网箱养殖大黄鱼假单胞菌病病原的分离与鉴定  

Isolation and identification of pseudomonassis pathogen from cultured Pseudosciaena crocea

作　　者：沈锦玉[1] 余旭平[2] 潘晓艺[1] 许文军[3] 尹文林[1] 曹铮[1] 

机构地区：[1]浙江省淡水水产研究所,湖州313001 [2]浙江大学动物科学学院,杭州310029 [3]浙江省海洋水产研究所,舟山316000

出　　处：《海洋水产研究》2008年第1期1-6,共6页Marine Fisheries Research

基　　金：浙江省科技厅重点项目(2005F12005);浙江省海洋与渔业局项目(2004-S-02)共同资助

摘　　要：浙江宁波、台州等地相继发生网箱养殖大黄鱼的大量死亡,主要症状为脾脏、肾脏有许多白色结节,大小为0.1～1mm。从病鱼体内分离出致病力极强的菌株GYCWS,人工感染试验证实为大黄鱼的病原菌,其半数致死量(LD50)为8.5×104CFU/ml。对该细菌进行了形态、生理生化特性的测定,并进行了全自动微生物分析系统(ID32GN)及16S rRNA分子鉴定。经PCR扩增获得了大小约1.5Kb的16S rRNA部分基因片段,产物经回收纯化,克隆到pMD18-T载体,转化大肠杆菌TG1,对阳性克隆子进行酶切及PCR鉴定确认阳性重组质粒,将测定的序列递交NCBI进行BLAST同源序列比对,与恶臭假单胞菌的16S rRNA基因(DQ201403、AY785244)具有较高的同源性(99%),该序列在Genbank上的登录号为DQ648602。结合细菌的生理生化特性,GYCWS菌株属于恶臭假单胞菌(Pseudo monas putida)。药敏试验结果表明,病原菌GYCWS对卡那霉素、庆大霉素、丁胺卡那、氟派酸、四环素、链霉素药物敏感。ABSTRACT Serious epidemic of great yellow croaker （Pseudosciaena crocea） cultured in net-cage in Ningbo and Taizhou, Zhejiang Province, broke out in spring 2004 and 2005. The main symptom of diseased fish was the appearance of many 0.1 - 1mm size white spots in the spleen and kidney. Strain GYCWS isolated from the diseased fish was confirmed to be the pathogen of the epidemic by challenge test, and its LD50 was 8.5×10^4 CFU/mL. The morphological, physiological and biochemical characters of the GYCWS colony isolate was then determined. About 1.5Kb fragments of the isolate was amplified by PCR using general primers of 16s ribosomal DNA. The PCR product was extracted and cloned into pMD 18-T vector. After the transformation into E. coli TG1, the recombination plasmid was identified by restriction enzyme digestion and by PCR. One selected clone was then sequenced. The BLAST results of the sequence showed that it has high homology （99%） with respective sequences of Pseudomonas putida. The accession number of 16S rRNA gene sequence was DQ648602 on Genbank. The results indicated that GYCWS isolate belonged to Pseudomonas putida. The test of sensitivities of strain GYCWS to 14 kinds of antibiotics revealed that the pathogen was sensitive to kanamytin, gentamicin, amikacin, norfloxacin, tetracycline and streptomycin.

关 键 词：大黄鱼 病原 恶臭假单胞菌 16S RRNA 

分 类 号：S941.42[农业科学—水产养殖]