Genome-Wide Sequence Variation among Mycobacterium avium Subspecies paratuberculosis Isolates: A Better Understanding of Johne?s Disease Transmission Dynamics

Abstract

Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne’s disease, infects many farmed ruminants, wild-life animals, and recently isolated from humans. To better understand the molecular pathogenesis of these infections, we analyzed the whole-genome sequences of several M. ap and M. avium subspecies avium (M. avium) isolates to gain insights into genomic diversity associated with variable hosts and environments. Using Next-generation sequencing technology, all six M. ap isolates showed a high percentage of similarity (98%) to the reference genome sequence of M. ap K-10 isolated from cattle. However, two M. avium isolates (DT 78 and Env 77) showed significant sequence diversity (only 87 and 40% similarity, respectively) compared to the reference strain M. avium 104, a reflection of the wide environmental niches of this group of mycobacteria. Within the M. ap isolates, genomic rearrangements (insertions/deletions) were not detected, and only unique single nucleotide polymorphisms (SNPs) were observed among M. ap isolates. While more of the SNPs (~100) in M. ap genomes were non-synonymous, a total of ~6,000 SNPs were detected among M. avium genomes, most of them were synonymous suggesting a differential selective pressure between M. ap and M. avium isolates. In addition, SNPs-based phylo-genomics had a enough discriminatory power to differentiate between isolates from different hosts but yet suggesting a bovine source of infection to other animals examined in this study. Interestingly, the human isolate (M. ap 4B) was closely related to a M. ap isolate from a dairy facility, suggesting a common source of infection. Overall, the identified phylo-genomes further supported the idea of a common ancestor to both M. ap and M. avium isolates. Genome-wide analysis described here could provide a strong foundation for a population genetic structure that could be useful for the analysis of mycobacterial evolution and for the tracking of Johne’s disease transmission among animals.

Ecology and genomic features of infection with Mycobacterium avium subspecies paratuberculosis in Egypt

“…For colony PCR, a quick protocol for DNA extraction was used as detailed before (Talaat et al, 1997). The target for PCR amplification included a 229 bp fragment of the IS900 sequence with specific forward (59-CCG CTA ATT GAG AGA TGC GAT TGG-39) and reverse primers (59-AAT CAA CTC CAG CAG CGC GGC CTC G-39), as described previously (Hsu et al, 2011). Amplification conditions included an initial denaturation at 94 uC for 3 min followed by 50 cycles of denaturation at 94 uC for 60 s, annealing at 60 uC for 60 s and extension at 72 uC for 60 s. A final extension step at 72 uC for 7 min was also employed.…”

“…PCRs consisted of 25 ml, containing 1 M betaine, 50 mM potassium glutamate, 10 mM Tris-HCl pH 8.8, 0.1 % Triton X-100, 2 mM magnesium chloride, 0.2 mM dNTPs, each primer at 0.5 mM, 0.5 U Taq DNA polymerase (Promega) and 5 ml genomic DNA. The amplification conditions included an initial denaturation step of 94 uC for 5 min, followed by 35 cycles of denaturation at 94 uC for 30 s, annealing at 55 uC for 30 s and extension at 72 uC for 1 min, and then followed by a final extension at 72 uC for 7 min (Hsu et al, 2011). Following PCR, amplicons were cleaned up and processed for sequencing with the BigDye Terminator v3.1 Cycle Sequencing kit (Hsu et al, 2011).…”

“…Fortunately, genome-wide approaches could improve the sensitivity and specificity of assays used to detect Johne's disease (Castellanos et al, 2012). Recently, several clinical isolates of M. paratuberculosis were subjected to whole genome sequencing, which provided an accurate estimate for strain geographical and host association (Ghosh et al, 2012;Hsu et al, 2011;Wu et al, 2006), with implications for our understanding of strain evolution (Bannantine et al, 2012). Genome-based techniques could also provide more targets for developing better markers for tracing the source of M. paratuberculosis infection.…”

Molecular Identification of Mycobacterium avium subsp. silvaticum by Duplex High-Resolution Melt Analysis and Subspecies-Specific Real-Time PCR

“…avium Env77, and 1,201 contigs of M. avium subsp. avium DT78 (21). Based on preliminary sequence and alignment studies, out of 2,800 nucleotide position differences, 50 potential sequence variations, such as single or multiple gaps, SNPs, or multiple mismatching (MM) base pairs, were selected.…”

Viable Mycobacterium avium ssp. paratuberculosis isolated from calf milk replacer

“…When MAP isolates were successfully cultured at Queen's University Belfast (QUB), genomic DNA was extracted from cells grown in Dubos broth according to a method supplied by Adel Talaat, University of Wisconsin-Madison (Hsu et al, 2011). Briefly, this involved heating at 80°C for 20 min to kill the mycobacterial cells, lysozyme, and proteinase K treatments to weaken the cell wall, and addition of 5 M NaCl and cetrimonium bromide/NaCl to lyse the MAP cells.…”