乌原鲤源致病性维氏气单胞菌分离鉴定及其毒力基因分析

Identification and virulence gene analysis of pathogenic Aeromonas veronii isolated from Procypris merus

【目的】明确引起乌原鲤(Procypris merus)发病死亡的病原菌,分析其毒力基因,为乌原鲤养殖中的病害诊断及有效防治提供理论依据。【方法】从患病乌原鲤肝脏和肾脏中分离病原菌,采用形态学观察、生理生化特性鉴定及16S rDNA序列分析对分离菌株进行鉴定,通过人工回归感染试验确定分离菌株的致病性,使用纸片扩散法进行药敏试验,并对18种毒力基因进行PCR检测。【结果】从患病乌原鲤肝脏和肾脏中分离纯化到1株革兰氏阴性短杆菌(命名为19042401),经形态学观察、生理生化特性鉴定和16S rDNA序列分析,确定菌株19042401为维氏气单胞菌(Aeromonas veronii);人工回归感染试验结果表明该菌株对乌原鲤具有较强的致病性,半数致死量(LD50)为5.75×107 CFU/kg;药敏试验结果表明,在19种常见抗生素中,菌株19042401对卡那霉素、阿奇霉素、多西环素等6种药物敏感,对链霉素、罗红霉素、庆大霉素等7种药物中度敏感,对青霉素、恩诺沙星、阿莫西林等6种药物耐药;毒力基因PCR检测结果表明,菌株19042401携带Act、Aer、GcaT、Acg、OmpAI、Alt、Fla、Exu、Ser和AhyB共10种毒力基因。【结论】维氏气单胞菌是引起此次乌原鲤发病的致病菌,携带10种毒力基因,对乌原鲤具有较强的致病性,仅对少数药物敏感,对多种常用抗生素具有不同程度的耐药性,养殖生产中可选用多西环素进行治疗。

【Objective】The pathogen causing Procypris merus disease and death was identified,and its virulence genes were analyzed to provide theoretical basis for disease diagnosis and effective prevention in future P.merus culture.【Method】Pathogenic bacteria were isolated from the liver and kidney of diseased P.merus,and the isolated strains were identified by morphological observation,physiological and biochemical characteristics identification and 16S rDNA sequence analysis.The pathogenicity of the isolated strain was determined by artificial regression infection test,and the drug sensitivity test was carried out by disk diffusion method,and 18 virulence genes were detected by PCR.【Result】A Gram negative short bacillus(named 19042401)was isolated and purified from the liver and kidney of diseased P.merus.After morphological observation,physiological and biochemical identification and 16S rDNA sequence analysis,it was identified as Aeromonas veronii.The results of artificial regression infection showed that the strain had strong pathogenicity,with a lethal dose(LD50)of 5.75×107 CFU/kg.The results of drug sensitivity test showed that among 19 kinds of common antibiotics,strain 19042401 was sensitive to 6 drugs such as kanamycin,azithromycin and doxycycline,mode-rately sensitive to 7 drugs such as streptomycin,roxithromycin and gentamicin,and resistant to 6 drugs such as penicillin,enrofloxacin and amoxicillin.The results of the virulence gene PCR indicated that strain 19042401 carried 10 virulence genes,including Act,Aer,GcaT,Acg,OmpAI,Alt,Fla,Exu,Ser and AhyB.【Conclusion】A.veronii was the pathogenic bacterium causing the disease of P.merus,which has strong pathogenicity and carries 10 virulence genes.It is only sensitive to a few drugs and has different degrees of resistance to many commonly used antibiotics.Doxycycline can be used for treatment in aquaculture production.