小黄鱼海豚链球菌的分离鉴定

Isolation and identification of Streptococcus iniae in Larimichthys polyactis

为探究浙江舟山地区网箱养殖小黄鱼暴发的以眼球突出、眼周充血、内脏出血等为特征症状的疾病病因,本研究采集发病小黄鱼的脑及内脏病变组织制作病理切片,观察结果显示,病鱼的脑、肝、脾、肾组织损伤严重,主要表现为淤血、细胞空泡变性、散在坏死,炎性细胞浸润等病理变化。将组织碎片于常规细菌培养基(TSA)和血琼脂培养基上划线培养,进行病原分离,结果显示,从患病鱼的脑和头肾组织中均分离到同一优势菌株DB2009001。经形态学观察、生理生化鉴定、16S rRNA 基因扩增及序列分析,并结合随机扩增多态性 DNA(RAPD)分型试验及海豚链球菌 lctO 基因的检测,确定菌株 DB2009001 为 Ⅰ 型海豚链球菌。毒力基因的 PCR 检测结果显示,DB2009001 同时携带海豚链球菌的 7 种毒力基因(pgmA、scpI、simA、cpsD、sagA、pdi、cfi)。药敏试验结果显示,菌株 DB2009001 对四环素类、喹诺酮类、β 内酰胺类、氯霉素类等药物敏感,对氨基糖苷类药物不同程度耐药。动物回归试验结果显示,菌株DB2009001可感染健康小黄鱼并引起死亡,对其半数致死剂量(LD)为1.66×10^(3)cfu/g,感染小黄鱼表现出与自然发病类似的症状,并从濒死的鱼体内再次分离到相同的菌株,表明菌株DB2009001为该养殖场小黄鱼暴发性疾病的致病菌。本研究首次报道了海豚链球菌能够感染小黄鱼,为小黄鱼链球菌病的防治提供了依据。

In order to find out the pathogen to cause the disease with the characterization of exophthalmos, eye congestion and visceral hemorrhage in the cage cultured small yellow croakers in Zhoushan, Zhejiang. The hisotpatholocial observation was carrid out by preparing the histopathological sections for the brain and viscera samples of sick fish. The results showed that the brain, liver, spleen, and kidney tissues of the sick fish were severely damaged, mainly manifested by histopathological changes such as congestion, vacuolar degeneration, scattered necrosis, and inflammatory cell infiltration. A kind of dominat strain, named DB2009001, was isolated from both the diseased fish’s brain and its head kidney tissues by plate streaking cultivation on conventional bacterial medium (TSA) and blood agar culture-medium. The isolated DB2009001 strain was identified as type Ⅰ Streptococcus iniae by the morphological observation, physiological and biochemical identification, 16S rRNA sequnece analysis,random amplified polymorphic DNA (RAPD) typing test, and Streptococcus iniae lctO gene detection. The results of PCR test on the virulence genes showed that the DB2009001 strain carried 7 virulence genes (pgmA, scpI, simA, cpsD, sagA, pdi, cfi) of Streptococcus iniae. The drug sensitivity test showed that DB2009001 strain was sensitive to tetracyclines, quinolones, β-lactams,chloramphenicol, etc. While it has resistant to aminoglycoside drugs in different degree. The results of animal regression test showed that the DB2009001 strain could infect the healthy L. polyactis and cause the death, with the half-lethal dose (LD) of 1.66×10^(3)cfu/g. The infected L. polyactis showed clinical symptoms, which were similar to the natural diseased fish;and the same strain was isolated again from the dying fish, it is suggested that DB2009001 was the pathogene which caused the outbreak of small yellow croaker. To our knowledge, this study is the first report of S. iniae infecting small yellow croaker, which provides a basis for the prevention and control of streptococcus disease on small yellow croaker.