Abstract

Molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought insights into the epidemiology of animal tuberculosis (TB) in Portugal, showing high genotypic diversity of circulating strains that mostly cluster within the European 2 clonal complex. The genetic relatedness of M. bovis isolates from cattle and wildlife have also suggested sustained transmission within this multi-host system. However, while previous surveillance highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric and provided valuable information on the prevalence and spatial occurrence of TB, links at the wildlife-livestock interfaces were established mainly via genotype associations. Therefore, evidence at a local fine scale of transmission events linking wildlife hosts and cattle remains lacking. Here, we explore the advantages of whole genome sequencing (WGS) applied to cattle, red deer and wild boar isolates to reconstruct the evolutionary dynamics of M. bovis and to identify putative pathogen transmission events. Whole genome sequences of 44 representative M. bovis isolates, obtained between 2003 and 2015 from three TB hotspots, were compared through single nucleotide polymorphism (SNP) variant calling analyses. Consistent with previous results combining classical genotyping with Bayesian population admixture modelling, SNP-based phylogenies support the branching of this M. bovis population into five genetic clades, three with geographic specificities, as well as the establishment of a SNPs catalogue specific to each clade, which may be explored in the future as phylogenetic markers. The core genome alignment of SNPs was integrated within a spatiotemporal metadata framework to reconstruct transmission networks, which together with inferred secondary cases, further structured this M. bovis population by host species and geographic location. WGS of M. bovis isolates from Portugal is reported for the first time, refining the spatiotemporal context of transmission events and providing further support to the key role of red deer and wild boar on the persistence of animal TB in this Iberian multi-host system.