Genetic diversity in the plasticity zone and the presence of the chlamydial plasmid differentiates Chlamydia pecorum strains from pigs, sheep, cattle, and koalas

Abstract

BackgroundChlamydia pecorum is a globally recognised pathogen of livestock and koalas. To date, comparative genomics of C. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms (SNPs) and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. No chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. Genomic analyses have not previously included C. pecorum from porcine hosts. We sequenced the genome of three C. pecorum isolates from pigs with differing pathologies in order to re-evaluate the genetic differences and to update the phylogenetic relationships between C. pecorum from each of the hosts.MethodsWhole genome sequences for the three porcine C. pecorum isolates (L1, L17 and L71) were acquired using C. pecorum-specific sequence capture probes with culture-independent methods, and assembled in CLC Genomics Workbench. The pairwise comparative genomic analyses of 16 pig, sheep, cattle and koala C. pecorum genomes were performed using several bioinformatics platforms, while the phylogenetic analyses of the core C. pecorum genomes were performed with predicted recombination regions removed. Following the detection of a C. pecorum plasmid, a newly developed C. pecorum-specific plasmid PCR screening assay was used to evaluate the plasmid distribution in 227 C. pecorum samples from pig, sheep, cattle and koala hosts.ResultsThree porcine C. pecorum genomes were sequenced using C. pecorum-specific sequence capture probes with culture-independent methods. Comparative genomics of the newly sequenced porcine C. pecorum genomes revealed an increased average number of SNP differences (~11 500) between porcine and sheep, cattle, and koala C. pecorum strains, compared to previous C. pecorum genome analyses. We also identified a third copy of the chlamydial cytotoxin gene, found only in porcine C. pecorum isolates. Phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of C. pecorum in livestock.Most surprising, we also discovered a plasmid in the porcine C. pecorum genome. Using this novel C. pecorum plasmid (pCpec) sequence, a) we developed a pCpec screening assay to evaluate the plasmid distribution in C. pecorum from different hosts; and b) to characterise the pCpec sequences from available previously sequenced C. pecorum genome data. pCpec screening showed that the pCpec is common in all hosts of C. pecorum, however not all C. pecorum strains carry pCpec.ConclusionsThis study provides further insight into the complexity of C. pecorum epidemiology and novel genomic regions that may be linked to host specificity. C. pecorum plasmid characterisation may aid in improving our understanding of C. pecorum pathogenesis across the variety of host species this animal pathogen infects.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2053-8) contains supplementary material, which is...

Identification of unusual Chlamydia pecorum genotypes in Victorian koalas (Phascolarctos cinereus) and clinical variables associated with infection

“…The presence of genotype L in this population, not previously detected in koalas and not found in other Victorian populations in this study, suggests this C. pecorum strain was not moved with the koalas when this population was established by translocation of koalas from French Island in the 1980s (Martin, 1989). The origin of genotypes L, M and N in koalas is unknown, although their close similarity to C. pecorum ompA sequences from livestock raises interesting questions over the potential for cross-host transmission from livestock, for which molecular evidence continues to grow (Jelocnik et al, 2013;Bachmann et al, 2015;Legione et al, 2016). Future sampling of livestock that occupy ranges overlapping those of koala populations would be invaluable in investigating this hypothesis further.…”

“…The prevalence of pCpec across the C. pecorum-positive samples identified in this study was detected by use of pCpec-specific conventional PCR, as described previously (Jelocnik et al, 2015).…”

“…Although a number of different methods are now available to genotype C. pecorum samples (Marsh et al, 2011;Jelocnik et al, 2013), the existing catalogue of ompA sequences from northern koala populations makes it a useful tool to undertake comparisons with Victorian koala populations. Interestingly, differences in the presence of a chlamydial plasmid, a known virulence factor in some chlamydial species (O'Connell et al, 2007), was also found to be a distinguishing feature of C. pecorum strains from northern populations compared with those from southern populations in South Australia (Jelocnik et al, 2015). Whilst some Victorian koala C. pecorum samples were found to be PCR-positive for this plasmid (pCpec), only a relatively small number of animals were sampled.…”

Chlamydia pecorum: successful pathogen of koalas or Australian livestock?

“…pecorum genome-derived phylogenies highlighted the polyphyletic history of the pathogen itself, and provided important clues about the evolutionary origins of koala strains, giving more confidence to our hypothesis that the origin of C. pecorum infections in Australia is associated with importation of domesticated animals into Australia with European colonisation 3,25,27 .…”

“…(a) Phylogenetic analyses of the concatenated seven MLST gene fragments sequences from the previously described C. pecorum STs available from Chlamydiales PubMLST (http://pubmlst.org/chlamydiales/) and newly generated C. pecorum STs from our database. The mid-point rooted phylogenetic tree was generated using FastTree, as implemented in 25 (Figures 2 and 3). The unexpected genomic finding in our studies was the identification of a chlamydial virulence plasmid (pCpec) (Figure 2).…”