Isolation and Identification of Aeromonas hydrophila from Alosa sapidissima

Aeromonas hydrophila (Family: Aeromonadaceae) is a traditional aquatic animal pathogen. It has been widely prevalent in our country since the 1980s and 1990s, causing diseases in a series of freshwater fish, leading to serious economic losses. It has been confirmed to be one of the main pathogens in freshwater aquaculture worldwide. The American shad (Alosa sapidissima) is one of the biggest shad in the world and grows much faster than other shad. Because of its delicious taste as reeves shad (Tenualosa reevesii), American shad were introduced into China from the USA by the Shanghai Fisheries Research Institute for local farming in 1998 and is widely welcomed in Shanghai, Jiangsu, and Zhejiang Provinces. In recent years, with the rapid development of the American shad culturing, fish diseases have become a major threat to fish farming. However, because most of the American shad are cultured in extensive ponds, few diseases have been reported in China. In this study, we reported a case of A. hydrophila infection in American shad. In 2021, the disease outbreak was observed in American shad cultured in Linyi City, Shandong Province, with severe mortality. The daily mortality could be up to 2.5%. The fish were cultured in indoor ponds for breeding and outdoor ponds when fish reached 300 g. The American shad were cultured with underground water and water was changed 1–3 times daily. The water temperature was 18–20 ℃. The fish were fed largemouth bass (Micropterus salmoides) commercial feed, and the daily feeding rate was approximately 2%. The disease broke out in the indoor ponds first, and then in outdoor ponds. The cumulative mortality was approximately 90% in 2 months. Enrofloxacin was administered orally, but no effects were observed and the disease continued to progress. The typical disease symptoms in the American shad were furunculosis or ulceration, with shedding scales and surface bleeding, especially on the tail, sometimes swollen and pus-filled. In autopsies of the diseased fish, ascites were found in the fish abdomen, and dark red necrosis on the liver, with sepsis and enteritis. The liver, spleen, and kidney of American shad with typical symptoms were collected and cut into about 1 cm3 tissue blocks, immersed in Davidson's Fixative (Davidson's AFA) for 24 h, and preserved in 70% ethanol. The tissues were mounted onto glass slides with hematoxylin and eosin staining for histological analysis. Histopathological results showed swollen liver cells, vacuolar degeneration, basophilia, and diffuse necrosis; the spleen showed hemorrhagic anemic necrosis, nuclear rupture, and atrophy. Glomerular atrophy of the renal corpuscle, cells in the proximal and distal tubules cytoarchitectural loss, and necrosis and shedding of kidney lymphocytes was also observed. No parasite was found on the fish surface, fins, in the gills, or internal organs with the naked eye and a light microscope. Freshwater viruses, such as Cyprinus herpesvirus type 2, largemouth bass ranavirus, megalocytivirus, and rhabdovirus, were checked by polymerase chain reaction (PCR), and no viruses were detected. The liver, spleen, and kidney of the diseased fish were sampled and cultured in tryptic soy agar medium (TSA) and Luria-Bertani agar medium (LB) plate medium at 28 ℃ for 24 h. Several pure and dominant colonies with the same morphology were observed on all the plates. These colonies were purified and cultured. The 16S rRNA gene sequencing results of all the purified colonies showed that the dominant strains were of the same species. The typical isolate was purified and named AS-AH2101. The results of biochemical identification with Biolog GenⅢ showed that the isolate AS-AH2101 was negative to gentiobiose, stachyose, D-raffinose, α-D-lactose, D-melibiose, 3-methyl glucose, D-fucose, D-sorbitol, D-arabitol, and Myo-inositol, while positive to dextrin, D-maltose, D-trehalose, D-cellobiose, sucrose, D-turanose, and β-methyl-D-glucoside. According to the Biolog GenⅢ identification system database, the biochemical characterization of AS-AH2101 was similar to that of A. hydrophila, with a confidence of 0.999. The 16S rRNA gene sequence of AS-AH2101 was submitted to GenBank databases under the accession number OP787967 and blasted in GenBank and EzTaxon. Comparison of the 16S rRNA gene sequences showed 99%−100% identity with those of A. hydrophila. The phylogenetic tree was constructed using Mega 7 with the Aeromonas typical strains 16S rRNA gene sequences obtained from GenBank, and the phylogenetic analysis also clustered AS-AH2101 with A. hydrophila. Thus, the molecular analysis results identified the SC18032201 strains as A. hydrophila, and the phenotype also supported this result. Because of the strong stress response of American shad, it is difficult to perform the experimental culturing and infection in the laboratory. As a classic pathogenic infection model organism in aquatic animals, blue gourami (Trichogaster trichopterus) is a traditional model for fish pathogen study and has been widely used in the research of A. hydrophilia and E. piscicida. Therefore, in this study, blue gouramis were used as the model organism for virulence evaluation of AS-AH2101 in the experimental infection. The results of the challenge experiment showed that the death of the blue gourami infected via intramuscular injection was observed on the third day post infection. The infected fish showed redness, bleeding, and scale shedding at the injection site, congestion or bleeding at the base of the fin, abdominal ascites, and liver necrosis, which were similar to the naturally infected American shad. The isolate strain AS-AH2101 showed high virulence to blue gouramis, with the median lethal dose (LD50) of 3.23×104 CFU/fish. The virulence genes of A. hydrophilia were also detected by PCR, and results indicated that AS-AH2101 possessed six virulence genes, including aerolysin (aerA), hemolysin (hlyA), extracellular protease (ahpA), anti-metalloproteinases (ast), enterotoxin (altA), and quorum sensing gene (luxS). Antibiotic sensitivity studies showed that AS-AH2101 was resistant to cefradine, amoxicillin, ampicillin, and erythromycin. These results provided important information for disease control and A. hydrophila prevention and control of American shad culturing in China.