Typing of Mycobacterium avium Subspecies paratuberculosis Isolates from Newfoundland Using Fragment Analysis

Abstract

Short Sequence Repeat (SSR) typing of Mycobacterium avium subspecies paratuberculosis (Map) isolates is one of the most commonly used method for genotyping this pathogen. Currently used techniques have challenges in analyzing mononucleotide repeats >15 bp, which include some of the Map SSRs. Fragment analysis is a relatively simple technique, which can accurately measure the size of DNA fragments and can be used to calculate the repeat length of the target SSR loci. In the present study, fragment analysis was used to analyze 4 Map SSR loci known to provide sufficient discriminatory power to determine the relationship between Map isolates. Eighty-five Map isolates from 18 animals from the island of Newfoundland were successfully genotyped using fragment analysis. To the best of our knowledge, this is the first report on Map SSR diversity from Newfoundland dairy farms. Previously unreported Map SSR-types or combinations were also identified during the course of the described work. In addition, multiple Map SSR-types were isolated from a single animal in many cases, which is not a common finding.

Mycobacterium avium ssp . paratuberculosis detection in animals, food, water and other sources or vehicles of human exposure: A scoping review of the existing evidence

“…cattle, sheep, bison) found by species category is shown in Figure 4. Multiple fingerprinting techniques were used to show the diversity of M. paratuberculosis within populations, and it was argued that it is possible to demonstrate strain sharing within and across species by the degree of homogeneity between isolates (Stevenson et al, 2009 (Singh et al, 2010b;Sohal et al, 2014;Ahlstrom et al, 2015;Podder et al, 2015).…”

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

“…We recently reported that on more than one occasion, Map with different SSR types could be isolated from single animals from dairy farms from Newfoundland (NL), Canada (Podder et al, 2015), suggesting mixed genotype infections. Our previous SSR study only analyzed four genetic loci/repeats to examine genetic diversity.…”

“…Sample collection, isolation/culture of Map isolates, DNA extraction and subsequent manipulations were carried out as described previously (Podder et al, 2015). Six genetically distinct Map isolates were selected based on SSR profiles (Podder et al, 2015) and DNA was sent for WGS to The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, Canada).…”

“…Six genetically distinct Map isolates were selected based on SSR profiles (Podder et al, 2015) and DNA was sent for WGS to The Centre for Applied Genomics (The Hospital for Sick Children, Toronto, Canada). Nextera XT libraries were prepared and sequence data was gathered using the Illumina HiSeq platform (Illumina, Inc. USA) with an average read depth of 1,000× coverage.…”

“…There have also been numerous WGS studies that have examined the genetic diversity of Map from dairy animals (Ahlstrom et al, 2015; Bryant et al, 2016; Ahlstrom et al, 2016; Yue et al, 2016; Leão et al, 2016), but none which have analyzed or addressed mixed Map genotype infections using multiple isolates from a single animal. We recently reported that Map with different s hort s equence r epeat (SSR) types could be isolated from individual dairy animals from the island of Newfoundland (Podder et al, 2015). Here we use WGS, single nucleotide polymorphism (SNP) calling and phylogenetics to analyze six Map isolates, three of which were isolated from a single animal.…”