Abstract
Over a decade ago Mycobacterium avium subspecies paratuberculosis (Map) specific genes were initially identified in a whole genome context by comparing draft genome sequences of Map strain K-10 with Mycobacterium avium subspecies hominissuis (Mah) strain 104. This resulted in identification of 32 Map specific genes, not including repetitive elements, based on the two-genome comparison. The goal of this study was to define a more complete catalog of M. avium subspecies-specific genes. This is important for obtaining additional diagnostic targets for Johne's disease detection and for understanding the unique biology, evolution and niche adaptation of these organisms. There are now over 28 complete genome sequences representing three M. avium subspecies, including avium (Maa), Mah, and Map. We have conducted a comprehensive comparison of these genomes using two independent pan genomic comparison tools, PanOCT and Roary. This has led to the identification of more than 250 subspecies defining genes common to both analyses. The majority of these genes are arranged in clusters called genomic islands. We further reduced the number of diagnostic targets by excluding sequences having high BLAST similarity to other mycobacterial species recently added to the National Center for Biotechnology Information database. Genes identified as diagnostic following these bioinformatic approaches were further tested by DNA amplification PCR on an additional 20 M. avium subspecies strains. This combined approach confirmed 86 genes as Map-specific, seven as Maa-specific and three as Mah-specific. A single-tube PCR reaction was conducted as a proof of concept method to quickly distinguish M. avium subspecies strains. With these novel data, researchers can classify isolates in their freezers, quickly characterize clinical samples, and functionally analyze these unique genes.
Highlights
Accurate diagnosis of mycobacterial infections is important for effective disease management within the livestock production industry
Mycobacterium avium Diagnostic Sequences subspecies comprise three predominant, closely-related subspecies, M. avium subspecies avium (Maa), M. avium subspecies hominissuis (Mah), and M. avium subspecies paratuberculosis (Map) and their genetic similarity has limited the identification of diagnostic targets that distinguish each subspecies
These genes have aided the specific detection of Map, but are singlecopy genes compared to IS900, a repetitive element that was known to be diagnostic for Map long before whole-genome comparisons were performed [9, 10]
Summary
Accurate diagnosis of mycobacterial infections is important for effective disease management within the livestock production industry. National and state veterinary laboratories are routinely confronted with unresolved cases of non-tuberculous mycobacterial (NTM) infections that are not further delineated due to complicated cross-reactive results as well as a lack of time and funds. Correct identification of M. avium subspecies is very important because of the divergent, yet prominent, clinical relevance that exists among these strains [1]. Mycobacterium avium Diagnostic Sequences subspecies comprise three predominant, closely-related subspecies, M. avium subspecies avium (Maa), M. avium subspecies hominissuis (Mah), and M. avium subspecies paratuberculosis (Map) and their genetic similarity has limited the identification of diagnostic targets that distinguish each subspecies. Map is the causative agent of Johne’s disease, which is a chronic intestinal ailment of ruminants that results in significant economic loss to dairy, meat and wool industries [3, 4]. Mah is an opportunistic pathogen of swine and humans that manifests most commonly as a pulmonary infection in humans [5], while Maa is primarily a pathogen of birds [6]
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