美洲鲥鱼嗜水气单胞菌病原的分离与鉴定

Isolation and identification of Aeromonas hydrophila from Alosa sapidissim

中文关键词: 美洲鲥鱼  嗜水气单胞菌  细菌鉴定  毒力基因

英文关键词: Alosa sapidissima  Aeromonas hydrophila  Bacteria identification  Virulence genes

中文摘要:

      2021年，山东省临沂市一养殖场养殖的美洲鲥鱼(Alosa sapidissima)突发疾病并出现严重死亡，日死亡率高峰期达到2.5%，累积死亡率约为90%。患病鱼主要症状为体表出血、溃疡，解剖可见腹腔腹水、肝脏暗红，并伴有肠炎。组织病理学检测发现，病鱼肝脏出现弥散性坏死，嗜碱性粒细胞增多和细胞肿胀空泡变性；脾脏出现出血性贫血性坏死灶、核破裂和核固缩；肾脏淋巴细胞坏死脱落，肾小体毛细血管球萎缩，近端小管和远端小管内的细胞出现不同程度的细胞结构消失。发病鱼肉眼和显微镜观察未见明显寄生虫，利用PCR方法检测鲤疱疹病毒2型(Cyprinid herpesvirus 2)、鲈鱼蛙病毒(largemouth bass ranavirus)等淡水鱼类常见病毒均为阴性。细菌分离培养结果显示，从发病鱼的肝脏、肾脏和脾脏中分离得到形态一致的优势菌，命名为AS-AH2101。经16S rRNA测序比对和生理生化鉴定，确定AS-AH2101为嗜水气单胞菌(Aeromonas hydrophila)。毒力基因检测结果显示，AS-AH2101携带气溶素(aerA)、溶血素(hlyA)、丝氨酸蛋白酶(ahpA)、热稳定细胞肠毒素(ast)、热敏感细胞肠毒素(altA)和密度感应系统(luxS)基因。人工感染实验结果显示，AS-AH2101能引起蓝曼龙(Trichogaster trichopterus)发病死亡，半数致死量为3.23×104 CFU/尾。药物敏感性研究表明，AS-AH2101对头孢拉啶、阿莫西林、氨苄西林和红霉素等4种抗生素具有耐药性，对四环素和多西环素等11种抗生素敏感。综上所述，本研究报道了我国养殖美洲鲥鱼感染嗜水气单胞菌的典型病例，为美洲鲥鱼在我国养殖过程中的疾病防控以及嗜水气单胞菌病的防治提供了参考和借鉴。

英文摘要:

      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.