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United States Department of Agriculture

Agricultural Research Service

2009 Annual Report

1a.Objectives (from AD-416)
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.

1b.Approach (from AD-416)
Objective 1 will evaluate tonsilar processing of M. bovis and lesion development using a combination of in vitro 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 evaluate 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.

3.Progress Report
Activities within the ARS Tuberculosis (TB) Project relate to the National Program Action Plan via development of tools for use in the TB eradication program. The approach is to evaluate vaccine strategies, improved diagnostic tools, and basic disease processes (i.e., immunopathogenesis of TB) in partnership with federal and state agencies, industry (veterinary biologics companies), and academia. Studies on TB vaccines include: evaluation of attenuated M. bovis mutants for safety and efficacy in the prevention of TB infection in cattle and deer, evaluation of vaccine delivery routes, and development of improved methods for determining vaccine efficacy and safety. Studies on TB diagnosis include: continued discovery and validation of improved antigens for use in cellular and humoral immune-based TB assays, determination of sensitivity/specificity values for emerging serologic TB assays for cattle and WTD, and development of improved determinants of infection. Immunopathogenesis studies include: determination of the kinetics of the specific cytokine response to infection by cattle; characterization of the normal bovine tonsilar lymphoepithelium (a major portal of entry for the TB bacillus; development of primer pairs for the evaluation of in vitro and in vivo alterations in cytokine, chemokine, and transcription factors of WTD in response to infection/vaccination; evaluation of methods to explore transcriptomes to discover new readouts of infection; and characterization of unique mononuclear cell types (e.g., CD172a+ cells) responsive to M. bovis antigens. Our project members participated in and co-organized a second international symposium with the NIH TB program staff, FDA, USDA, academia and other key TB researchers in Gettysburg PA and are organizing a third conference. Key outside collaborations for ongoing TB research include: APHIS TB program staff; Howard Hughes Medical Institute, Albert Einstein College of Medicine, Bronx, NY; Prionics Ag, Schlieren, Switzerland; Chembio Diagnostics, Medford, NY; Iowa State University, Ames, IA; University of Texas Medical Branch, Galveston, TX; New Mexico State University; IDEXX Laboratories; and the Michigan Department of Natural Resources.

1. Field evaluation of blood-based assays for the detection of tuberculous white-tailed deer. An obstacle to the United States Department of Agriculture tuberculosis eradication program is the presence of a wildlife reservoir (white-tailed deer), in Michigan. Detection of infection in these animals requires laborious methods. In this study, we evaluated the accuracy and feasibility of seven blood-based tests with hunter-collected samples in Michigan. Several of these tests proved highly specific for the disease with sensitivity acceptable to policy makers for detection of infected white-tailed deer. The impact is that a test was identified for use in an innovative strategy (capture, test and release/euthanize) proposed by the Michigan Department of Natural Resources. These findings provide a new tool for the control of tuberculosis in white-tailed deer and subsequently cattle in Michigan.

2. Assessment of a new antigen (Outer membrane protein A-TB) for the diagnosis of bovine tuberculosis. In most states, the Bovigam assay has replaced the comparative cervical test as the most widely used confirmatory test for the detection of tuberculous cattle. The test relies on differential reactivity to complex antigens from Mycobacterium avium and M. bovis. Specific antigens (e.g., peptides and/or recombinant proteins) could improve the specificity of the assay. In close collaboration with Prionics Ag (the owners of the Bovigam assay) and Veterinary Laboratory Agencies, Weybridge, United Kingdom; we defined a new antigen (Outer membrane protein A-TB) that can be used in combination with other specific antigens to improve the accuracy of the assay. The impact will be a more useful and specific test; thereby, decreasing costs to producers and regulatory agencies (e.g., Animal and Plant Health Inspection Service) accrued upon slaughter of animals falsely identified as tuberculosis reactors.

3. Evaluation of the immune response of white-tailed deer to M. bovis Bacille Calmette Guerin vaccination and M. bovis infection. Vaccination of white-tailed deer is a viable option for controlling the spread of tuberculosis in Michigan. Previously, we demonstrated that either oral or parenteral vaccination of white-tailed deer with the attenuated M. bovis vaccine strain, bacille Calmette Guerin, decreases colonization and pathology associated with subsequent experimental challenge with virulent M. bovis. In this study, we extended these findings by demonstrating an immune signature indicative of vaccine failure. While a correlate of infection upon vaccination was not identified, the impact of this study was the demonstration of an immune response indicative of disease progression.

6.Technology Transfer

Number of Active CRADAs1
Number of the New/Active MTAs (providing only)8

Review Publications
Endsley, J., Waters, W.R., Palmer, M.V., Nonnecke, B.J., Thacker, T.C., Jacobs, W.R., Larsen, M., Hogg, A., Shell, L., Mcalauy, M., Capinos-Scherer, C., Coffey, T., Howard, C., Villareal-Ramos, B., Estes, M. 2009. The Calf Model of Immunity for Development of a Vaccine Against Tuberculosis. Veterinary Immunology and Immunopathology. 128(1-3):199-204.

O'Brien, D.J., Schmitt, S.M., Lyashchenko, K.P., Waters, W.R., Berry, D.E., Palmer, M.V., Mcnair, J., Greenwald, R., Esfandiari, J., Cosgrove, M.K. 2009. Evaluation of Blood Assays for Detection of Mycobacterium Bovis in White-tailed Deer (Odocoileus Virginianus) in Michigan. Journal of Wildlife Diseases. 45(1):153-164.

Lyashchenko, K.P., Greenwald, R., Esfandiari, J., Chambers, M.A., Vicente, J., Gortazar, C., Santos, N., Correia-Neves, M., Buddle, B.M., Jackson, R., O'Brien, D.J., Schmitt, S., Palmer, M.V., Delahay, R.J., Waters, W.R. 2008. Animal-side Serologic Assay for Rapid Detection of Mycobacterium bovis Infection in Multiple Species of Free-ranging Wildlife. Veterinary Microbiology. 132(3-4):283-292.

Waters, W.R., Palmer, M.V., Nonnecke, B.J., Thacker, T.C., Capinos Scherer, C.F., Estes, D.M., Hewinson, R.G., Vordermeier, H.M., Barnes, S.W., Federe, G.C., Walker, J.R., Glynne, R.J., Hsu, T., Weinrick, B., Biermann, K., Larsen, M.H., Jacobs, W.R. 2009. Efficacy and Immunogenicity of Mycobacterium bovis Delta RD1 against Aerosol M. bovis Infection in Neonatal Calves. Vaccine. 27(8):1201-1209.

Zimmerman, D., Waters, W.R., Lyashchenko, K.P., Nonnecke, B.J., Armstrong, D.L., Jacobs, Jr., W.R., Larsen, M.H., Egan, E., Dean, G.A. 2009. Safety and Immunogenicity of the Mycobacterium tuberculosis {Delta}lysA {Delta}panCD Vaccine in Domestic Cats Infected with Feline Immunodeficiency Virus. Clinical and Vaccine Immunology. 16(3):427-429.

Greenwald, R., Lyashchenko, O., Esfandiari, J., Miller, M., Mikota, S., Olsen, J.H., Ball, R., Dumonceaux, G., Schmitt, D., Moller, T., Payeur, J.B., Harris, B., Sofranko, D., Waters, W.R., Lyashchenko, K.P. 2009. Highly Accurate Antibody Assays for Early and Rapid Detection of Tuberculosis in African and Asian Elephants. Clinical and Vaccine Immunology. 16(5):605-612.

Nol, P., Lyashchenko, K.P., Greenwald, R., Esfandiari, J., Waters, W.R., Palmer, M.V., Nonnecke, B.J., Keefe, T.J., Thacker, T.C., Rhyan, J.C., Aldwell, F.E., Salman, M.D. 2009. Humoral Immune Responses of White-tailed Deer (Odocoileus virginianus) to Mycobacterium bovis BCG Vaccination and Experimental Challenge with M. bovis. Clinical and Vaccine Immunology. 16(3):323-329.

Thacker, T.C., Palmer, M.V., Waters, W.R. 2009. T-cell mRNA Expression in Response to Mycobacterium bovis BCG Vaccination and Mycobacterium bovis Infection of White-tailed deer. Clinical and Vaccine Immunology. 16(8):1139-1145.

Capinos Scherer, C.F., Endsley, J.J., De Aguiar, J.B., Jacobs, W.R., Larsen, M., Palmer, M.V., Nonnecke, B.J., Waters, W.R., Estes, D.M. 2009. Evaluation of Granulysin and Perforin as Candidate Biomarkers for Protection Following Vaccination with Mycobacterium bovis BCG or M. bovis DeltaRD1. Transboundary and Emerging Diseases. 56(6-7):228-239.

Palmer, M.V., Thacker, T.C., Waters, W.R. 2009. Histology, Immunohistochemistry and Ultrastructure of the Bovine Palatine Tonsil with Special Emphasis on Reticular Epithelium. Veterinary Immunology and Immunopathology. 127(3-4):277-285.

Palmer, M.V., Thacker, T.C., Waters, W.R. 2009. Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White-tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis. Zoonoses and Public Health. 56(5):243-251.

Duncan, C., Ridpath, J., Palmer, M.V., Driskell, E., Spraker, T. 2008. Histophathologic and Immunohistochemical Findings in Two White-Tail Deer Fawns Persistently Infected with Bovine Viral Diarrhea Virus. Journal of Veterinary Diagnostic Investigation. 20(3):289-296.

Ridpath, J.F., Driskell, E.A., Chase, C.C., Neill, J.D., Palmer, M.V., Brodersen, B.W. 2008. Reproductive Tract Disease Associated with Inoculation of Pregnant White-Tailed Deer with Bovine Viral Diarrhea Virus. American Journal of Veterinary Research. 69(12):1630-1636.

Harrington, N.P., Surujballi, O.P., Prescott, J.F., Duncan, J.R., Waters, W.R., Lyashchenko, K., Greenwald, R. 2008. Antibody Responses of Cervids (Cervus elaphus) following Experimental Mycobacterium bovis Infection and the Implications for Immunodiagnosis. Clinical and Vaccine Immunology. 15(11):1650-1658.

Last Modified: 8/24/2016
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