Epidemiological Investigation Of Haemophilus Parasuis In Eastern China And Screening Of Subunit Vaccine Candidates

Haemophilusparasuis(H.parasuis)is an important respiratory-tract pathogen in swine and the etiological agent of porcine polyserositis,polyarthritis and meningitis,known as Glasser's disease.To date,15 H.parasuis serovars have been described,each with apparently different virulence.Serovars 1,5,10,and 12-14 are considered to be highly virulentandlead to high mortality in pigs.Serovars 2,4,8 and 15 are recognized as less virulent,and 6,7,9 and 11 are considered asavirulent.In addition,a large number of un-classifiable isolates are frequently reported.Because of its existence in a variety of serovars,the inactivated vaccine lacked cross protection.Antibiotics were still widely used all over the world.With the abuse of antibiotics,strain resistance was increasing year by year.Therefore,the potential virulence factors of Haemophilus parasuis had become the focus of research,which laid the foundation for the screening of subunit vaccines.In this study,30 clinical isolates of H.parasuis were isolated and identified.After PCR identification,antimicrobial susceptibility,a novel plasmid,designated pHPSGC,was extracted from GC stain with resistance to florfenicol.Proteome analysis of 2 avirulent strains BZ,SY(serovar 7)and 2 highly virulent strains BB,LC(serovar 13)was performed with Lable free.Finally,transferrin-binding protein 1,CdtB,htpG,Heme-binding protein A and tubulin binding protein were further confirmed as potential virulence factor for subsequent validation experiments.1.Epidemiological investigation and antimicrobial susceptibility patterns of haemophilus parasuis strains in Eastern ChinaTo reveal antimicrobial susceptibility of clinical isolates in eastern China,30 clinical isolates of H.parasuis were isolated and identified.Antimicrobial susceptibility showed that all isolates were sensitive to ceftiofur and 29 of them sensitive to florfenicol.However,3 to 8 antibiotics resistance were carried by all isolates,especially,all isolates carried resistance to tiamulin and kanamycin.Importantly,a novel plasmid,designated pHPSGC,was extracted from GC stain with resistance to florfenicol.Genome sequencing revealed that pHPSGC is 5297 bp in length with 44.82%GC content.Three open reading frames(ORF)were identified,corresponding to the genes rep,floR,and lysR.Sequence analysis showed that the homology of the rep gene of pHPSGC with that of pHPS1019 is 99.9%.At the same time,the gene fragment containing floR and lysR of pHPSGC is 90.2%homology with that of pHPSF1.So,we can conclude that pHPSGC maybe derived from the recombination between pHPS1019 and pHPSFl.Our results firstly demonstrated that florfenicol resistance have been spread by the recombination in different plasmids existed in H.parasuis,which will constitutes a serious challenge to the swine industry in China.2.Analysis of protein composition of different Haemophilus parasuis virulence isolates In order to screen subnuit vaccine candidates to effectively control this disease,proteome analysis of 2 avirulent strains BZ,SY(serovar 7)and 2 highly virulent strains BB,LC(serovar 13)was performed with Lable free.17,444 unique peptides were identified,which targeted to 2282 protein groups and 1199 of them were co-existed in 4 candidate strains and only 117 proteins were found in BB and LC strains.To better understand the function of proteins only identified in BB and LC strains,we performed gene ontology analysis using STRAP(Software Tool for Researching Annotations of Proteins).Finally,transferrin-binding protein 1,CdtB,htpG,Heme-binding protein A and tubulin binding protein were further confirmed as potential virulence factor for subsequent validation experiments.3.Virulence repertoire revealing of Haemophilus parasuis and Identification of immune protectionHaemophilus parasuis(H.parasuis)is the etiological agent of Glasser's disease in pigs.At least 15 serovars have been identified,each with varying virulence.In order to screen subunit vaccine candidates,we analyzed the proteomes of 2 avirulent strains BZ and SY(serovars 7),and 2 highly virulent strains BB and LC(serovar 13).2282 total proteins were identified,1199 of which are present in all four strains.117 proteins are unique to 2 virulent BB and LC strains.Five of the unique proteins,transferrin-binding protein 1,CdtB,htpG,heme-binding protein A and tubulin binding protein,were examined more closely.To our surprise,these proteins are present in all four strains,but heme-binding protein A(HbpA)is expressed at a higher level in the virulent strains.Thirteen field strains were used to confirm the correlation between HbpA expression and bacteria virulence.Finally,Balb/C mice were immunized with purified HbpA and subsequently challenged with 100-and 1000 LD50H.parasuis.Heme-binding protein A induces high titers of antibody and provided partial protection to the immunized mice.Our study demonstrates that heme-binding protein A is highly expressed in virulent H.parasuis strains,and may be pathogenesis associated.Our animal studies suggest that HbpA is a potential candidate for subunit vaccine development.