Virulence Genotyping And Population Analysis Of Streptococcus Suis Serotype 2 Isolates From China

Streptococcus suis is a one of the most important swine pathogen worldwide,belonging to Gram-positive bacterium and is responsible for massive economic losses to the swine industry.The infection of S.suis could lead to severe respiratory tract diseases and systemic diseases,including arthritis,pneumonia,septicaemia and meningitis.As a zoonotic pathogen,SS2 is also a major threat to public health.In China,outbreaks of human-associated infection occurred in Sichuan province in 2005 and in Jiangsu province in 1998.Currently,at least 29 true S.suis serotypes have been identified.Streptococcus suis serotype 2(SS2)is considered the most virulent serotype and is the serotype most frequently associated with disease.Research on the virulence of SS2 provide a better understanding of the SS2 infection mechanism and contribute to the prevention and treatment of streptococcosis.In this study,we selected 101 strains of SS2 from 357 S.suis field strains that we collected since 1998,with the help of species-specific PCR and serotype-specific antiserum.Based on their DNA sequence,we designed PCR primers for 22 virulence-related genes,namely,mrp,epf,sly,rgg,salKR,fbps,rgg,ofs,pgdA,gapdh,iga,endoD,ciaRH,manN,purD,srtA,dppIV,neuB,dltA,comR,permease and SspA.We performed individual PCR to detect distribution of virulence-related genes among 101 SS2 strains.The PCR result was visualized using clustering method to study similarity among those isolates.Two clusters of SS2 were apparent on the phylogenetic tree,namely,Clusters A and B.Statistical comparison between the two clusters was performed,and structure analysis demonstrated that the prevalences of epf,sly,endoD,rgg,comR and permease were significantly different in two clusters,therefore we assumed that strains in Cluster B is more virulent than that in Cluster A.To test our hypothesis,we randomly selected SS2 isolates from both Cluster A and Cluster B as representative isolates,and assess their virulence using animal infection model.A zebrafish infection model was used,with injection dose of 1.0 × 107,1.0 ×106 and 1.0 ×105 CFU/fish.The result of zebrafish infection experiment showed that,at each dose,the mortalities of Cluster A infected groups were lower than those of Cluster B groups;the distinguishable injection dose was 1.0×105 CFU.In addition,BALB/c mice infection model was also used,with injection dose of 2.0 × 109,2.0 ×108 and 4.0 × 107 CFU/fish..All Cluster B strains were considered to be virulent,except isolate JX081202;while all Cluster A strain tested were considered to be less virulent in BALB/c mice infection model.Both mice and zebrafish infection models revealed that strains in Cluster A were less virulent than those in Cluster B.Therefore,epf,sly,rgg,endoD and permease were possible predictive markers for SS2 virulence.The transcription levels of highly prevalent genes in both clusters were detected by qRT-PCR in representative strains.For Cluster A isolates,the transcription levels of neuB,dltA,fbps and pgdA were significantly lower,but the transcription level of iga was significantly higher than that in Cluster B isolates.Although encoded in the genomes of the selected Cluster A isolates,dppIV and mrp genes were not expressed.These results revealed the genetic differences between virulent and low-virulence SS2 isolates from China and provide a better understanding of the SS2 pathogenic mechanism.