南方鲇(Silurus meridionalis)豚鼠气单胞菌溶血素的分离、纯化与致病性研究

采用硫酸铵盐析、DEAE-Sephadex A50凝胶层析法对豚鼠气单胞菌的溶血素进行分离、纯化,获得分子量为32.2kDa的单一多肽溶血素,测定溶血价为25。将该溶血素倍比稀释后梯度接种南方鲇,研究其对南方鲇的致病性和组织病理损伤情况。结果表明:豚鼠气单胞菌溶血素具有较强的毒力,对南方鲇的半数致死剂量(LD50)为0.29mg蛋白/kg体重;病鱼表现为体表褪色,腹部膨大,肝、脾、肾肿大,肝、脾淤血、出血明显;胃肠道内充有大量粘液,黏膜充血、出血;组织病理学表现为骨骼肌水肿、变性,肝细胞变性、坏死,血窦扩张淤血,肾小管上皮细胞变性、坏死,脾脏淋巴细胞减少,胃肠道黏膜上皮变性、坏死,脱落。

ISOLATION, PURIFICATION AND PATHOGENICITY OF THE HAEMOLYSIN PRODUCED BY AEROMONAS CAVIAE FROM SILURUS MERIDIONALIS CHEN

The haemolysin produced by Aeromonas caviae was salted out by ammonium sulphate precipitation and purified by chromatography on Sephadex A50. It was estimated to a homogeneous polypeptide of 32.2kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Haemolysis assay showed that its haemolytic activity was 2⁵. To investigate its pathogenicity, double dilution haemolysin was intraperitoneally injected into Silurus meridionalis Chen. The results showed that the purified haemolysin was toxic towards S. meridionalis, with a half-lethal dose (LD₅₀) of 0.29mg/kg (protein/weight). Fish injected with this haemolysin exhibited symptoms of color fading, abdominal intumescence, congestion and enlargement of liver and spleen, renomegaly, gastrointestinal inflammation. Skeletal muscle appeared swollen and disruption with red stained sarcoplasm. Liver corroded to cell death and necrosis, and were congested with few sinusoid. Degeneration and necrosis was found in the renal cells, which could extend to some histolysis. Renal interstitium showed a reduced incidence of lymphocytes with numerous inflammatory cells infiltration. Spleen lymphocytes were reduction. The epithelial cells of gastrointestinal tract mucoa developed symptoms of degeneration, necrosis and desquamation.