Whole Genome Sequencing Refines Knowledge on the Population Structure of Mycobacterium bovis from a Multi-Host Tuberculosis System

Abstract

Classical molecular analyses of Mycobacterium bovis based on spoligotyping and Variable Number Tandem Repeat (MIRU-VNTR) brought the first 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. Previous surveillance provided valuable information on the prevalence and spatial occurrence of TB and highlighted prevalent genotypes in areas where livestock and wild ungulates are sympatric. However, links at the wildlife–livestock interfaces were established mainly via classical genotype associations. Here, we apply whole genome sequencing (WGS) to cattle, red deer and wild boar isolates to reconstruct the M. bovis population structure in a multi-host, multi-region disease system and to explore links at a fine genomic scale between M. bovis from wildlife hosts and cattle. 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 apparent geographic specificities, as well as the establishment of an SNP 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 further structure this M. bovis population by host species and TB hotspots, providing a baseline for network analyses in different epidemiological and disease control contexts. WGS of M. bovis isolates from Portugal is reported for the first time in this pilot study, refining the spatiotemporal context of TB at the wildlife–livestock interface and providing further support to the key role of red deer and wild boar on disease maintenance. The SNP diversity observed within this dataset supports the natural circulation of M. bovis for a long time period, as well as multiple introduction events of the pathogen in this Iberian multi-host system.

Use of Whole-Genome Sequencing to Unravel the Genetic Diversity of a Prevalent Mycobacterium bovis Spoligotype in a Multi-Host Scenario in Spain

“…Despite the small sample size included in this study, this picture is in agreement with findings of previous studies in Spain and elsewhere on the potential effect of local risk factors (i.e., spatial proximity, extensive management in beef herds, contact with other sources of infection) in bTB endemic areas (Green et al, 2008;Biek et al, 2012;Pozo et al, 2020). Additionally, our results are in agreement with a previous study conducted in Portugal, where the observed genetic diversity supported the natural circulation of M. bovis for a long time with multiple interactions of different host species (Reis et al, 2021). Interaction with wildlife reservoirs was identified as the second most important cause of herd breakdowns in Spain, after residual infection (Guta et al, 2014a).…”

“…These ambiguous calls occurred in non-informative SNPs (SNPs shared by all the isolates included in the study that were not relevant to define the clustering pattern presented here). Erroneous calls were meticulously verified with IGV, and reliable SNPs were manually corrected as suggested by the vSNP documentation ( Orloski et al, 2018 ) and performed in several studies ( Orloski et al, 2018 ; Salvador et al, 2019 ; Perea et al, 2021 ; Reis et al, 2021 ). In case erroneous calls fell in areas with low coverage or mapping issues these were manually filtered out.…”

IS6110 Copy Number in Multi-Host Mycobacterium bovis Strains Circulating in Bovine Tuberculosis Endemic French Regions

“…This category with a determined function is the most frequent in our analysis. Genes encoding replication, recombination, and DNA repair functions seem to play an important role in the evolution of highly clonal bacteria such as M. bovis ( Vultos et al, 2008 ; Mestre et al, 2011 ; Reis et al, 2021 ). Other frequent categories are “Intracellular trafficking, secretion, and vesicular transport” (7%, 26/369), “Cell wall/membrane/envelope biogenesis” (6%, 22/369), and “Transcription” (6%, 22/369) or “Cell motility” (5%, 20/369).…”

Genome-wide estimation of recombination, mutation and positive selection enlightens diversification drivers of Mycobacterium bovis

“…Mycobacterium bovis isolates dataset. Forty-two newly sequenced M. bovis genomes from an endemic multi-host TB scenario in Portugal (details below), previously characterized from an epidemiological point of view 36 , were at the centre of this work. Considering that the dataset from Portugal only has representatives of European 2 clonal complex and strains without complex assigned, publicly available whole genome sequencing data was added in order to enlarge the dataset with representatives from all M. bovis clonal complexes.…”