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 10 7 CFU of M. bovis BCG (n = 11); 1 SC dose of 10 7 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. Published by Elsevier Ltd.

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(48) and Zairians living in Belgium (35) compared to control individuals with less pigmented skin, presumably due to diminished synthesis. Tuberculosis, likewise, is common among individuals with heavily pigmented skin that relocate from equatorial regions to higher latitudes, in part due to deficiencies in vitamin D synthesis within the skin (65). In addition, patients with untreated tuberculosis often have lower concentrations of 25(OH)D 3 in plasma than do healthy subjects, and tuberculosis tends to occur during the winter when exposure to sunlight is reduced and production of cholecalciferol within the skin is diminished (16). Evidence for a clear correlation between vitamin D deficiency and susceptibility to tuberculosis, however, remains controversial. In vitro studies, however, provide more compelling evidence linking vitamin D status to susceptibility to tuberculosis.Addition of 1,25(OH) 2 D 3 to monocyte-macrophage cultures infected with Mycobacterium tuberculosis suppresses bacterial growth and viability (17,53,54). The mechanism of this suppression is mediated, at least partially, by nitric oxide (NO) (53). Induction of inducible NO synthase of macrophages and subsequent generation of reactive nitrogen intermediates (RNI) toxic to mycobacteria is a potent mechanism of killing (14,15,21,22,34). Cytokines (e.g., tumor necrosis factor alpha and gamma interferon [IFN-␥]) from antigen-specific T cells and/or from macrophages stimulated directly with mycobacterial antigens is responsible for RNI-mediated antimycobacterial defense (24,60). Production of RNI is crucial for controlling acute as well as latent infections in the mouse model of virulent M. tuberculosis infection (14,15,25,34). The role of RNI in mycobacterial killing within human macrophages is less clear. Alveolar macrophages of tuberculosis patients express high levels of inducible NO synthase, suggesting a role for RNI in disease pathogenesis and/or host defense (42). Nevertheless, recent evidence suggests that human but not mouse macro-* Corresponding author. Mailing address: United States Department

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Current assays used to detect Mycobacterium bovis infection lack accuracy, especially for recently infected animals, or are impractical for rapid field diagnostic applications. To overcome these limitations with serological assays, a synthetic peptide derived from early secretory antigenic target 6 (ESAT6-p) and a recombinant major secreted immunogenic protein (rMPB70) of M. bovis were used in an enzyme-linked immunosorbent assay (EIA), an immunochromatographic assay (ICGA), and a latex bead agglutination assay (LBAA). Sera from noninfected, M. bovis-infected, or M. avium subsp. Bovine tuberculosis (bTB) is caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex. bTB is one of the important bovine diseases causing great economic loss worldwide (21, 30). These are also diseases of concern for other ruminant species and humans (8, 21). The presence of M. bovis in wild-animal reservoirs (21,23,32,38,49) has also made control of the diseases more difficult.At present, the standard diagnostic assay for bTB is the single intradermal (SID) test (skin test) with purified protein derivative (PPD). There are many limitations to the SID test and other cell-mediated immunity (CMI)-based tests (e.g., gamma interferon [IFN-␥] and lymphocyte proliferation assays) for the diagnosis of bTB. The first limitation is that the delayed-type hypersensitivity response to the antigen has low sensitivity and specificity. It is not possible to determine if the response to PPD is attributable to exposure to M. bovis, M. avium subsp. paratuberculosis, or environmental mycobacteria, e.g., Mycobacterium avium subsp. avium. Second, there is a predominant CMI response during the early phases of infection with low bacillary loads which may be reduced or absent in animals at the advanced stages of disease with high bacterial loads. The animals become anergic to CMI-based tests (18,35,42,46). Third, animals need to be handled twice over a 72-h period to obtain results of the test. Fourth, other CMI-based tests are expensive and require trained staff to perform the tests and interpret the results. Fifth, false positives may result from exposure to environmental mycobacteria and/or vaccination with BCG. Therefore, there is a need to develop sensitive and easy-to-use M. bovis-specific serological tests that can be used to distinguish between animals exposed to M. bovis, M. avium subsp. paratuberculosis, and environmental mycobacteria.

Extensive studies have shown that the current assays used to identify cattle infected with Mycobacterium bovis or Mycobacterium avium subsp. paratuberculosis are not sufficiently sensitive and specific to detect all infected animals, especially animals recently infected with the pathogens. In the present report we show that these limitations might be overcome with a latex bead agglutination assay (LBAA). With the specific immunodominant epitope (ESAT6-p) of M. bovis, we developed an LBAA and enzyme immunoassay (EIA) for that purpose and compared them with the "gold standard" culture method and skin test for their efficacy in detecting bovine tuberculosis. When sera from control healthy cows (n ‫؍‬ 10), M. avium subsp. Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB) (13), and Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease (paratuberculosis [pTB]), are of economic importance worldwide and a potential health hazard for animals and humans (9). Efforts to control these diseases have been difficult because of the lack of effective vaccines and the lack of diagnostic assays that can identify infected animals before the appearance of clinical disease. This has been a major problem in the control of pTB because infected animals begin to shed bacteria in feces and contaminate the environment well before signs of clinical disease. Difficulty in controlling the diseases has also been compounded by the presence of reservoirs of M. bovis and M. avium subsp. paratuberculosis in wild animal reservoirs (9,11,15,17,20). To address these problems, there is a need for a continued effort to develop diagnostic assays with greater sensitivity (Se) and specificity (Sp) that can be used in the field and laboratory, ideally assays that can be formatted for use with multiple species. Since the ESAT6 protein is secreted at an early or active phase of mycobacterial infection but not from M. bovis 22,25,26), synthetic ESAT6 peptides (ESAT6-p) were prepared to establish early specific detection of M. bovis serologically. In this report we show that a latex bead agglutination assay (LBAA) with synthetic peptides containing species-specific mycobacterial epitopes, ESAT6-p, may provide an approach to developing rapid diagnostic assays for bTB and pTB. MATERIALS AND METHODS Animals. (i) M. bovis-infected cattle.Three different groups of bovine sera were used in this study. A herd surveillance program in Korea has been checking for bTB-positive cows among all the cattle in Korea by using skin tests and then following up with cultures from suspect cows at least two times a year. Within 10 days after a positive skin intradermal test reaction (skin thickness, over 5 mm) in this national herd check program, sera were obtained from 49 cows documented to be naturally infected with M. bovis, which was verified by culture of M. bovis from intestinal tissue or nasal and tracheal mucus at the time of necropsy.

In the search for better tools to control bovine tuberculosis, the development of diagnostic tests with improved specificity and sensitivity has a high priority. We chose to search for novel immunodiagnostic reagents. In this study, Rv0899 (outer membrane protein A of Mycobacterium tuberculosis [OmpATb]) was evaluated as a stimulation antigen in a gamma interferon (IFN-␥) release assay to diagnose bovine tuberculosis. OmpATb induced IFN-␥ responses in cattle experimentally infected with M. bovis as early and as persistently as ESAT-6 and CFP-10, the current lead diagnostic antigens. In naturally infected cattle, OmpATb stimulated IFN-␥ production in 22 of 26 animals (85%). Importantly, OmpATb detected a portion of M. bovis-infected cattle which did not respond to ESAT-6 and CFP-10 (five of six cattle). The combined diagnostic sensitivity of OmpATb, ESAT-6, and CFP-10 for a preselected group consisting of naturally infected cattle with an overrepresentation of ESAT-6/CFP-10 nonresponders was 96% (25 of 26 animals). The specificity of OmpATb for uninfected cattle was 100% (27 cattle were tested; 12 of them gave false-positive results with tuberculins). In summary, our results indicate that OmpATb has the potential to enhance the sensitivity of previously described diagnostic tests based on ESAT-6 and CFP-10 and that the combined use of OmpATb, ESAT-6, CFP-10, and other proteins may achieve at least equal sensitivity to that obtained with purified protein derivative, but at a higher specificity. Further studies evaluating the diagnostic performance of OmpATb in combination with other proteins are ongoing.Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), is also responsible for a proportion of human TB cases. Thus, infection of cattle with M. bovis constitutes both a human health hazard and an animal welfare problem, with economic implications in terms of trade restrictions, productivity losses, and massive annual expenditure on bTB eradication programs. The control of bTB is based mainly on a policy of test and slaughter. The persistence of this zoonotic disease combined with the loss of trade and the exponential costs for control justify a need not only for more sensitive but also for more specific diagnostic assays. The occurrence of false-positive results can be attributed, at least in part, to the fact that immune responses to purified protein derivative (PPD) are present not only in animals with TB but also in animals exposed to environmental mycobacteria (reviewed in reference 29). PPDs are prepared by precipitation from heat-killed cultures of mycobacteria and are poorly defined, complex antigens containing many proteins, some of which are shared by different mycobacterial species or even other bacteria. Coinfection of cattle with M. bovis and M. avium subsp. paratuberculosis has been reported to coincide not only with increased numbers of false-positive results in PPD-based diagnostic assays but also with an increased frequency of false-negative results due to a general depression of ce...

The range of susceptible hosts to M. bovis is extremely broad and includes humans, cattle, pigs, carnivores and deer. Deer are widely distributed and indigenous species and can be found in all continents with the exception of Antarctica and Australia. Today, of the approximately 16 genera and over 50 species of the family Cervidae, tuberculosis due to M. bovis is reported in at least 14 species. The route of infection, pathology and transmission; experimental infection; epidemiology; zoonotic concerns; diagnosis; disease control; genetic resistance and susceptibility; and vaccination programmes against tuberculosis in wild and captive deer are discussed in this book chapter.