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United States Department of Agriculture

Agricultural Research Service

Research Project: Prevention and Control Strategies for Tuberculosis in Cattle and Wildlife Reservoirs
2013 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:
USDA initiated an eradication campaign against bovine tuberculosis in 1917. Research activities within the ARS Tuberculosis (TB) project provide direct support for the USDA TB eradication program. Significant progress has been made towards eradication but eradication has been elusive. Two significant obstacles to eradication include, the lack of rapid and accurate diagnostic tests, and wildlife acting as a source of infection for cattle. The approach of the ARS TB project is to evaluate diagnostic (Objective.
2)and vaccine (Objective.
3)strategies, as well as basic disease processes (Objective 1). In FY13 progress was made on all project objectives. Most notably, continued discovery and validation of improved diagnostic tests for TB in cattle and deer (Objective 1), resulted in USDA licensing and approval of new diagnostic tests. During FY13, diagnostic tests investigated and evaluated by ARS scientists were licensed and approved for use in captive deer and cattle, and have received approval by the OIE World Organization for Animal Health for use in cattle. Project progress also included: further characterization of immune responses of cattle to Mycobacterium bovis (M. bovis) infection; continued development of novel methods to detect and measure vaccine-induced protection; increased understanding of ultrastructural and functional characterization of tonsils (a major portal of entry for the TB bacillus) in cattle and deer; evaluation of the human TB vaccine, M. bovis bacillus Calmette-Guerin (BCG) in white-tailed deer for efficacy and safety; assessment of the potential for unintended transfer of BCG from vaccinated deer to non-vaccinated cattle or humans; evaluation of large-scale production (with APHIS- National Veterinary Services Laboratory) of the BCG vaccine for use in experimental and field operations; evaluation of various “bait” delivery systems for vaccination of wildlife (in conjunction with USDA, APHIS Wildlife Services); sequencing and comparison of whole genomes of environmental Mycobacteria spp. that currently confound diagnostic tests; and field evaluation of blood-based tests for tuberculosis. In addition to supporting the missions of both USDA ARS and APHIS, these studies support stakeholder (beef, dairy and deer producer groups) interests as well as interests of State Departments of Agriculture and Natural Resources. This support is particularly relevant in states with continuing bovine tuberculosis problems (in particular – Michigan, Colorado, Nebraska, South Dakota). Research findings are used to create or modify state and federal regulations regarding TB testing of cattle and deer including approval of novel tests for use in the USDA Uniform Methods and Rules (UM&R) for the eradication of bovine tuberculosis.


4.Accomplishments
1. USDA licensing and approval of new tuberculosis (TB) diagnostic test for deer species. TB testing of deer requires at least 2 animal handling events. Each handling event risks injury to frightened deer and personnel. Moreover, TB tests currently used in deer require a minimum of three days for results. A rapid TB test requiring one handling event would significantly reduce risk to animals and personnel, as well as decrease costs through decreased labor and decreased loss of production. ARS researchers in Ames, Iowa, in cooperation with international collaborators, industry, and USDA, APHIS developed and validated a new, rapid, blood-based test for use in deer as part of the USDA tuberculosis eradication effort. A separate rapid, blood-based test was licensed and approved for use in cattle. Although this research spans a period of over 13 years, in FY13 both tests were licensed and approved by USDA. Use of the new test, with a single handling event, will save money for producers, decrease animal stress, decrease animal loss from injury, and enhance identification of TB-infected animals.

2. Identification of mycobacteria from samples that interfere with accurate diagnosis. In meat processing plants USDA inspectors identify tuberculosis (TB) suspect cattle and submit samples to the USDA’s National Veterinary Services Laboratories in Ames, Iowa, for confirmation. There are many species of non-disease producing bacteria related to the bacteria that cause tuberculosis. These are often found in the submitted samples. The presence of other bacterial species confounds and delays accurate diagnosis of TB. ARS scientists in Ames, Iowa, in collaboration with USDA, APHIS veterinarians did a comprehensive analysis of various bacterial species isolated from submitted samples over a 7-year period. Knowledge of the identity and frequency of occurrence of these confounding bacteria will greatly facilitate development of improved and rapid diagnostics. Improved diagnostics with greater specificity will decrease false positive results, resulting in fewer retained carcasses at meat inspection, fewer cattle herds under quarantine, and enhanced identification of animals truly infected with TB.


Review Publications
Joshi, D., Harris, B., Waters, W.R., Thacker, T.C., Mathema, B., Krieswirth, B., Sreevatsan, S. 2012. Single nucleotide polymorphisms in the Mycobacterium bovis genome resolve phylogenetic relationships. Journal of Clinical Microbiology. 50(12):3853-3861.

Palmer, M.V., Thacker, T.C., Waters, W.R., Robbe-Austerman, S., Aldwell, F.E. 2012. Persistence of Mycobacterium bovis bacillus Calmette-Guerin (BCG) Danish in White-tailed deer (Odocoileus virginianus) vaccinated with a lipid-formulated oral vaccine. Transboundary and Emerging Diseases. DOI.org/10.1111/tbed.12032.

Palmer, M.V., Thacker, T.C., Madison, R.J., Koster, L., Swenson, S., Li, H. 2013. Active and latent ovine Herpesvirus-2 (OvHV-2) infection in a herd of captive white-tailed deer (Odocoileus virginianus). Journal of Comparative Pathology. 149(2-3):162-166.

Lyashchenko, K.P., Greenwald, R., Esfandiari, J., O'Brien, D.J., Schmitt, S., Palmer, M.V., Waters, W.R. 2013. Rapid detection of serum antibody by dual-path platform VetTB assay in white-tailed deer infected with Mycobacterium bovis. Clinical and Vaccine Immunology. 20(6):907-911.

Thacker, T.C., Robbe-Austerman, S., Harris, B., Palmer, M.V., Waters, W.R. 2013. Isolation of Mycobacteria from clinical samples collected in the United States from 2004 to 2011. BioMed Central (BMC) Veterinary Research. 9(1):100.

Lyashchenko, K.P., Greenwald, R., Esfandiari, J., Mikota, S., Miller, M., Moller, T., Vogelnest, L., Gairhe, K.P., Robbe-Austerman, S., Gai, J., Waters, W.R. 2012. Field application of serodiagnostics to identify elephants with Tuberculosis prior to case confirmation by culture. Clinical and Vaccine Immunology. 19(8):1269-1275.

Last Modified: 9/10/2014
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