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United States Department of Agriculture

Agricultural Research Service

2007 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 invitro and in vivo methods in 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.

3.Progress Report
With both white-tailed deer (WTD) and cattle, we continue to evaluate the immunopathogenesis of tuberculosis (TB) infection, improved diagnostics for detection of tuberculous animals, and TB vaccines for efficacy and safety. 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 tonsilar explant culture techniques; 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; and characterization of unique mononuclear cell types (i.e., CD172a+ cells and gamma delta T cells) responsive to M. bovis antigens. 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 2-D gel technologies for antigen discovery. Studies on TB vaccines include: evaluation of novel M. bovis and M. tuberculosis attenuated live mutants for safety and efficacy in the prevention of TB infection in cattle and deer, evaluation of vaccine delivery routes, and further development of improved methods for determining vaccine efficacy and safety. Organized an international mini-symposia with the NIH TB program staff, FDA, USDA, academia and other key TB researchers to be held at NADC in September. Key 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; and the Michigan Department of Natural Resources.

Validation of serum-based tests for TB surveillance in free-ranging white-tailed deer (WTD) populations. The apparent prevalence of TB in free-ranging WTD in the “TB endemic zone” of Michigan has not significantly decreased over the past 2 years, despite intensive efforts. Additional control strategies such as capture, test (and euthanize positives), vaccinate, and release are being evaluated. In collaboration with the Michigan Department of Natural Resources and Chembio, we validated the use of several serum-based TB tests to adequately detect TB-infected free-ranging WTD. These findings will be critical for the control of TB in Michigan. This accomplishment addresses the “countermeasures to prevent and control zoonotic diseases” action plan of National Program 103, Animal Health.

Optimization of a whole blood interferon-gamma assay for the detection of tuberculous cattle. 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 M. 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), we defined a cocktail of antigens that improve the specificity of the assay without limiting the sensitivity - as compared to the current format of the test using complex antigens. We also standardized and simplified testing protocols. The impact will be a more useful and specific test; thereby, decreasing costs to producers and regulatory agencies (e.g., APHIS) for slaughter of animals falsely defined as TB reactors. This accomplishment addresses the “countermeasures to prevent and control zoonotic diseases” action plan of National Program 103, Animal Health.

Demonstrated that vaccination of WTD with an attenuated vaccine strain [i.e., M. bovis bacille Calmette Guerin (BCG)] significantly decreases the severity of disease upon experimental challenge. One of the greatest obstacles to the USDA TB eradication campaign is the presence of M. bovis-infected free-ranging WTD. A goal for vaccination of wildlife reservoirs of TB is to limit intra- and inter-species spread of infection. We demonstrated that either oral or parenteral vaccination of WTD with the attenuated M. bovis vaccine strain, BCG, decreases colonization and pathology associated with subsequent experimental challenge with virulent M. bovis. The impact is that use of this vaccine within free-ranging populations will most likely decrease the spread of TB, both between WTD cohorts and between WTD and cattle. One caveat to these findings is that the vaccine strain is not cleared efficiently by WTD and may spread between deer – raising potential food safety concerns. This accomplishment addresses the “countermeasures to prevent and control zoonotic diseases” action plan of National Program 103, Animal Health.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of new CRADAs and MTAs4
Number of active CRADAs and MTAs12
Number of newspaper articles and other presentations for non-science audiences7

Review Publications
Yarlett, N., Wu, G., Waters, W.R., Harp, J.A., Wannemuehler, M.J., Morada, M., Athanasopoulos, D., Martinez, M.P., Upton, S.J., Marton, L.J. 2007. Cryptosporidium parvum spermidine/spermine N1-acetyltransferase Exhibits Different Characteristics From the Host Enzyme. Molecular and Biochemical Parasitology. 152(2):170-180.

Eda, S., Bannantine, J.P., Waters, W.R., Mori, Y., Whitlock, R.H., Scott, M.C., Speer, C.A. 2006. A Highly Sensitive and Subspecies-Specific Surface Antigen Enzyme-Linked Immunosorbent Assay for the Diagnosis of Johne's Disease. Clinical and Vaccine Immunology.13(8)837-844.

Foote, M.R., Nonnecke, B.J., Beitz, D.C., Waters, W.R. 2007. High growth rate fails to enhance adaptive immune responses of neonatal calves and is associated with reduced lymphocyte viability. Journal of Dairy Science. 90(1):404-417.

Lyashchenko, K.P., Greenwald, R., Esfandiari, J., Olsen, J., Ball, R., Dumonceaux, G., Dunker, F., Buckley, C., Richards, M., Waters, W.R. 2006. Tuberculosis in Elephants: Antibody Responses to Defined Antigens of Mycobacterium tuberculosis, Potential for Early Diagnosis, and Monitoring of Treatment. Clinical and Vaccine Immunology. 13(7):722-732.

Waters, W.R., Nonnecke, B.J., Olsen, S.C., Palmer, M.V. 2007. Effects of pre-culture holding time and temperature on interferon-gamma responses in whole blood cultures from Mycobacterium bovis-infected cattle. Veterinary Microbiology. 119(2-4):277-282.

Waters, W.R., Palmer, M.V., Thacker, T.C., Payeur, J.B., Harris, N.B., Minion, F.C., Greenwald, R., Esfandiari, J., Andersen, P., Mcnair, J. 2006. Immune Responses to Defined Antigens of Mycobacterium bovis in Cattle Experimentally Infected with Mycobacterium kansasii. Clinical and Vaccine Immunology. 13(6):611-619.

Maue, A.C., Waters, W.R., Palmer, M.V., Nonnecke, B.J., Minion, F.C., Brown, W.C., Norimine, J., Foote, M., Scherer, C.F., Estes, D.M. 2007. An ESAT-6:CFP10 DNA vaccine Administered in Conjunction with Mycobacterium bovis BCG confers Protection to Cattle Challenged with Virulent M. bovis. Vaccine. 25(24):4735-4746.

Palmer, M.V., Waters, W.R. 2006. Advances in bovine tuberculosis diagnosis and pathogenesis: What policy makers need to know. Veterinary Microbiology. 112(2006):181-190.

Shigetoshi, E., Elliot, B., Scott, M.C., Waters, W.R., Bannantine, J.P., Speer, C.A. 2005. New Method of Serological Testing for Mycobacterium Avium subsp. Paratuberculosis (Johne's Disease) by Flow Cytometry. Foodborne Pathogens and Disease. 2(3):250-62.

Koo, H.C., Park, Y.H., Ahn, J., Waters, W.R., Palmer, M.V., Hamilton, M.J., Barrington, G., Abdelaziz, M.A., Cho, S., Shin, S. 2005. Use of rMPB70 Protein and ESAT-6 Peptide as Antigens for Comparison of the Enzyme-linked Immunosorbent, Immunochromatographic, and Latex Bead Agglutination Assays for Serodiagnosis of Bovine Tuberculosis. Journal of Clinical Microbiology. 43(9):4498-4506.

Koo, H.C., Park, Y.H., Hamilton, M.J., Barrington, G.M., Davies, C.J., Kim, J.B., Dahl, J.L., Waters, W.R., Davis, W.C. 2004. Analysis of the immune response to Mycobacterium avium subsp. paratuberculosis in experimentally infected calves. Infection and Immunity. 72:6870-6883.

Palmer, M.V., Stoffregen, W.C., Carpenter, J.G., Stabel, J.R. 2005. Isolation of Mycobacterium avium subsp paratuberculosis (MAP) from feral cats on a dairy farm with MAP-infected cattle. Journal of Wildlife Diseases. 41(3):629-635.

Yarlett, N., Waters, W.R., Harp, J.A., Wannemuehler, M.J., Morada, M., Bellcastro, J., Upton, S.J., Marton, L.J., Frydman, B.J. 2007. Activity of DL-alpha-Difluoromethylarginine and Polyamine Analogues against Cryptosporidium parvum Infection in a T-Cell Receptor Alpha-Deficient Mouse Model. Antimicrobial Agents and Chemotherapy. 51(4):1234-1239.

Last Modified: 11/28/2015
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