Limitations of variable number of tandem repeat typing identified through whole genome sequencing of Mycobacterium avium subsp. paratuberculosis on a national and herd level

Abstract

BackgroundMycobacterium avium subsp. paratuberculosis (MAP), the causative bacterium of Johne’s disease in dairy cattle, is widespread in the Canadian dairy industry and has significant economic and animal welfare implications. An understanding of the population dynamics of MAP can be used to identify introduction events, improve control efforts and target transmission pathways, although this requires an adequate understanding of MAP diversity and distribution between herds and across the country. Whole genome sequencing (WGS) offers a detailed assessment of the SNP-level diversity and genetic relationship of isolates, whereas several molecular typing techniques used to investigate the molecular epidemiology of MAP, such as variable number of tandem repeat (VNTR) typing, target relatively unstable repetitive elements in the genome that may be too unpredictable to draw accurate conclusions. The objective of this study was to evaluate the diversity of bovine MAP isolates in Canadian dairy herds using WGS and then determine if VNTR typing can distinguish truly related and unrelated isolates.ResultsPhylogenetic analysis based on 3,039 SNPs identified through WGS of 124 MAP isolates identified eight genetically distinct subtypes in dairy herds from seven Canadian provinces, with the dominant type including over 80% of MAP isolates. VNTR typing of 527 MAP isolates identified 12 types, including “bison type” isolates, from seven different herds. At a national level, MAP isolates differed from each other by 1–2 to 239–240 SNPs, regardless of whether they belonged to the same or different VNTR types. A herd-level analysis of MAP isolates demonstrated that VNTR typing may both over-estimate and under-estimate the relatedness of MAP isolates found within a single herd.ConclusionsThe presence of multiple MAP subtypes in Canada suggests multiple introductions into the country including what has now become one dominant type, an important finding for Johne’s disease control. VNTR typing often failed to identify closely and distantly related isolates, limiting the applicability of using this typing scheme to study the molecular epidemiology of MAP at a national and herd-level.

Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis

“…Techniques such as AFLP and RAPD employ PCR to detect smaller genomic DNA fragments but are less utilized for epidemiological studies due to difficulties in standardization and reproducibility and limited discriminative power. Other typing methods based on repetitive sequences, such as SSR and MIRU-VNTR, are popular due to their ease of use and rapidity but, again, are limited with respect to their ability to discriminate within the two major strain types, and the typing results may not reflect the evolutionary relationships between isolates (10,12,25,26).…”

“…Genetic diversity among M. avium subspecies paratuberculosis strains has been investigated using molecular techniques, such as restriction fragment length polymorphism (RFLP) and IS900 analysis (IS900 RFLP) (3), pulsed-field gel electrophoresis (PFGE) (4), amplified fragment length polymorphism (AFLP) analysis (5), random amplified polymorphic DNA (RAPD) analysis (6), mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) analysis (7), and short-sequence repeat (SSR) analysis (8). However, these techniques have limited discriminatory power when applied to M. avium subspecies paratuberculosis; although this power can be increased by combining complementary genotyping techniques, it is often insufficient for accurately determining relationships among isolates or global epidemiological studies (9, 10).Whole-genome sequencing (WGS) provides the ultimate resolution of isolates, and, unlike the techniques listed above, it can provide a phylogenetic context to facilitate global epidemiology studies and affirm epidemiological connections (10,11,12). Although WGS is becoming cheaper, it is still too expensive to be used for routine genotyping of M. avium subspecies paratuberculosis isolates and requires robust data handling and analysis processes.…”

“…Whole-genome sequencing (WGS) provides the ultimate resolution of isolates, and, unlike the techniques listed above, it can provide a phylogenetic context to facilitate global epidemiology studies and affirm epidemiological connections (10,11,12). Although WGS is becoming cheaper, it is still too expensive to be used for routine genotyping of M. avium subspecies paratuberculosis isolates and requires robust data handling and analysis processes.…”

Molecular typing of Mycobacterium avium subsp. paratuberculosis (MAP) isolated from dairy goats in Brazil

“…Those with intolerance and gastrointestinal diseases lactose (Fonseca and Bruschi, 2009). The agent can survive in milk after heat treatment (Dimareli-Malli, 2010;Carvalho et al, 2012) and there is a strong suspicion que MAP can be the agent zoonotic, although there is controversy (Ahlstrom et al, 2015). In addition, there are economic losses due to the Decreased production of meat and milk of animals contaminated (Stehman, 1996;Oliveira et al, 2010).…”

Examination ofMycobacterium aviumsubspeciesparatuberculosismixed genotype infections in dairy animals using a whole genome sequencing approach

“…It has also been shown that the early stages of Map infection can occur in asymptomatic hosts that continue to shed viable bacteria in their faeces, leading to subsequent infection of other susceptible individuals (Whitlock et al, 2000). Therefore, there has been a lot of recent interest in understanding the dissemination of Map using source tracking and epidemiological studies (Ahlstrom et al, 2015; Ahlstrom et al, 2016; Leão et al, 2016; Bryant et al, 2016). …”