Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen responsible for paratuberculosis or Johne’s Disease (JD) in ruminants, which is responsible for substantial economic losses worldwide. MAP transmission primarily occurs through the fecal-oral route, and the introduction of an MAP infected animal into a herd is an important transmission route. In the current study, we characterized MAP isolates from 67 cows identified in 20 herds from the provinces of Quebec and Ontario, Canada. Whole genome sequencing (WGS) was performed and an average genome coverage (relative to K-10) of ∼14.9 fold was achieved. The total number of SNPs present in each isolate varied from 51 to 132 and differed significantly between herds. Isolates with the highest genetic variability were generally present in herds from Quebec. The isolates were broadly separated into two main clades and this distinction was not influenced by the province from which they originated. Analysis of 8 MIRU-VNTR loci and 11 SSR loci was performed on the 67 isolates from the 20 dairy herds and publicly available references, notably major genetic lineages and six isolates from the province of Newfoundland and Labrador. All 67 field isolates were phylogenetically classified as Type II (C-type) and according to MIRU-VNTR, the predominant type was INMV 2 (76.1%) among four distinct patterns. Multilocus SSR typing identified 49 distinct INMV SSR patterns. The discriminatory index of the multilocus SSR typing was 0.9846, which was much higher than MIRU-VNTR typing (0.3740). Although multilocus SSR analysis provides good discriminatory power, the resolution was not informative enough to determine inter-herd transmission. In select cases, SNP-based analysis was the only approach able to document disease transmission between herds, further validated by animal movement data. The presence of SNPs in several virulence genes, notably for PE, PPE, mce and mmpL, is expected to explain differential antigenic or pathogenetic host responses. SNP-based studies will provide insight into how MAP genetic variation may impact host-pathogen interactions. Our study highlights the informative power of WGS which is now recommended for epidemiological studies and to document mixed genotypes infections.

Mixed strain infection (MSI) refers to the concurrent infection of a susceptible host with multiple strains of a single pathogenic species. Known to occur in humans and animals, MSIs deserve special consideration when studying transmission dynamics, evolution, and treatment of mycobacterial diseases, notably tuberculosis in humans and paratuberculosis (or Johne's disease) in ruminants. Therefore, a systematic review was conducted to examine how MSIs are defined in the literature, how widespread the phenomenon is across the host species spectrum, and to document common methods used to detect such infections. Our search strategy identified 121 articles reporting MSIs in both humans and animals, the majority (78.5%) of which involved members of the Mycobacterium tuberculosis complex, while only a few (21.5%) examined non-tuberculous mycobacteria (NTM). In addition, MSIs exist across various host species, but most reports focused on humans due to the extensive amount of work done on tuberculosis. We reviewed the strain typing methods that allowed for MSI detection and found a few that were commonly employed but were associated with specific challenges. Our review notes the need for standardization, as some highly discriminatory methods are not adapted to distinguish between microevolution of one strain and concurrent infection with multiple strains. Further research is also warranted to examine the prevalence of NTM MSIs in both humans and animals. In addition, it is envisioned that the accurate identification and a better understanding of the distribution of MSIs in the future will lead to important information on the epidemiology and pathophysiology of mycobacterial diseases.

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne’s Disease (JD) in ruminants, which is responsible for significant economic loss to the global dairy industry. Mixed strain infection (MSI) refers to the concurrent infection of a susceptible host with genetically distinct strains of a pathogen, whereas within-host changes in an infecting strain leading to genetically distinguishable progeny is called microevolution. The two processes can influence host-pathogen dynamics, disease progression and outcomes, but not much is known about their prevalence and impact on JD. Therefore, we obtained up to 10 MAP isolates each from 14 high-shedding animals and subjected them to whole-genome sequencing. Twelve of the 14 animals examined showed evidence for the presence of MSIs and microevolution, while the genotypes of MAP isolates from the remaining two animals could be attributed solely to microevolution. All MAP isolates that were otherwise isogenic had differences in short sequence repeats (SSRs), of which SSR1 and SSR2 were the most diverse and homoplastic. Variations in SSR1 and SSR2, which are located in ORF1 and ORF2 , respectively, affect the genetic reading frame, leading to protein products with altered sequences and computed structures. The ORF1 gene product is predicted to be a MAP surface protein with possible roles in host immune modulation, but nothing could be inferred regarding the function of ORF2. Both genes are conserved in Mycobacterium avium complex members, but SSR1-based modulation of ORF1 reading frames seems to only occur in MAP, which could have potential implications on the infectivity of this pathogen. IMPORTANCE Johne’s disease (JD) is a major problem in dairy animals, and concerns have been raised regarding the association of Mycobacterium avium subsp. paratuberculosis (MAP) with Crohn’s disease in humans. MAP is an extremely slow-growing bacterium with low genome evolutionary rates. Certain short sequence repeats (SSR1 and SSR2) in the MAP chromosome are highly variable and evolve at a faster rate than the rest of the chromosome. In the current study, multiple MAP isolates with genetic variations such as single-nucleotide polymorphisms, and more noticeably, diverse SSRs, could simultaneously infect animals. Variations in SSR1 and SSR2 affect the products of the respective genes containing them. Since multiple MAP isolates can infect the same animal and the possibility that the pathogen undergoes further changes within the host due to unstable SSRs, this could provide a compensative mechanism for an otherwise slow-evolving pathogen to increase phenotypic diversity for overcoming host responses.