【作者】 周晓杨；

【导师】 张其中；

【作者基本信息】 西南师范大学 ， 水生生物学， 2004， 硕士

【摘要】 实验证明南方鲶出血性败血症是由细菌引起的。从自然患病南方鲶的肝、肾、脾等脏器分离获得3株强致病性菌株，鉴定均为嗜水气单胞菌，但3株菌药物敏感性有差异。 患病南方鲶外周血液中红细胞和嗜中性粒细胞有较大变形，淋巴细胞个体增大。红细胞显著减少，不成熟红细胞增多。白细胞总数显著增加，白细胞分类百分比(DLC)发生变化，嗜中性粒细胞、单核细胞分类百分比明显增加，淋巴细胞和血栓细胞变化不显著。另外，红细胞脆性增加，血细胞沉降率增加。 病鱼肝实质结构严重受损，细胞索结构模糊，界限不清；肝细胞肿大，细胞核肿胀，空泡化，肝细胞大面积坏死解体，染成浅色一片。肾单位数量显著减少，肾间质所占比例增大，淋巴细胞增多；肾小囊腔扩大；肾小管肿胀，部分肾小管上皮细胞变性，甚至坏死。脾脏淋巴组织松散，淋巴细胞减少，衰老细胞增多。 嗜水气单胞菌灭活全菌苗免疫南方鲶，免疫后第2d免疫组实验鱼外周血白细胞数量剧增，白细胞分类百分比发生变化。单核细胞和嗜中性粒细胞分类百分比在第2～14d显著增加，以后逐渐减低，至第28d恢复到起始状态，其中在第7d均达到最大值。单核细胞的增殖幅度明显高于嗜中性粒细胞。淋巴细胞在第2～4d比对照组低，从第7d起开始增殖，第21d达到最大值47.29％；实验过程中免疫组血栓细胞分类百分比始终略低于对照组。吞噬细胞活性显著增强，吞噬百分比平均比对照组提高了17.66％，吞噬指数平均比对照组高出1.16。免疫后第4d吞噬百分比(PP)和吞噬指数(PI)均达到最高值，前者为48.40％，后者为4.82，第21d的吞噬百分比和吞噬指数开始下降，但仍高于对照组。 嗜水气单胞菌灭活全菌苗、脂多糖(lipopolysaccharide，LPS)、胞外产物(ECP)、菌细胞苗分别进行1次免疫和加强免疫南方鲶，各种疫苗在接种后第14d血清凝集抗体效价开始上升，第21d快速上升，至第28d达到峰值，后缓慢下降，但能以较高水平维持20d左右，之后快速下降。同种疫苗的加强免疫组均比1次免疫组获得的凝集抗体效价高，高效价抗体持续的时间长，全菌苗、LPS、ECP和菌细胞加强免疫组凝集抗体滴度的峰值分理学硕l学位论文:南方鳍出l(ll.性败](ll庄的炳原、病理及免疫颅防研究别为1024、256、512和128，1次免疫的峰值依次为128、64、128和32。LPS组凝集抗体滴度峰值出现较ECP组早，但ECP组的凝集抗体滴度更为稳定，能以较高水平持续到第8周。 全菌苗、脂多糖(LPS)和胞外产物(ECP)均能使鱼体获得较好的保护效果，菌细胞苗效果差。免疫后不同时间各疫苗对同源菌株注射攻击的保护效果不同，第2周时，各组的保护力均很低;第4周，各疫苗组的免疫保护率达到最高，分别如下:全菌苗一次免疫组保护率为50%，加强免疫组64.3%;LPS一次免疫为42.9%，加强免疫组为50%;ECP一次免疫64.3%，加强免疫为78.6%;第6周时，各组的保护率略低于第4周，但仍保持了较好的保护效果。各对照组死亡率均高于93.3%，且对照组死亡鱼表现出头部、眼睛、上下领充血等出血病典型症状。说明接种鱼获得了良好的免疫保护效果，能有效抵抗嗜水气单胞菌的攻击。更多还原

【Abstract】 Haemorrhagic Septicemia of catfish is a threatening disease with high incidence and mortality in fish-farms . At present, no effective measure has been developed to prevent this disease.In this paper, we have demonstrated that Haemorrhagic Septicemia of Southern Catfish is caused by bacterials. Three strains of bacterial pathogen with high virulence have been isolated from liver, kidney and spleen of diseased Southern Catfish with typical Haemorrhagic Septicemia. The three strains of the pathogen are all capable of causing Haemorrhagic Septicemia in healthy naive Southern Catfish and have been identified to be Aeromonas hydrophila with different biological characters and sensitivities to antibiotic drugs.After experimental infection with the isolated pathogens, the morphology of blood cells and hematological parameters of infected fish were monitored. In infected fish, the red blood cell counts were (1.44 + 0.16) X 106mm-3, which were significantly lower than that of control[ (2.28 +0.17) X 106mm-3], indicating that hemolysis was caused. On the contrary, the white blood cell counts were increased from (6.25 + 1.67) X 104mm-3 to (8.375 + 3.25) X 104mm-3 , suggesting that immunological response was induced. At the same time , the erythrocytes and neutrophils were deformed and the lymphocytes were enlarged. The DLCs (differential leukocyte counts) of neutrophils and monocytes were higher in the infected fish, but the levels of lymphocytes and thrombocytes remained unchanged.Obvious pathological changes were observed in liver, kidney and spleen of diseased catfish with a light microscope. The structure of the liver was significantly changed. The arrangement of liver cells was disturbed and the borderlines between cellls were invisible. The majority of liver cells swelled and became larger than that of healthy catfish, and were even degenerated. In kidney, fewer nephrons and more lymphocytes were observed. Renal capsule space became larger. Renal corpuscle swelled and renal tubule epithelial cells had significantly larger size, and some showed necrosis. In spleen, fewer and scattered lymphocytes were observed.The vaccines made of detox icated bacterial were all capable of inducing immunological response in immunized fish. The naked bacterial cell vaccine has some effect, but not so good as that of others. On 2nd day of immunization, a rapid increasing level of the white blood cell counts in peripheral blood was observed. On 4th day, the white blood cell counts were elevated to levels between (79.8 + 9.5472) X103mm-3and (84.2 + 7.3521) X 103mm-3, and remained on such higher levels until 28th day. The DLCs of white blood cells were also changed after immunization. From 2nd day to 14th day, increasing DLCs of monocyte and neutrophile were observed, and on 7th day both reached to peak points(13.35% and 10.06%), which were very siginificantly higher than that of control(3.58% and 5.99%). After 14th day, the DLCs of both were decreased gradually and on 26th day, returned back to the beginning. Although no increasing lymphocyte levels were observed between 2nd and 4th day, lymphocyte level began to be increased on 7th day, and on 21s’ day reached to peak point 47.29%. During the whole immunization process, the fhrombocyte level of immunized fish was slightly lower than that of control, but with no statistically significant difference.Phagocyte was significantly activated after immunization. The phagocytosis percentage(PP) and phagocytosis index(PI) in immunized group were 16.00%~48.40% and 1.36-4.82 respectively, which were both significantly higher than that of control(12.48%~19.57%). On 4th day of immunization, both the PP and PI reached to peak points 48.40% and 4.82 respectively. On 21st day, PP and PI began to be decreased, but both were still higher than those of control.Antibody levels were increased after immunization. Increasing antibody levels began to be observed on 14th day. Except the group immunized with naked cell vaccine, rapid increasing antibody levels were observed on 21st day, and reach更多还原