广东与海南养殖罗非鱼无乳链球菌的分离、鉴定与特性分析

Identification and Characterizations of Streptococcus agalactiae Isolated from Tilapia Cultured in Guangdong and Hainan Provinces

摘要:

从中国广东、海南罗非鱼主养区发生爆发性疾病的多个养殖场的罗非鱼病鱼体上, 分离到多株致病菌株。人工感染试验显示分离菌株具有较强的致病力, 有多株经腹部注射分离细菌浓度为 1 × 106 CFU/mL时可使100%的受感染鱼死亡, 选择其中7株强毒株进行药物敏感性实验与鉴定。不同菌株对药物敏感性存在一定的差异但与菌株来源无相关性, 29种抗生素中对13种敏感、7种不敏感、9种存在菌株的差异。各分离菌株均为革兰氏阳性菌, 呈β溶血。采用链球菌快速鉴定系统ID 32 STREP、Lancefield分析及多项补充生理生化鉴定结果, 初步判断为无乳链球菌Streptococcus agalactiae。PCR扩增16S rRNA基因和GBS-specific gene cfb (CAMP factor)基因的全长序列, BLAST分析显示所有菌株的16S rRNA基因与GenBank上登录的无乳链球菌的相应序列高度同源 (> 99.8%), 各分离菌株间的16S rRNA基因序列也高度同源 (≥99.9%-100%)。各菌株cfb基因序列高度同源 (100%), BLAST显示与已知无乳链球菌的相应序列也具有高度同源性 (≥99.0%)。综合上述实验结果, 可判定广东与海南罗非鱼主养区2009年夏季发生的罗非鱼爆发性疾病的病原菌为无乳链球菌。

Abstract:

Bacteria pathogens were isolated from diseased tilapia cultured in several farms of Guangdong and Hainan provinces. Artificial infection experiments showed that these isolates possessed strong virulence. Intraperitoneal injection (≥1 × 106 CFU/mL) of some isolates caused 100% mortality of healthy tilapia. 7 bacteria strains with strong virulence were chosen for antibiotics sensitivity test and identifications. Antibiotic sensitivity assays showed that among 29 antibiotics tested 13 were sensitive, 7 were resist, while 9 items showed various reactions among strains. The isolates were all gram-positive and β-hemolysis and were identified as Streptococcus agalactiae by biochemistry assays of ID 32 STREP test, Lancefield test and other assays. Molecular analysis of 16S rRNA and cfb genes (GBS-specific gene cfb, CAMP factor) were used for further identification. BLAST showed that 16S rRNA and cfb genes of all the strains possessed high similarities with their counterparts registered in GenBank, and with each other. Taken together, these experiment results showed that pathogen caused high mortality of tilapia in both provinces in 2009 was S. agalactiae. The result was helpful for tilapia disease control and prevention, and for development of streptococcus vaccine for tilapia.