| Pig Glasser's disease is a bacterial infectious disease with high morbidity and fatality caused by Haemophilus parasuis(HPS),which is characterized by polyserositis,arthritis and meningitis.Antibiotics and vaccination are common methods to prevent and control the disease.Due to the unreasonable use of all kinds of antibiotics,the drug resistance of HPS is becoming more and more serious,so vaccination has become an effective measure to prevent Glasser's disease and reduce mortality.However,there are more than 15 serotypes of HPS.Different serotypes of HPS lack mutual immunity and have obvious local characteristics,resulting in the uncertainty of vaccine immune effect.In addition,there are many kinds of epidemic diseases in pig production,and vaccination is frequent,which lead to many pig farms not using vaccination to prevent and control HPS infection.Hence,timely monitoring and analysis are important means to understand the immune effect of vaccines and HPS infection in pigs.Transferrin-Binding Proteins(Tbp)is one of the main virulence factors of HPS,including two polypeptide structures,Tbp A and Tbp B.Early results of our laboratory show that Tbp A has good immunogenicity and reactivity,and can provide good immune protection to homologous bacteria and different serotype strains.This study used HPS recombinant Tbp A as coating antigen to establish indirect ELISA antibody,which is a rapid,sensitive and specific technique for immune surveillance and Glasser's disease seroepidemiological investigation in pigs.The contents of this study include:(1)Purification and identification of the expression of recombinant Tbp A of HPS serotype 4 and 13,respectively,and their use as coating antigens,and the establishment of indirect ELISA methods for the detection of HPS antibodies,which are named as serotype 4 Tbp A-ELISA and serotype 13 Tbp A-ELISA,respectively.(2)To optimize the reaction conditions such as antigen concentration and coating conditions,types and time of Sealing solution,dilution and time of action of serum and HRP,reaction time of TMB.(3)30 HPS negative serums were tested using the optimized method to determine the critical value of the two methods.(4)Optimized methods were used for Streptococcus suis(SS),Escherichia coli(E.coli),Erysipelothrix rhusiopathiaes(ER),Actinobacillus pleuropneumoniae(APP),Porcine circovirus serotype2(PCV2),Porcine pseudorabies virus(PRV),Classical Swine Fever virus(CSFV),Porcine Reproductive and Respiratory Syndrome virus(PRRSV)positive serum to assess the specificity of the two methods.(5)HPS standard positive serum wasdiluted by doubling ratio of 1:200,1:400,1:800,1:1600,1:3200,1:6400,1:12800,1:25600,1:51200,1:102400.The optimized methods were used to detect HPS antibody simultaneously with the commercial ELISA kit to evaluate the sensitivity of the two methods.(6)The purified HPS recombinant Tbp A coated enzyme labeled plate was expressed by the same batch and three different batches,respectively.Four HPS positive serum samples and one HPS negative serum sample were tested in batch and batch by optimized methods,and the coefficient of variation was calculated to evaluate the repeatability of the two methods.(7)The two optimized methods were used to detect 60 porcine serum samples immunized by HPS vaccine respectively,and were compared with methods of indirect ELISA antibody detection using HPS serotype 4 and 13 whole cell as coating antigen,respectively,commercial HPS antibody ELISA detection kit and Western-blot to investigate the reliability of the two methods.(8)The serotype 4Tbp A-ELISA was used to detect 600 serum samples of pigs from 10 cities in Anhui province to verify the application of the method.Result display:(1)The recombinant Tbp A HPS both serotypes were successfully expressed and purified by SDS-PAGE and Western-blot identification,and had good reactivity.(2)The optimization of reaction conditions of serotype 4 Tbp A-ELISA and serotype 13 Tbp A-ELISA show that: Optimal antigen concentrations were 5?g/m L and7?g/m L,respectively,and all overnight at 4?.The types and action time of sealing solution were 5% skimmed milk powder sealed at 37? for 2 hours.The optimum dilution of serum was 1:1600,1:3200,respectively,and incubation time was 45 min,60 min at 37?,respectively.The best dilution of HRP was 1:5000,1:10000,respectively,and incubation time was 30 min,60 min at 37?,respectively.The optimal reaction time of TMB was5 min,10 minutes,respectively.(3)The positive critical values of two optimized methods were 0.215 and 0.292,respectively,and the negative critical values were 0.188 and 0.242,respectively.(4)The two indirect ELISA methods did not cross-react with SS,E.coli,ER,APP,PCV2,PRV,CSFV and PRRSV positive serum,and had strong specificity.(5)The sensitivity of the optimized serotype 4 Tbp A-ELISA method had the same sensitivity as the commercial HPS antibody ELISA kit,while the sensitivity of the optimized serotype 13 Tbp A-ELISA method was slightly than the kit.(6)The coefficient of variation of the two optimized methods inter-and intra-assay tests were less than 10%,and the repeatabilities are good.(7)The positive detection rate of serotype 4 Tbp A-ELISA was 80%,the coincidence rate was 90%,88.33%,86.67% and 90%,respectively,compared with parallel comparison methods.While the positive detection rate of serotype 13 Tbp A-ELISA was76.67%,and the coincidence rate which compared with parallel comparison methods was80%,90%,83.33% and 86.67%,respectively.(8)Using the screened serotype 4Tbp A-ELISA to detect 600 serum samples,117 positive samples were detected,and the total positive rate of HPS infection antibody was 19.5%.The results showed that :(1)In this study,the recombinant Tbp A of HPS serotype 4 and serotype 13 were used as coating antigen respectively to establish sensitive,specific and reproducible method for detecting HPS recombinant Tbp A indirect antibody.They were named serotype 4 Tbp A-ELISA and serotype 13 Tbp A-ELISA.in turn.(2)Two methods were used to detect the serum samples of pigs without HPS vaccine immunization,confirming that serotype 4 Tbp A-ELISA is more suitable for clinical HPS antibody detection and epidemiological investigation. |
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