宁海地区香鱼弧菌病病原菌鉴定

Characterization of Listonella anguillarum as the aetiological agent of vibriosis occurred in cultured ayu (Plecoglossus altivelis) in Ninghai country, China

摘要:

摘要：【目的】香鱼弧菌病对中国沿海地区的香鱼养殖业造成了巨大的危害，然而，病原不明导致了防治上的许多问题。本文鉴定了引起宁海地区香鱼爆发性弧菌病的病原。【方法】采用TCBS平板分离优势菌；采用回归感染试验确认病原菌，采用改进的寇氏法计算LD50；采用形态学观察、生理生化特征测定、细菌特异性引物PCR扩增检测及细菌16S rRNA和金属蛋白酶（MP）基因序列分析鉴定细菌；采用药敏实验测定它对部分抗生素的敏感性。【结果】分离并鉴定优势菌株ayu-H080701为宁海地区香鱼弧菌病的病原菌，它对香鱼的半致死量为1.2×104 CFU。形态学观察和生理生化特征测定表明，ayu-H080701与鳗利斯顿氏菌最为接近。PCR扩增检测表明，细菌16S rRNA 基因通用引物和鳗利斯顿氏菌MP基因特异引物均能扩增到预期大小的特异性条带。ayu-H080701与鳗利斯顿氏菌16S rRNA基因核苷酸序列同源性最高，为99.4%～99.5%，与同属的海弧菌和美人鱼发光杆菌分别为94.3%和91.9%；ayu-H080701与鳗利斯顿氏菌MP氨基酸序列同源性高达97.6%～98.8 %，与其它弧菌科成员则低于75.6 %，系统进化树分析也揭示ayu-H080701与鳗利斯顿氏菌进化相关性最高。【结论】引起宁海地区香鱼弧菌病的菌株ayu-H080701被鉴定为鳗利斯顿氏菌。

Abstract:

Abstrat: [Objective] Ayu (Plecoglossus altivelis) vibriosis threatens ayu aquaculture seriously caused by mass mortality due to severe infections. We characterized the vibriosis pathogen of ayu in Ninghai country. [Methods] A dominant strain was isolated and identified by a series of biochemical and physiological tests. The lethal dose 50% (LD50) was calculated by the modified Karber’s method. PCR amplification and sequence analysis were used to further identify the pathogen. [Results] LD50 of ayu-H080701 was 1.2×104 CFU to ayu. PCR amplification showed that the bacterial universal primers for 16S rRNA gene and the specific primers for the metalloprotease (MP) gene of Listonella anguillarum worked. 16S rRNA gene analysis showed that ayu-H080701 shared 99.4%-99.5 % nucleotide identical to L. anguillarum isolates, while 94.3% and 91.9% nucleotide identical to L. pelagius and Photobacterium damselae respectively. MP analysis showed that ayu-H080701 shared 97.6%-98.8 % amino acid sequence identical to L. anguillarum isolates, while lower than 75.6 % to other bacteria. Phylogenetic analysis showed that ayu-H080701 grouped constantly with L. anguillarum isolates. [Conclusion] The biochemical, physiological tests and sequence analysis all strongly supported the identification of the pathogen causing ayu vibriosis in Ninghai country, China, as an isolate of L. anguillarum.