| Haemophilus parasuis is a commensal organism of the upper respiratory tract of pigs,but under appropriate conditions can invade and cause severe systemic disease,characterized by fibrinous polyserositis, arthritis, meningitis, pneumonia, high fever andhigh mortality rate, known as Glasser’s disease. This disease has become one of the mostimportant bacterial diseases, being responsible for considerable economic losses in theswine industry worldwide. To date, fifteen serovars of H.parasuis, which typically have arange of different virulence potentials, have been described. In addition, a high percentageof strains are non-serotypeable. Limited cross-protection among strains and little knownabout virulence factors specific to H.parasuis have complicated the control of Glasser’sdisease and vaccine development. Therefore, it is important for the control of Glasser’sdisease to start relative research on HPS. The study reported here included:①Understand the antimicrobial susceptibility of Haemophilus parasuis in Anhui, and thecorrelations between antimicrobial resistance and virulence, serovars or genotypes, whichcan provide a scientific basis for effective Glasser’s disease prevention and control;②Sub cloning and expressing tbpA gene of HPS and discussing the role of sub-protectiveimmunity proteins for the prevention and control and the development of new HPS subunitvaccine basis. The main research included as below:1. Serological characterization and genotypes of Haemophilus parasuis isolates fromAnhui provinceFrom fourteen regions in Anhui province,24strains of Haemophilus parasuis wereisolated from samples of diseased pigs from2008to2011. All isolates were serotyped byKRG gel diffusion test and ERIC-RCR, PCR-RFLP.Three Haemophilus parasuis serovars were identified. Serovar4(29.17%) was themost prevalent serovar, followed by serovar14(12.5%) and serovar14(8.33%), while50%of the isolates could not be assigned to a serovar (nontypable).24Haemophilus parasuisisolates were identified to11distinct strains by enterobacterial repetitive intergeneicconsensus based-polymerase chain reaction (ERIC-PCR). Genotypes Ⅳ(20.83%,5/24) wasthe most prevalent genotypes, followed by genotypes Ⅰ(16.67%,4/24) and genotypesⅡ(12.5%,3/24)；24HPS isolates were analysed by using RFLP-PCR based on tbpA geneproduced6different patterns, the prevalent genotypes were DBP(37.5%,9/24) andBBB(20.83%,5/24). The strains which can not be divided were also obtained effectivetyping. 2. Study on the antimicrobial resistance of Haemophilus parasuis isolatesIn vitro susceptibility test of the24H.parasuis isolates was performed to24kinds ofdrug by Kirby-Bauer disk diffusion method, meanwhile common PCR technique was usedin this trial to detect28kinds of drug resistance genes in the isolates.All isolates were susceptible to ofloxacin, cefalexin, cefamezin, cefotaxime, rocephin,erythromycin, azithromycin, gentamicin, spectinomycin and florfenicol. Most of them weresusceptible to ampicillin(95.8%), amoxicillin(95.8%), chloromycetin(95.8%),penicillin(91.7%); the sensitivity rate of ofloxacin, enrofloxacin, cefradine and amikacinwere all83.3%; the sensitivity rate of trimethoprim and doxycycline were all75%.However, a high level of resistance was observed for sulfamethoxazole(58.3%) andstreptomycin(54.2%). The ciprofloxacin and tetracycline got the highest resistance rate of41.7%and37.5%.There were21antimicrobial antibiograms observed in the24H.parasuis isolates. Onlynine isolates(12.5%) were susceptible to all drugs tested; however,20.8%isolates beingresistant to only one or two antibiotics, multidrug resistant strains(≥3antimicrobial agents)were observed in66.7%of all isolates.5isolates(20.8%) were resistant to at least fiveantimicrobials; and only one isolate(4.2%) was resistant to seven antimicrobialsstimultaneously.PCR technique was used in this trial to detect28kinds of drug resistance genes.23strains of24H.parasuis isolates were positive with rate of95.8%. A total of13kinds ofdrug resistance genes were all detected on genomic and plasmid DNA. The phenotype ofmultidrug-resistance and the drug-resistant genes were consistent.Serovar4was the most prevalent serovar, followed by serovar14and serovar13, theirsensitivity rate were respectively14.3%,9.7%and0%. Although the resistance rate wasdifferent in different types of serovar, but it is impossible to observe the correlationbetween the antibiotic resistance and the serovars of H.parasuis because of the limitednumbers of the strains a number of nontypable strains. The prevalent genotypes wereDBP(37.5%4) and BBB(20.83%) in RFLP-PCR, their sensitivity rate were respectively19.9%and10%. The results showed that the resistance was high in the prevalent genotypes.Comparing the change of drug resistant in different period of isolates, the multiple drugresistance spectrum was broaden and the multiple drug-resistant phenomenon alsoincreasingly serious. 3. Cloning and expression the tbpA gene of HPS and the protection of therecombinant protein in guinea pigsPrimers were designed according to the tbpA gene sequence of HPS SH0165strainform GenBank number7277767, tbpA gene from H.parasuis serovar13(LJ3strain) wasamplified by PCR and cloned into a pET-SUMO expression vector, generating thepET-SUMO-tbpA construction. It was transformed into the Escherichia coil Rosettacompetent cells, recombinant protein was expressed highly after induced and sodiumdodecyl sulphate-polyacrylamide gel electrophoresis(SDS-PAGE) analysis showed that themolecular weight of the protein was approximately110kD in agreement with the expect.The results of western blotting analysis demonstrated that the fusion protein possessedantigenic activity and could be specifically recognized by antiserum against HPS(LJ3strain). The guinea pigs were vaccinated subcutaneously on1day,20days. The antiserumwas taken before the day of the first vaccine and20days after each vaccine. And theantibody of ELISA-rTbpB is determinded. A significant increase in IgG titer was detectedin all protein-immunized groups. The cytokines in the blood of the second immunized wereanalysed by quantitative real-time PCR, and the production of interferon gamma(IFN-γ),interleukin2(IL-2), interleukin5(IL-5), interleukin8(IL-8), tumour necrosis factorα(TNF-α) and monocyte chemoattractant protein-1(MCP-1) were increased significantly.Three weeks after the booster immunization, all groups were intraperitoneally challengedwith a lethal dose of H.parasuis serovar4and serovar13, the result indicated that TbpAshowed good protection against infection with HPS of the homologous serovar and alsoshowed good cross-protection against infection with the heterogenous serovar. |
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