Results Pathogenic isolates of M. bovis differed in their capacity to grow in the cultured macrophages To investigate the mechanisms employed by pathogenic Mbv to modulate MΦ activation, we selected for this study two clinical isolates of Mbv which showed significant difference in capacity of bacteria to grow in MΦ. As shown in Figure 1A, growth kinetics of one of the Mbv isolates,

strain B2, was similar to that of the reference Mtb strain H37Rv. In contrast, the Mbv strain MP287/03 grew in MΦ significantly faster (p < 0.001). After six days of incubation, an increase in the numbers of intracellular bacteria was 3-fold higher in cultures infected by the strain MP287/03, than those infected by strain B2. In contrast to the intracellular growth, growth rate of the tested

strains in specific Middlebrook 7H9 media was similar, demonstrating that screening assay the intrinsic abilities of the different strains to replicate were similar (Figure 1B). These data suggested that the observed differences in intracellular growth of these bacteria could be associated with differential resistance of the bacterial strains to microbicidal effects of MΦ. Figure 1 Evaluation of the growth properties of M. bovis isolates. Isolates obtained from animals with tuberculosis, strains MP287/03 and B2, and reference M. tuberculosis strain H37Rv, were used for infection of BMDM in Belnacasan vitro (A) or Fossariinae cultured in Middlebrook 7H9 broth (B). Growth rates of mycobacteria inside MΦ infected at MOI of 1 were determined using the colony count method. Intracellular CFU numbers were quantified immediately after infection (day 0) or at 3 or 6 days after infection (A). Growth rates of mycobacteria in 7 H9 Middlebrook broth were monitored by measurement of OD of the mycobacterial cultures by spectrophotometry. The growth curves of the mycobacterial strains within a 12 day period of incubation are presented. (B). Values are the means ± SD of three

independent experiments with samples in triplicate. The main cytokines regulating proinflammatory MΦ activity, IFN-γ [16] and IL-10 [17], are known to increase or decrease the bactericidal functions of these cells, respectively. To verify whether intracellular survival of the different mycobacterial strains are equally regulated by the effects of IFN-γ and IL-10 on MΦ, we tested intracellular growth rates of the studied bacterial strains in BMDM cultured in the presence of these cytokines. As shown in Figure 2, the treatment of macrophage cultures with recombinant IL-10 had no significant effect on the growth of the studied strains. Treatment with IFN-γ significantly reduced the growth rate of the strains B2 and H37Rv, but this effect was less pronounced in the cell cultures infected with the strain MP287/03.