Systems epidemiology to devise new interventions for multi-host tuberculosis

Animal tuberculosis (TB) is an infectious disease of livestock and wildlife mainly caused by Mycobacterium bovis and, in a minor extent, Mycobacterium caprae. In Portugal, animal TB is maintained in a multi-host system involving livestock and wild ungulates. The understanding of the processes driving transmission at this interface is key to inform control. In this work, M. caprae isolates (n=55) from Portugal were characterized by spoligotyping and MIRU-VNTR, supporting the clonal structure, co-infection and in vivo microevolution of this ecotype. M. bovis (n=948) from cattle, red deer and wild boar from TB hotspot regions were genotyped. Bayesian inference identified five ancestral populations and associated the most probable ancient M. bovis subpopulation with cattle and Beja, providing clues on the epidemics origin. A multinomial spatiotemporal probability model identified two significant TB clusters: one that persisted in 2004-2010 with Barrancos (Beja) at the centre, highlighting a significant higher risk associated to cattle; a second cluster, predominant in 2012-2016, holding the county Rosmaninhal (Castelo Branco) at the centre, for which wild boar contributed the most in relative risk. Whole-genome sequences (WGS) of 44 representative M. bovis distinguished five genetic clades and supported sustained transmission and multiple introductions in this multi-host system. Exploratory evolutionary analysis gave further support to pathogen transition between different hosts. Comparative genomics applied to M. bovis (n=70) representing the global clonal complex diversity predicted an open pan-genome and showed diversification of discrete subpopulations through core and accessory genomes. Consistent non-synonymous SNPs illustrated clade-specific virulence landscapes correlating with disease severity. Positive selection and weaker effects of recombination compared with mutation were evidenced as predominant evolutionary forces. Altogether, our results provide novel evidence on the population structure and evolution of M. caprae and M. bovis, delivering insights that could be used to inform adaptive TB control choices in different hosts and regions