鲫源迟缓爱德华氏菌的分离鉴定及其毒力基因的检测

摘要

[目的]探明上海某养殖场的异育银鲫(Carassius auratus gibelio)发生死亡的病因,研究致病菌分类地位和毒力基因携带情况.[方法]通过病原菌的筛查和回感试验,确定发病原因,对16S rRNA基因、gyrB和rpoB管家基因测序,建立病原菌系统进化树,结合API-32E细菌鉴定系统对菌株的生理生化进行鉴定,综合判定致病菌的种类及其分类地位;根据已报道的7个毒力基因fimA、citC、gadB、mukF、katB、esrB和sodB序列,设计引物进行PCR扩增,研究病原菌毒力基因携带情况.[结果]致病菌GY15是导致异育银鲫发病的原因,GY15的16S rRNA、gyrB和rpoB基因与已报道的迟缓爱德华氏菌(Edwardsiella tarda)相似性在99％以上,构建系统进化树和API鉴定确定该菌株为E.tarda,腹腔注射回感可导致异育银鲫死亡,半致死浓度(LDso)为4.26×105 CFU/mL;已报道的7种毒力基因在该致病菌中均能被检测到;药敏试验结果显示,该菌对恩诺沙星、氟苯尼考和氧氟沙星等15种药物敏感,对新霉素、四环素和复方新诺明等18种药物表现为耐药.[结论]首次报道E.tarda可感染异育银鲫,它对异育银鲫的养殖造成威胁.%[Objective] To confirm the pathogeny that causes Carassius auratus gibelio death in some fish farms in Shanghai,and characterize the virulence genes of the pathogenic bacteria.[Methods] The pathogenic bacteria were confirmed based on the pathogens screening and artificial challenge experiment,and identified according to 16S rRNA,gyrB and rpoB genes sequence and biochemical characteristics.Published nucleotide sequence methods were used to study the virulence genes (fimA,citC,gad,mukF,katB,esrB and sodB) by PCR amplification,and PCR primers for virulence genes.[Results] Strain GY15 isolated from infected fish was proved to be the pathogeny to Carassius auratus gibelio.Analysis of the 16S rRNA,gyrB and rpoB gene sequences of the GY15 gave the highest identity (99％) to Edwardsiella tarda.This was further supported by phylogenetic tree and biochemical characteristics.GY15 was identified as E.tarda.Intraperitoneal injection with GY15 could lead to Carassius auratus gibelio death,with a value of the median lethal dose (LDs0) of 4.26× 105 CFU/mL.GY15 gave positive PCR results for 7 virulence genes.Antibiotic sensitivity test showed that among 35 antibiotics tested,GY15 was sensitive to 15 antibiotics such as enrofloxacin,florfenicol and ofloxacin,but was resistant to 18 antibiotics such as neomycin,acheomycin and cotrimoxazole.[Conclusion] Carassius auratus gibelio was the susceptible host species of E.tarda,which is a new potential threat to carp's breeding.