USDA FUNDED PROJECTS FROM THE CRIS DATABASE
http://cris.csrees.usda.gov


ACCESSION NO: 0203516 SUBFILE: CRIS
PROJ NO: COLV-2005-06103 AGENCY: CSREES COLV
PROJ TYPE: SPECIAL GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 2005-34405-15758 PROPOSAL NO: 2006-06123
START: 15 AUG 2005 TERM: 14 MAY 2008 FY: 2006 GRANT YR: 2006
GRANT AMT: $755,088

Investigator: Salman, M. D.

Performing Institution:
Clinical Science
Colorado State University
Fort Collins, Colorado 80523

ENHANCEMENT OF THE PROGRAM FOR ECONOMICALLY IMPORTANT INFECTIOUS ANIMAL DISEASES

NON-TECHNICAL SUMMARY: A multidisciplinary research center is needed to study animal diseases of economic importance. Integration of studies covering a broad spectrum of disciplines is needed to prevent duplication of existing efforts and programs. A comprehensive study approach is expected to lead to improved disease surveillance, risk assessment, management, and control/prevention strategies. Furthermore, these studies will lead to the generation of fundamental knowledge concerning disease transmission, diagnosis, pathogenesis, and virulence of
economically important infectious animal diseases.

OBJECTIVES: The objectives are to: 1) Initiate, conduct, and promote research activities on infectious animal diseases that have impacts on trade issues. Following the mission of a land grant university, research in both a basic and applied arena are initiated and conducted with the focus on diseases that have local, regional, or national trade impacts. 2) Use a multidisciplinary, integrated approach to examine each disease studied. Experts from different disciplines, other institutions, governmental agencies, and local and regional laboratories collaborate under the aegis of PEIIAD in order to solve complex problems, thereby minimizing redundancy and promoting the expertise of individuals. By integrating information gathered through these collaborations, the effectiveness of each research project is maximized. 3) Prioritize research topics through the PEIIAD Advisory Group. Representatives from the livestock industry, animal health (including governmental) decision-makers, and researchers from other institutions are enlisted to prioritize critical research. In this way, issues of practical and timely importance, rather than issues of purely academic interest, are being addressed. 4) Disseminate results and information. Research results are available directly to the stakeholders for immediate implementation through the PEIIAD Advisory Group. Information and links related to PEIIAD research are also being made available on the APHI website (www.cvmbs.colostate.edu/aphi). 5) Provide training and graduate programs, including international study programs with a focus on important animal diseases. Industry, international, veterinary, and traditional students from many disciplines receive advanced, short-term or long-term training in a variety of areas through the APHIs position within Colorado State University.

APPROACH: PEIIAD will include three major sections which will be simultaneously integrated into the research approach: biology of infectious diseases, epidemiology of animal diseases, and risk analysis/assessment. An Advisory group will be composed of scientists from all involved disciplines, commodity representatives, state departments of agriculture, and consumer advocates.

PROGRESS: 2005/08 TO 2006/08
The focus of PEIIAD is the advancement of research and outreach activities that are related to economically critical infectious animal diseases in order to prevent the introduction and spread of infectious diseases in US animal populations. Research strategies unite appropriate diagnostic measurements and surveillance systems through an integrated, broad-based approach. Research
findings are synthesized so that an animal disease concern is pursued from its roots in basic science through to policy development. The five priority PEIIAD research areas are: I. Global, emerging infectious animal diseases include 1) Extensive involvement in the current global effort to control the spread of Avian Influenza in poultry through conducting national and international training programs, participating in risk modeling, and advising government agencies regarding control strategies. 2) Research on FMD in wildlife species to address transmission from wildlife to domestic species. 3) Participation in the global and national science-based policy making process for BSE and other TSE diseases in animal populations 4) Participation in global animal health and welfare through engagement with the European Union Animal Health Programs. II. Risk and decision analysis models include creation of a risk analysis model for describing the spread of highly contagious animal diseases. This model is currently being evaluated by Canadian and US animal health authorities for its application in FMD and AI situations. Validation of the model is underway using data collected from the most recent outbreaks of FMD in Uruguay and Exotic New Castle disease in California. III. Endemic animal diseases that impact animal movement, marketing and food safety studies include the development of a bovine tuberculosis (Mycobacterium bovis) assay that is currently used by USDA National Veterinary Services Laboratory and participation in the validation of the new, advanced diagnostic assays for animal diseases such as Vesicular Stomatitis (VSV) and FMD. IV. Biosecurity includes development of a nationally and internationally recognized program for objective assessment of the efficacy and value of biosecurity practices and initiation and continuation of an awareness program in foreign animal diseases for practicing veterinary professionals to train them to be first responders in the event of a disease introduction. V. Antimicrobial drug use and antimicrobial resistance includes development and publication of major "white paper" concerning use of antimicrobial drugs by veterinarians for treatment of disease and the development of both large-scale assessments of antimicrobial drug use patterns for treatment of animal diseases and large-scale investigations concerning association of antimicrobial drug use and antimicrobial resistance in livestock species (especially beef and dairy
cattle).

IMPACT: 2005/08 TO 2006/08
Establish a biosecurity model for the intentional or non-intentional introduction of exotic diseases such as AI to livestock premises & other facilities. Continue the training programs at local, national, & international levels in disease. Investigations, surveillance systems and control strategies for highly contiguous animal diseases. Contribute to the assessment of global surveillance for infectious animal diseases including AI, FMD, bovine TB, and BSE. Validate real time PCR for detection of vesicular stomatitis virus in cattle. Address the critical need for a sensitive & specific rapid screening test for Mycobacterium bovis by continuing serological & molecular studies. Implement the recommendations for establishing an FMD free zone region between Thailand, Myanmar & Malaysia in conjunction with the OIE regional office. This activity is a model of a risk assessment process to establish a disease free zone. Continue assessment of potential FMD disease transmission between domestic animals & wildlife. Continue Johnes Disease research to determine the association between infection status of dairy cows based on postmortem histopathology & culture of multiple tissues and previous results of fecal culture and multiple sera ELISA tests. Complete efforts documenting patterns of antimicrobial drug use in animals by veterinarians in the U.S. Continue research investigating associations among antimicrobial drug use, antimicrobial resistance and effects on animal health & production. Provide an outreach program in foreign animal diseases for practicing veterinary professionals.

PUBLICATIONS (not previously reported): 2005/08 TO 2006/08
1. Branscum AJ, Gardner IA, Wagner BA, McIntuff PS, Salman, MD. Effect of diagnostic testing error on intracluster correlation coefficient estimation. Prev Vet Med 2005 69:63to75.
2. Davidson AH, Traub-Dargatz JL, Rodeheaver RM, Ostlund EM, Pedersen DD, Moorhead RG, Stricklin JB, Young BD, Dewell RD, Salman MD, et al. Immunologic responses to West Nile virus in vaccinated and clinically affected horses. J Am Vet Med Assoc 2005 226:240to5.
3. Dewell GA, Ransom JR, Dewell RD, McCurdy K, Gardner IA, Hill AE, Sofos JN, Belk KE, Smith GC, Salman MD. Prevalence of and Risk Factors for Escherichia coli O157 in Market-Ready Beef Cattle from 12 U.S. Feedlots. Foodborne Pathogens and Disease 2005 2:70to76.
4. Dunowska M, Morley PS, Hyatt DR. The effect of Virkon S fogging on survival of Salmonella enterica and Staphylococcus aureus on surfaces in a veterinary teaching hospital. Vet Micro 2005; 105: 281 to 289.
5. Morley PS, Morris N, Hyatt DR, Van Metre DC. Evaluation of the efficacy of disinfectant footbaths as used in veterinary hospitals. J Am Vet Med Assoc 2005; 226:2053 to 2058.
6. Patterson G, Morley PS, Blehm KD, Lee DE, Dunowska M. Efficacy of directed misting application of a peroxygen disinfectant for environmental decontamination of a veterinary hospital. J Am Vet Med Assoc 2005; 227:597 to 602.
7. Zepeda C, Salman M, Thiermann A, Kellar J, Rojas H, Willeberg P. The role of veterinary epidemiology and veterinary services in complying with the World Trade Organization SPS agreement. Prev Vet Med 2005 67:125 to 140.
8. Cleveland SM, Salman MD, Van Campen H. Assessment of a bovine viral diarrhea virus (BVDV) antigen capture ELISA and a micro-titer virus isolation ELISA using pooled ear notch and serum samples. J Vet Diagn Invest. 2006 July; 18(4): 395to8.
9. Cleveland SM, Zagmutt-Vergara FJ, Cleveland MA, Salman MD, Van Campen H. 2006 A stochastic simulation model of control of bovine viral diarrhoea virus (BVDV) in western US beef herds based on identification and removal of persistently infected (PI) animals. Accepted in the Journal of Preventive Veterinary Medicine.
10. Herrero M. V., Peral J., Vazquez J., Navarro R., Salman MD, Rodriguez L.L. A Retrospective Study Of Vesicular Stomatitis In Mexico (1981 to 2000) Utilizing Geographic Information System (Gis). Accepted in the Journal of Preventive Veterinary Medicine.
11. Pedersen K, Clark L, Andelt WF, Salman MD. Prevalence of Shiga Toxin-producing Escherichia coli and Salmonella enterica in Pigeons Captured in Fort Collins, Colorado. J Wildl Ds. 2006 Jan; 426(1):46to55.
12. Stockton KA, Morley PS, Hyatt DR, Burgess BA, Patterson G, Dunowska M, Lee DE. Effects of footwear hygiene protocols on bacterial contamination of floor surfaces in an equine hospital. J Am Vet Med Assoc 2006 Apr; 228 (7): 1068to73.
13. Tanner JM, Traub-Dargatz JL, Hill AE, Van Campen H, Knight AP, Cunningham WE, Salman MD. 2006 Population Characteristics of Equids Tested for West Nile Virus Infection in Colorado in 2003. Accepted in the Journal of American Veterinary Medical Association.
14. Antognoli MC, Salman MD, Hill AE, Jemmi T, Ochs H. Comparison of Test Strategies for Control of Paratuberculosis in Switzerland. 2006
15. Immunologic responses and protection in elk vaccinated with Brucella abortus strain RB51over-expressing SOD and wboA. P. Nol, S.C. Olsen, W.S. Stoffregen, J.C. Rhyan, S.M. Boyle, G.G. Schurig. Clin Vaccine Immunol 2006 Oct; 13 (10):1098to1103.
16. Seminar-Efficacy of Oral and Parenteral bacille Calmette-Guerin (BCG) in Protecting White-tailed Deer Against Bovine Tuberculosis. USDA National Wildlife Research Center, Fort Collins, CO May 25, 2006
17. Oral Presentation Efficacy of Oral and Parenteral bacille Calmette-Guerin (BCG) in Protecting White-tailed Deer Against Bovine Tuberculosis. Wildlife Disease Association Annual Conference, Storrs, CT August 8, 2006
18. Goehring LS, van Winden SC, van Maanen C, Sloet van Oldruitenborgh-Oosterbaan MM. Equine Herpesvirus type1 associated myeloencephalopathy in The Netherlands: A 4 year retrospective study. J Veterinary Internal Medicine 2006, 20:601 to 7
19. Slater JS, Lunn DP, Horohov DW, Antczak DF, Babiuk L, Breathnach CC, Chang Y-W, Davis-Poynter N, Edington N, Ellis S, Foote C, Goehring L, Kohn CW, Kydd J, Matsumura T, Minke J, Morley P, Mumford J, Neubauer T, OCallaghan D, Osterrieder K, Reed S, Smith K, Townsend HGG, van der Meulen K, Whalley M, Wilson WD: Report of the Equine Herpesvirus-1 Havermeyer Workshop, San Gimignano, Tuscany, June 2004. Veterinary Immunology and Immunopathology, 2006,111: 3 to 13.
20. Goehring LS, Kessels BGF, van Maanen C, Voorbij HAM, Sloet van Oldruitenborgh-Oosterbaan MM. Evaluation of nephelometry for albumin measurement in serum and cerebrospinal fluid: experiences with an indwelling subarachnoidal catheter system for repetitive cerebrospinal fluid collection in horses. J Vet Diag Invest. 2006, 18: 251 to 6.
21. Rockx B, Van Asten L, Van Den Wijngaard C, Godeke GJ, Goehring L, Vennema H, Van Der Avoort H, Van Pelt W, Koopmans M. Syndromic surveillance in The Netherlands for the early detection of West Nile Virus epidemics. Vector Borne Zoonotic Dis. 2006, 6:161 to 9.
22. Dunowska M, Morley PS Traub-Dargatz JL, Davis MA, Patterson G, Frye JG, Hyatt DR, Dargatz DA. Comparison of Salmonella enterica serotype Infantis isolates from a veterinary teaching hospital. J Appl Microbiol 2007Jun; 102(6): 1527to36..
23. Dunowska M, Morley PS, Patterson G, Hyatt DR, Van Metre DC. Evaluation of the efficacy of a peroxygen disinfectant footmat for reduction of bacterial load on boots in veterinary hospital settings. J Am Vet Med Assoc 2006;228:1935 to 1939.
24. Traub-Dargatz JL, Weese JS, Rousseau J, Dunowska M, Morley PS, Dargatz DA. Evaluation of three hygiene protocols on the reduction of bacterial load on the hands of veterinary staff performing routine equine physical examinations. Can Vet J 2006;47:671 to676..
25. Morley PS, Strohmeyer RA, Tankson JD, Hyatt DR, Dargatz DA, Fedorka-Cray PJ. Evaluation of the association between feeding raw meat and Salmonella enterica infections at a Greyhound breeding facility. J Am Vet Med Assoc 2006; 228:1524 to 1532.
26. Stockton KA, Morley PS, Hyatt DR, Burgess BA, Patterson G, Dunowska M, Lee DE. Effects of footwear hygiene protocols on bacterial contamination of floor surfaces in an equine hospital. J Am Vet Med Assoc 2006;228:1068to1073.
27. Strohmeyer RA, Morley PS, Hyatt DR, Dargatz DA, Scorza VA, Lappin MR. Bacterial contamination of commercially available raw meat diets for canines. J Am Vet Med Assoc 2006; 228:537to 542.
28. Hill AE, Duarte P, Morley PS. Epidemiology of equine infectious disease. In: Sellon DR, eds. Equine Infectious Diseases. Elsevier, New York, 2005 [In Press].
29. Dunowska M, Morley PS, Traub-Dargatz JL, Van Metre DC. Biosecurity. In: Sellon DC, Long M, eds. Equine Infectious Diseases. Elsevier, New York, 2005 [In
Press].
30. Traub-Dargatz JL, Morley PS, Duarte PC, Salazar P, Salman MD, Kogan LR. The use of applied epidemiology studies in teaching veterinary professional students. Proceedings of the 11th Symposium of the International Society for Veterinary Epidemiology and Economics, Cairns, Australia, 2006. Number 82.
31. Duarte PC, Morley PS, Traub-Dargatz J, Creekmore L, Neubauer M, Schoenbaum M, Salman MD. Factors associated with the 2004 outbreak of vesicular stomatitis in the western United States. Proceedings of the 11th Symposium of the International Society for Veterinary Epidemiology and Economics, Cairns, Australia, 2006. Number 728.
32. Booker CW, Guichon PT, Jim GK, Schunicht OC, Wildman BK, Pittman TJ, Perrett T, Morley PS, Jones CW, Pollock CM, Janzen ED. Food animal veterinary student feedlot externship in Alberta. Proceedings of the 11th Symposium of the International Society for Veterinary Epidemiology and Economics, Cairns, Australia, 2006. Number 1106.
33. Effect of transportation and lairage on E. coli O157 and Salmonella enterica in finished U.S. beef cattle. Dewell GA, Dewell RD, Simpson CA, Patterson JG, Hyatt DR, Scanga JA, Morley PS, Belk KE, Grandin T, Smith GC, Salman MD. Proceedings of the 11th Symposium of the International Society for Veterinary Epidemiology and Economics, Cairns, Australia, 2006. Number 1109.
34. Dewell RD, Dewell GA, Simpson CA, Patterson JG, Hyatt DR, Scanga JA, Morley PS, Belk KE, Grandin T, Smith GC, Salman MD. Effect of stress in cattle during transportation or lairage on prevalence of O157 and Salmonella spp. Proceedings of the 11th Symposium of the International Society for Veterinary Epidemiology and Economics, Cairns, Australia, 2006. Number 1034.
35. Morley PS. Practical biosecurity considerations for beef operations. Proceedings of the 24th Annual American College of Veterinary Internal Medicine Forum, Louisville, KY, 2006. p 245.
36. Booker CW, Morley PS, Janzen ED, Guichon PT, Jim GK, Schunict OC, Wildman BK, Pittman TJ, T Perrett. Re-examination of fatal undifferentiated fever/bovine respiratory disease of feedlot cattle. Proceedings of the 7th Annual Phi Zeta Research Day, CSU College of Veterinary Medicine and Biomedical Science, Fort Collins, CO, 2006. p 71.
37. Analysis of mortality clustering: a new approach for investigating prevention methods. Invited presentation at the 20th annual Feedlot Health Management Services Research Seminar, Calgary, Canada, 2006.
38. Coping with Salmonella in Equine Patients, Populations, and Facilities. Invited presentation for the Equine Emerging Issues Luncheon at the 24th annual American College of Veterinary Internal Medicine Forum, Louisville, KY, 2006.
39. Branscum AJ, Gardner IA, Wagner BA, McIntuff PS, Salman, MD. Effect of diagnostic testing error on intracluster correlation coefficient estimation. Prev Vet Med 2005 69:63to75.
40. Davidson AH, Traub-Dargatz JL, Rodeheaver RM, Ostlund EM, Pedersen DD, Moorhead RG, Stricklin JB, Young BD, Dewell RD, Salman MD, et al. Immunologic responses to West Nile virus in vaccinated and clinically affected horses. J Am Vet Med Assoc 2005 226:240to5.
41. Dewell GA, Ransom JR, Dewell RD, McCurdy K, Gardner IA, Hill AE, Sofos JN, Belk KE, Smith GC, Salman MD. Prevalence of and Risk Factors for Escherichia coli O157 in Market-Ready Beef Cattle from 12 U.S. Feedlots. Foodborne Pathogens and Disease 2005 2:70to76.
42. Dunowska M, Morley PS, Hyatt DR. The effect of Virkon S fogging on survival of Salmonella enterica and Staphylococcus aureus on surfaces in a veterinary teaching hospital. Vet Micro 2005; 105: 281to289.
43. Morley PS, Morris N, Hyatt DR, Van Metre DC. Evaluation of the efficacy of disinfectant footbaths as used in veterinary hospitals. J Am Vet Med Assoc 2005; 226:2053to2058.
44. Patterson G, Morley PS, Blehm KD, Lee DE, Dunowska M. Efficacy of directed misting application of a peroxygen disinfectant for environmental decontamination of a veterinary hospital. J Am Vet Med Assoc 2005; 227:597to602.
45. Zepeda C, Salman M, Thiermann A, Kellar J, Rojas H, Willeberg P. The role of veterinary epidemiology and veterinary services in complying with the World Trade Organization SPS agreement. Prev Vet Med 2005 67:125to140.
46. Herrero M. V., Peral J., Vazquez J., Navarro R., Salman MD, Rodriguez L.L. A Retrospective Study Of Vesicular Stomatitis In Mexico (1981 to 2000) Utilizing Geographic Information System (GIS). Accepted in the Journal of Preventive Veterinary Medicine.
47. Tanner JM, Traub-Dargatz JL, Hill AE, Van Campen H, Knight AP, Cunningham WE, Salman MD. Population Characteristics of Equids Tested for West Nile Virus Infection in Colorado in 2003. Accepted in the Journal of American Veterinary Medical Association.
48. J. Rhyan, M. Shalev, T. Gidlewski, M. McCollum, G. Ward, M. Deng, T. McKenna, and M. Salman. 2006. Susceptibility of North American wild ungulates to foot-and-mouth disease virus: initial findings. Proc of Open Session of the Research Group of the European Commission for the Control of FMD. Paphos, Cyprus, October, 2006.
49. J.C. Rhyan, T. Gidlewski, M. McCollum, G.B. Ward, F.M.Mohamed, M.Y.Deng, T.S. McKenna, M. Shalev, M. Salman. Foot-and-mouth disease in pronghorn (Antilocapra Americana) and mule deer (Odocoileus hemionus): Susceptibility, clinical signs, and lesions. (Abstract) 49th Annual Conference of the American Association of Veterinary Laboratory Diagnosticians, Minneapolis, MN. Oct 12to18, 2006.p.103.

PROJECT CONTACT:
Name: SALMAN, M. D.
Phone: 970-491-7950
Fax: 970-291-1889
Email: m.d.salman@colostate.edu


ACCESSION NO: 0196720 SUBFILE: CRIS
PROJ NO: COLV-SALMAN AGENCY: CSREES COLV
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2003-34405-13795 PROPOSAL NO: 2003-06088
START: 15 AUG 2003 TERM: 14 AUG 2004 FY: 2004 GRANT YR: 2003
GRANT AMT: $696,539

Investigator: Salman, M.

Performing Institution:
Environmental Health Research
Colorado State University
Fort Collins, Colorado 80523

ENHANCEMENT OF THE PROGRAM FOR ECONOMICALLY IMPORTANT INFECTIOUS ANIMAL DISEASES

NON-TECHNICAL SUMMARY: A multidisciplinary research center is needed to study animal diseases of economic importance. Integration of studies covering broad spectrum of disciplines is needed to prevent duplication of existing efforts and programs. A comprehensive study approach is expected to lead to improved disease surveillance, risk assessment, management, and control/prevention strategies. Furthermore, these studies will lead to the generation of fundamental knowledge concerning disease transmission, diagnosis, pathogenesis, and virulence of economically important animal diseases.

OBJECTIVES: A multidisciplinary research center at CSU will be established to study animal diseases of economic importance. The Center will work collaboratively with universities, and state and federal agencies in order to produce results covering a broad spectrum of disciplines without duplication of existing efforts and programs. Expected results are: 1)The development of improved surveillance, risk assessment, management, and control/prevention strategies; 2)Generation of fundamental knowledge concerning transmission, diagnosis, pathogenesis, and virulence of economically important infectious animal diseases.

APPROACH: The Center will include three major sections which will be simultaneously integrated into the research approach: biology of infectiuos diseases, epidemiology of animal diseases, and risk analysis/assessment. An advisory goup will be composed of scientists from all involved disciplines, commodity representatives, state departments of agriculture, and consumer advocates.

PROGRESS: 2003/08 TO 2004/08
TSEs 6 commercial screening assays were evaluated for deer/elk. A project was initiated which could result in diagnostic tests and prevention methods for prionic infections. Personnel were trained on the western blot test for the presence of CNS tissue in food products. PEIIAD hosted a conference TSE in Animal Populations: Facts & Fiction to address research and policy issues. Participation in international organizations in risk assessment and classification of countries for BSE status: Dr. Salman was re-appointed to the scientific working group of the GBR. West Nile Virus (WNV) Epidemiology: A survey with the goal of determining the long-term outcome of WNV-affected horses was initiated. A study has been initiated to compare antibody titers of WNV in vaccinated horses to those recovering from natural infection. APHI personnel also investigated the possibility of development of an ELISA test for detection of IgG to WNV. Vesicular Stomatitis (VS) Participation in the field validation of the newly developed real-time PCR for VSV detection Test development: A one-step single tube multiplex reverse-transcriptase PCR test for detection of the VSV in biological samples and insects was developed and validated. Molecular epidemiology: Molecular fingerprinting is being used in conjunction with GIS analysis to understand the spread of the VSV. Serological data: The team has demonstrated serological evidence of VSV during non-outbreak years. Equine Infectious Diseases Equine clostridiosis Toxoid development: Development of a toxoid against equine clostridiosis for use in broodmares prior to foaling has been investigated. Test development and validation: A test to identify clostridial beta 1 and beta 2 toxins in clinical samples was developed and is now being validated. Treatment: The use of metranidazone and the subsequent development of resistance were investigated. Mycobacterial work M bovis and M tuberculosis Serological testing PCR development and testing Non-domestic species Molecular epidemiology M avium ssp paratuberculosis (Johnes disease) Diagnostic strategies PCR development and testing Assessment of the presence of M avium ssp paratb in selected lymph nodes & other tissues Food Safety and Risk Analysis E. coli O157 testing Fecal sampling protocols Analytical methods Modeling prevalence in clusters Modeling low prevalence Modeling test dependence Modeling transmission Global Vet Epidemiology Researchers have continued to collaborate with several animal health researchers and regulators in the design & implementation of projects related to surveillance and risk analysis. Other Topics Researchers have secured funding to gather corresponding antimicrobial resistance data from intensive livestock raising units in South America to compare to data from the USA New RB51 brucellosis vaccine are being developed and tested for use in bison and wild ungulates. A nationally-recognized biosecurity program for livestock operations, including teaching hospitals, was developed and initiated. PEIIAD personnel provide teaching expertise for the delivery of epidemiology training to USDA VMOs. A new initiative involves the creation of on-line course.

IMPACT: 2003/08 TO 2004/08
The PEIIAD will continue to support detection and prevention of animal diseases that have impact on movement and trade of animals and animal products.

PUBLICATIONS (not previously reported): 2003/08 TO 2004/08
1. Paul S. Morley, Josie L. Traub-Dargatz, et. al. Availability of Antimicrobial Drugs for Use in Animals Without a Prescription. College of Veterinary Medicine and Biomedical Sciences, Colorado State University: Fort Collins, Colorado. Poster presentation, Phi Zeta, Research Day, 2004.
2. Young S, Dunowska M, Hyatt DR, Morley PS. Evaluation of the environmental cleanliness in a veterinary teaching hospital. Poster presented at the Workshop on Nosocomial Infections and Biosecurity for Equine Hospitals. Dorothy Russell Havemeyer Foundation, Lexington, KY, 2003
3. Serena Young, Magda Dunowska, Doreene R. Hyatt, Paul S. Morley. Evaluation of the Environmental Cleanliness in a Veterinary Teaching Hospital. College of Veterinary Medicine and Biomedical Sciences, Colorado State University: Fort Collins, Colorado. Poster presentation, Phi Zeta Research Day, 2004.
4. Antimicrobial use and resistance in enteric bacteria. Invited presentation presented at the 2003 FDA/CSFSAN-CVM Research Meeting. Baltimore, MD, 2003.
5. Morley PS, Hyatt DR, Dunoswka M. The Effect of Virkon Fogging on Survival of Salmonella enterica on Surfaces in a Veterinary Teaching Hospital. Presentation at the CSU, Phi Zeta research day, January 2004.
6. Evaluation of the Efficacy of Disinfectant Footbaths. Nanea Morris, Paul S. Morley, Doreene R. Hyatt. College of Veterinary Medicine and Biomedical Sciences, Colorado State University: Fort Collins, Colorado. Poster Presentation, Phi Zeta Research Day, 2003.
7. Antognoli, M.C., Hirst H.L, Goodell G., and Salman M.D. Evaluation of Cell Mediated Immunity-based tests for detection of Paratuberculosis in young cattle. Tenth International Symposium of Veterinary Epidemiology and Economics. Abstract325. Vina del Mar, Chile, November 17-21, 2003
8. Antognoli, M.C., Hirst H.L, Goodell G, and Salman M.D. Evaluation of three methods for direct diagnosis of Paratuberculosis in dairy cattle. Tenth International Symposium of Veterinary Epidemiology and Economics. Abstract # 326. Vina del Mar, Chile, November 17-21, 2003
9. Morley PS. Infectious Disease Monitoring and Surveillance at the James L. Voss Veterinary Teaching Hospital. Invited presentation at the Workshop on Nosocomial Infections and Biosecurity for Equine Hospitals. Dorothy Russell Havemeyer Foundation, Lexington, KY, 2003.
10. Dunowska M, Morley PS. Surveillance for Salmonella shedding in large animal patients. Poster presented at the Workshop on Nosocomial Infections and Biosecurity for Equine Hospitals. Dorothy Russell Havemeyer Foundation, Lexington, KY, 2003.
11. Surveillance for Salmonella Shedding in Large Animal Patients. Magda Dunowska and Paul S. Morley, College of Veterinary Medicine and Biomedical Sciences, Colorado State University: Fort Collins, Colorado. Poster presentation, Phi Zeta Research Day, 2003.
12. Burgess BA, Morley PS, Hyatt DR. 2004 Environmental Surveillance for Salmonella in a Veterinary Teaching Hospital. J Am Vet Med Assoc [Submitted].
13. Burgess BA, Morley PS, Hyatt DR. Environmental surveillance for Salmonella in a veterinary teaching hospital. Poster presented at the Workshop on Nosocomial Infections and Biosecurity for Equine Hospitals. Dorothy Russell Havemeyer Foundation, Lexington, KY, 2003.
14. Burgess BA, Morley PS, Hyatt DR. Salmonella surveillance in a large veterinary teaching hospital. Poster presented at the 4th Annual Phi Zeta Research Day, CSU College of Veterinary Medicine and Biomedical Science, Fort Collins, CO, 2003.
15. Salazer P, Traub-Dargatz JL, Morley PS, et al. Characterization of the 2002 West Nile Virus Epidemic in Nebraska and Colorado equids. J Am Vet Med Assoc [In Press].
16. Geiser S, Seitzinger A, Salazar P, et al. Economic Impact of West Nile Virus on the Colorado and Nebraska Equine Industries: 2002. [Government Report]. USDA:APHIS:VS, Centers for Epidemilogy and Animal Health. Fort Collins, CO, 2003. [available
http://www.aphis.usda.gov/vs/ceah/cahm/Equine/wnv-info-sheet.pdf] #N394.0403. 4 pp.
17. Traub-Dargatz JL, Salazer P, Morley PS, et al. Vaccination status and outcome of cases of equine west Nile virus cases in Colorado and Nebraska in 2002. Proceedings of the 3rd International Veterinary Vaccines and Diagnostics Conference, Guelph, Ontario, 2003, p 68.
18. Salazer P, Traub-Dargatz JL, Morley PS, et al. Characterization of the 2002 West Nile Virus Epidemic in Nebraska and Colorado equids. Scientific Proceedings of the 4th Annual Phi Zeta Research Day of the Theta Chapter, CSU College of Veterinary Medicine and Biomedical Science, Fort Collins, CO, 2003, p 34.
19. Traub-Dargatz JL, Morley PS, Salazer P, et al. Characterization of the 2002 West Nile Virus Epidemic in Nebraska and Colorado equids. Presented at the 10th International Society for Veterinary Epidemiology and Economics Symposium, Vina del Mar, Chile, 2003.
20. Geiser S, Seitzinger A, Salazar P, Traub-Dargatz J, Morley P, Salman M, Wilmot D, Steffen D, Cunningham W. Economic Impact of West Nile Virus on the Colorado and Nebraska Equine Industries: 2002. Presented at the 10th International Society for Veterinary Epidemiology and Economics Symposium, Vina del Mar, Chile, 2003.
21. International Society for Veterinary Epidemiology and Economics Abstract and Oral Presentation by Dr. Elizabeth Mumford, November 2003 and Abstract and Oral Presentation at Phi Zeta Research Day January 2003.
22. Hoover, EA. 2003 Chronic wasting disease: Rocky Mountain Virology Conference. November.
23. Mathiason, CK, Sigurdson, CJ, Foos, T, Eliason, G, and Hoover, EA. 2003. Expression of cervid PrPc in Tissues of Deer. Keystone Conference TSEs. Beaver Creek, CO, April.
24. Sigurdson, CJ, Mathiason, CK, Perrott, MR, Eliason, GA, and Hoover, EA: 2003 Transmission of Chronic Wasting Disease in the Ferret. I

PROJECT CONTACT:
Name: Turney, L.
Phone: 970-491-6229
Email: lturney@cvmbs.colostate.edu
URL: http://www.colostate.edu/CVEADSS


ACCESSION NO: 0097775 SUBFILE: CRIS
PROJ NO: COLV05420 AGENCY: CSREES COLV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 1998 TERM: 30 SEP 2004 FY: 2004

INVESTIGATOR: Niswender, G.

PERFORMING INSTITUTION:
College Administration
Colorado State University
Fort Collins, Colorado 80523

BACTERIAL DISEASES AND THE IMMUNE SYSTEM.

OBJECTIVES: 1) To continue development of new procedures for the rapid and reliable detection of the causative agents of bacterial diseases; 2) To develop new, more efficacious methods for the prevention of diseases caused by bacterial agents and 3) to study the pathogenic processes and epidemiology of bacterial diseases.

APPROACH: Improved diagnostic procedures will be developed for the detection of several important pathogenic bacterial (for example, Brucella ovis; Mycobacterium ovis; Mycobacterium paratuberculosis and Clostridium perfringens). Tests will include improved enzyme immunoassays, polymerase chain reaction to detect bacterial DNA and/or RNA, and use of fluorescently labeled recombinant DNA probes and antibodies to specific coat proteins. These reagents will be used to study the disease process and the epidemiology of individual infections. Finally, in some cases more effacacious recombinant vaccines will be developed to prevent the disease.

PROGRESS: 1998/10 TO 2004/09
Mice of the CBA inbred strain background expressing the well characterized mutation designated xid in the cytoplasmic signalling enzyme Bruton's protein kinase have been previously noted to illustrate shifts in T helper type 1 (Th1)/Th2 immunity which is underlined by an apparent failure to produce the regulatory cytokine interleukin-10. This study examined if this extended to infection with Mycobacterium tuberculosis, which also depends on Th1 immunity. Contrary to expectations, xid mice showed evidence of a transient early susceptibility to pulmonary infection, changes in macrophage morphology, and decreased activation of lung natural killer cells, while showing evidence of substantial IL-10 production and accumulation in lung lesions macrophages, but paradoxically this did not influence the course of the chronic disease. In addition, macrophages from the lungs of xid mice also expressed high levels of CD14. These observations suggest that the xid mutation in cellular signalling has much wider effects on the immune system than previously thought. In a separate study, major histocompatibility complex class I tetramer reagent was used to track antigen-specific CD8 T cells in the lungs of mice immunized with the tuberculosis vaccine candidate Mtb72F. The results show that CD8 T cells recognizing an immunodominant Mtb32-specific epitope could be detected in significant numbers over the course of infection in mice exposed to low-dose aerosol challenge with Mycobacterium tuberculosis and that prior vaccination substantially increased the numbers of these cells early in the lungs. The effector phenotype of the cells was shown by the demonstration that many secreted gamma interferon, but very few contained granzyme B. As the course of the infection progressed, many activated CD8 T cells down-regulated expression of CD45RB and upregulated expression of the interleukin-7 receptor alpha chain, indicating a transition of these cells to a state of memory. These data support the hypothesis that M. tuberculosis-specific CD8 T cells can be targeted by vaccination with the Mtb72F polyprotein.

IMPACT: 1998/10 TO 2004/09 Mycobacterial infection in dairy cattle remains a significant economic loss to producers. How the immune system responds to these infections is poorly understood. This limitation has severely hampered the development of a vaccine effective for eliminating Mycobaterial infections (Johne's Disease)in dairy cattle. Some of these studies show that specific bacterial proteins can be used to specifically target components of the immune system, a mechanism that may lead to an effective vaccine for prevention of the multi-billion loss to the
dairy industry caused by Johne's disease.

PUBLICATIONS (not previously reported): 1998/10 TO 2004/09
1. Junqueira-Kipnis AP, Kipnis A, Henao Tamayo M, Harton M, Gonzalez Juarrero M, Basaraba RJ, Orme IM. 2005. Interleukin-10 production by lung macrophages in CBA xid mutant mice infected with Mycobacterium tuberculosis. Immunology. 115:246-52.
2. Irwin SM, Izzo AA, Dow SW, Skeiky YA, Reed SG, Alderson MR, Orme IM. 2005. Tracking antigen-specific CD8 T lymphocytes in the lungs of mice vaccinated with the Mtb72F polyprotein. Infect Immun. 73:5809-16.
3. Perry JA, Olver CS, Burnett RC, Avery AC. 2005. Cutting edge: the acquisition of TLR tolerance during malaria infection impacts T cell activation. J Immunol.
174:5921-5.


ACCESSION NO: 0007172 SUBFILE: CRIS
PROJ NO: CONS00118 AGENCY: SAES CONS
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 JUL 1942 TERM: 01 JAN 2009 FY: 2006

INVESTIGATOR: Van Kruiningen, H.

PERFORMING INSTITUTION:
Pathobiology & Veterinary Science
Univ of Connecticut
Storrs, Connecticut 06268
Survey of Animal Diseases in Connecticut

OBJECTIVES: Determine the occurrence of various animal diseases in Connecticut. Laboratory diagnostic facilities are provided. The relative economic importance of various diseases is being determined. The project serves as a source of leads on diseases needing extensive investigation in the State. ]

APPROACH: Mammals and birds are submitted to the Department of Animal Diseases for necropsy by agricultural interests and veterinarians. Staff veterinarians of the Department do the necropsy and may save time for histopathology, bacteriology, virology, biochemistry, and serology. All cases are reported to the Commissioner of Agriculture and Natural Resources, and the clinical signs and our work-ups are filed here. This adds to the file which is continuous since 1930. If tissues are studied, slides and blocks are also saved. This approach results in an unusual museum of histopathologic material. In recent years gross color transparencies have been made of interesting cases adding to the significance of the file.

PROGRESS: 2006/01 TO 2006/12
The Connecticut Veterinary Medical Diagnostic Laboratory is a division of the Department of Pathobiology and Veterinary Science, University of Connecticut. There were 1660 animal cases (avian, mammalian, and aquatic) examined by the pathology services of the diagnostic laboratory. Included were 255 surgical specimens, which were generally biopsies of canine and feline neoplasms or multiple tissues from autopsies conducted by practicing veterinarians. There were 323 avian and 1337 mammalian/aquatic submissions; multiple animals were often submitted. Species examined included cats, dogs, horses, cattle, swine, goats, sheep, llamas, alpacas, wildlife, laboratory animals, aquatic mammals, fish, shellfish, domestic poultry, game birds and other avian species. Important diagnoses included infectious laryngotracheitis, pasteurellosis, salmonellosis, avian encephalomyelitis, ectoparasitism, metritis, Streptococcus zooepidemicus infection, coronavirus enteritis, dermatophytosis, inclusion body hepatitis, avian infectious bronchitis, air sacculitis, West Nile encephalitis, polioencephalomalacia, mastitis, pyelonephritis, colibacillosis, histomoniasis, infectious bursal disease, caseous lymphadenitis, herpesvirus infection, intussusception, pancreatitis, listeriosis, rotavirus enteritis, acute gastric dilatation, coccidiosis, aspiration pneumonia, ventricular impaction, aspergillosis, dirofilariasis, cryptosporidiosis, canine distemper, scabies, osteomyelitis, Mareks disease, myocarditis, cardiomyopathy, traumatic reticulitis, equine laminitis, cauda equina neuritis, hemorrhagic bowel syndrome of cattle, pheohyphomycosis of seadragons, bovine virus diarrhea, staphylococcal dermatitis, gunshot trauma, feline infectious peritonitis, toxoplasmosis, enthylene glycol poisoning, feline panleukopenia, mycobacteriosis, avian tuberculosis, eastern equine encephalitis, chlamydiosis and equine protozoal myelitis.

IMPACT: 2006/01 TO 2006/12
This diagnostic laboratory serves an important function for the veterinary medical and animal-owning community, explaining deaths, rendering biopsy diagnoses, understanding current diseases in Connecticut and standing ready to identify new ones.

PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
No publications reported this period


ACCESSION NO: 0406145 SUBFILE: CRIS
PROJ NO: ISM-102 AGENCY: ERS MTED
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 01 AUG 2003 TERM: 30 SEP 2007 FY: 2006
INVESTIGATOR: Mathews, K.

PERFORMING INSTITUTION:
Economic Research Service
USDA/ERS
1800 M Street NW
Washington, District Of Columbia 20036

ISM ECONOMIC IMPACTS OF ANIMAL DISEASES

NON-TECHNICAL SUMMARY: The objective of the research is to measure the costs and benefits of different animal disease mitigation measures in domestic and international markets for meat and other animal products.

OBJECTIVES: The objective of the research is to measure the costs and benefits of different animal disease mitigation measures in domestic and international markets for meat and other animal products.

APPROACH: The approach will be a combination of descriptive analyses, econometric estimation of parameters, and case studies of mitigation techniques already in place.

PROGRESS: 2005/10 TO 2006/09
The objective of the research is to measure the costs and benefits of different animal disease mitigation measures in domestic and international markets for meat and other animal products. The spread of infectious disease among and between wild and domesticated animals has become a major problem worldwide. We analyze the socially optimal management of wildlife and livestock, including choices involving environmental habitat variables and on-farm biosecurity controls, when wildlife and livestock can spread an infectious disease to each other. The model is applied to the problem of bovine tuberculosis among Michigan white-tailed deer. The optimum is a cycle in which the disease remains endemic in the wildlife, but in which the cattle herd is depleted when the prevalence rate in deer grows too large. A second project presents a modeling framework designed to estimate the economic impacts of livestock disease outbreaks. The framework (1) combines a disease-spread model with an economic model, (2) introduces supply, demand, and trade shocks resulting from epidemiological model results into a model of the U.S. agricultural sector, and (3) the disaggregation of time into 16 quarters. A number of papers have been presented, a number of articles have been published, several more presentations and journal articles are in progress. The most recent acceptance is Fenichel, E.P., and R.D. Horan, âGender-Based Harvesting in Wildlife Disease Managementâ, American Journal of Agricultural Economics, whic is in press. Two ERS Economic Reports are in final draft stages and an ERS Policy Brief is in progress. The projects were each given no cost extentions of 1 year to facilitate publication of the numerous products.

IMPACT: 2005/10 TO 2006/09
The approach will be a combination of descriptive analyses, econometric estimation of parameters, and case studies of mitigation techniques already in place. For the case of bovine tuberculosis in Michigan deer populations, we found that the ability to mitigate damages via changes in on-farm choices results in greater disease prevalence rates in deer and a smaller likelihood that eradication of deer will be an optimal strategy. In addition, a second cooperative agreement examines a hypothetical outbreak of foot-and-mouth disease (FMD) under the destruction of direct-contact herds, destruction of direct-contact and indirect-contact herds, and slaughter of all animals within a 1-km ring. Relatively few animals are destroyed, but large losses for beef, beef cattle, hogs, and pork tied to the loss of trade sharply lower prices. Other sectors experience small losses or small gains. Ring destruction always reduces the duration of an outbreak to less than 1 quarter. Because of lower prices, consumers benefit when exports are embargoed.

PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
1. Richard Horan, Christopher A. Wolf, Eli P. Fenichel, and Kenneth H. Mathews, Jr., 2005, "Spatial Management of Wildlife Diseases", Review of Agricultural Economics (proceedings isue), Vol. 27/Issue 3, pp. 483-490
2. Horan, R.D. and C.A. Wolf, 2005, "The Economics of Managing Infectious Wildlife Disease", American Journal of Agricultural Economics, Vol. 87/Issue 3, pp. 537-551

PROJECT CONTACT:
Name: Mathews, K.
Phone: 202-694-5183
Fax: 202-694-5183
Email: KMATHEWS@ers.usda.gov


ACCESSION NO: 0200070 SUBFILE: CRIS
PROJ NO: GEOV-0471 AGENCY: CSVM GEOV
PROJ TYPE: STATE PROJ STATUS: TERMINATED
START: 01 JUL 2003 TERM: 30 SEP 2003 FY: 2004

INVESTIGATOR : Quinn, F. D.

PERFORMING INSTITUTION:
College Of Vet Medicine
University Of Georgia
110 Riverbend Road
Athens, Georgia 30602

ZEBRAFISH AS A MODEL FOR MYCOBACTERIUM SHOTTSII PATHOGENESIS

NON-TECHNICAL SUMMARY: An epizootic of mycobacteriosis in striped bass is occurring in the Chesapeake Bay and other areas on the eastern sea coast. These infections are caused primarily by Mycobacterium shottsii, a close relative of M. tuberculosis and a newly characterized species. We need to understand the etiology of this disease due to its potential impact on commercial fishing and effects on workers and residents near the affected waterways. We will begin to elucidate virulence mechanisms of M. shottsii using the zebrafish model system.

OBJECTIVES: Mycobacterial infections within a host are controlled through phagocytosis and intracellular killing by activated macrophages. If this initial control step is unsuccessful, the infected macrophages prime the formation of differentiated structures called granulomas that function to contain the infection and facilitate the persistence or latency of the pathogen in a decreased metabolic state for years. Mycobacterial granulomas analyzed from various mammalian, amphibian and fish species have shown that these structures are composed of similar cell types, primarily macrophages, and in similar cellular proportions; a possible indication of the evolutionary association between mycobacterial pathogens and their vertebrate hosts. Striped bass, a primary target species of M. shottsii infection, produce a significant granulomatous response in the skin and internal organs after infection with this agent. Experimentally, striped bass are not a convenient model system to examine M. shotsii pathogenesis and the host immune response due primarily to its large size and the lack of available immunological and genetic tools and reagents. Alternatively, zebrafish, which also produce granulomas in response to M. shottsii infection and whose macrophages actively phagocytose the invading pathogen, is becoming the preeminent fish model system for infectious disease investigations. Thus, our hypothesis is that the mycobacterial disease process for M. shottsii in zebrafish mimics the analogous (innate) immune response in striped bass. With this in mind, we will: 1) detail the infectious process in the zebrafish, focusing on tissue destruction and granuloma formation, and 2) isolate zebrafish primary macrophages, infect them, and examine the ability of the mycobacteria to replicate intra- or extracellularly.

APPROACH: The experimental approach will establish an in vivo infection model in zebrafish. Fish will be injected with virulent mycobacteria and lesions will be identified and characterized. The second project is to infect short term cultured zebrafish macrophages. This will enable studies of bacterial adherence, internalization and intracellular growth. M. shottsii will be grown at 23degreesC to logarithmic phase, diluted and viable count determined. Groups of 30 adult zebrafish will be inoculated intraperitoneally with 105, 103, or 102 colony-forming units (CFU) of M. shottsii in PBS in 50 l volumes in PBS. Samples of liver and spleen will be collected at various time points, homogenized in PBS with 0.05% Tween 80 and bacterial counts determined by plating on agar medium. Bacteria will be confirmed as M. shottsii using species-specific PCR primers. This procedure will determine in vivo distributions of infected zebrafish. In order to determine if M. shottsii will attach, enter and multiply within zebrafish macrophages in a manner and at a rate analogous to that observed for pathogenic mycobacteria in mammalian macrophages, the following standard assays will be performed. Primary zebrafish macrophage cells will be removed from the peritoneal cavity and identified by flow cytometry. Cells of appropriate size and granularity will be (sterile) sorted. Sorted cells will be seeded at a density of 5X104 cells per well in 48-well plates and left at 25oC for 24 hours. The medium will be replaced before use and bacteria will be added to achieve a multiplicity of infection (MOI) of 10. The infection will proceed for 2 hours at 25oC after which the macrophages will be washed and incubated in fresh medium plus 200 g/ml amikacin for 1 hour to kill extracellular bacteria (MIC of amikacin for M. shottsii is 30 g/ml). In order to determine the number of intracellular bacteria at this basal time point, the cells will be washed once in PBS and lysed by adding 1 ml of 0.1% Triton X-100 for 10 min. Dilutions will be plated to determine the number of intracellular CFU. For long term intracellular growth assays, fresh medium plus 30 g/ml of amikacin will be added after the medium containing the 200 g/ml amikacin is removed. The cells will be incubated for various times up to 14 days before being lysed, plated and percent survival determined for each time point. Adherence assays will be carried out in a similar fashion except that bacteria will be added to the cells and within 15 min. will be washed five times with PBS prior to lysis. Microscopic observations will be performed using infections at a MOI of 10 with and without amikacin on coverslips in 24-well dishes. Ziehl-Neelsen stained cells will be examined. Transmission electron microscopy also will be used to examine the ultrastructure and membrane trafficking of M. shottsii-infected macrophages.

PROGRESS: 2003/07 TO 2003/09
We conducted experiments to determine if macrophages in the zebrafish embryo responded to mycobacterial infection with the formation of granulomas. We injected approximately 50 viable M. marinum or M. shottsii bacilli (expressing green fluorescent protein) into the brain cavities of 1-day old embryos, or the heart cavities of 2-day old embryos. Injected embryos were examined 24 or 48 h post-injection under a compound microscope with Fluorescent and DIC optics. Whereas Escherichia coli and Bacillus subtlis bacilli were cleared from embryos within 5-6 hours after injection, mycobacteria persisted in embryos for at least 48 hours, the latest time-point examined. This indicated that, as in adults, M. marinum and M. shottsii bacilli avoid destruction by the embryonic immune system. The fluorescent bacteria were associated with cells approximately 10 micrometers in diameter, the size of macrophages in zebrafish embryos. Cells associated with bacteria appear in clumps, ranging from 3 cells to more than a dozen. Our data strongly suggest that the zebrafish immune system is capable of responding to M. marinum and M. shottsii infection by forming granuloma-like structures. Zebrafish embryos are convenient for making in vivo microscopic observations, however, the numbers of macrophages that can be acquired for routine study is low. We, therefore, developed a technique for acquiring large numbers of macrophages infected in vivo from adult zebrafish. Adult zebrafish (2 centimeters or more in length) were injected with a PBS:Mineral Oil and Dextran-rhodamine suspension along with 50 viable M. shottsii or M. marinum bacilli (expressing green fluorescent protein). Fifty microliters of solution was injected per fish peritoneal cavity. Seventy-two hours later the fish were euthanized and contents of the peritoneal cavity were harvested. The fluid was then analyzed using a Coulter EPICS XL flow cytometer. Approximately 10% of the peritoneal exudates cells contained Dex-Red and green fluorescent protein. This experiment demonstrated that with a signal as strong as that occurring in the second decade of a log scale, it will be possible not only to enumerate total perotineal phagocytes, but also to sort out a collection of the Dex-Red positive cells infected with bacilli will be feasible.

IMPACT: 2003/07 TO 2003/09
Mycobacterial granulomas analyzed from various mammalian, amphibian and fish species have shown that these structures are composed of similar cell types, primarily macrophages, and in similar cellular proportions; a possible indication of the evolutionary association between mycobacterial pathogens and their vertebrate hosts. Striped bass, a primary target species of Mycobacterium shottsii infection, produce a significant granulomatous response in the skin and internal organs after infection with this agent. Experimentally, striped bass is not a convenient model system to examine M. shotsii pathogenesis and the host immune response due primarily to its large size and the lack of available immunological and genetic tools and reagents. Alternatively, zebrafish, which also produce granulomas in response to M. shottsii infection and whose macrophages actively phagocytose the invading pathogen, is becoming the preeminent fish model system for infectious disease investigations. The research presented here will ultimately benefit mycobacterial disease investigators and the zebrafish research community. The former by elucidating basic mechanisms of cellular immunity that should lead to new methods of diagnosis, treatment, or control of fish populations with M. shottsii infections and the latter through the sharing of novel reagents and techniques for this important animal model.

PUBLICATIONS (not previously reported): 2003/07 TO 2003/09
No publications reported this period

PROJECT CONTACT:
Name: Quinn, F. D.
Phone: 706-542-5790
Fax: 706-542-5771
Email: fquinn@vet.uga.edu


ACCESSION NO: 0405020 SUBFILE: CRIS
PROJ NO: 3625-32000-063-00D AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 27 OCT 2001 TERM: 26 OCT 2006 FY: 2006

INVESTIGATOR: Palmer M V; Waters W R

PERFORMING INSTITUTION:
Agricultural Research Service
Ames, Iowa 50010

DIAGNOSIS AND CONTROL OF TUBERCULOSIS IN LIVESTOCK AND WILDLIFE

OBJECTIVES: Develop and evaluate improved tests for diagnosis of Mycobacterium bovis infection in different animal species; Develop improved methods for differentiation of M. bovis isolates; Characterize M. bovis infection in domestic livestock and wildlife. Identify vaccine strategies to elicit protective immunity in cattle and relevant wildlife against Mycobacterium bovis.

 

APPROACH: Sensitivity and specificity of tests for detection of M. bovis infection in live animals will be determined. We will determine if new antigens can be used to improve skin tests and in-vitro diagnostic tests. Improved PCR assays for direct detection of M. bovis in various specimens will be developed by modification of existing tests. Improved methods for isolation of M. bovis from various samples will be developed by changing processing methods and decontaminants. Improved methods for DNA fingerprinting of M. bovis isolates will be developed by adapting published methods. Animals will be exposed to M. bovis by different routes and clinical signs, immune system parameters, and lesion distribution will be monitored. Routes of transmission of M. bovis from infected animals to uninfected animals will be assessed by periodic sampling of oral and nasal secretions, urine, and feces. BSL-3; (IBC-#0239) canceled 10/05/04. BSL-2/BSL-2N; Recertified May 11, 2006. IBC #0278. BSL-Exempt; Recertified June 8, 2006. IBC #0269. BSL-2/BSL-2N;Recertified May 14, 2006. IBC#0264. BSL-Exempt; (IBC-#0283) 01/12/06. BSL-2/BSL-1N/BSL-3N; Certified June 1, 2006. IBC#0285.

PROGRESS: 2001/10 TO 2006/10
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? The "Diagnosis and Control of Tuberculosis in Livestock and Wildlife" Research Project is aligned with NP 103 Animal Health. Bovine tuberculosis, which is caused by Mycobacterium bovis, is an infectious disease that affects many species of animals as well as human beings. Animals infected with M. bovis can shed organisms as they exhale or cough and in various secretions including saliva, milk, feces and urine. Elimination of animals infected with M. bovis is important to prevent the spread of disease among animals and to human beings. The US initiated a program to eradicate tuberculosis from cattle in 1917 when the prevalence of disease was approximately 5%. In general, the eradication program has been successful and today, less than 0.002% of cattle are infected with M. bovis. However, a low prevalence of disease has persisted and it has not been possible to eradicate bovine tuberculosis from the US using available technology. Moreover, in the last 5 years agriculturally important states such as California, Texas, Minnesota and Michigan have lost their TB-free status due to the presence of M. bovis infected cattle herds. It is estimated that loss of TB free status will cost livestock producers in these states from $2.55 billion in Texas to $156 million in Michigan over the next 10 years. Improved diagnostic tests and control measures are needed to detect and eliminate cattle that have bovine tuberculosis. Tuberculosis has been detected in captive deer and elk and in wild white- tailed deer, coyotes, raccoons, bear, bobcat, opossum, and fox. There is epidemiological evidence to demonstrate that tuberculosis has been transmitted between deer to cattle. The presence of tuberculosis in wildlife poses a serious threat to the national eradication program due to spillover of disease from wildlife to domestic animals and the difficult of eliminating an infectious disease from a wild population. Other countries with tuberculosis in wildlife have been unable to eradicate tuberculosis from domestic livestock. The Bovine Tuberculosis Research Project at the National Animal Disease Center (NADC) is conducting research to develop a better understanding of the interactions between various host species and M. bovis, including immune response, disease progression and interspecies transmission. This information will be used to develop improved diagnostic tests, vaccines and strategies to minimize disease transmission. Bovine tuberculosis is considered a public health threat because human beings can become infected with M. bovis through contact with infected animals or by ingestion of contaminated food and milk. Elimination of tuberculosis from cattle is important to provide food and milk to the public that is free of M. bovis. Elimination of bovine tuberculosis from domestic livestock is also important to maintain free international trade. Trade restrictions between the US and Canada and between Mexico and the US have occurred because of bovine tuberculosis. Eradication of tuberculosis from wildlife is important to prevent transmission of disease between wildlife and domestic livestock. The Bovine Tuberculosis Research Project is assigned to the Animal Health National Program 103 (100%) and relates to the vision of this program to ensure animal health through improved disease detection and control. Specifically, the objectives of the project are to: 1)develop and evaluate improved tests for diagnosis of tuberculosis in cattle, deer, and other species, which relates to the component on Pathogen Detection; 2)develop improved methods for differentiation of M. bovis isolates, which relates to the component on Epidemiology of Disease; 3)define interactions between various host species and M. bovis, which relates to the component on Host/Pathogen Interaction; and 4) develop and evaluate vaccines for control and prevention of tuberculosis in animals, which relates to the component on Disease Control Strategies. Cattle as well as bison and all species of Cervidae are subject to testing for tuberculosis under the guidelines of the USDA uniform rules and methods for the eradication of bovine tuberculosis. The most common means of testing is the tuberculin skin test. Tuberculin skin testing lacks specificity and has not been fully validated in all species of Cervidae. Especially problematic is the absolute lack of specificity associated with tuberculin skin testing reindeer. The Animal and Plant Health Inspection Service (APHIS) of the USDA has provided funds for NADC to conduct research on tuberculosis in various species of Cervidae (3625- 32000-063-01R). The objectives of such research are to study immune responses of Cervidae and develop improved methods of antemortem diagnosis of tuberculosis in these species. Such objectives are consistent with the National Program as stated above. 2. List by year the currently approved milestones (indicators of research progress) Year 1 (FY 2002) Develop aerosol model of inoculation of M. bovis for use in large animal species. Evaluate blood based assays of cell mediated immunity for diagnosis of M. bovis infection in both cattle and deer. Continue evaluation of mechanisms of transmission of M. bovis between wildlife and cattle. Year 2 (FY 2003) Validate aerosol model of inoculation of M. bovis for cattle and deer. Compare resulting disease in cattle with that seen naturally. Begin evaluation of blood based assay measuring gamma interferon (Cervigam assay) as a means of diagnosis of M. bovis infection in white- tailed deer. Evaluate other promising blood based assays of cell mediated immunity of diagnosis of M. bovis infection in cattle and deer such as the nitric oxide (NO) assay. Examine deer to deer transmission of M. bovis by investigating doe to fawn transmission through milk. Continue evaluation of shared feed as a means of transmission of M. bovis between wildlife and cattle. Begin study of reindeer immune system to aid in development of improved diagnostic test for M. bovis infection in reindeer. Year 3 (FY 2004) Use aerosol inoculation model of cattle in evaluation of efficacy of experimental vaccine candidates. Continue evaluation of aerosol inoculation of white-tailed deer with M. bovis. Compare disease with naturally occurring tuberculosis. Continue evaluation and validation of gamma interferon assay, nitric oxide assay and others as methods of antemortem diagnosis of M. bovis infection in cattle and deer. Experimentally infect reindeer with M. bovis to evaluate accuracy of current and experimental diagnostic tests in know M. bovis infected reindeer. Year 4 (FY 2005) Continue use of aerosol inoculation model of cattle in evaluation of efficacy of experimental vaccine candidates and investigation of immune responses of cattle to infection and vaccination. Evaluate alternative antigens for use in blood based assays for diagnosis of M. bovis infection in cattle and deer. Evaluate their specificity by examining cattle infected with other species of mycobacteria. Evaluate serological assays as a method of diagnosis of M. bovis infection in cattle and deer. Complete evaluation of diagnostic tests for M. bovis infection in reindeer. Begin evaluation of potential vaccines for prevention of M. bovis infection in wildlife (white-tailed deer) by evaluating the safety and efficacy of the human tuberculosis vaccine, M. bovis strain BCG Year 5 (FY 2006) Continue use of aerosol inoculation model of cattle in evaluation of efficacy of experimental vaccine candidates and investigation of immune responses of cattle to M. bovis infection or vaccination. Continue evaluation of serological assays as a method of diagnosis of M. bovis infection in cattle and deer. Continue evaluation of alternative antigens to increase sensitivity and/or specificity in both in vivo and in vitro diagnostic tests for M. bovis infection in cattle and deer. Continue evaluation of potential vaccines for prevention of M. bovis infection in wildlife (white-tailed deer) by evaluating the efficacy of various vaccine preparations and adjuvants. 4a List the single most significant research accomplishment during FY 2006. This accomplishment aligns with the Disease Control Strategies component of the NP103 Animal Health Action Plan. The first trial was completed to examine the efficacy of M. bovis BCG as a vaccine to prevent tuberculosis in white-tailed deer. Results of the trial are encouraging suggesting that vaccination with BCG may be effective in decreasing the severity of disease in deer and consequently decreasing the ability of deer to transmit M. bovis to other deer or to livestock. The completion of this first trial is significant as these results will serve as the basis for subsequent trials that will investigate other parameters such as comparative efficacy of various strains of M. bovis BCG, oral vs parenteral routes of vaccination, vaccine safety, etc. 4b List other significant research accomplishment(s), if any. This accomplishment aligns with both the Host/Pathogen Interaction and Pathogen Detection components of the NP103 Animal Health Action Plan. We continue evaluation of several serological tests for M. bovis infection in cattle and deer. Preliminary findings have identified several tests that may prove useful for diagnosis of tuberculosis in various species including cattle and deer. These serological tests will be faster, cheaper and technically less difficult than blood based assays of cell mediated immunity. We are currently evaluating the sensitivity and specificity of these tests in both cattle and deer. The only approved test for tuberculosis in deer is the intradermal tuberculin test. Currently less than 6% of the captive deer in the US are tested annually for tuberculosis. This is due in part to difficulties using the intradermal tuberculin test in this species. An accurate blood-based assay would significantly improve producer participation in the testing program and increase the ability of USDA to detect tuberculosis in captive deer. This test is also being evaluated in free-ranging deer in a trap and test program in Michigan. This work is being done in collaboration with scientists at Chembio Diagnostics, Medford, NY, Iowa State University, Ames, IA, and the Michigan Department of Natural Resources, with financial assistance from the subordinate CRIS, 3625- 32000-063-01R, a reimbursable agreement with USDA/APHIS. 4c List significant activities that support special target populations. Presentations were made to the Reindeer Owners and Breeders Association, North America Elk Breeders Association and North American Deer Farmers Association on advances in the diagnosis of tuberculosis in farmed deer. Significant advances have been made in development of improved blood- based tests to diagnose tuberculosis in deer. Some tests have received conditional approval through USDA's Center for Veterinary Biologics. Since 1990, deer have been tested for tuberculosis in a manner similar to that used for cattle as part of the USDA's bovine tuberculosis eradication program. These tests were not specifically designed for deer, but rather the tests used for cattle were applied to deer. These tests, however, have not performed as well in deer as they have in cattle creating 2 primary problems for deer farmers; 1) There are many more false positive test results in deer than there are in cattle. This results in the unnecessary euthanasia of deer that do not really have tuberculosis, creating a hardship for producers due to animal losses, financial losses, prolonged quarantines, animal movement restrictions and lost market opportunities. 2) Because the testing procedure currently used involves handling the animals multiple times, there is significant opportunity for injuries to deer due to their fractious nature. Blood- based assays designed specifically for deer would enhance the accuracy of testing in deer species and eliminate the need for repeated animal handling. Such tests would decrease overall costs to producers as well as expedite the USDA's bovine tuberculosis eradication program, which was implemented in large part due to public health concerns, as bovine tuberculosis is a zoonotic disease transmissible to humans. Additional research on blood-based tests for tuberculosis in deer is planned as part of our 5 year project plan. Updates will be provided to deer producer groups as needed. 5. Describe the major accomplishments to date and their predicted or actual impact. These accomplishments align with the pathogen detection, epidemiology of disease, host/pathogen interaction, and disease control strategies components of the NP103 Animal Health Action Plan. A new test, the polymerase chain reaction (PCR) for detection and identification of M. bovis in tissue samples collected for microscopic examination was developed. When animals are slaughtered, meat inspectors collect tissue samples from animals that are suspected of having tuberculosis. Tissue samples are examined for microscopic evidence of tuberculosis and for the presence of organisms. Stains used to detect M. bovis in a tissue sample also stain other organisms making it impossible to identify the organism in the sample. The new test detects a specific piece of DNA that is present only in organisms that cause tuberculosis. The new test permits more accurate and rapid identification of animals with tuberculosis than was previously possible. The test is used extensively at the request of state and federal regulatory officials to confirm suspected cases of tuberculosis in animals. This technology has been transferred to USDA, APHIS and has allowed more rapid confirmation of tuberculosis, allowing epidemiologic investigation to start much earlier than was previously possible. Quarantine times for producers are reduced and continued spread of disease through animal movements is minimized. A method to differentiate strains of M. bovis was developed. Differences among various strains of M. bovis can be identified by using specific genetic markers. Using these markers, it is possible to determine if different animals are infected with a common strain or different strains of M. bovis. This technology has been transferred to USDA, APHIS. This information is used by epidemiologists to determine possible sources of infection in outbreaks of tuberculosis in animals. An interferon gamma blood based assay for the diagnosis of tuberculosis in cattle is currently available. NADC scientists, in cooperation with USDA, APHIS and various state agencies evaluated the accuracy of this assay compared to traditional skin testing. In FY03 such research allowed the approval of the interferon gamma assay for use in cattle. In many cases this test has replaced the previously used comparative cervical skin test, thereby decreasing animal handling costs, decreasing time to diagnosis, decreasing quarantine time and expediting epidemiological investigations. An experimental model of tuberculosis in white-tailed deer was developed using intratonsilar inoculation. The resulting disease is very similar to that observed in naturally infected deer. This model has been used extensively by NADC scientists to study the transmission of tuberculosis among deer and between deer and cattle and has demonstrated that deer can transmit M. bovis to other deer or cattle through indirect contact such as the sharing of feed. Subsequent epidemiological investigations have supported these findings and shown an association between supplemental feeding and disease prevalence. This information has been used by Michigan wildlife and agricultural officials to lobby for the banning of supplemental feeding of wildlife to control the tuberculosis outbreak in northern Michigan. This model has also been used to develop and evaluate improved tests for diagnosis of tuberculosis in deer and to determine the effectiveness of vaccines to prevent infection. This model is currently being used by investigators in other countries ( Canada) to examine tuberculosis in red deer and elk. Current surveys to determine the prevalence of tuberculosis in wild white-tailed deer are based on examination of tissues in the head to detect lesions. In a study that involved detailed examination of the entire deer carcass, we determined that about 35-50% of the naturally infected deer did not have lesions in the head. This underestimation of disease prevalence should be considered when estimating prevalence through surveillance efforts that focus on examination of grossly visible lesions of the head only. A safe, reliable, and reproducible method of aerosol delivery of M. bovis to cattle, or other large ruminants was developed and validated by scientists at NADC. Aerosol exposure is believed to be a primary means of M. bovis transmission between cattle. NADC scientists conducted the first studies on aerosol exposure of M. bovis in cattle. These studies have shown that this method of exposure results in lesions similar to those seen in naturally infected cattle. Such a delivery method is being used at NADC to investigate disease pathogenesis, transmission, immune responses, and vaccine efficacy and could be used to deliver other pathogens of cattle where aerosol delivery is critical. In contrast, aerosol exposure of deer to M. bovis did not result in disease similar to that seen in naturally infected deer, further supporting the hypothesis that transmission of M. bovis between deer involves routes such as sharing of feed and that aerosol transmission may be less important. The outbreak of tuberculosis in white-tailed deer in Michigan, identified in 1995, represents the first wildlife reservoir of M. bovis in North America and a serious threat to the federal bovine tuberculosis eradication effort. ARS scientists at NADC demonstrated that M. bovis can be transmitted between deer in close contact, through the sharing of feed and to deer fawns through milk containing M. bovis. These results document that contaminated milk, in addition to saliva, nasal secretions, urine and feces may be involved in transmission of M. bovis between deer. Such information is vital for state and federal officials to make educated decisions concerning disease control strategies. Currently, tuberculin skin testing is the most common method of antemortem testing of deer for tuberculosis; however, tuberculin skin testing of deer lacks specificity and requires the capture and handling of animals at least twice for testing. ARS scientists at NADC have investigated various blood-based assays for the diagnosis of tuberculosis in deer species common in North America. Such tests would decrease overall cost to producers, animal stress and time to diagnosis. Serological assays are particularly appealing and may be useful for screening animals in field situations. Research on improved diagnosis of tuberculosis in Cervidae has resulted in a change in the interpretation of tuberculin skin testing results in reindeer by state and federal veterinarians. Based on ARS research a new scattergram was adopted by USDA, APHIS for interpretation of the skin test in reindeer that results in decreased numbers of false positive results, decreased destruction of non-infected animals, and decreased cost to USDA. Research on blood based diagnostic assays has resulted in the recommendation by the USAHA in 2003 that the gamma interferon assay for deer (Cervigam) be evaluated side by side with skin testing to determine if this blood based assay may be a suitable replacement for skin testing. Serological assays developed or evaluated by NADC scientists are being used to assist Michigan wildlife officials in screening of large numbers of trapped wild deer as an approach to selectively remove tuberculous deer. Research on an assay to detect nitric oxide as a means of antemortem diagnosis of tuberculosis was patented by ARS. Research on alternative antigens for use in tuberculosis diagnostics has yielded several potential candidates that may increase specificity of these tests, thereby decreasing the number of false positive results caused by exposure of animals to environmental non-tuberculous mycobacteria. ARS has identified one candidate, the fusion protein ESAT- 6:CFP-10. Field trials in Michigan have begun to evaluate the ability of the fusion protein to improve the specificity of the existing test (Bovigam). NADC conducted the first efficacy trials of vaccines to prevent tuberculosis in white-tailed deer. The first trial tested the human TB vaccine, M. bovis BCG. Results were encouraging suggesting that vaccination may decrease disease severity, thus decreasing the potential for disease transmission between deer or from deer to cattle. This first trial will serve as the foundation for various subsequent studies. Diseases at the interface of wildlife and domestic animals have become increasingly important. The study of infectious diseases in wildlife is costly and challenging and very few institutions are involved in such research involving agents requiring bio-containment. NADC has been visited by scientists from the Foreign Animal Disease Lab at Plum Island, Canadas National Center for Foreign Animals Diseases in Winnipeg, and other laboratories to observe how NADC scientists conduct such research. The impact of the overall wildlife research at NADC has been that NADC scientists are consulted regularly on research in wildlife in bio- containment involving BL-3 agents, foreign animal diseases, etc. 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? In FY05-FY06, a field study to evaluate ESAT-6:CFP-10 was initiated with technology transferred to USDA/APHIS and Michigan State University. In FY06, USDA/APHIS continued the use of a modified scattergram for interpretation of skin testing in reindeer based on research findings of NADC scientists. This modified interpretation has been adopted nation- wide as the current approved method of interpretation. During FY06, NADC scientists presented research findings on improved diagnostic tests for cattle and Cervidae to producer groups at international, national and regional meetings. During FY06 NADC scientists presented research findings on improved diagnosis of tuberculosis through use of the interferon gamma assay and serological assays in cattle and deer at various meetings of wildlife and agricultural officials. During FY06 NADC scientists presented research findings on the transmission of M. bovis between deer and from deer to cattle at various meetings of wildlife and agricultural officials. These officials have used such findings to lobby legislators for a ban on supplemental feeding of wildlife to control tuberculosis. During FY06 NADC scientists presented research findings on vaccination of white-tailed deer with M. bovis BCG to the Michigan Departments of Agriculture and Natural Resources. 7.

List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).

PUBLICATIONS:
1.
Palmer M.V., T.C. Thacker, and W.R. Waters. 2006. Vaccination of white- tailed deer with Mycobacterium bovis bacillus Calmette Guerin (BCG). Michigan Bovine Tuberculosis 11th Annual Meeting, Lansing MI (Invited Lecture).
2.Waters W.R. 2006. Tuberculosis tests for Cervids: Looking past the new rules. North American Deer Farmers Association Annual Meeting, Pittsburgh, PA (Invited Lecture).
3.Update on tuberculosis research in Cervidae. Reindeer Owners and Breeders Association National Meeting. July 2006.
4.Update on tuberculosis diagnostics research in cattle. National Milk Producers Federation Animal Health Meeting. April 2006.
5.Diagnosis of M. bovis infection in white-tailed deer using IFN-gamma RNA. United States Animal Health Association, Tuberculosis Scientific Advisory Subcommittee. Nov 2005.
6.Using white-tailed deer as laboratory animals. American Association of Laboratory Animal Science. May 2005.
7.Foote M.R., B.J. Nonnecke, W.R. Waters, and D.C. Beitz. 2006. High Growth Rate Fails to Enhance Adaptive Immune Responses in Neonatal Calves and Decreases Immune Cell Viability. Iowa State Animal Industry Report, (Technical Note).
8.Waters W.R. 2006. Tuberculosis: Basic Immunology and Diagnostic Tests, Basic TB Epidemiology Course, Ames, Iowa (Invited Lecture).
9.Waters W.R., T.C. Thacker, and M.V. Palmer. 2006. ARS Research Activities Diagnostic Tests and Vaccines, Designated TB Epidemiology Course, Estes Park, CO (Invited Lecture).
10.USDA, VS, APHIS Memo 552.40. Evaluation of provisional tests for bovine tuberculosis.
11.TB tests for Cervids: Looking past the new rules. The Reindeer Owners and Breeders Association Review. May/June 2006.


PUBLICATIONS (not previously reported): 2001/10 TO 2006/10
1. Lyashchenko, K.P., Greenwald, R., Esfandiari, J., Vordermeier, H.M., Palmer, M.V., Waters, W.R. 2006. Rapid lateral-flow for bovine tuberculosis [abstract]. In: Proceedings of Veterinary Vaccines and Diagnostics Conference. 4th International Veterinary Vaccines and Diagnostics Conference 2006, June 25-29, 2006, Oslo, Norway. 2006 CDROM.
2. Palmer, M.V., Waters, W.R., Thacker, T.C., Minion, C.F., Greenwald, R., Esfandiari, J., Andersen, P., Mcnair, J., Pollock, J., Lyashchenko, K. 2005. Effects of different skin testing regimens on interferon gamma and antibody responses in cattle experimentally infected with Mycobacterium bovis [abstract]. American Association of Veterinary Laboratory Diagnosticians. p. 7.
3. Waters, W.R., Palmer, M.V., Thacker, T.C., Payeur, J.B., Minion, F.C., Greenwald, R., Esfandiari, J., Andersen, P., Mcnair, J., Pollock, J.M. 2005. Mycobacterium Kansasii Infection of Cattle Confounds Interpretation of Tuberculosis Diagnostic Tests [abstract]. International Conference on Mycobacterium bovis. p. 26.
4. Waters, W.R., Palmer, M.V., Thacker, T.C., Harris, N.B., Payeur, J.B., Minion, F.C., Mcnair, J., Pollock, J.M., Lyashchenko, K.P. 2006. Interpretation of tuberculosis diagnostic test results may be confounded in cattle infected with Mycobacterium kansasii [abstract]. Research Workers in Animal Diseases Conference Proceedings. p. 81.
5. Harrington, N.P., Waters, W.R., Surujballi, O.P., Prescott, J.F. 2006. Development of a multispecies real-time RT-PCR whole blood assay to detect interferon-gamma response to mycobacterial antigens and evaluation in Mycobacterium bovis-infected elk (Cervus elaphus)[abstract]. Research Workers in Animal Diseases Conference Proceedings. p. 72.
6. Foote, M.R., Nonnecke, B.J., Waters, W.R., Beitz, D.C., Fowler, M.A., Johnson, T.E., Miller, B.L. 2006. High growth rate fails to enhance adaptive immune responses of neonatal calves and is associated with decreased T cell viability [abstract]. American Dairy Science Association- American Society of Animal Science 2006 Joint Annual Meeting. p. 163.
7. Harrington, N.P., Prescott, J.F., Waters, W.R., Lyashchenko, K.P., Surujballi, O.P. 2006. Serological Responses of cervids (Cervus elaphus) experimental infected with Mycobacterium bovis: Potential for serodiagnostics [abstract]. In: Proceedings of 4th International Veterinary Vaccines and Diagnostics Conference, June 25-29, 2006, Oslo, Norway. 2006 CDROM.
8. Prescott, J.F., Harrington, N.P., Waters, W.R., Lyashchenko, K.P., Greenwald, R., Surujballi, O. 2006. Serological Responses of cervids (Cervus elaphus) experimentally infected with Mycobacterium bovis: Potential for serodiagnosis [abstract]. In: Proceedings of American Association of Wildlife Veterinarians. 2006 Annual Conference of the Wildlife Disease Association and the American Association of Wildlife Veterinarians, August 6-10, 2006, Storrs, CT. 2006 CDROM.
9. Waters, W.R., Palmer, M.V., Thacker, T.C., Bannantine, J.P., Vordermeier, H.M., Hewinson, R.G., Greenwald, R., Esfandiari, J., Mcnair, J., Pollock, J.M. 2006. Early Antibody Responses to Experimental Mycobacterium bovis Infection of Cattle. Clinical and Vaccine Immunology. 13(6):648-654.
10. Thacker, T.C., Palmer, M.V., Waters, W.R. 2006. Correlation of Cytokine Gene Expression with Pathology in White-Tailed Deer (Odocoileus virginianus) Infected with Mycobacterium bovis. Clinical and Vaccine Immunology. 13(6):640-647.
11. Palmer, M.V., Waters, W.R., Thacker, T.C., Lyashchenko, K. 2006. Effect of Different Tuberculin Skin-Testing Regimens on Gamma Interferon and Antibody Responses in Cattle Experimentally Infected with Mycobacterium bovis. Clinical and Vaccine Immunology. 13(3):387-394.
12. Palmer, M.V., Thacker, T.C., Waters, W.R. 2006. Vaccination of white- tailed deer with Mycobacterium bovis bacillus Calmette Guerin (BCG). [abstract]. In: Michigan Bovine Tuberculosis 11th Annual Meeting, June 7- 8, 2006, Lansing, Michigan, 2006 CDROM.
13. Nol, P., Palmer, M.V., Waters, W.R., Thacker, T.C., Rhyan, J., Aldwell, F., Buddle, B., Dunbar, M., Salman, M. 2006. Oral bacille Calmette-Guerin (BCG) Vaccination of White-tailed Deer against Bovine Tuberculosis [abstract]. In: Michigan Bovine Tuberculosis 11th Annual Meeting, June 7- 8, 2006, Lansing, Michigan, 2006 CDROM.
14. Foote, M.R., Nonnecke, B.J., Waters, W.R., Palmer, M.V., Beitz, D.C., Fowler, M.A., Miller, B.L., Johnson, T.E., Perry, H.B. 2005. Effects of increased dietary protein and energy on composition and functional capacities of blood mononuclear cells from vaccinated, neonatal calves. International Journal for Vitamin and Nutrition Research. 75(5):357-368.


ACCESSION NO: 0408013 SUBFILE: CRIS
PROJ NO: 3625-32000-063-02R AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 09 DEC 2003 TERM: 30 SEP 2004

INVESTIGATOR: Palmer M V; Waters W R; Whipple D L

PERFORMING INSTITUTION:
Agricultural Research Service
Ames, Iowa 50010

IMPROVED DIAGNOSIS OF TUBERCULOSIS IN CERVIDAE

OBJECTIVES: Evaluate immune responses of reindeer sensitized to Mycobacterium bovis BCG; Evaluate immune responses and diagnostic tests in reindeer experimentally infected with pathogenic M. bovis; Evaluate lesions in reindeer experimentally infected with M. bovis; Evaluate immune responses and lesion development in white-tailed deer experimentally infected with M. bovis.

APPROACH: A group of reindeer will be sensitized with M. bovis BCG and matched with a group of non-sensitized control animals. Blood samples will be collected and skin tests will be conducted periodically throughout the study period. Various immune function assays will be conducted to monitor immune responses. In the second study, reindeer will be challenged with virulent M. bovis. Blood samples will be collected and skin tests will be conducted similar to the first study. In addition, tissue samples will be collected at various times to characterize the progression of disease in reindeer. White-tailed deer will be experimentally challenged with M. bovis using two routes of inoculation and three dosages. Immune responses will be monitored by evaluating blood collected at various times and conducting skin tests. Lesions will be characterized at the conclusion of the study. BSL 3 recertified through 10/15/04. (IBC 239)

PROGRESS: 2003/12 TO 2004/09
4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and USDA-Animal and Plant Health Inspection Service (APHIS)- Veterinary Services. Additional details of research can be found in the report for the parent project 3625-32000-063-00D Diagnosis and control of tuberculosis in livestock and wildlife. Cattle, as well as bison, and all species of Cervidae are subject to testing for tuberculosis under the guidelines of the USDA uniform rules and methods for the eradication of bovine tuberculosis. The most common means of testing is the tuberculin skin test. Tuberculin skin testing lacks specificity and has not been fully validated in all species of Cervidae. Moreover, handling of deer multiple times for skin testing results in unacceptable high losses due to stress and injury to these wildlife species. The APHIS of the USDA has provided funds for NADC to conduct research on improved methods of diagnosis of tuberculosis in Cervidae. The objectives of such research are to study the immune response of tuberculous deer and develop an improved method of antemortem diagnosis of tuberculosis for use in multiple deer species. White-tailed deer and reindeer have been experimentally infected with M. bovis and skin testing as well as blood- based assays were evaluated. Results show that several blood based assays that measure either cell mediated or humoral immunity have promise and may have an application in TB testing of captive as well as free-ranging deer populations. Using non-infected deer from private producers the specificity is being evaluated of one particular assay (Cervigam). The goal is to evaluate 200 samples from each of 4 deer species (white-tailed, elk, fallow, reindeer) to determine the level of false positive responses in non-infected deer. Currently we are at approximately 25% of our goal.

PUBLICATIONS (not previously reported): 2003/12 TO 2004/09
No publications reported this period.


ACCESSION NO: 0409750 SUBFILE: CRIS
PROJ NO: 3625-32000-063-03R AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: TERMINATED
START: 31 OCT 2004 TERM: 30 SEP 2005 FY: 2005

INVESTIGATOR: Palmer M V; Waters W R
PERFORMING INSTITUTION:
Agricultural Research Service
Ames, Iowa 50010

BLOOD BASED ASSAYS FOR DIAGNOSIS OF TUBERCULOSIS IN CERVIDAE

OBJECTIVES: Evaluate immune responses and diagnostic tests in white-tailed deer experimentally infected with pathogenic M. bovis. Evaluate immune responses and lesion development in white-tailed deer experimentally infected with M. bovis.

APPROACH: White-tailed deer will be experimentally challenged with M. bovis. Immune responses will be monitored by evaluating blood collected at various times and conducting skin tests. Lesions will be characterized at the conclusion of the study. Various blood-based assays of cell-mediated and humoral immune responses will be evaluated. BSL 3 recertified through 10/15/05 (IBC 239)

PROGRESS: 2004/10 TO 2005/09
Progress Report 4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and USDA-Animal and Plant Health Inspection Service (APHIS)- Veterinary Services. Additional details of research can be found in the report for the parent project 3625-32000-063-00D Diagnosis and control of tuberculosis in livestock and wildlife. Cattle, as well as bison, and all species of Cervidae are subject to testing for tuberculosis under the guidelines of the USDA uniform rules and methods for the eradication of bovine tuberculosis. The most common means of testing is the tuberculin skin test. Tuberculin skin testing lacks specificity and has not been fully validated in all species of Cervidae. Moreover, handling of deer multiple times for skin testing results in unacceptable high losses due to stress and injury to these wildlife species. The APHIS of the USDA has provided funds for NADC to conduct research on improved methods of diagnosis of tuberculosis in Cervidae. The objectives of such research are to study the immune response of tuberculous deer and develop an improved method of antemortem diagnosis of tuberculosis for use in multiple deer species. White-tailed deer and reindeer have been experimentally infected with M. bovis and skin testing as well as blood- based assays were evaluated. Results show that several blood based assays that measure either cell mediated or humoral immunity have promise and may have an application in TB testing of captive as well as free-ranging deer populations. Using non-infected deer from private producers the specificity of various tests is also being evaluated. The goal is to evaluate 200 samples from each of 4 deer species (white-tailed, elk, fallow, reindeer) to determine the level of false positive responses in non-infected deer. Currently we are at approximately 25%-75% of our goal for most species.

PUBLICATIONS (not previously reported): 2004/10 TO 2005/09
No publications reported this period.


ACCESSION NO: 0407520 SUBFILE: CRIS
PROJ NO: 3625-32000-070-05G AGENCY: ARS 3625
PROJ TYPE: USDA CONTRACT PROJ STATUS: TERMINATED
START: 29 JUL 2003 TERM: 30 SEP 2003 FY: 2003

INVESTIGATOR: Ridpath J F; Roth J A; Palmer M V; Murray P K

PERFORMING INSTITUTION:
College Of Veterinary Medicine
Iowa State University
Ames, Iowa 50011

DISEASES AT THE INTERFACE BETWEEN DOMESTIC LIVESTOCK AND WILDLIFE SPECIES

OBJECTIVES: Support for a national meeting entitled "Diseases at the Interface Between Domestic Livestock and Wildlife Species," July 17 and 18, 2003, held by the Institute for International Cooperation in Animal Biologics (IICAB), at Iowa State University in Ames, Iowa. The transmission of diseases between domestic animals and wildlife species is concern for both those involved in agricultural production and those involved in wildlife conservation. The purpose of this meeting is to promote the exchange of information and foster discussion regarding wildlife-livestock disease interaction.

APPROACH: Abstracts of meeting presentations will be available in the conference proceedings and on the NADC website. This will enable scientist, producers, diagnosticians, wildlife managers and government official to access the information presented. Representatives from the veterinary biologics industry, producer groups, government agencies and veterinary colleges are expected to attend the meeting. The organizing committee is anticipating 150 to 200 attendees.

PROGRESS: 2003/07 TO 2003/09
4. What were the most significant accomplishments this past year? D. Progress Report. This report serves to document support for a conference under an outgoing grant between ARS and Iowa State University. Additional details of research can be found in the report for the parent project 3625-32000-070- 00X, Tools for Differentiation of High Consequence Pathogens and Endemic Viruses. This agreement funded a meeting held in Ames, Iowa on July 17 and 18 of 2004. This meeting was organized because the transmission of diseases between domestic animals and wildlife species is concern for both those involved in agricultural production and those involved in wildlife conservation. The objective was to promote the exchange of information and foster discussion regarding wildlife-livestock disease interaction. There were a total of 183 attendees. Of these, 39 were from educational institutions, 112 were from U.S. federal agencies, 27 were from state agencies or zoos, 3 were from industry and 1 was from a foreign veterinary agency. A program for the meeting may be viewed at the following web site: http://www.nadc.ars.usda. gov/events/wilddom/index.asp.

PUBLICATIONS (not previously reported): 2003/07 TO 2003/09
No publications reported this period.


ACCESSION NO : 0407026 SUBFILE: CRIS
PROJ NO: 3625-32000-071-01G AGENCY: ARS 3625
PROJ TYPE: USDA CONTRACT PROJ STATUS: NEW
START: 07 JUL 2003 TERM: 31 DEC 2003 FY: 2003

INVESTIGATOR: Lager K M; Roth J A

PERFORMING INSTITUTION:
College Of Veterinary Medicine
Iowa State University
Ames, Iowa 50011

INTERNATIONAL CONFERENCE ON EMERGING ZOONOSES

OBJECTIVES: Support for an international meeting entitled "4th International Conference on Emerging Zoonoses," September 18-21, 2003, held by the Institute for International Cooperation in Animal Biologices (IICAB), at Iowa State University in Ames, Iowa. The objective of this meeting is to present research and gather information on the transmission, impact, diagnosis, and control of emerging zoonotic diseases: West Nile virus, Borna disease, rabies, hantavirus, Ebola, Nipah, Hepatitis E, BSE/vCJD, trypanosomiasis, cryptosporidiosis, Salmonella, E. coli 0157:H7, tuberculosis, lyme disease, plague, tularemia, and others.

APPROACH: Abstracts of the presentations that will be given during the meeting will be published in the Conferece Proceedings. This information will also be placed on a web site at ISU/NADC. This will enable scientists and government officials to have access to the important information that will be presented. International representatives from the veterinary vaccine industry, government agencies, veterinary colleges, producer groups, and industry associations are expected to attend the meeting. The organizing committee is anticipating an attendance of 200 scientists.

PROGRESS: 2003/07 TO 2003/12
4. What were the most significant accomplishments this past year? D. Progress Report: This report serves to document the preparation for the meeting "4th International Conference on Emerging Zoonoses" to be held in Ames, Iowa on September 18-21, 2003. Additional details of research can be found in the report for the parent project 3625-32000-071-00D, Emerging Viral Diseases of Swine. The objective of this meeting is to present research and gather information on the transmission, impact, diagnosis, and control of emerging zoonotic diseases. The abstracts will be published in a conference proceedings and will be placed on an Iowa State University/National Animal Disease Center (USDA-ARS-National Animal Disease Center (NADC), Ames, IA,) website providing this information for public viewing.

PUBLICATIONS (not previously reported): 2003/07 TO 2003/12
No publications reported this period.


ACCESSION NO: 0411438 SUBFILE: CRIS
PROJ NO: 3625-32000-082-00D AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 27 OCT 2006 TERM: 26 OCT 2011

INVESTIGATOR: Waters W R; Palmer M V; Thacker T C

PERFORMING INSTITUTION:
Agricultural Research Service
Ames, Iowa 50010

COUNTERMEASURES TO PREVENT AND CONTROL TUBERCULOSIS IN CATTLE AND WILDLIFE RESERVOIRS

OBJECTIVES: 1) Characterize the immunopathogenesis of Mycobacterium bovis infection in domestic livestock and wildlife. 2) Develop and evaluate improved tests for diagnosis of M. bovis infection in different animal species. 3) Identify vaccine strategies to elicit protective immunity in cattle and relevant wildlife species.

APPROACH: Objective 1 will evaluate tonsilar processing of M. bovis and lesion development using a combination of invitro and in vivo methods and both non-infected and experimentally infected cattle and deer. Objective 2 will utilize blood samples from both naturally and experimentally infected cattle and deer to evaluated test sensitivity as well as normal cattle and deer to evaluate test specificity. Vaccine trials in Objective 3 will be limited to efficacy studies utilizing experimentally infected animals and a combination of quantitative and semi-quantitative analysis to evaluate vaccine efficacy. BSL-2/BSL-2N; Recertified May 11, 2006. IBC #0278 BSL-Exempt; Recertified June 8, 2006. IBC #0269 BSL-2/BSL-2N; Recertified May 14, 2006. IBC#0264. BSL-Exempt; (IBC-#0283) 01/12/06. BSL-2/BSL-1N/BSL-3N; Certified June 1, 2006. IBC#0285. BSL-Exempt; Certified November 8, 2006. IBC #0288. BSL-Exempt; Recertified January 12, 2007. IBC #0284.


ACCESSION NO: 0405543 SUBFILE: CRIS
PROJ NO: 3625-32000-082-01R AGENCY: ARS 3625
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 01 FEB 2002 TERM: 31 JAN 2007 FY: 2006

INVESTIGATOR: Whipple D L; Waters W R; Palmer M V

PERFORMING INSTITUTION:
Agricultural Research Service
Ames, Iowa 50010

DIAGNOSIS AND PATHOGENESIS OF TUBERCULOSIS IN ANIMALS

OBJECTIVES: Evaluate immune responses of reindeer sensitized to Mycobacterium bovis BCG; Evaluate immune responses and diagnostic tests in reindeer experimentally infected with pathogenic M. bovis; Evaluate lesions in reindeer experimentally infected with M. bovis; and Evaluate immune respones and lesion development in white-tailed deer experimentally infected with M. bovis.

APPROACH: A group of reindeer will be sensitized with M. bovis BCG and matched with a group of non-sensitized control animals. Blood samples will be collected and skin tests will be conducted periodically throughout the study period. Various immune function assays will be conducted to monitor immune responses. In the second study, reindeer will be challenged with virulent M. bovis. Blood samples will be collected and skin tests will be conducted similar to the first study. In addition, tissue samples will be collected at various times to characterize the progression of disease in reindeer. White-tailed deer will be experimentally challenged with M. bovis using two routes of inoculation and three dosages. Immune responses will be monitored by evaluating blood collected at various times and conducting skin tests. Lesions will be characterized at the conclusion of the study. BSL-1-3-N; Certified through October 15, 2004.

PROGRESS: 2005/10 TO 2006/09
Progress Report 4d Progress report. This report serves to document research conducted under a reimbursable agreement between ARS and USDA-Animal and Plant Health Inspection Service (APHIS)- Veterinary Services (3625-32000-063-01R, Diagnosis and pathogenesis of tuberculosis in animals. Additional details of research can be found in the report for the parent project 3625-32000-063-00D Diagnosis and control of tuberculosis in livestock and wildlife. Cattle, as well as bison, and all species of Cervidae are subject to testing for tuberculosis under the guidelines of the USDA uniform rules and methods for the eradication of bovine tuberculosis. The most common means of testing is the tuberculin skin test. Tuberculin skin testing lacks specificity in deer and cattle and has not been fully validated in all species of deer. Research on modifications to existing blood bases tests for cattle such as the Bovigam are being conducted to improve test specificity. In FY06 ARS began a field study in collaboration with USDA/APHIS to evaluate the specificity of a modified version of the Bovigam in Michigan, USA. Results of the study are being collected and will be evaluated. Preliminary results suggest that modifications can improve test specificity, thus decreasing the number of false positive test results, decreasing the number of cattle euthanized unnecessarily, and decreasing costs to cattle producers and USDA.

PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
No publications reported this period.


ACCESSION NO: 0083779 SUBFILE: CRIS
PROJ NO: IOWV-400-23-09 AGENCY: CSVM IOWV
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 AUG 1980 TERM: 17 SEP 2007 FY: 2006

INVESTIGATOR: Thoen, C. O.

PERFORMING INSTITUTION:
Veterinary Medicine
Iowa State University
Ames, Iowa 50011

MYCOBACTERIAL INFECTIONS IN ANIMALS

OBJECTIVES: Characterization of mycobacterial isolates from domestic and exotic
animals.

APPROACH: In vitro investigations will include biochemical and drug susceptibility tests; ELISA will be conducted to select humoral antibodies in experimentally or naturally infected animals.

PROGRESS: 2003/01 TO 2003/12
Mycobacteriologic examinations were conducted on tissues and fecal specimens submitted from 497 animals. Mycobacterium avium ss paratuberculosis was isolated from 35 (7%) of the specimens. M. avium ss avium was isolated from 8 specimens. Rapidly growing nonphotochromogenic mycobacteria were isolated from 11 specimens. M. tuberculosis was isolated from 2 specimens.

IMPACT: 2003/01 TO 2003/12
The isolation of M. avium ss paratuberculosis is in support of research to develop improved diagnostic tests for Johne's disease in cattle. The disease is widespread in dairy herds in Iowa and causes significant economic losses to producers.
PUBLICATIONS (not previously reported): 2003/01 TO 2003/12
No publications reported this period

PROJECT CONTACT:
Name: Thoen, C. O.
Phone: 515-294-7608
Fax: 515-294-8500
Email: cthoen@iastate.edu


ACCESSION NO: 0205410 SUBFILE: CRIS
PROJ NO: LAB03774 AGENCY: SAES LA.B
PROJ TYPE: STATE PROJ STATUS: NEW
START: 01 OCT 2005 TERM: 30 SEP 2010 FY: 2006

INVESTIGATOR: Elzer, P.; Sanders, D.; Enright, F.; French, D.; Bastian, F.

PERFORMING INSTITUTION:
Veterinary Science
Louisiana State University
Baton Rouge, Louisiana 70893

INVESTIGATION OF EMERGING INFECTIOUS DISEASES IN SMALL RUMINANTS, INCLUDING WHITE-TAILED DEER

NON-TECHNICAL SUMMARY: In order to reach valid statistical conclusions with adequate numbers, the cost of large animal research is prohibitive. The characterization of a small ruminant model to evaluate the pathogenesis of and immune response to various infectious agents and the development of potential vaccine candidates for these pathogens will benefit animal researchers and producers.

OBJECTIVES: To expand the established brucellosis small ruminant model system to study emerging infectious diseases of domestic livestock and wildlife. This small ruminant model system has been tested using three important regulatory diseases of cattle: brucellosis, tuberculosis and Johne's disease. Two of these diseases are caused by zoonotic bacterial pathogens, one of which is classified as a biological terrorism agent. The goal of this project is to expand the capacity of the model system to look at Transmissible Spongiform Encephalopathies (TSE) and other future regulatory/emerging pathogenic diseases of domestic livestock and/or wildlife species.

APPROACH: In this phase of the project, we plan to inoculate neonatal goats, sheep, and white-tailed deer with Spiroplasma in an attempt to induce TSE. Neonates will be used due to their probable increased susceptibility and reduced incubation period. Inoculation will be accomplished via intracranial injection in the right hemisphere between the fontanels using a 21-gauge needle. Groups of animals will be experimentally inoculated with bacteria and equal numbers will be injected with media to serve as controls. Animals will be euthanatized upon onset of clinical signs or at the termination of the study if no signs develop. Control animals will be sacrificed concurrently with experimental animals. For acute studies animals will be sacrificed monthly to monitor changes in brain pathology prior to manifestation of clinical signs.

PROGRESS: 2006/01 TO 2006/12
Neonatal goats and sheep were inoculated via intracranial injections in the right hemisphere between the fontanels using a 21-gauge needle with Spiroplasma in an attempt to induce Transmissible Spongiform Encephalopathies (TSE). Control groups received injections of media. Animals were euthanatized one year post inoculation since no clinical signs developed. Upon necropsy, numerous histological lesions similar to TSE like lesions were observed in the brains of the Spiroplasma injected animals compared to none in the control animals. White tailed-deer have currently been injected with Spiroplasma in an attempt to induce TSE like symptoms and or lesions.

IMPACT: 2006/01 TO 2006/12
The goal of this project is to expand the capacity of the model system to look at TSE and other future regulatory/emerging pathogenic diseases of domestic livestock and/or wildlife species. The results of this project will lead to the development of a ruminant model and diagnostic tests to assist in the control and or eradication of these diseases.

PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
No publications reported this period

PROJECT CONTACT:
Name: Elzer, P. H.
Phone: 225-578-4763
Fax: 225-578-4890
Email: pelzer@agcenter.lsu.edu


ACCESSION NO: 0189284 SUBFILE: CRIS
PROJ NO: LAB93527 AGENCY: CSREES LA.B
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 MAY 2001 TERM: 30 OCT 2006 FY: 2006

INVESTIGATOR: Elzer, P. H.

PERFORMING INSTITUTION:
Veterinary Science
Louisiana State University
Baton Rouge, Louisiana 70893

EVALUATION OF B. ABORTUS RB51 AS A MULTIVALENT VACCINE TO GENERATE IMMUNE RESPONSES AGAINST BRUCELLOSIS, TUBERCULOSIS AND JOHNE'S IN CATTLE.

NON-TECHNICAL SUMMARY : Brucellosis, Tuberculosis and Johne's Disease are three major bacterial diseases which have a negative impact on the cattle industry in the United States. The long term goal of our research program is to develop a recombinant RB51 strain that would function as a highly efficacious live multivalent vaccine against three important chronic intracellular bacterial diseases: brucellosis, tuberculosis (Tb), and paratuberculosis (Johne's).

OBJECTIVES: 1.Determine the localization and number (colony forming units CFU) of Brucella abortusRB51 constructs expressing Mycobacterium bovis and M. avium paratuberculosis antigens in the lymphoid tissues of Brucella , Mycobacterum naive, sexually mature, female cattle at 7, 14, 21, 28 and 42 days post inoculation. 2. Determine the localization of selected rough B. abortus mutants expressing the mycobacterial antigens and their pathogenic potential when administered to late gestational cattle.

APPROACH: Sixty beef cattle, which are not vaccinated for brucellosis and tuberculosis negative, will be used in this study and will be divided into 6 groups. 1. Strain RB51 overexpressing SOD and expressing the 85A antigen from M. bovis BCG (strain RB51SOD/85A) 2. Strain RB51 overexpressing SOD and expressing the 35 kDa antigen from M. paratuberculosis (strain RB51SOD/35) 3. Strain RB51 overexpressing SOD and expressing the ESAT6 antigen from wild type M. bovis (strain RB51SOD/ESAT6) 4. Strain RB51 overexpressing SOD and expressing the three antigens, ESAT-6, 85A, and 35 kDa antigen (strain RB51SOD/3xAG) 5. RB51 overexpressing SOD (vector control) 6. Saline (negative control) All animals will receive intramuscularly (im) 1-3 x1010 CFU of RB51 expressing the different antigens. At 7, 14, 21, 28, and 42 days post-vaccination, 2 animals from each group will be sacrificed using a captive bolt. Forty-eight hours prior to necropsy, skin tests will be performed using Brucella and Tb antigens. The animals will then be necropsied, and sera and tissues will be removed for the culturing of Brucella and for histology. The skin test site and a small section of each tissue will be removed and placed in 10% buffered formalin for histology. The remaining tissues will be individually homogenized and plated on Brucella-selective media. After 3 to 14 days of incubation at 37C in 5% CO2, the plates will be counted; and selective isolates will be tested using molecular techniques to make sure the mutant strains have not changed. Fixed tissues will be stained using H&E and morphological changes will be recorded compared to control tissues. Prevaccination and necropsy serum samples will be tested using standard brucellosis diagnostic tests (all which should remain negative), Western blot analysis, and ELISA. Thirty sexually mature beef cattle, which are not vaccinated for brucellosis and tuberculosis negative, will be used in this study and will be divided into 6 groups. The animals will be bred, and pregnancies will be time-dated. At 200 days pregnancy, 5 animals from each experimental group will be injected intramuscularly with1-3x1010 cfu of the different RB51 strains and 5 animals will be infected conjunctivally with 1x107 cfu of the virulent field strain 2308.Pregnancies will be monitored; and abortions, premature or live births will be recorded. At the time of birth or abortion, the fetus, fetal membranes, and maternal membranes will be collected. A portion of the fetal and maternal membranes will be taken and placed in 10% buffered formalin and the remainder will be cultured. The following tissues will be taken for histology and Brucella culture from the fetus: lungs, liver, spleen, adrenal glands, thymus, internal iliac ln, and blood. The cows will be sacrificed and necropsied as described in Specific Aim 1. Histological and immunological analyses will also be performed as described above.

PROGRESS: 2001/05 TO 2006/10
Vaccinating animals against brucellosis, specifically cattle and swine, with a vaccine that is safe and efficacious, aids in the protection of domestic and wild animals from this zoonotic or potential agroterrorist pathogen. Rough Brucella abortus vaccine derivatives of strain RB51 were used to express heterologous antigen preparations from Mycobacterium bovis (MB), M. avium paratuberculosis (MAP) and Pseudorabies virus (PRV). Cattle vaccinated with RB51 expressing MB or MAP antigens generated the appropriate cell mediated and or humoral responses to the antigens. These vaccines provided significant protection against virulent brucellae challenge. When RB51-MB vaccinated cattle were challenged with MB, significant protection was observed with the vaccine strain expressing Esat-6 of MB. These results were also confirmed with histological observations. Swine vaccinated with RB51-PRV or a rough strain of B. suis expressing PRV antigens (VTRS-PRV) generated humoral immune responses to the PRV antigen, and both vaccines provided significant protection against virulent brucellae challenge in swine. When used in pregnant animals, none of the above vaccines induced abortions or any negligible gross pathological lesions. Vaccination with RB51 expressing antigens from other facultative intracellular pathogens provides protective immunity against both homologous and heterologous organisms. A duel purpose vaccine will be of benefit to producers in areas where the above mentioned diseases pose a risk of transmission to traditional livestock populations from feral or wild animals.

IMPACT: 2001/05 TO 2006/10
A disease-free food animal population is imperative to the well-being of all individuals. The regulatory disease addressed in this study deleteriously impacts the economics of cattle and swine producers, directly affecting the market price and interstate and international import/export potential of the animals, which in turn influences all consumers. As zoonotic organisms, Brucella species pose a human health threat, hence a protected animal population benefits the general public. Brucellosis animal vaccine work has a significant impact in protecting the human population since Brucella species are also known as bioterrorist agents or "agents of mass destruction."

PUBLICATIONS (not previously reported): 2001/05 TO 2006/10
1. Nielsen, K, P. Smith, W. Yu, P. Nicoletti, P.H. Elzer, C. Robles, R. Bermudez, T. Renteria, A. Ruiz, C. Massengill, Q. Muenks, G. Jurgersen, T. Tollersrud, L. Samartino, S. Conde, L. Forbes, D.Gall, B. Perez, X. Rojas, and A. Minos (2005). Towards a single screening test for brucelloisis. Res. Sci. Off. Int. Epiz 24(3):1027-1038.
2. Zygmunt, M.S., S.D. Hagius, J.V. Walker, and P.H. Elzer. (2006). Signature tagged mutagenesis identification of Brucella melitensis 16M genes required for bacterial survival in the caprine host. Microbes Infect. 2006 Oct 16; [Epub ahead of print]
3. Roux, C.M., N.J. Booth, B.H. Bellaire, J.M. Gee, R.M. Roop, M.E. Kovach, R.M. Tsolis, P.H. Elzer, and D.G. Ennis. (2006). RecA and RadA proteins of Brucella abortus do not perform overlapping protective DNA repair functions following oxidative burst. J. Bacteriology, 188(14):5187-95.
4. Kahl-McDonagh, M.M., P.H. Elzer, S.D. Hagius, J.V. Walker, Q.L. Perry, C.M. Seabury, R.M. Tsolis, L.G. Adams, D.S. Davis and T. Ficht. Evaluation of novel Brucella melitensis unmarked deletion mutants for safety and efficacy in the goat model of brucellosis. (2006) Vaccine. Jun 12;24(24):5169-77.

PROJECT CONTACT:
Name: Elzer, P. H.
Phone: 225-578-4763
Fax: 225-578-4890
Email: pelzer@agctr.lsu.edu


ACCESSION NO: 0209503 SUBFILE: CRIS
PROJ NO: LAB93841 AGENCY: CSREES LA.B
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: NEW
START: 01 NOV 2006 TERM: 31 OCT 2011

INVESTIGATOR: Elzer, P. H.; French, D. D.

PERFORMING INSTITUTION:
Veterinary Science
Louisiana State University
Baton Rouge, Louisiana 70893

EVALUATION OF B. ABORTUS RB51 AND B. SUIS VTRS1 AS MULTIVALENT VACCINES TO GENERATE IMMUNE RESPONSES AGAINST BRUCELLOSIS, TUBERCULOSIS AND P [ title incomplete from database]

NON-TECHNICAL SUMMARY: The eradication of bovine and swine brucellosis and tuberculosis from cattle and pigs within the United States remains a major goal of the USDA. Although eradication is essentially complete, isolated pockets of disease continue to plague both programs. The long term goal of our research program is to develop recombinant sp. strains that would function as highly efficacious live multivalent vaccines against three important chronic intracellular diseases: brucellosis, tuberculosis, and pseudorabies.

OBJECTIVES: The long term goal of our research program is to develop recombinant Brucella sp. strains that would function as highly efficacious live multivalent vaccines against three important chronic intracellular diseases: brucellosis, tuberculosis, and pseudorabies.

APPROACH: 1. Determine the localization and number of colony forming units (cfu) of Brucella abortus and B. suis VTRS1 constructs in the lymphoid tissues of Brucella, Mycobacterium-naive, sexually mature female cattle and swine at 7, 14, 21, 28 and 42 days post-inoculation. 2. Determine the localization of selected rough B. abortus and B. suis mutants expressing brucella, mycobacterial or pseudorabies antigens and their pathogenic potential when administered to late-gestational cattle or swine. 3. Determine the vaccine efficacy of rough B. abortus and B. suis mutants in pregnant swine against virulent B. suis challenge.

PROJECT CONTACT:
Name: Elzer, P. H.
Phone: 225-578-4763
Fax: 225-578-4890
Email: pelzer@agcenter.lsu.edu


ACCESSION NO: 0194591 SUBFILE: CRIS
PROJ NO: MICL02050 AGENCY: CSREES MICL
PROJ TYPE: HATCH PROJ STATUS: TERMINATED
START: 01 NOV 2002 TERM: 31 DEC 2004 FY: 2005

INVESTIGATOR: Woods, M. D.; Beckwith, J.
PERFORMING INSTITUTION:
Dept. of Community, Agriculture, Recreation & Resource Studies
Michigan State University
East Lansing, Michigan 48824

THE IMPACT OF COMMUNICATING ANR RISKS ON STAKEHOLDER PARTICIPATION AND PUBLIC POLICY

NON-TECHNICAL SUMMARY: As public perceptions and concerns within Michigan towards ANR risks continue to increase, so has the need to expand research on the impact of communicating ANR risks on stakeholder participation and public policy. This project investigates the proposition that wider stakeholder participation in Michigan ANR risk issues will be beneficial to building both trust relationships and public understanding, while mapping possible unintended consequences of public participation.

OBJECTIVES: ANR risk communication and perception research is a complex and often controversial activity that is both a product of analysis and dependent on the processes of defining and conducting analysis. This MAES umbrella research project will assess opportunities to improve the communication of risk to better inform decision-making and enhance the resolution of controversies over risk in Michigan ANR industries. The project will address: 1) technical issues such as the representation of uncertainty; 2) issues relating to translating the outputs of conventional risk analysis into non-technical language; and 3) the impact of communicating ANR risks on stakeholder participation and public policy. Specifically, the long-term goal of this research project is to contribute knowledge of risk communication and perception in a way that is genuinely interdisciplinary and attains theoretical integration across several levels of analysis. A key concern will be to understand the logic and dynamics of risk and trust and how stakeholder participation relates to: institutional behavior, public policy, the social and cultural networks that people inhabit, and new forms of risk communication. The objective of this project is to investigate the proposition that wider stakeholder participation in Michigan ANR risk issues will be beneficial to building both trust relationships and public understanding, while mapping possible unintended consequences of public participation. Specifically four projects over a five-year period will be conducted, each addressing a distinct set of issues (see objective section for specific aims of each project). At the core of the research proposal (Project A) will be a set of major in-depth empirical investigations of public perceptions of key biological, behavioral, and societal risk issues in Michigan (i.e. land use, bovine tuberculosis, food safety, etc.). The data from these core cases will be used to inform parallel projects on institutional handling of risk (Project B) on public expectations, risk communication and new institutional forms (Project C) and on stakeholder involvement in public policy decision making (Project D).

APPROACH: For this project, the researcher will examine the research objectives and specific aims through Q modeling. Q is a technique for studying human subjectivity. Every person perceives the world differently, and Q uses these subjective viewpoints to construct typologies of different perspectives. Moreover, through the use of Q methodology, the PI will be allowed the opportunity to blend both quantitative and qualitative methods in order to gain both the breadth and depth of the population's perspectives towards ANR risks. Q is different from typical correlation methodology. Correlation statistics with which most researchers are familiar are the main types of statistical techniques used to measure inter-individual differences. Q, in contrast, excels at measuring intra-individual differences or subjectivity, but not at the expense of group comparison. The PI sees the uniqueness of Q as its power to blend knowledge created through qualitative analysis and quantitative analysis. Q, as the PI intends to use it, would begin with a qualitative or interpretive phase, then cycle into a quantitative phase. After statistical analysis, the PI will then cycle into another interpretive phase. Theoretically, Q differs fundamentally from correlation methodology. In contrast to R, Q measures an individual's conceptualization of an issue from his or her point of view rather than the subjective interpretation of a construct as defined by a researcher. It may seem counter-intuitive that subjective statements can be administered in an objective fashion to people, each assumed to possess a unique worldview. However, one key to the subjectivity/objectivity dilemma lies in the origin and treatment of the statements themselves. The Q statements are placed by people in order of agreement in relation to one another. The result is a scale that is anchored in the respondent's own subjective reality as opposed to one that is constructed and anchored for the respondent by the researcher. One of the basic tenets of Q lies in the treatment of individuals as variables and conceptualizations as traits. The individuals are then factor-analyzed.

PROGRESS: 2002/11 TO 2004/12
This project helps build understanding of ways to engage citizens in dialogue about possible policy directions when risk is uncertain. This project used a National Issues Forum style of deliberation to engage citizens in small-group dialogue meetings about cleanup choices for a hazardous contamination problem in mid-Michigan. Citizens who participated live in the area affected by dioxin contamination in the Tittabawassee River sediment and floodplain soils. The research is significant to understanding how citizens who are not affiliated with activist or other stakeholder groups engage in an issue that affects them. It provides citizens with educational support on the topic, facilitated discussion groups, and surveys their knowledge and opinions before and after the forum. Progress this year included the development of an Issue Guide that was used by forum participants. This guide was the result of researcher investigation of the topic and stakeholder interviews. Forum participants were recruited by door-to-door efforts that surveyed citizens and screened those ineligible due to an affiliation. Nine forums were conducted in July and August of 2004. Surveys were repeated 30-days after the forum to determine stability of attitudes and opinions. The data are qualitative and analysis is underway. It is too soon to report results.

IMPACT: 2002/11 TO 2004/12
Expected impacts from the funded project associated with my Experiment Station project include: 1.The proposed methodology will measure citizen preference with respect to choices and trade-offs associated with a specific site remediation 2.The proposed methodology will utilize local citizen knowledge. 3.A key component of the proposed project is a methodology for evaluating the effectiveness of the public issues forums. 4.Another key component of the methodology is the preparation of unbiased and accurate background documents that are written in a manner appropriate for ordinary citizens to engage in deliberative discourse on policy choices associated with the environmental cleanup in their community. 5.An output of the study will be an outreach tool that could be used by the EPA and others to conduct similar public involvement exercises in other communities with environmental cleanups.

PUBLICATIONS (not previously reported): 2002/11 TO 2004/12
1.United State Environmental Protection Agency, & Michigan State University 2004. Dioxin Contamination and the Tittabawassee River and Floodplain. Managing the Risks: An Issue Guide.

PROJECT CONTACT:
Name: Woods, M. D.
Phone: 517-355-6580
Fax: 517-353-4981
Email: mwoods@msu.edu


ACCESSION NO: 0181070 SUBFILE: CRIS
PROJ NO: MICL06891 AGENCY: CSREES MICL
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 1998 TERM: 30 SEP 2003 FY: 2003

INVESTIGATOR: Scribner, K.

PERFORMING INSTITUTION:
Fisheries and Wildlife
Michigan State University
East Lansing, Michigan 48824

PREDICTIVE GENEALOGICAL MODEL FOR TRANSMISSION OF TUBERCULOSIS IN FREE-RANGING MICHIGAN DEER

OBJECTIVES: The long-term goals are to use molecular genetic techniques, detailed data on animal ecology and movement patterns, and spatial statistical methods to provide critical data which would elucidate the mechanism(s) of transmission of Bovine Tuberculosis in free-ranging white-tailed deer in Michigan and to aid in elimination. The general objectives are to characterize the extent of spatial genetic structuring and degree of genetic relatedness among deer from areas of high and low TB prevalence. Estimate of inter-individual relatedness will be examined as a potential predictor of incidence of TB infection.

APPROACH: Subsamples of the entire white-tailed deer hunt in NE Michigan will be selected for genetic analysis. All deer which have been tested positive for TB will be genotyped. Uninfected deer will be chosen to provide samples of comparable geographic dispersion as the TB+ deer. Each deer will be genotyped for each of 15-20 polymorphic microsatellite loci. Estimate of the degree of genetic similarity among individuals will be quantified using the coefficient of relationship, which is based on the number of alleles individuals share. This analysis will explicitly test whether co-infection is significantly correlated to geographic proximity and to genealogical relationship (and inferentially kinship).

PROGRESS: 1998/10 TO 2003/09
Zoonnoses are of increasing importance to wildlife conservation and human health. Ecological attributes of wildlife species are increasingly recognized as playing a key role in disease transmission in natural populations. In domestic populations, contacts among individuals are controlled by humans and disease transmission is often density dependent. Unlike domestic animals, natural wildlife populations often have complex social systems that can plan an important role in the transmission and maintenance of disease in a density-independent manner. White-tailed deer (Odocoileus virginianus) in the northeast lower peninsula of Michigan (MI) are infected with bovine tuberculosis (Mycobacterium bovis)(TB). Wide-spread use of artificial feeding brought large numbers of deer into contact and likely facilitated the transmission of TB. White-tailed deer ecology may also play an important role in the probability of infection with TB. Deer have a complex social system in which females live in related groups (matrilines). The rate of contact among individuals within matrilines is high relative to contact rates among individuals from different matrilines. Estimates of genealogical relationships were used to infer the role that white-tailed deer social structure played in the risk of TB infection. TB-infected deer were significantly more closely related than were non-infected deer, suggesting that matrilines serve as reservoirs of TB within free-ranging deer populations. White-tailed deer matrilineal social structure would be expected to result in spatial hetergeneity in allele frequencies. Artificial feeding of deer in MI, however, resulted in the congregation of large numbers of individuals, potentially from large areas, at artficial feeding sites. Molecular markers were used to characterize the impact of artificial feeding on deer spatial genetic structure in the northeast lower peninsula of MI. Spatial autocorrelation analyses revealed that when artificial feeding occurred, no sigificant relationship between degree of genetic differentiation and geographic distance was observed. The aggregation of multiple matrilines at feeding sites likely homogenized spatial genetic structure. Following the ban on artificial feeding, there was significant heterogeneity in allele frequencies among groups of deer as a function of genetic distance. The significant microgeographic genetic structure that exists within the deer population following the ban on artificial feeding indicates that transmission of TB across genetically differentiated groups is likely to be limited.

IMPACT: 1998/10 TO 2003/09
Results of this study have been widely cited across the country. Information is being used by federal and state agencies to develop better predictive capabilities in areas of disease prevalence and distribution.

PUBLICATIONS (not previously reported): 1998/10 TO 2003/09Blanchong, J.A. 2003. Genealogical relationships influence the probability of infection with bovine tuberculosis and microgeographic genetic structure in free-ranging white-tailed deer. PhD dissertation, Michigan State University, 102pp.


ACCESSION NO: 0188199 SUBFILE: CRIS
PROJ NO: MICL07664 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2001-34427-10444 PROPOSAL NO: 2001-03570
START: 01 JUL 2001 TERM: 30 JUN 2004 FY: 2004 GRANT YR: 2001
GRANT AMT: $303,339

INVESTIGATOR: Kaneene, J. B.; Fitzgerald, S. D.; Bartlett, P. C.; Griffore, F.;
Bolin, S.; Bolin, C.

PERFORMING INSTITUTION:
Population Medicine Center
Michigan State Univ
East Lansing, Michigan 48824

EPIDEMIOLOGY AND RISK ANALYSIS OF MYCOBACTERIUM BOVIS IN WILD AND DOMESTIC ANIMALS IN MICHIGAN

NON-TECHNICAL SUMMARY: Bovine tuberculosis (TB) in Michigan is being recognized as more prevalent in wildlife and livestock than originally thought. We have seen bovine TB in beef and dairy cattle and in a number of wildlife species. The current combination of active and passive surveillance programs, as well as control and eradication efforts, have shown that more research is needed to provide information needed to deal with the problem. The first focus of these projects is to identify factors that will influence whether a cattle herd will develop TB. Next, these projects will look at the effectiveness of tests used to detect TB in cattle. Finally, the projects will look at the impact of bovine TB on farm families and communities in the TB-affected area.

OBJECTIVES: Specific objectives are to: 1) conduct epidemiological studies to determine major risk factors influencing TB transmission in livestock and deer, 2) refine and validate preliminary risk analysis models, 3) evaluate the effect of M. paratuberculosis status on the reliability of the CFT in cattle, 4) use new risk analysis approaches to estimate the rate of false positives on the CFT and CCT, 5) determine whether wild rodents are possible TB reservoirs, and 6) determine the social impact of bovine TB on farm families and farm communities.

APPROACH: Objective 1: A retrospective epidemiological study will examine the association between M. bovis infection and physical landscape factors. Environmental samples will be taken from livestock operations with confirmed M. bovis infections and processed for bacterial isolation, identification, and typing. A cross-sectional study will identify different wildlife species with bovine TB, determine the most likely routes of infection for each species, and estimate the potential of each species to be hosts for M. bovis by describing pathogenicity and assessing the possibility of shedding through different routes. A retrospective epidemiological analysis will be conducted to examine the association between the occurrence of M. bovis on farms with M. bovis in deer, deer-related supplemental feeding, and physical landscape factors. Objective 2: A stochastic simulation model, using cattle herd factors, deer factors, deer feeding factors, and land use factors, will be developed to estimate the risk of a dairy or beef cattle herd developing TB infection in a year. Objective 3: From dairy herds in the Michigan Johne's Control Program, three herds will be selected: one high prevalence herd (> 15%); one low prevalence herd (< 2%); and one Johne's-free herd. Each animal will receive a bovine TB caudal fold test, comparative cervical test, and gamma-interferon tests, and TB test results will be compared by Johne's disease status. Objective 4: A database and data entry template for infected Michigan cattle herds will be developed for CFT and CCT data. Risk analysis software will be used to generate frequency distributions of tuberculosis prevalence in different sub-populations of domestic ruminants. Risk analysis software will be used to generate frequency distributions of rates of false positives and negatives, and true positives and negatives, from the Michigan TB testing program for livestock. Objective 5: M. bovis of deer-origin from Michigan will be used to inoculate prairie voles by oral gavage and intranasal installation. Groups of oral-inoculated, nasal-inoculated, and control rodents will be euthanized at days 30 and 60 post-inoculation. Necropsies, histopathology, acid-fast staining and mycobacterial isolation will be evaluated for rodent susceptibility to infection with M. bovis and ability to shed the organism. Objective 6: Followup interviews with farm families affected by bovine TB in northeastern Michigan will be conducted to examine patterns of adaptive behavior and long-term impact of bovine TB on farm families. Families with new TB herds, and farmers in the area who have not yet been directly affected by TB will be interviewed. Interviews have been conducted with members of various stakeholder groups, and a subset of these interviewees will be contacted to aid in clarification of the results. A public opinion survey will collect attitudes about the Michigan TB situation from key personnel in state-level agricultural and natural resource agencies across the US. Data collected by the questionnaire will be evaluated to assess respondents' reactions to the TB situation and areas where additional information or education are needed.

PROGRESS: 2001/07 TO 2004/06
The first aim of this project was to determine the spatial relationships of bovine TB (bTB) in white-tailed deer, relating to factors in the physical landscape and location-specific human activity. Spatial clusters of TB were detected in areas that encourage deer to congregate for long periods of time. One paper has been submitted for publication. To identify factors that may influence whether a cattle herd will develop TB, a matched case-control study of herds was conducted to identify herd management factors and environmental conditions associated with TB (Kaneene et al., 2002). A stochastic risk assessment model for herd TB status was developed, based on results of this study, and has been integrated with economic data to create a management tool to develop recommendations to reduce the TB risk for individual cattle farms. The on-farm program is undergoing field testing. The study on the effect of Johnes disease (JD) on the caudal fold tuberculin test (CFT) in herds without TB was completed and submitted for publication. Fecal culture and antibody ELISA for M. avium ssp. paratuberculosis, were performed on cattle from 10 herds. Blood samples were taken and subjected to gamma-interferon (GI) tests for M. bovis and JD. Cattle positive for JD by fecal culture, ELISA, or GI appear to be more likely to be false + on CFT than were negative cattle, and no associations were found between + fecal culture or ELISA with GI for M. bovis. To determine the effectiveness of current TB testing in Michigan cattle, cattle from TB-infected herds were examined by gross necropsy, histopathologic exam, mycobacterial culture, and PCR. Bayesian inference was used to estimate the sensitivity and specificity of the CFT and comparative cervical tuberculin test (CCT) using two-population-two-tests latent-class models. Bayesian estimates of the sensitivity and specificity of the CFT and CCT were 85 and 94%, and 76 and 99%, respectively, which agrees with reports from other studies in the U.S. Two papers have been submitted for publication. To evaluate the role of rodents as possible reservoirs of M. bovis, an experimental study was conducted to study the relative susceptibility of 'wild-type' rodents to inoculation with M. bovis by the oral and intranasal routes. M. bovis was cultured from the feces of 9 oral inoculates and 8 intranasal inoculates on day 1 post-inoculation, and from fecal samples of 3 intranasal inoculates at day 30 post-inoculation, and also from pooled tissue samples. Results of this study are being prepared for publication. In-person interviews were conducted with farm families to measure the social impacts of bTB, and study findings indicate that families can adapt to the changes imposed by the presence of TB on their farms, but they experienced problems in receiving information in a timely fashion, and inconsistency and inequity in the application of government policies and procedures. To lessen negative impacts, families should be accorded more attention and consideration when policies are made, and should have a more substantial role in decision-making as it relates to their own farms. Two manuscripts are being submitted for publication.

IMPACT: 2001/07 TO 2004/06
Control and eradication of TB from the Michigan livestock industry requires an understanding of the disease and how it spreads, efficient disease detection methods, and the development of tools for control of the disease at the farm level. Risk assessment models can be used to develop sound, cost-effective disease control programs. The reliability of the caudal fold skin test, used for TB testing in livestock, may be affected by an animal's disease or vaccination status. These factors should be taken into consideration when interpreting TB skin test results, or designing a TB surveillance program. Current TB tests require great time, effort and expense, and false results are common with existing skin and blood tests. Even with TB lesions, many times associated with acid-fast bacilli, bacteria cannot be cultured because of poor samples, freeze-thaw, or lack of available fresh tissues. New DNA-based testing methods (cDNA microarray analysis of gene expression and laser-capture microscopy for isolating DNA for PCR) have the potential to become rapid, sensitive methods to identify TB in tissue samples in an efficient and reliable way. With a better understanding of the stresses and social impacts of bovine TB on Michigan farm families, programs can be designed to reduce the negative impact of TB control and surveillance programs on families' lives.

PUBLICATIONS (not previously reported): 2001/07 TO 2004/06
1. Dunn, J.R., Kaneene, J.B., Grooms, D.L., Bolin, S.R., Bolin, C.A., Bruning-Fann, C.S. Testing positive for Mycobacterium avium subsp. paratuberculosis and the lack of significant effect on the caudal fold tuberculin (CFT) and gamma interferon tests for bovine tuberculosis. Am J Vet
Res, accepted for publication 2004.
2. Fitzgerald, S.D., Boland, K.G., Clarke, K.R., Wismer, A., Kaneene, J.B., Berry, D.E., Church, S.V., Hattey, J.A., C.A. Bolin. Experimental Inoculation of Mallard Ducks (Anas platyrhynchos) with Mycobacterium bovis. Avian Dis. submitted 2004.
3. Griffore, R., Phenice, L. The Impact of Bovine TB on the Farm Family Ecosystem. In preparation for submission to Family Relations, 2004.
4. Griffore, R., Phenice, L; Kaneene, J.B. Veterinarians: A Complex Role of Mediation Between the State and Farm Families. In preparation for submission to Journal of the American Veterinary Medical Association, 2004.
5. Miller, R., Kaneene, J.B., Schmitt, S.M., Lusch, D.P., Fitzgerald, S.D.
Geographic distribution and spatial analysis of Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus) in Michigan. Prev Vet Med. accepted
2004.
6. Norby, B., Bartlett, P.C., Fitzgerald, S.D., Granger, L., Bruning-Fann, C.,
Whipple, D.L., J.B. Payeur. The Sensitivity of Gross Necropsy, Caudal Fold and Comparative Cervical Tests for the Diagnosis of Bovine Tuberculosis. J Vet Diagn Invest. 16:126-131, 2004.
7. Norby, B., Bartlett, P.C., Grooms, D.L., Kaneene, J.B., Bruning-Fann, C.S. Herd-level sensitivity, specificity, and predictive values of bovine tuberculosis skin tests in Michigan. Am J Vet Res. submitted 2004.
8. Norby, B. Tempelman, R.J., Hansonc, T.E., Kaneene, J.B., Bartlett, P.C. Estimation of sensitivity and specificity of bovine tuberculosis skin tests in Michigan when a perfect reference test is not available. Prev Vet Med. submitted 2004.
9. O'Brien, D.J., Schmitt, S.M., Berry, D.E., Fitzgerald, S.D., Vanneste, J.R., Lyon, T.J., Church, S.V., Fierke, J.S., Schooley, A.M., Cooley, T.M., Magsig, D., Zwick, L., and B.V. Thomsen: Estimating the True Prevalence of M. bovis in Hunter-harvested White-tailed Deer in Michigan. J Wildl Dis. 40:42-52, 2004.

PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-353-5941
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu


ACCESSION NO: 0191695 SUBFILE: CRIS
PROJ NO: MICL07676 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2002-34427-11829 PROPOSAL NO: 2002-06051
START: 01 SEP 2002 TERM: 31 AUG 2004 FY: 2006 GRANT YR: 2002
GRANT AMT: $297,445

INVESTIGATOR: Kaneene, J. B.; Fitzgerald, D. D.; Bolin, S. R.; Bartlett, P. C.;
Bolin, C. A.

PERFORMING INSTITUTION:
Population Medicine Center
Michigan State University
East Lansing, Michigan 48824

BOVINE TUBERCULOSIS: EPIDEMIOLOGY, DIAGNOSIS, AND PATHOGENESIS

NON-TECHNICAL SUMMARY: Since bovine TB control programs for livestock have significant costs, there is a need to improve the performance of these programs. The proposed research will evaluate the costs of current TB control programs, the reliability of TB tests being used, and factors that influence the effectiveness of these programs. In addition, the study will try to identify new, more efficient methods of identifying bovine TB in cattle.

OBJECTIVES: There are six projects with specific objectives: Project 1 Objective: to develop a predictive risk assessment model, combining epidemiological and economic risk factors, for the effects of bovine TB infection on the Michigan cattle industry; Project 2 Objectives: 1) determine the sensitivity, specificity and predictive values and reliability of the CFT and the gamma-interferon blood test in Johne's-positive cattle herds, using the comparative cervical skin test (CCT) and mycobacterial culture for validation; 2) compare results of the CFT from Johne's-positive cattle with results from Johne's-negative cattle, using the CCT and mycobacterial culture for validation; and 3) to determine if cattle with advanced Johne's Disease are immunologically competent to respond appropriately to M. bovis antigens; Project 3 Objectives: 1) study the relative susceptibility of wild-type rodents to oral inoculation with M. bovis; 2) attempt to isolate M. bovis from inoculated rodents through fecal culture at multiple times post-inoculation, and from various organs at necropsy; 3) evaluate various tissues microscopically at multiple times post-inoculation to understand the pathogenesis of M. bovis in these rodents; and 4) evaluate rodents for the threat they pose in re-introduction of M. bovis to cattle farms, and their potential as sentinel species for infection on farms; Project 4 Objectives: 1) construct probability distributions of the prevalence of bovine TB in different sub-populations of Michigan domestic ruminants; 2) construct probability distributions of the false and true positives and false and true negatives resulting from the current serial testing (CFT/CCT) program in Michigan; and 3) estimate the rates of human injuries and deaths suffered during the bovine TB testing program in Michigan; Project 5 Objectives: 1) identify altered gene expression for bovine cytokines in cattle sensitized to M. bovis, using lymphocytes stimulated in vitro with purified protein derivative (PPD) from M. bovis or M. avium; and 2) identify additional genes from bovine lymphocytes that show altered expression attributable to exposure with M. bovis, using a cDNA microarray made from bovine lymphocytes and mRNA from lymphocytes stimulated in vitro with PPD from M. bovis or M. avium.

APPROACH: Project 1: Herd-based risk assessment models and models of TB in wild white-tailed deer in northeastern Michigan will be used to develop industry-level predictive models of levels of TB and its economic consequences to the state's cattle industry, so that the impact of herd-level or industry-level TB control measures, and their attendant costs, can be projected over periods of time and be used to determine which measures would be most efficient and cost-effective for the industry. Project 2: Objectives 1 & 2: To test effect of Johne's Disease (JD) on the caudal fold and comparative cervical skin tests and the gamma-interferon test for Mycobacterium bovis, cattle from dairy herds with no/low/high levels of JD will have TB skin tests administered. Blood and fecal samples will be collected to compare animal JD status (by fecal culture, ELISA and gamma-interferon for M. paratuberculosis) with results of TB skin tests and gamma-interferon testing. Objective 3: TB-negative cattle with and without JD will be injected with killed M. bovis antigen in a dose response study to compare the immune response of cattle with and without JD to respond to the M. bovis antigen. Project 3: Norway rats and wild rodents will be orally inoculated with high and low doses of M. bovis, with some sham-inoculated controls. Fecal cultures and body weights will be collected throughout the study. Groups will be euthanatized at different intervals, and results of gross necropsy, histopathology, acid-fast staining and bacterial culture will be assessed. Project 4: Objectives 1 & 2: Probabilistic risk assessment models will be developed to assess the prevalence of bovine TB on cattle operations in Michigan. Risk analysis software will be used to generate frequency distributions of rates of false-positives and -negatives from the Michigan TB livestock testing program. Objective 3: A survey on worker injuries will be administered to a random sample of the veterinarians involved with TB testing. Project 5: Objective 1: Whole blood will be collected from calves sensitized to M. bovis, and stimulated by incubation with PPD made from M. bovis or M. avium. Lymphocytes will be harvested and total cellular RNA will be obtained. RT-PCR and a real-time PCR detection system will be used to monitor up- and down-regulation, and altered product-ratios for bovine cytokines. Results from sensitized cattle will be compared to results from animals with natural infection. Objective2: Available cDNA microarrays made from bovine lymphocytes will be used to identify genes that are up-regulated or down-regulated after in vitro exposure of lymphocytes with either M. bovis or proteins derived from M. bovis. Total cellular RNA will be harvested from lymphocytes after stimulation with antigen, and reverse transcribed using an oligo (dT)15 primer to incorporate aminoallyl-modified dUTP into the single strand product. The fluorescent-labeled probe cDNAs will be hybridized to microarrays, the microarrays will be washed several times, dried, and scanned to create reports of spot intensity ratios to identify genes that have altered levels of expression.

PROGRESS: 2002/09 TO 2004/08
Project 1: A stochastic risk assessment model for herd TB status was developed, based on results from a case-control study to identify herd management factors and environmental conditions associated with TB status. A method to examine trade-offs between expected benefits and expected costs of biosecurity management practices and investments was developed. The model is being updated with additional data from over 10 new TB-positive herds, and is being implemented in a form for on-farm use. Project 2: A prospective study was designed to determine if cattle infected with Mycobacterium. avium subsp. paratuberculosis (JD) have a higher proportion of false positive caudal fold tuberculin test (CFT) results for TB when compared to uninfected cattle. Blood and fecal samples from 1043 cattle were subjected to M. bovis and M. avium gamma-interferon (INF-gamma) and JD antibody ELISA testing, and fecal culture. The overall false positive rate on the CFT was 17%. The high (>15%) and low (<15%) prevalence herds averaged 21 and 14% positive on the CFT, respectively, and 32 and 19% of JD positive (+) cows from high and low prevalence herds, respectively, were CFT positive. These results indicate an association between JD disease and false positive CFT. Project 3: The experimental infection study of wild house mice was completed, and mice were found to be highly susceptible to M. bovis and may pose a real threat to infected farms that are depopulated and later repopulated. The final analysis, combining results of several studies, shows that voles were the most susceptible to infection, mice were highly susceptible, and rats being highly resistant to both infection and shedding. A manuscript for publication comparing and summarizing these findings is in preparation. Project 4: The sensitivities of the CFT, CFT and comparative cervical tests (CCT) in series, and gross necropsy were 93, 88, and 86%, respectively. Sensitivities of skin tests were slightly higher when at least 2 lesions were found at gross necropsy. If 1 TB+ animal is enough to declare a herd TB+ and the tests used to classify a single animal as TB+ has a specificity of 1.0, the herd level performance of the TB skin tests in Michigan is very good. When prevalence is low, herd level sensitivity is correlated with TB prevalence and the size of the tested herd. Herd negative predictive value is very high and decreases slightly when herd size decreases. These results show that attention should be paid to smaller herds to meet the goal of TB eradication. Project 5: Whole blood from 5 false-positive CCT reactors, 1 lesion+ animal, 1 INF-gamma reactor, and 1 cow sensitized with sensitinogen was stimulated with bovine PPD before harvest of total cellular RNA for determination of levels of cytokine gene expression compared with levels of 2 housekeeping genes. There was an increase in gene expression for 6 cytokines and INF-gamma, and decreased expression of IL-4 in the CCT reactors. The lesion+ animal showed an increase in expression of TNF-alpha, and IL-10 or INF-gamma. Additional RNA has been collected for further study, from a TB positive herd and a negative herd located outside of the TB endemic area.

IMPACT: 2002/09 TO 2004/08
Control and eradication of TB from the Michigan livestock industry requires an understanding of the disease and how it spreads, efficient disease detection methods, and the development of tools for control of the disease at the farm level. Risk assessment models can be used to develop sound, cost-effective disease control programs. The reliability of the caudal fold skin test, used for TB testing in livestock, may be affected by an animal's disease or vaccination status. These factors should be taken into consideration when interpreting TB skin test results, or designing a TB surveillance program. Current TB tests require great time, effort and expense, and false results are common with existing skin and blood tests. Even with TB lesions, many times associated with acid-fast bacilli, bacteria cannot be cultured because of poor samples, freeze-thaw, or lack of available fresh tissues. New DNA-based testing methods (cDNA microarray analysis of gene expression and laser-capture microscopy for isolating DNA for PCR) have the potential to become rapid, sensitive methods to identify TB in tissue samples in an efficient and reliable way. Determining what wildlife species can serve as reservoirs of M. bovis is fundamental to understanding the epidemiology of TB, which is necessary to develop effective disease eradication programs

PUBLICATIONS (not previously reported): 2002/09 TO 2004/08
No publications reported this period

PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-355-2269
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu


ACCESSION NO: 0195202 SUBFILE: CRIS
PROJ NO: MICL07681 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2003-34427-13292 PROPOSAL NO: 2003-06030
START: 01 JUL 2003 TERM: 30 JUN 2006 FY: 2006 GRANT YR: 2003
GRANT AMT: $323,193

INVESTIGATOR : Kaneene, J. B.; Fitzgerald, S. D.; Grooms, D.; Bolin, S. R.;
Bolin, C. A.; Wolf, C. A.; Fine, A.

PERFORMING INSTITUTION:
Large Animal Clinical Sciences
Michigan State University
East Lansing, Michigan 48824

BOVINE TUBERCULOSIS: EPIDEMIOLOGY, DIAGNOSIS, AND PATHOGENESIS

NON-TECHNICAL SUMMARY: Since bovine TB control programs for livestock have significant costs, there is a need to improve the performance of these programs. The proposed research will evaluate the costs of current TB control programs, the reliability of TB tests being used, and factors that influence the effectiveness of these programs. In addition, the study will try to identify new, more efficient methods of identifying bovine TB in cattle.

OBJECTIVES: There are four projects with specific objectives. Project 1 Objectives: 1) to validate the epidemiological and economic risk assessment models for a cattle herd becoming infected with bovine tuberculosis; and 2) develop a user-friendly software package to predict a herd's risk for the bovine tuberculosis, and estimate the costs associated with changing management practices identified as contributing to the herd risk for bovine tuberculosis. Project 2 Objectives: 1) determine the level of association between false positive rates in the caudal fold skin tests and levels of M. paratuberculosis infection in the cattle herd; 2) determine the incidence of M. paratuberculosis infection in cattle that are classified as suspects or reactors by the comparative cervical tuberculin test; 3) determine whether the administration of a vaccine containing a modified-live bovine viral diarrhea virus changes the response to the caudal fold tuberculin test or a-interferon test for bovine tuberculosis; and 4) determine whether the administration of a new L. borgpetersenii serovar Hardjo bacterin changes the response to the caudal fold tuberculin test or a-interferon test for bovine tuberculosis. Project 3 Objectives: 1) compare patterns of gene expression in WBC RNA from comparative cervical suspects or reactors from tuberculosis free herds, and cattle naturally infected with M. bovis; 2) optimize the DNA extraction process and the PCR reaction conditions to increase the sensitivity for detection of M. bovis in lesions that lack observable acid fast organisms; 3) develop and standardize DNA extraction and PCR techniques for detection of M. bovis in known positive animal tissues; 4) compare the sensitivity and specificity of this new technique to existing PCR on formalin-fixed tissue techniques; and 5) determine the utility of this new technique for wildlife and domestic animal tuberculosis surveillance, as well as experimental inoculation study application. Project 4 Objectives: 1) use bacteriologic culture and DNA-based testing techniques to identify M. bovis in environmental substrates from TB-affected cattle farms and areas with identified clusters of TB-infected white-tailed deer; 2) assess the effect of environmental conditions (humidity, temperature, and light) on the probability and duration of M. bovis survival in the environment; 3) study the relative susceptibility of mallard ducks to oral and intra-tracheal inoculation with M. bovis and attempt to isolate M. bovis from inoculated birds through fecal culture at multiple times post-inoculation, and from various organs at necrospy; 4) evaluate various tissues microscopically at multiple times post-inoculation to understand the pathogenesis of M. bovis in these birds; and 5) evaluate wild bird species for the threat they pose in re-introduction of M. bovis to cattle farms.

APPROACH: Project 1: Herd-based risk assessment models and economic models of TB in cattle herds in northeastern Michigan will be used to develop predictive models for a herd's risk for TB and its economic consequences, so that the impact and costs of herd-level TB control measures can be projected over periods of time and be used to determine which measures would be most efficient and cost-effective. Project 2: Objectives 1-2: Cattle from dairy herds with no/low/high levels of Johne's Disease (JD) will receive caudal fold skin tests (CFT). Blood and feces will be collected to compare animal JD status with results of CFT and a-interferon testing. Objective 3: Calves free of BVDV will be sensitized to M. bovis, vaccinated with a modified-live virus vaccine containing attenuated BVDV, bovine herpesvirus-1, bovine respiratory syncytial virus, and parainfluenza-3. The CFT will be given after vaccination, and comparisons made between vaccinated and unvaccinated calves. Objective 4: Calves free of Leptospira will be sensitized to M. bovis, vaccinated with a monovalent serovar Hardjo vaccine, and given CFTs after vaccination. Comparisons will be made between CFT results from sensitized and unsensitized calves. Project 3: Objectives 1-2: Whole blood collected from calves sensitized to M. bovis will be stimulated with M. bovis or M. avium PPD to obtain total cellular RNA. RT-PCR and real-time PCR will monitor up- and down-regulation, and altered product-ratios for bovine cytokines. Available cDNA microarrays made from bovine lymphocytes will be used to identify genes that are up- or down-regulated after in vitro exposure of lymphocytes with M. bovis or proteins derived from M. bovis. Objectives 3-5: Extraction of DNA and PCR will be used on sections of formalin fixed, paraffin embedded tissue with microscopic lesions consistent with TB. A laser capture microsdissection system will be used to dissect out microscopic granulomatous lesions for capture using HS Caps, DNA will be extracted, and real-time PCR will be conducted. Project 4: Objective 1: Soil, hay, and water samples will be inoculated with M. bovis. Processed samples will be analyzed for the presence of M. bovis by culture and PCR, and the most efficient method for processing environmental samples will be determined. Objectives 2-3: Locations on TB-infected cattle farms will be identified and samples of feces, feeds, open water, and pasture grass will be taken. Similar samples will be taken from sites in areas with high TB-prevalence in wildlife. Analysis for M. bovis will be done by culture, DNA probe, and IS6110 primer-based PCR. Objectives 3-5: Mallard ducks will be orally inoculated with high and low doses of M. bovis, with some sham-inoculated controls. Fecal cultures and body weights will be collected throughout the study. Groups will be euthanatized at different intervals, and results of gross necropsy, histopathology, acid-fast staining and bacterial culture will be evaluated.

PROGRESS: 2003/07 TO 2006/06
Project 1: The test version of a user-friendly software package to predict herd risk for TB, identify conditions on the farm associated with increasing TB risk, and estimate the economic costs associated with changing management practices identified as contributing to the herd risk for TB for on-farm use is undergoing field-testing and refinement, and the predictive risk assessment model for a herd becoming reinfected with TB after depopulation is being completed. Project 2: Four groups of 9 cattle each were sensitized with antigen from M. bovis to stimulate an immune response that would mimic natural infection when the cattle were tested for tuberculosis, using currently approved testing methods. One group was vaccinated with a modified live virus commonly used to control respiratory pathogens, another group was vaccinated with a commonly used inactivated bacterin for Leptospirosis, and 2 groups served as nonvaccinated controls. Neither vaccine affected the caudal fold test (CFT), and only the respiratory vaccine negatively affected the whole blood gamma interferon assay for tuberculosis. Project 3: Blood was collected 2 TB positive cattle, 5 skin test positive/lesion negative cattle, and 1 cow experimentally sensitized with inactivate antigen from M. bovis to determine if using cDNA microarrys to analyze gene regulation in response to stimulation of white blood cells with antigens derived from M. bovis would identify gene targets for secondary testing for TB. Altered gene expression patterns among cattle were detected using a cDNA microarry created from differentially expressed genes in bovine lymphocytes, but no clear targets were found. To determine if M. avium ssp paratuberculosis (MAP) infection affects currently used tests for TB, formalin fixed, paraffin embedded tissues from 300 cattle were tested for DNA from MAP. The cattle were lesion negative for TB on post mortem examination and tested as TB suspects on the CFT or were CFT negative. MAP was not associated with a positive reaction on the CFT. Project 4: Methods for processing environmental samples capable of detecting small numbers of M. bovis were established. Based on a cross-sectional study of environmental substrates collected on TB-affected cattle farms, M. bovis was not isolated from any samples (soil, water, feed) collected from 13 TB-affected cattle farms and 5 wildlife areas with known TB. The study to determine the effect of substrate (water, soil, hay, grain) and environmental conditions (humidity, temperature, sunlight) on the persistence of viable M. bovis in the environment found that M. bovis can persist for 6-10 weeks in cooler seasons. These results are being prepared in 3 papers for publication in scientific journals. Mallard ducks are highly resistant to oral infection with TB, and do not shed the organism in feces. Results of this study were published in 2005.

IMPACT: 2003/07 TO 2006/06
Control and eradication of TB from the Michigan livestock industry requires an understanding of the disease and how it spreads, efficient disease detection methods, and the development of tools for control of the disease at the farm level. Risk assessment models can be used to develop sound, cost-effective disease control programs. The reliability of the caudal fold skin test, used for TB testing in livestock, may be affected by animal disease or vaccination status. These factors should be taken into consideration when interpreting TB skin test results, or designing a TB surveillance program. Current TB tests require great time, effort and expense, and false results are common with existing skin and blood tests. Even with TB lesions, many times associated with acid-fast bacilli, bacteria cannot be cultured because of poor samples, freeze-thaw, or lack of available fresh tissues. New DNA-based testing methods (cDNA microarray analysis of gene expression and laser-capture microscopy for isolating DNA for PCR) have the potential to become rapid, sensitive methods to identify TB in tissue samples in an efficient and reliable way. Determining where in the environment and ecosystem M. bovis exists, and the length of time it survives and remains infective, is fundamental to understanding the epidemiology of TB, which is necessary to develop effective disease eradication programs. Experiments have shown that some wild bird species may contribution to the maintenance and spread of TB in wildlife and livestock.

PUBLICATIONS (not previously reported): 2003/07 TO 2006/06
Fine, A.E. The role of indirect transmission in the epidemiology of bovine tuberculosis in cattle and white-tailed deer in Michigan. Ph.D. Thesis, Michigan State University, East Lansing, Michigan, 2006.

PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-353-5941
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu


ACCESSION NO: 0203138 SUBFILE: CRIS
PROJ NO: MICL07691 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 2005-34427-15887 PROPOSAL NO: 2005-06033
START: 15 SEP 2005 TERM: 14 SEP 2008 FY: 2006 GRANT YR: 2005
GRANT AMT: $328,726

INVESTIGATOR: Kaneene, J. B.; Fitzgerald, S. D.; Bolin, S. R.; Griffore, R.;
Phenice, L.

PERFORMING INSTITUTION:
Large Animal Clinical Sciences
Michigan State University
East Lansing, Michigan 48824

BOVINE TUBERCULOSIS: EPIDEMIOLOGY, DIAGNOSIS, AND PATHOGENESIS

NON-TECHNICAL SUMMARY: Bovine tuberculosis (TB) in Michigan is being recognized as more prevalent in wildlife and livestock species than originally thought. The current combination of active and passive surveillance programs, as well as TB control and eradication efforts, have shown that more research is needed to provide information needed to deal with the problem. From previous funding, we are conducting studies to determine the major factors influencing TB transmission in wild white-tailed deer and cattle, and to determine how well TB surveillance and TB control programs for wildlife and cattle are working. In conducting these studies, we observed that there is great need to improve the efficiency and accuracy of livestock TB testing, and that the TB outbreak has had serious financial and psychological consequences for affected farm families. It is also apparent that, while current disease control programs for wildlife are reducing TB in deer, additional disease control methods to eliminate TB from wildlife are becoming necessary. The purpose of this study is to address these research needs.

OBJECTIVES: There are four projects with specific objectives in this study. Project 1 objectives: 1) Identify factors associated with risk of finding CCT reactor cattle on the farm, including data collected via questionnaire during routine TB testing, wildlife surveillance data (cervid and non-cervid surveillance), and geological and ecological factors around the farm; 2) Describe and quantify risk into distinct levels for use in TB testing, develop testing protocols for each specific risk level, and develop a risk calculator based on results from Objective a, for use on laptop or PDA, to calculate farm risk for TB and recommend testing. Project 2 objectives: 1) Vaccinate a Mycobacterium-susceptible mouse strain (BALBc) with either a RB51 vector vaccine or a BCG vaccine; 2) challenge those mice with deer-origin M. bovis; 3) evaluate those animals over several months for fecal shedding of the organism, clinical signs, and terminal necropsy for histologic evaluation and mycobacterial isolation and titration; 4) compare results unvaccinated, M. bovis challenged mice to evaluate vaccine efficacy in increasing disease resistance, decreasing lesion development and mycobacterial organism replication, and controlling shedding of M. bovis. Project 3 objectives: 1) Compare proportions of false positive whole blood gamma interferon assays with and without ESAT-6/CFP-10 stimulation; 2) Compare proportions of false positive whole blood gamma interferon assays with and without ESAT-6/CFP-10 stimulation by geographic region and season. Project 4 objectives: 1) Collect information about relevant farm characteristics, and family attitudes and behavior patterns via self-descriptions; 2) incorporate these into regression models to identify factors that differ between farm families that have and have not been directly affected by bovine TB.

APPROACH: Project 1: A retrospective and nested case-control study will be conducted to identify factors that are associated with positive CCT test in individual CCT reactor cattle. Results of this analysis, combined with existing data on TB risk from our previous work, will be used to develop distribution curves for TB risk based on risk factors, and risk levels will be categorized for use in designing testing protocols based on levels of risk. Project 2: An experimental study will be conducted to compare the efficacy of a new recombinant M. bovis vaccine with the existing BCG vaccine in a mouse model. Vaccinated mice will be challenged with different levels of M. bovis cultured from Michigan wildlife, and levels of bacterial shedding, clinical signs, lesion development and histopatholgical results will be compared between the two vaccine groups and challenge levels. Project 3: An experimental study will be conducted to determine if use of ESAT-6/CFP-10 improves the whole blood gamma interferon assay for M. bovis by reducing false positive results. Blood samples collected during TB surveillance will be subjected to the currently used whole blood gamma interferon assay and to an additional stimulation phase with ESAT-6/CFP-10. False positive rates will be computed for samples with and without additional stimulation to determine if use of ESAT-6/CFP-10 reduces the numbers of false positive results. Project 4: A case-control study will be conducted to determine if there are differences in the ways that farm families interact with their farm ecosystems between farms that have and have not been infected with M. bovis. This will be a post-test-only control group design, using survey methodology to collect data. Questionnaires will be administered to both groups to collect data on farm family attitudes and behaviors, physical characteristics of the farm, and the farm's location. These data will be used to determine if there are any significant differences in attitudes and behaviors of those families whose farms have been affected by TB with those of families in the region whose farms have not been directly affected by TB.

PROGRESS: 2005/09 TO 2006/09
Project 1: The retrospective and nested case-control study for factors associated with positive CCT tests in individual reactors is ongoing. Completed surveys of farm risk factors has been collected and are being entered into a computerized database to add to existing risk factor information to complete the study. Project 2: This project has been completed. Twenty-eight BALBc mice were vaccinated twice with one of two vaccines, including standard BCG vaccine (n=12), and a new recombinant vaccine (n=16), then challenged intranasally with live Mycobacterium bovis. All of twelve unvaccinated control mice lost weight, became moribund, and were sacrificed within 4 weeks of challenge; results were similar with the recombinant vaccine mice. BCG-vaccinated mice maintained activity and body weight, and only two of twelve mice needed to be sacrificed seven weeks post challenge. The subunit vaccine showed marked reduction in mortality, gross and microscopic lesions compared to unvaccinated mice, which shows good promise for future development of a deer or cattle vaccine that will not interfere with current testing methods. Project 3: A total of 1,912 field samples of whole blood from CFT+ cattle, and 315 samples from cattle examined post mortem that were CFT+ and CCT and/or gamma-interferon positive, were stimulated with recombinant ESAT-6/CFP-10 and tested in the gamma interferon assay for bovine tuberculosis. The samples tested included whole blood from 5 lesion positive cattle. The purpose was to determine if ESAT-6/CFP-10 (E/C) as a Mycobacterium bovis specific antigen would stimulate white blood cells collected under field conditions to produce sufficient gamma interferon to be detected in the gamma interferon ELISA for bovine tuberculosis. Work is currently under way to match these test results with data from individual cattle that are in USDA whole-herd testing records. Preliminary results indicate that E/C vastly reduces the rates of false positives tests (increases test sensitivity). These results were communicated to the Scientific Advisory Committee of the Bovine Tuberculosis Committee of the USAHA in 2006. Project 4: We conducted focus groups in northern Michigan with four County Extension Directors and five veterinarians, and a focus group at Michigan State University with six epidemiologists, to obtain information that could be used in developing the questionnaire to collect data from farm families on TB-positive farms and control farm families. The questionnaire, based on results from the focus group, has been finalized. A database of potential farm family participants is being gathered, and the questionnaire and a revised research protocol have been submitted to the MSU Social, Behavioral, Education Institutional Review Board (SIRB). We are currently awaiting approval from SIRB to begin data collection.

IMPACT: 2005/09 TO 2006/09
By making TB testing for cattle more reliable (Projects 1 and 3) and conducting the basic scientific research (Project 2) needed to develop a vaccine to prevent TB in cattle, this research will reduce the economic costs of TB in Michigan by preventing the disease and wasting less time and money on false TB test results. Comparing farm families with and without TB (Project 4) will give us information that can be used to raise awareness of the social and emotional costs of TB, and to create programs to help affected families deal with social and emotional problems cause by having TB on their farms.

PUBLICATIONS (not previously reported): 2005/09 TO 2006/09
No publications reported this period

PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-355-2269
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu


ACCESSION NO: 0199414 SUBFILE: CRIS
PROJ NO: MICL07692 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2004-34427-14585 PROPOSAL NO: 2004-06033
START: 01 SEP 2004 TERM: 31 AUG 2007 FY: 2006 GRANT YR: 2004
GRANT AMT: $288,804

INVESTIGATOR: Kaneene, J. B.; Fitzgerald, S. D.; Bolin, S. R.; Grooms, D. L.;
Bolin, C. A.

PERFORMING INSTITUTION:
Large Animal Clinical Sciences
Michigan State University
East Lansing, Michigan 48824

BOVINE TUBERCULOSIS:EPIDEMIOLOGY, DIAGNOSIS, AND PATHOGENESIS

NON-TECHNICAL SUMMARY: Since bovine TB control and prevention programs for livestock have significant costs, there is a need to improve the performance of these programs. The proposed research will evaluate current TB control and prevention programs, the reliability of TB tests being used, factors that influence the effectiveness of these tests and programs, and the efficacy of two TB vaccines for animals.

OBJECTIVES: There are four projects with specific objectives. Project 1 Objectives: 1) Identify risk factors associated with reacquiring TB after repopulation, including factors associated with herd biosecurity, cattle movement to and from affected herds, cattle feeding practices, cattle housing, and wildlife access to livestock and livestock feed; 2) Evaluate the spatial relationship between risk for reacquiring TB and proximity to other herds affected by TB, deer habitat, surface water features, and other ecological features in the region; and 3) Determine association between herd TB status and herd-related management factors, geographic location of environmental conditions, proximity to TB positive herds, levels of TB in deer. Project 2 Objectives: 1) Determine the level of association between false positive rates in the caudal fold skin tests (CFT) and levels of M. paratuberculosis infection in the cattle herd; and 2) Determine the prevalence of M. paratuberculosis infection in cattle that are classified as suspects or reactors by the comparative cervical tuberculin test (CCT); 3) determine whether the administration of a vaccine containing a modified-live bovine viral diarrhea virus changes the response to the caudal fold tuberculin test or gamma interferon test for bovine tuberculosis; 4) determine whether the administration of a new L. borgpetersenii serovar Hardjo bacterin changes the response to the caudal fold tuberculin test or gamma-interferon test for bovine tuberculosis; 5) Retrospectively apply PCR to formalin-fixed, paraffin-embedded sections of ileum and determine the prevalence of M. paratuberculosis infected cattle that falsely test as suspects or reactors by 1) the CFT, 2) the CCT, and 3) cattle that are skin test negative; and 6) Compare resulting prevalences of false reactors by the two skin tests with the negative test population as a negative control to determine effect of Johnes infection on test results. Project 3 Objectives: 1) Identify targets of altered gene expression after 0, 1, 2, 4 or 20 hours of antigen stimulation to determine optimal time for whole blood stimulation; and 2) compare patterns of gene expression in WBC RNA from comparative cervical suspects or reactors from tuberculosis free herds, and cattle naturally infected with M. bovis. Project 4 Objectives: 1) Evaluate BALB/c mice vaccinated with a RB51 vector vaccine or a BCG vaccine over several months after challenge with deer-origin M. bovis for fecal shedding of the organism, clinical signs, and terminal necropsy for histologic evaluation and mycobacterial isolation and titration; and 2) Compare results with unvaccinated, M. bovis-challenged mice to evaluate vaccine efficacy in increasing disease resistance, decreasing lesion development and mycobacterial organism replication, and controlling shedding of M. bovis.

APPROACH: Project 1, Part A: A case-control study will compare risk factors (farm location, ecological data, herd management practices before and after restocking) between herds diagnosed with TB that were depopulated and repopulated (controls) with repopulated herds that have become reinfected with TB (cases). Project I, Part B: A retrospective case-control study will compare spatial risk factors between herds diagnosed with TB since the beginning of the current disease outbreak (cases) and herds that have not been diagnosed with TB (controls) from the five county area. Case herds will be those. Risk factors include livestock housing location data (located by Global Positioning Satellite (GPS) systems), specific ecological conditions in cattle housing areas, and herd management practices. Project 2, Part A: The effect of infection with M. paratuberculosis on results of the bovine TB caudal fold test (CFT) test and gamma interferon assay will be assessed by comparing the false negative rate for these two assays in Johnes disease high prevalence dairy herds and herds known to be free of Johnes disease. Project 2, Part B: The effect of use of select veterinary vaccines on the reliability of the CFT for TB in cattle will be tested by comparing CFT test results and M. bovis gamma interferon production between groups of calves sensitized to M. bovis that are unvaccinated or vaccinated with a commercially available vaccine containing modified-live bovine viral diarrhea virus (BVDV), infectious bovine rhinotracheitis (IBR), PI-3 and bovine respiratory syncital virus (BRSV) (Pyramid, Ft. Dodge Animal Health, Fort Dodge, IA). Project 2, Part C: A retrospective study of the role of M. paratuberculosis in false reactor response on the CFT and the comparative cervical test (CCT) will compare levels of M. paratuberculosis between CFT positive/M. bovis culture negative cattle and CFT negative cattle by PCR for M. paratuberculosis from sections of ileum and ileal-cecal lymph nodes. Project 3, Part A: The optimal time of exposure of whole blood to bovine ppd for detection of diagnostic gene targets in caudal fold suspect cattle will be determined by comparing levels of altered expression of 10 genes after 0, 1, 2, 4 or 20 hours of antigen stimulation, between cattle that have been sensitized to M. bovis and cattle that have not been sensitized. Project 3, Part B: Optimal gene targets for detection of bovine tuberculosis will be identified by stimulating blood with phosphate-buffered saline (PBS) (negative control), M. avium purified protein derivative (PPD), and M. bovis PPD, and harvesting total cellular RNA to generate cDNA for use in real-time quantitative polymerase chain reaction (RT-QPCR). Project 4: The efficacy of BCG vaccine and a recombinant vector vaccine (RB-51) for M. bovis in laboratory mice will be tested by comparing lesion development and mycobacterial isolation results from vaccinated and unvaccinated mice challenged with deer-origin M. bovis.

PROGRESS: 2004/09 TO 2007/08
Project 1: The predictive risk assessment model for reacquiring TB and proximity to other herds affected by TB, deer habitat, surface water features, and other ecological features in the region has been finalized. Updated information on data from newly infected cattle herds (biosecurity, cattle movement to and from affected herds, cattle feeding practices, cattle housing, and wildlife access to livestock and livestock feed) and additional retrospective data collection was used to refine the existing predictive model, which is undergoing testing. Project 2: Dairy cattle with Johne's disease (JD, infection with M. avium ssp. paratuberculosis [n=11]) and age-matched cattle without JD (n=8) were sensitized with antigen from M. bovis to stimulate an immune response that would cause the cattle to test positive for TB using currently approved testing methods. JD did not affect the result of the caudal fold test (CFT), as all sensitized cattle showed a positive reaction, but did adversely affect the results of the whole blood gamma interferon test in some cattle. Project 3: To identify altered gene expression patterns in cattle that are false positive on current TB tests as potential diagnostic targets for TB detection, whole blood was collected from 60 lesion negative cattle suspect for TB on the CFT and the comparative cervical test (CCT) or the whole blood gamma interferon test, and 19 microarray analyses have been completed for RNA samples from blood pre-stimulated with M. bovis antigen for 4 hrs (n=15; 7 to CFT reactors, 4 double reactors, 4 TB positive) and 0-hr (n=4; all double reactors). Altered gene expression of 5-fold or greater was seen in each group: 10 up- and 2 down-regulated genes from CFT reactors, 223 up- and 5 down-regulated genes from double reactors, and 12 up- and 5 down-regulated genes from TB positives. No genes demonstrating altered expression levels were shared among or between these groups of cattle. The 0-hr microarrays showed only 4 genes with altered expression and none showed altered levels that were greater than 5-fold above or below control values. Project 4: BALBc mice were vaccinated twice with standard BCG or a new recombinant vaccine, then challenged intranasally with live M. bovis. 12 unvaccinated controls and 12 recombinant vaccine mice lost weight, became moribund, and were sacrificed within 4 weeks of challenge. BCG vaccinates maintained activity and body weight, and only 2 of 12 mice were sacrificed 7 weeks post challenge. The recombinant vaccine showed marked reduction in mortality, gross and microscopic lesions compared to controls, which shows promise for development of a vaccine that will not interfere with current tests. A second trial was performed using a modified subunit vaccine:weight loss, lesions development, and mortality were reduced compared to controls, but slightly higher compared to BCG vaccinates. Final summarization of the study is pending on final mycobacterial isolation and titration. A study was conducted on Michigan strains of M. bovis isolates from 1999 and 2004, to look for evidence of antimicrobial resistance, and no evidence of antimicrobial resistance development was seen.

IMPACT: 2004/09 TO 2007/08
Infection of cattle with Johne's disease does not appear to have major influence on the rate of false positive skin TB tests in Michigan, but may cause cattle to test negative on some secondary laboratory diagnostic assays. Results of gene expression profiling showed that there are promising gene targets that may be used for developing diagnostic tools to detect TB in live cattle. The intranasal challenge in BALBc mice is an efficient system for testing efficacy of tuberculosis vaccines. BCG vaccine demonstrated good protection to challenge, and the redesigned recombinant vaccine was much more effective that earlier recombinant vaccines. Lack of antimicrobial resistance in M. bovis from Michigan is important for the treatment of TB if cases in humans occur.

PUBLICATIONS (not previously reported): 2004/09 TO 2007/08
1. Clarke, K.R. 2005. Effects of Mycobacterium bovis inoculation in select potential reservoir or spillover wildlife host species. Ph.D. Thesis, Michigan State University, East Lansing, Michigan, 2005.
2. Clarke, K.R., Fitzgerald, S.D., Zwick, L.S., Church, S.V., Kaneene, J.B., Wismer, A.R., Bolin, C.A., Hattey, J.A., Yuzbasiyan-Gurkan, V. Experimental inoculation of meadow voles (Microtus pennsylvanicus), and Norway rats (Rattus norvegicus) with Mycobacterium bovis. J. Wildl. Dis., 43(3): 353-365, 2007.
3. Daly, M., Diegel, K.L., Fitzgerald, S.D., Schooley, A., Berry, D.E., Kaneene, J.B., Patterns of antimicrobial susceptibility in Michigan wildlife and bovine isolates of Mycobacterium bovis . J Vet Diagn Invest, Vol 18: 401-404, 2006.
4. Miller, R. and Kaneene, J.B. Evaluation of historical factors influencing the occurrence and distribution of Mycobacterium bovis infection among wildlife in Michigan. AJVR, Vol. 67 (4): 604-615, 2006.
5. Miller, R., Kaneene, J.B., Schmitt, S.M., Lusch, D.P., Fitzgerald, S.D. Spatial analysis of Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus) in Michigan, USA. Prev. Vet. Med., doi:10.1016/j.prevetmed.2007.05.011, 2007.
6. Norby, B., Bartlett, P.C., Grooms, D.L., Kaneene, J.B., Bruning-Fann, C.S. Use of simulation modeling to estimate herd-level sensitivity, specificity, and predictive values of diagnostic tests for detection of tuberculosis in cattle. Am. J. Veterinary Research, 66(7): 1285-1291, 2005.

PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-355-2269
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu


ACCESSION NO: 0206672 SUBFILE: CRIS
PROJ NO: MICL07708 AGENCY: CSREES MICL
PROJ TYPE: SPECIAL GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2006-34427-17171 PROPOSAL NO: 2006-06046
START: 01 SEP 2006 TERM: 31 AUG 2008 FY: 2006 GRANT YR: 2006
GRANT AMT: $329,022

INVESTIGATOR: Kaneene, J. B.; Fitzgerald, S. D.; Bolin, S. R.; Bolin, C. A.;
Fine, A. E.

PERFORMING INSTITUTION:
Large Animal Clinical Sciences
Michigan State University
East Lansing, Michigan 48824

BOVINE TUBERCULOSIS: EPIDEMIOLOGY, DIAGNOSIS, AND PATHOGENESIS

NON-TECHNICAL SUMMARY: Since bovine TB control programs for livestock have significant costs, there is a need to improve the performance of these programs. A risk calculator has been designed for farmers, to identify problem areas that can be corrected to reduce TB risk to their farms. The proposed research will also work to improve testing to detect TB in farm environments, and in wild deer, cattle, and in farm cats.

OBJECTIVES: To aid in the development of risk-based surveillance system, the objectives of the first project are to continue testing of the on-farm risk calculator to estimate farm risk for TB, adding enhancements to improve ease of use in the field, and collecting additional risk factor data to the current body of data used to generate risk model parameters, and to continue model development and refinement, by validating the existing model with data from new TB-positive herds from Michigan, and from TB-positive herds from Minnesota and any other states if present. The second project seeks to demonstrate that molecular detection techniques will improve our ability to identify Mycobacterium bovis in soil, hay, water and similar substrates and enable an accurate characterization of the persistence and distribution of M. bovis in farm environments. The objectives for this project are to test and validate the molecular detection techniques with an extensive set of environmental samples experimentally inoculated with M. bovis and previously processed for mycobacterial culture, and then apply the validated molecular detection technique to environmental substrates collected from bovine TB transmission sites in Michigan The third project is designed to determine whether a rapid (30-minute or less), easy-to-use, and sensitive card test can be applied directly to lesioned deer or cattle tissues to screen for the detection of M. bovis under field conditions, with minimal equipment or specialized training. The first objective of this project is to apply a new smartDNA technique to one season of hunter-harvested deer (est. 50 animals) to quickly screen lesioned lymph nodes, and compare results with traditional methods of histopathology, acid-fast staining, mycobacterial isolation and PCR techniques, to determine sensitivity and specificity. The same lesioned deer will have the new smartDNA test applied to tonsillar or oral cavity tissues to try to detect M. bovis shedding, and later correlate these results with histopathology, culture, and lesion distribution to gain insight into what gross lesions may indicate a shedding animal. The next objective is to apply the smartDNA technique to TB-reactor cattle submitted to the MI state Diagnostic Center for Population and Animal Health (DCPAH) to suspect lesions, and again correlate with histopathology, acid-fast staining, culture and PCR results, to evaluate its suitability for use in USDA-inspected slaughterhouse cattle surveillance setting. The fourth project is to investigate whether free-ranging domestic cats can serve as sentinels for TB in circumstances where they frequently come into contact with potentially tuberculosis large animals. The objectives of this project are to sensitize a group of cats using killed M. bovis to stimulate an antibody response, and evaluate several different ante-mortem tuberculosis testing techniques (ELISA, MAPIA, TB Rapid Test) under both laboratory and field conditions in order to choose the optimal method for on-farm surveillance.

APPROACH: Project 1: The on-farm risk calculator, based on a retrospective and matched case-control study to identify factors that are associated with positive CCT tests in individual CCT reactor cattle, will be field tested and refined for ease of use on the farm. We plan to use data from recently-infected herds in Michigan, and other states such as Minnesota, to refine distribution curves for TB risk based on risk factors, and to modify risk level categories for use in designing testing protocols based on levels of risk. Project 2: To circumvent problems of contamination when attempting to culture M. bovis from environmental samples, we will test for the presence of M. bovis using PCR procedures that target antigen genes unique to the M. tuberculosis complex: mpb70 and mpb64. Soil, corn, hay, and water samples that were inoculated with M. bovis and subjected to environmental conditions will be used. DNA or RNA will be extracted from soil samples using specialized kits from Mobio Laboratories. For other samples, the MasterPure DNA/RNA Extraction Kit from Epicentre will be used. To confirm that the M. bovis detected by PCR is viable, we will use reverse transcription PCR to detect 16s ribosomal RNA, confirmed by nucleic acid sequencing. Project 3: SmartDNA(TM) test cards, containing strips that react to the presence of target nucleic acid sequences without the need for amplification, will be used on samples collected from white-tailed deer during TB surveillance, and from TB suspect and reactor cattle. Lesions detected in deer heads or whole carcasses in TB surveillance will be loaded into the detection card by sterile disposable swabs, and read after 30 minutes. Swabs of the oral cavity and tonsillar area will be loaded into a detection card, and results used to determine whether or not this lesioned deer is shedding M. bovis. All cattle with gross lesions suggestive of TB will be tested using the detection cards, and standard histopathology and acid fast staining, mycobacterial isolation and identification and PCR testing will be done according to current USDA protocols. Card results will be compared to isolation/PCR results to evaluate sensitivity and specificity of the assay. Project 4: We will develop and assess 4 serologic tests for detection of M. bovis in cats (ELISA, multi-antigen print immunoassay (MAPIA), lateral-flow antibody detection test (TB Rapid Test) to be developed in collaboration with Chembio Diagnostics, Inc). Six cats will be sensitized by intradermal injection with inactivated M. bovis (sensitinogen) 2x, 3 weeks apart. Four ml of blood will be drawn from the jugular veins of anesthetized cats, and 0.5 ml of sera will be shipped to Iowa State University for use in another ELISA that has shown promise in limited field use. Another 1.0 ml of sera will be shipped to Chembio Diagnostics for development and application of their MAPIA and TB Rapid Tests. These tests will be performed at 30-day intervals (0-150 days pi) until each cat converts positive or is euthanized. Two non-sensitized control cats will be similarly sampled. Cats from TB-positive farms will be used to validate the MAPIA and TB Rapid Tests.

PROJECT CONTACT:
Name: Kaneene, J. B.
Phone: 517-353-5941
Fax: 517-432-0976
Email: kaneene@cvm.msu.edu, rmiller@cvm.msu.edu


ACCESSION NO: 0203692 SUBFILE: CRIS
PROJ NO: MICL08381 AGENCY: CSREES MICL
PROJ TYPE: SERD GRANT PROJ STATUS: EXTENDED
CONTRACT/GRANT/AGREEMENT NO: 2005-38411-15862 PROPOSAL NO: 2005-03319
START: 01 SEP 2005 TERM: 31 AUG 2008 FY: 2006 GRANT YR: 2005
GRANT AMT: $71,880

INVESTIGATOR: Kramer, D.; Christoffel, R. A.; Felix, A. B.; Lamp, N. E.;
Wolfson, L. G.; Campa, I. R.; Hayes, D.; Millenbah, K. F.; Fine, A.

PERFORMING INSTITUTION:
Fisheries and Wildlife
Michigan State University
East Lansing, Michigan 48824

DEVELOPMENT OF A NATURAL RESOURCES FIELD INSTITUTE: SHAPING FUTURE PROFESSIONALS THROUGH EXPERIENTIAL LEARNING AND TEACHING

NON-TECHNICAL SUMMARY: A Graduate students in natural resources fields have few teaching opportunities because research supports most graduate appointments. B Increasing numbers of urban students possessing great experience with computers than the outdoors are enrolling in NR programs, and they lack outdoor skills needed for future academic and professional success. C Agency personnel observe that recent student job applicants do not have adequate backgrounds in natural history, field skills, etc., to be effective employees. Our project goal is to strengthen NR student preparation through experiential learning and teaching enhancement.

OBJECTIVES: Objective 1 is to develop a sophomore level field-based institute designed to reduce student anxieties and correct misconceptions about working outdoors. Objective 2 is to provide undergraduate and graduate students with professional contacts and experiences to strengthen skills needed for future internships or jobs. Objective 3 is to evaluate the course's success as it relates to student learning and teaching enhancement.

APPROACH: The MSU Natural Resources Field Institute (NRFI) is being developed by 3 Ph.D. students (Teaching Fellows) and 6 faculty. We have developed objectives and a syllabus and identified skills students will obtain during the NRFI. Activities will give students hands-on, outdoor experiences, data collection and analysis participation and discussions of ongoing research with NR professionals. When funding is secured, equipment will be purchased and logistical arrangements made. Course advertising begins in the fall of 2005. Prior to the course (May 06), co-PDs will have specific responsibilities for its implementation and evaluation. Teaching Fellows will facilitate the course and remain for its duration. Faculty will provide a session(s) and advise and evaluate Teaching Fellows. We have identified important skills for students to develop prior to degree completion such as orienteering, map reading, communication, networking, critical thinking, problem solving, data collection and analysis, contextual thinking, and natural history knowledge. The NRFI is designed to strengthen proficiencies in these areas. For example, we will utilize peer to facilitate orienteering and map reading skills development in students by pairing up low scoring (<80%) students with individuals having demonstrated orienteering and map reading competency by scoring >80% on a quiz. Activities to strengthen communication skills and provide networking opportunities include informal and formal interactions between students and instructors. For example, evening fireside chats will provide opportunities for participants to de-brief and reflect upon activities, get to know instructors in an informal setting, and give instructors opportunities to interact with students and become familiar with their educational and professional goals. Several activities are included to develop student understanding of scientific methods. Field exercises will strengthen application skills; prompting them with questions will build contextual thinking skills. Learner-centered activities will help students develop technical skills needed for careers, explore new ideas and become critical thinkers. To assess fulfillment of our objectives, we will conduct post-course evaluations. Students will rate course enjoyment, effectiveness of instructors, specific skills enhancement, and provide recommendations for future NRFIs. The final project and its presentation will indicate how well students met NRFI learning objectives. One year post-course, we will administer a survey to two student groups, those who have completed our NRFI and a control group. Students will assess how well they felt their previous courses prepared them for upper-level courses, exposed them to different directions they could follow in the NR field and provided them with experiences to facilitate career development. This is critical for assessing curriculum effectiveness and identifying ways to improve NR programs to meet career/professional development needs of students. Faculty will evaluate instructional capabilities of Teaching Fellows.

PROGRESS: 2005/09 TO 2006/09
We developed a field course to help students overcome fear or misconceptions about natural resource (NR) management fieldwork. We identified important skills for students to develop and designed our course to include activities to build these proficiencies. We partnered with a hunt club, Mid-Forest Lodge (MFL) and worked with MFLs forester and biologist on course logistics and field research project identification. We advertised the course by: distributing brochures at 3 conferences; presenting to student clubs; enlisting faculty in course promotion; and sending announcements to CIC institutions and nearby colleges and posting it on listservs. Teaching Fellows (TF) purchased course equipment; finalized the class syllabus, teaching duties, and course details; reviewed applications; created, collected, copied, and collated course materials; and held a pre-course meeting with students to make introductions, give a course overview, complete forms, and answer course-related questions. During the course, TFs facilitated, instructed and led field teams. Faculty co-PDs and 4 external professionals provided a classroom and/or field session(s) and advised TFs. Seven female and 7 male students participated, nine from urban or suburban backgrounds. Fireside chats provided students with professional contacts and networking opportunities. The Assistant Chief of the Wildlife Division, MDNR led a chat about local emerging NR issues. MI State Director for USDA-APHIS, Wildlife Services led a chat about his agency and the growing need for professionals that can effectively deal with human-wildlife conflicts. He prepared students for a field trip to explore the bovine tuberculosis (TB) issue. Students had many opportunities to enhance their communication skills on their 2 teams: 1) a fieldwork data collection team and 2) a research team that analyzed data, wrote a report and gave an oral presentation to their peers, TFs, and interested MFL members. Students developed (1) a better understanding of and gained hands-on experience with scientific research methods, skills that agencies have identified as important for employees and (2) an understanding of how and what data to record and why through field research projects and entering data into a computer program and analyzing data for research presentations. Classroom sessions, available resources including publications and specimens, and field sessions provided species identification and natural history information. Students were encouraged to reflect on their experiences and question what they observed. A journaling assignment helped students work through course concerns and alerted TFs to such concerns and points that had not been fully grasped by students. TFs worked with and got to know their field teams quite well. This led to many conversations and facilitated student-TF interactions to clarify instructions or ambiguities from classroom sessions. Students provided suggestions for future course iterations and completed assessments regarding course enjoyment, visiting professionals, and the importance of what they learned about NR management and themselves. PRODUCTS: OUTCOMES: DISSEMINATION ACTIVITIES: FUTURE INITIATIVES:

IMPACT: 2005/09 TO 2006/09
Several recent changes have occurred in NR-related fields: (1) most natural history and identification classes have been cut from requirements; (2) recent students include more females and individuals from suburban and urban backgrounds; and (3) there is increasing demand by NR employers to hire people with field experience, critical thinking and problem-solving skills, knowledge of ecosystem structure, species life-history traits, and abilities to use and apply new technology to address management issues. The sheer magnitude of changes facing academics makes preparation of undergraduates to be well-qualified professionals challenging. We designed and implemented a two-week field-intensive course as a method to address these changes and better prepare undergraduate and graduate students for careers in NR management and research. The 14 students that attended the 2006 course iteration indicated that they learned very positive things about themselves, especially their abilities to work with others and conduct fieldwork; half indicated that they would take advantage of the professional contacts they had made during the course; and the importance of people and inclusion of so many elements besides science in NR management were the most important things that most students indicated they had learned. Students will be re-queried about their experiences one year after the courses conclusion and academic performance of these students in upper-level FW classes will be compared with the performance of a group of students that did not take our course but were otherwise similar.

PUBLICATIONS (not previously reported): 2005/09 TO 2006/09
No publications reported this period

PROJECT CONTACT:
Name: Christoffel, R. A.
Phone: 517-432-4943
Fax: 517-432-1699
Email: chris317@msu.edu


ACCESSION NO: 0208028 SUBFILE: CRIS
PROJ NO: MICL08393 AGENCY: CSREES MICL
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: NEW
CONTRACT/GRANT/AGREEMENT NO: 2006-55204-17459 PROPOSAL NO: 2006-01725
START: 01 SEP 2006 TERM: 31 AUG 2009 FY: 2006 GRANT YR: 2006
GRANT AMT: $231,886

INVESTIGATOR: Horan, R. D.; Wolf, C. A.

PERFORMING INSTITUTION:
Agri Economics
Michigan State University
East Lansing, Michigan 48824

BIOECONOMICS OF MANAGING PATHOGENS IN MULTI-HOST, LIVESTOCK-WILDLIFE SYSTEMS

NON-TECHNICAL SUMMARY: The spread of infectious disease among and between wild and domesticated animals has become a major problem worldwide, threatening the economic well-being of farmers and ranchers, wildlife conservation efforts, and human health, and posing a potential threat to the safety of the American food production system. Human actions can intensify or mitigate these risks. We examine the design of strategies to sustainably manage infectious disease risks posed by livestock and wildlife systems.

OBJECTIVES: The purpose of this research is to improve the understanding of the economics of infectious wildlife and livestock disease prevention and management. This goal will be accomplished by incorporating recent ecological developments on multi-host species-pathogen dynamics into a bioeconomic modeling framework. Bioeconomic models can be used to understand how ecological and economic factors jointly determine how livestock production systems, wildlife ecosystems, and human activities interact in affecting disease transmission risks among and between species, and also the economic outcomes of these risks. These models can be used to gauge economic-ecological tradeoffs that are useful in developing prevention, control, and mitigation strategies, and for assessing the economic and ecological implications of the disease and the associated human responses. Specific research questions that will be addressed include 1. How do economic and ecological feedbacks between wildlife hosts and livestock and human systems matter? 2. What is the appropriate allocation of economic resources (e.g., ex ante vs. ex post, on-farm vs. off-farm) to deal with multi-host disease problems? 3. How do the answers to these questions differ for different types of disease systems, including bTB in Michigan white-tailed deer and cattle, and brucellosis (Brucella abortus) in Wyoming bison, elk, and cattle. Developing this knowledge will ultimately lead to more efficient and sustainable livestock production and wildlife systems, as disease management is ultimately an economic problem of how to allocate finite resources to manage infection
risks.

APPROACH: Our research approach will address research questions 1-3 (from OBJECTIVES) through the conceptual development and numerical application of dynamic bioeconomic models to the problem of wildlife-livestock diseases. A bioeconomic model is an economic decision model that takes into account the ecological impacts of economic choices, thereby modeling the endogenous feedbacks between economic and ecological systems. This approach is novel because ecological models of multi-host-pathogen systems are relatively new and have not yet been incorporated into a bioeconomic framework. There will be two inter-related components applied to each research question. The first is the development of conceptual models to investigate theoretical results related to economic and ecological tradeoffs associated with the management of multi-host systems. The second will be the development and application of numerical simulations that can lend additional insight into the management problem, as we expect many theoretical results will depend on the specific details of particular problems. These models will be applied to the cases of bovine tuberculosis in Michigan white-tailed deer and cattle, and brucellosis in Wyoming bison, elk, and cattle.

PROJECT CONTACT:
Name: Horan, R. D.
Phone: 517-355-1301
Fax: 517-432-1800
Email: horan@msu.edu



ACCESSION NO: 0098315 SUBFILE: CRIS
PROJ NO: MINV-65-006 AGENCY: CSVM MINV
PROJ TYPE: STATE PROJ STATUS: EXTENDED
START: 01 OCT 1983 TERM: 30 JUN 2008 FY: 2006

INVESTIGATOR: Collins, J. E.; Goyal, S.

PERFORMING INSTITUTION:
Veterinary Diagnostic Medicine
University of Minnesota
St Paul, Minnesota 55108

ANIMAL DISEASE DIAGNOSTIC LABORATORY

OBJECTIVES: Provide laboratory diagnostic service to poultry and livestock producers and veterinary practitioners for State of Minnesota, conduct preliminary investigations and initiate minor research on new animal disease problems, develop improved diagnostic procedures for selected disease problems.

APPROACH: Case histories and diagnostic laboratory data will be recorded for each case presented. Appropriate laboratory procedures will be employed for each case or specimen. Field diagnostic investigations will be made for animal disease problems as necessary. Efforts will be made to improve existing diagnostic laboratory procedures through minor research studies and new diagnostic procedures will be utilized when developed.

PROGRESS: 2006/01 TO 2006/12
The number of cases submitted to the Minnesota Veterinary Diagnostic Laboratory (MVDL) increased from 66,801 in 2005 to 69,123 in 2006. The number of procedures also increased from 1,364,618 in 2005 to 1,474,680 in 2006. Paratuberculosis (Johne's disease), bovine viral diarrhea, and enteritis by rotavirus, coronavirus, and Salmonella were the major problems. Tuberculosis was also detected in five Minnesota beef herds and one wild white-tailed deer. In pigs the major problems were caused by porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus, and swine influenza virus. Avian pneumovirus continues to be a problem for the turkey industry. Fewer Minnesota deer were tested for chronic wasting disease this year because no cases of this disease were found in the last 4 years of testing.

IMPACT: 2006/01 TO 2006/12
The Veterinary Diagnostic Laboratory plays an important role in protecting the health of animals as well as to protect the public from food borne and zoonotic diseases.

PUBLICATIONS (not previously reported): 2006/01 TO 2006/12
1. Goyal, S.M. (Ed.). 2006. Viruses in Foods. Springer, New York, NY, 345 pp.
2. Tiwari, A., Patnayak, D.P., and Goyal, S.M. 2006. Attempts to improve on a challenge model for subtype C avian pneumovirus. Avian Pathol. 35:117-121.
3. Patnayak, D.P., and Goyal, S.M. 2006. Duration of immunity engendered by a single dose of cold adapted strain of avian pneumovirus. Can. J. Vet. Res.
70:65-67.
4. Allwood, P.B., Malik, Y.S., Maherchandani, S., Hedberg, C.W., and Goyal, S.M. 2006. Effect of temperature on the survival of F-specific RNA coliphage, feline calicivirus, and Escherichia coli in chlorinated water. Int. J. Environ. Res.
Publ. Hlth. 2:442-446.
5. Clay, S., Maherchandani, S., Malik, Y.S., and Goyal, S.M. 2006. Survival on uncommon fomites of feline calicivirus, a surrogate of noroviruses. Am. J. Infect. Control. 34:41-43.
6. Malik, Y.S., Allwood, P.B., and Goyal, S.M. 2006. Disinfection of fabrics and carpets artificially contaminated with calicivirus: relevance in institutional and health care centers. J. Hosp. Infect. 63:205-210.
7. Malik, Y.S., and Goyal, S.M. 2006. Virucidal efficacy of sodium carbonate against FCV, a Norovirus surrogate. Int. J. Food Microbiol. 109:160-163.
8. Chander, Y., Goyal, S. M. and Gupta, S. C. 2006. Antimicrobial resistance of Providencia sp. isolated from animal manure. Vet. J. 172:188-191.
9. McMartin, S., Godden, S., Metzger, L., Feirtag, J., Bey, R., Stabel, J., Goyal, S., Fetrow, J., Wells, S., and Chester-Jones, H. 2006. Heat-Treatment of Bovine Colostrum. I: Effects of temperature on viscosity and immunoglobulin G
level. J. Dairy Sci. 89:2110-2118.
10. Tiwari, A., Patanayak, D.P. and Goyal, S.M. 2006. Survival of two avian respiratory viruses on porous and nonporous surfaces. Avian Dis. 50:284-287.
11. Farnsworth, J.E., Goyal, S.M., Kim, S.W., Kuehn, T.H., Raynor, P.C., Ramakrishnan, M.A., Anantharaman, S., and Tang, W. 2006. Development of a method for bacteria and virus recovery from Heating, Ventilation, and Air Conditioning (HVAC) filters. J. Environ. Monit. 8:1006-1013.
12. Godden, S., McMartin, S., Feirtag, J., Stabel, J., Bey, R., Goyal, S.,
Metzger, L., Fetrow, J., Wells, S., and Chester-Jones, H. 2006. Heat-Treatment of Bovine Colostrum II: Effects of Heating Duration on Pathogen Viability and Immunoglobulin G. J. Dairy Sci. 89:3476-3483.
13. Vincent AL, Lager KM, Ma W, Lekcharoensuk P, Gramer MR, Loiacono C, Richt JA. 2006. Evaluation of hemagglutinin subtype 1 swine influenza viruses from the United States. Vet Microbiol. 118:212-22.
14. Ma W, Gramer M, Rossow K, Yoon KJ. 2006. Isolation and genetic characterization of new reassortant H3N1 swine influenza virus from pigs in the midwestern United States. J Virol. 80:5092-6.
15. Chou J, Wunschmann A, Hodzic E, Borjesson DL. 2006. Detection of Borrelia
burgdorferi DNA in tissues from dogs with presumptive Lyme borreliosis. J Am Vet Med Assoc. 229:1260-5.
16. Wunschmann A, Ziegler A. 2006. West Nile virus-associated mortality events in domestic Chukar partridges (Alectoris chukar) and domestic Impeyan pheasants (Lophophorus impeyanus). Avian Dis. 50:456-9.
17. Finno CJ, Valberg SJ, Wunschmann A, Murphy MJ. 2006. Seasonal pasture myopathy in horses in the midwestern United States: 14 cases (1998-2005). J Am Vet Med Assoc. 229:1134-41.
18. Dean J, Latimer KS, Oaks JL, Schrenzel M, Redig PT, Wunschmann A. 2006. Falcon adenovirus infection in breeding Taita falcons (Falco fasciinucha). J Vet
Diagn Invest. 18:282-6.
19. Fano E, Jiang Y, Faaberg K, Murtaugh MP, Guedes A, Collins JE, Joo HS. 2006. The impact of animal age, bacterial coinfection, and isolate pathogenicity on the shedding of porcine reproductive and respiratory syndrome virus in aerosols from experimentally infected pigs. Can J Vet Res. 70:297-301.
20. Wells SJ, Collins MT, Faaberg KS, Wees C, Tavornpanich S, Petrini KR, Collins JE, Cernicchiaro N, Whitlock RH. 2006. Evaluation of a rapid fecal PCR test for detection of Mycobacterium avium subsp. paratuberculosis in dairy cattle. Clin Vaccine Immunol. 13:1125-30.
21. Cho JG, Dee SA, Deen J, Guedes A, Trincado C, Fano E, Jiang Y, Faaberg K, Collins JE, Murtaugh MP, Joo HS. 2006. Evaluation of the effects of animal age, concurrent bacterial infection, and pathogenicity of porcine reproductive and respiratory syndrome virus on virus concentration in pigs. Am J Vet Res. 67:489-93.


ACCESSION NO: 0403282 SUBFILE: CRIS
PROJ NO: 1940-32000-039-05S AGENCY: ARS 1940
PROJ TYPE: USDA COOPERATIVE AGREEMENT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 58-1940-0-008
START: 01 MAR 2000 TERM: 28 FEB 2005 FY: 2001 GRANT YR: 2000
GRANT AMT: $1,266,886

INVESTIGATOR: Rock D L; Mcintosh M; Riley L

PERFORMING INSTITUTION:
University of Missouri
Columbia, Missouri 65211

PROGRAM FOR PREVENTION OF ANIMAL INFECTIONS AND ADVANCED TECHNOLOGIES FOR VACCINES AND DIAGNOSTICS

OBJECTIVES: (1) To identify and characterize vaccine targets for important viral and bacterial pathogens of livestock; (2) To identify and characterize important factors involved in optimizing host responses to intracellular and extracellular pathogens of livestock; and (3) To perform epidemiologic and other surveys sufficient to identify and design enhanced diagnostic capabilities for economically important pathogens of livestock.

APPROACH: Conduct collaborative research on development of vaccine materials and diagnostics for economically important pathogens of livestock.

PROGRESS: 2000/03 TO 2005/02
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? The infectious agents under study cause significant production losses to domestic food animals and include agents of foreign animal diseases that threaten a key segment of the agricultural economy. Mycoplasmas, mycobacteria, PRRSV and other viruses, and nematodes cause serious animal health problems in the United States and around the world. In some cases, these agents may also pose a significant health risk to humans. The economic impact of these agents to domestic producers exceeds 100 million dollars per annum. Strategies for selective detection and vaccine protection are hampered by a lack of understanding regarding the infectious strategies, genetic diversity and the immunological properties of these agents. We are developing strategies for improved diagnostics and vaccine approaches to meet this challenge. These projects were defined by relevant infectious disease and animal immunology expertise at the University of Missouri, in collaboration with scientists at the Plum Island Animal Disease Center and the Center for Excellence in Vaccine Research at the University of Connecticut. 2. List the milestones (indicators of progress) from your Project Plan. Year 1 (1999-2000) 1. Collaborations were formed and agreements put in place for a three way consortium (USDA-ARS, University of Missouri, and University of Connecticut) to address problems in foreign and domestic animal diseases, both in the design and development of new and improved vaccines as well as diagnostic tests. 2. Collaborators were actively engaged for sequencing the genomes of the mycoplasma pathogens of interest to all groups. 3. SCID-bovine mouse model used for trial of DNA vaccines for bovine tuberculosis. 4. Linkages were developed with USDA-ARS at both Plum Island and NADC for reagent and technique development and animal studies to begin testing new materials and approaches. DNA vaccine study extended to cattle. Year 2 (2000-2001) 5. Project was initiated for sequencing genome of model organism from the mycoides cluster, Mycoplasma capricolum subspecies capricolum. DNA libraries made and contracted with Institute for Systems Biology (ISB) in Seattle for primary sequence determination. 6. Studies in heterochimeric SCID-bovine mice completed and demonstrated efficacy of materials and approach. 7. Full-length PRRSV clone constructed and expressed RNA transfected into tissue culture cells and subsequently inoculated into naïve pigs. Fully infectious virus was not recovered however. Year 3 (2001-2002) 8. Completion of >80% of genome sequence of M. capricolum subsp capricolum, a critical model organism representing a cluster of ruminant pathogens with similar genetic origins; computer and human annotation underway 9. Identification of multiple gene families encoding variable surface proteins of significance to mycoplasma pathogenesis and immune evasion 10. In comparison with data from the foreign threat agent MmySC, identification of refined DNA-based targets for species differentiation and of distinctive patterns of mobile genetic elements that might confound DNA-based diagnostic efforts 11. Studies initiated with NADC-TB unit to evaluate DNA-based constructs for bovine TB constructs in cattle. 12. Alternative strategies for PRRSV infectious clone developed; transfection produced visible cytopathic effects indicating infectious particles. Deletion strategy developed to identify key regions of genome involved in cytopathic effects. Year 4 (2002-2003) 13. Completed genome sequence of M. capricolum subsp capricolum strain Kid. Computational and human annotation was in full swing. Gained access through collaborators to the still unpublished genome sequence of M. mycoides subsp mycoides SC, a foreign threat agent pathogen of cattle. This first preliminary view of a genomic framework for the closely related M. mycoides cluster of bovine and caprine mycoplasma species revealed genomic similarities at multiple levels and underscored the need for detailed comparisons of the genomes from multiple species in this cluster, along with selective other mycoplasmal pathogens, particularly those of cattle. 14. Identified antigen targets in mycoides cluster of mycoplasmas, and in swine pathogenic mycoplasmas, for prototype development of FRET-based platform for biological sensors (in collaboration with faculty in MU Bioengineering). 15. Initiated sequence analysis of bovine pathogen Mycoplasma bovis. DNA libraries prepared and sequence contracted to TIGR. 16. Initial trials were completed to evaluate costimulatory molecules as a means to enhance the development of memory and effector cytolytic T cells in cattle. An aerosol challenge model with Mycobacterium bovis was used to determine the potential of this approach using a subunit DNA vaccine platform. Initial studies demonstrated reduced pathology in costimulatory molecule treated animals. Additional studies began to evaluate the effector pathways involved in direct killing of intracellular mycobacteria. Granulysin and perforin homologs were identified for the bovine. 17. Initiated discovery project using RNA interference for in vivo sterilization of the parasitic nematode Ascaris and prevent infection of swine. Target genes identified and proof of concept established using the nonpathogenic surrogate C. elegans. 18. Initiated 2 additional discovery projects to characterize viral pathogenicity of porcine Circovirus 2 and viral latency of bovine herpes virus infection of cattle. Year 5 (2003-2004) 19. Computer and human annotation of M. capricolum subsp capricolum strain Kid essentially finished and sequence prepared for public release. MmySC sequence from European collaborators was publicly released and published (http://www.genome.org/cgi/content/full/14/2/221). 20. Comparative genomics identified DNA and antigen targets for diagnostic reagent development for the mycoides cluster of pathogens. Continued development of immobilized FRET biosensor to detect mycoplasmas. 21. Mycoplasma bovis DNA sequence close to completion; computer and human annotation begun a. http://www.tigr.org/tdb/mdb/mdbinprogress.html. b. http://www.ncbi.nlm.nih.gov/entrez/query.fcgi? db=genomeprj&cmd=Retrieve&dopt=Overview&list uids=12525 22. Initiated work on sequencing project for Gladysdale strain of MmySC; DNA to be isolated at Plum Island by MU investigators; sequence acquisition contracted with TIGR. 23. Mycobacterium bovis DNA-based vaccine in cattle project curtailed because the principle scientist moved to UTMB in Galveston. Future collaborations on this and related project will be sought. 24. Chimeric PRRS viruses constructed between gene fragments of virulent and attenuated strains to define regions important to virulence. Growth studies were identical and a candidate region was identified that produced reduced numbers and severity of lung lesions. This region is target for additional studies. Quantitative RT-PCR methods developed for virus detection. 25. RNAi effective in sterilizing model nematodes and target genes established for the swine parasite Ascaris. Next step is to move this technology to clinical trials with infected animals. 26. Porcine Circovirus was characterized for expression of viral RNAs and the role of nonstructural proteins in viral growth and pathogenesis. 27. Transgenic mice constructed expressing BHV-1 transcriptional regulators. Neurons expressing these regulators differentially express viral proteins; viral reactivation studies underway. 28. New discovery project initiated to mutate surface protein genes in Francisella tularensis and assess role in cytadherence and pathogenesis of tularemia. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Sequencing analysis of pathogenic mycoplasma strains and identification of specific nucleic acid and protein targets for the further development of diagnostic reagents. Milestone Substantially Met 2. Demonstrated efficacy of materials and approach to DNA-based vaccines and immunostimulatory molecules for tuberculosis in cattle. Milestone Substantially Met 3. Identification and characterization of PRRSV genomic determinants for viral pathogenesis and as diagnostic reagents. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? This agreement has come to an end and has been replaced with a new agreement (58-1949-5-519), Project Number 58-1940-32000-039-08S. This project continues many of the milestones initiated under the previous agreement and establishes the following objectives for the FY 2005 and FY 2006 funding periods. 4a What was the single most significant accomplishment this past year? The single most important accomplishment during FY 2005 was the identification and characterization of PRRSV genomic deteriminats for viral pathogenesis and as diagnostic reagents. 4d Progress report. Computer and human annotation of M. capriocolum subsp capriocolum straid Kid was essetially finished and sequence was prepared for public release. The MmySC sequence from European collaborators was pubicly released and published. The comparative genomics identified DNA and antigen targets for diagnostic reagent development for the mycoides cluster of pathogens. Development continued of the immobilized FRET biosensor to detect mycoplasmas. Mycoplasma bovis DNA sequence is close to completion and computer and human annotation has begun. Work was initiated on the sequencing project for Gladysdale strain of MmySC; DNA to be isolated at PIADC; and sequence acquistion contracted with TIGR. Mycobacterium bovis DNA-based vaccine in cattle project has been curtailed as PI moved. Future collaborations on this and related project will be sought. Chimeric PRRS viruses were constructed between gene fragments of virulent and attenuated strains to define regions important to virulense. Growth studies were identical and a candidate region was identified that produced reduced numbers and severity of lung lesions. This region is targeted for additional studies. Quantitiave RT-PCR methods were developed for virus detection. RNAi effective in sterilizing model nematodes and target genes were established for the swine parasite Ascaris. The next step is to move this technology to clincial trials with infected animals. Porcine Circovirus was characterized for expression of viral RNAs and the role of nonstructural proteins in viral growth and pathogenesis. Transgenic mice were construced expressing BHV- 1 trnascriptional regulators. Neurons expressing these regulators differentially express viral proteins; viral reactivation studies are underway. A new discovery project was also initiated to mutate surface protein genes in Francisella tularensis and assess role in cytadherence and pathogenesis of tularemia. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The major accomplishment of this project is the demonstration of the efficacy of materials and approach to DNA-based vaccines and immunostimulatory molecules for tuberculosis in cattle.

PUBLICATIONS (not previously reported): 2000/03 TO 2005/02
Carter D.B., Lai, L., Park, K.W., Samuel, M., Lattimer, J.C., Jordan, K.R.,
Estes, D.M., Besch-Williford, C., Prather, R.S. Phenotyping of transgenic cloned
pigs. Cloning and Stem Cells. 2002. v. 4. p. 131-145.


ACCESSION NO: 0193834 SUBFILE: CRIS
PRkOJ NO: MOV-4-FF31 AGENCY: CSREES MO.V
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2002 TERM: 30 SEP 2004 FY: 2003

INVESTIGATOR: Estes, D. M.

PERFORMING INSTITUTION:
Veterinary Pathology
University Of Missouri
Columbia, Missouri 65211

CHARACTERIZATION OF THE CYTOLYTIC ACTIVITY OF GAMMA DELTA T LYMPHOCYTES

NON-TECHNICAL SUMMARY: The organism that causes bovine tuberculosis (Mycobacterium bovis) presents a significant biosecurity risk to the US cattle industry and can also cause tuberculosis in humans. The objective of the proposed research is to study a cell type that may have a very important role for helping the immune system fight disease caused by tuberculosis. The gamma delta T lymphocyte is an immune cell that can kill cells that are infected with Mycobacterium bovis. Identifying the molecules that gamma delta T lymphocytes use to kill Mycobacterium bovis infected cells is the focus of the proposed research.

OBJECTIVES: To characterize the cytotoxic mechanisms that bovine gamma delta T lymphocytes use to kill cells infected with Mycobacterium bovis (M. bovis). The specific goals are to identify cytolytic proteins produced by gamma delta T cells after exposure to M. bovis infected cells, and to demonstrate production of these proteins in forming a mycobacterium granuloma.

APPROACH: Production of cytolytic proteins by gamma delta T lymphocytes will initially be determined in vitro. Peripheral blood mononuclear cells will be collected from a healthy bovine donor. Gamma delta T cells will be separated from the blood sample using magnetically labeled antibody and exposed to bovine macrophages that have been infected with Mycobacterium bovis (M. bovis). Total cell lysates will be collected from the gamma delta T lymphocytes following exposure to the infected macrophages and electrophoresed in a polyacrylamide gel. Western blotting will be performed on the separated proteins to determine the production of cytolytic molecultes as a result of M. bovis exposure. The ability of gamma delta T lymphocytes to induce apoptosis in macrophages infected with M. bovis will also be determined using a commercially available kit for detection of cell apoptosis and death. To determine the tissue localization of cytolytic gamma delta T lymphocytes during infection, scid-bo mice will be infected with a virulent strain of M. bovis. The scid-bo mice will be generated by engrafting scid/beige mice with fetal bovine tissue, and thus reconstituting the mice with a bovine immune system. Tissues will be collected following infection and fixed in formalin. The location of cells producing cytolytic proteins, relative to M. bovis granulomas, will be determined using immunohistochemistry. The phenotype of T cells in the M. bovis granuloma will also be determined using immunohistochemistry. The location of cells infected with M. bovis in infected tissues will be determined using the Ziehl-Nielson technique for visualization of acid fast bacilli.

PROGRESS: 2002/10 TO 2004/09
Completed analysis of cytotoxic protein expression in M. bovis granulomas from infected animals and determined the effect of depleting the WC1+ gamma/sigma T cell population on expression of these proteins. Left MU; appointment at University of Texas Medical Branch, Department of Pediatrics.

IMPACT: 2002/10 TO 2004/09
These cells comprise a major portion of the T cell population in ruminants yet their functions are largely uncharacterized. On completion of this series of studies, we will have a much greater insight into their direct antimicrobial activities.

PUBLICATIONS (not previously reported): 2002/10 TO 2004/09
No publications reported this period

PROJECT CONTACT:
Name: Estes, D. M.
Phone: 573-882-1385
Email: estesd@missouri.edu


ACCESSION NO: 0192602 SUBFILE: CRIS
PROJ NO: MOV-NRI ENDSLEY AGENCY: CSREES MO.V
PROJ TYPE: NRI COMPETITIVE GRANT PROJ STATUS: TERMINATED
CONTRACT/GRANT/AGREEMENT NO: 2002-35204-12307 PROPOSAL NO: 2002-02183
START: 01 SEP 2002 TERM: 31 AUG 2004 FY: 2003 GRANT YR: 2002
GRANT AMT: $90,000

INVESTIGATOR: Endsley, J.; Estes, D.

PERFORMING INSTITUTION:
Veterinary Pathology
University Of Missouri
Columbia, Missouri 65211

CHARACTERIZATION OF THE CYTOLYTIC ACTIVITY OF GAMMA DELTA T LYMPHOCYTES

NON-TECHNICAL SUMMARY: The organism that causes bovine tuberculosis (Mycobacterium bovis) presents a significant biosecurity risk to the US cattle industry and can also cause tuberculosis in humans. The objective of the proposed research is to study a cell type that may have a very important role for helping the immune system fight disease caused by tuberculosis. The immune system is composed of many different cells and molecules that protect the body against infection by microorganisms. While the importance of many different immune cell types is known, the means used by these cells to protect against infection is not completely understood. The knowledge gained by characterizing the mechanism these cell types use to fight disease may greatly advance the development of protective vaccines. The gamma delta T lymphocyte is an immune cell that can kill cells that are infected with Mycobacterium bovis. Identifying the molecules that gamma delta T lymphocytes use to kill Mycobacterium bovis infected cells is the focus of the proposed research. Gamma delta T lymphocytes will be taken from a cow blood sample and allowed to interact with Mycobacterium bovis infected cells in an artificial environment. The molecules produced by the gamma delta T lymphocytes in response to the infected cell will be identified. Tissues from infected animals will then be examined to determine if production of these molecules by gamma delta T lymphocytes prevents the spread of disease. This information is very important for designing vaccines that activate immune cells with a primary role in preventing tuberculosis.

OBJECTIVES: The overall objective of the proposed research is to characterize the cytotoxic mechanisms that bovine gamma delta T lymphocytes use to kill cells infected with Mycobacterium bovis (M. bovis). The specific goals are to identify cytolytic proteins produced by gamma delta T cells after exposure to M. bovis infected cells, and to demonstrate production of these proteins in a forming mycobacterium granuloma.

APPROACH: The production of cytolytic proteins by gamma delta T lymphocytes will initially be determined in vitro. Peripheral blood mononuclear cells will be collected from a healthy bovine donor. Gamma delta T cells will be separated from the blood sample using magnetically labeled antibody and exposed to bovine macrophages that have been infected with Mycobacterium bovis (M. bovis). Total cell lysates will be collected from the gamma delta T lymphocytes following exposure to the infected macrophages and electrophoresed in a polyacrylamide gel. Western blotting will be performed on the separated proteins to determine the production of cytolytic molecules as a result of M. bovis exposure. The ability of gamma delta T lymphocytes to induce apoptosis in macrophages infected with M. bovis will also be determined using a commercially available kit for detection of cell apoptosis and death. To determine the tissue localization of cytolytic gamma delta T lymphocytes during infection, scid-bo mice will be infected with a virulent strain of M. bovis. The scid-bo mice will be generated by engrafting scid/beige mice with fetal bovine tissue, and thus reconstituting the mice with a bovine immune system. Tissues will be collected following infection and fixed in formalin. The location of cells producing cytolytic proteins, relative to M. bovis granulomas, will be determined using immunohistochemistry. The phenotype of T cells in the M. bovis granuloma will also be determined using immunohistochemistry. The location of cells infected with M. bovis in infected tissues will be determined using the Ziehl-Nielson technique for visualization of acid fast bacilli.

PROGRESS: 2002/09 TO 2004/08
The objective of the proposed research was to characterize the cytotoxic mechanisms of bovine WC1 gamma delta T cells against M. bovis infected macrophages and provide information regarding the in vivo relevance of this response during infection. To date we have used real time PCR to establish baseline kinetics of expression of cytokine genes and cytotoxic protein genes by purified WC1 gamma delta T following stimulation in culture. The expression of cytotoxic proteins by WC1 gamma delta T and WC1 gamma delta T depleted lymphoctyes has also been determined. A manuscript titled Expression of Immunoregulatory Cytokines and Cytotoxic Effector Molecules by Activated gamma delta T Lymphocytes is currently being prepared for submission to the Journal of Leukocyte Biology. In order to analyze expression of cytotoxic proteins by T lymphocytes at sites of infection in M. bovis infected animals, we have optimized an immunohistochemistry technique for antigen retrieval of cell surface markers and cytotoxic proteins in formalin fixed tissue sections. We are currently evaluating the production of cytolytic proteins and identifying the T cell subset producing cytolytic proteins at the site of a M. bovis liver granuloma in the presence and absence of WC1 gamma delta T cells. A manuscript titled WC1 gamma delta T cells Indirectly Regulate Chemokine Production during Mycobacterium bovis Infection in SCID-bo Mice is in preparation for submission to the Journal of Immunology. We have also cloned a previously uncharacterized bovine cytotoxic granule protein (granulysin), established activity of this protein against gram negative and gram positive bacteria, and determined the expression of bovine granulysin in CD4 , CD8 , and WC1 gamma delta T cells. This work titled Characterization of Bovine Homologues of Granulysin and NK-lysin was published this year (J. Immunology. Aug 15;173(4):2607-14). Our laboratory has developed antibody to bovine granulysin for use in the analysis of effector mechanism used by bovine T lymphocytes in antigen specific cell mediated immune responses. Disclosure documents have been filed for the bovine molecule and antibody for subsequent patent application. Research results from this project were presented at two national meetings (American Association of Immunogists, Denver, CO 2003 and The Changing Landscape of Vaccine Development, Galveston, TX, 2004) and one international meeting (The International Veterinary Immunology Symposium, Quebec, 2004).

IMPACT: 2002/09 TO 2004/08
The potential for the bovine gamma delta T lymphocyte subset to express cytotoxic proteins and produce cytokines that may have roles in regulating effector activity of other T lymphocytes was characterized as a result of this research. The abundance of the gamma delta T lymphocyte subset, along with the ability to contribute to cytotoxicity against infected cells, indicates the need to consider gamma delta T cell activation when designing vaccines. The enhancement of T cell mediated immune responses by vaccine design (choice of epitope, adjuvant, etc.) is important for protecting cattle against many pathogens. The availability of effective vaccines is very relevant to reducing economic losses in the dairy and beef cattle industries and for preventing agroterrorism related food supply instability.

PUBLICATIONS (not previously reported): 2002/09 TO 2004/08
1. Endsley, J.J. and D. M. Estes. 2004. Expression of Immunoregulatory Cytokines and Cytotoxic Effector Molecules by Activated gamma delta T Lymphocytes. J. Leukocyte Biology. (in preparation).
2. Alvarez, A., J.J. Endsley, D. Werling, and D.M. Estes. 2004. WC1 gamma delta T Cells Indirectly Regulate Chemokine Production During Mycobacterium bovis Infection in SCID-bo Mice. J. Immunology. (in preparation).
3. Endsley, J.J. , J.L. Furrer, M.A. Endsley, M.A. McIntosh, A.C. Maue, W.R. Waters, D.R. Lee, and D. M. Estes. 2004. Characterization of Bovine Homologues of Granulysin and NK-lysin. J. Immunology. Aug 15;173(4):2607-14.

PROJECT CONTACT:
Name: Endsley, J.
Phone: 573-884-8966
Fax: 573-884-5414
Email: endsleyj@missouri.edu


ACCESSION NO: 0194275 SUBFILE: CRIS
PROJ NO: MONB00032 AGENCY: CSREES MONB
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 OCT 2002 TERM: 30 SEP 2006 FY: 2005

INVESTIGATOR: Quinn, M. T.

PERFORMING INSTITUTION:
Veterinary Molecular Biology
Montana State University
Bozeman, Montana 59717

ANALYSIS OF BISON INNATE DEFENSE AGAINST MICROBIAL PATHOGENS

NON-TECHNICAL SUMMARY: The American Bison (Bison bison) is a wild/semi-domesticated ruminant that encounters serious infectious diseases, such as tuberculosis and brucellosis. This project studies the types of antimicrobial proteins present in bison neutrophils and how active they are against several relevant pathogens. A better understanding of these proteins could potentially lead to practical applications to controlling infectious disease in bison and other wildlife.

OBJECTIVES: We hypothesize that bison neutrophils contain mobilizable proteins, which have direct antimicrobial properties with therapeutic potential against persistent bison diseases such as tuberculosis. To address this hypothesis, we propose the following aims: 1) Characterize biochemically and functionally the types of antibacterial proteins present in bison neutrophils and 2) Probe for and clone selected bison neutrophil antimicrobial proteins.

APPROACH: Objective 1 will require the collection of blood from captive bison and further isolation and purification of neutrophils. We will then use one or more procedures to extract a subset of proteins, which is likely to contain neutrophil antimicrobial proteins (AMPs), as determined by bacterial killing assays. Finally, we will systematically screen the proteins we have extracted, for killing activity against E. coli, S. aureus, and M. bovis BCG. Following completion of objective 1, we hope to have identified one or more proteins that have interesting antimicrobial activity; we then propose to find and clone the gene(s) for these proteins, so that they can be produced as recombinant proteins for further detailed mechanistic and microbiological testing. One possibility is that the protein of interest is one of the bison bactenecins, which we have already begun to characterize, and we will continue cloning these genes as described below. If the protein does not appear to be one of the bactenecins, we will use N-terminal sequencing to obtain sufficient code to design primers for further sequencing/cloning.

PROGRESS: 2002/10 TO 2006/09
Bison can become infected with bacteria that linger as intracellular parasites, where they are sheltered from neutrophil defenses. This project studied the types of antimicrobial proteins and processes present in bison neutrophils and how active they are against several relevant pathogens. A protein of interest, one of the bison bactenecins, has been characterized and sequencing/cloning efforts are underway. Bison neutrophil granule extracts were found to have potent killing activity against E. coli. Conversely, the neutrophil extracts did not kill S. aureus and, in fact, had a permissive effect. Analysis of the extracts showed that the granules possessed many low molecular weight proteins. Further analysis showed that bovine and bison neutrophil granule proteins may have differences in molecular weight, which may or may not translate to functional differences. In addition to these antimicrobial proteins, bison neutrophils also possess a number of microbicidal functions. Since not much is known about the system responsible for oxidant production in bison (the NADPH oxidase), we characterized this bison neutrophil function and found unique differences that may allow bison to respond to the distinct host defense challenges that they encounter. We also cloned and sequenced the genes for six bison NADPH oxidase components. When compared to other species, the bison proteins were most similar to those of bovine, but were less similar to those of the other species. Overall, these studies show that the bison and bovine NADPH oxidase genes are highly conserved between these two species, despite their divergence from a common ancestor over 1 million years ago. Extension of this work into understanding the role of neutrophil function in brucella infection could potentially lead to better ways to control the spread of Brucellosis from bison to cattle.

IMPACT: 2002/10 TO 2006/09
Bison can become infected with bacteria that linger as intracellular parasites, where they are sheltered from neutrophil defenses. This project studies the types of antimicrobial proteins present in bison neutrophils and how active they are against several relevant pathogens. A better understanding of these proteins could potentially lead to practical applications to controlling infectious disease in bison and other wildlife.

PUBLICATIONS (not previously reported): 2002/10 TO 2006/09
No publications reported this period

PROJECT CONTACT:
Name: Quinn, M. T.
Phone: 406-994-5721
Fax: 406-994-4303
Email: mquinn@montana.edu


ACCESSION NO: 0404496 SUBFILE: CRIS
PROJ NO: 5438-32000-023-00D AGENCY: ARS 5438
PROJ TYPE: USDA INHOUSE PROJ STATUS: NEW
START: 17 NOV 2001 TERM: 16 NOV 2006 FY: 2006

INVESTIGATOR: Heaton M P; Clawson M L; Harhay G P; Chitko Mckown C G; Laegreid W W

PERFORMING INSTITUTION:
Agricultural Research Service
Clay Center, Nebraska 68933

GENETIC PREDISPOSITION OF LIVESTOCK TO INFECTION BY MUCOSAL PATHOGENS

OBJECTIVES: 1) Identify nucleotide sequence variation in host genes that play a fundamental role in the infection process and 2) identify DNA sequences that are associated with susceptibility or resistance to infectious disease, 3) provide a secure, well-annotated database of nucleotide seqence data, and associated biological data, of relevant viral pathogens affecting livestock.

APPROACH: Infectious diseases in livestock are a significant source of economic loss and represent a potential risk to human health. Improvements in herd health and food safety may result, for example, if individuals with the highest risk for infectious disease are eliminated from the production cycle. The ultimate goal of functional genomics with regard to animal health is to read an animal's DNA sequence and estimate its risk of acquiring or maintaining infections. Before this can be accomplished, there are two key issues to address: 1) the identification of nucleotide sequence variation in host genes that play a fundamental role in the infection process and 2) the identification of DNA sequences that are associated with susceptibility of resistance to infectious disease. This project is designed to address these issues in commercial populations of livestock. As these objectives are achieved for selected candidate genes, new information and technology will be developed that will facilitate reading an animal's DNA sequence for estimating its risk of acquiring or maintaining infections. In addition a database containing a set of well-annotated genomic sequences of viral pathogens currently on the threat list, and sequences of known pathogens that produce disease similar to those caused by threat list agents. Beyond providing the basis for analysis of pathogen isolates, this high quality core of reference sequences will enable the discovery of association between pathogen sequence and phenotypes on a scale not yet approached in livestock infectious disease research.

PROGRESS: 2005/10 TO 2006/09
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Infectious diseases in livestock are a significant source of economic loss and represent a potential risk to human health. Improvements in herd health and food safety may result if animals with the highest risk for infectious disease are eliminated from the production cycle. The ultimate goal of this project is to read an animal's DNA sequence (genotype) and estimate its risk of acquiring or maintaining infections. This requires the identification of host genes and DNA sequence variation associated with infectious diseases in livestock. Important outcomes of this research are accurate and economical genetic tests that are accessible by managers of U.S. livestock populations. In cattle, the total number of death losses from respiratory and digestive diseases is about 2 million animals per year and cost about $800 million. Although these figures include non-infectious diseases, it is acknowledged that many (if not most) are caused by infectious agents. With regard to human health, the top six foodborne bacterial pathogens are estimated to cost about $5 billion annually due to illness. Lastly, reducing the impact of infectious agents is important in maintaining consumer confidence through all phases of production and has significant effects on domestic and export markets for fresh meat. In lost beef export markets alone, the economic impact of the Washington State bovine spongiform encephalopathy (BSE) case announced December 23, 2003, was estimated to be between $3.2 and $4.7 billion in revenue loss to the U.S. beef industry in the first year alone. This research addresses several goals of the National Program 103 Action Plan for Animal Health including: genetic resistance to disease, epidemiology of disease, and strategies to control infectious and non- infectious diseases. 2. List by year the currently approved milestones (indicators of research progress) There are two objectives in the Project Plan: 1) discover DNA sequence differences in livestock and 2) evaluate these alleles for association with infectious disease. Milestones from Objective 1 in the Project Plan: FY 2001: Develop a DNA-based test for disease traceback in beef cattle. FY 2001: Identify DNA sequence variation for the bovine neonatal Fc receptor, e.g. the IgG alpha chain transporter (FCGRT). FY 2002: Adapt the traceback test for parentage determination in beef cattle. FY 2005: Identify DNA sequence variation in the bovine beta-2- microglobulin (B2M) gene. FY 2005: Identify DNA sequence variation in other disease-related genes. FY 2006: Collect case-control material for ovine progressive pneumonia (OPP) in sheep. FY 2006: Identify 75 new single nucleotide polymorphisms (SNPs)(200 total). FY 2006: Complete the sequence analysis for a 25-kb bovine prion gene region in 192 animals representative of U.S. beef and dairy populations. FY 2006: Identify 60 additional DNA markers for disease traceback in North American beef and dairy populations. Milestones from Objective 2 in the Project Plan: FY 2003: Assemble diverse germplasm for U.S. sheep populations. FY 2004: Evaluate FCGRT alleles for association with failure of passive transfer (FPT) phenotype in neonatal calves. FY 2005: Evaluate B2M alleles for association with FPT in neonatal calves. FY 2005: Assemble USMARC case-control material for OPP in sheep. FY 2005: Identify new DNA-markers for disease traceback in beef and dairy populations. FY 2006: Risk factor analysis for OPP in sheep. 4a List the single most significant research accomplishment during FY 2006. Discovery of new genetic variation in the bovine prion gene: Bovine spongiform encephalopathy (BSE) is a fatal neurological disorder characterized by abnormal deposits of a protease resistant isoform of the prion protein. Characterizing the bovine prion gene is important for understanding its role in BSE susceptibility. Our aim was to sequence a 25.2-kb genomic region containing the prion gene in 192 diverse U.S. beef and dairy cattle. Sequence analyses identified 388 total DNA differences in cattle, of which 287 have not previously been reported. This number of DNA sequence differences in the prion gene region of U.S. cattle is nearly four times greater than all the previously described differences combined. Our research defined the patterns of DNA differences that may influence BSE susceptibility in cattle. 4b List other significant research accomplishment(s), if any. Discovery of new genetic types of scrapie susceptibility: Genetic variation within the ovine prion gene of domestic sheep is associated with predisposition to scrapie, a transmissible spongiform encephalopathy disease of sheep. We identified a new pattern of DNA differences associated with scrapie susceptibility. The chromosomal DNA sequence encoding scrapie-susceptible "ARQ" type, was further subdivided into nine different classes. The existence of multiple classes of the "ARQ" type raises the question of whether sheep bearing these different versions of the prion gene are equally susceptible to scrapie. The results are important for a higher resolution analysis of genetic contributions to scrapie susceptibility. Traceback of a tuberculosis-positive steer: At the request of APHIS, we used gender-specific DNA markers to confirm the identity of a male tuberculosis-positive animal from a cohort of otherwise female bovine DNA samples. Our laboratory was able to report conclusive results to APHIS within 24 hours of receiving the samples. Assays for the markers were developed in our lab and are described in peer-reviewed scientific publications. 4d Progress report. Major accomplishment during FY 2006: In cattle, the second set of 30 highly-informative SNPs in U.S. beef and dairy populations are publicly available in GenBank. This represents 60 total files viewable at GenBank from our project entitled "A single nucleotide polymorphism (SNP) marker set for DNA-based traceback in North American beef and dairy cattle." The target SNPs have an average minor allele frequency of 0.424 (beef) and 0.433 (dairy) in diverse groups of U.S. cattle. In this set of 60 files, we have identified and annotated 847 flanking polymorphisms (important for assay designs) from 64,107 total bp sequenced through each of 216 diverse beef and dairy cattle. This amounts to 539,038 total bp of annotated sequence including: 127 amplicons, 110 exons, 92 CDSs, and 854 bovine repetitive elements. With the 60 highly-informative markers, the average probability that the genotypes from two unrelated individuals are identical by state is about 7 x 10E-26. The average probability of paternity exclusion to be 0. 999996 and 0.9995 for all 60 SNPs with one or zero parents known, respectively. These are markers specially selected for optimum power, genome-wide distribution, accuracy in genotyping, and high-throughput "multiplexibility." The target (parentage) SNP is flagged with the note: "highly polymorphic in diverse populations of U.S. beef and dairy cattle. " An NCBI nucleotide query with "heaton & contig" at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide&itool=toolbar will retreive these files: DQ381153, DQ451555, AY773474, DQ404150, DQ404149, AY761135, DQ404151, DQ404152, AY776154, AY841151, DQ422949, DQ786757, DQ422950, DQ647187, AY842472, AY842474, AY842473, DQ435443, DQ489377, DQ839235, DQ647186, AY842475, DQ485413, DQ647188, DQ470475, DQ500958, DQ647189, DQ468384, DQ846688, AY844963, DQ647190, DQ789028, AY849380, AY849381, DQ786758, DQ650635, DQ786759, DQ650636, AY850194, DQ837644, DQ674265, DQ846689, DQ786765, DQ786766, DQ786760, DQ786761, DQ786762, DQ837646, DQ837645, AY851162, DQ837643, AY851163, DQ786763, DQ786764, DQ832700, AY853302, AY853303, DQ846690, DQ846691, DQ846692. 5. Describe the major accomplishments to date and their predicted or actual impact. Host-pathogen expression analysis. Bacterial infections in cattle represent a potential risk to animal and human health. At USMARC, we reported the identification and mapping of two bovine genes that are expressed in response to Escherichia coli O157:H7 exposure. Transcripts encoding interleukin 8 (IL-8) and a novel chemokine gene (ECIP-1) were markedly elevated when measured by two independent methods. Our results were important because this family of chemokine genes became the target of subsequent genomic efforts and was the first example of mapping bovine genes with DNA sequencing--SNP technology. SNP linkage mapping is now standard practice in U.S. cattle genetics. DNA sequence diversity in bovine cytokine genes: Dissection of complex traits in commercial populations of cattle will require the ability to genotype significant numbers of animals and an abundant supply of informative DNA markers. At USMARC, we reported the first estimation of DNA sequence diversity in bovine genes and established MALDI-TOF MS as an accurate high-throughput automated genotype scoring platform for use in cattle. The average number of SNP haplotype alleles was 4.4 per 600-bp bovine PCR amplicon and these haplotypes could be correctly deduced without use of pedigree information. Our results were important because they indicated that a wide range of genetic studies in commercial populations of cattle was possible where genotypic information from relatives may not be available. Haplotype analysis of bovine IL8 gene: Efficient use of gene-based SNP markers in commercial populations of cattle requires the identification of the majority of haplotype alleles for each gene and an estimation of the allele frequencies in relevant breeds. At USMARC, we assembled a panel of 96 sires that reflect the breadth of genetic diversity in U.S. beef cattle and reported the nucleotide sequence diversity and haplotype structures of IL-8. The five IL-8 haplotypes identified are estimated to be present in more than 98% of U.S. beef cattle. Our results were important because they showed that a diverse population sampling, combined with high-resolution tests within candidate gene regions, will provide molecular tools required for genetic epidemiology. DNA-based animal ID and parentage in beef cattle: Although SNP marker technology represents a promising means for determining the genetic identity and kinship of an animal, in cattle, the challenge has been to identify SNPs with sufficient power for use in many popular breeds and crossbred populations. At USMARC, we have described a carefully selected set of 32 SNP markers that is useful for both "fingerprinting" animals and determining paternity. The selection criteria were critical since randomly selected sets of SNP markers had little power. Our results were important because they represented the first SNP-based test for identity and paternity testing in any animal species other than human, and allowed the design of robust accurate genotype assays on a variety of economical high-throughput SNP genotyping platforms. DNA marker for failure of passive transfer (FPT), a health-related trait in cattle: Newborn calves afflicted with FPT do not posses immuno-protective maternal antibodies and they are highly prone to illness and death from infectious diseases. At USMARC, we identified the first maternal DNA marker for FPT in beef calves. A gene (FCGRT) encoding part of the neonatal immunoglobulin transfer protein (FcRn) was sequenced in populations of cattle and specific nucleotide sequence changes were associated with low or high concentrations of immunoglobulin in calves. Our results are important because they suggest a genetic component to the problem of low antibody levels in young calves, and they provide DNA markers on the dam's side for predicting FPT. This facilitates developing genetic strategies for controlling this important problem. Washington State BSE index case: Reducing the impact of BSE is important in maintaining consumer confidence through all phases of production and has significant effects on domestic and export markets for fresh meat. At USMARC, we described novel prion gene sequence variation in U.S. cattle, sheep, and deer. These results were published in a peer-reviewed scientific journal and deposited into GenBank one month prior to the December 23, 2003, Washington State BSE index case. Our results are important because they provided a reference map of the common prion gene sequence variation present in U.S. populations. The reference variation in U.S. beef cattle was compared with that from the U.S. and Canadian BSE cases to show that the latter sequences were unremarkable. Moreover, we used our DNA-based traceback tests to verify pedigrees associated with the Washington State BSE case and, thereby, confirmed its Canadian origin. Identification of new FPT risk factor in calves: Calves affected with FPT are at risk for morbidity and mortality from infectious disease. At USMARC, we described the association of a B2M haplotype with FPT in newborn calves. These results were published in a peer-reviewed scientific journal and deposited into GenBank. Our results are important because FPT is a serious problem in U.S. beef cattle that can result in calf illness or death. The identification of this DNA marker may facilitate new genetic programs that reduce the prevalence of FPT in U.S. beef cattle by selective breeding programs. Developed a new DNA test for sheep scrapie susceptibility: Scrapie is a fatal, transmissible disease affecting the central nervous system of sheep and its eradication is a national priority in the U.S. and Europe. At USMARC, we developed a novel and highly accurate DNA test for scrapie susceptibility in sheep. These results were made public by deposition in GenBank. Our results are important because they facilitate accurate and efficient analysis of scrapie susceptibility in sheep. Genotyping companies are now offering this low-cost DNA test to the public as part of the National Scrapie Eradication Program. Developed new DNA markers for gender testing: Embryo transfer is widely used in the beef and dairy industries and a cornerstone of many breeding programs. Determining an embryo's sex prior to implantation may advance the rate of genetic improvement without compromising the calf's productivity. At USMARC, we described a new DNA- based gender-specific test for U.S. beef cattle. The results were published in a peer-reviewed scientific journal and deposited into GenBank. Our results are important because accurate characterization of gender from bovine cells, tissues, or embryos is essential for many beef and dairy improvement programs and for DNA traceback situations. Our results have been employed in the development of commercially available bovine sex-typing assays. DNA-based carcass tracking in slaughter plants: Accurate food animal identification is essential for improving disease control and enhancing food safety. Our study showed that a selected set of 20 beef cattle DNA markers will verify sample tracking in a large, federally inspected, Northeastern slaughter facility that primarily processes culled dairy cows. Blood was collected from random animals just prior to slaughter and the purported corresponding liver samples were collected during beef processing. DNA tests were run on each sample and the results were compared. Results showed that the chance of a coincidental genotype match between two animals was estimated to be 1 in 23 million. DNA testing confirmed the matches for more than 90% of the purported blood-liver pairs and also revealed the mismatched samples. Our results are important for two reasons: 1) these DNA markers are able to accurately match bovine carcasses to their tissues and 2) the carcass mismatch rates may be significant in slaughter plants. This has implications for food safety "test and hold" programs that rely on holding the correct carcass until a particular food safety-related test result is obtained. Developed a set of 15 PRNP genotyping controls for sheep scrapie eradication programs: As part of ongoing sheep genetic experiments at the USMARC, we have developed a genetic test for scrapie susceptibility. The details of this novel prion gene (PRNP) genotyping test have not yet been published but were made publicly available via GenBank submissions. This genotyping assay detects PRNP haplotype combinations and has been adopted by a number of genotyping companies. Collectively, these companies are conducting the majority of commercial PRNP genotyping assays conducted in the U.S. Additionally, we recognized that DNA controls for all 15 possible combinations of the five common PRNP haplotypes (ARR, ARQ, ARH, AHQ, and VRQ) are needed as a quality control check for our genotyping production runs. Moreover, several members of the PRNP genotyping community have expressed a similar need for these DNA controls. Thus, we have identified 15 USMARC sheep, each having one of these 15 genotype combinations, and created a set of 15 PRNP DNA genotyping controls. To facilitate use of these DNA controls across a variety of genotyping platforms, we have sequenced the complete PRNP coding sequence for each of the 15 genotype combinations and made them publicly available in GenBank (accession numbers AY907681-AY907694 and AY909542) and distributed the genotyping controls to APHIS and other institutions around the world. 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? In total, more than 2000 SNPs have been identified, made publicly available, and transferred to more than 43 companies/institutions in more that 22 countries. Moreover, we have assisted more than a dozen commercial genotyping companies, forensic laboratories, research institutions, and universities in the adaptation of our assays to their particular genotyping platforms. The genetic tests include those for health, animal identity, parentage, gender, species, and prion diseases. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). "Genetics of disease resistance in sheep," American Sheep Industry Association Research Committee, November 24, 2003. "Beef cattle genomics and animal health," ARS TSE Scientific Committee/Working Group at the Annual NCBA Conference, Phoenix, AZ, January 28, 2004. "Using DNA evidence to test a pedigree: The Washington State BSE case, December 23, 2003," U.S. Meat Export Federation-European Union Meat Representatives, June 7, 2004. "TSEs touch off ARS research," Agricultural Research magazine, December 2004 issue, pp. 4-9. "The best DNA markers pennies can buy." Article written for cattle breed associations. June 2006.

PUBLICATIONS (not previously reported): 2005/10 TO 2006/09
1. Isler, B.J., Freking, B.A., Thallman, R.M., Heaton, M.P., Leymaster, K.A. 2006. Evaluation of associations between prion haplotypes and growth, carcass, and meat quality traits in a Dorset x Romanov sheep population. Journal of Animal Science. 84:783-788.
2. Harhay, G.P., Sonstegard, T.S., Keele, J.W., Heaton, M.P., Clawson, M.L., Snelling, W.M., Wiedmann, R.T., Van Tassell, C.P., Smith, T.P. 2005. Characterization of 954 bovine full-CDS cDNA sequences. Biomed Central (BMC) Genomics 6:166 (11 pp).
3. Clawson, M.L., Heaton, M.P., Keele, J.W., Smith, T.P., Laegreid, W.W. 2006. Linkage disequilibrium and haplotype structure in the prion gene of U.S. beef and dairy cattle (abstract). Plant and Animal Genome Conference Proceedings. p. 145.
4. Donthu, R., Larkin, D.M., Heaton, M.P., Lewin, H.A. 2006. In silico discovery, mapping, and genotyping of 1,039 cattle SNPs on a panel of eighteen breeds [abstract]. Plant and Animal Genome Conference Proceedings. p. 235.
5. Heaton, M.P., Leymaster, K.A., Clawson, M.L., Freking, B.A., Laegreid, W.W. 2006. A freely-available set of reference DNAs for genotyping the major scrapie susceptibility alleles in sheep [abstract]. Plant and Animal Genome Conference Proceedings. p. 239.
6. Isler, B.J., Freking, B.A., Heaton, M.P., Thallman, R.M., Leymaster, K.A. 2005. Effects of prion haplotype on growth and carcass traits in sheep [abstract]. Midwestern Section of the American Society of Animal Science. 83(Suppl. 2):44-45


ACCESSION NO: 0186991 SUBFILE: CRIS
PROJ NO: NYC-433333 AGENCY: CSREES NY.C
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 JUN 2000 TERM: 30 SEP 2004 FY: 2004

INVESTIGATOR: Russell, D. G.

PERFORMING INSTITUTION:
Veterinarian Microbiology & Immunology
Cornell University
Ithaca, New York 14853

PATHOGEN MYCOBACTERIA: M.TUBERCULOSIS, M. BOVIS, AND M. AVIUM

NON-TECHNICAL SUMMARY: Mycobacterial species are important pathogens in both animals and humans. Study into the lifestyles of mycobacteria species pathogenic to animals and humans such as M. tuberculosis, M. leprae, M. bovis and M. avium reveals extensive parallels in their mechanisms of intracellular survival and persistence. The laboratory is devoted to the study of microbial pathogens that exploit the macrophage as their host cell.

OBJECTIVES: The laboratory is dedicated to the study of pathogen mycobacteria. The primary area of interests are as follows: 1. Analysis of the biology of the interaction between the macrophage and the bacillus with respect to the intracellular environment and the regulation of host cell function. 2. The elucidation of the metabolism of the intracellular bacillus and its exploitation as possible targets of drug action. 3. The appreciation of the modulation of the infection foci and the role of the granuloma in the persistence of infection.

APPROACH: Cell Biology of Intracellular Infections by Mycobacterium avium: This delicate interplay between the bacterium, and its potentially microbicidal host cell is little understood. Previous work in the laboratory has fostered the belief that, with respect to the interaction with macrophages and the immune responses at site of infection, there are many parallels between pathogenic mycobacteria species, including Mycobacterium bovis. For this reason emphasis is also placed on exploitation of genetic approaches available for other mycobacterial species, notably M. tuberculosis and M. bovis, for the resolution of mechanisms common to all pathogenic mycobacteria. 2. Elucidation of Intermediate Metabolism and Carbon Source Acquisition by M. bovis and M. tuberculosis: We have extensive experience in modeling intracellular infections by both prokaryote and eukaryote pathogens and propose to apply this expertise to determining the contribution of the glyoxylate shunt pathway to infection. 3. Formation and Maintenance of the Granuloma and it role in Infection by Mycobacterium spp: We have carried out a systematic analysis of bacterial lipidoglycans released and trafficked through infected macrophages. These comprise 7 major species of lipids some, or all, capable of inducting granulomas in mice. The ability of these lipids to expand the influence of the bacteria beyond the infected macrophages and induce granuloma formation suggest that they play roles key to the evolution of this response. Our recent development of an in vivo model exploiting these lipids will enable the functional determination of the roles of these molecules in granuloma induction.

PROGRESS: 2000/06 TO 2004/09
Mycobacterium tuberculosis is a phenomenally successful pathogen that infects approximately a third of its host species, mankind. Its success lies in its capacity to establish and maintain its infection within the hosts phagocytes. In the short term this is achieved though arresting the normal maturation of its phagosome and blocking its fusion with acidic, hydrolytic lysosomes. We have been studying this aspect of the pathogens biology through the isolation of transposon-mutagenized bacteria defective in arresting the maturation of the phagosome. The majority of realtime assays for phagosome maturation exploit ratio fluorometry to measure the pH of the phagosome lumen. These methods provide a very sensitive assay for the first 15-20 minutes following internalization of the particle but do not provide any insights beyond the number and activity of vacuolar ATPase (V-ATPase) complexes acquired by the phagosome. Over the past year we have developed two novel realtime assays of phagosome maturation that are linked directly to the biological function of the phagosome as a degradative compartment. Importantly these assays measure independently, and with differing kinetics, processes exhibited by all phagosomes during their maturation. The first assay is a FRET-based assay for phagosome/lysosome fusion, and the second assay provides realtime analysis of the rate of degradation of a fluorogenic cysteine proteinase substrate bound to the surface of the experimental particle. The FRET-based assay for phagosome/lysosome fusion effectively measures the mixing of the phagosome with the lumenal cargo of late endosomal/lysosomal compartments as a function of time. This provides a dynamic readout extending to 2 hours post-internalization. Treatment of the cells with inhibitors known to affect phagosome maturation produce profiles consistent with published reports. The proteinase assay measures the rate of hydrolysis of a synthetic cysteine proteinase substrate, (biotin-Phe-Arg)2-Rhod 110. This substrate is cleaved by cysteine proteinases, although it has been reported that this sequence is recognized preferentially by cathepsin L over cathepsin B. The kinetics of degradation shows an endpoint equilibrium reached at 17-18 minutes after which the free fluor slowly leaches out of the cell. Furthermore, combination of these assays and the pH phagosome maturation assay with inhibitors of different cellular processes demonstrate that these assays have the capacity to resolve certain key decision points in phagosome maturation. These data reveal a series of checks and balances decision points that are easy to rationalize when one considers how the phagosomal system is manipulated by different intracellular pathogens. The data are described in a manuscript we have submitted to Nature Methods.

IMPACT: 2000/06 TO 2004/09
Infections by Mycobacterium spp. continue to be a serious problem for the health of both humans and livestock. Results of these studies will provide a better understanding of the pathogen's biology through the isolation of transposon-mutagenized bacteria defective in arresting the maturation of the phagosome.

PUBLICATIONS (not previously reported): 2000/06 TO 2004/09
No publications reported this period

PROJECT CONTACT:
Name: Wood, J. R.
Phone: 607-253-3759
Fax: 607-253-3756
Email: jrw7@cornell.edu


ACCESSION NO : 0187063 SUBFILE: CRIS
PROJ NO: NYCV-433338 AGENCY: CSREES NYCV
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: EXTENDED
START: 01 JUN 2000 TERM: 30 SEP 2005 FY: 2005

INVESTIGATOR: Russell, D. G.

PERFORMING INSTITUTION:
Microbiology and Immunology
Cornell University
Ithaca, New York 14853

PATHOGEN MYCOBACTERIA: M. TUBERCULOSIS, M. BOVIS, AND M. AVIUM

NON-TECHNICAL SUMMARY: Study into the lifestyles of mycobacteria species pathogenic to animals and humans such as M. tuberculosis, M. leprae,M. bovis and M. avium reveals extensive parallels in their mechanisms of intracellular survival and persistence. The laboratory is devoted to the study of microbial pathogens that explit the macrophage as their host cell.

OBJECTIVES: The laboratory is dedicated to the study of pathogen mycobacteria. The primary area of interests are as follows: 1. Analysis of the biology of the interaction between the macrophage and the bacillus with respect to the intracellular environment and the regulation of host cell function. 2. The elucidation of the metabolism of the intracellular bacillus and its exploitation as possible targets of drug action. 3. The appreciation of the modulation of the infection foci and the role of the granuloma in the persistence of infection.

APPROACH: Cell Biology of Intracellular Infections by Mycobacterium avium: This delicate interplay between the bacterium, and its potentially microbicidal host cell is little understood. Previous work in the laboratory has fostered the belief that, with respect to the interaction with macrophages and the immune responses at site of infection, there are many parallels between pathogenic mycobacteria species, including Mycobacterium bovis. For this reason emphasis is also placed on exploitation of genetic approaches available for other mycobacterial species, notably M. tuberculosis and M. bovis, for the resolution of mechanisms common to all pathogenic mycobacteria. 2. Elucidation of Intermediate Metabolism and Carbon Source Acquisition by M. bovis and M. tuberculosis: We have extensive experience in modelling intracellular infections by both prokaryote and eukaryote pathogens and propose to apply this expertise to determining the contribution of the glyoxylate shunt pathway to infection. 3. Formation and Maintenance of the Granuloma and it role in Infection by Mycobacterium spp: we carried out a systematic analysis of bacterial lipidoglycans released and trafficked through infected macrophages. These comprise 7 major species of lipids some, or all, capable of inducting granulomas in mice. The ability of these lipids to expand the influence of the bacteria beyond the infected macrophages and induce granuloma formation suggest that they play roles key to the evolution of this response. Our recent development of an in vivo model exploiting these lipids will enable the functional determination of the roles of these molecules in granuloma induction.

PROJECT CONTACT:
Name: Wood, J. R.
Phone: 607-253-3759
Fax: 607-253-3756
Email: jrw7@cornell.edu


ACCESSION NO: 0185687 SUBFILE: CRIS
PROJ NO: NYCV-433394 AGENCY: CSVM NYCV
PROJ TYPE: STATE PROJ STATUS: NEW
START: 01 JUL 2000 TERM: 30 JUN 2005 FY: 2006

INVESTIGATOR: Russell, D. G.

PERFORMING INSTITUTION:
Microbiology And Immunology
Cornell University
Ithaca, New York 14853
Intramacrophage Infections

NON-TECHNICAL SUMMARY: Our lab is devoted to the study of microbial pathogens that exploit the macrophage as their host cell.

OBJECTIVES: The macrophage is the primary defence against microbial invasion. Despite its microbicidal capabilities several pathogens have evolved to live inside it. We work predominantly with the intracellular pathogens Mycobacterium and Leishmania. The Mycobacterium species on which we work include pathogens of humans (M. leprae and M. tuberculosis), cattle (M. bovis) and birds (M. avium). The Leishmania species include pathogens of humans ( L. mexicana) and dogs (L. infantum). Both pathogens reside in membrane bound vacuoles within their host cells and cause chronic persistent infections capable of reactivation. My lab is interested in the nature of this interaction as a longterm relationship between two organisms. Our objectives are the following: 1)Understand the nature of the intracellular compartment that supports growth of these pathogens. 2)How do these pathogens ensure their longterm persistence despite residing in a host cell capable of stimulating a cellular immune response and subsequently responding to macrophage-activiting cytokines and killing the pathogens. 3)Understanding the metabolism of intracellular pathogens, with particular reference to their carbon source utilization.

APPROACH: 1)For characterization of the vacuoles and identification of the mechanisms of vacuole modification we have employed cell biological techniques for analysis of the vacuoles in situ. These are underpinned with cell fractionation and biochemical identification of the vacuolar constituents. 2)We have used both metabolic-labeling and fluorochrome-tagging methods for labelling pathogen surface glycolipids to track their release and trafficking through their host cells. 3)The metabolic studies are being conducted through screens of transposon mutagenized bacteria to identify mutations in specific steps of fatty acid uptake and catabolism, in the glyoxylate shunt pathway, and in gluconeogenesis. The identified genes are analyzed by replacement and complementation, growth on different carbon source and analysis of their phenotypes in macrophage cultures and in the mouse. We are studying the activities and structures of recombinant enzymes from these pathways and using these enzymes for drug screens.

PROJECT CONTACT:
Name: Russell, D. G.
Phone: 607-253-3401
Fax: 607-253-4058
Email: mdl1@cornell.edu


ACCESSION NO: 0190583 SUBFILE: CRIS
PROJ NO: ORE00031 AGENCY: CSREES ORE
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 JUL 2003 TERM: 30 SEP 2005 FY: 2004

INVESTIGATOR: Kent, M. L.

PERFORMING INSTITUTION:
Microbiology
Oregon State University
Corvallis, Oregon 97331

CHARACTERIZATION OF MYCOBACTERIUM SPP. FROM FISHES OF OREGON

NON-TECHNICAL SUMMARY: Diseases impact both wild and captive fishes. One of these is a bacterial disease called 'fish mycobacteriosis' or 'fish tuberculosis.' Many strains or species of Mycobacterium occur in fish, and we will use both traditional methods and molecular biology to resolve their identity. The project examines the taxonomy of Mycobacterium pathogenic bacteria in fishes in Oregon This study has demonstrated that several more Mycobacterium spp. infect fishes than previously thought. Some of these strains may be related to pathogens of humans.

OBJECTIVES: Our overall objectives are 1) to continue to resolve the taxonomy of Mycobacterium species infecting various commercially important fishes in Oregon, focusing on a more variable region of the rDNA gene (ITS), 2) to evaluate the virulence of selected strains using macrophage assays, and 3) to develop PCR-based diagnostic tests using these isolates. Such tests would ultimately be employed by fish health researchers and diagnostic laboratories in the Department of Microbiology, the College of Veterinary Medicine, and Oregon Department of Fisheries & Wildlife. Moreover, with we will evaluate the potential virulence of the strains in culture to mammals and fish using in vitro macrophage models. Using human, mouse and fish cell lines we will evaluate whether strains from fish are pathogens to both mammals and humans, fish pathogens only, or merely opportunists

APPROACH: rDNA sequences.. We will obtain rDNA sequences using both direct PCR from tissues and PCR from isolated colonies In addition, we will continue to use our new primers for amplification of problematic samples, especially fish tissue samples, as they amplify smaller products and thus appear to be more sensitive for certain samples These primer sets provide SSU sequence, and to more precisely characterize and differentiate our isolates (e.g., the closely related salmonid isolates), we will examine a more variable region of the gene (the ITS) using published primers After the various isolates are sequenced, we will compare sequences using BLAST Search to compare with existing strains in GenBank. Furthermore, we will conduct phylogenetic comparisons using standard molecular systematics programs available through PAUP, etc. Virulence. We will characterize the virulence of representative strains obtained from our epidemiological and taxonomy studies using in vitro macrophage assays. To evaluate the ability of the bacterium to infect and survive in macrophages, we propose to employ the systems currently in use in Dr. Bermudez's laboratory. We plan to determine: (1) efficiency of invasion and (2) the ability of survive and replicate inside macrophages. For those experiments we propose to use carp macrophage cell line, the zebrafish macrophage cell line we have recently established from zebrafish spleens, and mouse macrophage cell line RAW 246.7. We also plan to use as controls for the experiments a human isolate of Mycobacterium avium, a fish derived Mycobacterium marinum (also a human isolate) and a non-virulent Mycobacterium smegmatis. The strain to be tested and the controls will be cultured and then used to infect macrophages in a ratio of 0.1 to 1, 1 to 1 and 10 to 1 (bacteria:macrophage). Invasion will be determined after 1 hour. The inoculum will be plated onto 7H11 agar plates to determine the number of bacteria. Infected macrophage monolayers will be washed several times with buffer in order to remove extracellular bacteria and then the monolayers will be lysed by incubating them with water for 10 min as described (Bermudez and Young 1988; Bermudez et al. 1994). Then 0.025% SDS will be added to the suspension for 10 additional minutes to prevent clumping. The suspension will be plate onto 7H11 plates to determine the number of viable intracellular bacteria. The efficiency of invasion will be calculated as a percentage of the initial inoculum used to infect the monolayers. To determine the ability of the bacteria to grow intracellularly, monolayers will be established and infected in a similar manner as described above. The intracellular bacteria will be allowed to grow and 4 and 7 days after infection the monolayers will be lysed and the number of intracellular bacteria quantified as reported (Wagner et al. 2002). The bacterial growth or decrease of the number of intracellular bacteria will be calculated as the variation of the number of bacteria inside macrophages 1 h after infection.

PROGRESS: 2003/07 TO 2005/09
Bacteria in the genus Mycobacterium are responsible for many diseases in humans (tuberculosis and leprosy) and in domestic animals (i.e., pseudotuberculosis of sheep and cattle). Infections by various species of Mycobacterium bacteria are also common in a wide variety of both wild and captive fishes. Mycobacteriosis of fishes is of importance to agriculture (aquaculture) in the state of Oregon as it is common in our hatchery reared salmonids, aquarium fishes and commercially important marine fishes (i.e. rockfishes). Mycobacterium species of fishes are also of concern because they infect humans. Mycobacterium marinum is ubiquitous in the aquatic environment. The bacterium can cause systemic infection in fishes, and usually skin infection in humans. Most strains grow better at temperatures lower than 37C, and this characteristic has been used to explain the rarity of deep tissue or systemic infections in humans. We investigated the ability of a strain from humans and 3 strains from fish to grow in various media at 30C and 37C, and in macrophage cell lines from carp, humans, and mouse. We also tested the ability of the 3 fish isolates to infect mice by both foot pad and intravascular injections. Significant discrepancies in the ability of some strains to grow in vitro versus in macrophages and in vivo were observed. Only one fish isolate grew well at 37 C, but all fish strains were capable of causing systemic infections in mice. Recent studies have suggested that isolates from fish would not cause disease in humans, and our findings suggest that certain strains from fish are indeed capable of growth at 37C in mammals even if they do not grown on culture plates at this temperature.

IMPACT: 2003/07 TO 2005/09
Mycobacterium marinum is a common environmental bacterium that often causes disease in captive or wild fishes. It occasionally infects humans, but is usually confined to the skin. The hypothesis for the location of the infection has been that the bacterium is that most strains of the bacterium are not capable of growing at human body temperatures. This project demonstrated that certain strains of Mycobacterium marinum are capable of growing at human body temperatures under the appropriate circumstances, i.e. within human or mouse macrophages in culture or within live mice. This suggests that the bacterium is more capable of causing systemic infections in humans than previously thought.

PUBLICATIONS (not previously reported): 2003/07 TO 2005/09
1. Kent, ML , V. Watral , M. Wu , L. Bermudez. 2005. In vivo and in vitro growth of Mycobacterium marinum at homoeothermic temperatures. FEMS Microbiol. Lett. (in press).

PROJECT CONTACT:
Name: Kent, M. L.
Phone: 541-737-5088
Fax: 541-737-2166
Email: Michael.Kent@orst.edu


ACCESSION NO: 0209159 SUBFILE: CRIS
PROJ NO: TEN00344 AGENCY: CSREES TEN
PROJ TYPE: HATCH PROJ STATUS: NEW
START: 01 OCT 2006 TERM: 30 SEP 2011


INVESTIGATOR: Hickling, G. J.; Muller, L.; Gray, M. J.; Eda, S.; Henry, T. B.;
Scott, M. C.

PERFORMING INSTITUTION:
Forestry Fisheries & Wildlife
University of Tennessee
569 Dabne Hall
Knoxville, Tennessee 37996

NEW APPROACHES TO WILDLIFE HEALTH: MONITORING AND MANAGING DISEASE SPREAD BETWEEN FREE-RANGING WILDLIFE, LIVESTOCK AND HUMANS.

NON-TECHNICAL SUMMARY: Throughout the U.S., and internationally, wildlife have extensive and growing contact with livestock and human populations. Aquaculture initiatives continue to expand and diversify. Consequently, there are increasing problems with emerging and resurging fish and wildlife disease that have significant implications for human health. Contributing factors include the recovery of previously over-exploited wildlife populations, the intermingling of suburban development with wildlife habitat, and the development of new farming and aquaculture techniques. Emergence of new and introduced pathogens compounds the problem. This project represents a multi-disciplinary approach to the investigation of disease problems arising at the interface between wildlife, livestock and human populations. Laboratory and field approaches are combined to investigate specific disease problems. A particular focus is on ecological perspectives on pathogen-host interactions that are often overlooked in by more traditional veterinary and public health approaches. The project also builds capacity in the areas of cellular and molecular biology of wildlife and veterinary diseases.

OBJECTIVES: 1. Determine the influence of cattle in wetlands on pathogen transmission and prevalence in amphibian communities; 2. Determine whether amphibians could serve as spill-over hosts of zoonotic pathogens; 3. Investigate the effects of environmental stressors on fish physiology in laboratory experiments using zebrafish; 4. Use results of laboratory experiments with zebrafish to direct mesocosm toxicology studies on environmentally relevant native fish species; 5. Link results obtained in laboratory research and mesocosm studies to guide toxicology research on native fishes in Tennessee watersheds; 6. Investigate the spread and emergence of wildlife-vectored, tick-borne disease in Tennessee; 7. Model the spatial dynamics of bovine tuberculosis in wild ungulates; 8. Develop improved chemical immobilization techniques for capturing and collaring deer and other mammals; 9. Develop innovative immunological assays that will allow assessment of reproductive function in free-ranging deer and elk using blood and fecal samples; 10. Develop and evaluate improved diagnostic tests for mycobacterial diseases in wildlife, livestock and humans.

APPROACH: 1-2. Experimental challenges will be performed, involving oral inoculations of tadpoles with amphibian and zoonotic pathogens under various immunocompromised scenarios that represent field conditions in cattle-access and non-access wetlands. 3-5. Laboratory experiments will involve exposure of specific life stages of fish to a toxicant (or an electric field) and include examination of various endpoints, including gene expression, collection of tissues for histopathology, assessment of reproduction, and assessment of behavior. Field research involves collection of fish from aquatic environments stressed by presence of toxicants, or exposure of fish in cages to conditions present at field sites. 6. A spatially explicit simulation model of the spatial dynamics of tick, pathogens, reservoir host species (mice, raccoons, birds, deer) and human risk potential will be developed. The model will be interfaced with data stored in a geographic information system to use realistic landscape composition, relative abundance and spatial distribution characteristics of the Tennessee landscape. 7. A spatially-explicit, stochastic model of TB dynamics in white-tailed deer will be developed by adapting an existing model of TB in brushtail possums (Trichosurus vulpecula) in New Zealand. The base model is a mixed deterministic/stochastic type, with the number of infectious contacts influenced by deer abundance, herd immunity, vaccination, prevalence of TB, and other relevant factors. 8-9. Chemical immobilization techniques developed by Miller et al. (2004) will be refined. Radioimmunoassays and enzyme-linked immunosorbent assays (ELISA) for protein hormones will be used to study reproductive function in deer and elk using blood and fecal samples. 10. Serum samples from various M. bovis-infected animals (cattle, sheep and wildlife) will be obtained from collaborators. Flow Cytometry and the UT-ELISA will then be used to determine diagnostic sensitivity, specificity and cut-off values for diagnosis of bovine TB.

PROJECT CONTACT:
Name: Hickling, G. J.
Phone: 865-974-6173
Fax: 865-974-4714
Email: ghicklin@tennessee.edu
URL: http://wildlifehealth.tennessee.edu


ACCESSION NO: 0193020 SUBFILE: CRIS
PROJ NO: WNV-00155 AGENCY: CSREES WN.V
PROJ TYPE: ANIMAL HEALTH PROJ STATUS: TERMINATED
START: 01 JUL 2002 TERM: 30 JUN 2003 FY: 2003

INVESTIGATOR: Davis, W. C.; Barrington, G. M.; Park, Y. H.

PERFORMING INSTITUTION:
Animal Health Research Center
Washington State University
Pullman, Washington 99164

RAPID UNIVERSAL DIAGNOSTIC ASSAYS FOR DETECTION AND DIFFERENTIATION OF ANIMALS INFECTED WITH M. BOVIS AND/OR M. PARATUBERCULOSIS

NON-TECHNICAL SUMMARY: Two mycobacteria are of economic importance and a potential health hazard for animals and humans: M. bovis, the causative agent of bovine tuberculosis and M. paratuberculosis, the causative agent of Johne's disease (paratuberculosis). The lack of effective vaccines and rapid diagnostic tests for early detection has made eradication of these diseases difficult. There is an essential need to develop better assays to control these diseases. The primary objective of this research is to develop rapid simple diagnostic assays that can be used for early diagnosis and control of these diseases.

OBJECTIVES: 1. Demonstrate an anti-RBC monoclonal antibody conjugated with peptide sequences containing an epitope derived from species-specific early antigens expressed by Mbv and Map can be used in an autologous hemagglutination assay to detect animals infected with either pathogen. 2. Demonstrate a commercial latex bead coated with peptide sequences containing an epitope derived from species-specific early antigens expressed by Mbv and Map can be used in an agglutination assay to detect animals infected with either pathogen.

APPROACH: 1. A non-hemagglutinating mAb, TH17A (IgM), known to recognize a highly conserved determinant on RBCs and leukocytes, will be used in the initial studies to prepare ESAT-6 and a362 peptide conjugates. Peptides containing the immunodominant determinants of ESAT-6 and a362 will be prepared with an extra lysine for conjugation with the mAb. Following purification, the mAb will be treated with sodium periodate to generate aldehyde groups on the carbohydrate sidechains. The peptides will bind through an amine group to the aldehyde. The binding of mAb-peptide conjugates to RBC will be demonstrated by flow cytometry. Reactivity of the peptide will be determined by ELISA before and after coupling to the antibody, using immune sera from animals infected with Mbv or Map. Nonspecific peptides will be coupled to the mAb and used as negative controls. The mAb conjugates will be tested for their capacity to agglutinate in the presence of antibody. 2. A commercial source of sulfated 0.8 mM latex microspheres will be used in initial trials to develop bead assays to detect ESAT-6 and a362 peptide antigens. The beads will be prepared according to the manufacturer's protocol and reacted with serial dilutions of ESAT-6 and a362 peptides. Comparable sets of latex beads will be coated with nonspecific peptides to serve as negative controls. Following validation of the capacity of the mAb and latex bead conjugates to form antibody-antigen aggregates, studies will be performed with sets of antisera from known infected animals and uninfected animals to establish the sensitivity and specificity of the assays.

PROGRESS: 2002/07 TO 2003/06
The objective of the study was to develop and compare the sensitivity and specificity of two antigenic peptide based assays for use in the diagnosis and control of bovine tuberculosis and paratuberculosis. A red blood cell (rbc) based antigen-antibody capture assay and antigen coated latex bead assay were developed and compared with the sensitivity and specificity of an existing enzyme linked immunosorbent assay (EIA). An antigenic peptide from Mycobacterium bovis, ESAT-6, was used as the first test antigen. The rbc based antigen assay proved to be difficult to use and not sufficiently sensitive so further efforts to develop the assay were stopped. Comparison of the latex bead agglutination assay with the EIA, with a panel of sera obtained from infected and uninfected animals, showed both assays yield comparable levels of sensitivity, 94% and 96% respectively. The data suggest it will be possible to develop a rapid latex bead based diagnostic assay for use in the diagnosis of animals infected with M. bovis. Studies are now needed with blind panels of sera from known infected and uninfected animals to verify the sensitivity and specificity of the assay. Studies are also needed to determine if a comparably sensitive latex bead agglutination assay can be developed for paratuberculosis.

IMPACT: 2002/07 TO 2003/06
Diagnostic assays that improve our ability to detect infected animals at early stages of disease will help control disease and the movement of infected animals into clean dairy and cattle operations.

PUBLICATIONS (not previously reported): 2002/07 TO 2003/06
No publications reported this period

PROJECT CONTACT:
Name: Davis, W. C.
Phone: 509-335-6051
Fax: 509-335-8328
Email: davisw@vetmed.wsu.edu

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