养殖大菱鲆中牙鲆肠弧菌的分离鉴定及组织病理学

Isolation and identification of Vibrio ichthyoenteri in farmed turbot(Scophthalmus maximus)and histopathology study of the diseased fish

2007年1月,山东省胶南某养殖场人工养殖的大菱鲆发生严重病害并大批死亡。病鱼的主要症状是体表溃疡,腹腔积液,肠道肿胀,肝脏萎缩,胆囊暗绿色等。从病鱼胆囊中分离纯化得到优势菌株,命名为da3。人工感染试验证实,该菌株对大菱鲆有较强的致病性。对体重为25 g的大菱鲆的半数致死量为每尾鱼2×106CFU。通过细菌16S rDNA序列测定及形态学和生理生化特征研究确定,该病原菌为牙鲆肠弧菌(Vibrio ichthyoenteri)。组织病理学观察表明,病鱼的肝脏、肾脏、脾脏、肠道和脑的微观结构发生了明显的病理变化,由此引起的器官功能衰竭可能是病鱼死亡的主要原因。

In January 2007, an epizootic occurred among cultured turbot （Scophthalmus maximus） in a fish farm in Jiaonan, China. The diseased turbot exhibited hemorrhagic ulcer on the body, abundant yellowish ascetic fluid filled in the body cavity, swollen intestine, necrosis of liver and darkened cholecyst. Samples were taken aseptically from the hepatopancreas, spleen, gall bladder, kidney and brain, and inoculated immediately onto LBN （LB medium supplement with 2 % NaCl） plates, which were incubated at 28℃ for 24 h. A predominant strain, which was designated as da3, was isolated from the gall bladder of the infected fish. Pathogenicity assays revealed that da3 was virulent to turbot by intraperitoneal injection challenge. The LD50 of da3 to turbot was calculated as 2 × 10^6 CFU/fish to the 25 g weight turbot. Strain da3 was a Gram- negative, curved bacterium with single polar flagellum, and could grow on TCBS medium and produce yellow colonies. Strain da3 was similar to Vibrio ichthyoenteri in morphological, most of the physiological, biochemical characteristics and 16S rDNA sequence except in lysinedecarboxylase （LDC） and the ability of using D-Gluconic acid. Strain da3 was identified to be V. ichthyoenteri. Portions of liver, spleen, kidney, intestine, brain and heart from the naturally diseased turbot were sampled, fixed in Davidson＇ s fixative for 12 h, and then were processed for paraffin embedding. Paraffin blocks of the tissues were cut at 4 p,m thickness, and the sections were stained by the procedure of H. E. The slides were examined using an Olympus microscope, and images were captured with digital camera. There were distinctive histopathological changes in liver, kidney, spleen and intestine of the diseased turbot, and to a less extent in the brain. In the liver, necrosis of the hepatocytes were observed, and erythrocytopenia in the vein of the liver illustrated a septicemia. In the kidney, lesions of histocytes membrane, necrosis of some renal tubules, and decreased number of erythrocytes was observed. In the spleen, the number of erythrocytes decreased sharply, indicating the damage to the hematopoiesis. In the intestine, the intestinal villus goblet cells expanded to round, and the lumina popria necrosed severely. Examination of fixed tissues from healthy turbot showed structure integrity in liver, kidney, spleen and intestine. The histopathological changes in the diseased turbot may lead to the dysfunction of the organs. The results of this study may be important for controlling the bacterial disease of turbot.