Molecular characterisation of Mycobacterium avium subsp. paratuberculosis in Australia

Abstract

Background Mycobacterium avium subsp. paratuberculosis (Map) causes Johne’s disease (JD), a chronic enteritis widespread in ruminants, resulting in substantial economic losses, especially to the dairy industry. Understanding the genetic diversity of Map in Australia will assist epidemiological studies for tracking disease transmission and identify subtype characteristics for use in development of improved diagnostic typing methods. Here we investigated the phylogenetic relationships of 351 Map isolates and compared different subtyping methods to assess their suitability for use in diagnostics and accuracy. Results SNP-based phylogenetic analysis of 228 Australian isolates and 123 publicly available international isolates grouped Type S and Type C strains into two distinct lineages. Type C strains were highly monomorphic with only 20 SNP differences separating them. Type S strains, when aligned separately to the Telford strain, fell into two distinct clades: The first clade contained seven international isolates while the second clade contained one international isolate from Scotland and all 59 Australian isolates. The Australian Type B strain clustered with US bison strains. IS1311 PCR and Restriction Enzyme Analysis (REA) intermittently generated incorrect results when compared to Long Sequence Polymorphism (LSP) analysis, whole genome SNP-based phylogenetic analysis, IS1311 sequence alignment and average nucleotide identity (ANI). These alternative methods generated consistent Map typing results. A published SNP based assay for genotyping Map was found to be unsuitable for differentiating between Australian and international strain types of Map. Conclusion This is the first phylogenetic analysis of Australian Map isolates. The Type C lineage was highly monomorphic, and the Type S lineage clustered all Australian isolates into one clade with a single Scottish sheep strain. The Australian isolate classified as Type B by IS1311 PCR and REA is likely to be descended from bison and most closely related to US bison strains. Limitations of the current typing methods were identified in this study.

Characterisation of IS1311 in Mycobacterium avium subspecies paratuberculosis genomes: Typing, continental clustering, microbial evolution and host adaptation

“…The bison-type cluster was separated from the S strains by a branch containing a single genome, ERR037950/MAPMRI074 ( Fig 3 , purple branch). This genome has been reported to be an IS 1311 S strain [ 25 ] but it clustered most closely with C strains in this investigation and in previous studies [ 25 , 32 ].…”

“…The two major branches of the phylogeny represent the S and C strains of MAP, supported by the reported IS 1311 types, host species and locations of reference genomes. Some previously reported IS 1311 types (n = 10/805) did not match the clade that this WGS SNP analysis placed them in [ 32 , 59 ]. The C strain clade ( Fig 3 , green clade) appeared to have some clusters that were highly similar, indicated by long, “flat” clades.…”

“…Evidence of geographical clustering of MAP isolates has not been described previously. This may be due to previous investigations having focussed on specific countries or regions with few representative genomes from other locations, or due to smaller datasets [ 25 , 32 ]. Although exceptions were found, Australian C and S strain MAP isolates clustered within the respective C and S lineages in the phylogeny.…”

“…A total of 496 previously curated MAP genomes were sourced from the Sequence Read Archive (SRA) [ 36 ]. Additionally, approximately 223 genomes from a recent Australian study [ 32 ] and an earlier global MAP study containing approximately 206 genomes [ 25 ] were sourced from the SRA after the original public genome dataset had been curated. A further 67 MAP isolates from the University of Sydney sample archive were sequenced at the University of Sydney.…”

“…In the case of IS, variants can be under selective pressure and subject to rapid evolution [ 31 ]. Recently, conflicting LSP and IS 1311 types were reported for some MAP isolates but without any attempt to resolve the discrepancies [ 25 , 32 ]. Another concern with reliance on SNP mutations in IS 1311 is the inability to differentiate a mixed C and S strain infection from a pure C strain infection because S strains do not have a unique SNP.…”