副鸡禽杆菌毒力质粒缺失株的构建及其致病性的研究

Construction and pathogenicity of plasmid deletion strain of Avibacterium paragallinarum

为研究副鸡禽杆菌(APG)质粒与菌株致病力的关系,本研究采用42℃诱导的方法缺失APG分离株APG-Hu中的毒力质粒,经PCR鉴定质粒缺失菌株后将其命名为APG-HuΔ。分别采用分光光度计测定OD600nm值法、微量生化鉴定管法、K-B纸片法和雏鸡感染试验测定APG-HuΔ的生长曲线、生化特性、药物敏感性和对雏鸡的致病性。结果显示,在42℃诱导条件下,能够有效缺失APG-Hu中的毒力质粒p250和p A14,而质粒p YMH5未被缺失;APG-HuΔ生化特性、生长曲线及耐药性与APG-Hu相比均无明显差异。雏鸡感染试验结果显示,多数APG-HuΔ感染鸡无明显临床症状,总体发病情况明显弱于同等剂量APG-Hu感染的鸡,表明APG-HuΔ毒力显著降低。本研究结果表明,APG毒力与质粒p250和p A14相关,而质粒p YMH5可能增加APG耐受多种抗生素的风险。本研究为APG致病机理及耐药机制研究奠定基础,也为挖掘APG质粒的应用潜力提供一定参考。

In order to study the relationship between the plasmid and the pathogenicity of of Avian paragallinarum(APG)strain,the virulence plasmid of APG isolate APG-Hu was deleted by maintain at 42℃.The plasmid-deleted strain was identified by PCR and named APG-HuΔ.The physiological and biochemical characteristics,growth curve,drug sensitivity and pathogenicity of APG-HuΔwere determined by biochemical micro-identification tube method,spectrophotometer at OD600nm,test paper method and chicken infection test respectively.The results showed that plasmids p250 and pA14 were deleted in APG-HuΔ,except for pYMH5.There were no significant difference in the physiological and biochemical characteristics,growth curve,and drug resistance between APG-HuΔand the parent strain of APG-Hu.The results of chicken infection experiments showed that most of chickens inoculated with APG-HuΔhad no obvious clinical manifestations,which was similar as normal,and the overall incidence was significantly weaker than that of the same dose of APG-Hu infected chickens,indicating the virulence of APG-HuΔwas much lower than that of APG-Hu.The results suggested that the virulence of APG was related to plasmid of p250 and pA14,and the potential tolerance to a variety of antibiotics might associated with the presence of pYMH5.This study not only lays a foundation for the study of the pathogenesis and drug resistance mechanism of APG,but also provides some reference for exploring the application potential of APG plasmid.