Population structure of Mycobacterium bovis in Germany: A long-term study using Whole Genome Sequencing combined with conventional molecular typing methods

Mycobacterium bovis (Mbov) is the primary cause of bovine tuberculosis (bTB), and also infecting a wide range of domestic animal and wildlife species and humans. In Germany, bTB still emerges sporadically in cattle herds, free-ranging wildlife, diverse captive animal species, and humans. In order to understand the underlying population structure and estimate the population size fluctuation through time, we analyzed 131 Mbov strains from animals (n = 38) and humans (n = 93) in Germany from 1999 to 2017 by whole genome sequencing (WGS), MIRU-VNTR typing, and spoligotyping. Based on WGS data analysis, 122 out of the 131 Mbov strains were classified into 13 major clades, six contained strains from both human and animal cases, and seven only from human cases. Bayesian analyses suggest that the Mbov population went through two sharp anticlimaxes, one in the middle of the 18th century and another one in the 1950's. WGS based cluster analysis grouped 46 strains into 13 clusters ranging in size from 2-11 members and involving strains from distinct host types, e.g. only cattle, and also mixed hosts. Animal strains of four clusters were obtained over a nine-year time span, pointing towards autochthonous persistent bTB infection cycles. As expected, WGS had a higher discriminatory power than spoligotyping and MIRU-VNTR typing. In conclusion, our data confirm that WGS and suitable bioinformatics is the method of choice to implement a prospective molecular epidemiological surveillance of Mbov. The population of Mbov in Germany is diverse, with subtle, but existing interactions between different host groups.