我国罗非鱼源新型无乳链球菌的分离、鉴定及其分子特征

Isolation, identification, and molecular characteristics of a new genotype of Streptococcus agalactiae from cultured tilapia in China

2014年海南省文昌市多个养殖场的罗非鱼出现暴发性疾病，患病罗非鱼表现出体色发黑、打转游动、眼球突出或混浊等典型的链球菌病症状。从患病罗非鱼的肝、肾、脾、眼球和脑等组织中分离到19株病原菌，即TC-1、TC-2、BL1441~BL1448和WT1451~WT1459。通过形态观察、生理生化特征和16S rRNA基因序列分析等方法对病原菌进行鉴定，结果表明，这些病原菌均为无乳链球菌。溶血试验结果表明， TC-1、TC-2和BL1441~BL1448菌株为β-溶血性无乳链球菌，而 WT1451~WT1459菌株为不溶血无乳链球菌。进一步通过 MLST、分子血清型和毒力相关基因检测等技术对这些分离菌株进行遗传特征分析，结果表明TC-1、TC-2和BL1441~BL1448菌株是常见的 Ia-ST7型，其毒力基因型为 bac＋-bca＋-bibA＋-cfb＋-hylB＋-iagA＋-fbsB＋-lmb–-scpB–-cylE＋-gbs20186–。而 WT1451~WT1459菌株则是Ib-ST261型，其毒力基因型为bac–-bca–-bibA＋-cfb＋-hylB＋-iagA＋-fbsB＋-lmb–-scpB–-cylE–-gbs20186＋。将分离菌株 BL1441和 WT1451分别对罗非鱼进行攻毒试验，结果表明， WT1451菌株是强毒株，当其攻毒剂量为4.5×103 CFU/mL 时，罗非鱼累积死亡率可达85%。本研究将为我国罗非鱼无乳链球菌的流行病学、疫苗研制以及疾病防控等研究奠定基础。

A Streptococcosis outbreak in cultured tilapia caused by Streptococcus agalactiae occurred in Wenchang, Hainan Province, China in 2014. In this study, the moribund tilapias were collected from different farms for bac-teria isolation. Most of the moribund tilapias displayed typical clinical signs, such as melanosis, corneal opacity, and swimming abnormalities. A total of 19 isolates （TC-1, TC-2, BL1441–BL1448, and WT1451–WT1459） were isolated from the liver, kidney, spleen, eye, and brain of the diseased tilapia;then, these isolates were identified by morphological observation, physiological, and biochemical characteristics, and sequence analysis of the 16S rRNA gene. All of the isolates were identified as S. agalactiae, and the WT1451–WT1459 strains grew slowly and could not utilize trehalose and ribose. In addition, TC-1, TC-2, and BL1441–BL1448 isolates were positive forβ-hemolysis on sheep’s blood agar plates, but WT1451–WT1459 isolates did not exhibit hemolysis. The genetic characteristics of these isolates were analyzed by multilocus sequence typing （MLST）, molecular serotyping, and virulence-related gene sequencing. The results indicated that TC-1, TC-2, and BL1441–BL1448 isolates were common types of Ia-ST7 S. agalactiae in tilapia in China. However, the WT1451–WT1459 isolates were rare types of Ib-ST261 S. agalactiae in tilapia worldwide. PCR revealed that the virulence-related genes bac, bca, bibA, cfb, hylB, iagA, fbsB, and cylE were present in TC-1, TC-2, and BL1441–BL1448 isolates, and the virulence-related genotype of these strains was bac＋-bca＋-bibA＋-cfb＋-hylB＋-iagA＋-fbsB＋-lmb–-scpB–-cylE＋-gbs20186–. However, the WT1451–WT1459 isolates were positive for bibA, cfb, hylB, iagA, fbsB, and gbs20186, and the virulence-related genotype was bac–-bca–-bibA＋-cfb＋-hylB＋-iagA＋-fbsB＋-lmb–-scpB–-cylE–-gbs20186＋. Despite the fact that the number of the virulence-related genes of the WT1451 strain was less than that of the BL1441 strain, the former was more virulent than the latter in tilapia. In this study, the virulence tests were conducted by intraperitoneal in-jection of the BL1441 and WT1451 strains in tilapia. The results of the challenge experiments showed that the WT1451 strain possessed stronger pathogenicity to tilapia than that of BL1441 strain. The WT1451 strain caused 85%mortality in tilapia at a dose of 4.5×103 CFU/mL. The BL1441 strain showed weak virulence in tilapia, with cumulative mortality of 65%at a dose of 4.5×108 CFU/mL postchallenge. In summary, the 19 S. agalactiae strains isolated from tilapia in Wenchang were divided into two genotypes based on physiological and biochemical char-acteristics, 16S rRNA gene sequences, hemolysis activity, molecular serotyping, and PCR screening of viru-lence-related genes. Although reports on the Ib-ST261 S. agalactiae strains in tilapia are rare worldwide, these strains were highly virulent in tilapia. Therefore, it is very important to obtain data on the epidemiology of Ib-ST261 S. agalactiae strains in tilapia in China. To the best of our knowledge, this is the first report on the hy-pervirulent Ib-ST261 strain of S. agalactiae, which was isolated from tilapia in China. This study provides a framework for the exploration of epidemiological analysis, vaccine development, prevention, and treatment of S. agalactiae in tilapia.