The principal surveillance tool used to control bovine tuberculosis in cattle is the removal of animals that provide a positive response to the tuberculin skin-test. In this study we performed a longitudinal investigation of the immunological and diagnostic consequences of repeated short-interval skin-tests in cattle naturally infected with Mycobacterium bovis. Tuberculin skin-test positive cattle were subjected to up to four further intradermal comparative cervical skin-tests at approximately 60-day intervals. A significant progressive reduction in the strength of the skin-test was observed after successive tests. In contrast, the magnitude of interferon-γ (IFN-γ) responses was not influenced by repeat skin-testing either transiently around the time of each skin-test or longitudinally following repeated tests. A significant boost in blood interleukin-10 (IL-10) production was observed within 3 days following each skin-test although the magnitude of this boosted response returned to lower levels by day 10 post-test. The application of a novel multiplex assay to simultaneously measure seven cytokines and chemokines also identified that skin-testing resulted in a significant and progressive reduction in antigen specific interleukin-1β (IL-1β) whilst confirming stable IFN-γ and elevated IL-10 responses in the blood. Therefore, we have demonstrated that in cattle naturally infected with M. bovis, repeat short-interval skin-testing can lead to a progressive reduction in skin-test responsiveness which has potential negative consequences for the detection of infected animals with marginal or inconclusive skin-test responses. The desensitising effect is associated with decreased IL-1β and elevated IL-10 responses, but importantly, does not influence antigen specific IFN-γ responses.

Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 106 CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-γ and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97–infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis–infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis of host preference, and in particular the molecular basis of virulence in M. bovis.

Bovine tuberculosis caused by Mycobacterium bovis is a worldwide animal health problem and remains a major threat to public health in the countries in which people live in close contact with their cattle and milk is not pasteurized (7, 11). Early experimental studies (5, 15) suggested that the principal route of M. bovis transmission is most likely to be aerogenous (rather than oral). These experiments indicated that lower doses of M. bovis could be used to infect cattle intranasally compared with the larger doses required when an oral route of delivery was used. Furthermore, tuberculous lesions in the gut, a relatively rare event in naturally infected animals, were common only in cattle that were experimentally infected via the oral route (10, 15). Observations from more recent experimental infections confirmed that infection of cattle via the intranasal route results in pathology that is largely confined to the upper respiratory tract, while intratracheal infection tends to cause lesions in the lower respiratory tract. Naturally infected field reactor cattle most commonly have lesions in the lower respiratory tract and pulmonary lymph nodes, while involvement of the upper respiratory tract occurs more rarely (2,6,17,19,30).Bovine tuberculosis may spread by cattle-to-cattle transmission and also through the involvement of wildlife reservoirs (16). However, experimental intranasal and intratracheal M. bovis infections in cattle have shown that bacterial shedding (the presence of viable M. bovis in the nasal mucus) is, at best, transient and involves extremely low numbers (approximately 70 CFU) of bacilli (14). Such a low dose of M. bovis has not previously been considered relevant in the bovine model of tuberculosis, even though a low infection dose for Mycobacterium tuberculosis in humans has been accepted for many decades (21, 24, 25, 27). These historical studies showed that the numbers of primary calcified M. tuberculosis lesions in otherwise healthy people were low (between one and three lesions per individual) and also established that the infectious dose of M. tuberculosis could be as low as 1 to 10 bacilli.Previous experimental infections of cattle with M. bovis have suggested that the infective dose can have a profound influence on the severity of the disease that follows. For example, in the intranasal model, 5 ϫ 10 5 to 10 6 CFU resulted in multiple respiratory lesions, while 5 ϫ 10 2 to 10 4 CFU resulted in a more variable pathology (some animals had multiple lesions, and some animals had no lesions) and 10 2 CFU resulted in no visible lesions at all. Although the latter group remained skin test negative, M. bovis was isolated from the nasal mucus of one animal 100 days after infection (17). We and other workers have shown that in the intratracheal model low doses of M. bovis (800 to 6 ϫ 10 3 CFU) can result in animals that are skin test negative, have no visible lesions at post mortem, and are M. bovis culture negative (2,4,20,23). Interestingly, Rhodes et al. (23) also measured specific cytokine re...

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