omaha, NE), and a combination of an ELISA assay with follow-up fecal culture of seropositive cows (EIFC).  Based on field data relevant to California herd demographics and Map prevalence, 4 herd profiles of 700 cows and 2500 cows with within-herd prevalences of 5% and 30% were simulated.  The 7 different testing methods were applied to detect Map-infected cows in the simulated herds.  In the 700-cow herd with 5% within-herd prevalence, for the same number of samples tested, the testing method that yielded the highest herd sensitivity was ENV followed by PFC, PCR, ELISA A, IFC, ELISA B, and EIFC, respectively.  To achieve > 95% herd sensitivity, 2, 10, 55, 60, 90, 100 samples per herd were required for ENV, PFC, PCR, ELISA A, IFC, and ELISA B, respectively.  Testing of 30 samples using EIFC yielded only 13% herd sensitivity, and required > 180 samples to achieve 95% herd sensitivity.  In the 700-cow herd with 30% within-herd prevalence, ENV and PFC also yielded the highest herd sensitivity.  However, of the methods applied to individual cows, IFC yielded a higher herd sensitivity than PCR followed by ELISA A, ELISA B, and EIFC, respectively.  Testing of 30 samples using EIFC method yielded 82% herd sensitivity, but only 1 sample of ENV provided 100% herd sensitivity in this population.  The effect of herd size on the herd sensitivity was minimal.  The 7 testing methods were compared in terms of their monetary cost per unit of effectiveness (herd sensitivity).  ENV was overall the most cost-effective testing method in the herd with 5% within-herd prevalence followed by PFC, ELISA A, ELISA B, PCR, IFC, and EIFC, respectively.  The ratio of cost per unit of herd sensitivity for the PFC, ELISA A, ELISA B, PCR, and IFC were approximately 2, 4, 7, 16, and 20 times higher than that of ENV for 95% herd sensitivity level.  In high prevalence herd (30%), although ENV and PFC remained the 2 most cost-effective testing methods, ELISA A and ELISA B were indistinguishable, and IFC was more cost-effective than either EIFC or PCR.
IMPACT: 2005/01 TO 2005/12.  Environmental sampling (ENV) was the most cost-effective testing method for detection of Map-infected California dairy herds of unknown Map status.  As few 2 ENV samples of ENV yielded > 95% herd sensitivity when the within-herd prevalence was 5%.  The herd sensitivity for ENV did not decrease substantially when herd size was large.  The CSL ELISA was the preferable testing method when individual infected cows needed to be identified because of its high specificity and low cost.
PUBLICATIONS: 2005/01 TO 2005/12
No publications reported this period
PROJECT CONTACT:
     Name: Gardner, I. A.  Phone: 530-752-6992.  Fax: 530-752-0414
     Email: iagardner@ucdavis.edu

 

PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 27 SEP 2002 TERM: 26 SEP 2003
INVESTIGATOR: Gardner, I. A.
PERFORMING INSTITUTION:  Medicine and Epidemiology.  University of California (Vet-Med)  Davis, California 95616
SUBJECT:  Field evaluation of fecal pool cultures for detection of Mycobacterium paratuberculosis in large dairy herds.
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.  This project will apply new epidemiologic approaches to test evaluation and prevalence estimation for Mycobacterium paratuberculosis in dairy herds using field data.
OBJECTIVES: Pooled fecal culture for Mycobacterium paratuberculosis has potential application to national Johne's disease control programs in 3 ways: 1 - It can be used to define M. paratauberculosis herd status (infected or non-infected) as an alternative to ELISA testing and/or individual fecal culture.  2 - It can be applied as an initial screening tool to identify groups of cows warranting further testing by other methods, e.g. individual fecal culture.  3 - Individual-animal prevalence can be estimated from pooled test results provided that multiple pools are collected per herd.  An additional benefit of this project is that it will provide culture-based estimates of the proportion of M. paratuberculosis infected dairy herds and within-herd prevalence in typical well-managed large California dairies.  Such data have not been available to date.  Estimates of prevalence in California have been based on ELISA data collected as part of the NAHMS program and as part of other disease surveys.
APPROACH: 1. Determine the sensitivity of pooled fecal culture by 2 methods (TREK and traditional HEY culture) relative to individual fecal culture.  2. Estimate the prevalence of infected herds and within-herd prevalence in California by 2 methods - a) Based on pooled fecal culture results, b) Based on individual animal results (ELISA and fecal culture).  3. Determine the sensitivity and specificity of pooled fecal culture and ELISA tests using latent-class analysis.
PROGRESS: 2002/09 TO 2003/09  We evaluated the sensitivity of pooled fecal culture for detection of Mycobacterium avium subsp. paratuberculosis (Map) in large dairy herds and assessed the utility of the method for estimation of Map prevalence.  We used a cross-sectional study design that involved random sampling of 60 cows in each of 29 California dairy herds.  The herds ranged from 285 to 2233 cows and had no or minimal previous testing for Map. Blood and fecal samples were collected from each cow.  Sera were tested using a commercial ELISA test kit.  Fecal samples were individually cultured and used for making fecal pools.  Each fecal pool consisted of 10 randomly-selected individual fecal samples from cows in the same herd for a total of 6 pooled samples per herd. Herrolds egg yolk agar (HEY) and Trek-ESP para-JEM system II (ESP) were used for culture of individual and pooled fecal samples.  Sensitivity of pooled fecal culture was estimated using individual fecal culture results for samples within a pool as the definitive test (gold standard) and specificity of pooled fecal culture was considered perfect.  McNemars test was used to compare sensitivities of Map detection between HEY and ESP culture methods.  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 / 38 pools that were Map positive) which was consistent with a prior experimental study by Wells and Whitlock.  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 (P-value = 0.145) and the mean days-to-detection of ESP was 24 compared with 12-16 weeks by HEY.  ELISA, individual fecal culture and pooled fecal culture results for the 29 herds are shown in Table 1.  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 (Table 2).  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 (Table 2).
IMPACT: 2002/09 TO 2003/09.  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.
PUBLICATIONS: 2002/09 TO 2003/09
Saraya Tavornpanich, Ian A. Gardner, Randall J. Anderson, Sang Shin, Robert H. Whitlock, Terry Fyock, John M. Adaska, Richard L. Walker, Sharon K. Hietala. 2004. Evaluation of pooled fecal culture for detection of Mycobacterium avium
subsp. paratuberculosis in large dairy herds. American Journal of Veterinary Research 2004 (in press)
PROJECT CONTACT:
     Name: Gardner, I. A.  Phone: 530-752-1363.  Fax: 530-752-0414
     Email: iagardner@ucdavis.edu

 

PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JAN 2003 TERM: 31 DEC 2004
INVESTIGATOR: Adaska, J. M.
PERFORMING INSTITUTION: CA Animal Health and Food Safety Laboratory System (CAHFS).  University of California (Vet-Med).  Davis, California 95616
SUBJECT:  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 Johne’s 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 165degreesF 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 Johne’s 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 (165 degrees F 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 Johne’s 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 Johne’s disease in the calves they raise.
PUBLICATIONS: 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

 

PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JAN 2002 TERM: 31 DEC 2002 FY: 2003
INVESTIGATOR: Cullor, J. S.; Smith, W.; Kirk, J.
PERFORMING INSTITUTION:  Population Health & Reproduction.  University of California (Vet-Med).  Davis, California 95616
SUBJECT:  A modified culturing procedure using spiral plating and microscopic screening for early detection of M. paratuberculosis from bovine manure.
NON-TECHNICAL SUMMARY: The detection of M. paratuberculosis (Johne's disease) in young cattle continues to be difficult using current tests.  There is a lack of good screening assays that are fast, inexpensive and sensitive enough to regularly and frequently screen cattle.  This project seeks to improve Johne's disease screening assays by modifying the conventional media culturing method in ways to shorten the time required for results, while maintaining low costs, ease of performance and the sensitivity/specificity which is characteristic of conventional culturing methods.
OBJECTIVES: Conventional media culturing for M. paratuberculosis involves decontaminating the sample with various agents and antibiotics followed by subculturing onto solid agar slants which are incubated for many weeks to months before visible colonies appear.  This project examines the hypothesis that the culturing step can be modified by the substitution of spiral plating on Middlebrook agar and the addition of micro-colonial morphology screening to obtain primary culture results more rapidly.
APPROACH: The first objective is to assess the ability of the spiral plating micro-colony morphology screening method (SP/MCM) to return results more rapidly than the conventional culture method using 'spiked' manure samples.  Once this is confirmed the second objective is to assess the SP/MCM method on actual field samples from herds with high incidence of Johne's disease.
PROGRESS: 2002/01 TO 2002/12.  Previous results funded by other grants, suggested that a modification in the way that manure samples were plated and screened could provide a more rapid detection of Mycobacterium paratuberculosis, however these results were obtained from manure samples containing very high numbers of the organism.  In order to compare the modified culture technique to conventional culture methods, additional studies were necessary to optimize the sensitivity (lower limit of detection) of the assay.  Because M. paratuberculosis has an extremely prolonged growth rate, clinical samples are first "decontaminated" with agents that will suppress the growth of other faster growing bacteria.  However these agents can also suppress the growth of mycobacteria to varying degrees.  Several methods to decontaminate the samples prior to plating are being investigated and optimized so that the very low numbers of mycobacteria that may be present in the naturally infected animal can be detected.  Results to date suggest that a commonly used decontamination agent, HPC (hexadecylpyridinium chloride) continues to suppress the growth of mycobacteria after being plated and during incubation, thus hindering the detection of very low numbers in the sample.
IMPACT: 2002/01 TO 2002/12.  Johne's is a contagious bacterial disease of ruminants that has significant animal health, economic, and potential human health consequences.  This grant provides additional funding to investigate and optimize the sensitivity of a more rapid culture method for the detection of the M. paratuberculosis from bovine manure.
PUBLICATIONS: 2002/01 TO 2002/12
No publications reported this period
PROJECT CONTACT:
     Name: Cullor, J. S.  Phone: 559-688-1732.  Fax: 559-688-4231
     
Email: jscullor@ucdavis.edu

 

PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JAN 2002 TERM: 31 DEC 2002 FY: 2003
INVESTIGATOR: Gardner, I. A.; Johnson, W.
PERFORMING INSTITUTION: Medicine & Epidemology.  University of California (Vet-Med).  Davis, California 95616
SUBJECT:  Certification of California cattle herds as free of Johne's disease using Bayesian methods.
NON-TECHNICAL SUMMARY: Johne's Disease (JD) is a chronic infectious bacterial disease caused by Mycobacterium paratuberculosis.  Infections spreads between herds primarily by trade in animals and it is estimated that at least 22f herds are infected.  There is an industry need for science based research on Johne's Disease.  This study will contribute to tests that can be used by industry to set standards for the trade of cattle.
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 pretest (prior) probability of JD.  
APPROACH: This will be accomplished by developing and expanding an existing Bayesian model for certification of Johne's disease.  This will include addressing multiple stages of disease, hypergenometric sampling and use of multiple tests.
PROGRESS: 2002/01 TO 2002/12.  We have developed a Bayesian computer model to allow improved inferences about the infection status of dairy herds with Mycobacterium paratuberaculosis, the causative agent of Johne's disease, based on herd testing.  The model links laboratory test results from a sample of cows or the entire cow herd with test accuracy data and prior information from the herd to provide updated inferences about the probability that the herd is infected and within-herd prevalence.  In the model, uncertainty in all model inputs is modeled as a probability.  The model currently is being validated with serum enzyme-linked immunosorbent assay (ELISA) and fecal culture data from 29 Central Valley dairies.  These field data indicated that more than most herds have Mycobacterium paratuberculosis infection although within-herd prevalences are typically low.  The model should be useful in the Voluntary Johne's Disease Herd Status Program and for other certification programs that are used nationally and internationally, and for decision making by dairymen with regard to Johne's disease control.  In addition, findings from the study indicate that a sample size of sixty lactation-2 or older cows with ELISA appears to provide a reasonable balance between cost of testing and failing to detect low prevalences of M. paratuberculosis infection.
IMPACT: 2002/01 TO 2002/12.  The Bayesian model will allow for improve