【作者】 胡广洲；

【导师】 李登峰；

【作者基本信息】 宁波大学 ， 水产养殖， 2010， 硕士

【摘要】 对中华鳖(Trionyx sinensis)暴发性败血症进行了研究,该病是2005年来发生于浙江宁波等地区中华鳖生态养殖场的一种暴发性疾病,病鳖主要的症状为出血性败血,死亡严重。对中华鳖暴发性败血症流行情况,主要组织病理变化和超微病理变化进行了研究,确定了该病病原,并对病原进行了研究。流行病学调查表明,中华鳖暴发性败血症主要流行浙江宁波等地区的中华鳖生态养殖场,病鳖主要为体重150-200 g的幼鳖,成、亲鳖也有患病感染。发病季节为6-10月份,其中6-9月份为发病高峰期,发病池塘10天内累积死亡率可达90-100%,主要的症状为出血性败血,常规药物进行治疗无效,给养殖生产带来很大经济损失。对病鳖组织涂片,镜检观察未发现寄生虫。病鳖组织通过电镜检测病原,观察可见病鳖心、肝、肠及胃等组织内存在大量90-100 nm的球状病毒颗粒,未发现气单胞菌等的感染,且从病鳖组织中分离纯化到了大小、形态相同的病毒颗粒。用病鳖组织匀浆液和组织匀浆滤液对健康鳖进行回归感染,都可引起鳖死亡,死亡的中华鳖解剖观察结果与之前患病中华鳖症状基本一致,其中组织匀浆液攻毒组中华鳖死亡率比组织匀浆滤液组高15%。上述结果表明病毒是原发性病原,细菌加重病情。组织病理研究结果表明,患病中华鳖肝脏、肠、胃均表现出非常明显的病理学变化。肝脏、肠、胃等组织细胞肿胀充血,排列松散,并可见破坏的血细胞,呈败血症状,胃、心等组织细胞出现空泡化,心脏中血细胞减少,肾小球失血萎缩,还有部分肾小球坏死解体、消失,肾小管破裂,脾髓中部分血细胞破裂。超微病理研究结果表明,患病中华鳖的肝脏组织细胞病变最为严重,在各组织细胞中均观察到了球状病毒颗粒。从各组织细胞的病理变化来看,主要表现为线粒体减少,嵴断裂,甚至呈空泡化,溶酶体增多,细胞发生坏死、凋亡。使用SPF鸡胚对败血症病毒进行培养研究,采用卵黄囊接种法和羊膜腔接种法接种鸡胚,36℃培养。收获接种后第2-7天死亡的鸡胚,7天内不死的鸡胚第8天停止培养。对鸡胚的培养时间进行方差分析可知,实验组和对照组的鸡胚培养时间差异极显著(p<0.01),两种方法接种的鸡胚培养时间无显著性差异(p>0.05);在实验组鸡胚的卵黄囊膜中经电镜观察可见大量病毒,和病鳖组织中的病毒大小、形态基本相同;将实验组鸡胚制成攻毒液对中华鳖进行回归感染成功,死亡的中华鳖症状与该病例基本相同;并采用卵黄囊接种法对病毒进行两次传代培养,结果实验组和对照组的鸡胚培养时间差异均极显著(p<0.01)。上述结果表明鸡胚培养中华鳖球状病毒成功,卵黄囊膜接种法和羊膜腔接种法接种效果基本相同,病毒能够在鸡胚中传代培养。对病毒的理化特性进行研究,结果表明病毒对热(56℃)、酸(pH 3.0)和碱(pH 11.0)表现不稳定;对脂溶剂(氯仿)敏感;紫外线照射30 min可使大部分病毒灭活。对病毒进行了初步纯化,提取病毒DNA,采用6条随机引物,对病毒的核酸进行了扩增和测序初步研究,除两条引物没有扩增和测序成功外,共测得4条基因序列,Blastn结果表明,4条序列与Genebank中的序列同源性或覆盖率不高。4条序列中仅有1条氨基酸序列可以通读,Blastp结果表明该序列和甲基化酶家族基因同源。测序结果结合电镜观察结果,说明所测病毒应该是一种新病毒。从病鳖体内分离到8株细菌,对细菌进行形态观察,结合16S rDNA序列分析鉴定8株细菌分别为为格氏乳球菌、假单胞菌、海氏肠球菌、屎肠球菌、人苍白杆菌、惰性嗜血杆菌、弗氏柠檬酸杆菌和迟钝爱德华氏菌。对格氏乳球菌、弗氏柠檬酸杆菌和迟钝爱德华氏菌进行药敏试验发现,格氏乳球菌对青霉素等4种药物敏感,弗氏柠檬酸杆菌对头孢唑啉等13种药物敏感,迟钝爱德华氏菌对新霉素等3种药物敏感,药敏结果可对该病的治疗起到帮助。采用16S rDNA分析方法,构建了病鳖体表和体内细菌克隆文库。结果表明,中华鳖体表和体内文库克隆子分别归为15和11类OTU,体表和体内菌群多样性较强,优势细菌类群分别为拟杆菌类群和变形菌类群,并在文库中检测到了几株益生菌和条件致病菌。更多还原

【Abstract】 Fulminant septicaemia of soft-shelled turtle (Trionyx sinensis) originally occurred in Zhejiang Eco-Farm in 2005, breaked out frequently these years. This disease causes mass mortality and makes a severe hazard. The main symptoms were hemorrhagic septicemia. Epidemiology, pathology and ultrastructural studies were carried out. The pathogen was determined and studied.Epidemiological survey showed that the fulminant septicaemia of soft-shelled turtles mainly outbreaked at eco-farms in Zhejiang province. Both larvae and adult turtles can be infected and die, while sick turtles mainly weight 150-200 g. Epidemic season was from June to October, especially from June to September. Mortality within 10 days of the pond can reach 90-100%, main symptoms were hemorrhagic septicemia. Conventional drugs were not effectual. The contagion has caused great economic losses.Parasites were not found under microscopic observation. But under TEM, a large number of spherical enveloped viruses, with diameter of 90-100 nm, were observed in the tissues of sick turtles. Virus particles were isolated and purified from the sick turtle tissues. The tissue fluid and tissue homogenate filtrate from sick turtles were injected to health turtles, and caused classical hemorrhagic septicemia symptom and mortality to the turtles. Mortality rate of the tissue fluid attacking group was 15% higher than the tissue homogenate filtrate group. The above results showed that the disease was mainly caused by virus, and aggravated by bacteria.Histopathological observation found obvious pathological changes in the liver, intestine, stomach, kidney and spleen of sick turtles. Cells of liver, intestine, stomach were tumid and congestive, they were loosely and disorderly arranged. Destroyed blood cells were observed in tissues. Tissue cells of stomach and heart appeared vacuolization, blood cells in the heart decreased. Glomerular was atrophic, disintegrated and disappeared. Renal tubular and partial blood cells in spleen were ruptured. Ultrastructural results showed that liver had most severe lesions. There were spherical viruses in most tissue cells. The main ultrastructural changes were the reduction, rupture of mitochondrial cristae; vacuolization of mitochondrial, increasation of lysosomes; putrescence and apoptosis of cells.The virus cultivation in SPF chicken embryos was studied, using yolk sac and amniotic inoculation methods. Variance analysis of the survival time of inoculated chick embryos showed that experimental groups and control group, which were injected with PBS, had a significant difference (p <0.01). No significant difference appeared between the two experimental groups using different inoculation methods (p>0.05); a large number of virus, which were similar with the virus found in tissues in sick turtles,were observed in chick embryo yolk sac membrane of the experimental group under TEM; tissue homogenate filtrate of infected chicken embryo were injected to health turtles and caused infection, hemorrhagic septicemia symptom and death, similar with natural case. Virus was cultured two passages in chick embryo, and the culture time of experimental and control group had a significantly difference (p<0.01). The above results showed that culturing spherical virus of soft-shelled turtles was succeeded. The two inoculation methods were equivalent. Virus can be stable passaged in chick embryo.Physicochemical properties of the spherical virus were studied. The result showed that the virus was instability to heat (56℃), pH 3.0 and pH 11.0; the virus was sensitive to chloroform; the majority of virus could be inactivated by ultraviolet irradiation for 30 min.DNA of crudely purified virus was extracted. Six random primers were used to the virus nucleic acid amplification and preliminary sequencing. Four fragment sequences were acquired. Blastn results showed that four sequences had a low homology or coverage with the sequences of Genebank. Only one amino acid sequence could be read in the four sequences. Blastp results showed that the sequence was homologous to methylase family genes. Sequencing results associated with electron microscopy results indicated that the partially sequenced virus should be a new virus.Eight bacteria were isolated from sick soft-shelled turtles (Trionyx sinensis) associated with fulminant septicaemia. Morphology of the bacteria was characterized by LM, SEM and TEM. 16S rDNA of the bacteria were amplified and sequenced, Results showed that they were Lactococcus garvieae, Pseudomonas sp., Enterococcus hirae, Enterococcus faecium, Ochrobactrum anthropii, Haemophilus segnis, Citrobacter freundii and Edwardsiella tarda. Antibiotic sensitivity patterns of L. garvieae、C. freundii and E. tarda were evaluated against 20 antibiotics. L. garvieae was sensitive to 4 antibiotics including Penicillin. C. freundii was sensitive to 13 antibiotics including Cefazolin. E. tarda was sensitive to 3 antibiotics including Neomycin. Antibiotic sensitivity results could help curing this disease.Diversity of external and internal bacterium associated with the Soft-shelled turtle (Trionyx sinensis) was determined by 16S rDNA gene sequences. Results showed cloned partial sequences of external and internal bacterium clone library were belonged to 15 and 11 OUT respectively. The phylogenetic trees showed that, they mainly related to Bacteroidetes and Proteobacteria respectively. Flavobacterium sp. were detected in both cloned librarys, they were dominate species. Also several beneficial bacteria (Lactococcus lactis and Pseudomonas sp.) and conditional pathogens (E. tarda, C. freundii) were detected. Results demonstrated prolific diversity of external and internal bacterium associated with the soft-shelled turtle.更多还原