Mycobacterium bovis with different genotypes and from different hosts induce dissimilar immunopathological lesions in a mouse model of tuberculosis

Abstract

With 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)-gamma and inducible nitric oxide synthase (iNOS). In contrast, the more virulent strains induced lower expression of IFN-gamma, tumour necrosis factor-alpha 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.

Fig. 2

Representative mouse lung histopathology after 1 month of infection with selected Mycobacterium bovis strains with different genotypes and isolated from diverse hosts. (a) Reference strain AN5 induced large granulomas (arrows), moderate pneumonic patches and abundant perivascular inflammation. (b) In contrast, animals infected with the high virulent strain 04-303 showed massive pneumonia with extensive necrosis at the same infection day. (c) Mice infected with strain 04-302 also showed extensive pneumonia but mild necrosis. (d) In comparison, the lungs of mice after 4 months of infection with attenuated strain 02-2B showed small patches of pneumonia (all micrographs 100×, haematoxylin and eosin staining).

Fig. 1

Survival, lung bacillary loads and morphometry (% of lung surface area affected by pneumonia and granuloma size) in BALB/c mice infected by an intratracheal injection (2·5 × 10⁵ bacilli) of Mycobacterium bovis with different genotypes and isolated from different hosts. Reference strain AN5 was used as control. Survival curves were constructed with 20 infected mice. Bacillary loads and morphometry curves were performed with four animals per time-point in two different experiments. Asterisks represent statistical significance (P < 0·005) compared with infection with the AN5 strain.

Fig. 3

Expression of cytokine genes determined by real-time reverse transcription–polymerase chain reaction in the infected lungs. BALB/c mice were infected with the indicated Mycobacterium bovis strain and killed at different time-points. The lungs from four different animals at each time-point were used to determine the gene expression of the indicated factor. In comparison with the control strain AN5 (black and white bars), the clinical isolate 04-303 (black bars) induced lower expression of cytokines, inducible nitric oxide synthase (iNOS) and murine beta defensin-4 (mBD-4). Strain 04-302 (grey bars) also induced similar low interferon (IFN)-γ, iNOS and mBD-4 expression but with high interleukin (IL)-4 levels. In contrast, less virulent strains 02-2B (white bars), V2 (diagonal bars) and 534 (hatched bars) induced progressive increase of IFN-γ and iNOS, and high expression of IL-4 and mBD-4 during the first month followed by a sharp decrease during late infection, and constant high expression of tumour necrosis factor-α. Data are the means and standard deviations. Asterisks represent statistical significance (P < 0·05) when compared with mice infected with the AN5 strain.