Molecular Epidemiology, Antigenicity And Detection Of The Outer Membrane Protein P2Gene Of Haemophil Us Parasuis

Haemophilus parasuis （H.parasuis）, the causative agent of Glasser’s disease, is a member of the family Pasteurellaceae and commensal organism of the upper respiratory tract of pigs.Under appropriate conditions it can invade and cause severe systemic disease, characterized by fibrinous polyserositis, arthritis and meningitis. Currently, H.parasuis infection, which causing significant mortality and morbidity in piglets and great economic losses in pig industry, became one of the most important swine diseases in China. And it has demonstrated that a high antigenic heterogeneity exists among H. parasuis strains. But the virulence factors and protective antigens of H.parasuis are not clear, and there is no universal vaccine to prevent and control the outbreaks of this disease.In this study,111H.parasuis field isolates from several provinces or municipality of China were genotyped by ERIC-PCR and the biological characters of H.parasuis isolates were detected. And the OMP（out membrane protein） P2gene of H.parasuis was cloned and expressed, and the antigenicity of the OMP P2protein was identified. The protective efficacy of the recombinant P2proteins against highly pathogenic H.parasuis strain HB070214infection was examined in Balb/c mice, which provided new strategies for the diagnosis and development of H.parasuis genetic engineering vaccine. The contents of the paper including five parts as following:1. Isolation, identification and biological characters of Haemophilus parasuisFor isolation of Haemophilus parasuis several media, including trypone soya agar （TSA）, chocolate agar, pleuropneumonia-like organism（PPLO） agar and proteose peptone tryptone agar or into liquid media including tryptone soya broth（TSB）, Brian heart infusion（BHI）, Martin’s broth, pleuropneumonia-like organism（PPLO） broth and peptone tryptone, and the optimal media TSA and TSB were selected and compared in this experiment. The results showed that TSA and TSB could be used to culture the H.parasuis isolates. And then111strains of Haemophilus parasuis were isolated from clinical ill piglets. Haemophilus parasuis HB070214was inoculated into TSB and cultured for10-12hours under the condition of37℃,180r/min, the bacterial number was up to109cfu/mL. The morphological diversity and diference of capsules and pili of HPS were observed under electron microscopy. Susceptibility of the isolates was defected by using22antimicrobial. And the results showed that the drugs, which were susceptible to more than50%isolates, were cefoperazone, cefoxitin, ceftazidime, cotrimoxazole, polymyxin, tilmicosin, ceftiofur and ceftriaxone. Acetyl flumequine, doxycycline, florfenicol, rifampicin, enrofloxacin and sulfa six SDM appeared no susceptibility to more than50%isolates. Pig experiment results showed that the HPS serumtype6,7,8and11had very low or no virulence, while the HPS serumtype4,5,12and13has very strong virulence. And HPS serumtype1,2,9,10,11,14and15showed moderate pathogenicity. The standard Haemophilus parasuis which were maintained at Nanjing Agricultural University were cultured and inactived antigens were prepared, and the antibodies to HPS were made by the antibody titers were more than1:8Gel Diffusion （GD） test. And the cross-immune responses existed between the15serotypes of HPS and their hyperimmunized serum.2. Genotype analyzing of Haemophilus parasuis field isolates with ERIC-PCRThe fifteen serovars of H. parasuis reference strains were analyzed by Enterobacterial Repetitive Intergenic Consensus PCR （ERIC-PCR）. The results showed that all the15serovars of reference strains gave unique, reproducible ERIC-PCR fingerprints. The results of the111field isolates from South-east of China showed that they presented23special fingerprints. The first3prevalence of ERIC-PCR fingerprint type were ⅩⅩ（20/111）, ⅩⅩⅢ（9/111） and Ⅳ（8/111）. And all provinces had different fingerprint types of H. parasuis. A gene about1100bp in the ERIC-PCR fingerprints was found in all15serotypes HPS reference strains and111HPS isolates. It indicated that H. parasuis has existed widely in South-east of China with large genotypes, and ERIC-PCR could be used to evaluate the molecular Epidemiology of H. parasuis and it provides theory basis to find HPS common antigen gene.3. Sequencing and genoping of Haemophilus Parasuis based on OMP P2GeneBased on the results of the ERIC-PCR, the fragment of about1100bp of strain HB070214of HPS was cloned into pMD18-T. The sequencing results indicated that this gene had1077bp length and the sequence was snbmitted to Genbank（GU323686）. The result of sequence analysis showed that it has95.5-98.1%homology with OMP P2gene. A pair of specific primers were designed based on the sequences of the OMP P2gene of Haemophilus Parasuis in Genbank, and the OMP P2genewere amplified from22isolates. And the sequences results showed that the OMP P2genes of the isolates could be divided into2genotypes: genotype Ⅰ and genotype Ⅱ. The sizes of genotype Ⅰ OMP P2gene was1077bp to1095bp, and genotype Ⅱ gene was1167bp to1203bp. The genotype Ⅰ and genotype Ⅱ have92.8%homology at the amino acid level and96.6%homology at the nucleotide level; Genotype Ⅰ had27-42amino acid deletion compared with Genotype Ⅱ at the protein level. The pathogenic strains serotype4,5,12,13belong to genotype I strains and the no pathogenic strain serotypes6,7,8and11belong to genotype Ⅱ. IIt indicated that this genotyping might be related to the virulence of the isolates.4. Expression and antigenicity of OMP P2fusion protein in E.coli.The OMP P2gene was ligated into pGEX-6p-1to get the expressing vector of pGEX-P2which was then transformed into E. col i BL21. The result of SDS-PAGE shows the expressed protein is about65kD, and the recombinant protein occupid persent50%of total cell protein and in inclusion body form. Western-blot results indicate that this protein possesses biological activity.A pair of specific primers was designed and synthesized according to the OMP P2gene of HPS in GenBank （ZP₀2477753）, And the OMP P2P2gene（1077bp） was amplified by P2-PCR from HPS strain HB070214, and cloned into pET-32a vector. pET-P2was identified by PCR and sequencing. Then the plasmid pET-P2was transformed into E.coli BL21（DE3）. The P2-His fusion protein with molecular weight of,60500Da was expressed by the induction of1.0mmol/L IPTG. It could specifically react to the antiserum against HPS by Western blotting. The P2-His fusion protein was further purified by nickel-agarose gel resin chromatographic column with300mmol/L imidazole phosphate elution buffe. The purity of P2-His was up to92.5%. The P2-His fusion protein was used as a packaging antigen, and a novel HPS P2-ELISA was established. The comparison results between P2-ELISA and kit Synbiocits-ELISA showed that they had93.4%agreement in the detecting results of196sera samples. It indicated that the indirect P2-ELISA had the same specific and sensitive as the Synbiocits-ELISA kit. In addition, The animal test results showed that this recombinant protein had80%protection rate in Balb/c mice.5. Application and Development of PCR Assay for detection of OMP P2Gene of Haemophilus parasuisA PCR assay for amplifying OMP P2gene of Haemophilus parasuis（HPS） was developed for quick and accurate detection of HPS. The results showed that the size of the amplified PCR product was approximately1080bp. The required minimum dose of HPS was1000bacteria. The specificity of the PCR was tested on Actinobacillus pleuropnemoniae, Pasteurella multocida, E coli, Salmonella enterica, Staphylococcus aureus and Streptococcus, the results showed that it did not recognize these bacteria. The positive results were obtained by amplifying the extracted DNA from the artificial challenged piglets. And the negative results were obtained from the natural infection. Twelve clinical samples （6from experimentally infected piglets with HPS isolates and6from naturally infected piglets） were tested by PCR. The results showed that the PCR assay could be used to detect HPS from lung sample. The lung samples of sick piglets from116pig farms were detected, the results showed that the110samples of them were positive to HPS, which were consist with the results as16S rRNA PCR test. It indicated that the P2-PCR assay could be used to detect HPS, specially in the Glasser’s disease.