红尾皇冠鱼(Aequidens rivulatus)病原无乳链球菌的分离、鉴定与特性分析

Biological Characteristics of Pathogenic Streptococcus agalactiae Isolated From Aequidens rivulatus

关键词:  红尾皇冠鱼  无乳链球菌  生物学特性  16S rRNA  cfb

Key words:  Aequidens rivulatus  Streptococcus agalactiae  Biological characteristics  16S rRNA gene  cfb gene

摘要:

为查明天津地区养殖红尾皇冠鱼(Aequidens rivulatus)大量死亡的原因，取患病红尾皇冠鱼进行细菌分离，从病鱼肾脏中分离到优势菌株071901，人工感染试验证实其对红尾皇冠鱼有较强的致病性；采用形态学观察、生理生化特性、16S rRNA基因序列系统发育树分析及cfb (GBS-specific gene cfb, CAMP factor)基因检测对菌株071901进行鉴定。结果显示，菌株071901为革兰氏阳性球菌，生化特性与链球菌属的无乳链球菌(Streptococcus agalactiae)相近，基于16S rRNA基因序列的系统发育分析将菌株071901与无乳链球菌聚为一支，以071901为模板特异性扩增无乳链球菌的cfb基因，也得到了预期的900 bp的核酸片段，进一步证实菌株071901为无乳链球菌。对30种抗生素的药敏结果显示，菌株071901对红霉素、阿奇霉素等19种药物敏感，对多粘霉素、罗红霉素、呋喃唑酮等11种药物耐药。

Abstract:

The cultivation of ornamental fish has been gaining more and more popularity and commercial importance. Consequently ornamental fish diseases have also caught attentions. Aequidens rivulatus is one of the most important ornamental fish in commercial fisheries in Tianjin of China. In recent years mass death of A. rivulatus has caused heavy economic losses. In this study we aimed to identify the biological characteristics of the pathogens which caused diseases in A. rivulatus. A dominant bacterium 071901 was isolated from the kidney of diseased A. rivulatus and was confirmed to be pathogenic to A. rivulatus in artificial challenge test. We examined the morphological, physiological and biochemical characteristics of this bacterium, and analyzed 16S rRNA and cfb gene (GBS-specific gene cfb, CAMP factor) for further identification. The 16S rRNA gene and cfb gene were partially sequenced and compared with data from the GenBank database. BLAST showed that 16S rRNA gene and cfb gene of strain 071901 were highly similar to their counterparts registered in GenBank. Based on the morphological observation and physiological and biochemical characterization, strain 071901 was identified as Streptococcus agalactiae. Phylogenetic trees of S. agalactiae based on 16S rRNA gene indicated that strain 071901 was branched into the same cluster as S. agalactiae and showed the highest similarity to S. agalactiae. The results of molecular analysis of cfb gene were consistent with the previous results from the biochemical assays. We then performed antimicrobial susceptibility assay for 30 antimicrobial agents and found that strain 071901 was susceptible to 19 agents including erythromycin, azithromycin and florfenicol, and was highly resistant to 11 agents including enrofloxacin, bacitracin and furazolidone. In conclusion our results demonstrated that S. agalactiae was the pathogen that caused high mortality in A. rivulatus.