Mycobacterium bovis–Infected White-Tailed Deer (Odocoileus Virginianus): Detection of Immunoglobulin Specific to Crude Mycobacterial Antigens by ELISA

White-tailed deer (Odocoileus virginianus) have recently emerged as a source of Mycobacterium bovis infection for cattle within North America. The objective of this study was to evaluate the antibody response of M. bovis–infected deer to crude mycobacterial antigens. Deer were experimentally inoculated with M. bovis strain 1315 either by intratonsilar instillation or by exposure to M. bovis–infected (i.e., in contact) deer. To determine the time course of the response, including the effects of antigen administration for comparative cervical skin testing, serum was collected periodically and evaluated by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (i.e., IgG heavy and light chains) reactivity to mycobacterial antigens. The reactivity to M. bovis purified protein derivative (PPDb) exceeded (P < 0.05) the reactivity to M. avium PPD (PPDa) only after in vivo administration of PPDa and PPDb for comparative cervical testing of the infected deer. The mean immunoglobulin response, as measured by ELISA, of intratonsilar-inoculated deer to a proteinase K–digested whole-cell sonicate (WCS-PK) of M. bovis strain 1315 exceeded (P < 0.05) the mean of the prechallenge responses to this antigen at approximately 1 month after inoculation and throughout the remainder of the study (i.e., ~11 months). This response also exceeded (P < 0.05) that of the uninfected deer. Although this is encouraging, further studies are necessary to validate the use of the proteinase K–digested M. bovis antigens in the antibody-based assays of tuberculosis.

Emergence of Diseases From Wildlife Reservoirs

Interest in the epidemiology of emerging diseases of humans and livestock as they relate to wildlife has increased greatly over the past several decades. Many factors, most anthropogenic, have facilitated the emergence of diseases from wildlife. Some livestock diseases have ‘‘spilled over’’ to wildlife and then ‘‘spilled back’’ to livestock. When a population is exposed to an infectious agent, depending on an interaction of factors involving the host, agent, and environment, the population may be resistant to infection or may become a dead-end host, a spillover host, or a maintenance host. Each exposure is unique; the same species of host and agent may respond differently in different situations. Management actions that affect the environment and behavior of a potential host animal may allow the emergence of a new or as yet undetected disease. There are many barriers in preventing, detecting, monitoring and managing wildlife diseases. These may include political and legal hurdles, lack of knowledge about many diseases of wildlife, the absence of basic data on wildlife populations, difficulties with surveillance, and logistical constraints. Increasing interaction between wildlife and humans or domestic animals may lead to disease emergence and require innovative methods and strategies for disease surveillance and management in wildlife.

Vaccination with Mycobacterium bovis BCG Strains Danish and Pasteur in White-tailed Deer (Odocoileus virginianus) Experimentally Challenged with Mycobacterium bovis

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock and the cause for many faltering bovine tuberculosis eradication programs. One approach in dealing with wildlife reservoirs of disease is to interrupt inter-species and intraspecies transmission through vaccination of deer or cattle. To evaluate the efficacy of BCG vaccination in white-tailed deer, 35 deer were assigned to one of three groups; one s.c. dose of 107 CFU of M. bovis BCG Pasteur (n = 12); 1 s.c. dose of 107 CFU of M. bovis BCG Danish (n = 11); or unvaccinated deer (n = 12). After vaccination, deer were inoculated intratonsilarly with virulent M. bovis. Lesion severity scores of the medial retropharyngeal lymph node, as well as all lymph nodes combined, were reduced in vaccinated deer compared to unvaccinated deer. BCG Danish vaccinated deer had no late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli compared to BCG Pasteur vaccinated or unvaccinated deer where such lesions were present. Both BCG strains were isolated as late as 250 days after vaccination from deer that were vaccinated but not challenged. In white-tailed deer, BCG provides protection against challenge with virulent M. bovis. Issues related to vaccine persistence, safety and shedding remain to be further investigated.

Vaccination of White-Tailed Deer (Odocoileus virginianus) with Mycobacterium bovis Bacillus Calmette Guerin

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis in domestic livestock. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to cattle. One approach in dealing with this wildlife reservoir is to vaccinate deer in order to interrupt the cycle of deer to deer and deer to cattle transmission. Thirty-one white-tailed deer were assigned to one of three groups; 2 SC doses of 107 CFU of M. bovis BCG (n = 11); 1 SC dose of 107 CFU of M. bovis BCG (n = 10); or unvaccinated deer (n = 10). After vaccination, deer were inoculated intratonsilarly with 300 CFU of virulent M. bovis. Gross lesion severity scores of the medial retropharyngeal lymph node were significantly reduced in deer receiving 2 doses of BCG compared to unvaccinated deer. Vaccinated deer had fewer lymph node granulomas than unvaccinated deer, and most notably, fewer late stage granulomas characterized by coalescent caseonecrotic granulomas containing numerous acid-fast bacilli. BCG was isolated from 7/21 vaccinated deer as long as 249 days after vaccination. In one case BCG was transmitted from a vaccinated deer to an unvaccinated deer. In white-tailed deer BCG provides measurable protection against challenge with virulent M. bovis. However, persistence of vaccine within tissues as well as shedding of BCG from vaccinates remain areas for further investigation.

Evaluation of an In Vitro Blood-Based Assay to Detect Production of Interferon-γ By Mycobacterium Bovis–Infected White-Tailed Deer (Odocoileus Virginianus)

Tuberculosis due to Mycobacterium bovis in captive Cervidae was identified as an important disease in the United States in 1990 and prompted the addition of captive Cervidae to the USDA Uniform Methods and Rules for eradication of bovine tuberculosis. As well, M. bovis infection was identified in free-ranging white-tailed deer in northeast Michigan in 1995. Tuberculosis in both captive and free-ranging Cervidae represents a serious challenge to the eradication of M. bovis infection from the United States. Currently, the only approved antemortem tests for tuberculosis in Cervidae are the intradermal tuberculin skin test and the blood tuberculosis test (BTB). At present, the BTB is not available in North America. Tuberculin skin testing of Cervidae is time-consuming and involves repeated animal handling and risk of injury to animals and humans. This study evaluated the potential of a new blood-based assay for tuberculosis in Cervidae that would decrease animal handling, stress, and losses due to injury. In addition, a blood-based assay could provide a more rapid diagnosis. Twenty 6–9-month-old white-tailed deer, male and female, were experimentally inoculated by instillation of 300 colony-forming units of M. bovis in the tonsillar crypts. Seven, age-matched uninfected deer served as controls. Blood was collected on days 90, 126, 158, 180, 210, 238, 263, and 307 after inoculation and was analyzed for the production of interferon-γ (IFN-γ) in response to incubation with M. bovis purified protein derivative (PPDb), M. avium PPDa, pokeweed mitogen (PWM), or media alone. Production of IFN-g in response to PPDb was significantly greater (P < 0.05) at all time points in samples from M. bovis–infected deer as compared with uninfected control deer, whereas IFN-γ production to PWM did not differ significantly between infected and control deer. Measurement of IFN-γ production to PPDb may serve as a useful assay for the antemortem diagnosis of tuberculosis in Cervidae.

Antibody Responses in Reindeer (Rangifer tarandus) Infected with Mycobacterium bovis

Despite having a very low incidence of disease, reindeer (Rangifer tarandus) are subject to tuberculosis (TB) testing requirements for interstate shipment and herd accreditation in the United States. Improved TB tests are desperately needed, as many reindeer are falsely classified as reactors by current testing procedures. Sera collected sequentially from 11 (experimentally) Mycobacterium bovis-infected reindeer and 4 noninfected reindeer were evaluated by enzyme-linked immunosorbent assay (ELISA), immunoblotting, and multiantigen print immunoassay (MAPIA) for antibody specific to M. bovis antigens. Specific antibody was detected as early as 4 weeks after challenge with M. bovis. By MAPIA, sera were tested with 12 native and recombinant antigens, which were used to coat nitrocellulose. All M. bovis-infected reindeer developed responses to MPB83 and a fusion protein, Acr1/MPB83, and 9/11 had responses to MPB70. Other antigens less commonly recognized included MPB59, ESAT-6, and CFP10. Administration of purified protein derivatives for skin testing boosted serum antibody responses, as detected by each of the assays. Of the noninfected reindeer, 2/4 had responses that were detectable immediately following skin testing, which correlated with pathological findings (i.e., presence of granulomatous lesions yet the absence of acid-fast bacteria). The levels of specific antibody produced by infected reindeer appeared to be associated with disease progression but not with cell-mediated immunity. These findings indicate that M. bovis infection of reindeer elicits an antibody response to multiple antigens that can be boosted by skin testing. Serological tests using carefully selected specific antigens have potential for early detection of infections in reindeer.

2006 Michigan Dairy Farm Business Analysis Summary

This report summarizes the financial and production records of 139 dairy farms from throughout Michigan in 2006. To be included, the farms must have produced at least 50 percent of gross cash farm income from milk and dairy animal sales. The records came from Michigan State University’s TelFarm project and the Farm Credit Service system in Michigan. The values were pooled into averages for reporting purposes. The farms are larger than would be the average of all dairy farms in Michigan. While considerable variation in the data exists, average values are reported in the summary tables and discussion that follows.

Experimental Inoculation of Coyotes with Mycobacterium bovis Susceptibility and Shedding

Several wildlife species have tested positive for bovine tuberculosis in Michigan and may potentially transmit the disease to other animals. Coyotes have the highest known prevalence in the endemic area and thus, our objective was to investigate the shedding of Mycobacterium bovis by coyotes. Four coyotes were orally inoculated with 1 ml of 1 x 105 CFU/ml of M. bovis. Oral and nasal swabs, and feces were collected regularly and tested by culture. Fecal samples were also tested by exposing guinea pigs to the coyotes' feces. All animals were necropsied to determine if infection occurred. All swabs, feces and tissues were negative on culture. The dosage of M. bovis given to these coyotes was considered biologically relevant, but was insufficient for causing infection. Due to the lack of infection, we still do not know the risk coyotes pose for shedding M. bovis.

Livestock Protection Dogs for Deterring Deer From Cattle and Feed

Disease transmission between wildlife and livestock is a worldwide issue. Society needs better methods to prevent interspecies transmission to reduce disease risks. Producers have successfully used livestock protection dogs (LPDs) for thousands of years to reduce predation. We theorized that LPDs raised and bonded with cattle could be used to also reduce risk of bovine tuberculosis (Myobacterium bovis; TB) transmission between white-tailed deer (Odocoileus virginianus) and cattle by minimizing contact between the 2 species and use of cattle feed by deer. We evaluated 4 LPDs over 5 months, utilizing 2 data collection methods (direct observation and motion-activated video) on deer farms that supported higher densities than wild populations. Dogs were highly effective in preventing deer from using concentrated cattle feed (hay bales), likely the greatest risk factor of TB transmission on farms. Dogs also prevented deer from approaching cattle in core areas of pastures (near hay bales) and were very effective throughout pastures. Our research supports the theory that LPDs, specifically trained to remain with cattle, may be a practical tool to minimize potential for livestock to contract TB from infected deer in small-scale cattle operations. Where disease is present in deer, it may be possible to reduce the potential for disease transmission by employing LPDs.

Epizootiologic Survey of Mycobacterium Bovis in Wildlife and Farm Environments in Northern Michigan

Bovine tuberculosis (bovine TB), caused by Mycobacterium bovis, has reemerged in northern Michigan, USA, with detections in white-tailed deer (Odocoileus virginianus) in 1994 and in cattle in 1998. Since then, significant efforts have been directed toward reducing deer densities in the area in the hopes of reducing the bovine TB prevalence rate in deer and eliminating spillover of the disease into cattle. Despite the success of the efforts to reduce deer densities, additional cattle herds have become infected. Other mammals can be infected with M. bovis, and some carnivores and omnivores had been found to be infected with the disease in northern Michigan, USA. We conducted a multiyear surveillance effort to detect bovine TB in wild species of mammals in the Michigan, USA, outbreak area. From 2002 to 2004, tissue samples from 1,031 individual animals of 32 species were collected, processed, and cultured for M. bovis. Only 10 (1.0%) were culture-positive for M. bovis (five raccoons [Procyon lotor], four opossums [Didelphis virginiana], and one grey fox [Urocyon cinereoargenteus]). We also found two raccoons and four opossums to be positive for Mycobacterium avium. We collected 503 environmental samples from cattle farms recently identified as bovine TB positive; none yielded positive M. bovis culture results. Finally, we used infrared cameras to document wildlife use of four barns in the area. Many avian and mammalian species of wildlife were observed, with raccoons being the most commonly observed species. This surveillance study identified no new wildlife species that should be considered significant reservoirs of bovine TB in the outbreak area in northern Michigan, USA. However, the relatively high, apparent bovine TB prevalence rates in some carnivorous and omnivorous species, their relatively long life spans, and their frequent use of barns, suggests that removal of raccoons, opossums, foxes, and coyotes (Canis latrans) should be considered when a newly infected farm is depopulated of cattle.