BACKGROUND
chlamydia pecorum is a globally distributed animal pathogen of domesticated and wild ruminants, and the iconic native australian marsupial, the koala.
expansion of the number of available whole genome sequences of c. pecorum from sheep, cattle, and koala hosts has provided insights into its lifestyle, associated virulence factors, and evolution .
the genetic differences that may influence host specificity and/or pathogenicity in c. pecorum remain largely unknown, however, with only  single nucleotide polymorphisms ;  a limited number  of pseudogenes; and  variable numbers of tandem repeats in certain c. pecorum genes , differentiating strains from different hosts or associated with different diseases .
in other chlamydial species, the almost ubiquitous non-integrative chlamydial plasmid  <cit>  has been recognised as a major chlamydial virulence factor  <cit> .
this plasmid also has value as a target for molecular typing  <cit>  and as an immunogenic vaccine candidate both for humans and animals such as ducks, pigeons, cats and pigs  <cit> .
two studies have previously detected a c. pecorum plasmid in isolates from three koalas and one bovid  <cit> , but further evidence for a c. pecorum plasmid has remained absent despite the growing number of genomics studies for this species .
although c. pecorum infects a wide range of hosts, including some other australian marsupials, european wild ruminants, and pigs, c. pecorum comparative genomics studies thus far have focused only on sheep, cattle, and koala strains .
porcine c. pecorum infections are underestimated as a source of on-farm economic loss for producers, manifesting as a variety of diseases such as pneumonia, pleuritis, polyserositis, polyarthritis, conjunctivitis, and enteritis.
as with other hosts, c. pecorum subclinical infections in pigs are also common .
in an effort to broaden our knowledge of the genetic diversity of c. pecorum in other hosts, in the present study, we sequenced three porcine c. pecorum genomes and compared them to the available sheep, cattle, and koala c. pecorum genomes.
in doing so, we also provide the first detailed description of the genetic structure and distribution of the c. pecorum plasmid, a genetic feature associated with virulence in other chlamydial species.
methods
descriptions of pig c. pecorum isolates, clinical samples and other c. pecorum strains used in this study
the three c. pecorum strains l <dig>  l <dig> and l <dig> isolated from pigs presenting with pneumonia  and polyarthritis  were utilised for whole genome sequencing and plasmid analyses in the present study.
these strains were one of the first pig chlamydial strains, isolated together with many others in  <dig> in austria, from pigs presenting with chlamydiosis during a mass chlamydial outbreak  <cit> .
plasmid screening was performed on a large collection of  previously tested c. pecorum pcr positive clinical samples from sheep, cattle and koalas ;  and a small collection of cultured c. pecorum isolates from a variety of hosts ;  additional previously untested but c. pecorum pcr positive sheep and koala samples identified in this study .
this latter testing, combined with our previous screening for c. pecorum dna in clinical samples, resulted in   <dig> c. pecorum pcr positive ocular and urogenital swab samples from  <dig> koalas from different populations across australia;   <dig> ocular, joint, vaginal and rectal swabs collected from  <dig> sheep from nine different flocks from the central nsw region in australia  <cit> ; and  ocular, nose, internal organs, and rectal samples  from four cattle from two herds, one previously described from western australia   <cit>  and a second from central nsw .
cultured c. pecorum isolates from pigs, sheep, cattle, and koalas screened are listed in additional file 4: table s <dig>
genomic dna extraction and c. pecorum-specific qpcr screen
new sheep, cattle and koala clinical swab samples used in this study were processed as previously described  <cit> .
the cultures of three c. pecorum porcine isolates l <dig>  l <dig> and l <dig> were propagated in chicken embryos and purified, as previously described  <cit> , while the cultures of  <dig> koala isolates were propagated in hep- <dig> and/or mccoy cells and semi-purified using a probe sonication and density gradient centrifugation, as previously described  <cit> .
dna extraction for both samples and cultures was performed using the qiamp dna mini kit , as per the manufacturer’s instructions.
c. pecorum infectious load and/or presence were determined using a c. pecorum species-specific qpcr assay targeting 202 bp of 16s rdna  <cit> .
samples with <  <dig> copies c. pecorum 16s rdna were considered negative.
dna concentration for each sample was also measured in duplicates on a nanodrop.
additionally, 1 μl of extracted, screened and quantified gdna of three porcine  c. pecorum samples were run on a 1 % tbe agarose gel to confirm high molecular weight dna prior to whole genome sequencing.
sequence capture, whole genome sequencing and assembly
sequence capture was performed on total dna extracted from c. pecorum l <dig>  l <dig> and l <dig> cultures with the c. pecorum custom-designed probes   <cit> , using a dna hybridisation capture and amplification process based on the methods described elsewhere  <cit> .
l <dig>  l <dig> and l <dig> genomes were sequenced at the institute for genome sciences , baltimore, usa, using the illumina hiseq  <dig> platform producing paired-end  <dig> base-pair reads.
read quality, de novo assembly and read-mapping of paired-end reads from l <dig>  l <dig> and l <dig> was performed using clc genomics workbench , after filtering and trimming for size and quality.
the read mapping was visualised with blast ring image generator  software  <cit> .
after de novo assembly, l <dig>  l <dig> and l <dig> contigs were blasted to confirm identity using blastn  and ordered against complete c. pecorum e <dig> genome  with progressive mauve  <cit>  to produce single genome scaffold.
genome annotations were done using rast  <cit>  and the igs prokaryotic annotation pipeline, assisted by mapping onto the c. pecorum e <dig> type strain sequence, as previously described  <cit> .
number and distribution of snps was determined using the probabilistic variant detection plug-in with default settings with l <dig>  l <dig> and l <dig> reads mapped to a reference genome, as implemented in clc genomics workbench.
general descriptions and accession numbers of l <dig>  l <dig> and l <dig> genomes were outlined in table  <dig>
macroscopic comparative genomics and phylogenetic analyses using new porcine genomes
alongside the newly sequenced three porcine c. pecorum genomes, we also used publicly available genome sequences of c. pecorum from: cattle encephalomyelitis e <dig>  <cit>  ; sheep arthritis p <dig> , sheep fecal w <dig> , cattle metritis pv3056/ <dig>   <cit> ; koala cystitis mc/marsbar  and dbdeug , koala conjunctivitis iptale , sheep polyarthritis ipa    <cit> ; cattle encephalomyelitis nsw/bov/sbe , sheep polyarthritis mer/ovi1/jnt , sheep conjunctivitis nar/s22/rec , koalas with cystitis gun/koa1/ure  and sa/k2/ugt   <cit>  for whole genome comparative analyses.
pairwise comparative genomic analyses were performed in act, the artemis comparison tool  <cit>  and geneious  <dig> .
<dig>  <cit>  using alignments generated with progressive mauve  <cit> .
polymorphic loci and/or regions of interest in the genomes were extracted and further aligned with clustalx  <cit> .
dnasp  <dig>   <cit>  was used to analyse the level of sequences polymorphisms by: determining the ratio of non-synonymous to synonymous substitutions  ; calculating the number of polymorphic  sites and haplotype diversity.
further, if a polymorphism resulted in a non-synonymous amino acid change or premature stop codon in the loci of interest in each of the analysed c. pecorum genome sequence, the translated sequence was further analysed in blast for comparison ,uniprot  and conserved domains database  to assess protein functionality.
figures of the genomic regions of interest, such as the pz was generated using easyfig  <cit> , based on tblastx comparison.
construction of phylogenetic relationships between the loci was performed using mrbayes  <cit>  as implemented in geneious  <dig> .
<dig>
in addition to the subset of  <dig> c. pecorum genomes used for comparative analyses, for phylogenetic analyses we have included:  sheep nar/s22/re ;  as well as the two secondary koala c. pecorum strains  identified in the same study  <cit> .
conserved regions were identified in  <dig> c. pecorum genomes using all-versus-all blast search.
syntenic regions were individually aligned using muscle  <cit>  and concatenated to produce a ~  <dig> kbp core genome alignment.
poorly aligned regions were removed from the alignment using gblocks with the minimum length of a block set to  <dig> and no gap positions were allowed.
recombination regions were predicted using gubbins  <cit>  and subsequently filtered from the alignment.
a mid-point rooted phylogenetic tree was constructed from the genome alignment with phyml  <dig>  using the gtr model.
bootstrap values were calculated using  <dig> replicates.
pcr amplification and phylogenetic analyses of toxc
during pairwise comparative genomic analyses, the presence of a third copy of the cytotoxin gene  in porcine l <dig> genome was identified.
primers  and toxc rev ) were designed to amplify a 644 bp fragment of the toxc gene in order to confirm the presence of this sequence.
using purified l <dig> genomic dna as a template , and purified c. pecorum koala mc/marsbar and iptale, cattle e <dig>  porcine l <dig>  l <dig> and sheep ipa genomic dna as negative controls, conventional pcr assay was performed to amplify the 644 bp toxc fragment.
pcr assays for 644 bp toxc fragment were prepared to a total reaction volume of 50 μl including 1x amplitaq gold®  <dig> master mix ,  <dig>  μm of forward and reverse primer each  and 3 μl dna template.
the cycling conditions included an initial denaturation  followed by 35 cycles of denaturation , annealing  and extension , followed by a final extension .
the amplicon was then purified and dideoxy sequenced .
after successful amplification and confirmation of the toxc fragment in l <dig> only, we tested the remaining eight porcine, seven cattle, two sheep  and five koala c. pecorum isolates  for the presence of the toxc 644 bp fragment sequence.
identification of the full length l <dig> toxc sequence was checked in blast and the midpoint rooted bayesian phylogeny was constructed using alignment of all three cytotoxin  genes from a subset of  <dig> c. pecorum analysed in this study.
the phylogenetic tree was constructed with mrbayes as implemented in geneious  <dig> .
<dig>  using the hky + i model with four markov chain monte carlo  chains with a  <dig>  <dig> generations, sampled every  <dig> generations and with the first  <dig>  <dig> trees were discarded as burn-in.
c. muridarum nigg  three cytotoxin gene sequences were also included in the alignment for phylogenetic analyses, with the c. muridarum nigg tox  <dig> used as an out-group.
c. pecorum plasmid contig identification
the l <dig>  <dig> kbp plasmid contig was annotated with rast  <cit> , and further curated in geneious  <dig> .
<dig>  additionally, pcpec l <dig> sequence was also blasted against raw reads of c. pecorum porcine l <dig> and l <dig>  sheep ipa, cattle nsw/bov/sbe, and koala mc/marsbar, dbdeug, iptale, sa/k2/ugt and gun/k1/ure.
pcpec  <dig> kbp sequence was only identified in the three koala c. pecorum mc/marsbar, dbdeug and iptale raw reads and also used in this study.
pcpec conv.
pcr confirmatory assay design
newly identified pcpec sequences from a porcine l <dig> and three koala c. pecorum genomes were aligned using clustalx.
primer pair  was designed to amplify a 522 bp plasmid fragment which includes a conserved intergenic region and a part of the plasmid gene cds <dig>  primer sequences were tested in silico for secondary structures and dimerisations at integrated dna technologies, inc oligoanalyzer  <dig>  online tool .
additionally, plasmid isolation was performed from fresh available koala c. pecorum mc/mars, dbdeug, and iptale cultures.
plasmid isolation and purification was performed using genejet plasmid midiprep  <dig> reactions, thermofisher scientific, victoria, australia, as per manufacturer’s instructions.
freshly extracted plasmids were digested with neb saci  and visualised on the ethidium bromide stained  <dig>  % gel, using neb 2-log dna ladder .
using purified porcine l <dig> genomic dna and extracted plasmid dna from koala c. pecorum mc/mars, dbdeug, and iptale as templates and positive controls , conventional pcr to amplify 522 bp plasmid fragment was performed.
in the same assay we used purified pig l <dig> and l <dig> genomic dna as negative controls .
the plasmid primers were additionally tested against genomic avian c. psittaci dna extracted from a cultured isolate in order to test for unspecified primer binding.
after successful amplification of the 522 bp plasmid fragments from templates only, the presence of the amplicon was confirmed on  <dig>  % tbe gel, purified and sent off for confirmatory dideoxy sequencing.
we tested the detection limit of our pcpec screening assay, based on a conventional pcr using: i) serially diluted pcpec from  <dig> to  <dig> copies/μl as a template in triplicate; and ii) c. pecorum positive clinical sample bella ugt, with 106c.
pecorum genome copy number/μl diluted to 101c.
pecorum genome copy number/μl, as a template in duplicate.
the detection limit for pcpec assay used in the present study was from  <dig> to  <dig> pcpec fragment copies/μl, as determined by amplicon visualisation on  <dig>  % tbe gel.
pcr-based pcpec screening
after confirming the 522 bp amplicon sequence, we applied the above described assay to screen for plasmid presence in koala and livestock c. pecorum samples.
the full lists of screened samples are outlined in additional file 4: table s <dig> and additional file 6: table s <dig>  the testing of these swab samples, collected as a part of routine diagnostic investigations of koalas and livestock, has been considered by the university of sunshine coast  animal ethics committee and the requirement for ethics approval was waived .
pcr assays for screening 522 bp pcpec fragment were prepared to a total reaction volume of 50 μl including 1x amplitaq gold®  <dig> master mix ,  <dig>  μm of forward and reverse primer each  and 3 μl dna template.
negative  and positive  controls were included in each amplification assay.
the cycling conditions included an initial denaturation  followed by 35 cycles of denaturation , annealing  and extension , followed by a final extension .
upon amplification, pcr products were detected on a 2 % ethidium bromide agarose gel and visualised under an uv transilluminator and purified, using a high pure pcr product purification kit .
a subset of  <dig> pcr products was directly sequenced using a bigdye terminator v <dig>  cycle sequencing kit  and subsequently purified according to the manufacturer’s instructions.
sequencing was performed at the institute for future environments , queensland university of technology , brisbane, australia using the applied biosystems abi <dig> gene analyser.
RESULTS
porcine c. pecorum l <dig>  l <dig> and l <dig> genome assemblies
using c. pecorum-specific sequence capture probes  <cit> , we acquired whole genome sequences for three porcine c. pecorum isolates.
c. pecorum strains l <dig> and l <dig> were isolated from the lungs of two different pigs presenting with pneumonia, while the l <dig> strain was a joint isolate from a case of polyarthritis  <cit> .
consistent with the high average read depth , de novo assembly resolved the genomes of these c. pecorum isolates into  <dig> –  <dig> contigs for each isolate .
read-mapping to a reference c. pecorum e <dig> genome  <cit>  confirmed almost whole chromosome coverage  for all three genomes.
similar to the previously described c. pecorum genomes , our annotation pipelines predicted ~  <dig> coding dna sequences , including the three rrna and  <dig> trna genes.
the draft genome sizes of ~ <dig>  mbp are almost identical to all other available c. pecorum genomes, with the exception of l <dig> which has a slightly larger draft genome size of  <dig>  mbp .table  <dig> general description of pig c. pecorum l <dig>  l <dig> and l <dig> samples and their genomes
interestingly, we also identified a distinct  <dig> kbp contig , assembled only from c. pecorum l <dig> strain reads.
blast searches revealed that this contig shared 70 % sequence identity to other related chlamydial species plasmids and 99 % sequence identity to a 710 bp sequence previously identified from a suspected c. pecorum plasmid   <cit> , based on blastn and discontiguous megablast blast searches .
the annotation of this l <dig>  <dig> kbp contig resulted in eight cdss specific for chlamydial plasmids.
sequence capture methodology continues to be an effective tool for acquiring high quality whole genome sequence data  <cit> , especially when cultured isolate gdna of high concentration and genome copy number is used as a template, as in the present study.
high read depths, complete genome coverage, and sequence homogeneity for our three pig strains was not surprising, as they were all pure cultured isolates, contrasting the sequence heterogeneity previously seen in c. pecorum-positive clinical swab studies  <cit> .
interestingly, design of the c. pecorum species-specific probes  <cit>  also allowed for identification of distinct and/or novel sequences, such as the full length plasmid contig detected in the l <dig> draft genome.
now that whole genome sequences for porcine c. pecorum are available, the use of c. pecorum-specific rna probes for sequence capture of c. pecorum dna can be further improved in order to reveal features not previously observed or investigated, and to improve coverage of the polymorphic genomic regions in this species.
re-evaluating the genetic differences between c. pecorum strains
the previously described c. pecorum gene order and synteny  <cit>  remained conserved in the porcine c. pecorum genomes with nearly all variation, once again, limited to snps .
comparison of the porcine l <dig>  l <dig> and l <dig> genome sequences against those from other hosts revealed the most snps between our c. pecorum porcine strains and the genomes of c. pecorum strains previously sequenced from sheep .
the closest c. pecorum strains from other hosts, on the other hand, were from a cow  and a koala  with approximately  <dig> and  <dig> snps, respectively.
interestingly, the majority of koala c. pecorum strains differed from porcine strains by ~  <dig> snps, less than sheep and cattle strains .
pairwise comparison of the genome sequences revealed that the majority of snps were found in two major clusters, including  the plasticity zone ; and  the major polymorphic membrane  gene cluster .fig.
<dig> snp distribution and identification of genomic elements across the porcine l <dig> genome in comparison to cattle nsw/bov/sbe and pv <dig>  sheep mer/ovi1/jnt, porcine l <dig> and l <dig>  and koala iptale genomes.
all genomes start from malate dehydrogenase  gene.
genomic location of type  <dig> secretion system gene clusters , major outer membrane protein , pyrimidine genes , polymorhic membrane protein  gene clusters, inclusion proteins genes , tryptophan  and biotin operon  genes are denoted on the horizontal line above the histograms.
variants, represented as histograms of a 10kbp size, were determined in clc genomics  platform
not surprisingly, averaging at ~ <dig> snps/10kbp, the pmp gene regions were a major contributor of single nucleotide differences .
overall sequence similarity of different c. pecorum pmp gene families from the porcine, sheep, cattle, and koala strains ranged from  <dig> – 100 % .
the bayesian phylogenetic analyses resolved the pmp a, b, e, e’, d, and h gene families in clades respective to their designated gene family, while the pmpg family was the most divergent, resolving each of the eight pmpg subtypes into its own diverse clade .
porcine c. pecorum l <dig>  l <dig>  and l <dig> strains had an identical genomic organisation, subtype and number of predicted pmp genes  as the previously characterised sheep, cattle, and koala strains  <cit>  .
sequence analyses of each of the  <dig> pmp genes from the porcine c. pecorum strains, indicated that only pmpg <dig> and pmpg <dig> type appear to be under positive selection pressure, with observed dn/ds ratios of  <dig> , and  <dig> , respectively .
subsequent sequence analyses of the pmpg <dig>  and pmpg <dig> from all  <dig> c. pecorum strains analysed in this study, also returned dn/ds ratios >  <dig>  interestingly, pmp g <dig> and g <dig> phylogenies were similar to the phylogenetic relationships constructed from the core genomes of the  <dig> c. pecorum strains analysed in this study , as well as the previous study  <cit>  .
in the recent sheep, cattle and koala c. pecorum comparative genomic study  <cit> , the pmpg region was also identified as the most diverse genomic region, however, most of the genetic variation observed resulted in synonymous substitutions.
in our study, variation in the pmp genes also mainly resulted in synonymous snps, with the exception of c. pecorum pmp genes g <dig> and g <dig>  which appeared to be under positive selection.
predicted to have a major role in chlamydial infection due to their adhesive function in the interaction with host  <cit> , pmp genes are predicted to maintain some hypervariability in order to evade immune defences  <cit> .
rapid diversification and evolution of the pmpg genes family has been previously observed in the genomic studies of the related ovine pathogen c. abortus  <cit> , and avian pathogen c. psittaci, where it has been suggested that these genes may play a role in adaptation to different hosts and environments  <cit> .
the observed positive selection on these genes in the c. pecorum genomes may support a role in host adaptation for this pathogen, an aspect that may warrant extended investigation.
while the remaining genetic variation was evenly distributed across the porcine c. pecorum chromosomes, we identified additional eight genes under positive selection in the polymorphic regions.
as outlined in additional file 4: table s <dig>  cpl1_ <dig> and cpl1_ <dig>  were both identified as effectors of the chlamydial type  <dig> secretion system , a system of structural, chaperone and secreted effector proteins with its main function in host cell manipulation and subversion of the host cellular processes  <cit> .
the cpl1_ <dig> was predicted to be orthologous to a c. psittaci secreted t3ss protein   <cit> , while the cpl1_ <dig> was a well-studied chlamydial t3ss translocated actin-recruiting protein  homologue  <cit> .
in the porcine c. pecorum genomes, although polymorphic, the remaining t3ss genes were under negative selection.
positive selection on the c. pecorum tarp homologue is perhaps not surprising, as the chlamydial tarp was previously recognised as an important antigenic protein  <cit> .
further, genomic studies of the c. psittaci and c. trachomatis revealed that the variation in the t3ss effector genes  could also contribute to the differences in virulence, and host and/or tissue tropism associated with these pathogens  <cit> .
presently the exact role of t3ss effectors in c. pecorum infections remains largely unknown, and will require more comprehensive in silico as well as cell biology analyses comprising of isolates from healthy, as well as diseased hosts to fully dissect t3ss effector roles.
the remaining genes under positive selection were:  cpl1_ <dig>  identified as a homologue of a previously described c. pecorum surface binding protein gene , also under positive selection in koala strains  <cit> ;  cpl1_ <dig>  a homologue of a macro domain  protein; and  four genes encoded conserved chlamydial hypothetical proteins of unknown function .
variation in the c. pecorum pz
in addition to the analysed pmp genes, the pz, spanning from inosine-5'-monophosphate dehydrogenase  to acetyl-coa carboxylase , also displayed significant variability .
the pz is a region of focus in chlamydial comparative genomic studies due to the presence and/or absence of a range of established chlamydial virulence factors .
in our study, like other c. pecorum strains , the porcine c. pecorum pzs were found to contain:  three purine biosynthesis genes,  a mac/perforin,  a variable number of phospholipase d  genes,  two copies of cytotoxin genes,  and two acetyl-coa-carboxylase genes .
some notable genetic variation, however, was also observed in these porcine c. pecorum strains including  major differences in the number of pld genes and;  the presence of a third copy of the cytotoxin gene in the l <dig> strain, as summarised in fig.
<dig>  in terms of pld genes, five were observed in l <dig>  a comparable number to that identified in the genome sequences of c. pecorum strains from sheep , koalas  and cattle .
the previously sequenced bovine pv3056/ <dig> isolate also had four plds like l <dig>  while the porcine l <dig> isolate is presently the only c. pecorum strain with six plds .
the variable number of plds is not unique to c. pecorum  <cit> , as related species such as c. trachomatis and c. muridarum have also been shown to have a variable number of pld genes among strains  <cit> .
it has been suggested that the number of pld genes could influence virulence  <cit> , but whether this is the case for c. pecorum is still unclear, as the majority of the sequenced c. pecorum were sampled from hosts with evidence of chlamydial disease.fig.
<dig> graphical representation of the c. pecorum cattle nsw/bov/sbe and pv <dig>  sheep mer/ovi1/jnt, pigs l <dig>  l <dig> and l <dig>  and koala sa/k2/ugt plasticity zones  tblastx comparisons.
the coloured arrows represent pz genes according to their function, as outlined in the legend.
the intensity of grey shaded areas corresponds to the sequence identity.
the image was generated with easyfig  <cit>
in terms of cytotoxin genes, almost all of the analysed strains had two copies of the cytotoxin gene, with observed toxa truncation in c. pecorum cattle nsw/bov/sbe and koala sa/k2/ugt strains .
the porcine l <dig> strain however, had three copies of the cytotoxin gene  .
among the genus chlamydia, only c. muridarum has three copies of the cytotoxin gene  <cit> .
sequences from this latter species were included as an out-group in the tox phylogenetic tree to investigate whether the third tox copy identified in the l <dig> is similar to the c. muridarum third tox copy .
analyses of the c. pecorum cytotoxin genes revealed that l <dig> toxc is more similar to c. pecorum toxa  than toxb .
a mid-point rooted bayesian phylogenetic tree of  <dig> c. pecorum toxa and toxb sequences with tox sequences from l <dig> and c. muridarum further revealed that the l <dig> toxc may be a duplication of the c. pecorum toxa, as evidenced by the clustering of this gene with c. muridarum tox <dig> and other c. pecorum toxa genes.
to see if toxc could be found in strains from other hosts, we designed a toxc-specific pcr  to screen c. pecorum strains from pigs, sheep, cattle, and koalas .
this toxc fragment was detected only in three more isolates: porcine pneumonia l <dig>  l <dig> and hslurz .
these strains were isolated from the same chlamydial outbreak in  <dig> among austrian pigs as the l <dig>  l <dig> and l <dig> strains and the remaining five porcine strains   <cit> .
thus, based on our genomic and preliminary toxc analyses, it appears that the  <dig> c. pecorum outbreak among austrian pigs was polyclonal, with strains involved having two or three cytotoxin genes.
variability in the number of tox genes and our previously noted truncation of certain tox genes in some c. pecorum strains  <cit> , raises the question over the function and impact of this gene in c. pecorum virulence.
expansion on c. pecorum phylogenetic relationships
using a c. pecorum multi locus sequence typing  scheme  <cit> , we recently showed that porcine c. pecorum strains clustered in a clade separate to that of c. pecorum strains from a diverse range of hosts including sheep, koalas, and deer  <cit> .
in the current study, core genome alignment, including all  <dig> available c. pecorum sequences, resolved six clades in the maximum likelihood tree .
the three porcine c. pecorum strains clustered with cattle pv <dig> and a koala sa/k2/ugt strains, forming the first well supported clade .
four primary koala strains resolved into their own exclusive second clade, while the third clade consisted of the european sheep c. pecorum isolates w <dig> and p <dig>  usa polyarthritis c. pecorum ipa isolate formed its own fourth clade, as did the australian sheep rectal nar/s22/rec strain.
the final clade mainly consisted of australian cattle and sheep strains, along with two koala c. pecorum strains  detected as mixed infections with genetically distinct c. pecorum strains  <cit> , and the usa cattle encephalomyelitis e <dig> isolate .fig.
<dig> a mid-point rooted phylogenetic tree, constructed from the 280kbp core genome alignment with phyml  <dig>  using the gtr model, using  <dig> c. pecorum pig, cattle, sheep and koala strains.
bootstrap values were calculated using  <dig> replicates.
bootstrap values >  <dig> are displayed on the tree nodes.
hosts are indicated by the colouring in the legend
sequence-based detection of the c. pecorum plasmid
no plasmids were reported in any of the c. pecorum genomes published prior to this study .
the unexpected finding of a c. pecorum  <dig>  kbp plasmid  in the l <dig> genome prompted us to re-investigate the presence of the plasmid in other sequenced livestock and koala c. pecorum, using the raw reads available in our collection  <cit> .
interestingly, full length plasmid sequences were detected only in the genomic data of c. pecorum mc/marsbar, dbdeug and iptale strains from koala hosts  <cit> .
these additional three plasmid contigs were confirmed based on blast homology search and read mapping to the originally identified l <dig> plasmid.
all identified plasmids shared an identical annotation with eight cdss and four 22 bp tandem repeats , as previously described in related chlamydial plasmids  <cit> .
briefly, cdss  <dig> ,  <dig> ,  <dig> , and  <dig>  were denoted as putative integrase, helicase, and a partioning plasmid proteins, while the cdss  <dig> ,  <dig> ,  <dig> , and  <dig>  were denoted as putative chlamydia-specific plasmid virulence proteins.
the  <dig>  kbp pcpec sequences were  <dig>  % similar to each other and shared an overall sequence similarity of  <dig> – 70 % to other chlamydial plasmids  <cit>  .
the exact function of the pcpec genes and/or their predicted products will require further in vitro investigations.fig.
<dig> sequence alignment of the annotated characterised pcpecs from a pig and three koala c. pecorum strains.
snps are highlighted in black.
on the identity heat map above the alignment, green denotes 100 % sequence similarity while yellow denotes between 30 % to less than 100 % sequence similarity
distribution of the c. pecorum plasmid
to investigate the plasmid distribution in c. pecorum strains, we developed a specific pcr assay that targets a 522 bp pcpec fragment to screen:   <dig> c. pecorum pcr positive samples collected from  <dig> livestock , and   <dig> c. pecorum pcr positive samples from  <dig> koalas from different populations .
as summarised in table  <dig>   <dig>  % of the livestock c. pecorum strains sampled had a plasmid, with the highest detection of  <dig>  % in the porcine isolates .
pcpec was present in  <dig>  % of the sheep c. pecorum strains sampled, with plasmids detected in c. pecorum strains detected at a range of anatomical sites .
in sheep strains, we were further able to observe that  genetically diverse strains  infecting different sites in the same host ) can carry pcpec; and  both pcpec-positive and pcpec-negative genetically diverse strains can circulate in a single flock ).table  <dig> plasmid distribution in c. pecorum positive clinical samples and cultured isolates from pigs, sheep and cattle
in cattle, pcpec was detected in ocular, brain, joint and faecal c. pecorum isolates .
pcpec was less common in the limited number of clinical samples analysed .
a subset of these c. pecorum positive clinical samples, collected from two calves , with sporadic bovine encephalomyelitis  were described previously in a case study  <cit>  where c. pecorum mlst  <cit>  indicated that brain, liver and lung c. pecorum wa/b <dig> and wa/b <dig> strains were of the same c. pecorum genotype  as other sbe isolates such as nsw/bov/sbe and e <dig>  in the present study we observed that all of these samples were pcpec negative, but that a genetically distinct c. pecorum positive ileal sample was pcpec positive.
pcpec was similarly absent in several other c. pecorum sbe isolates .
the absence of pcpec from sbe-associated c. pecorum strains based on this limited pcpec screening may imply that the pcpec is not a virulence factor that affects sbe pathogenesis.
the plasmid was more commonly detected in the koala c. pecorum samples studied, with  <dig>  %,  <dig>  % and  <dig>  % distribution in the samples collected from queensland , new south wales  and victoria , respectively .
in contrast, pcpec was less common in the south australian  koala samples analysed  .
together, this data indicates that the c. pecorum plasmid is not ubiquitous, in contrast to c. trachomatis, where nearly all naturally occurring strains contain the plasmid  <cit> .
in terms of confidence in our detection, we believe that the sensitivity of our pcpec assay was sufficient to detect plasmid even in low c. pecorum  positive samples as the chlamydial plasmid is thought to have between two and  <dig> copies/per chlamydial genome, based on other studies  <cit> .
as outlined in additional file 4: table s <dig> and additional file 6: table s <dig>  the number of c. pecorum genomes for each screened sample ranged from a  <dig> x <dig> to  <dig> x106c.
pecorum genome copies/μl of the extracted dna , while the tested pcpec fragment lower detection limit was 1x <dig> plasmid copies/μl.
indeed, in the present study, there was no apparent relationship between ability to detect plasmid and the number of genome copies in a sample, with plasmid detected in koala a <dig>  r <dig>  r1- <dig> ugt and sheep cur/l236/vag samples with only ~1x102c.
pecorum genome copies .
nevertheless, we do also acknowledge that it is possible that we may have missed detection of pcpec in samples with < 1x102c.
pecorum genome copies if each c. pecorum genome were to be associated with only single pcpec copy.
the reliance on archived samples in the present study precluded observation of trends in disease, tissue and/or host specificity for plasmid distributions; most pcr positive samples or isolates were from diseased animals, sample sizes were small, and sampling was spatially clustered .
as prevalence among such samples may not accurately reflect prevalence in wider populations and small sample sizes would confer very wide confidence intervals on prevalence estimates, it was not possible to compare prevalence across geographic locations, hosts, or disease states with any certainty.
however, the markedly lower apparent prevalence of pcpec in south australian koala samples   relative to koala samples from qld, vic, and nsw  appears worthy of further investigation.
anecdotal evidence and preliminary pcr-based screening studies  <cit>  , suggest that c. pecorum infections in sa may be less common and pathogenic than those described in eastern and northern australian states   <cit> .
similarly low prevalence of chlamydial disease has been proposed for victorian koalas.
in other chlamydial species, there is strong evidence linking the chlamydial plasmid to pathogenic potential.
for example, in the mouse model, it was demonstrated that plasmid deficient c. trachomatis isolates are less infective and less virulent  <cit> .
similarly, c. muridarum studies demonstrated critical role of the plasmid in the development and severity of intrauterine infections  <cit> .
given the multifactorial nature of disease pathogenesis, however, the picture is unlikely to be simple, particularly in the koala.
as such, further studies are required to examine the impact of the presence or absence of this plasmid on c. pecorum infection, growth and pathogenicity.
as an interesting sidenote, in c. trachomatis, it was also observed that the chlamydial plasmids are correlated with the accumulation of glycogen granules inside the inclusion  and that some plasmid loci  may regulate transcription of the corresponding chromosomal genes involved in glycogen pathways  <cit> .
in their  <dig> study  <cit> , kaltenboeck and storz examined the biological properties of the same pig strains that we used for our genomic and plasmid analyses.
inclusions of l <dig>  r <dig>  1710s, and 1920brz pig strains were observed to be aberrant, coarse and patchy, with pleomorphic reticulate bodies, in contrast to other pig strains  with regular inclusions.
in our study we detected plasmid in the same strains , with plasmid fully characterised from l <dig>  while the regular inclusion strains l <dig> and l <dig> were plasmid negative.
given the differing distribution of the c. pecorum plasmid and the otherwise conserved coding sequences of this plasmid relative to the plasmid in other chlamydial species, this study raises important questions over the function of the c. pecorum plasmid.
these questions can only be answered with additional in vitro and in vivo investigations.
CONCLUSIONS
in the present study, we sequenced three c. pecorum genomes from pigs presenting with pneumonia  and polyarthritis , and compared them to genomes of other c. pecorum strains from sheep, cattle and koala.
the main genetic differences were limited to the highly variable pmp region and the c. pecorum pz.
besides the variable number of phospholipase d genes, we also observed a third full copy of the cytotoxin gene  in the pz of the porcine l <dig> genome.
preliminary screening for a toxc fragment in other c. pecorum strains revealed that this additional tox gene could only be found in other porcine strains .
the most significant and novel genomic feature described in this study was the identification of the chlamydial plasmid in c. pecorum.
based on our pcr-based screening approach, this plasmid does not appear to be ubiquitous, raising questions over its function and impact on chlamydial pathogenesis in light of the important role it plays in other chlamydial species such as c. muridarum  <cit> .
it appears possible that the plasmid is less common in strains infecting koalas in south australia, relative to those in other states.
although no association was observed between the presence or absence of the plasmid in association with tissue, host or disease in the present study, differences in the distribution of this plasmid among koala populations and individuals with different levels of chlamydial disease appears to warrant further investigations.
whether the c. pecorum plasmid is a virulence factor or just a “harmless accessory” otherwise, remains to be elucidated.
availability of supporting data
all of the available supporting data from this manuscript is provided as additional files.
additional files
additional file 1: table s <dig>
c. pecorum l <dig> plasmid sequence identity comparisons based on discontiguous megablast hits.
additional file 2: table s <dig>  sequence analyses of the porcine l <dig>  l <dig>  and l <dig> c. pecorum pmp gene families.
additional file 3: figure s <dig>  bayesian phylogenetic analyses of the c. pecorum pmp gene subtypes.
posterior probabilities >  <dig>  are displayed on the tree nodes.
a: c. pecorum pmps a,b, d, e, e, h gene family phylogenies from the  <dig> porcine, sheep, cattle, and koala c. pecorum strains analysed in this study; b: c. pecorum pmpg gene family phylogeny from the  <dig> porcine, sheep, cattle, and koala c. pecorum strains analysed in this study; c: pmpg <dig> phylogenetic tree constructed from the 2406 bp alignment of  <dig> porcine, sheep, cattle, and koala c. pecorum strains analysed in this study; and d: pmpg <dig> phylogenetic tree constructed from the 2841 bp alignment of  <dig> porcine, sheep, cattle, and koala c. pecorum strains analysed in this study.
hosts are indicated by the colouring in the legend.
additional file 4: table s <dig>  characteristics of the polymorphic chromosomal genes under positive selection analysed in the present study from three porcine c. pecorum l <dig>  l <dig>  and l <dig> strains.
additional file 5: figure s <dig>  bayesian phylogenetic analyses of the cytotoxin genes from the subset of  <dig> porcine, sheep, cattle, and koala c. pecorum strains.
posterior probabilities >  <dig>  are displayed on the tree nodes, while c. muridarum nigg tox  <dig> is used as an out-group.
additional file 6: table s <dig>
c. pecorum isolates and clinical samples additional file 4: table s <dig> and additional file 6: table s <dig>
abbreviations
bpbase pairs
cdscoding dna sequence
kbpkilo base pairs
mbpmega base pairs
mlstmulti locus sequence typing
pcrpolymerase chain reaction
pmppolymorphic membrane protein
pzplasticity zone
qpcrquantitative polymerase chain reaction
rrnaribosomal rna
snpsingle nucleotide polymorphism
trnatransfer rna
competing interests
the authors declare that they have no competing interests.
authors’ contributions
mj performed the experimental studies, comparative genomics analyses and prepared the manuscript.
nb assisted with bioinformatics and phylogenetic analyses.
bk and dph maintained the chlamydial strains used in this study.
cw performed samples processing and pcr screening.
lw, kns, ag and cf collected koala samples used in the present study.
gsm assisted with c. pecorum probe design and performed culture-independent genome sequencing.
pt and ap conceived and designed the study and helped write the paper.
all authors have read and approved the final manuscript.
this work was funded by an arc discovery project grant  and a queensland government department of environment and heritage protection koala research grant  awarded to ap and pt.
we thank  alyce taylor-brown and marion desclozeaux for helpful discussions and technical assistance,  mike humphreys, institute of genome sciences, university of maryland for assistance with c. pecorum sequence capture,  tamieka fraser for assisting with koala swab processing and screening,  evelyn walker for providing sheep c. pecorum positive dna samples used in our analyses; and  eileen roulis for her advice on bioinformatics analyses.
we acknowledge the queensland department of transport and main roads, specifically the moreton bay rail link project team, and the dedicated staff at endeavour veterinary ecology for collecting samples from queensland koalas.

 chlamydia pecorum is a globally distributed animal pathogen of domesticated and wild ruminants, and the iconic native australian marsupial, the koala. expansion of the number of available whole genome sequences of c. pecorum from sheep, cattle, and koala hosts has provided insights into its lifestyle, associated virulence factors, and evolution. the genetic differences that may influence host specificity and/or pathogenicity in c. pecorum remain largely unknown, however, with only single nucleotide polymorphisms; a limited number of pseudogenes; and variable numbers of tandem repeats in certain c. pecorum genes, differentiating strains from different hosts or associated with different diseases. in other chlamydial species, the almost ubiquitous non-integrative chlamydial plasmid has been recognised as a major chlamydial virulence factor. this plasmid also has value as a target for molecular typing and as an immunogenic vaccine candidate both for humans and animals such as ducks, pigeons, cats and pigs. two studies have previously detected a c. pecorum plasmid in isolates from three koalas and one bovid, but further evidence for a c. pecorum plasmid has remained absent despite the growing number of genomics studies for this species. although c. pecorum infects a wide range of hosts, including some other australian marsupials, european wild ruminants, and pigs, c. pecorum comparative genomics studies thus far have focused only on sheep, cattle, and koala strains. porcine c. pecorum infections are underestimated as a source of on-farm economic loss for producers, manifesting as a variety of diseases such as pneumonia, pleuritis, polyserositis, polyarthritis, conjunctivitis, and enteritis. as with other hosts, c. pecorum subclinical infections in pigs are also common. in an effort to broaden our knowledge of the genetic diversity of c. pecorum in other hosts, in the present study, we sequenced three porcine c. pecorum genomes and compared them to the available sheep, cattle, and koala c. pecorum genomes. in doing so, we also provide the first detailed description of the genetic structure and distribution of the c. pecorum plasmid, a genetic feature associated with virulence in other chlamydial species. methods descriptions of pig c. pecorum isolates, clinical samples and other c. pecorum strains used in this study the three c. pecorum strains l l and l isolated from pigs presenting with pneumonia and polyarthritis were utilised for whole genome sequencing and plasmid analyses in the present study. these strains were one of the first pig chlamydial strains, isolated together with many others in in austria, from pigs presenting with chlamydiosis during a mass chlamydial outbreak. plasmid screening was performed on a large collection of previously tested c. pecorum pcr positive clinical samples from sheep, cattle and koalas; and a small collection of cultured c. pecorum isolates from a variety of hosts; additional previously untested but c. pecorum pcr positive sheep and koala samples identified in this study. this latter testing, combined with our previous screening for c. pecorum dna in clinical samples, resulted in c. pecorum pcr positive ocular and urogenital swab samples from koalas from different populations across australia; ocular, joint, vaginal and rectal swabs collected from sheep from nine different flocks from the central nsw region in australia; and ocular, nose, internal organs, and rectal samples from four cattle from two herds, one previously described from western australia and a second from central nsw. cultured c. pecorum isolates from pigs, sheep, cattle, and koalas screened are listed in additional file 4: table s genomic dna extraction and c. pecorum-specific qpcr screen new sheep, cattle and koala clinical swab samples used in this study were processed as previously described. the cultures of three c. pecorum porcine isolates l l and l were propagated in chicken embryos and purified, as previously described, while the cultures of koala isolates were propagated in hep- and/or mccoy cells and semi-purified using a probe sonication and density gradient centrifugation, as previously described. dna extraction for both samples and cultures was performed using the qiamp dna mini kit, as per the manufacturer’s instructions. c. pecorum infectious load and/or presence were determined using a c. pecorum species-specific qpcr assay targeting 202 bp of 16s rdna. samples with <  copies c. pecorum 16s rdna were considered negative. dna concentration for each sample was also measured in duplicates on a nanodrop. additionally, 1 μl of extracted, screened and quantified gdna of three porcine c. pecorum samples were run on a 1 % tbe agarose gel to confirm high molecular weight dna prior to whole genome sequencing. sequence capture, whole genome sequencing and assembly sequence capture was performed on total dna extracted from c. pecorum l l and l cultures with the c. pecorum custom-designed probes, using a dna hybridisation capture and amplification process based on the methods described elsewhere. l l and l genomes were sequenced at the institute for genome sciences, baltimore, usa, using the illumina hiseq platform producing paired-end base-pair reads. read quality, de novo assembly and read-mapping of paired-end reads from l l and l was performed using clc genomics workbench, after filtering and trimming for size and quality. the read mapping was visualised with blast ring image generator software. after de novo assembly, l l and l contigs were blasted to confirm identity using blastn and ordered against complete c. pecorum e genome with progressive mauve to produce single genome scaffold. genome annotations were done using rast and the igs prokaryotic annotation pipeline, assisted by mapping onto the c. pecorum e type strain sequence, as previously described. number and distribution of snps was determined using the probabilistic variant detection plug-in with default settings with l l and l reads mapped to a reference genome, as implemented in clc genomics workbench. general descriptions and accession numbers of l l and l genomes were outlined in table  macroscopic comparative genomics and phylogenetic analyses using new porcine genomes alongside the newly sequenced three porcine c. pecorum genomes, we also used publicly available genome sequences of c. pecorum from: cattle encephalomyelitis e; sheep arthritis p, sheep fecal w, cattle metritis pv3056/; koala cystitis mc/marsbar and dbdeug, koala conjunctivitis iptale, sheep polyarthritis ipa; cattle encephalomyelitis nsw/bov/sbe, sheep polyarthritis mer/ovi1/jnt, sheep conjunctivitis nar/s22/rec, koalas with cystitis gun/koa1/ure and sa/k2/ugt for whole genome comparative analyses. pairwise comparative genomic analyses were performed in act, the artemis comparison tool and geneious. using alignments generated with progressive mauve. polymorphic loci and/or regions of interest in the genomes were extracted and further aligned with clustalx. dnasp was used to analyse the level of sequences polymorphisms by: determining the ratio of non-synonymous to synonymous substitutions; calculating the number of polymorphic sites and haplotype diversity. further, if a polymorphism resulted in a non-synonymous amino acid change or premature stop codon in the loci of interest in each of the analysed c. pecorum genome sequence, the translated sequence was further analysed in blast for comparison,uniprot and conserved domains database to assess protein functionality. figures of the genomic regions of interest, such as the pz was generated using easyfig, based on tblastx comparison. construction of phylogenetic relationships between the loci was performed using mrbayes as implemented in geneious. in addition to the subset of c. pecorum genomes used for comparative analyses, for phylogenetic analyses we have included: sheep nar/s22/re; as well as the two secondary koala c. pecorum strains identified in the same study. conserved regions were identified in c. pecorum genomes using all-versus-all blast search. syntenic regions were individually aligned using muscle and concatenated to produce a ~  kbp core genome alignment. poorly aligned regions were removed from the alignment using gblocks with the minimum length of a block set to and no gap positions were allowed. recombination regions were predicted using gubbins and subsequently filtered from the alignment. a mid-point rooted phylogenetic tree was constructed from the genome alignment with phyml using the gtr model. bootstrap values were calculated using replicates. pcr amplification and phylogenetic analyses of toxc during pairwise comparative genomic analyses, the presence of a third copy of the cytotoxin gene in porcine l genome was identified. primers and toxc rev ) were designed to amplify a 644 bp fragment of the toxc gene in order to confirm the presence of this sequence. using purified l genomic dna as a template, and purified c. pecorum koala mc/marsbar and iptale, cattle e porcine l l and sheep ipa genomic dna as negative controls, conventional pcr assay was performed to amplify the 644 bp toxc fragment. pcr assays for 644 bp toxc fragment were prepared to a total reaction volume of 50 μl including 1x amplitaq gold® master mix,  μm of forward and reverse primer each and 3 μl dna template. the cycling conditions included an initial denaturation followed by 35 cycles of denaturation, annealing and extension, followed by a final extension. the amplicon was then purified and dideoxy sequenced. after successful amplification and confirmation of the toxc fragment in l only, we tested the remaining eight porcine, seven cattle, two sheep and five koala c. pecorum isolates for the presence of the toxc 644 bp fragment sequence. identification of the full length l toxc sequence was checked in blast and the midpoint rooted bayesian phylogeny was constructed using alignment of all three cytotoxin genes from a subset of c. pecorum analysed in this study. the phylogenetic tree was constructed with mrbayes as implemented in geneious. using the hky + i model with four markov chain monte carlo chains with a generations, sampled every generations and with the first trees were discarded as burn-in. c. muridarum nigg three cytotoxin gene sequences were also included in the alignment for phylogenetic analyses, with the c. muridarum nigg tox used as an out-group. c. pecorum plasmid contig identification the l kbp plasmid contig was annotated with rast, and further curated in geneious. additionally, pcpec l sequence was also blasted against raw reads of c. pecorum porcine l and l sheep ipa, cattle nsw/bov/sbe, and koala mc/marsbar, dbdeug, iptale, sa/k2/ugt and gun/k1/ure. pcpec kbp sequence was only identified in the three koala c. pecorum mc/marsbar, dbdeug and iptale raw reads and also used in this study. pcpec conv. pcr confirmatory assay design newly identified pcpec sequences from a porcine l and three koala c. pecorum genomes were aligned using clustalx. primer pair was designed to amplify a 522 bp plasmid fragment which includes a conserved intergenic region and a part of the plasmid gene cds primer sequences were tested in silico for secondary structures and dimerisations at integrated dna technologies, inc oligoanalyzer online tool. additionally, plasmid isolation was performed from fresh available koala c. pecorum mc/mars, dbdeug, and iptale cultures. plasmid isolation and purification was performed using genejet plasmid midiprep reactions, thermofisher scientific, victoria, australia, as per manufacturer’s instructions. freshly extracted plasmids were digested with neb saci and visualised on the ethidium bromide stained  % gel, using neb 2-log dna ladder. using purified porcine l genomic dna and extracted plasmid dna from koala c. pecorum mc/mars, dbdeug, and iptale as templates and positive controls, conventional pcr to amplify 522 bp plasmid fragment was performed. in the same assay we used purified pig l and l genomic dna as negative controls. the plasmid primers were additionally tested against genomic avian c. psittaci dna extracted from a cultured isolate in order to test for unspecified primer binding. after successful amplification of the 522 bp plasmid fragments from templates only, the presence of the amplicon was confirmed on  % tbe gel, purified and sent off for confirmatory dideoxy sequencing. we tested the detection limit of our pcpec screening assay, based on a conventional pcr using: i) serially diluted pcpec from to copies/μl as a template in triplicate; and ii) c. pecorum positive clinical sample bella ugt, with 106c. pecorum genome copy number/μl diluted to 101c. pecorum genome copy number/μl, as a template in duplicate. the detection limit for pcpec assay used in the present study was from to pcpec fragment copies/μl, as determined by amplicon visualisation on  % tbe gel. pcr-based pcpec screening after confirming the 522 bp amplicon sequence, we applied the above described assay to screen for plasmid presence in koala and livestock c. pecorum samples. the full lists of screened samples are outlined in additional file 4: table s and additional file 6: table s the testing of these swab samples, collected as a part of routine diagnostic investigations of koalas and livestock, has been considered by the university of sunshine coast animal ethics committee and the requirement for ethics approval was waived. pcr assays for screening 522 bp pcpec fragment were prepared to a total reaction volume of 50 μl including 1x amplitaq gold® master mix,  μm of forward and reverse primer each and 3 μl dna template. negative and positive controls were included in each amplification assay. the cycling conditions included an initial denaturation followed by 35 cycles of denaturation, annealing and extension, followed by a final extension. upon amplification, pcr products were detected on a 2 % ethidium bromide agarose gel and visualised under an uv transilluminator and purified, using a high pure pcr product purification kit. a subset of pcr products was directly sequenced using a bigdye terminator v cycle sequencing kit and subsequently purified according to the manufacturer’s instructions. sequencing was performed at the institute for future environments, queensland university of technology, brisbane, australia using the applied biosystems abi gene analyser.  porcine c. pecorum l l and l genome assemblies using c. pecorum-specific sequence capture probes, we acquired whole genome sequences for three porcine c. pecorum isolates. c. pecorum strains l and l were isolated from the lungs of two different pigs presenting with pneumonia, while the l strain was a joint isolate from a case of polyarthritis. consistent with the high average read depth, de novo assembly resolved the genomes of these c. pecorum isolates into – contigs for each isolate. read-mapping to a reference c. pecorum e genome confirmed almost whole chromosome coverage for all three genomes. similar to the previously described c. pecorum genomes, our annotation pipelines predicted ~  coding dna sequences, including the three rrna and trna genes. the draft genome sizes of ~ mbp are almost identical to all other available c. pecorum genomes, with the exception of l which has a slightly larger draft genome size of mbp.table general description of pig c. pecorum l l and l samples and their genomes interestingly, we also identified a distinct kbp contig, assembled only from c. pecorum l strain reads. blast searches revealed that this contig shared 70 % sequence identity to other related chlamydial species plasmids and 99 % sequence identity to a 710 bp sequence previously identified from a suspected c. pecorum plasmid, based on blastn and discontiguous megablast blast searches. the annotation of this l kbp contig resulted in eight cdss specific for chlamydial plasmids. sequence capture methodology continues to be an effective tool for acquiring high quality whole genome sequence data, especially when cultured isolate gdna of high concentration and genome copy number is used as a template, as in the present study. high read depths, complete genome coverage, and sequence homogeneity for our three pig strains was not surprising, as they were all pure cultured isolates, contrasting the sequence heterogeneity previously seen in c. pecorum-positive clinical swab studies. interestingly, design of the c. pecorum species-specific probes also allowed for identification of distinct and/or novel sequences, such as the full length plasmid contig detected in the l draft genome. now that whole genome sequences for porcine c. pecorum are available, the use of c. pecorum-specific rna probes for sequence capture of c. pecorum dna can be further improved in order to reveal features not previously observed or investigated, and to improve coverage of the polymorphic genomic regions in this species. re-evaluating the genetic differences between c. pecorum strains the previously described c. pecorum gene order and synteny remained conserved in the porcine c. pecorum genomes with nearly all variation, once again, limited to snps. comparison of the porcine l l and l genome sequences against those from other hosts revealed the most snps between our c. pecorum porcine strains and the genomes of c. pecorum strains previously sequenced from sheep. the closest c. pecorum strains from other hosts, on the other hand, were from a cow and a koala with approximately and snps, respectively. interestingly, the majority of koala c. pecorum strains differed from porcine strains by ~  snps, less than sheep and cattle strains. pairwise comparison of the genome sequences revealed that the majority of snps were found in two major clusters, including the plasticity zone; and the major polymorphic membrane gene cluster.fig. snp distribution and identification of genomic elements across the porcine l genome in comparison to cattle nsw/bov/sbe and pv sheep mer/ovi1/jnt, porcine l and l and koala iptale genomes. all genomes start from malate dehydrogenase gene. genomic location of type secretion system gene clusters, major outer membrane protein, pyrimidine genes, polymorhic membrane protein gene clusters, inclusion proteins genes, tryptophan and biotin operon genes are denoted on the horizontal line above the histograms. variants, represented as histograms of a 10kbp size, were determined in clc genomics platform not surprisingly, averaging at ~ snps/10kbp, the pmp gene regions were a major contributor of single nucleotide differences. overall sequence similarity of different c. pecorum pmp gene families from the porcine, sheep, cattle, and koala strains ranged from – 100 %. the bayesian phylogenetic analyses resolved the pmp a, b, e, e’, d, and h gene families in clades respective to their designated gene family, while the pmpg family was the most divergent, resolving each of the eight pmpg subtypes into its own diverse clade. porcine c. pecorum l l and l strains had an identical genomic organisation, subtype and number of predicted pmp genes as the previously characterised sheep, cattle, and koala strains. sequence analyses of each of the pmp genes from the porcine c. pecorum strains, indicated that only pmpg and pmpg type appear to be under positive selection pressure, with observed dn/ds ratios of, and, respectively. subsequent sequence analyses of the pmpg and pmpg from all c. pecorum strains analysed in this study, also returned dn/ds ratios >  interestingly, pmp g and g phylogenies were similar to the phylogenetic relationships constructed from the core genomes of the c. pecorum strains analysed in this study, as well as the previous study. in the recent sheep, cattle and koala c. pecorum comparative genomic study, the pmpg region was also identified as the most diverse genomic region, however, most of the genetic variation observed resulted in synonymous substitutions. in our study, variation in the pmp genes also mainly resulted in synonymous snps, with the exception of c. pecorum pmp genes g and g which appeared to be under positive selection. predicted to have a major role in chlamydial infection due to their adhesive function in the interaction with host, pmp genes are predicted to maintain some hypervariability in order to evade immune defences. rapid diversification and evolution of the pmpg genes family has been previously observed in the genomic studies of the related ovine pathogen c. abortus, and avian pathogen c. psittaci, where it has been suggested that these genes may play a role in adaptation to different hosts and environments. the observed positive selection on these genes in the c. pecorum genomes may support a role in host adaptation for this pathogen, an aspect that may warrant extended investigation. while the remaining genetic variation was evenly distributed across the porcine c. pecorum chromosomes, we identified additional eight genes under positive selection in the polymorphic regions. as outlined in additional file 4: table s cpl1_ and cpl1_ were both identified as effectors of the chlamydial type secretion system, a system of structural, chaperone and secreted effector proteins with its main function in host cell manipulation and subversion of the host cellular processes. the cpl1_ was predicted to be orthologous to a c. psittaci secreted t3ss protein, while the cpl1_ was a well-studied chlamydial t3ss translocated actin-recruiting protein homologue. in the porcine c. pecorum genomes, although polymorphic, the remaining t3ss genes were under negative selection. positive selection on the c. pecorum tarp homologue is perhaps not surprising, as the chlamydial tarp was previously recognised as an important antigenic protein. further, genomic studies of the c. psittaci and c. trachomatis revealed that the variation in the t3ss effector genes could also contribute to the differences in virulence, and host and/or tissue tropism associated with these pathogens. presently the exact role of t3ss effectors in c. pecorum infections remains largely unknown, and will require more comprehensive in silico as well as cell biology analyses comprising of isolates from healthy, as well as diseased hosts to fully dissect t3ss effector roles. the remaining genes under positive selection were: cpl1_ identified as a homologue of a previously described c. pecorum surface binding protein gene, also under positive selection in koala strains; cpl1_ a homologue of a macro domain protein; and four genes encoded conserved chlamydial hypothetical proteins of unknown function. variation in the c. pecorum pz in addition to the analysed pmp genes, the pz, spanning from inosine-5'-monophosphate dehydrogenase to acetyl-coa carboxylase, also displayed significant variability. the pz is a region of focus in chlamydial comparative genomic studies due to the presence and/or absence of a range of established chlamydial virulence factors. in our study, like other c. pecorum strains, the porcine c. pecorum pzs were found to contain: three purine biosynthesis genes, a mac/perforin, a variable number of phospholipase d genes, two copies of cytotoxin genes, and two acetyl-coa-carboxylase genes. some notable genetic variation, however, was also observed in these porcine c. pecorum strains including major differences in the number of pld genes and; the presence of a third copy of the cytotoxin gene in the l strain, as summarised in fig.  in terms of pld genes, five were observed in l a comparable number to that identified in the genome sequences of c. pecorum strains from sheep, koalas and cattle. the previously sequenced bovine pv3056/ isolate also had four plds like l while the porcine l isolate is presently the only c. pecorum strain with six plds. the variable number of plds is not unique to c. pecorum, as related species such as c. trachomatis and c. muridarum have also been shown to have a variable number of pld genes among strains. it has been suggested that the number of pld genes could influence virulence, but whether this is the case for c. pecorum is still unclear, as the majority of the sequenced c. pecorum were sampled from hosts with evidence of chlamydial disease.fig. graphical representation of the c. pecorum cattle nsw/bov/sbe and pv sheep mer/ovi1/jnt, pigs l l and l and koala sa/k2/ugt plasticity zones tblastx comparisons. the coloured arrows represent pz genes according to their function, as outlined in the legend. the intensity of grey shaded areas corresponds to the sequence identity. the image was generated with easyfig in terms of cytotoxin genes, almost all of the analysed strains had two copies of the cytotoxin gene, with observed toxa truncation in c. pecorum cattle nsw/bov/sbe and koala sa/k2/ugt strains. the porcine l strain however, had three copies of the cytotoxin gene. among the genus chlamydia, only c. muridarum has three copies of the cytotoxin gene. sequences from this latter species were included as an out-group in the tox phylogenetic tree to investigate whether the third tox copy identified in the l is similar to the c. muridarum third tox copy. analyses of the c. pecorum cytotoxin genes revealed that l toxc is more similar to c. pecorum toxa than toxb. a mid-point rooted bayesian phylogenetic tree of c. pecorum toxa and toxb sequences with tox sequences from l and c. muridarum further revealed that the l toxc may be a duplication of the c. pecorum toxa, as evidenced by the clustering of this gene with c. muridarum tox and other c. pecorum toxa genes. to see if toxc could be found in strains from other hosts, we designed a toxc-specific pcr to screen c. pecorum strains from pigs, sheep, cattle, and koalas. this toxc fragment was detected only in three more isolates: porcine pneumonia l l and hslurz. these strains were isolated from the same chlamydial outbreak in among austrian pigs as the l l and l strains and the remaining five porcine strains. thus, based on our genomic and preliminary toxc analyses, it appears that the c. pecorum outbreak among austrian pigs was polyclonal, with strains involved having two or three cytotoxin genes. variability in the number of tox genes and our previously noted truncation of certain tox genes in some c. pecorum strains, raises the question over the function and impact of this gene in c. pecorum virulence. expansion on c. pecorum phylogenetic relationships using a c. pecorum multi locus sequence typing scheme, we recently showed that porcine c. pecorum strains clustered in a clade separate to that of c. pecorum strains from a diverse range of hosts including sheep, koalas, and deer. in the current study, core genome alignment, including all available c. pecorum sequences, resolved six clades in the maximum likelihood tree. the three porcine c. pecorum strains clustered with cattle pv and a koala sa/k2/ugt strains, forming the first well supported clade. four primary koala strains resolved into their own exclusive second clade, while the third clade consisted of the european sheep c. pecorum isolates w and p usa polyarthritis c. pecorum ipa isolate formed its own fourth clade, as did the australian sheep rectal nar/s22/rec strain. the final clade mainly consisted of australian cattle and sheep strains, along with two koala c. pecorum strains detected as mixed infections with genetically distinct c. pecorum strains, and the usa cattle encephalomyelitis e isolate.fig. a mid-point rooted phylogenetic tree, constructed from the 280kbp core genome alignment with phyml using the gtr model, using c. pecorum pig, cattle, sheep and koala strains. bootstrap values were calculated using replicates. bootstrap values >  are displayed on the tree nodes. hosts are indicated by the colouring in the legend sequence-based detection of the c. pecorum plasmid no plasmids were reported in any of the c. pecorum genomes published prior to this study. the unexpected finding of a c. pecorum kbp plasmid in the l genome prompted us to re-investigate the presence of the plasmid in other sequenced livestock and koala c. pecorum, using the raw reads available in our collection. interestingly, full length plasmid sequences were detected only in the genomic data of c. pecorum mc/marsbar, dbdeug and iptale strains from koala hosts. these additional three plasmid contigs were confirmed based on blast homology search and read mapping to the originally identified l plasmid. all identified plasmids shared an identical annotation with eight cdss and four 22 bp tandem repeats, as previously described in related chlamydial plasmids. briefly, cdss,,, and were denoted as putative integrase, helicase, and a partioning plasmid proteins, while the cdss,,, and were denoted as putative chlamydia-specific plasmid virulence proteins. the kbp pcpec sequences were  % similar to each other and shared an overall sequence similarity of – 70 % to other chlamydial plasmids. the exact function of the pcpec genes and/or their predicted products will require further in vitro investigations.fig. sequence alignment of the annotated characterised pcpecs from a pig and three koala c. pecorum strains. snps are highlighted in black. on the identity heat map above the alignment, green denotes 100 % sequence similarity while yellow denotes between 30 % to less than 100 % sequence similarity distribution of the c. pecorum plasmid to investigate the plasmid distribution in c. pecorum strains, we developed a specific pcr assay that targets a 522 bp pcpec fragment to screen: c. pecorum pcr positive samples collected from livestock, and c. pecorum pcr positive samples from koalas from different populations. as summarised in table   % of the livestock c. pecorum strains sampled had a plasmid, with the highest detection of  % in the porcine isolates. pcpec was present in  % of the sheep c. pecorum strains sampled, with plasmids detected in c. pecorum strains detected at a range of anatomical sites. in sheep strains, we were further able to observe that genetically diverse strains infecting different sites in the same host ) can carry pcpec; and both pcpec-positive and pcpec-negative genetically diverse strains can circulate in a single flock ).table plasmid distribution in c. pecorum positive clinical samples and cultured isolates from pigs, sheep and cattle in cattle, pcpec was detected in ocular, brain, joint and faecal c. pecorum isolates. pcpec was less common in the limited number of clinical samples analysed. a subset of these c. pecorum positive clinical samples, collected from two calves, with sporadic bovine encephalomyelitis were described previously in a case study where c. pecorum mlst indicated that brain, liver and lung c. pecorum wa/b and wa/b strains were of the same c. pecorum genotype as other sbe isolates such as nsw/bov/sbe and e in the present study we observed that all of these samples were pcpec negative, but that a genetically distinct c. pecorum positive ileal sample was pcpec positive. pcpec was similarly absent in several other c. pecorum sbe isolates. the absence of pcpec from sbe-associated c. pecorum strains based on this limited pcpec screening may imply that the pcpec is not a virulence factor that affects sbe pathogenesis. the plasmid was more commonly detected in the koala c. pecorum samples studied, with  %,  % and  % distribution in the samples collected from queensland, new south wales and victoria, respectively. in contrast, pcpec was less common in the south australian koala samples analysed. together, this data indicates that the c. pecorum plasmid is not ubiquitous, in contrast to c. trachomatis, where nearly all naturally occurring strains contain the plasmid. in terms of confidence in our detection, we believe that the sensitivity of our pcpec assay was sufficient to detect plasmid even in low c. pecorum positive samples as the chlamydial plasmid is thought to have between two and copies/per chlamydial genome, based on other studies. as outlined in additional file 4: table s and additional file 6: table s the number of c. pecorum genomes for each screened sample ranged from a x to x106c. pecorum genome copies/μl of the extracted dna, while the tested pcpec fragment lower detection limit was 1x plasmid copies/μl. indeed, in the present study, there was no apparent relationship between ability to detect plasmid and the number of genome copies in a sample, with plasmid detected in koala a r r1- ugt and sheep cur/l236/vag samples with only ~1x102c. pecorum genome copies. nevertheless, we do also acknowledge that it is possible that we may have missed detection of pcpec in samples with < 1x102c. pecorum genome copies if each c. pecorum genome were to be associated with only single pcpec copy. the reliance on archived samples in the present study precluded observation of trends in disease, tissue and/or host specificity for plasmid distributions; most pcr positive samples or isolates were from diseased animals, sample sizes were small, and sampling was spatially clustered. as prevalence among such samples may not accurately reflect prevalence in wider populations and small sample sizes would confer very wide confidence intervals on prevalence estimates, it was not possible to compare prevalence across geographic locations, hosts, or disease states with any certainty. however, the markedly lower apparent prevalence of pcpec in south australian koala samples relative to koala samples from qld, vic, and nsw appears worthy of further investigation. anecdotal evidence and preliminary pcr-based screening studies, suggest that c. pecorum infections in sa may be less common and pathogenic than those described in eastern and northern australian states. similarly low prevalence of chlamydial disease has been proposed for victorian koalas. in other chlamydial species, there is strong evidence linking the chlamydial plasmid to pathogenic potential. for example, in the mouse model, it was demonstrated that plasmid deficient c. trachomatis isolates are less infective and less virulent. similarly, c. muridarum studies demonstrated critical role of the plasmid in the development and severity of intrauterine infections. given the multifactorial nature of disease pathogenesis, however, the picture is unlikely to be simple, particularly in the koala. as such, further studies are required to examine the impact of the presence or absence of this plasmid on c. pecorum infection, growth and pathogenicity. as an interesting sidenote, in c. trachomatis, it was also observed that the chlamydial plasmids are correlated with the accumulation of glycogen granules inside the inclusion and that some plasmid loci may regulate transcription of the corresponding chromosomal genes involved in glycogen pathways. in their study, kaltenboeck and storz examined the biological properties of the same pig strains that we used for our genomic and plasmid analyses. inclusions of l r 1710s, and 1920brz pig strains were observed to be aberrant, coarse and patchy, with pleomorphic reticulate bodies, in contrast to other pig strains with regular inclusions. in our study we detected plasmid in the same strains, with plasmid fully characterised from l while the regular inclusion strains l and l were plasmid negative. given the differing distribution of the c. pecorum plasmid and the otherwise conserved coding sequences of this plasmid relative to the plasmid in other chlamydial species, this study raises important questions over the function of the c. pecorum plasmid. these questions can only be answered with additional in vitro and in vivo investigations.  in the present study, we sequenced three c. pecorum genomes from pigs presenting with pneumonia and polyarthritis, and compared them to genomes of other c. pecorum strains from sheep, cattle and koala. the main genetic differences were limited to the highly variable pmp region and the c. pecorum pz. besides the variable number of phospholipase d genes, we also observed a third full copy of the cytotoxin gene in the pz of the porcine l genome. preliminary screening for a toxc fragment in other c. pecorum strains revealed that this additional tox gene could only be found in other porcine strains. the most significant and novel genomic feature described in this study was the identification of the chlamydial plasmid in c. pecorum. based on our pcr-based screening approach, this plasmid does not appear to be ubiquitous, raising questions over its function and impact on chlamydial pathogenesis in light of the important role it plays in other chlamydial species such as c. muridarum. it appears possible that the plasmid is less common in strains infecting koalas in south australia, relative to those in other states. although no association was observed between the presence or absence of the plasmid in association with tissue, host or disease in the present study, differences in the distribution of this plasmid among koala populations and individuals with different levels of chlamydial disease appears to warrant further investigations. whether the c. pecorum plasmid is a virulence factor or just a “harmless accessory” otherwise, remains to be elucidated. availability of supporting data all of the available supporting data from this manuscript is provided as additional files. additional files additional file 1: table s c. pecorum l plasmid sequence identity comparisons based on discontiguous megablast hits. additional file 2: table s sequence analyses of the porcine l l and l c. pecorum pmp gene families. additional file 3: figure s bayesian phylogenetic analyses of the c. pecorum pmp gene subtypes. posterior probabilities >  are displayed on the tree nodes. a: c. pecorum pmps a,b, d, e, e, h gene family phylogenies from the porcine, sheep, cattle, and koala c. pecorum strains analysed in this study; b: c. pecorum pmpg gene family phylogeny from the porcine, sheep, cattle, and koala c. pecorum strains analysed in this study; c: pmpg phylogenetic tree constructed from the 2406 bp alignment of porcine, sheep, cattle, and koala c. pecorum strains analysed in this study; and d: pmpg phylogenetic tree constructed from the 2841 bp alignment of porcine, sheep, cattle, and koala c. pecorum strains analysed in this study. hosts are indicated by the colouring in the legend. additional file 4: table s characteristics of the polymorphic chromosomal genes under positive selection analysed in the present study from three porcine c. pecorum l l and l strains. additional file 5: figure s bayesian phylogenetic analyses of the cytotoxin genes from the subset of porcine, sheep, cattle, and koala c. pecorum strains. posterior probabilities >  are displayed on the tree nodes, while c. muridarum nigg tox is used as an out-group. additional file 6: table s c. pecorum isolates and clinical samples additional file 4: table s and additional file 6: table s abbreviations bpbase pairs cdscoding dna sequence kbpkilo base pairs mbpmega base pairs mlstmulti locus sequence typing pcrpolymerase chain reaction pmppolymorphic membrane protein pzplasticity zone qpcrquantitative polymerase chain reaction rrnaribosomal rna snpsingle nucleotide polymorphism trnatransfer rna competing interests the authors declare that they have no competing interests. authors’ contributions mj performed the experimental studies, comparative genomics analyses and prepared the manuscript. nb assisted with bioinformatics and phylogenetic analyses. bk and dph maintained the chlamydial strains used in this study. cw performed samples processing and pcr screening. lw, kns, ag and cf collected koala samples used in the present study. gsm assisted with c. pecorum probe design and performed culture-independent genome sequencing. pt and ap conceived and designed the study and helped write the paper. all authors have read and approved the final manuscript. this work was funded by an arc discovery project grant and a queensland government department of environment and heritage protection koala research grant awarded to ap and pt. we thank alyce taylor-brown and marion desclozeaux for helpful discussions and technical assistance, mike humphreys, institute of genome sciences, university of maryland for assistance with c. pecorum sequence capture, tamieka fraser for assisting with koala swab processing and screening, evelyn walker for providing sheep c. pecorum positive dna samples used in our analyses; and eileen roulis for her advice on bioinformatics analyses. we acknowledge the queensland department of transport and main roads, specifically the moreton bay rail link project team, and the dedicated staff at endeavour veterinary ecology for collecting samples from queensland koalas.
using this novel c. pecorum plasmid 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. to date, comparative genomics of c. pecorum strains from sheep, cattle and koalas has revealed that only single nucleotide polymorphisms and a limited number of pseudogenes appear to contribute to the genetic diversity of this pathogen. comparative genomics of the newly sequenced porcine c. pecorum genomes revealed an increased average number of snp differences between porcine and sheep, cattle, and koala c. pecorum strains, compared to previous c. pecorum genome analyses. pcpec screening showed that the pcpec is common in all hosts of c. pecorum, however not all c. pecorum strains carry pcpec. the pairwise comparative genomic analyses of 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. 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 were acquired using c. pecorum-specific sequence capture probes with culture-independent methods, and assembled in clc genomics workbench. we also identified a third copy of the chlamydial cytotoxin gene, found only in porcine c. pecorum isolates. chlamydia pecorum is a globally recognised pathogen of livestock and koalas. three porcine c. pecorum genomes were sequenced using c. pecorum-specific sequence capture probes with culture-independent methods. 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 c. pecorum samples from pig, sheep, cattle and koala hosts. no chlamydial plasmid has been detected in these strains despite its ubiquitous presence in almost all other chlamydial species. c. pecorum plasmid characterisation may aid in improving our understanding of c. pecorum pathogenesis across the variety of host species this animal pathogen infects. pecorum comparative genomicscytotoxin genephylogenetic relationshipsissue-copyright-statement© the author 2015 phylogenetic analyses clustered porcine isolates into a distinct clade, highlighting the polyphyletic origin of c. pecorum in livestock. genomic analyses have not previously included c. pecorum from porcine hosts. most surprising, we also discovered a plasmid in the porcine c. pecorum genome. this study provides further insight into the complexity of c. pecorum epidemiology and novel genomic regions that may be linked to host specificity.
 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 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. methods whole genome sequences for the three porcine c. pecorum isolates 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 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 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 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 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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords c. pecorum plasmidporcine hostsc. pecorum comparative genomicscytotoxin genephylogenetic relationshipsissue-copyright-statement© the author 2015 