肉鸡源产气荚膜梭菌的分离鉴定及耐药性分析

Isolation,Identification and Drug Resistance Analysis of Clostridium perfringens from Broiler Chickens

【目的】对江苏某大型白羽肉鸡场疑似产气荚膜梭菌感染造成大规模死亡的病例进行确诊。【方法】无菌采集病死鸡肝脏、脾脏、肺脏、肾脏及肠道等病变组织,通过厌氧培养进行细菌分离和纯化,革兰染色镜检对分离菌株进行初步鉴定,通过16S rRNA基因序列分析确定属和演化关系,通过PCR扩增毒素基因确定分离株毒素型,通过动物致病性试验和药敏试验,确定其致病性和耐药表型并检测耐药基因。【结果】从4只病死鸡不同脏器中分离到了9株疑似菌,分离菌在产气荚膜梭菌鉴定培养基上呈黑色,16S rRNA分析得出9株分离菌与产气荚膜梭菌相似性最高可达99.60%。16S rRNA基因系统进化树显示,4株分离株与巴基斯坦鸡源产气荚膜梭菌(GenBank登录号:MN365136.1)具有较近的亲缘性,5株分离株与南非鸡源产气荚膜梭菌(GenBank登录号:OR494055)具有较近的亲缘性。毒素分型结果显示,5株为A型,2株为F型,2株为E型。致病性试验结果显示,小鼠全部死亡,表明该菌具有致死性。药敏试验结果显示,9株分离株均对头孢类抗生素敏感,耐药基因分析表明大环内酯类erm(B)(77.8%)和四环素类tetA(P)(55.6%)、tetB(P)(66.7%)为主要耐药基因。【结论】本研究从同一只病死鸡中分离到不同毒素型产气荚膜梭菌,且分离菌耐药情况较严重。研究结果为兽医临床鸡产气荚膜梭菌病防治提供了新的思路。

【Objective】The study was aimed to identify the pathogens of the death of broiler chickens suspected to be infected by Clostridium perfringens in a farm in Nantong,Jiangsu.【Method】The livers,spleens,lungs,kidneys and contents of intestines of the dead chickens were collected aseptically.Bacterial isolation and purification were carried out through anaerobic cultivation,and the isolated strains were preliminarily identified by Gram staining microscopy.Genus and evolutionary relationship were determined by 16S rRNA gene sequence analysis,toxin type was determined by PCR amplification of toxin genes,pathogenicity and drug resistance phenotypes were determined by animal pathogenicity test and drug susceptibility test,and drug resistance genes were detected.【Result】9 suspected strains were isolated from different organs of 4 dead chickens,and the isolates were black on the identification medium of Clostridium perfringens.16S rRNA analysis showed that the similarity between the 9 isolates and Clostridium perfringens was up to 99.60%.The 16S rRNA gene phylogenetic tree showed that 4 isolates were closely related to chicken-derived Clostridium perfringens of Pakistan(GenBank accession No.:MN365136.1),and 5 isolates were closely related to chicken-derived Clostridium perfringens of South Africa(GenBank accession No.:OR494055).The results of toxin typing showed that 5 strains were type A,2 strains were type F and others were type E.The results of pathogenicity test showed that the bacteria were lethal.The drug sensitivity tests showed that 9 strains were sensitive to cephalosporin antibiotics.Resistance genes analysis showed that macrolide erm(B)(77.8%),tetracycline tetA(P)(55.6%),tetB(P)(66.7%)genes were the main resistance genes.【Conclusion】In this study,different toxin-type Clostridium perfringens were isolated from the same dead chicken,and the drug resistance of the isolated bacteria was serious.This result provided a new idea for the prevention and treatment of chicken Clostridium perfringens in veterinary clinic.