Establishment Of An Indirect ELISA For Detecting Haemophilus Parasuis And Screening Of Subunit Vaccine Candidates

Haemophilus parasuis is a non-motile,small,pleomorphic rod-shaped,and nicotinamide adenine dinucleotide(NAD)-dependent bacterium of the Haemophilus genus of the family Pasteurellaceae.This bacterium is the etiological agent of Glasser's disease in swine,which is characterized by polyserositis,arthritis,and meningitis.Haemophilus parasuis infection is causing a significant increase in mortality and morbidity in swine,leading to major economic losses in the pig industry.So far,15 serovars of Haemophilus parasuis have been defined,with apparent differences in virulence,but a high precentage of the evaluated clinical isolates are nontypeable.The clinical symptoms of the acute and chromic forms of the disease differ significantly,possibly depending on the strain's virulence and the host's immune status.This brings a lot of difficulties for diagnoses and prevention of Glasser's disease.Three strains of Haemophilus parasuis,AH1108(serovar 4),JS1023(serovar 5)and ZJ0906(serovar 12),were proteomic analyzed and stably expressed proteins were screened.Based on the above results,Three stably expressed proteins were chosed as coating antigens and immonogens.The main contents of the research are as following:1.Screening and identification of stably expressed proteins in different serovars of Haemophilus parasuisThree genes of stably expressed proteins were amplified using Haemophilus parasuis serovar 12 DNA as template.The purified genes were ligated into prokaryotic expression vector pET-28a(+),then the recombinant plasmids were transformed into E.coli BL21 competent cells.The expressed proteins were purified by Ni-NTA affinity chromatography resin and purified protein concentration is high.Moreover,the polyclonal antibody prepared through immunized mice was purified by Protein A affinity chromatography resin.Western-blot showed that the three proteins expressed at the same amount in the three serovars of Haemophilus parasuis.The study indicated that the three stably expressed proteins will lay a foundation for creating an ELISA for detecting various serovars of Haemophilus parasuis and using as candidate of subunit vaccines.2.Establishment of a general indirect ELISA using stably expressed proteins and epidemiological investigationThree stably expressed proteins in serotypes 4,5 and 12 expressed in E.coli and lysis products of bacteria were used as coating antigens to coat ELISA plates.Compared with the results of ELISA using lysis products of bacteria as antigens,the recombinant manganese-dependent superoxide dismutase(MnSOD)protein was confirmed to be the optimal coating antigen to establish ELISA assay.The optimized ELISA conditions were as follows:antigen protein was coated at 4? overnight with a concentration of 1.5?g/mL;The plates were blocked with 5%skim milk at 37? for 2h;The sera samples were incubated at 37? for 1.5h with 1:160 dilution and secondary antibody was incubated at 37? for 45min with 1:15000 dilution.The cutoff values of positive and negative results were OD450nm>0.2451 and OD450nm<0.2083,respectively.The specificity results showed that this ELISA method did not react with positive sera of other 8 diseases(porcine reproductive and respiratory syndrome virus,classical fever virus,pseudorabies virus,porcine circovirus type 2 virus,foot-and-mouth disease virus,Erysipelothrix thusiopathiae,Streptococcus equi,Streptococcus suis,Escherichia coli,Salmonella enteritidis)and the repeatability of intra-and inter-assay were excellent.A total of 114 sera samples obtained from swine farms were detected by indirect ELISA using MnSOD protein and lysis products of serotypes 4,5 and 12 Haemophilus parasuis as antigen,the positive coincidence rates were 80.77%,80.00%and 90.00%,the total positive coincidence rate was 90.63%.1610 swine serum samples from 8 provinces were detected by established ELISA,the results showed that the total positive rate was 54.41%and the highest in Jiangsu province and the lowest in Jiangxi province.Moreover,the positive rate in autumn and winter were higher than other seasons.So the assay can be used as a tool for diagnosis and epidemiological surveys on serotypes 4,5 and 12 Haemophilus parasuis.3.Screening of recombinant subunit vaccine candidate factor in Haemophilus parasuisThe causative agent of Glasser's disease in swine is Haemophilus parasuis.Commercial bacterins are widely used for protection of the swine population.However,cross protection is limited because Haemophilus parasuis has more than 15 serovars.Here three stable proteins expressed in serovars 4,5 and 12 were chosed as vaccine candidates.The immunogenicity and protective efficacy of the three proteins were evaluated in guinea pigs by two subcutaneous immunizations with ISA 15A adjuvant.Antibody titers were tested by ELISA.AH1108(serovar 4),JS1023(serovar 5)and ZJ0906(serovar 12)were used to challenge the immunized guinea pigs and nonvaccinated controls on 2 weeks after secondary immunization,respectively.All immunized groups showed serum antibody titers higher than those of negative-control groups.Furthermore,the cytokine and chemokine levels were evaluated at the transcriptional level by the real-time PCR analysis of six cytokines and chemokines.The cytokine and chemokine levels of all immunized groups were higher than those of negative-control groups.The protection rates of the group immunized with protein CyaY were 60%,40%,80%after a challenge with strains AH 1108 AH1108(serovar 4),JS1023(serovar 5)and ZJ0906(serovar 12)respectively.The protection rates of another group vaccinated with protein MnSOD showed 40%,40%,80%after a challenge with strains AH1108(serovar 4),JS1023(serovar 5)and ZJ0906(serovar 12)respectively.The guinea pigs immunized with protein GPD were protected at levels of 60%,40%,80%against AH1108(serovar 4),JS1023(serovar 5)and ZJ0906(serovar 12),respectively.The results indicate that the three stable proteins have cross protection for guinea pigs against serovars 4,5,and 12 of Haemophilus parasuis infection.Taken together,these results suggest that the three stable protein is a promising vaccine candidate that needs to be confirmed in a swine population.