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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Research Project #422014


Location: Infectious Bacterial Diseases Research

2012 Annual Report

1a. Objectives (from AD-416):
Objective 1: Characterize the immuopathogenesis of M. bovis infection in cattle and white-tailed deer and the role of the tonsils in transmission of the organism. Subobjective 1.1. Describe relevant aspects of tonsilar lymphoepithelium in cattle and deer. Subobjective 1.2. Evaluate M. bovis BCG interaction with tonsilar lymphoepithelium and associated cells of the innate immune system by using ex vivo tonsil organ cultures. Objective 2: Determine new strategies for the detection of M. bovis infection, including strategies to differentiate from other environmental mycobacteria, using evaluation of the transcriptome and/or proteome to discover new proteins that can be used in diagnostic assays. Subobjective 2.1. Sequence environmental mycobacteria commonly isolated from diagnostic samples to ascertain regions unique to M. bovis that may be exploited for development of improved diagnostic tests. Subobjective 2.2. Evaluate the proteome/transcriptome of M. bovis expressed in vivo to discover proteins to facilitate discovery of improved diagnostic reagents and vaccine targets. Objective 3: Develop new vaccines based on novel platforms and determine immune parameters that correlate to protection. Subobjective 3.1. Evaluate the efficacy and safety of oral BCG for use in white-tailed deer. Subobjective 3.2. Determine the efficacy of M. bovis DeltanuoG x DeltaRD1 against aerosol M. bovis infection in neonatal calves. Subobjective 3.3. Determine immune parameters elicited by vaccination that correlate to protection from challenge with virulent M. bovis.

1b. Approach (from AD-416):
The approach is to evaluate local M. bovis interactions within the tonsilar lymphoepithelium to provide basic knowledge relevant to biomarker discovery and protective immune responses. Specific host responses detected via transcriptomics / proteomics studies will provide targets for discovery of novel correlates of protection, diagnostic biomarkers, and reagents / knowledge necessary for immunopathogenesis studies. With vaccine studies, the approach with white-tailed deer is to finalize research to optimize delivery of BCG to deer in the field, including safety and efficacy studies with the final product. With cattle, a novel pro-apoptotic attenuated live tuberculosis vaccine will be tested for efficacy and correlates of protection evaluated. Each of the studies are intimately linked to optimize resources.

3. Progress Report:
The USDA initiated a bovine tuberculosis eradication campaign in 1917. Significant progress has been achieved, but eradication has proved elusive. Obstacles to eradication include lack of rapid diagnostic tests and wildlife reservoirs of tuberculosis serving as a source of infection for cattle. In FY12, progress was made on all project objectives. Advancement relevant to Objective 1 included further characterization of immune responses of cattle to M. bovis and ultrastructural characterization of tonsils (a major portal of entry for the TB bacillus) in cattle and deer. In support of Objective 2 there was continued effort toward improved diagnostic tests for cattle and deer, as well as the sequencing of 5 different environmental mycobacteria that currently confound existing diagnostic tests. Progress pertinent to Objective 3 incorporates evaluation of both efficacy and safety of the human TB vaccine, M. bovis bacillus Calmette-Guerin (BCG), in white-tailed deer, and the potential for unintended, and undesirable transfer of BCG from vaccinated deer to cattle. In cooperation with Animal and Plant Health Inspection Service (APHIS)-National Veterinary Services Laboratories we have initiated large-scale production of BCG for use in experimental and field operations, while at the same time, in collaboration with APHIS, Wildlife Services, have evaluated various bait delivery systems for vaccination of wild deer. In sum, this research supports the missions of USDA, ARS and APHIS, and interests of State Departments of Agriculture and Natural Resources (in particular – Michigan, Colorado, Nebraska, South Dakota) as well as beef, dairy, and deer producer groups. Research findings continue to be used to create or modify state and federal regulations regarding TB testing of cattle and deer, including approval of novel diagnostic tests for use in the USDA effort to eradicate bovine tuberculosis. For example during FY12 diagnostic tests investigated by ARS scientists have been approved for use in captive deer, and received approval by the World Organization for Animal Health (OIE) for use in cattle.

4. Accomplishments
1. Persistence of the human tuberculosis (TB) vaccine bacillus Calmette-Guerin (BCG) in tissues of deer vaccinated with oral baits containing BCG. Vaccination is proposed as one method to control tuberculosis in wildlife reservoirs such as white-tailed deer. ARS researchers in Ames, IA found that deer orally vaccinated with the human TB vaccine, BCG, demonstrated persistence of BCG for up to 12 months, suggesting that hunters handling deer carcasses could potentially be exposed to live BCG vaccine. Importantly BCG was never found in tissues commonly used for food (i.e. muscle), suggesting a low risk of oral exposure of humans to BCG. State and Federal animal health and public health officials will need to consider the potential of BCG persistence in deer when contemplating wildlife vaccination efforts.

2. Mycobacterial genome sequencing. Although incidence is low, cattle infected with the tuberculosis continue to be identified in a number of states. As tuberculosis in cattle can spread to people, this is a public health concern. ARS researchers at Ames, Iowa, have sequenced the genomes of 5 environmental mycobacteria isolated from clinical sources. By comparing the sequences in these nonpathogenic mycobacteria to virulent strains of Mycobacterium bovis, genomic differences were identified that may be used to develop assays that are specific for M. bovis. Development of better diagnostic assays that rapidly differentiate M. bovis from environmental mycobacteria will ultimately reduce the number of ‘false positive’ tests. Development of rapid and more sensitive diagnostics will enhance surveillance for tuberculosis in cattle, reduce cost to producers and regulatory agencies, and directly benefit public health.

Review Publications
Waters, W.R., Thacker, T.C., Nonnecke, B.J., Palmer, M.V., Schiller, I., Oesch, B., Vordermeier, H.M., Silva, E., Estes, D.M. 2012. Evaluation of gamma interferon (IFN-gamma)-induced protein 10 responses for detection of cattle infected with Mycobacterium bovis: comparisons to IFN-gamma responses. Clinical and Vaccine Immunology. 19(3):346-351.

Lyashchenko, K.P., Greenwald, R., Esfandiari, J., Lecu, A., Waters, W.R., Posthaus, H., Bodmer, T., Janssens, J., Aloisio, F., Graubner, C., Grosclaude, E., Piersigilli, A., Schiller, I. 2012. Pulmonary disease due to Mycobacterium tuberculosis in a horse: zoonotic concerns and limitations of antemortem testing. Veterinary Medicine International [serial online]. 2012:Article 642145. Available:

Thacker, T.C., Harris, B., Palmer, M.V., Waters, W.R. 2011. Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR. BioMed Central (BMC) Veterinary Research [serial online]. 7(50). Available:

Waters, W.R., Buddle, B.M., Vordermeier, H.M., Gormley, E., Palmer, M.V., Thacker, T.C., Bannantine, J.P., Stabel, J.R., Linscott, R., Martel, E., Milian, F., Foshaug, W., Lawrence, J.C. 2011. Development and evaluation of an enzyme-linked immunosorbent assay for use in the detection of bovine tuberculosis in cattle. Clinical and Vaccine Immunology. 18(11):1882-1888.

Waters, W.R., Palmer, M.V., Buddle, B.M., Vordermeier, H.M. 2012. Bovine tuberculosis vaccine research: historical perspectives and recent advances. Vaccine. 30(16):2611-2622.

Palmer, M.V., Thacker, T.C., Waters, W.R., Gortazar, C., Corner, L.A. 2012. Mycobacterium bovis: a model pathogen at the interface of domestic livestock, wildlife, and humans. Veterinary Medicine International [serial online]. 2012:Article ID 236205. Available:

Palmer, M.V., Whipple, D.L., Payeur, J.B., Bolin, C.A. 2011. Use of the intradermal tuberculin test in a herd of captive elk (Cervus elaphus nelsoni) naturally infected with Mycobacterium bovis. Journal of Veterinary Diagnostic Investigation. 23(2):363-366.

Palmer, M.V., Stasko, J.A., Waters, W.R., Thacker, T.C. 2011. Examination of the reticular epithelium of the bovine pharyngeal tonsil. The Anatomical Record. 294(11):1939-1950.