Detailed chronological analysis of microevolution events in herds infected persistently by Mycobacterium bovis

•A refined fingerprinting scheme together with a systematic chronological sampling revealed that microevolution in the infection by M. bovis occurred in half of the chronically infected herds analyzed.•Emergence of clonal variants did not require long infection periods or a high number of infected animals in the herd.•The genetic locations of the subtle genotypic variations recorded in the clonal variants indicated potential functional significance.•Some of the variants, once emerged, outcompeted the original strains, suggesting an advantageous phenotype.•Our data constitute a first step in defining the thresholds of variability to be tolerated in molecular epidemiology studies of M. bovis.

Various studies have analyzed microevolution events leading to the emergence of clonal variants in human infections by Mycobacterium tuberculosis. However, microevolution events in animal tuberculosis remain unknown. We performed a systematic analysis of microevolution events in eight herds that were chronically infected by Mycobacterium bovis for more than 12 months. We analyzed 88 animals using a systematic screening procedure based on discriminatory MIRU-VNTR genotyping at sequential time points during the infection. Microevolution was detected in half of the herds studied. Emergence of clonal variants did not require long infection periods or a high number of infected animals in the herd. Microevolution was not restricted to strains from specific spoligotypes, and the subtle variations detected involved different MIRU loci. The genetic locations of the subtle genotypic variations recorded in the clonal variants indicated potential functional significance. This finding was consistent with the dynamics of some clonal variants, which outcompeted the original strains, suggesting an advantageous phenotype. Our data constitute a first step in defining the thresholds of variability to be tolerated in molecular epidemiology studies of M. bovis. We could therefore ensure that related clonal variants emerging as a result of microevolution events are not going to be misinterpreted as unrelated isolates.