Genetic structure of Mycobacterium avium subsp. paratuberculosis population in cattle herds in Quebec as revealed by using a combination of multilocus genomic analyses

Abstract

Mycobacterium avium subsp. paratuberculosis is the etiological agent of paratuberculosis, a granulomatous enteritis affecting a wide range of domestic and wild ruminants worldwide. A variety of molecular typing tools are used to distinguish M. avium subsp. paratuberculosis strains, contributing to a better understanding of M. avium subsp. paratuberculosis epidemiology. In the present study, PCR-based typing methods, including mycobacterial interspersed repetitive units/variable-number tandem repeats (MIRU-VNTR) and small sequence repeats (SSR) in addition to IS1311 PCR-restriction enzyme analysis (PCR-REA), were used to investigate the genetic heterogeneity of 200 M. avium subsp. paratuberculosis strains from dairy herds located in the province of Quebec, Canada. The majority of strains were of the "cattle type," or type II, although 3 strains were of the "bison type." A total of 38 genotypes, including a novel one, were identified using a combination of 17 genetic markers, which generated a Simpson's index of genetic diversity of 0.876. Additional analyses revealed no differences in genetic diversity between environmental and individual strains. Of note, a spatial and spatiotemporal cluster was evidenced regarding the distribution of one of the most common genotypes. The population had an overall homogeneous genetic structure, although a few strains stemmed out of the consensus cluster, including the bison-type strains. The genetic structure of M. avium subsp. paratuberculosis populations within most herds suggested intraherd dissemination and microevolution, although evidence of interherd contamination was also revealed. The level of genetic diversity obtained by combining MIRU-VNTR and SSR markers shows a promising avenue for molecular epidemiology investigations of M. avium subsp. paratuberculosis transmission patterns.

FIG 1

Rarefaction curves with 95% confidence intervals comparing the numbers of genotypes observed in 66 strains (isolates) from environmental sampling and 114 from individual case sampling.

FIG 2

Geographical distribution of the 64 herds included in the study. Point size is proportional to the number of herds (1 to 4) located in the postal code area. Distribution of MapGnt 16 is illustrated in blue, as are the distributions of the two most common genotypes (MapGnt 5 and MapGnt 6, in red and green, respectively). The large blue dotted line circle represents the significant spatial cluster of MapGnt 16, and the small dotted line blue circle represent herds included in the significant spatiotemporal cluster of MapGnt 16.

FIG 3

Minimum spanning tree based on combined VNTR and SSR profiles of a set of 17 loci from 181 M. avium subsp. paratuberculosis strains. Node sizes are proportional to the number of strains sharing a given genotype (cluster). Each cluster is represented as a uniquely colored and numbered pie chart, where the number of subdivisions illustrates the number of strains. The number of loci differing between the nodes is represented by the style of the connecting lines: thick and short, 1 difference; thin and intermediate length, 2 differences; thin and long, 3 differences.

FIG 4

Minimum spanning tree based on combined VNTR and SSR profiles of M. avium subsp. paratuberculosis strains originating from herds with more than one strain. Node sizes are proportional to the number of strains sharing a given genotype (cluster). A distinct color and its corresponding letter or number placed outside the node represent each herd within a cluster. For each cluster, the number of subdivisions represents the number of strains. The corresponding genotype is indicated inside the node. The number of loci differing between the nodes is represented by the style of the connecting lines: thick and short, 1 difference; thin and intermediate length, 2 differences; thin and long, ≥3 differences.