斑点叉尾鮰源致病性维氏气单胞菌的分离与生物学特性鉴定

为鉴定一起致斑点叉尾鮰突然发病死亡的病原,本研究从发病斑点叉尾鮰中分离到1株致病菌GZTL2017,通过临床解剖观察、细菌分离培养、革兰氏染色镜检、动物回归试验、生化试验、16S rDNA序列分析、部分毒力基因检测和药敏试验进行鉴定。临床解剖观察结果显示,患病斑点叉尾鮰呈现体表溃疡、鳍根部出血、烂腮、肠管充血等症状;分离菌在培养基中呈现表面湿润凸起、边缘光滑半透明、形态均一的乳白色菌落;革兰氏染色镜检显示,分离菌为单个或成双存在、两端钝圆的短直阴性杆菌;动物回归试验显示,该分离菌有较强的致病性;生化试验结果显示,分离菌具有运动性,且氧化酶、V-P、赖氨酸脱氢酶等反应阳性,精氨酸双水解酶、硫化氢等反应阴性;16S rDNA基因序列系统进化树显示,该菌与维氏气单胞菌聚为一支,同源性均>99%;毒力基因检测结果显示,该菌能检出气溶素基因（Aer）、黏附素基因（Aha）、外膜蛋白基因（OmpA）3种毒力基因;药敏试验结果显示,该菌对氟苯尼考、强力霉素、氧氟沙星等14种药物敏感;对诺氟沙星、新霉素、万古霉素等7种药物中度敏感;对麦迪霉素、苯唑西林、头孢拉定等8种药物耐药,且对氟苯尼考、强力霉素的药物最小抑菌浓度分别为1和2 μg/mL。本试验成功分离到1株维氏气单胞菌,为斑点叉尾鮰维氏气单胞菌病防治提供参考依据。

Isolation and Biological Characterization of the Pathogenic Aeromonas veronii from Ictalurus punctatus

In order to identify the agent of a serious disease occurred in Ictalurus punctatus,a dominant bacteria (GZTL2017) was isolated from diseased Ictalurus punctatus.It was identified by clinical anatomy observation,bacterial isolation and culture,gram stain microscopy,animal regression test,biochemical test,16S rDNA sequence analysis,partial virulence gene detection and drug susceptibility analysis.Clinical anatomical observations showed that the diseased Ictalurus punctatus showed surface ulcers,fin root bleeding,gills and symptoms of intestinal congestion.The isolate showed milky white colonies with moist surface,smooth and translucent edges,and uniform morphology in the medium.Gram stain microscopy showed that the isolate was single or double-existent,with straight-negative bacilli at both ends.Animal regression tests showed that the isolate had strong pathogenicity.Biochemical tests showed that the isolate was mobilized,with positive reactions such as oxidase,V-P and phenylalanine deaminase,and negative reactions such as arginine dihydrolase and H₂S.The 16S rDNA sequence of the isolate was more than 99% homology with that of Aeromonas veronii,and the isolate formed a cluster with other Aeromonas veronii reference strains by phylogenetic tree.Three virulence genes were detected from the isolate,including aerolysin (Aer),adhesion (Aha) and outer membrane protein A (OmpA) genes.The drug sensitivity test showed that the strain was sensitive to 14 drugs such as florfenicol,doxycycline and ofloxacin;It was moderately sensitive to 7 drugs such as norfloxacin,neomycin and vancomycin;It was resistant to 8 drugs such as medimycin,oxacillin and cefradine;And the minimal inhibitory concentrations of florfenicol and doxycycline were 1 and 2 μg/mL,respectively.This experiment successfully isolated a single strain of Aeromonas veronii,which provided a scientific basis for the prevention and treatment of Ictalurus punctatus infected with Aeromonas veronii.