发病鲆鲽类分离菌株的16S rRNA基因测序分析

细菌性疾病是中国海水养殖鲆鲽类的主要病害,为全面了解病原菌种类,本研究对1999~2012年从山东、江苏、河北、天津等沿海地区养殖场发病鲆鲽鱼类中分离得到的124株优势菌株进行了16S rRNA基因测序和系统发育学分析。将基因序列与GenBank核酸序列数据库进行相似度比对分析,结果显示,有83株与弧菌属(Vibriosp.)细菌相似度最高,11株与气单胞菌属(Aeromonas sp.)细菌相似度最高,4株与爱德华氏菌属(Edwardsiella sp.)细菌相似度最高,26株为其他15种属的细菌。根据系统发育学分析结果,进一步将66株菌鉴定为16个种,优势种为溶藻弧菌(V.alginolyticus)、哈氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)、杀鲑气单胞菌(A.salmonicida)和迟缓爱德华氏菌(E.tarda)。选择其中的9株鳗弧菌和4株迟缓爱德华氏菌进行人工感染实验,结果显示,其中7株鳗弧菌和3株迟缓爱德华氏菌对大菱鲆(Scophthalmus maximus)有较强的致病性。研究结果可为阐明中国海水养殖鲆鲽类的流行病发生历史、病原种类、病原监测及疾病控制提供重要参考。

Sequencing and Phylogenetic Analysis of the 16S rRNA Genes of Bacterial Strains Isolated from Diseased Flatfish

Flatfish are the major industrial aquaculture marine fish species bred in North China. During the culturing process, members of this species are exposed to infection from a variety of pathogens. Diseases caused by bacterial pathogens are the major cause of harm to cultured flatfish in China. To investigate the bacterial pathogens that affect flatfish, we focused on 124 strains isolated from organs of diseased flatfish in Shandong, Jiangsu, Hebei, Tianjin and other similar places between 1999 and 2012. The 16S rRNA gene of the isolates was sequenced, analyzed using BLAST (GenBank), and subjected to phylogenetic analysis by using Mega 5.05. The results identified 66.90% of isolates as Vibrio (83 strains), 8.90% as Aeromonas (11 strains), 3.20% as Edwardsiella (4 strains), and other 15 genera (26 strains). According to the phylogenetic tree, 66 strains were identified as 16 species; the dominant species were V. alginolyticus, V. harveyi, V. anguillarum, V. damselae, V. furnissii, V. splendidus, A. allosaccharophila, A. salmonicida, and E. tarda. To evaluate the pathogenicity of isolates, the virulence of V. anguillarum and E. tarda strains isolated from diseased fish was further determined by experimental infection based on the 50% lethal dose (LD50) in turbot (Scophthalmus maximus). The results showed that seven V. anguillarum strains and three E. tarda strains were pathogenic to fish. The LD50 values were 105.1 to 106.8 CFU/fish for pathogenic V. anguillarum and 103.4 to 104.1 CFU/fish for pathogenic E. tarda. There were also two strains of V. anguillarum and one strain of E. tarda that showed low virulence to turbot, with an LD50 higher than 107 CFU/fish. The presented results provide significant information to ascertain the bacterial pathogens of flatfish, which is important for establishing strategies for the epidemiology, monitoring, and control of bacterial diseases.