In a previous study carried out in a murine model of tuberculosis, different M. bovis strains showed wide virulence variability . In that work, the strain M. bovis 04-303 isolated from a wild boar in Argentina showed to be the most virulent, killing the mice soon after infection and producing high lung bacillary loads and extensive pneumonia accompanied by massive necrosis. Similar results of survival and bacillary loads were obtained in another work with the hypervirulent M. tuberculosis Beijing strain 9501000 . The Beijing strain, however, did not produce necrosis as massive as that observed with M. bovis 04-303 .
In this study, we assessed the virulence of the wild boar M. bovis isolate and compared it with that of the M. bovis NCTC 10772 in a cattle model of infection. This M. bovis NCTC 10772 collection strain was selected as a virulent control strain based on previous findings demonstrating that this strain is capable of replicating in mouse organs  and producing lesions compatible with tuberculosis in cattle after intratracheal inoculation of 109 CFU (data not shown). In our conditions, experimental inoculation produced fewer lesions as observed by other authors [18, 19]. We think that this situation is caused by non tuberculous mycobacteria exposure and helminth infections that provoked an increased resistance to M. tuberculosis complex pathogenesis, as postulated by other authors .
Detectable immune responses and reactivity in skin tuberculin test were observed in both groups of animals after the bacterial infection and throughout the study, indicative of bTB infection. As expected, all the infected animals were IS 6110 PCR-positive in nasal swabs at the end of the experiment. Animals infected with M. bovis 04-303 became positive earlier after inoculation, whereas the M. bovis NCTC 10772-infected animals were positive at 30 dpi. In our knowledge this is the first report on the presence of M. bovis in bovine blood and nasal secretions along the infective cycle. We have previously observed that PCR results to detect M. bovis are variable from animal to animal and in the same animal along time . There are scarce papers about the presence of mycobacteria in blood from cattle. The intermittent M. avium susbp paratuberculosis bacteremia has been reported [22, 23]. These and our results suggest that mycobacteria may dispersed in blood, perhaps on phagocytic cell, as proposed by Juste et al . The detection of M. bovis in nasal swab is expected as it is respiratory pathogen and it has been reported previously [13, 21, 25–27].
ESAT6 elicited high levels of IFN-γ at 15 dpi in animals infected with M. bovis 04-303 and the levels were statistically significantly different compared to those of animals infected with M. bovis NCTC 10772. In the late infection, the stimulation of blood with either ESAT6 or CFP10 recombinant antigens did not show differences in IFN-γ production between animal groups.
Therefore, the presence of lesions compatible with bTB in lungs of infected animals and the early detection of bacilli in blood samples and swabs demonstrate that the M. bovis strains used in this study effectively infected bovines. However, the experimental infection of cattle with the M. bovis 04-303 strain causes lesion in lungs, while infection with M. bovis NCTC 10772 causes lesion only in lymphnodes. The short time period studied and the lower virulence of M. bovis NCTC 10772 could explain the reduced number of animals with lesion in the group infected with this strain. In addition, the outcome of guinea pigs intratracheally inoculated supports the conclusion of the higher virulence of the strain from wild boar.
The results of this study suggest that tuberculosis infection of wild boars is a risk factor associated with the propagation of M. bovis. This hypothesis is supported by the study of Aguilar et al. , who, by using a model of TB in mice, demonstrated the hypervirulent behaviour of two M. bovis strains isolated from wild boars of Argentina, one of which was the M. bovis 04 303 strain. Wild boars are numerous in Argentina, and it is postulated that the presence of a reservoir of bTB in these animals may frustrate efforts to reduce the incidence of bTB by the test and slaughter approach. If this is the case, ways to reduce the burden of disease in these animals will need to be contemplated, like that being done in badgers in Europe and in brushtail possums in New Zealand .