| Chlamydia spp.are the obligate intracellular gram-negative bacteria with unique biphasic developmental cycle,which have ribosomes and cell walls,containing DNA and RNA.The family Chlamydiaceae with its single genus Chlamydia?C.?currently contains 11 recognized species,namely C.trachomatis,C.abortus,C.pneumoniae,C.psittaci,C.suis,C.pecorum,C.muridarum,C.caviae,C.felis,C.avium and C.gallinacea.Chlamydia spp.have wide host spectrum and can cause significant economic losses to the livestock and poultry industry,which are also associated with enormous influences on public health security for potential zoonotic transmission to human.The Chlamydia spp.which can potentially infect pigs include C.suis,C.pecorum,C.abortus and C.psittaci.Swine chlamydial disease in China replies on mainly serology tests,including IHA,ELISA and CFT,which can only determine if there is any Chlamydia infection,however,can not differentiate different chlamydial species.Researchers in China have begun to detect Chlamydia by molecular biological methods,such as multiple nested PCR and fluorescent quantitative PCR.C.suis infections in pigs are mainly associated with subclinical infections;however,the infections have also been found in association with respiratory disease,diarrhea,conjunctivitis and reproductive failure.To analyze genetic diversity of C.suis in China,2,137 samples of pigs collected in 19 regions from ten provinces of China was detected by the Chlamydia FRET-qPCR.C.suis was found to be the only chlamydial species in all samples in this study with an overall positivity of 62.4%?1,334/2,137?.In this study,three sets of primers were designed to specifically amplify the ompA gene of C.suis,C.abortus and C.pecorum,along with a set of specific primers for C.psittaci established by our laboratory before,which can be used to further study the polymorphism of Chlamydia and verify our findings.We obtained 77 distinct partial ompA sequences encompassing the variable domain 1 and 2?VD1-2?from 108 pig C.suis strains.When compared with 50 global ompA sequences,phylogenetic analyses showed that the Chinese C.suis ompA VD1-2 sequences could be resolved into four major clades of the global ompA VD1-2 phylogram,suggesting that the global diversity in C.suis strains is captured in the Chinese pig population.In this study,a C.suis-specific MLST typing scheme based on a previously publishedChlamydiales MLST scheme was developed.Seven sets of primers for gatA,oppA3,hflX,gidA,enoA,hemN and fumC were designed to specifically amplify and sequence 27 C.suis isolates.In this study,we obtained 110 novel MLST alleles and 26 novel STs,and each ST has a length of 3,080bp.Total number of polymorphic sites for concatenated MLST sequences was 136 with most resulting mainly in synonymous substitution.C.suis house-keeping genes are under purifying selection with dN/dS ratios<1,indicating that 7 house-keeping genes were suitable for the MLST typing system.Each MLST allele had 3 to 8 predicted putative recombination events.To assess the phylogenetic relationships between the Chinese and other global C.suis strains,a mid-point rooted Bayesian phylogenetic tree was constructed from an alignment of a total of 40 strains from the 26 C.suis strains from this study,and 14 additional strains from Switzerland,USA,Italy and Austria.Using C.trachomatis as an out-group,the Chinese isolates resolved into seven diverse larger clades,clustering on their own or with European and US isolates.In their own well-supported clades,Chinese C.suis strains formed many distinct lineages.When clustered with European and the USA isolates within a larger clade,Chinese isolates also grouped in separate but diverse sub-clades.Using a relaxed fit where STs must share at least 4/7 alleles,the 26 STs clustered into five clonal complexes.The C.suis STs cluster analyses and minimum spanning tree also confirmed their high ST diversity.In order to further compare phylogenetic relationships of C.suis strains,we produced a tanglegram of both MLST and ompA trees to estimate phylogenetic positioning for each taxa?strain?.Results showed that only six taxa,including the USA R1 and R16,Chinese P619 and P621;and Swiss 5-22b and 10-26b strains,maintained the same phylogenetic clustering in both trees,whereas all other taxa clustered differently in each tree.We additionally tested the recombination break points and changes in topology in the MLST and ompA alignments using DualBrothers.Based on observed breakpoints,we can infer at least one putative recombination event in the C.suis MLST alignment,and at least four in the ompA alignment.In summary,we found that Chlamydia infecting pigs was C.suis,not C.psittaci which has been thought the species to infect pigs in China.The unambiguous conclusion from this study corrected a misunderstanding of Chlamydia infections in pigs in China.Furthermore,phylogenetic analyses showed that partial ompA VD1-2 sequences from C.suis strains from China identified in this study are highly polymorphic.We firstly established a C.suis-specific MLST scheme and made phylogenetic and cluster analysis of C.suis MLST worldwide,further illustrating high genetic diversity of C.suis,which enriched the PubMLST chlamydia database and laid a theoretical foundation for C.suis genetic evolution.Tanglegrams based on MLST and ompA sequences provided evidence of C.suis recombination amongst the strains analyzed.As it stands,the potential pathogenic effect of C.suis on health and production of pigs remains unclear and will be the subject of further investigations.Further study is also required to address the transmission of C.suis between pigs and the risk of 'spill-over' and 'spill-back' of infections to wild animals and humans. |
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