患病北极红点鲑的病原分离与鉴定

Isolation and Identification of Causative Agents of Sick Arctic Charr(Salvelinus alpinus)

对2004年5月四川省某养殖基地养殖的北极红点鲑患病鲑进行了病理学检查和病原分离,从病灶处分离到细菌和真菌。分离到的细菌经API生化鉴定为杀鲑气单胞菌杀鲑亚种(Aeromonas salmonicidasubsp.salmonicida)。用引物AP1和AP2对纯化后的细菌进行PCR扩增,结果扩增出长度为421 bp的DNA片段,对扩增片段进行克隆、测序,用NCBI-BLASTn在GenBank中搜寻相似序列,结果与杀鲑气单胞菌各株A层蛋白部分编码基因有99%以上的序列同源性。用引物EUS-F和EUS-R对分离到的真菌进行PCR扩增,结果扩增出长度为755 bp的DNA片段,对扩增片段进行克隆、测序,用NCBI-BLASTn在GenBank中搜寻相似序列,结果与水霉属各株的DNA片段(包括18S核糖体RNA基因部分序列、内转录间隔区1全序列、5.8S核糖体RNA基因全序列、内转录间隔区2全序列和28S核糖体RNA部分序列)有93%-100%的同源性;而与属于丝囊霉菌属的EUS各株相对应的DNA片段仅有60%的序列同源性。因此判定所分离的真菌为水霉。杀鲑气单胞菌是引起疖疮病和溃疡病等病的病原菌,而水霉广泛存在于水中,当鱼受伤后,水霉孢子容易从患病部位侵入鱼体,加重感染。因此判断该病主要是由杀鲑气单胞菌引起,而水霉则引起继发感染。

Severe ulceration occurred on the head and back of cultured arcticcharr（Salvelinus alpinus） in a farm of Sichuan Province in May, 2004. In order to study the aetiology and prevention measures, bacteriological and molecular biology methods were used in the identification of the bacteria and fungi isolated from the focus of sick fish. The isolated bacteria were identified as Aeromonas salmonicida with API kit. A fragment of 421 bp extracted from of the isolated bacteria with the specific primers of Aeromonas salmonicida, AP1 and AP2 was amplified in PCR. The amplified product was sequenced and compared with NCBI-Blastn in GenBank and found to have the homogenicity of 99% with the fragment of partial vapA gene of Aeromonas salmonicida. Another pair of primers, EUS-F and EUS-R were Used in the PCR and a fragment of 755 bp was amplified from the extraction of the isolated fungi. The ampler fled product was sequenced and compared with NCBI-Blastn in GenBank and found to have the homogenicity of 93%-100% with the fragment of 18S ribosomal RNA gene, partial sequence internal transcribed spacer 1, 5.8S ribosomal RNA gene, and internal transcribed spacer 2, complete sequence; and 28S ribosomal RNA gene, partial sequence of Saprolegnia, and have the homogenicity of 60% with the relevant fragment of Aphanomyces invadans. Saprolegnia is common in water, and the fish are easily infected by Saprolegnia if they were infected and hurt by bacteria first. It was deduced that Aeromonas salmonicida was the main pathogen in infected arctic charr and Saprolegnia played an important role as the cause of secondary infection.