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United States Department of Agriculture

Agricultural Research Service

2008 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
With both white-tailed deer (WTD) and cattle, we evaluated the immunopathogenesis of tuberculosis (TB) infection, improved diagnostics for detection of tuberculous animals, and TB vaccines for efficacy and safety. Immunopathogenesis studies included: 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 (e.g., CD172a+ cells) responsive to M. bovis antigens. Studies on TB diagnosis included: 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 included: 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. A second international symposium with the NIH TB program staff, FDA, USDA, academia and other key TB researchers will be held in Gettysburg PA this year as a follow-up to last years successful meeting in Ames, IA. 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; and the Michigan Department of Natural Resources. This project adresses NP103, component 3, problem statement 3C.

1. Evaluation of TB Vaccines in White-tailed Deer. Demonstrated that an attenuated live TB vaccine strain [i.e., M. bovis bacille Calmette Guerin (BCG)] persists for up to 9 months in white-tailed deer (WTD) and induces microgranulomas. One of the greatest obstacles to the USDA TB eradication campaign is the presence of Mycobacterium bovis-infected free-ranging WTD. A goal for vaccination of wildlife reservoirs of TB is to limit intra- and inter-species spread of infection. Previously, 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, induces small lesions (i.e., microgranulomas), and may spread between deer – raising potential food safety concerns. This accomplishment addresses NP 103, component 3, problem statement 3C.

2. 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) and Veterinary Laboratories Agency; Weybridge, UK, 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. Findings have been particularly timely, as inconsistencies in testing procedures (i.e., between US laboratories and states) have recently been identified by the TB program staff (APHIS). This accomplishment addresses NP 103, component 3, problem statement 3C.

3. Efficacy and immunogenicity of Mycobacterium bovis delta-RD1 against aerosol M. bovis infection in neonatal calves. Control of M. bovis in cattle is particularly difficult due to wildlife reservoirs, such as white-tailed deer. While the mainstay of bovine TB control has been abattoir inspection plus targeted test/cull campaigns, vaccines are now being considered as an additional tool for control, both in cattle and wildlife. In collaboration with Howard Hughes Medical Institute, AECOM, Bronx, NY, Veterinary Labories Agency, Weybridge, UK, and others, we demonstrated: (1) vaccination of neonatal calves with delta RD1 (an attenuated strain of M. bovis) provides equivalent efficacy as does BCG against aerosol challenge with virulent M. bovis, (2) central memory responses elicited by either delta RD1 or BCG prior to challenge correlate with reduced pathology and bacterial colonization, (3) vaccination with either delta RD1 or BCG does not interfere with an IFN-gamma-based TB assay using ESAT-6 and CFP-10 antigens, and (4) reduced in vitro recall responses after challenge are associated with effective delta RD1 and BCG vaccines. The impact is the characterization of a modern attenuated vaccine with a defined phenotype development of a safe and effective vaccine for bovine TB. Planned studies will evaluate additional genetic modifications of delta RD1 to enhance safety and immunogenicity. This accomplishment addresses NP 103, component 3, problem statement 3C.

4. Histology, immunohistochemistry and ultrastructure of the bovine palatine tonsil with special emphasis on tonsilar reticular epithelium. Tonsils are composed of lymphoid tissue organized both diffusely and in lymph nodules. Tonsils serve as a site for antigen uptake and processing leading to the generation of antigen specific immune responses, particularly relevant for the initiation of responses to pathogens such as M. bovis. Few studies have been performed to characterize these tissues in cattle. In this study, we intimately characterized the cell types associated with the bovine tonsilar lympho-epithelium using dual color immunofluorescence, electron microscopy, and functional assays for evaluation of antigen uptake capability. A key finding is that bovine palatine tonsilar reticular epithelium consists of cells capable of microparticle uptake (i.e., M-cells). These cells are closely associated with lymphoid and other mononuclear cells; thus, defining the bovine palatine tonsil as an important site for antigen uptake and initiation of immune responses. The impact is a better understanding of the palatine tonsilar epithelium, which will be important for elucidating the pathogenesis of various bovine diseases where the tonsil plays a critical role as well as aiding in the development of vaccines that could target palatine tonsilar M-cells for efficient vaccine uptake and processing. This accomplishment addresses NP 103, component 3, problem statement 3C.

5.Significant Activities that Support Special Target Populations

6.Technology Transfer

Number of Active CRADAs1
Number of the New MTAs (providing only)1
Number of Non-Peer Reviewed Presentations and Proceedings3

Review Publications
Palmer, M.V. 2008. Mycobacterium bovis Shuttles between Domestic Animals and Wildlife. Microbe. 3(1):27-34.

Waters, W.R., Sacco, R.E. 2007. Veterinary Applications for Monitoring Mononuclear Cell Proliferation Using Cell Tracking Dyes. Immunological Investigations. 36(5-6):887-908.

Nol, P., Palmer, M.V., Waters, W.R., Aldwell, F.E., Buddle, B.M., Triantis, J., Linke, L., Phillips, G., Thacker, T.C., Rhyan, J., Dunbar, M., Salman, M.D. 2008. Efficacy of Oral and Parenteral Routes of Mycobacterium bovis bacille Calmette-Guerin Vaccination Against Experimental Bovine Tuberculosis in White-tailed deer (Odocoileus virginianus): A Feasibility Study. Journal of Wildlife Diseases. 44(2):247-259.

Foote, M.R., Nonnecke, B.J., Beitz, D.C., Waters, W.R. 2007. Antigen-specific B-cell responses by neonatal calves after early vaccination. Journal of Dairy Science. 90(11):5208-5217.

Palmer, M.V., Thacker, T.C., Waters, W.R. 2007. Vaccination of White-tailed Deer (Odocoileus virginianus) with Mycobacterium bovis bacillus Calmette Guerin. Vaccine. 25(36):5489-97.

Endsley, J.J., Hogg, A., Shell, L.J., Mcaulay, M., Coffey, T., Howard, C., Capinos-Scherer, C.F., Waters, W.R., Nonnecke, B.J., Estes, D.M., Villarreal-Ramos, B. 2007. Mycobacterium bovis BCG Vaccination Induces Memory CD4+ T cells Characterized by Effector Biomarker Expression and Anti-mycobacterial Activity. Vaccine. 25(50):8384-8394.

Thacker, T.C., Palmer, M.V., Waters, W.R. 2007. Associations Between Cytokine Gene Expression and Pathology in Mycobacterium bovis Infected Cattle. Veterinary Immunology and Immunopathology. 119(3-4):204-213.

Palmer, M.V., Waters, W.R., Thacker, T.C. 2007. Lesion Development and Immunohistochemical Changes in Granulomas from Cattle Experimentally Infected with Mycobacterium bovis. Veterinary Pathology. 44(6):863-874.

Waters, W.R., Palmer, M.V., Thacker, T.C., Orloski, K., Nol, P., Harrington, N.P., Olsen, S.C., Nonnecke, B.J. 2008. Blood Culture and Stimulation Conditions for the Diagnosis of Tuberculosis in Cervids by the Cervigam Assay. Veterinary Record. 162(7):203-208.

Palmer, M.V., Stabel, J.R., Waters, W.R., Bannantine, J.P., Miller, J.M. 2007. Experimental Infection of White-tailed Deer (Odocoileus virginianus) with Mycobacterium avium subsp. paratuberculosis. Journal of Wildlife Diseases. 43(4):597-608.

Waters, W.R., Palmer, M.V., Nonnecke, B.J., Thacker, T.C., Scherer, C.F., Estes, D.M., Jacobs, W.R., Glatman-Freedman, A., Larsen, M.H. 2007. Failure of a Mycobacterium tuberculosis DeltaRD1 DeltapanCD Double Deletion Mutant in a Neonatal Calf Aerosol M. bovis Challenge model: Comparisons to Responses Elicited by M. bovis bacille Calmette Guerin. Vaccine. 25(45):7832-7840.

Last Modified: 12/1/2015
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