双棘黄姑鱼源致病发光杆菌的分离与鉴定

Isolation and identification of a pathogenic Photobacterium luminescent bacterium from Protonibea diacanthus

【目的】分离鉴定双棘黄姑鱼(Protonibea diacanthus)源致病发光杆菌,为查明引起双棘黄姑鱼出现大批量死亡的病因及科学指导防控该病的规范用药提供参考依据。【方法】对从广西北海市铁山港网箱养殖发病双棘黄姑鱼肾脏中分离到的菌株(编号20210120)进行人工感染试验,确定其是否为双棘黄姑鱼致病菌;并对该菌株进行形态学观察、多基因(gapA、topA、mreB、ftsZ、gyrB和16S rRNA)序列分析及药敏试验。【结果】分离菌株与托鲁尼发光杆菌(Photobacterium toruni)16S rRNA序列在系统发育进化树上聚类为一支,其序列相似性达99.93%,与5个管家基因(gyrB、gapA、topA、ftsZ和mreB)序列的相似性分别为99.50%、99.86%、100.00%、99.83%和99.44%,因此确定菌株20210120为托鲁尼发光杆菌。人工感染双棘黄姑鱼病鱼出现与自然发病相同的病症,且再次从其肾脏中分离到与自然发病相同的细菌。药敏试验结果表明,菌株20210120对恩诺沙星、氧氟沙星、环丙沙星、复方新诺明、头孢氨苄、链霉素和庆大霉素7种药物敏感。结合农业农村部《水产养殖用药明白纸》文件中批准使用的药物目录,建议选用恩诺沙星对双棘黄姑鱼托鲁尼发光杆菌进行防治。【结论】托鲁尼发光杆菌是引起广西北海双棘黄姑鱼发病死亡的病原菌,实际生产中可考虑将恩诺沙星、氧氟沙星、环丙沙星、复方新诺明、头孢氨苄、链霉素和庆大霉素7种药物(尤其是恩诺沙星)作为托鲁尼发光杆菌引起双棘黄姑鱼相关疾病的防治用药。

【Objective】This study was for identifying the pathogen caused a large number of Protonibea diacanthus dead and guiding the standardization of medication by isolation and identification of pathogenic bacillus Photobacterium.【Method】The bacterial strain(20210120)was isolated from the kidney of the diseased P.diacanthus raised in Tieshangang,Beihai,Guangxi.The strain 20210120 was confirmed to be the pathogens of P.diacanthus through artificial infection test.Morphological observation,sequence analysis of multiple genes(gapA,topA,MreB,FtsZ,gyrB and 16 S rRNA)and drug sensitivity analysis were carried out.【Result】The similarity of 16 S rRNA sequence between 20210120 and P.toruni was 99.93%,and clustered to one group in the phylogenetic tree.The sequence similarity to the five housekeeping genes(gyrB,gapA,topA,ftsZ,and mreB)was 99.50%,99.86%,100.00%,99.83%,and 99.44%,respectively,so the pathogenic bacteria were identified as P.toruni.Artificial infection with P.diacanthus disease fish appeared the same symptoms as natural diseased fish,and the same bacteria were isolated as natural diseased kidneys.The drug sensitivity test showed that the strain was sensitive to enrofloxacin,ofloxacin,ciprofloxacin,compound sulfamethoxazole,cephalexin,streptomycin and gentamicin.In combination with the list of drugs approved in the document of Guidelines of Aquaculture Drugs by the Ministry of Agriculture and Rural Affairs,it was recommended to choose enrofloxacin for the prevention and control of P.toruni.【Conclusion】P.toruni is the pathogen causing the death of P.diacanthus in Beihai,Guangxi.Enrofloxacin,ofloxacin,ciprofloxacin,compound sulfamethoxazole,cephalexin,streptomycin and gentamicin(especially enrofloxacin)can be used in the diseased P.diacanthus caused by P.toruni.