Establishment And Application Of Indirect ELISA For Detection Of Antibody Against Streptococcus Suis Serotype 9

Streptococcus suis is an important zoonotic pathogen that can cause disease in swine and human.The main clinical symptoms are meningitis,septicemia and arthritis in pigs.The epidemic of the disease in the world has brought huge economic losses to the global pig industry.Streptococcus suis includes 29 serotypes,which can be divided into high pathogenic,low pathogenic and non-pathogenic strains by their different virulence.In general,Streptococcus suis serotype 2(SS2)is considered to be the most virulent serotype,as well as the highest isolation rate in clinical diseased pigs.The isolation rate of Streptococcus suis serotype 9(SS9)in pig farms in China,Australia,Belgium,Holland and Germany has gradually increased,and it may has become one of the dominant serotypes of Streptococcus suis in recent years.At present,the detection of the bacteria only depends on the clinical isolation and cultivation as well as molecular biological identification,however,these detection methods are not only time-consuming and laborious but also not suitable for large-scale epidemiological investigation.In addition,many domestic research institutions are developing the inactivated vaccine of SS9 and the evaluation of the immune level of the vaccine also needs to detect the antibody against SS9.Capsular polysaccharide(CPS)is one of the main virulence factors of SS9.It has serotype specific antigenicity and is also an important protective antigen,which can be used for the diagnosis of swine streptococcal diseases caused by SS9 and the preparation of subunit vaccine.This study aims to provide a new method for the effective prevention and control of the disease caused by Streptococcus suis,by extracting the capsular polysaccharide of SS9 as antigen to establish an indirect ELISA method for detect the antibody of SS9.1.Extraction and purification of SS9 capsular polysaccharideIn this study,the crude extract of CPS was obtained by ethanol precipitation,and further CPS of SS9 was purified by phenol extraction and dialysis.The concentration of purified polysaccharide measured by phenol-sulfuric acid method was 65.5?g/mL.A single maximum absorption peak was obtained at 200 nm,but no absorption peak was found at 260 nm and 280 nm after full wavelength scanning by ultraviolet spectrophotometer,which indicated that high purity capsular polysaccharide was successfully extracted.The purified CPS can bind with SS9 positive serum and has good immunogenicity by agar double diffusion test.Therefore,it can be used as the coating antigen for indirect ELISA.2.Establishment and application of indirect ELISA for detection of antibody against SS9An indirect ELISA method for the detection of antibody against SS9 was established by coating ELISA plate with purified CPS.The optimized reaction system is as follows:The best coating concentration of CPS was 0.2 ?g/mL,the coating condition was at 37? for 2 h and the optimal blocking solution was PBST containing 5%skim milk;The optimal dilution ratio of antiserum against SS9 was 100 times,and the reaction at 37? for 45 min;The optimal dilution ratio of HRP labeled Goat anti-pig IgG was 5000 times,and the reaction condition was at 37? for 60 min;The TMB substrate developed color at 37? for 15 min in dark.Then,the method was used to measure the OD450 of 33 SS9 negative serum and these samples average value X and standard deviation SD were statistically processed,to determine the critical value of the method was X+3SD=0.341.Also,with the method to detect 4 SS9 positive serum,although the serum was diluted with 1:800,the results were still positive,which indicated that the established method has good sensitivity.The positive serum of Actinobacillus pleuropneumoniae,Mycoplasma pneumoniae,Haemophilus parasuis,Salmonella,Escherichia coli,Streptococcus suis serotype 2,Streptococcus equi subsp.zooepidemicus also were detected and these results all negative;The coefficient of variation interval in batch repeatability test of this method is 4.25%-8.72%and coefficient of variation interval of inter batch repeatability test is 4.05%-9.47%that demonstrated good specificity and repeatability of the method.The method was used to detect 410 serum samples randomly collected from five different pig farms,159 of which were antibody positive,and the antibody positive rate was 38.8%.In order to verify the effectiveness of this method,the serum of piglets immunized with SS9 inactivated vaccine was detected.The results showed that the serum antibody level of the immunized piglets increased significantly,while the serum antibody of the nonimmunized piglets did not change significantly,which indicated that this method has high reliability.In this study,the indirect ELISA method for the detection of antibody against SS9 has been established successfully which used extraction and purification of SS9 CPS as antigen.With the collected clinical serum samples were detected,which further proved that this method has better sensitivity,specificity and repeatability,and can be used for the detection of clinical antibody against SS9,which has a certain clinical value.Through further optimization and standardization,the detection method can provide a new diagnosis strategy for swine streptococcal diseases caused by SS9.