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.

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.

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

We investigated the efficacy of oral and parenteral Mycobacterium bovis bacille Calmette-Guerin Danish strain 1331 (BCG) in its ability to protect white-tailed deer (Odocoileus virginianus) against disease caused by M. bovis infection. Twenty-two white-tailed deer were divided into four groups. One group (n=5) received 109 colony-forming units (cfu) BCG via a lipid-formulated oral bait; one group (n=5) received 109 cfu BCG in culture directly to the oropharynx, one group (n=6) was vaccinated with 106 cfu BCG subcutaneously, and one group served as a control and received culture media directly to the oropharynx (n=6). All animals were challenged 3 mo after vaccination. Five months postchallenge the animals were examined for lesions. Results indicate that both oral forms of BCG and parenterally administerd BCG offered significant protection against M. bovis challenge as compared to controls. This study suggests that oral BCG vaccination may be a feasible means of controlling bovine tuberculosis in wild white-tailed deer populations.

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.

Wildlife Tuberculosis in South African Conservation Areas: Implications and Challenges

Tuberculosis, caused by Mycobacterium bovis, was first diagnosed in African buffalo in South Africa’s Kruger National Park in 1990. Over the past 15 years the disease has spread northwards leaving only the most northern buffalo herds unaffected. Evidence suggests that 10 other small and large mammalian species, including large predators, are spillover hosts. Wildlife tuberculosis has also been diagnosed in several adjacent private game reserves and in the Hluhluwe-iMfolozi Park, the third largest game reserve in South Africa. The tuberculosis epidemic has a number of implications, for which the full effect of some might only be seen in the long-term. Potential negative long-term effects on the population dynamics of certain social animal species and the direct threat for the survival of endangered species pose particular problems for wildlife conservationists. On the other hand, the risk of spillover infection to neighboring communal cattle raises concerns about human health at the wildlife–livestock–human interface, not only along the western boundary of Kruger National Park, but also with regards to the joint development of the Greater Limpopo Transfrontier Conservation Area with Zimbabwe and Mozambique. From an economic point of view, wildlife tuberculosis has resulted in national and international trade restrictions for affected species. The lack of diagnostic tools for most species and the absence of an effective vaccine make it currently impossible to contain and control this disease within an infected free-ranging ecosystem. Veterinary researchers and policy-makers have recognized the need to intensify research on this disease and the need to develop tools for control, initially targeting buffalo and lion.

Reducing the Public Health Impact of Bovine Tuberculosis by Controlling Disease Transmission Between Cattle and White-Tailed Deer in Northwestern Minnesota

Bovine tuberculosis, caused by infection with Mycobacterium bovis, is a re-emerging zoonotic disease. It has staged a comeback by establishing infections in wildlife and cattle, creating the potential for human disease in locations where it was thought to be under control. In northwestern Minnesota, infected cattle and white-tailed deer were first discovered in 2005. A major bovine tuberculosis eradication campaign is underway in the state, with multiple efforts employed to control M. bovis infection in both cattle and deer populations. In order to effectively eradicate bovine tuberculosis in Minnesota, there is a need for better understanding of the factors that increase the risk of deer and cattle interacting in a way that facilitates tuberculosis transmission. By reducing the risk of disease transmission within the animal populations, we will also reduce the risk that bovine tuberculosis will again become a common disease in human populations. The purpose of this study is to characterize the risk of interactions between cattle and white-tailed deer in northern Minnesota in order to prevent M. bovis transmission. A survey originally developed to assess deer-cattle interactions in Michigan was modified for use in Minnesota, introducing a scoring method to evaluate the areas of highest priority at risk of potential deer-cattle interaction. The resulting semi-quantitative deer-cattle interaction risk assessment was used at 53 cattle herds located in the region adjacent to the bovine tuberculosis “Core Area”. Two evaluators each scored the farm separately, and then created a management plan for the farm that prioritized the areas of greatest risk for deer-cattle interactions. Herds located within the “Management Zone” were evaluated by Minnesota Board of Animal Health staff, and results from these surveys were used as a point of comparison.

Environmental and Farm Management Factors Associated with Tuberculosis on Cattle Farms in Northeastern Michigan

Objective—To identify major environmental and farm management factors associated with the occurrence of tuberculosis (TB) on cattle farms in northeastern Michigan. Design—Case-control study. Sample Population—17 cattle farms with infected cattle and 51 control farms. Procedure—Each case farm (laboratory confirmed diagnosis of Mycobacterium bovis infection) was matched with 2 to 4 control farms (negative whole-herd test results within previous 12 months) on the basis of type of farm (dairy or beef) and location. Cattle farm data were collected from in-person interviews and mailed questionnaires. Wildlife TB data were gathered through state wildlife surveillance. Environmental data were gathered from a satellite image-based geographic information system. Multivariable conditional logistic regression for matched analysis was performed. Results—Major factors associated with increased farm risk of TB were higher TB prevalence among wild deer and cattle farms in the area, herd size, and ponds or creeks in cattle housing areas. Factors associated with reduced farm risk of TB were greater amounts of natural open lands in the surrounding area and reducing deer access to cattle housing areas by housing cattle in barns, barnyards, or feedlots and use of electrified wire or barbed wire for livestock fencing. Conclusions and Clinical Relevance—Results suggest that certain environmental and management factors may be associated with risk of TB on cattle farms.

Hidden Effects of Chronic Tuberculosis in African Buffalo

Infectious diseases can bring about population declines and local host extinctions, contributing significantly to the global biodiversity crisis. Nonetheless, studies measuring population-level effects of pathogens in wild host populations are rare, and taxonomically biased toward avian hosts and macroparasitic infections. We investigated the effects of bovine tuberculosis (bTB), caused by the bacterial pathogen Mycobacterium bovis, on African buffalo (Syncerus caffer) at Hluhluwe-iMfolozi Park, South Africa. We tested 1180 buffalo for bTB infection between May 2000 and November 2001. Most infections were mild, confirming the chronic nature of the disease in buffalo. However, our data indicate that bTB affects both adult survival and fecundity. Using an age-structured population model, we demonstrate that the pathogen can reduce population growth rate drastically; yet its effects appear difficult to detect at the population level: bTB causes no conspicuous mass mortalities or fast population declines, nor does it alter host-population age structure significantly. Our models suggest that this syndrome—low detectability coupled with severe impacts on population growth rate and, therefore, resilience—may be characteristic of chronic diseases in large mammals.

Wildlife-Cattle Interactions in Northern Michigan: Implications for the Transmission of Bovine Tuberculosis

Bovine tuberculosis (Mycobacterium bovis) was discovered in northern Michigan white-tailed deer (Odocoileus virginianus) in 1994, and has been known to exist in Michigan cattle herds since 1998. Despite efforts to eradicate the disease in cattle, infection and re-infection of farms continues to occur, suggesting transmission among cattle, deer, or other wildlife reservoirs. The goals of this study were to document wildlife activity on farms and evaluate the possible role wildlife play in the ecology of bovine tuberculosis (TB) in Michigan. Visual observations were conducted on farms in a 5-county area of northern Michigan to document direct wildlife-cattle interactions (i.e., <5 m between individuals) and indirect interactions (e.g., wildlife visitations to food stores and areas accessible to cattle). Observations were conducted primarily during evening and early morning hours between January and August, 2002, and on a 24-hour schedule between January and August, 2003. Total observation time accumulated through the duration of the study was 1,780 hours. Results indicated that direct interaction between deer and cattle was a rare event; no direct interactions were observed during the first year, and only one direct interaction was observed during the second year. However, through the duration of the study 21 direct interactions were documented between cattle and turkey, and 11 direct interactions were documented between cattle and mammals other than deer. In total, 273 indirect interactions by deer, 112 indirect interactions by turkeys, and 248 indirect interactions by mammals other than deer were observed during the 2 field seasons combined. These data supported the hypothesis that indirect interactions among wildlife and cattle are a potential mechanism for the transmission of TB in Michigan. If direct interactions were important mechanisms of TB transmission to cattle in northern Michigan, my data suggested that feral cats were the species of most concern, even though there were more observations between turkey and cattle. Unlike cats, which can become infected with and transmit TB, there is no evidence for such pathogenesis in turkey.

Naturally Occurring Tuberculosis in White-Tailed Deer

Objective—To determine the distribution of lesions and extent of tissues infected with Mycobacterium bovis in a captive population of white-tailed deer. Design—Cross-sectional study. Animals—116 captive white-tailed deer. Procedure—Deer were euthanatized, and postmortem examinations were performed. Tissues with gross lesions suggestive of tuberculosis were collected for microscopic analysis and bacteriologic culture. Tissues from the head, thorax, and abdomen of deer with no gross lesions were pooled for bacteriologic culture. Tonsillar, nasal, oral, and rectal swab specimens, fecal samples, and samples of hay and pelleted feed, soil around feeding sites, and water from 2 natural ponds were collected for bacteriologic culture. Results—Mycobacterium bovis was isolated from 14 of 116 (12%) deer; however, only 9 of 14 had lesions consistent with tuberculosis. Most commonly affected tissues included the medial retropharyngeal lymph node and lung. Five of 14 tuberculous deer had no gross lesions; however,M bovis was isolated from pooled tissue specimens from the heads of each of these deer. Bacteriologic culture of tonsillar swab specimens from 2 of the infected deer yielded M bovis. Mean (± SEM) age of tuberculous deer was 2.5 ± 0.3 years (range, 0.5 to 6 years). Mycobacterium bovis was not isolated from feed, soil, water, or fecal samples. Conclusions and Clinical Relevance—Examination of hunter-killed white-tailed deer for tuberculosis commonly includes only the lymph nodes of the head. Results of such examinations may underestimate disease prevalence by as much as 57%. Such discrepancy should be considered when estimating disease prevalence.

Tuberculin Skin Testing in White-Tailed Deer (Odocoileus virginianus)

The comparative cervical skin test for antemortem diagnosis of tuberculosis was done 169 times on 116 different white-tailed deer of known Mycobacterium bovis infection status. The sensitivity and specificity were 97 and 81%, respectively. The magnitude of change in skin thickness at test sites was not significantly influenced by dosage of inoculum, dissemination of the disease process, or repeated skin testing. However, the magnitude of change in skin thickness was significantly greater in deer infected for less than 109 days than in deer infected for more than 109 days. As used in the present study, the comparative cervical skin test is a sensitive method of antemortem diagnosis of M. bovis infection in white-tailed deer.

Monitoring of Transmission of Tuberculosis between Wild Boars and Cattle: Genotypical Analysis of Strains by Molecular Epidemiology Techniques

An epidemiological survey for the monitoring of bovine tuberculosis transmission was carried out in western Liguria, a region in northern Italy. Fifteen Mycobacterium bovis strains were isolated from 63 wild boar samples (62 from mandibular lymph nodes and 1 from a liver specimen). Sixteen mediastinal lymph nodes of 16 head of cattle were collected, and 15 Mycobacterium bovis strains were subsequently cultured. All M. bovisstrains isolated from cattle and wild boars were genotyped by spoligotyping and by restriction fragment length polymorphism (RFLP) analysis with the IS6110 and IS1081 probes. All M. bovis strains showed the typical spoligotype characterized by the absence of the 39 to 43 spacers in comparison with the number in M. tuberculosis. A total of nine different clusters were identified by spoligotyping. The largest cluster included 9 strains isolated from wild boars and 11 strains isolated from cattle, thus confirming the possibility of transmission between the two animal species. Fingerprinting by RFLP analysis with the IS6110 probe showed an identical single-band pattern for 29 of 30 strains analyzed, and only 1 strain presented a five-band pattern. The use of IS1081 as a second probe was useful for differentiation of M. bovis from M. bovis BCG but not for differentiation among M. bovis strains, which presented the same undifferentiated genomic profile. In relation to the epidemiological investigation, we hypothesized that the feeding in pastures contaminated by cattle discharges could represent the most probable route of transmission of M. bovis between the two animal species. In conclusion, our results confirmed the higher discriminatory power of spoligotyping in relation to that of RFLP analysis for the differentiation of M. bovis genomic profiles. Our data showed the presence of a common M. bovis genotype in both cattle and wild boars, confirming the possible interspecies transmission of M. bovis.