从患出血病草鱼的肝脏病灶中分离筛选出2株致病菌。取病鱼样品组织过滤液接种CIK细胞、培养, 电镜下观察到细胞质中含有草鱼呼肠孤病毒样颗粒和包涵体, 病毒颗粒大小65 nm~ 70 nm, 包涵体0.46 μm~1.81 μm。人工回归感染实验显示分离的菌株及细胞毒悬液均能使草鱼致病死亡。对分离菌株进行细胞形态学、理化特性分析及药敏试验, 初步判定所分离的2株菌均为嗜水气单胞菌。进一步对菌株进行DNA分子鉴定, 结果显示2株菌的16S rRNA基因、促旋酶亚单位蛋白(gryB)基因均与GenBank上的嗜水
Two strains of infectious bacteria and a GCRV-like virus were isolated from grass carp which seemed to be infected by both virus and bacteria in Guangdong and Fujian provinces. CIK cells were inoculated with tissue fluid filtrated from the diseased fish. GCRV-like virus particles and virus inclusion body were observed in the cytoplasm of the infected cells under electron microscope. Artificial infection experiments showed that healthy grass carp died after infected with both the bacteria strains and virus-infected cell culture supernatant. Bacteria morphology, characteristics, physiology and biochemistry assays were used to identify the isolated bacteria. The characteristics of the two strains isolated were in accordance with those of Aeromonas hydrophila. PCR was performed to identify the bacteria. Both 16S rRNA gene and gryB gene of the two strains possessed high similarity with those of A. hydrophila registered in GenBank. AerA and ahpA genes could be amplified in both strains which indicated that both of them possessed strong pathogenicity. The two strains were therefore identified to be virulent strains of A. hydrophila. The diseased grass carp analyzed was supposed to be mixed infected by both A. hydrophila and virus.
广东省农业领域重点项目(No. 2008A020100016); 广东省海洋渔业科技项目(No. A200899F01); 国家科技基础条件平台建设项目(No. 2006DKA30470-008)