The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world.

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SummaryWith the hypothesis that genetic variability of Mycobacterium bovis could influence virulence and immunopathology, five M. bovis strains were selected from an epidemiological study in Argentina on the basis of their prevalence in cattle and occurrence in other species. We then determined the virulence and the immunopathology evoked by these strains in a well-characterized mouse model of progressive pulmonary tuberculosis. The reference strain AN5 was used as a control. BALB/c mice infected with this M. bovis reference strain showed 50% survival after 4 months of infection, with moderate bacillary counts in the lung. Two weeks after inoculation, it induced a strong inflammatory response with numerous granulomas and progressive pneumonia. In contrast, strain 04-303, isolated from a wild boar, was the most lethal and its most striking feature was sudden pneumonia with extensive necrosis. Strain 04-302, also isolated from wild boar but with a different spoligotype, induced similar pathology but to a lesser extent. In contrast, strains 534, V2 (both from cattle) and 02-2B (from human) were less virulent, permitting higher survival after 4 months of infection and limited tissue damage. Strain AN5 and the cattle and human isolates induced rapid, high and stable expression of interferon (IFN)-g and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN-g, tumour necrosis factor-a and iNOS. Interestingly, these more virulent strains induced very low expression of murine beta defensin 4 (mBD-4); whereas, the control strain AN5 induced progressive expression of this anti-microbial peptide, peaking at day 120. The less virulent strains induced high mBD-4 expression during early infection. Thus, as reported with clinical isolates of M. tuberculosis, M. bovis also showed variable virulence. This variability can be attributed to the induction of a different pattern of immune response.

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Paratuberculosis (Ptbc) has a high prevalence in Argentina, that affects dairy and beef cattle. The culture is the gold standard to the diagnosis of the disease. Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis), the aetiological agent, is difficult to isolate and grow in culture. In this study, 24 randomly selected cows of the Fresian breed from a dairy herd with a history of Ptbc were used to evaluate the performance of different diagnostic techniques. These animals did not show clinical signs of the disease. However, another animal from this herd presented evidence of clinical disease at the moment of the present study. This animal was necropsied and one strain of M. paratuberculosis was isolated from faeces, lymph nodes and intestine. Serum for indirect absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) tests and whole blood samples to perform gamma interferon (gammaIFN) release assays were obtained from each animal. Faeces and milk samples to carry out bacteriological cultures, PCR identification of M. paratuberculosis, and direct examinations of smears with Ziehl-Neelsen's (ZN) stain were also collected. Tuberculin test with bovine purified protein derivative (PPD) in the caudal fold was performed. The results showed that 10 out of 24 animals (41.6%) were positive to ELISA. Eight strains of M. paratuberculosis were isolated, six from faeces, two from milk. Five of the animals that excreted the bacteria through faeces were ELISA-positive, whereas the excreters through milk were negative to ELISA. No positive samples by AGID were obtained in clinical asymptomatic animals. Seven samples gave positive gammaIFN results with avian PPD, but only two of these animals were confirmed with culture. Direct PCR, to detect IS900 (M. paratuberculosis) in faeces and milk samples, was negative, but PCR using material taken from faecal and milk cultures gave positive results before visualizing the colonies. No sample was positive by PCR directed to IS6110 (M. tuberculosis complex). There was not always agreement between isolations and ZN in the studied samples. In conclusion, the absorbed ELISA was useful to detect positive animals and excreters through faeces but not through milk. PCR applied to cultures with incipient development before the visualization of colonies was effective to specifically determine the presence of M. paratuberculosis. The gammaIFN test was not able to detect the most positive animals confirmed by culture. The importance of using ELISA and cultures is emphasized by this study but it is necessary to continue with the gammaIFN test development for early detection of the disease.

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Faecal samples from 76 diarrhoeic calves belonging to 36 farms located in the Pampas plain, Argentina, were examined for Shiga toxin-producing Escherichia coli (STEC). A total of 15 STEC strains were isolated from 12 (15.8%) calves which came from six different farms. All stx positive strains assayed by PCR were also positives in the Vero cell cytotoxicity test. The majority (60.0%) of the STEC strains carried the stx(1) gene. Twelve (80.0%) of the STEC isolates which belonged to serotypes O5:H- (n = 4), O26:H11 (n = 4), O26:H- (n = 1), O111:H- (n = 2), and O123:H38 (n = 1) were also enterohaemolysin (EHly) positive and carried the gene encoding for intimin (eae). All the stx positive strains were negative for the bfpA gene. Localized adherence to HEp-2 cells were observed in 83.3% of the eae+ STEC strains. STEC belonging to serotype O5:H- showed atypical biochemical properties, including urease production. Urease was also produced by two strains belonging to serotypes O153:H? and non-typeable, respectively. Resistance to three or more antibiotics was observed in 12 (80.0%) of the STEC isolates. Most of the serotypes of STEC recovered in this survey carried virulence traits that are associated with increased human and bovine pathogenicity. The present study shows that highly virulent STEC strains are being shed by diarrhoeic calves from farms located in a high incidence area of human STEC infections.