Molecular Epidemiology Of S.Suis In Middle Area Of Jiangsu Province And Construction Of Revsknock-Out Mutant Of S.Suis Type 2

Streptococcus suis is an important pathogen, that cause meningitis, septicemia, arthritis, and pneumonia. So far, thrity-five different serotypes(type 1/2 and 1 through 34) of S. suis have been described, of which type 2 is the most frequently isolated serotypes from pigs with disease. Isolates belonging to other serotypes including types 1,7, and9have also been associated with disease in pigs. Presently, the detection of S. suis has been limited to serotype 2 strains in China, little is known about other pathogenic serotypes. The mechanisms by which S. suis invades and infects the host are still unclear even though numerous studies on potential virulence factors have been carried out over the past decade. This study was undertaken to determine the prevalence, capsular serotype, the distribution of virulence factor and antimicrobial susceptibility of S. suis isolated from slaughter pigs in the middle of Jiangsu Province. And at the same time construction of RevS knock-out mutant of S. suis type 2 was also done.1 Molecular Epidemiology of Major Pathogenic S. suis types in Middle Area of Jiangsu ProvinceTonsillar specimens of 303 slaughtered pigs in middle area of Jiangsu Province in 2005 and 2006 were investigated for the presence of S. suis and major pathogenic serotypes by the method of PCR using species-specific 16S rRNA primer and serotype-specific primer based on the S. suis capsular polysaccharide(cps) genes specific for serotypes 1,2,7and9. Among 200 tonsillar specimens in 2005, 176 positive samples was detected based on 16S rRNA, the carrier rate of S. suis was up to 88%; and among 176 positive samples, typel, 2, 7 and 9 accounted for 17, 15, 20, 52 respectively, the percentage was 9.6%, 8.5%, 11.3% and 29.5%. In 2006, 85 of 103 tonsillar samples was positive on 16s rRNA, the prevalence of S. suis was 82.5%, and among 85 positive samples, capsular types 1, 2, 7 and 9 accounted for 15, 2, 22, 17, and the percentage was 17.6%, 2.4%, 25.8% and 20%. The percentage of the S. suis among the healthy herds in the areas was very high, various capsule types of S. suis seem to be involved in the same herds. 2 Isolation and Identification of S. suis Type 2, S. suis Type 7 and S. suis Type 9Three isolates of S. suis type 2, three isolates of S. suis type 7 and thirteen isolates of S. suis type 9 were isolated based on the bacteriological examination compared with molecular characterization, including the morphological, gram stain, cultural, biochemistry characters and PCR assays based on specific cps2 gene, cps7 gene and cps9 gene, detection of virulent factors and drug sensitivity test were also done. Biochemical identification shows that all the isolates belong to S. suis, the percentage of identification was above 85%. All isolates are sensitive to a penicillin-G, erythromycin, vancomycin and resistant to tetracycline, among these isolates 58c are resistant to several commonly used antimicrobials agents including tetracycline, streptomycin, sulphonamides, erythromycin and Clindamycin. Detection about the major virulent factors was that gdh exist in every isolates, and mrp, epf, sly, fbp was absent in these isolates, the distribution of orf2 among these isolates was different., the distribution of putative virulence traits of these isolates in China were different from both the same serotype isolates of Europe and the epidemic S. suis type 2 isolates of China.3 Construction of RevS Gene Knock-out Mutant of S. suis Type 2It is reported by Greeff in 2002 that an isogenic knock-out mutant of RevS was shown to be attenuated in colonising the S. suis specific organs in a competitive assay in four piglets, indicating that RevS plays a role in the pathogenesis of S. suis infections. To determin if the RevS is involved in the pathogenesis of wild-type isolates 05ZYH33, a recombinant gene knock-out vector was constructed consisting of Spc~r cassette with flanking homology regions to the target genes named RevS, the isogenic RevS-defieient mutant was screened by allelic replacement. PCR analysis confirmed that the coding genes of RevS were replaced completely by Spc~r cassette and the basic biological characters of 05ZYH33 including growth stability, colonial morphology, haemolysis, Gram staining, growth curve and protein expression did not undergo any apparent change. Balb/c mice infection assay indicate that RevS plays a role in the pathogenesis of S. suis infections, no remarkable difference was observed in the piglets pathogenesis infection between mutant and wild-type isolates.