Isolation and Identification of Aeromonas salmonicida from Thamnaconus septentrionalis and Sebastes schlegeli

The greenfin horse-faced filefish (Thamnaconus septentrionalis) and rockfish (Sebastes schlegeli) occupy important positions in the offshore net fishery in Shandong Province. Interest in their mariculture has been developing rapidly in recent years as candidates for submerged cage open-sea aquaculture. With the development of breeding techniques and the expansion of large- scale farming, fish disease may become a serious constraint that limits sustainable aquaculture and leads to great economic losses. Epidemiological investigation is the basis of disease control and should be carried out throughout the culture process. In this study, we describe the diseases of T. septentrionalis and S. schlegeli caused by Aeromonas salmonicida subsp. masoucida. In November 2018, an outbreak of T. septentrionalis disease was observed in a farm located in Penglai, Shandong Province, and an outbreak of S. schlegeli disease occurred in the same farm in April 2019, with daily mortalities of 0.4%~1% and about 1%, respectively. The main symptoms in the diseased fish were ulcers, redness, swelling, and bleeding in the mouth. Most diseased fish in the ponds showed "red mouth". No parasites were observed by the naked eye or light microscope. From the liver, spleen, and kidney of all the diseased fish, many homogeneous colonies were observed after three days incubation on TSA and 2216E agar plates. All strains had the same shape, color, and size, and the 16S rRNA genes of all strains were the same, with high identity with A. salmonicida. The virulence of the isolates was tested experimentally via injection with T. septentrionalis (infected by 2018TS-1) and S. schlegeli (infected by 2019SS-1) in the laboratory to calculate the median lethal dose (LD50). The results showed that the LD50 of 2018TS-1 to T. septentrionalis was 1.78×105 CFU/fish, and that of 2019SS-1 to S. schlegeli was 0.89×105 CFU/fish. The dead fish of the experimentally infected group showed ulcers and red mouth, the same symptoms as in 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 pathogens of T. septentrionalis and S. schlegeli, respectively. Bacterial identification was carried out by 16S rRNA gene analysis and Biolog Gen Ⅲ characterization. The 16S rRNA gene sequences of 2018TS-1 and 2019SS-1 (Gene Bank: OK258319 and OK258320) isolated from T. septentrionalis and S. schlegeli were analyzed using 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 two 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 encodes the outer membrane protein (A-layer protein) and causes the auto-aggregation of bacteria, is a conserved gene with some variation region in A. salmonicida. vapA gene typing is an effective and important method for classifying the molecular types and subspecies of this fish. vapA gene typing was also used in this study to identify subspecies of strains isolated from T. septentrionalis and S. schlegeli. The vapA gene sequences of 2018TS-1 and 2019SS-1 (Gene Bank: OK300094 and OK300095) were analyzed using MEGA5 with type strains obtained from Gene Bank. The phylogenetic tree derived from the 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, similar to the A-layer type Ⅶ strains, which are all from the northeast Asian and Canadian coasts in the Pacific Ocean. Based on the experimental infection, 16S rRNA sequence analysis, Biolog Gen Ⅲ characterization, and vapA gene typing, we confirmed that A. salmonicida subsp. masoucida is the pathogen of T. septentrionalis and S. schlegeli, and the cause of these two diseases on the farm. This is the first report of T. septentrionalis and S. schlegeli infected by A. salmonicida in industrial aquaculture, as well as the first report of a disease of T. septentrionalis in culture. It has been reported that A. salmonicida subsp. masoucida can infect Atlantic salmon (Salmo salar), turbot (Scophthalmus maximus), sablefish (Anoplopoma fimbria), and tongue sole (Cynoglossus semilaevis) cultured in Shandong Province. In this study, we expanded the host list of A. salmonicida subsp. masoucida to include two new species in aquaculture, T. septentrionalis and S. schlegeli, on 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 caused by A. salmonicida in fish culture, the prevention of A. salmonicida subsp. masoucida should be an important objective for mariculture in the future.