文章摘要
绿鳍马面鲀(Thamnaconus septentrionalis)与许氏平鮋(Sebastes schlegeli)杀鲑气单胞菌(Aeromonas salmonicida)病原的分离和鉴定
Isolation and Identification of Aeromonas salmonicida from Thamnaconus septentrionalis and Sebastes schlegeli
投稿时间:2021-08-31  修订日期:2021-10-04
DOI:
中文关键词: 绿鳍马面鲀  许氏平鮋  细菌鉴定  杀鲑气单胞菌杀日本鲑亚种
英文关键词: Thamnaconus septentrionalis  Sebastes schlegeli  Bacteria identification  Aeromonas salmonicida subsp. masoucida
基金项目:国家重点研发计划(2019YFD0900103)、国家自然科学基金-山东省人民政府联合基金(U1706205)、中国水产科学研究院基本科研业务费(2020XT0406)共同资助
作者单位邮编
晋怀远 天津农学院 水产学院中国水产科学研究院黄海水产研究所 266071
刘耀宽 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所 
高晔 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所 
夏苏东 天津农学院 水产学院 
陈四清 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所 
莫照兰 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所中国海洋大学 海洋生命学院 
边力 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所 
李杰 青岛海洋科学与技术国家实验室海洋渔业科学与食物产出过程功能实验室 农业农村部海水养殖病害防治重点实验室 中国水产科学研究院黄海水产研究所 266071
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中文摘要:
      绿鳍马面鲀(Thamnaconus septentrionalis)和许氏平鮋(Sebastes schlegeli)是近年来发展迅速的海水养殖品种,在山东省近海网箱养殖中占据了重要地位,也是适合深远海养殖发展的候选品种。2018年和2019年,山东省烟台市蓬莱市一养殖场工厂化养殖的绿鳍马面鲀和许氏平鮋出现发病死亡,主要症状为嘴部溃疡、红肿和出血。从发病鱼内脏中均可分离到大量形态一致的优势菌,分别命名为2018TS-1和2019SS-1,分离菌株经16S rRNA测序、生理生化鉴定和vapA基因分析确定为杀鲑气单胞菌杀日本鲑亚种(Aeromonas salmonicida subsp. masoucida)。人工感染实验证明2018TS-1和2019SS-1分别能引起绿鳍马面鲀和许氏平鮋的死亡,被感染鱼呈嘴部红肿症状,与自然发病症状一致,其半数致死量分别为1.78 × 105 cfu/尾和0.89 × 105 cfu/尾。本研究首次报道了国内工厂化养殖绿鳍马面鲀和许氏平鮋感染杀鲑气单胞菌的病例,是目前人工养殖绿鳍马面鲀的首个疾病报道,也是继大西洋鲑(Salmo salar)、大菱鲆(Scophthalmus maximus)、裸盖鱼(Anoplopoma fimbria)等品种后,在山东海水养殖鱼类中又一次发现杀鲑气单胞菌杀日本鲑亚种的感染,本研究结果丰富了杀鲑气单胞菌杀日本鲑亚种的感染宿主范围,也为绿鳍马面鲀和许氏平鮋养殖过程中病害防控提供了依据。,也为养殖过程中病害防控提供了依据。
英文摘要:
      The mariculture of greenfin horse‐faced filefish (Thamnaconus septentrionalisI) and rockfish (Sebastes schlegeli), which occupied an important position in offshore netcage in Shandong province, is developing rapidly in recent years as the candidates for submerging open-sea aquaculture. With the development of breeding techniques and expansion of farming scale, fish disease may become a serious restriction that limits sustainable aquaculture and leads to great economic losses. Epidemiological investigation is the basis of disease control, which should be carried out throughout the culture process. In this research, we described the disease of T. septentrionalis and S. schlegeli caused by Aeromonas salmonicida subsp. masoucida. In Nov. 2018, T. septentrionalis disease outbroke was observed in a farm located in Penglai city, Shandong province, and S. schlegeli disease outbroke was also occurred in the same farm in Apr. 2019, with daily mortality of 0.4%-1% and about 1%, respectively. The main symptoms of diseased fish were ulcers, redness, swelling, and bleeding in the mouth. Most diseased fish in the ponds showed “red mouth”. No parasite was observed by naked eye and light microscope. From the liver, spleen, and kidney of all the diseased fish, a large number of homogeneous colonies were observed after 3 days of incubation on TSA and 2216E agar plates. All strains had the same shape, color, and size, and the 16S rRNA genes of all the strains were the same, high identity with A. salmonicida. The virulence of the isolates was tested experimental infection via injection with T. septentrionalis (infected by 2018TS-1) and S. schlegeli (infected by 2019SS-1) in the lab to calculate the lethal dose 50% (LD50). Results showed that the LD50 of 2018TS-1 to T. septentrionalis was 1.78 × 105 cfu/fish, 2019SS-1 to S. schlegeli was 0.89 × 105 cfu/fish. The dead fish of experimental infection showed ulcers and red mouth, the same symptoms as the naturally infected fish. Dominant colonies isolated from experimentally infected fish were all identified as A. salmonicida by 16S rRNA gene sequencing, which indicated that 2018TS-1 and 2019SS-1 were the pathogen of T. septentrionalis and S. schlegeli, respectively. Bacteria identification was carried out by 16S rRNA genes analysis and Biolog Gen III characterization. The 16S rRNA gene sequences of 2018TS-1 and 2019SS-1 (Gene Bank: J40YB1EZ01R and J40X1DR4016) isolated from T. septentrionalis and S. schlegeli were analyzed with MEGA5, and the phylogenetic tree derived from 16S rRNA gene sequences clustered the isolates with A. salmonicida. Among the Biolog Gen III tests, 31 produced positive reactions or weak positive reactions for both strains (Dextrin, D-Maltose, D-Trehalose, D-Cellobiose, Sucrose, β-Methyl-D-Glucoside, D-Salicin, α-D-Glucose, D-Mannose, D-Fructose, D-Mannitol, Glycerol, Gelatin, Glycyl-L-Proline, L-Arginine, D-Gluconic Acid, Methyl Pyruvate, L-Malic Acid, Bromo-Succinic Acid, Tween 40, α-Keto-Butyric Acid, Acetoacetic Acid, Propionic Acid, Acetic Acid, pH 6, 1% NaCl, 1% Sodium Lactate, L-Aspartic Acid, L-Glutamic Acid, L-Histidine, and L-Serine), and 2 weak positive reactions for 2019SS-1, while the others were negative. According to the Biolog database, both strains were identified as A. salmonicida. Based on the molecular analysis of 16S rRNA genes and Biolog Gen III phenotype results, the isolates were identified as A. salmonicida. The vapA gene which coding the outer membrane protein (A-layer protein) and causing the auto aggregation of bacteria, is a conserved gene with some variation region in A. salmonicida. The vapA gene typing is an effective and important method to classify the molecular types and subspecies of A. salmonicida. The vapA gene typing was also used in this research to identify the subspecies of strains isolated from T. septentrionalis and S. schlegeli. The vapA gene sequences of 2018TS-1 and 2019SS-1 (Gene Bank: GA3DYRYE016 and GA3DYRYE016) were analyzed using MEGA5 with type strains obtained from Gene Bank. The phylogenetic tree derived from vapA gene sequences clustered 2018TS-1 and 2019SS-1 with type strain ATCC 27013, indicating that the strains isolated from T. septentrionalis and S. schlegeli belonged to A. salmonicida subsp. masoucida, the same as the A-layer type VII strains which are all from the northeast Asia and Canada coast in the Pacific Ocean. Based on the experimental infection, 16S rRNA sequences analysis, Biolog Gen III characterization, and vapA genes typing, we confirmed that A. salmonicida subsp. masoucida is the pathogen of T. septentrionalis and S. schlegeli, and the cause of these two diseases in the farm. This is the first report of T. septentrionalis and S. schlegeli infected by A. salmonicida in industrial aquaculture, and also the first report of T. septentrionalis farming diseases. It has been reported that A. salmonicida subsp. masoucida could infect Atlantic salmon (Salmo salar), turbot (Scophthalmus maximus), sablefish (Anoplopoma fimbria), and tongue sole (Cynoglossus semilaevis) cultured in Shandong province. In this research, we expanded the host of A. salmonicida subsp. masoucida to 2 new species in China aquaculture, T. septentrionalis and S. schlegeli in the same farm, indicating that A. salmonicida subsp. masoucida may translate and adapt to a new host in a short period. Considering the increasing host and economic losses that A. salmonicida caused in fish culture, the prevention of A. salmonicida subsp. masoucida should be an important work for mariculture in the future.
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