Tuberculosis in wild animals

Transcription

1 INT J TUBERC LUNG DIS 14(12): The Union EDUCATIONAL SERIES: MYCOBACTERIUM BOVIS Tuberculosis in wild animals SERIALISED ARTICLE Tuberculosis: a re-emerging disease in animals and humans NUMBER 4 IN THE SERIES J. B. Kaneene,* R. Miller,* I. N. de Kantor, C. O. Thoen * Center for Comparative Epidemiology, Michigan State University, East Lansing, Michigan, USA; World Health Organization Tuberculosis Consultants Panel, Buenos Aires, Argentina; Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, Iowa, USA Serialisation of article Thoen C O, LoBue P A, Enarson D A, Kaneene J B, de Kantor I N. Tuberculosis: a re-emerging disease in animals and humans. In: Kaplan B, Kahn L H, Monath T P, ed. One Health - One Medicine : linking human, animal and environmental health. [Special Issue] Vet Ital 2009; 45: Previous articles in the series LoBue P A, Enarson D A, Thoen C O. Tuberculosis in humans and animals: an overview. Int J Tuberc Lung Dis 2010; 14(9): LoBue P A, Enarson D A, Thoen T C. Tuberculosis in humans and its epidemiology, diagnosis and treatment in the United States. Int J Tuberc Lung Dis 2010; 14(10): de Kantor I N, LoBue P A, Thoen C O. Human tuberculosis caused by Mycobacterium bovis in the United States, Latin America and the Caribbean. Int J Tuberc Lung Dis 2010; 14(11): THERE HAS BEEN INCREASED INTEREST in tuberculosis (TB) due to infection with Mycobacterium tuberculosis complex organisms, including M. tuberculosis and M. bovis, in wild and captive wild animals following outbreaks of the disease in animals in zoos, primate centers, animal colonies, and game parks. The importance of these occurrences of TB is emphasized by the difficulty of replacing some rare and endangered species, by the economic losses, and by the public health hazard. 1 In the United States and in several other countries, M. bovis infection in wild animals has interfered with the eradication of TB in domestic livestock. 1 5 In several areas where bovine TB has been eradicated, it was observed that M. bovis infection in cervids was responsible for infection in cattle. 1,5 In Michigan, M. bovis outbreaks were traced to several cattle herds, resulting in herd depopulation and the loss of valuable blood lines and income related to the export of breeding animals and semen. Estimates indicate that the overall cost of the M. bovis outbreaks in deer and cattle in Michigan was in excess of US$100 million (M J Gilsdorf, personal communication). Efforts to control and eradicate TB in wild animals is hampered by a lack of validated in vivo and in vitro diagnostic tests of suitable sensitivity and specificity, and procedures for handling and restraining animals under field conditions to conduct tuberculin skin tests (TSTs) and/or collect specimens for laboratory examination to confirm a diagnosis, as well as by the lack of suitable regulations to limit the sale and transportation of infected or exposed animals. The criteria for conducting available diagnostic tests, such as TSTs, gamma interferon assays, enzyme-linked immunosorbent assays (ELISA), and immunochromatographic tests in wildlife have not been systematically evaluated in many species, but positive tuberculin reactions usually indicate infection or previous exposure to mycobacteria. Therefore, standardized approaches need to be developed to effectively identify and eradicate M. bovis infection in wild animals. 1 DISTRIBUTION Some early reports of TB describe observations of the disease in wild animals in zoos or animal parks. 1 In view of the reduction of TB in domestic animals and humans, one might expect a decrease in the disease in zoo animals. However, there is evidence to indicate that foci of infection may persist in some wild animal populations for long periods of time without the presence of livestock reservoirs. 1,2,6 In many countries there are no regulations that require reporting of TB outbreaks in wild or captive wild animals, and in many instances necropsies are not conducted. In addition, there is often a reluctance to publicize the occurrence of TB in these animals due to public relations impacts and loss of revenue. Tuberculosis in wild animals is most often reported in cases where they have been exposed to domestic animals or to humans that have disease. The majority of these are considered spill-over cases, where infection is present but not sustainable in the wildlife population. When TB in cattle in the same geographic Correspondence to: John B Kaneene, Center for Comparative Epidemiology, Michigan State University, East Lansing, MI 48824, USA. kaneene@cvm.msu.edu

2 Tuberculosis in wild animals 1509 areas was eliminated or reduced, comparable declines in the prevalence in wildlife were also seen. Tuberculous humans have been the source of M. tuberculosis infection in Asian elephants (Elephas maximus), while M. tuberculosis has caused TB in non-human primates. M. tuberculosis has been isolated from captive wild animals: black rhinoceros (Diceros bicornis), Arabian oryx (Oryx leucoryx), addax (Addax nasomaculatus), Asian elephants, and Rocky Mountain goats (Oreamnos americanus), among others. Several other species of the genus Mycobacterium have been isolated from wild animals maintained in captivity. The widespread occurrence of outbreaks of mycobacterial infections is of concern to public health officials and to veterinarians responsible for the health care of wild animals in zoos, animal parks and primate colonies. Several species of wildlife are now recognized as reservoirs of M. bovis. The main wildlife reservoirs for M. bovis are ungulates: bovid reservoirs include African buffalo (Syncerus caffer), wood bison (Bison bison athabascae) and North American bison (Bison bison). 6 Several species of cervids susceptible to M. bovis, such as the white-tailed deer (Odocoileus virginianus), mule deer (Odocoileus hemonius), lechwe (Kobus lechwe), and red deer/elk/wapiti (Cervus elaphus) subspecies, are recognized as reservoirs for M. bovis. 1,7 13 European badgers (Meles meles) are a reservoir species in the United Kingdom, 1,11,14 and brushtail possums (Trichosurus vulpecula) are a recognized reservoir in New Zealand. 1,4 Feral swine and wild boar (Sus scrofa) were once considered to be spill-over hosts for M. bovis; however, mounting evidence in Europe indicates that wild boar have now become reservoirs of M. bovis for other wildlife and domestic animal species. 1,7,12 In areas where more than one wildlife reservoir species is present, a multi-host system of M. bovis transmission may develop, making disease control more complex than dealing with a single wildlife reservoir. 2,4 Other wildlife species reported with M. bovis infection have a potential to be reservoirs of M. bovis, but have not been implicated as reservoirs of the disease. Among these are free-ranging axis deer (Axis axis) in Hawaii, fallow deer (Dama dama), 1,7 roe deer (Capreolus capreolus), 1,11,12 greater kudu (Tragelaphus strepsiceros), llama (Llama glama), muntjac (Muntiacus spp.), 1,11 giraffe (Giraffa camelopardalis), feral water buffalo (Bubalis bubalis), Arabian oryx, Sika deer (Cervus nippon), tapir (Tapirus terrestris), European wild goat (Capra aegagrus), impala (Aepyceros melampus), sitatunga (Tragelaphus spekii), Bactrian camel (Camelus ferus), wildebeest (Connochaetes spp.), lesser kudu (Tragelaphus imberbis), topi (Damalisus korrigum), yak (Bos grunniens), eland (Taurotragus spp.), bush pig (Potamochoerus porcus), and warthogs (Phacochoerus africanus). Carnivores and scavengers can acquire M. bovis under natural conditions through the consumption of infected carcasses. These species include the fennec fox (Fennecus zerda), coyote (Canis latrans), wolf (Canis lupus), fox (Vulpes vulpes), lion (Panthera leo), tiger (Panthera tigris), leopard (Panthera pardus), cheetah (Acinonyx jubatus), snow leopard (Uncia uncia), Iberian lynx (Lynx pardinus), bobcat (Felix rufus), hyena (Crocuta spp.), large spotted genet (Genetta tigrina), raccoon (Procyon lotor), black bear (Ursus americanus) and opossum (Didelphis virginiana), mustelids (Mustela spp.), stoats (Mustela erminea), ferrets (Mustela furo), and meerkats (Suricata suricatta). 1,7,11 15 M. bovis has been isolated from moles (Scalopus aquaticus), voles (Clethrionomys spp., Microtus spp.), rats (Rattus spp.), yellow-necked and wood mice (Apodemus spp.), grey squirrel (Sciurus carolinensis), hedgehogs (Erinaceous europaeus), hyraxes (Procavia capensis), rabbits (Orytolagus cuniculus cuniculus), hares (Lepus europaeus), white rhinoceros (Ceratotherium simum), primates, including a Siamang gibbon (Symphalangus syndactylus), Mayotte lemur (Lemur fulvus mayottensis), lion-tailed macaque (Macaca silenus), Patas monkey (Erythrocebus patas), Colobus monkeys (Colobus guereza caudatus), baboon (Papio spp.), otters (Lutra lutra), and Arctic marine mammals. 1,11 Tuberculosis caused by other members of the M. tu berculosis complex has been identified in several wildlife species. Tuberculosis due to M. pinnipedii, a new species within the M. tuberculosis complex, has been observed in fur seals (Arctocephalus spp.) and sea lions (Neophoca cinerea). 16 M. caprae, originally described as causative agent of TB in goats, also affects cattle in several European countries and has been recovered from red deer, wild boar, dromedary camel (Camelus dromedarius) and North American bison. M. africanum, commonly found in West African countries, can affect cattle, humans and other mammalian species. It has been isolated from monkeys and hyrax (Procavia capensis). 17 M. microti typically causes disease in voles, wood mice, and shrews. It has also been isolated from llama, cats, pigs, rock hyrax (P. capensis), ferrets, wild voles (Microtus agrestis) and wood mice (Apodemus sylvaticus). 1 AETIOLOGY Tubercle bacilli grow aerobically on in vitro culture. Pyruvate, when added to culture medium, enhances the growth of M. bovis, whereas glycerol inhibits it. Automated liquid culture methods (e.g., BACTEC, MGIT960) provide for more rapid detection than standard culture methods, which can take months for mycobacterial growth. Identification of mycobacterial isolates is accomplished by the use of appropriate biochemical, drug susceptibility and supplemental tests or by molecular techniques. Gene probes have

3 1510 The International Journal of Tuberculosis and Lung Disease been used to confirm M. tuberculosis complex organisms from wildlife samples. Polymerase chain reaction (PCR) conducted on formalin-fixed tissues and/ or cultures can provide for rapid identification of M. tuberculosis complex, but has met with limited success with gross lesions from some wildlife species. 1,10,12 TRANSMISSION AND RESISTANCE Infection with M. tuberculosis complex organisms can produce extensive disease involving the parenchyma of the lung as well as extra-pulmonary tissues. When animals with advanced disease cough, the organism may be transmitted by aerosol or droplets of exudate containing the bacilli. Animals may also be infected by ingestion of feed and water contaminated with urine, fecal material, or exudates from diseased animals that contain tubercle bacilli, and this has been found to be a critical route for inter-species transmission of M. bovis. 1,3,5,11 Fomites, such as thermometers, are a definite source of spread, as are cages, masks, and containers used for food and water. 3 Biting is another route of infection; in a study of TB in European badgers under a routine culling program, adult badgers with M. bovis were four times more likely to have bite wounds than non-tuberculous adults, and badgers with bite wounds were more likely to have tuberculous lesions in multiple body parts than tuberculous badgers with no bite wounds. 14 CLINICAL SIGNS It is important to emphasize that clinical signs are only rarely apparent in wild animals. The extensiveness of disease is related to the virulence of the organisms, the route of infection, the stage of infection, and several host-related factors. The obvious difficulty with observing and handling animals in the wild, in farmed herds, or in confined colonies, is that animals with TB remain in these populations when no signs of disease are present. It is essential to conduct suitable diagnostic procedures, including delayed type hypersensitivity and in vitro tests, while animals are held in isolation prior to introduction into a herd or exhibit. PATHOLOGY Wild mammals found to be tuberculous at necropsy after natural death are often without prior suspicion of TB. Gross lesions may be extensive, involving entire organs of one or both body cavities; however, the anatomical sites of lesions, the extent of pathological involvement and the consistency of nodular formations with some caseous necrosis are often present before failure to thrive is apparent. 1 In several instances, M. bovis has been cultured from specimens with no visible lesions. 1,12,17 Tuberculous lesions observed at necropsy usually have an appearance of yellowish caseous necrotic areas in nodules of firm, white to light gray fibrous tissue. Tubercles may not appear discrete in instances where lesions become diffuse with existing tissues. Other bacteria and agents are often present within tuberculous lesions, which may affect the gross appearance. Some lesions observed in lymph nodes of the head, particularly the medial retropharyngeal nodes or thoracic cavity of cervids, may have a purulent consistency, whereas others may be partially dry. Microscopically, tuberculous lesions from camelids and wild bovines closely resemble those in other bovidae. Baboons and several species of monkeys usually demonstrate microscopic similarities to those in other wild mammals. Histologically, tubercles from nonhuman primates infected with M. bovis do not differ significantly from lesions caused by M. tuberculosis. DIAGNOSIS Ante-mortem diagnostic tools Tuberculin skin testing The TST has been used successfully in captive and free-ranging cloven-hoofed animals, non-human primates, and other zoo animal species. Purified protein derivative (PPD) from M. tuberculosis, M. bovis, or M. avium (depending on the mycobacterial species of interest) is injected subcutaneously into a selected site on the animal to be tested, and the site is observed for induration at 24, 48, and 72 h. In situations where infection with M. avium complex organisms can result in false positives, comparative TSTs are conducted: using biologically balanced M. avium PPD and M. bovis PPD tuberculin injected at separate sites, comparison of responses from each site enables sensitization due to M. tuberculosis complex to be differentiated from that due to M. avium complex and other acid-fast organisms. 18 However, the sensitivity and specificity of these tests in some species may vary from those seen in domestic cattle. Tuberculin skin tests in bovines are most often conducted in the cervical region; in camelids, the tests are conducted in the axillary region, just behind the front leg. The TST for the diagnosis of TB in non-human primates is administered to the upper eyelid for a single PPD injection, or at separate sites on the abdomen lateral to the linea alba for the comparative TST. Adequate dosage is an important factor in performing TSTs in different species, and the PPD tuberculins used must be standardized in the species to be tested. Factors that may influence TST responsiveness include desensitization following repeated tuberculin skin testing and infection with certain viruses, such as rubeola. Blood and serological tests Blood and serological tests have some advantages over TSTs, and there is considerable research into the

4 Tuberculosis in wild animals 1511 development of new tests that should be rapid, easy to use, and highly sensitive and specific. 9,19,20 In vitro cellular techniques using specific mycobacterial antigens, such as the gamma interferon a ssay, are designed to measure cell-mediated immunity and are a promising test modality. These tests have an advantage in that the immune status of the animal is not altered by repeated testing, and they have been used successfully for the detection of M. bovis in several wildlife species. Other new approaches for in vitro testing for bovine TB are being developed, including microarray analysis of gene expression profiles, real-time reverse transcription-pcr (RT-PCR) to detect gamma interferon mrna in infected animals, and immunoblot assays for M. bovis antigens. 3,8,20 22 Serological tests to detect mycobacterial antibodies in wild animals with disease have been developed but do not permit definitive diagnoses of TB in certain animals, due to less-than-optimal test sensitivity and their inability to detect animals in the early stages of infection. 1,9 However, their ability to detect antibodies after markers of cell-mediated immunity have elapsed can be useful, particularly in conjunction with other mycobacterial tests. 1,20,21 The potential use of multiple antigens in detecting TB in wild animals is an emerging area of research. Rapid chromatographic immunoassays for the detection of M. bovis antibodies in wildlife serum, including tests such as the multiple antigen print immunoassay (MAPIA) and lateral flow rapid tests, are faster and easier to use in the field than the currently accepted skin tests, and do not have the same laboratory facility requirements for lymphocyte stimulation assays. These rapid tests are showing good results in livestock but are not sufficiently sensitive for use in wildlife surveillance programs unless accompanied by other tests. 15,19 21,23,24 Genotyping techniques are useful in conducting epidemiological investigations to obtain information on the source(s) of M. tuberculosis complex infection. 1,10,12 Restriction fragment length polymorphism (RFLP) and spoligotyping have been extensively applied in strain differentiation of M. tuberculosis and M. bovis. Mycobacterial interspersed repetitive unit variable number of tandem repeats (MIRU-VNTR) analysis, when used alone or in combination with other genomic analyses, may lead to a greater differentiation of M. bovis strains for use in epidemiological investigations, and online genomic databases of these strains have become available for use by researchers and diagnosticians. 1,7,12 Post-mortem diagnostic tools Slaughter surveillance is useful in some animals, such as cervids, to identify gross typical TB lesions. Tissues should be collected for culture or PCR whenever possible, because this is the only way to confirm a diagnosis of TB. PCR and RFLP are currently available in reference diagnostic laboratories in several countries. Histopathologic examination of tissue collected at necropsy or on biopsy is useful to establish a TB diagnosis, and has been used to establish the prevalence of mycobacterial infection in different wildlife species. 1,11,12 Lesions may vary for different animals; however, typical lesions are usually characterized by the presence of epithelioid cells and multinucleated giant cells. Caseation necrosis and mineralization may be present. PREVENTION AND CONTROL In instances where the reservoir of TB for wildlife is domestic cattle, control and eradication of the disease in cattle has reduced spill-over into susceptible wildlife species. Conversely, measures to reduce interaction between wildlife and livestock, either by increasing biosecurity measures to keep wildlife off farms, or by reducing factors that attract wildlife onto farms, have reduced disease transmission risk for both domestic and wild animals. 5,13 Research on vaccines for potential use in wildlife is ongoing, but problems exist regarding the efficacy of vaccines to prevent disease. Different vaccine strains of M. bovis (M. bovis bacille Calmette-Guérin [BCG]) result in differing levels of protection, and some animals shed BCG after vaccination, which can hamper surveillance and control efforts. 25 Moreover, guidelines have not been developed for effective vaccine delivery systems for wildlife. Wild animals acquired for zoos, animal parks, or primate colonies should come from sources known to be free of disease. Quarantines should be imposed on imported animals; the period of quarantine should be a minimum of 60 days and preferably 120 days, but in some instances currently accepted quarantine periods for some species may not be sufficient to detect infected animals. 17 When TB is diagnosed in an animal colony or holding facility, it is necessary to thoroughly clean and decontaminate the facilities, preferably by using substituted cresylic acid preparations. Control of TB in wildlife populations is extremely difficult when there is a wildlife reservoir of disease. The disease is most common among animals kept in close contact in exhibit pens or barns, or in freeranging wildlife where large numbers of animals come into close contact, such as when wildlife compete for limited food resources or where supplemental feeding is practiced. Efforts have been directed at reducing levels of disease in the wildlife species, reducing wildlife population numbers, and keeping infected animals from potential hosts. 1,2,4,13 Treatment of infected wildlife has been limited to animals in captivity. Anti-tuberculosis drugs used in humans, such as isoniazid and rifampin, or ethambutol, have been used to treat disease in elephants,

5 1512 The International Journal of Tuberculosis and Lung Disease monkeys, oryx, addax, giraffes and great apes in captivity. The dosage and period of time for which animals are maintained on drugs appear to be variable to obtain and maintain suitable blood levels to inhibit tubercle bacilli. As exacerbations may occur, it is necessary to periodically evaluate the health status of the animal(s), (e.g., trunk washes). Therefore, treatment is not recommended for animals that cannot be monitored. CONCLUSIONS Wild animals can serve as reservoirs and sources of tuberculosis infections to humans and livestock, resulting in both public health and economic costs. The bacteria of the M. tuberculosis complex have an extremely wide host range, their pathogenicity varies greatly from species to species, ante-mortem diagnosis is complicated, and the transmission of mycobacteria between domestic and wild species makes control in one species contingent on controlling the infection in all potential hosts. In addition, there are problems inherent in disease monitoring and control in freeranging wildlife populations, where handling animals is difficult and there is a lack of information about affected populations in the field. Efforts to control the disease in wildlife need to be strengthened, and areas that need particular attention include improved antemortem diagnostic tests, vaccine development, and improvements to programs to prevent inter-species interaction (including between domestic animals and wildlife) where disease transmission can occur. References 1 Thoen C O, LoBue P A, Enarson D A, et al. Tuberculosis: a reemerging disease in animals and humans. Vet Ital 2009; 45: Gortázar C, Torres M J, Vicente J, et al. Bovine tuberculosis in Doñana Biosphere Reserve: the role of wild ungulates as disease reservoirs in the last Iberian lynx strongholds. PLoS ONE 2008; 3(7): e Palmer M V, Waters W R, Whipple D L. Investigation of the transmission of Mycobacterium bovis from deer to cattle through indirect contact. Am J Vet Res 2004; 65: Porphyre T, Stevenson M A, McKenzie J. Risk factors for bovine tuberculosis in New Zealand cattle farms and their relationship with possum control strategies. Prev Vet Med 2008; 86: Kaneene J B, Bruning-Fann C S, Granger L M, et al. Environmental and farm management factors associated with tuberculosis on cattle farms in northeastern Michigan. J Am Vet Med Assoc 2002; 221: Wobeser G. Bovine tuberculosis in Canadian wildlife: an updated history. Can Vet J 2009; 50: Romero B, Aranaz A, Sandoval Á, et al. Persistence and molecular evolution of Mycobacterium bovis population from cattle and wildlife in Doñana National Park revealed by genotype variation. Vet Microbiol 2008; 132: Cross P C, Heisey D M, Bowers J A, et al. Disease, predation and demography: assessing the impacts of bovine tuberculosis on African buffalo by monitoring at the individual and population levels. J Appl Ecol 2009; 46: Himsworth C G, Elkin B T, Nishi J S, et al. Comparison of test performance and evaluation of novel immunoassays for tuberculosis in a captive herd of wood bison naturally infected with Mycobacterium bovis. J Wildl Dis 2010; 46: Olsen I, Barletta R G, Thoen C O. Mycobacterium. In: Gyles C L, Prescott J F, Songer J G, et al., eds. Pathogenesis of bacterial infections in animals. 4th ed. Ames, IA, USA: Wiley-Blackwell, 2010: pp Delahay R J, Smith G C, Barlow A M, et al. Bovine tuberculosis infection in wild mammals in the South-West region of England: a survey of prevalence and semi-quantitative assessment of the relative risks to cattle. Vet J 2007; 173: Zanella G, Durand B, Hars J, et al. Mycobacterium bovis in wildlife in France. J Wildl Dis 2008; 44: Miller R, Kaneene J B, Schmitt S M, et al. Spatial analysis of Mycobacterium bovis infection in white-tailed deer (Odocoileus virginianus) in Michigan, USA. Prev Vet Med 2007; 82: Jenkins H E, Morrison W I, Cox D R, et al. The prevalence, distribution and severity of detectable pathological lesions in badgers naturally infected with Mycobacterium bovis. E pidemiol Infect 2007; 136: Drewe J A, Dean G S, Michel A L, et al. Accuracy of three diagnostic tests for determining Mycobacterium bovis infection status in live-sampled wild meerkats (Suricata suricatta). J Vet Diagn Invest 2009; 21: Cousins D V, Bastida R, Cataldi A, et al. Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov. Int J Syst Evol Microbiol 2003; 53: Gudan A, Artuković B, Cvetnić Z, et al. Disseminated tuberculosis in hyrax (Procavia capensis) caused by Mycobacterium africanum. J Zoo Wildl Med 2008; 39: Thoen C O, Williams W J, Miller L D, Stackhouse L L, Newcomb B F, Ferrell J M. Mycobacterium bovis infection in North American elk (Cervus elaphus). J Vet Diagn Invest 1992: 4: Chambers M A. Review of the diagnosis and study of tuberculosis in non-bovine wildlife species using immunological methods. Transbound Emerg Dis 2009; 56: O Brien D J, Schmitt S M, Lyashchenko K P, et al. Evaluation of blood assays for detection of Mycobacterium bovis in whitetailed deer (Odocoileus virginianus) in Michigan. J Wildl Dis 2009; 45: Harrington N P, Surujballi O P, Waters W R, et al. Development and evaluation of a real-time reverse transcription-pcr assay for quantification of gamma interferon mrna to diagnose tuberculosis in multiple animal species. Clin Vacc Immunol 2007; 14: Meade K G, Gormley E, Doyle M B, et al. Innate gene repression associated with Mycobacterium bovis infection in cattle: toward a gene signature of disease. BMC Genomics 2007; 8: Gowtage-Sequeira S, Paterson A, Lyashchenko K P, et al. Evaluation of the CervidTB STAT-PAK for the detection of Mycobacterium bovis infection in wild deer in Great Britain. Clin Vacc Immunol 2009; 16: Himsworth C G, Elkin B T, Nishi J S, et al. Comparison of test performance and evaluation of novel immunoassays for tuberculosis in a captive herd of wood bison naturally infected with Mycobacterium bovis. J Wildl Dis 2010; 46: Palmer M V, Thacker T C, Waters W R. Vaccination with Mycobacterium bovis BCG strains Danish and Pasteur in whitetailed deer (Odocoileus virginianus) experimentally challenged with Mycobacterium bovis. Zoonoses Publ Hlth 2009; 56: