Identification Of Streptococcus Suis Strains In Anhui And Immunoprotecive Of RfeA Protein In Mice

Streptococcus suis disease is a kind of zoonotic infection disease worldwide caused by streptococcus suis, defined as one of the second class animal epidemic diseases by law, with the main clinical symptoms of acute hemorrhagic septicemia, meningitis, endocarditis, arthritis, pregnant pig abortion,dysentery of piglets and so on. The specially contacted people can be infected and lead to death seriously,streptococcus suis diseases cause enormous economic loss, and serious threat to public health in China and the world. The pathogens of swine streptococcus diseases can be divided into 35 serotype(1～34,1/2) basiced on the capsular polysaccharide,of which,the most popular clinical separation is streptococcus suis type 2, and types 1,7 and 9 also have strong pathogenic worldwide. Because the virulence factors and pathogenic mechanism are not very clear, poor cross-protection between different types becomes the barrier of the control of the swine streptococcus suis diseases and development of vaccine, it’s very important to search for good protective antigens. This research mainly included the following parts:1 The detection of the distribution of serotypes and virulence genotypes of Streptococcus suis strains isolated from Anhui province.We detected and analyzed the distribution of serptypes and virulence genotypes, and pathogenicity through the morphology, cultural characteristics and the PCR technology on the basis of 87 strains of Streptococcus suis in Anhui region from 2009 to 2013. The strain number of Streptococcus suis typel,2 and7 were 10,24,11 and percentage of Streptococcus suis serotype 1,2 and 7 werell.5%,27.6% and 12.6%, respectively.There was no Streptococcus suis serotype 9 strains detected, in which S. suis 2 was the dominant serotype in Anhui province.The result showed that there was genotype for mrp+epf+sly+in 16 strains SS2, HJ25 of SS1, without in SS7, the analysis suggested the the distribution of virulence genes of SS2 isolated from brains and joint fluid were similar to reports before, genotype of mrp+epf+sly+ was recognized as essential for strong virulent strain now.2 Determination of the virulence and pathogenic study of SS1,SS2,SS7.Select Streptococcus suis with genotype of mrp+epf+sly+, including type 1 (HJ25), type 2 (HJ15) and type 7 (CZ1), to determine the median lethal dose (LD50) by Kou’s method. The results indicated that the virulence of SS2(HJ15) was the highest (LD504.47x107CFU) and SS1 was the lowest (LD501.41×109 CFU). Pathogenic ity of three types of Streptococ--cus suis was studied by pathological section after toxicity attack test in mices. Three diffe--rent types of Streptococcus suis all injured lung primarily, which indicated Streptococcus suis mainly infected the respiratory system. The mechanism was possibly connected with lung infection of Streptococcus suis. The infection caused lobular pneumonia leading to breathing difficulties, which finally caused death.3 Clone and expression of the RfeA gene of secretory protein of Streptococcus suis, and study on the immunoprotecive between different types of Streptococcus suis.A pair of specific primers were designed according to the gene sequences of SS2 05ZYH33 from GenBank. All genes of SS1, SS2 and SS7 were amplified by PCR The results showed that amplification of SS7 did not get prupose gene. By cloning the purpose gene into pET-32a expression vector, the pET-32a-RfeA construction generated was transformed into the Escherichia coil BL21. Then the RfeA gene was expressed highly after induced and sodium dodecyl subhate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis showed that the molecular weight of the protein was approximately 38kD in agre--ement with the expected. The results of western blotting analysis demonstrated that the recombinant protein possessed antigenic activity. After emulsification win Freund’s adjuv--ant, the purified protein was vaccinated subcutaneously to guinea mice on day 1, day 14 and day 21. Before and after every vaccine, the IgG antibody levels and cytokines levels of IFN-γ,IL-4,IL-10,IL-2 in blood were detected with indirect ELISA method. Increases in IgG antibody levels were detected in all protein-immunized groups. The tier of IgG reachedl 2097075200. IFN-y,IL-4,IL-10, and IL-2 significant increased compared with the control group.After the second immunization, one week post the third immunization, all groups received intraperitoneal injection of lethal dose of SS1, SS2, SS7. The RfeA recombinant protein group showed 100% protection to SSI,80% to SS2 and 20% to SS7. The results indicated recombinant protein RfeA showed that the protein only had certain protective to Streptococcus suis with RfeA gene, and little protective to Streptococcus suis without RfeA.