笋壳鱼源柱状黄杆菌的分离鉴定及致病性和药物敏感性

Isolation,identification,pathogenicity and drug susceptibility of Flavobacterium columnare from Oxyeleotris marmoratus

【目的】明确2022年5月广东省珠海市某养殖场笋壳鱼烂鳃病的病原,为防控笋壳鱼相关病害提供参考依据。【方法】从患病笋壳鱼的腐烂鳃丝、肾脏和体表病灶等部位分离细菌,并纯化优势菌落,通过PCR方法扩增分离菌株的16S rRNA序列并进行测序分析,同时构建该基因的系统发育进化树,鉴定患病笋壳鱼的病原菌。选取代表菌株Om2202进行毒力基因检测和斑马鱼人工感染试验。测定中草药和消毒剂对分离菌株Om2202的抑菌和杀菌活性,分析菌株Om2202的致病性和药物敏感性。【结果】从患病鱼鳃部和肾脏分离到的优势菌落呈细长的弯曲或直杆状,菌体表面未见鞭毛和菌毛,有荚膜,有胞外分泌物;通过特异性PCR检测和16S rRNA序列的系统发育进化树分析,结合菌落形态分析结果,确定分离菌株Om2202为柱状黄杆菌(Flavobacterium columnare)。毒力基因检测结果显示,菌株Om2202携带gldD、hly、cslA、ompA和chiA等11种毒力相关基因。斑马鱼致病性试验结果显示,菌株Om2202对斑马鱼具有较强致病性,其LC50为7.50×106 CFU/mL。药物敏感性试验结果显示,Om2202菌株对芦荟大黄素、大黄素和苯扎溴铵等药物敏感,对连翘精油、二氧化氯、蛋氨酸碘和过氧化钙等药物不敏感。【结论】引起笋壳鱼烂鳃的主要致病菌为柱状黄杆菌,属于广泛流行的基因Ⅱ型菌株,且携带大量的毒力相关基因,对斑马鱼具有较强的致病性,可用芦荟大黄素、大黄素和苯扎溴铵等药物进行防控。

【Objective】In order to clarify the pathogen of gill rot of Oxyeleotris marmoratus in a farm in Zhuhai City,Guangdong Province in May 2022,and to provide reference for prevention and control of the related diseases of O.marmoratus.【Method】Bacteria were isolated from the rotten gill,kidneys and surface ulceration of the diseased fish,and the dominant pathogen was purified,and the 16S rRNA gene of the isolate was amplified by PCR,and then was sequenced and analyzed.Furthermore,phylogenetic tree of the 16S rRNA gene was constructed to identify pathogenic bacteria.A representative strain Om2202 was selected for detection of virulence genes and artificial infection test of Danio rerio.In addition,the antibacterial and bactericidal activities of Chinese herbal medicines and disinfectants against the isolate Om2202 were determined,and the pathogenicity and drug susceptibility of strain Om2202 were analyzed.【Result】The dominant strains isolated from the gill and kidney of the diseased fish were slender curved or straight rod-like,without flagella and fimbriae,with capsules and extracellular secretions.The isolate Om2202 was identified as Flavobacterium columnare based on specific PCR,phylogenetic tree analysis of 16S rRNA gene,and colony morphology analysis.The results of detection of virulence genes showed that the strain Om2202 carried 11 virulence-related genes including gldD,hly,cslA,ompA and chiA.The results of pathogenicity test showed that Om2202 strain had strong pathogenicity to D.rerio,and its LC50 was 7.50×106 CFU/mL.The results of drug susceptibility test showed that strain Om2202 was sensitive to aloe-emodin,emodin and benzalkonium bromide,but not sensitive to Forsythia suspensa essential oil,chlorine dioxide,methionine iodine and calcium peroxide.【Conclusion】F.columnare is the main pathogenic bacteria causing the gill rot of O.marmoratus,which belongs to the widespread genotype II and carries a large number of virulence-related genes.It has a strong pathogenicity to D.rerio and can be controlled with aloe-emodin,emodin and benzalonium bromide.