2002 Annual Report 1.What major problem or issue is being resolved and how are you resolving it? Bovine tuberculosis, which is caused by Mycobacterium bovis, is an infectious disease that affects many species of animals as well as human beings. Animals infected with M. bovis can shed organisms as they exhale or cough and in various secretions including saliva, milk, and urine. Elimination of animals infected with M. bovis is important to prevent the spread of disease among animals and to human beings. The U.S. initiated a program to eradicate tuberculosis from cattle in 1917 when the prevalence of disease was approximately 5%. In general, the eradication program has been successful and today, less than 0.002% of cattle are infected with M. bovis. However, a low prevalence of disease has persisted over the last 10-15 years and it has not been possible to eradicate bovine tuberculosis from the U.S. using available technology. Improved diagnostic tests and control measures are needed to detect and eliminate the few remaining cattle and herds that have bovine tuberculosis. In addition, tuberculosis has been detected in captive deer and elk and in wild white-tailed deer, coyotes, raccoons, bear, bobcat, opossum, and fox. In some cases, there is epidemiological evidence to suggest that tuberculosis has been transmitted from deer to cattle. The presence of tuberculosis in wildlife poses a serious threat to the national eradication program. Other countries with tuberculosis in wildlife species have been unable to eradicate the disease from the domestic livestock population. The Bovine Tuberculosis Research Project at the National Animal Disease Center (NADC) is conducting research to develop a better understanding of the interactions between various host species and M. bovis. This information will be used to develop improved diagnostic tests and vaccines. Methods to identify and differentiate strains of M. bovis are being developed and used to aid in epidemiological investigations of outbreaks of tuberculosis in animals. 2.How serious is the problem? Why does it matter? Bovine tuberculosis is considered a public health threat because human beings can become infected with M. bovis through contact with infected animals or by ingestion of contaminated food and milk. Elimination of tuberculosis from cattle is important to provide food and milk to the public that is free of M. bovis. Elimination of bovine tuberculosis from domestic livestock also is important to maintain free trade with the many trading partners of the U.S. Trade restrictions between the U.S. and Canada and between Mexico and the U.S. have occurred because of bovine tuberculosis in animals. Eradication of tuberculosis from wildlife is important to prevent transmission of disease from wildlife to domestic livestock. 3.How does it relate to the national Program(s) and National Program Component(s) to which it has been assigned? The Bovine Tuberculosis Research Project is assigned to the Animal Health National Program 103 (100%) and relates to the vision of this program to ensure animal health through improved disease detection and control. The objectives of the project are to: . 1)develop and evaluate improved tests for diagnosis of tuberculosis in cattle, deer, and other species, which relates to the National Program component on Pathogen Detection;. 2)develop improved methods for differentiation of M. bovis isolates, which relates to the National Program component on Epidemiology of Disease;. 3)define the interactions between various host species and M. bovis, which relates to the National Program component on Host/Pathogen Interactions; and. 4)develop and evaluate vaccines for control and prevention of tuberculosis in animals, which relates to the National Program component on Disease Control Strategies. Cattle as well as bison and all species of Cervidae are subject to testing for tuberculosis under the guidelines of the USDA uniform rules and methods for the eradication of bovine tuberculosis. The most common means of testing is the tuberculin skin test. Tuberculin skin testing lacks specificity and has not been fully validated in all species of Cervidae. Especially problematic is the absolute lack of specificity associated with tuberculin skin testing reindeer. The Animal and Plant Health Inspection Service (APHIS) of the USDA has provided funds for NADC to conduct research on reindeer (3625-32000-063-01R). The objectives of such research are to study the reindeer immune response and develop an improved method of antemortem diagnosis of tuberculosis in reindeer. Such objectives are consistent with the National Program as stated above. 4.What was your most significant accomplishment this past year? A. Single Most Significant Accomplishment during FY 2002: For decades it has been believed that tuberculosis in animals is transmitted by aerosol; however, aerosol delivery of M. bovis to large animals, such as cattle and deer, had not been done due to obvious logistical and safety concerns. A safe, reliable, and reproducible method of aerosol delivery of Mycobacterium bovis to cattle, or other large ruminants was developed and validated by ARS scientists at NADC. Studies completed during the past fiscal year have shown that this method of aerosol delivery of M. bovis results in lesions similar to those seen in naturally infected cattle. Such a delivery method is now being used at NADC to investigate disease pathogenesis, transmission, immune responses, and vaccine efficacy and could be used to deliver other pathogens of cattle where aerosol delivery is critical. B. Other Significant Accomplishment(s), if any: The outbreak of tuberculosis in white-tailed deer in Michigan, identified in 1995, represents the first wildlife reservoir of M. bovis in North America and a serious threat to the federal bovine tuberculosis eradication effort. During the last fiscal year, ARS scientists at NADC demonstrated the susceptibility of white-tailed deer fawns to oral infection through milk containing M. bovis. These results document that contaminated milk, in addition to saliva, nasal secretions, urine and feces may be involved in transmission of M. bovis between deer. Such information is vital for state and federal officials to make educated decisions concerning disease control strategies. Currently, tuberculin skin testing is the most common method of antemortem testing of deer for tuberculosis; however, tuberculin skin testing of deer lacks specificity and requires the capture and handling of animals at least twice for the testing procedure. During the past year, ARS scientists at NADC have investigated a commercial blood-based assay for the diagnosis of tuberculosis in deer species common in North America. ARS scientists have shown that the assay may be useful in white-tailed deer, reindeer, and possibly elk. After further work, this assay could potentially supplement or replace skin testing in captive Cervidae, decreasing animal handling and testing time. C. Significant Activities that Support Special Target Populations: none. 5.Describe your major accomplishments over the life of the project, including their predicted or actual impact? A new test for detection and identification of M. bovis in tissue samples that are collected for microscopic examination was developed. When animals are slaughtered, meat inspectors collect tissue samples from animals that are suspected of having tuberculosis. Tissue samples are examined for microscopic evidence of tuberculosis and for the presence of organisms. Stains used to detect M. bovis in a tissue sample also stain other organisms so that it is not possible to identify the organism in the sample. The new test detects a specific piece of DNA that is present only in organisms that cause tuberculosis. The new test permits more accurate and rapid identification of animals with tuberculosis than was previously possible. The test is used extensively at the request of state and federal regulatory officials to confirm suspected cases of tuberculosis in animals. We adapted and standardized methods for differentiation of strains of M. bovis. Differences among various strains of M. bovis can be identified by using specific genetic markers. Using these markers, it is possible to determine if different animals are infected with a common strain or different strains of M. bovis. This information is used by epidemiologists to determine possible sources of infection in outbreaks of tuberculosis in animals. An experimental model of tuberculosis in white-tailed deer was developed. The disease produced in deer experimentally infected with M. bovis was very similar to the disease that is observed in naturally infected deer. This model has been used to study the transmission of tuberculosis among deer and between deer and cattle. In addition, this model will be used to evaluate tests for diagnosis of tuberculosis in deer and to determine the effectiveness of vaccines to prevent infection. Current surveys to determine the prevalence of tuberculosis in wild white-tailed deer are based on examination of tissues in the head to detect lesions. In a study that involved detailed examination of the entire deer carcass, we determined that about 50% of the infected deer did not have lesions in the head. These results indicate current methods to detect tuberculosis in wild white-tailed deer underestimate the prevalence of disease by 50% and that improved methods for detection are needed. 6.What do you expect to accomplish, year by year, over the next 3 years? FY 2003: . 1)Further characterize the disease produced when cattle and deer are experimentally challenged with aerosolized M. bovis; . 2)Further characterize immune response of white-tailed deer to M. bovis; . 3)Determine the sensitivity and specificity of new diagnostic tests for the antemortem diagnosis of tuberculosis in Cervidae; . 4)Continue research to develop improved methods for differentiation of M. bovis isolates. FY 2004: . 1)Further characterize the progression of disease and immune responses of white-tailed deer infected with M. bovis; . 2)Continue research to determine the sensitivity and specificity of new diagnostic tests for the antemortem diagnosis of tuberculosis in Cervidae; . 3)Identify potential vaccines that can be used for prevention and control of tuberculosis in wildlife and domestic animals; . 4)Continue research to develop improved methods for differentiation of M. bovis isolates. FY 2005: . 1)Determine the safety and effectiveness of candidate vaccines; . 2)Continue research to develop and evaluate improved diagnostic tests. 7.What technologies have been transferred and to whom? When is the technology likely to become available to the end user (industry, farmer other scientist)? What are the constraints, if known, to the adoption durability of the technology? ARS scientists assigned to the Bovine Tuberculosis research project routinely presented research findings to scientists, state and federal action agencies, and industry groups. In some cases, results of research conducted by ARS scientists are used as the basis for regulatory action. ARS scientists received invitations to present results of research to specific groups, such as the Michigan Department of Natural Resources, because of the direct impact that the research findings have on recommendations of the group. ARS scientists are members of several organizations, committees, and working groups and provide scientific expertise on a broad range of issues relating to bovine tuberculosis. A patent has been approved for a new test that was developed by ARS scientists for diagnosis of tuberculosis (Invention Report No. 0152.01 "Diagnosis of tuberculosis infection through analysis of nitrite production of leukocytes stimulated with mycobacterial antigens.") 8.List your most important publications and presentations, and articles written about your work (NOTE: this does not replace your review publications which are listed below) Presented talks entitled "Studies on doe-to-fawn transmission of Mycobacterium bovis in white-tailed deer," and "Transmission of Mycobacterium bovis from white-tailed deer to cattle" to participants of Bovine Tuberculosis in Michigan, conference 2002, March 12-13, 2002, Lansing, Michigan. Registered participants of the conference included state and federal animal health officials, departments of natural resources and community health, livestock industry groups, private landowners, hunt clubs, state legislators, congressional representatives, and members of the press. Presented a talk entitled "Tuberculosis in wildlife: Impact on animal agriculture and public health" to Des Moines University Osteopathic Medical Center, Des Moines, IA. Participants included physicians and public health professionals. Presented a talk entitled "Johne's disease of cattle, potential impact on human health and the need for more research" at the CASE media fellowship seminar on animal disease outbreaks and their impact on society in Ames, IA. Review Publications Palmer, M.V., Waters, W.R., Whipple, D.L. Lesion development in white-tailed deer (Odocoileus virginianus) experimentally infected with Mycobacterium bovis. Veterinary Pathology. 2002. v. 39. p. 334-340. Palmer, M.V., Waters, W.R., Whipple, D.L. Susceptibility of raccoons (Procyon lotor) to infection with Mycobacterium bovis. Journal of Wildlife Diseases. 2002. v. 38. p. 266-274. Palmer, M.V., Whipple, D.L., Waters, W.R. Tuberculin skin testing in white-tailed deer (Odocoileus virginianus). Journal of Veterinary Diagnostic Investigation. 2001. v. 13. p. 530-533. Waters, W.R., Palmer, M.V., Sacco, R.E., Whipple, D.L. Nitric oxide production as an indication of Mycobacterium bovis infection in white-tailed deer. Journal of Wildlife Diseases. 2002. v. 38. p. 338-343. Waters, W.R., Sacco, R.E., Fach, S.J., Palmer, M.V., Olsen, S.C., Kreeger, T.J. Analysis of mitogen-stimulated lymphocyte subset proliferation and nitric oxide production by peripheral blood mononuclear cells of captive elk (Cervus elaphus). Journal of Wildlife Diseases. 2002. v. 38. p. 344-351. Waters, W.R., Nonnecke, B.J., Rahner, T.E., Palmer, M.V., Whipple, D.L., Horst, R.L. Modulation of Mycobacterium bovis-specific responses of bovine peripheral blood mononuclear cells by 1,25 dihydroxyvitamin D3. Clinical and Diagnostic Laboratory Immunology. 2001. v. 8. p. 1204-1212. Waters, W.R., Harkins, K.R., Wannemuehler, M.J. Five-color flow cytometric analysis of swine lymphocytes for detection of proliferation, apoptosis, viability, and phenotype. Cytometry. 2002. v. 48. p. 146-152. Kaneene, J.B., VanderKlok, M., Bruning-Fann, C.S., Palmer, M.V., Whipple, D.L., Schmitt, S.M., Miller, R.A. Prevalence of Mycobacterium bovis infection in cervids on privately owned ranches. Journal of the American Veterinary Medical Association. 2002. v. 220(5). p. 656-659. Palmer, M.V., W.R. Waters, Whipple, D.L. Lesion development in white-tailed deer experimentally infected with Mycobacterium bovis. Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. 2001. Abstract p. 50. Palmer, M.V., W.R. Waters, D.L. Whipple. Tuberculin skin testing in white-tailed deer (Odocoileus virginianus). Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. 2001. Abstract p. 110. Waters, W.R., Palmer, M.V., Sacco, R.E., Whipple, D.L. Nitric oxide production as an indication of Mycobacterium bovis infection of white-tailed deer (Odocoileus virginianus). Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. 2001. Abstract p. 111. Palmer, M.V., Waters, W.R., Whipple, D.L. Susceptibility of raccoons (Procyon lotor) to infection with Mycobacterium bovis. Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. 2001. Abstract p. 51. Waters, W.R., Palmer, M.V., Whipple, D.L. Lymphocyte subset proliferative responses of tuberculous cattle and white-tailed deer to Mycobacterium bovis purified protein derivative. Proceedings of the Keystone Symposia Molecular and Cellular Aspects of Tuberculosis Research in the Post Genome Era. 2001. Abstract p. 98. Waters, W.R., Nonnecke, B.J., Rahner, T.E., Palmer, M.V., Whipple, D.L., Horst, R.L. Modulation of Mycobacterium bovis specific responses of bovine peripheral blood mononuclear cells by 1,25-dihydroxyvitamin D3. Annual Meeting of the Conference of Research Workers in Animal Diseases. 2001. Abstract p.72. Fach, S.J., Waters, W.R., Palmer, M.V., Davis, W.C., Sacco, R.E. Phenotypic analysis of lymphocytes isolated from white-tailed deer (Odocoileus virginianus) fawns using three-color flow cytometry. Annual Meeting of the Conference of Research Workers in Animal Diseases. 2001. Abstract p. 84.