Establishment Of Typing Sethod Of Streptococcus Suis Serotype 2,3,7 And 9 And Screening Of Common Immunogens

Swine streptococcosis is clinically manifested as acute septicemia,meningitis,arthritis and lymph node abscess,which has brought great harm to the pig industry.Streptococcus suis(S.suis)can be divided into 29 serotypes according to the difference of Capsular polysaccharide(CPS).At present,S.suis serotypes 2,3,7 and 9 are prevalent serotypes,and among which S.serotype 2 is the most common serotype.Due to the large number of serotypes in S.suis and the different serotype prevalence in various countries and regions,it is of great significance to establish a typing method for epidemiological surveillance of S.suis.At present,there is a lack of universal vaccines agaisnt different serotypes of S.suis,and the existing commercial vaccines including inactivated vaccines and attenuated vaccines have some shortcomings,such as strong immune side effects and low broad-spectrum immune protection.Therefore,it is urgent to screen out vaccine candidate antigens with good immunogenicity to lay the foundation for the development of effective universal vaccines against S.suis.1.Establishment of indirect ELISA for detection of antibodies against Streptococcus suis serotypes 2,3,7 and 9In order to establish an indirect ELISA method for rapid detection of serum antibodies against S.suis serotypes 2,3,7 and 9.the whole cell antigen,ultrasonic lysis antigen and capsular polysaccharide antigen of S.suis serotypes 2,3,7 and 9 were prepared and incubated with the antisera induced by whole cell inactivated vaccine to analyses the specificity and determine the ELISA coating antigen.The ELISA conditions were optimized accordingly.The results showed that the purity of purified CPS was high and the antigenic specificity determined by cross-ELISA of CPS was also high.Therefore,CPS was selected as the coating antigen to establish and optimize the indirect ELISA antibody detection method.The optimum results showed that the reaction conditions of the four CPS antigens were consistent.The optimal coating antigen concentration was 15 μg/mL,the optimal coating condition was 4℃ overnight for 14 h.the optimal blocking condition was 1%gelatin blocking,37℃ 2h,the serum dilution was 1:50,and the incubation time was 37℃ 20min.The optimal dilution of HRP-goat anti-pig IgG secondary antibody was 1:15000,the incubation time was 20 min at 37℃,and the optimal coloration condition was 10 min at 37℃.The negative and positive cutoff values of the indirect ELISA method for serum antibodies against S.suis serotypes 2,3,7 and 9 were 0.341,0.351,0.387 and 0.331,respectively.The method has good specificity,sensitivity and repeatability.The coincidence rates between the indirect ELISA method and the slide agglutination test were 80%,88%,88%and 92%,respectively,indicating that the coincidence rate was high.The positive rates of 170 serum samples collected from pig farms in some areas of Jiangsu were 61.27%,84.39%,84.97%and 84.97%,respectively,indicating that the mixed infection rate of Streptococcus in pig farms in this area was high.The above results showed that the established indirect ELISA method could be used to detect the serum antibodies against S.suis serotypes 2,3,7 and 9,which provided a reliable technical means for the seroepidemiological investigation of S.suis.2.Preparation of Streptococcus suis serotype 2,3,7 and 9 capsular polysaccharide antiserum and its application in typingCPS2,CPS3,CPS7 and CPS9 were coupled with bovine serum albumin(BSA)by ADH inter-bridge method,and then the CPS-BSA conjugate,CPS alone and the mixture of CPS and BSA were used as immunogens to immunize BALB/c female mice according to the immunization procedure,The antiserum was obtained on the 7th day after the third immunization.At the same time,the antisera against the whole bacteria inactivated vaccine was used as a control.The specificity of the mouse antisera was identified by indirect ELISA and slide agglutination test.The results showed that the antisera induced by CPS-BSA conjugate were type-specific,while the CPS alone and the mixture of CPS and BSA could not induce the production of IgG antibody.The antisera induced by whole bacteria inactivated vaccine had a certain degree of cross-reaction with some S.suis strains.Therefore,the CPS antiserum can be used as an antibody for the serotype typing of S.suis,and also provides an experimental basis for the development of S.suis subunit vaccine.3.Screening and immunogenicity study of universal vaccine candidate antigens of Streptococcus suis serotype 2,3,7 and 9Based on the five candidate antigen proteins screened by reverse vaccinology technology,the homology analysis of gene sequence and protein sequence,protein expression and purification,and Western Blot analysis were carried out in this study.After immunizing BALB/c female mice,the IgG antibody titer was determined and the type of cellular immune response was determined.When the mouse challenge model was established,the challenge protection experiment was carried out.The results showed that the homologies among five gens were more than 92%,and the five expressed proteins reacted with the antiserum against the whole bacteria inactivated vaccine.The serum IgG antibody titer after the second immunization was significantly higher than that of the primary immunization.Except for LPXTG protein,the other proteins could induce effective humoral and cellular immune response.The five candidate vaccine antigen proteins could provide cross-protection against S.suis serotypes 2,3,7 and 9,and the protection rate was 40%100%.The results laid a foundation for the development of universal subunit vaccine against S.suis.In summary,this study successfully established an indirect ELISA method for the detection of antibodies against S.suis serotypes 2,3,7 and 9 by using CPS with high purity as diagnostic antigen,which has certain clinical application value.CPS was coupled with BSA to immunize mice,and capsular polysaccharide antiserum was successfully obtained,which could be used for typing and identification of S.suis strains.Five vaccine candidate proteins,EzrA,AdcA,LPXTG,LepA and MltG,were successfully screened by universal reverse vaccinology technology.