Studies on Pathology and Pathogenicity of Aeromonas hydrophila in Cultured Ayu

• Main Author: 呂學群
• Other Authors: 劉秉忠
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/05251612189309231680

碩士 === 國立海洋大學 === 水產養殖學系 === 91 === This thesis investigated the pathogenesis and pathology of Aeromonas hydrophila, originally isolated from diseased ayu, on ayu. The bacterium was isolated from surface ulcer of diseased ayu using TSA agar, and the fish were fixed with formalin for histology. The isolated bacterial strain AH910521 was characterized and identified as Aeromonas hydrophila by conventional biochemical tests, API 20-E and Biolog system. A reference strain ATCC13444 was tested in parallel for comparision. Live cells and extracellular products （ECP）of strain AH910521 were lethal to ayu （weighing 6g）reared at 15,20 and 25℃ with LD50 values（observed for 7 days）of 3.5×107 CFU and 1.78μg protein, 8.3×105 CFU and 1.53μg protein, and 1.6×104 CFU and 0.98μg protein/g fish，respectively. This result indicated that ayu may be stressed at higher temperature and more susceptible to the bacterium. The dead or moribund fish exhibited ulcer signs at the site i.p.injected with bacteria or ECP. The ECP were precipitated,dialyzed and applied onto Fast Protein Liquid Chromatography（FPLC）, using HIC, RESOURCE Q and Mono Q columns for protein purificatinon. A 94kDa protein band （SDS-PAGE）in a fraction was visualized. The purified protein exhibited protease activity using casein- overlay technique. The purified 94kDa protease was inhibited by L-cystein, iodoacetic acid, PCMB and N-ethylamleimide indicating that it belongs to cystein protease. The optimum pH and temperature of the purified protease were 6 and 60℃, respectively. The purified protease was lethal to ayu （reared at 20℃）with a LD50 value of 0.12μg protein/g fish. The diseased ayu was hemorrhage on the back and tail. The pectoral, pelvic and tail fin was hyperemia, the anus was swelling. From histopathological observation the muscular tissue had serious muscle fiber degeneration, necrosis and vacuolar degeneration. Challenge test by live cells and extracellular products （ECP）, the muscle fiber atrophy and necrosis , macrophage appear in heart tissue, necrosis in liver. After injection the purified protease the macrophage appear in the abdominal muscle and loose connective tissue, the muscle fiber was necrosis , intestinal ligment connective tissue inflammation, tissue infiltrates in leukemia, macrophage appear in the focal myocarditis,the spleen tissue was deposited with hemosiderin, also.