一株虾夷扇贝病原——科氏希瓦氏菌的分离鉴定及其致病性

Isolation, identification and pathogenicity of Shewanella colwelliana, a pathogen of Yesso scallop Patinopecten yessoensis

为探究近年来养殖虾夷扇贝Patinopecten yessoensis脓疱病的发病原因,取病灶部位涂布培养,挑取优势单菌落,进行分离纯化、生理生化试验、药敏试验、溶血试验、人工感染试验和组织病理检验等研究。结果表明:从扇贝病灶部位中共分离获得12株优势菌株(YD-1~YD-12),经16S rDNA基因序列比对发现,12株优势菌株与科氏希瓦氏菌Shewanella colwelliana一致性均较高,选取序列一致性最高(99.86%)的分离菌株YD-1进行细菌培养和透射电镜观察发现,该菌株于培养基中呈圆润光滑的乳黄色菌落(直径1.0~1.5 mm),为具有端生鞭毛的杆状菌;菌株YD-1革兰氏染色为阴性;综合生理生化试验和分子生物学试验结果,鉴定分离菌株YD-1为科氏希瓦氏菌;药物敏感性试验显示,菌株YD-1对头孢噻肟、头孢哌酮、氯霉素和阿奇霉素等药物高度敏感;菌株YD-1于28、37℃时出现α溶血性;人工感染试验显示,菌株YD-1对虾夷扇贝具有较高致病性,半数致死浓度(LD50)为2.08×106CFU/mL;人工感染的扇贝主要表现为外套膜萎缩脱落、闭壳肌收缩无力、对外界刺激反应迟缓及壳边缘有褐色沉积等,但未出现粉橘色脓疱,与脓疱病病征不完全相同。研究表明,科氏希瓦氏菌作为虾夷扇贝的潜在病原菌,参与脓疱病的发生。

In order to investigate the pathogenesis of abscess disease in cultured Yesso scallop Patinopecten yessoensis, dominant single colonies were selected from the lesions sited by coating culture, and the dominant strains were studied through isolation and purification, drug sensitivity test, hemolysis test, artificial infection test and histopathological examination. The results showed that a total of 12 dominant bacterial strains( from YD-1 to YD-12) were obtained from Yesso scallop lesions. The 16S rDNA gene sequence alignment analysis showed that the 12 strains had the maximal similarity with Shewanella colwelliana. The YD-1 strain with the maximal sequence identity(99.86%) with S.colwelliana was cultured and observed by transmission electron microscopy. It was found that the YD-1 strain was a creamy yellow colony with a diameter of 1.0 mm to 1.5 mm and was rod-shaped with one terminal flagella and negative response by Gram staining. After physiological, biochemical tests and 16S rRNA analysis, YD-1 was identified as S.colwelliana. The results of antibiotic susceptibility revealed that the YD-1 strain was highly susceptible to cefotaxime, cefoperazone, chloramphenicol and azithromycin, with α haemolytic activity at 28 ℃ and 37 ℃. The results of injection test showed that the YD-1 strain was highly pathogenic, with median lethal dose(LD50) of 2.08×10~6 CFU/mL. The infective symptoms included capsid atrophy and detachment, weak obturator muscle contraction, slow response to external stimulation and brown deposition on the shell edge, without pink orange pustule, which were not exactly similar to the symptoms of abscess. The finding indicated that S.colwelliana YD-1 strain was a potential pathogen of Yesso scallop and participated in the occurrence of abscess.