Revisiting host preference in the Mycobacterium tuberculosis complex: experimental infection shows M. tuberculosis H37Rv to be avirulent in cattle

Abstract

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 10(6) CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-gamma 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.

Figure 1. M. bovis and M. tuberculosis infection induces comparable bovine cellular immune responses.

Panels A and B; Blood was collected at regulated intervals from cattle following experimental infection with either M. bovis (n = 5) or M. tuberculosis (n = 5). PBMCs were isolated and stimulated with bovine-PPD (panel A) or a cocktail of peptides derived from ESAT-6 and CFP-10 (Panel B). The number of antigen-specific cells expressing IFN-γ was measured using an ELISPOT assay. Data for each time point is presented as the mean response ± SEM with the no-antigen response subtracted. The response in the M. tuberculosis infected cattle is shown as open circles with a dotted line, and for the M. bovis infected cattle as closed triangles with a solid line. Panel C; A tuberculin skin-test was performed at week 16 post-infection. The increase in skin induration was measured 72 hr after administration of bovine (black bars) or avian (grey bars) PPD and is presented as the mean ± SEM.

Figure 2. Elevated TNF-α responses in cattle infected with M. bovis.

Blood was collected from cattle following experimental infection with either M. bovis (n = 5) or M. tuberculosis (n = 5). PBMCs were isolated, stimulated with bovine-PPD and the expression of TNF-α measured in the culture supernatant by ELISA. The response in the M. tuberculosis infected cattle is shown as open circles with a dotted line, and for the M. bovis infected cattle as closed triangles with a solid line. Data for each time point is presented as the mean response ± SEM with the no-antigen response subtracted. Significant differences between groups was determined using 2-way ANOVA with Bonferroni post-test analysis (*** p<0.001).

Figure 3. Differential recognition of Rv3879c by M. bovis infected cattle.

Blood was collected from cattle following experimental infection with either M. bovis (n = 5) or M. tuberculosis (n = 5). Whole blood was stimulated with a pool of peptides derived from Rv3879c and expression of IFN-γ was measured by ELISA. The response in the M. tuberculosis infected cattle is shown as open circles with a dotted line, and for the M. bovis infected cattle as closed triangles with a solid line. Data for each time point is presented as the mean response (OD_450nm) ± SEM with the no-antigen response subtracted. Significant differences between groups was determined using 2-way ANOVA with Bonferroni post-test analysis (** p<0.01).