【作者】 徐进；

【导师】 龚世园； 曾令兵；

【作者基本信息】 华中农业大学 ， 水产养殖， 2012， 博士

【摘要】 斑点叉尾鮰自1984年引进我国养殖以来,成为我国主要出口创汇的名优养殖鱼类,已在我国20多个省市推广养殖,每年产量达22万吨以上。随着产量的增长,集约化程度的提高以及种质的退化,使得病害问题也越来越严重。近年来全国各地爆发疑似病毒性的出血性疾病,给斑点叉尾鮰养殖造成了巨大的经济损失。为了能分离病毒性病原,本研究从斑点叉尾鮰组织细胞系入手,建立了斑点叉尾鮰肾脏细胞系,并应用该细胞系针对斑点叉尾鮰出血病开展了病原学和疫苗研究,研究结果如下：1.斑点叉尾鮰肾脏细胞系的建立：采用组织块移植培养技术,对来源于斑点叉尾鮰肾脏组织的细胞进行原代培养,建立了斑点叉尾鮰肾脏组织细胞系,已稳定传代培养70多次,定名为CCK。斑点叉尾鮰肾脏组织细胞为成纤维样细胞,最佳培养基为M199,最适培养温度范围为28-32℃,培养基血清浓度为10%,此条件下CCK细胞的倍增时间约为38.9-41.0h。斑点叉尾鲴肾脏细胞的集落形成效率为74.16±3.54%,第9代传代细胞的染色体数目为正常二倍体2n=58,第33代传代细胞的染色体众数为2n=60。液氮冷冻保存6个月后的细胞经台盼兰染色检验,86.69±1.04%仍保持活性,细胞复苏后培养生长旺盛。通过对离体培养细胞的核糖体28S基因进行PCR扩增及序列分析与比对,表明该细胞系来源于斑点叉尾鲴。2.斑点叉尾鲴出血病的病原学研究：从湖北省一爆发出血病的斑点叉尾鲴鱼种养殖场分离到一株呼肠孤病毒,命名为CCRV-730.人工感染试验证实该病毒即为斑点叉尾鲴出血病的病原。斑点叉尾鲴出血病的主要症状包括腹部膨大,眼睛凸出,鳃盖、下颌、皮肤以及鳍条基部出血。用CCK细胞进行了病毒培养与分离,电子显微镜下可见大量呼肠孤病毒样颗粒以晶格状排列于细胞质中,直径60-70nm,提取病毒基因组总RNA,并用SDS-PAGE电泳分析其电泳型,克隆并测序了基因组S组节段基因的完整序列。将S组节段基因序列在NCBI基因库中进行比对分析,发现CCRV-730和水生呼肠孤病毒属的成员相似性最高,特别是和草鱼呼肠孤病毒873株(GCRV-873)相应片段的相似性高达99%～100%。这些结果表明GCRV-873可能在自然界发生基因变异,这些变异改变了其宿主专一性,扩大了宿主范围,使其成为斑点叉尾鮰的病原。3.斑点叉尾鮰出血病细胞培养灭活疫苗研究：以β丙内酯作为灭活剂,制备了斑点叉尾鮰细胞培养灭活疫苗,对疫苗的安全性、免疫效果进行了评估。结果显示,以终浓度为0.025%(V/V)的β内酯于4℃灭活CCRV-730病毒液24h,即可达到理想的灭活效果,灭活疫苗的细胞感染试验与鱼体感染试验均证实病毒已被灭活,无菌检验显示疫苗无细菌污染,表明此法制备的疫苗安全性好。免疫保护效果试验结果显示,在实验室条件下,规格为8～10cm的斑点叉尾鮰鱼种每尾腹腔注射0.2mL效价为105TCID50/mL的疫苗,可获得高达89.1%的保护力。池塘养殖试验免疫保护效果结果显示,采用浸泡的方式免疫4～6cm小规格鱼种也可获得72.6%的保护力。上述结果表明,无论是通过注射免疫还是浸泡免疫,β丙内酯灭活疫苗免疫的斑点叉尾鮰鱼种能获得很好的保护力,此疫苗可在生产上进行广泛应用以预防斑点叉尾鮰出血病。更多还原

【Abstract】 Since channel catfish(Ictalurus punctatus) was introduced into China in1984, it has become an important export species farmed in nearly20provinces in China. According to production estimates, the annual output of farmed channel catfish in China was about220,000metric tons. With the increasing production there has been more epizootic diseases detected in farmed channel catfish in China because of high density stocking and genetic depression. Recently, a hemorrhagic disease in cultured channel catfish fingerlings broke out and spread in the most part of China. It caused large economic losses to channel catfish industry. We attempted to establish a cell line from channel catfish tissues for the isolation of viral pathogen from the hemorrhagic diseased fish. Fortunately, a channel catfish kidney cell line was obtained and it was apllied in the pathology and vaccine research of channel catfish hemorrhagic disease. The results were present as below:1. Establishment of channel catfish kidney cell lineA channel catfish kidney cell line (CCK), was established from the kidney of channel catfish, Ictalurus punctatus, Rafinnesque by using explant techqiques. CCK cell line had been continuously subcultured over70times and been fully characterized in the aspects of morphology observation, optimal growth kinetics, plating efficiency, karyotyping, cryopreservation and28S ribosome RNA genotyping, etc. The CCK cell monolayer was consisted of fibroblast-like cells and the optimal growth condition for CCK cells was:medium, M199; temperature,28-32℃; FBS,10%. The plating efficiency of CCK cells was about74%. Following cryopreservation in liquid nitrogen, thawed cells exhibited a viability of86.69±1.04%after6months storage period. Chromosome typing of CCK cell line revealed that at9th pasage the modal diploid chromosome numeber was2n=58, and at33rd passage the modal number was60. Polymerase chain reaction amplification of partial28S ribosome RNA and sequence analysis indicated94.0%identity to the sequence found in Genbank from channel catfish source, confirming that the cell line was of Channel catfish origin.2. Pathology of channel catfish hemorrhagic disease.A reovirus, designated as CCRV-730, was isolated from channel catfish, Ictalurus punctatus, fingerlings suffering a severe hemorrhage in Hubei province in China. Experimental infection confirmed the pathogenicity of the virus and proved it to be the causative pathogen. Signs of the diseased channel catfish included abdominal distention, eyes bulging and hemorrhages of operculum, lower jaw, skin and fin bases. The CCK cell line was used for viral pathogen isolation. The electron microscopy observation of the virus infected cells revealed that there were a large number of reovirus-like particles measuring60～70nm in diameter in cytoplasm arrayed in crystalline. The viral genomic RNA was extracted and analyzed by SDS-PAGE and the complete S-class (S7-S11) segments of genome RNA were cloned and sequenced. The sequence alignment analysis of the S-class segments of CCRV-730with corresponding sequences of other Aquareovirus members in the genetic sequence database of NCBI indicated that the virus had high similarity to grass carp reovirus, especially shared99%-100%nucleotide sequence identity with the grass carp reovirus873strain (GCRV-873). The results implied that the genetic variation of GCRV-873potentially arose in natural environment and resulted in the viral host conversion or expansion and made it pathogenic to channel catfish.3. Inactivated vaccine of channel catfish hemorrhagic diseaseCell culture inactivated vaccine of channel catfish hemorrhagic disease was produced by using (3propiolactone as the inactivator. The safety and immune effect of the vaccine were assessed.The results showed that CCRV-730virus could be completely inactivated within a final concentration of0.025%(V/V) β-propiolactone at4℃for24h. The vaccine was test by infecting cells and fish to confirm the virus had been completely inactivated. Bacterial test showed no bacterial contamination of the vaccine. These results confirmed the safty of the produced vaccine. Immune protective effect test results show that, under laboratory conditions, the8～10cm channel catfish fingerlings intraperitoneal injection0.2mL105TCID5o/mL vaccine could obtain up to89.1%protective rate. Immune protective effect test in the fish cultured in ponds showed that4～6cm small size fingerlings, which were immunized by immersion, could obtain72.6%protective rate. These results indicate that channel catfish fingerlings immunized with P-propiolactone inactivated channel catfish hemorrhagic disease vaccine by injection or immersion could obtain good protective force. This vaccine can be widely used in the production in order to prevent the channel catfish hemorrhagic disease.更多还原