Establishment And Application Of Multi-PCR Assay For Streptococcus Suis, And It's Prevelent Study Of Samples In Slaughters

Streptococcus suis (SS) is an important zoonotic pathogen of many diseases such as mengingitis, septicemia, arthritis and sudden death in human and pigs,which causes huge losses to the pig industry and is a substantial threat to human public health security. Thirty-five serotypes (type 1 to 34 and 1/2) of SS have been described, of which type 2 is considered to be the most-virulent and prevalent in worldwide, then is serotype 7 and 9. Studies suggest that the pathogenicity of SS is closely related with the carrying and expression of virulence-associated factors.First of this study, made use of standard strains to establish a multi-PCR assay and evaluated the assay, then, according to the assay, detection kit was preliminary designed. The multi-PCR assay was used to detect samples, isolated from tonsil, for typing of Streptococcus suis and detection of virulence-associated factors. Combined with the detection results of 12 SS2, the pathogenicity of 12 SS2 were compared ,using zebrafish model.1. Establishment and application of Multi-PCR assay for Streptococcus suis type 2, 7, 9Ten pairs of primers were designed and synthesized according to the nucleotide sequences of Streptococcus suis. A Multi-PCR assay, intending to detect the seven virulence-associated factors mrp, epf, sly, gdh, gapdh, orf2, fbps and major soretypes (type 2, 7, and 9) of Streptococcus suis, was developed by optimizing and combinating the primers in three multiplex PCR reactions. Results showed that the method was highly specific, and the sensitivity of multiple PCR reached 103CFU. Finally, a detection kit was established on the basis of the developed Multi-PCR assay. The reproducibility of the detection kit is good. The efficiency of the kit was stable after being stored at -20℃for four months.2. The investigation of Streptococcus suis in tonsil samples and comparison of the pathogenicity of type 2 isolatesFrom 2008 to 2009, tonsil swabs of slaughtered pig were collected in Shanghai, Anhui and Guangdong. Results showed 37 of the 399 tested samples were SS positive (9.27%) by using PCR assay, of which, 23 SS strains were isolated (16 strains belong to serotype 2, 3 strains belong to serotype 7 and 4 strains belong to type 9). The serotype of remaining 14 strains of 16S rRNA positive were undetermined. There were 9 strains showed the phenotype of mrp+epf+fbps+gdh+orf2+sly+gapdh+. Then, 800 tail zebrafish were selected (Danio rerio) to compare the pathogenicity of 12 SS2 isolates, including HA9801, ZY05719,SH-07YH and 9 isolation strains. Twelve hours post-infection with certain bacteria strains, zebrafish showed sepsis lesions. The 50% lethal dose(LD50) were in the range of 9.07×103 CFU to 1.02×105 CFU within 96 h. The bacteria, re-isolated from death zebrafish, were confirmedas SS2 by Gram staining and PCR.