Serological And Molecular Epidemiological Survey On Streptococcus Suis In Zhejiang Province

Streptococcus suis type 2 (SS2) can cause meningitis, endocarditis, arthritis and other diseases, is an important zoonotic pathogen. There are 35 serotypes of Streptococcus suis accordding to its capsular polysaccharide antigen. Streptococcus suis type 2 is isolated most frequently from pigs and humans, the other serotypes 1/2, 9,7,14 and 5 also can cause illness. In this study, we conducted seroepidemiological and molecular epidemiological surveys on SS2 in order to find out prevalence of streptococcus suis in pigs in Zhejiang province.MRP has been recognized as a major virulence factor of SS2. Although some foreign reports show that some virulent isolates were absent of MRP, in Chinese strains, it is conserved. SAO proteins were confirmed to be conserved in most Streptococcus suis serotypes. Therefore, conserved segment of MRP and SAO genes were selected to express proteins as diagnostic antigens, establish the corresponding ELISA detection methods, and applied to serological surveys. The truncated protein of SAO and MRP could be readily expressed in E coli BL21, Western-blot result shows that the recombinant protein SAO and MRP reacted with SS2 positive sera. The antigen and serum were diluted for ELISA experiments by chessboard titration. After several attempts, we determined the SAO and MRP truncated proteins's best coating concentration was 20ug/ml, mixed coating concentration was 10ug/ml,1:100 dilution of the serum was the best, the time of TMB reaction was 2-3.5 minutes. Application of this ELISA method,644 serum samples collected from Zhejiang province were tested. The results show that the positive rate of SAO, MRP and their mixture was 56.5%,38.0%and 50.2%respectively, indicating that Streptococcus suis was prevalent in swine of Zhejiang province.To further understand the epidemiological characteristics of SS2 isolated from Zhejiang province in recent years, multilocus sequence typing (MLST) method was adopted, seven pairs of housekeeping gene primers (aroA, cpn, dpr, gki, mutS, recA, thrA) were designed,44 strains collected in different periods (2006-2010) were sequenced. Then DNASTAR, MEGA4, DNASP4.9 and other bioinformatics tools were applied to analyze the genetic relationship among the isolates. We found that the sequenced from our strains were of high homology, forming two major branches. Upload the sequences to streptococcus suis MLST database (http://www.mlst.net/) for the sequence comparison, we found that 14 strains belonged to ST1,19 to ST7,6 to ST28,1 to ST86 and 1 to ST162 which is actually a new sequence type. Strains in ST1 were founders. Strains in ST7 derived from ST1 are mostly pathogenic. The isolates from Sichuan outbreak in 2005 belonged to ST7.ELISA method developed in this study could be used for serosurveillance of streptococcus suis infection. MLST revealed the genetic struncture of SS2 isolated from Zhejiang province. The results thus obtained provide useful data for further research on the control of Streptococcus suis type 2 infections.