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

Martina Jelocnik, Nathan L. Bachmann, Bernhard Kaltenboeck, Courtney Waugh, Lucy Woolford, K. Natasha Speight, Amber Gillett, Damien P. Higgins, Cheyne Flanagan, Garry S. A. Myers, Peter Timms, Adam Polkinghorne

Chlamydia 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. Whole 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. Three 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. This 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.