【作者】 林晶；

【导师】 俞道进；

【作者基本信息】 福建农林大学 ， 基础兽医学， 2016， 硕士

【摘要】 目的:目前H.parasuis在世界范围内的广泛流行,给规模化猪场的生产养殖造成了巨大的经济损失。由于H.parasuis血清型众多,而且它的毒力因子非常复杂,到现在各种毒力因子的作用机理以及各种毒力因子之间的相互作用关系还不是很清楚,使得副猪嗜血杆菌病的防治更加困难。因此对H.parasuis毒力因子的研究已经成为现在的重点。本试验的主要目的是为了筛选出有效的H.parasuis毒力因子抑制剂,为该病的预防和控制提供新的方向和理论基础。方法:临床采集病料,对H.parasuis进行分离鉴定和培养;根据CLSI提供的标准方法,检测所选的十四种中药单体对H.parasuis的最小抑菌浓度(MIC)和最小杀菌浓度(MBC);通过荧光定量PCR研究十四种单体对H.parasuis的生长及潜在的毒力相关基因表达量的影响。结果:从福建地区临床采集的45份病料中分离到4株H.parasuis菌株。本研究选择两株标准的H.parasuis强毒株及两株临床分离株作为研究对象,对所选择的十四种中药单体进行了体外抑菌试验,发现黄芩苷、连翘苷、没食子酸和姜黄素的抑菌活性较弱。其余十种中药单体对H.parasuis都有抑菌作用,MIC范围在8-128μg/mL之间。尤其是木犀草素和盐酸小檗碱的抑菌活性最强,MIC范围在8-32μg/mL之间。十四种中药对两株标准株及两株临床分离株的最小杀菌浓度显示,4株H.parasuis对十四种中药单体均没有产生耐药性。通过荧光定量PCR分析发现,同一种单体对3株不同菌株的作用不同,其中两株为标准株,一株为临床分离株。试验筛选出的四种中药单体,黄萼苷、槲皮素、木犀草素和没食子酸对三株H.parasuis毒力基因表达都有良好的抑制作用。能够有效抑制外膜蛋白基因(OmpP-5和OmpP-2)、溶血操纵子基因(hhdA和hhdB)、细胞致死膨胀素基因(cdtA和cdtC)、荚膜形成相关基因(capD)、生物膜形成相关基因(gaIU和mviN)、转铁结合蛋白(tbpA)基因的表达,通过抑制基因的表达,降低H.parasuis毒力。结论:本试验临床分离到了 4株H.parasuis。木犀草素和盐酸小檗碱对H.parasuis的抑菌活性最强,MIC范围在8-32μg/mL之间。4株H.parasuis对十四种中药均没有产生耐药性。所选十四种中药单体对不同H.parasuis菌株毒力相关基因的表达影响各不相同,推测三种不同菌株的主要毒力因子不同。黄芩苷、槲皮素、木犀草素和没食子酸对三株H.parasuis毒力基因表达都有良好的抑制作用。可以作为候选H.parasuis毒力抑制剂进行更深入的研究。更多还原

【Abstract】 Objective:The widespread of H.parasuis has caused huge economic losses to the swine industry.The virulence factors of H.Parasois are very complex,the role of virulence factors and the interaction among the mechanism of virulence factors are still unclear,which make the prevention and control of H.parasuis in pig farm more difficult.Therefore,the research on virulence factors of H.parasuis has become the focus of the study.This study focused on the selection of effective inhibitor of virulence factors of H.parasuis.and providing of a new theoretical basis for the prevention and control of the disease.Method:H.parasuis were isolated from clinical and identificated;the minimum inhibitory concentration(MIC)and the Minimum bactericidal concentration(MBC)of H.parasuis to fourteen kinds of traditional Chinese medicine monomer was detected according to the susceptibility method described by CLSI;The effect of fourteen monomers on the growth of H.parasuis and the expression of the potential virulence associated genes was studied by real-time PCR methods.Results:4 strains of H.parasuis were isolated and identified.In vitro susceptive study on 14 kinds of traditional Chinese medicine monomer to H.parasuis found that the antibacterial activity of baicalin,forsythin,gallic acid and curcumin were relativly weak,meanwhile,other ten kinds of traditional Chinese medicine monomer had antibacterial effect on H.parasuis,MIC value ranged between 8-128 μg/mL.Luteolin and berberine hydrochloride have the MIC ranged between 8-32 μg/mL.Four strains of H.parasuis showed no resistance to fourteen kinds of traditional Chinese medicine monomer.Further real-time PCR research found that the effect of the same monomer on selected H.parasuis strains was different,among them,2 were standard strains and 1 was clilical isolated strain.But the effect of Baicalin,quercetin,luteolin and gallic acid on the relative expression level of virulence genes of three strains have good inhibitory effect,which can effectively inhibit the expression of the outer membrane protein genes(OmpP-5 and OmpP-2),Hemolytic operon genes(hhdA and hhdB),Cytolethal distending toxin(cdtA and cdtC),Capsular formation related genes(capD),Biofilm formation related genes(gaIU and mviN),Transferrin binding protein(tbPA).Reducing the H.parasuis toxicity by inhibiting the expression of diferrent genes.Conclusions:4 strains of H.parasuis were isolated and identified in this study.Luteolin and berberine hydrochloride had antibacterial effect on H.parasuis with the MIC ranged between 8-32 μg/mL.Four strains of H.parasuis showed no resistance to fourteen kinds of traditional Chinese medicine monomer.Fourteen kinds of Chinese medicinal herbs had different effects on the expression of virulence related genes of different H.parasuis strains.Main virulence factors of the three different strains may be different.Baicalin,quercetin,luteolin and gallic acid had good inhibitory effects on the expression of virulence related genes of three H.parasuis strains.Which could be used as candidate virulence inhibitors in H.parasuis study.更多还原