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United States Department of Agriculture

Agricultural Research Service

2010 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 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.

3.Progress Report
Activities within the Agriculture Research Service Tuberculosis (TB) Project relate to the National Program Action Plan through development of tools for use in the TB eradication program. The approach is to evaluate vaccine and diagnostic strategies, as well as basic disease processes (i.e., immunopathogenesis studies) in partnership with federal and state agencies, industry (veterinary biologics companies), and academia. Ongoing studies this year include: continued discovery and validation of improved antigens for use in immune-based TB assays, characterization of immune responses of cattle to Mycobacterium bovis (M. bovis) infection, development of novel methods to detect determinants of infection, ultrastructural characterization of tonsil lymphoid and epithelial tissues (a major portal of entry for the TB bacillus) in cattle and white-tailed deer (WTD), detection and description of novel mycobacterial isolates from skin lesions in cattle, further evaluation of M. bovis bacillus Calmette-Guerin (BCG) persistence in WTD, sequencing and comparison of genomes of environmental Mycobacteria spp. that potentially confound current diagnostic tests, and virulence evaluation of targeted deletion mutants of M. bovis in cattle. Over the past year, we have initiated challenge studies in our new large animal high containment facility. Our project members participated in several national and international meetings and are involved in the preparation of a bovine TB coordinated agricultural research project proposal.

1. Demonstrated persistence of M. bovis bacillus Calmette-Guerin (BCG) in white-tailed deer (WTD). The Michigan Departments of Agriculture and Natural Resources and APHIS, Veterinary Services have indicated that vaccines may be critical for the control of bovine Tuberculosis (TB) in Michigan (i.e., the TB core area). ARS researchers at Ames, Iowa, have previously demonstrated that M. bovis BCG reduces disease severity in WTD upon experimental challenge with virulent M. bovis. In an extension of these studies, ARS researchers at Ames, Iowa, demonstrated that M. bovis BCG persists in tissues of WTD for up to 9 months after vaccination. The attenuated live vaccine was primarily detected in lymphoid tissues without evidence of colonization of muscle (i.e., meat potentially consumed by humans); thus, the risk of transmission to humans is minimal. These findings underscore the necessity of detailed safety studies for use of vaccines intended for wildlife that may be consumed by humans.

2. Characterized an attenuated live M. bovis (Ravenel Delta RD1) vaccine in cattle. Vaccines are being considered for control of bovine Tuberculosis (TB), especially in countries with wildlife reservoirs that have been unable to control the spread of disease using traditional approaches (e.g., United Kingdom, New Zealand). An attenuated live vaccine with more consistent efficacy and/or improved safety in cattle as compared to M. bovis bacillus Calmette-Guerin (BCG) should prove invaluable as a bovine TB vaccine, either alone or in combination with subunit vaccines. In collaboration with others, ARS researchers at Ames, Iowa, constructed, characterized, and sequenced an attenuated live vaccine for use in cattle. These studies demonstrated the utility of the neonatal calf aerosol challenge model for determination of TB vaccine efficacy. The M. bovis Delta RD1 may prove useful for bovine TB vaccine programs, particularly if additional mutations are included to improve safety and immunogenicity. Also, these findings are directly applicable for the development of vaccines intended for human use.

3. Characterized the bovine palatine tonsil. A detailed understanding of the bovine tonsil is important as this set of structures represents the first line of defense against pathogens entering either by the respiratory or digestive tract. These lymphoid organs play an important role in diseases of economic significance to the cattle industry, including bovine Tuberculosis (TB). ARS researchers at Ames, Iowa, characterized the morphology, cellular composition, and anatomic distribution of lymphoid tissues as related to epithelial components within this locale. A better understanding of the bovine tonsil, as defined in this study, is crucial for elucidating the pathogenesis of various livestock diseases and for the development of vaccines to target these structures.

Review Publications
Schiller, I., Vordermeier, H.M., Waters, W.R., Whelan, A.O., Coad, M., Gormley, E., Buddle, B.M., Palmer, M.V., Thacker, T.C., Mcnair, J., Welsh, M., Hewinson, R.G., Oesch, B. 2010. Bovine Tuberculosis: Effect of the Tuberculin Skin Test on In vitro Interferon gamma Responses. Veterinary Immunology and Immunopathology. 136(1-2):1-11.

Waters, W.R., Whelan, A.O., Lyashchenko, K.P., Greenwald, R., Palmer, M.V., Harris, N.B., Hewinson, R.G., Vordermeier, H.M. 2010. Immune Responses in Cattle Inoculated with Mycobacterium bovis, Mycobacterium tuberculosis, or Mycobacterium kansasii. Clinical and Vaccine Immunology. 17(2):247-252.

Waters, W.R., Palmer, M.V., Nonnecke, B.J., Thacker, T.C., Estes, D.M., Larsen, M.H., Jacobs, W.R., Andersen, P., Mcnair, J., Minion, F.C., Lyashchenko, K.P., Hewinson, R.G., Vordermeier, H.M., Sacco, R.E. 2009. Signal Regulatory Protein alpha (SIRPalpha)+ Cells in the Adaptive Response to ESAT-6/CFP-10 Protein of Tuberculous Mycobacteria. PLoS One [serial online]. 4(7):e6414. Available:

Schiller, I., Waters, W.R., Vordermeier, H.M., Nonnecke, B.J., Welsh, M., Keck, N., Whelan, A., Sigafoose, T., Stamm, C., Palmer, M.V., Thacker, T.C., Hardegger, R., Marg-Haufe, B., Raeber, A., Oesch, B. 2009. Optimization of a Whole-Blood Gamma Interferon Assay for Detection of Mycobacterium bovis-Infected Cattle. Clinical and Vaccine Immunology. 16(8):1196-1202.

Schiller, I., Vordermeier, M., Waters, W.R., Palmer, M.V., Thacker, T.C., Whelan, A., Hardegger, R., Raeber, A., Oesch, B. 2009. Assessment of Mycobacterium tuberculosis OmpATb as a Novel Antigen for the Diagnosis of Bovine Tuberculosis. Clinical and Vaccine Immunology. 16(9):1314-1321.

Nonnecke, B.J., Reinhardt, T.A., Waters, W.R. 2009. Short Communication: The Preruminant Calf as a Model for Characterizing the Effects of Vitamin D Status in the Neonate. Journal of Dairy Science. 92(11):5692-5696.

Green, L.R., Jones, C.C., Sherwood, A., Garkavi, I., Cangelosi, G.A., Thacker, T.C., Palmer, M.V., Waters, W.R., Rathe, C. 2009. Single-Antigen Serological Testing for Bovine Tuberculosis. Clinical and Vaccine Immunology. 16(9):1309-1313.

Waters, W.R., Nonnecke, B.J., Gibbs, S.E., Yabsley, M.J., Schmitt, S.M., Cosgrove, M.K., Palmer, M.V., Thacker, T.C., Olsen, S.C., Horst, R.L., Reinhardt, T.A. 2009. Serum 25-Hydroxvitamin D Concentrations in Captive and Free-Ranging, White-Tailed Deer (Odocoileus virginianus). International Journal for Vitamin and Nutrition Research. 79(3):180-187.

Herzig, C., Waters, W.R., Baldwin, C., Telfer, J. 2010. Evolution of the CD163 Family and its Relationship to the Bovine Gamma Delta T Cell Co-receptor WC1. BMC Evolutionary Biology. 10(1):181.

Miller, L.C., Zanella, E.L., Waters, W.R., Lager, K.M. 2010. Cytokine Protein Expression Levels in Tracheobronchial Lymph Node Homogenates of Pigs Infected with Pseudorabies Virus. Clinical and Vaccine Immunology. 17(5):728-734.

Loving, C.L., Brockmeier, S.L., Vincent, A.L., Palmer, M.V., Sacco, R.E., Nicholson, T.L. 2010. Influenza Virus Coinfection with Bordetella bronchiseptica Enhances Bacterial Colonization and Host Responses Exacerbating Pulmonary Lesions. Microbial Pathogenesis. 49(5):237-245.

Last Modified: 7/29/2016
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