【作者】 刘志强；

【导师】 王新华； 钟发刚；

【作者基本信息】 石河子大学 ， 预防兽医学， 2008， 硕士

【摘要】 犬细小病毒(Canine parvovirus,CPV)是一种引起犬出血性肠炎或心肌炎的单链线状DNA病毒。据石河子兽医防疫部门统计,在当地犬的各种传染病中,犬细小病毒性肠炎发病率、死亡率均为最高,为犬的主要传染病之一。目前该病还没有有效的治疗药物,开发CPV抗体及其诊断、治疗试剂将有助于该病的防治。本研究从临床疑似犬细小病毒感染病死犬的内脏中分离CPV,应用F81猫胎肾传代细胞增毒,Sepharose-4B层析与Fractogel EMD TMAE(M)强阴离子交换层析相结合的方法纯化病毒,对分离病毒用常规方法血凝及血凝抑制、免疫电镜和PCR法予以确认。继而以纯化的CPV作为抗原免疫健康产蛋鸡,收集鸡蛋,用改良PEG6000法和水稀释法提取卵黄抗体(IgY)。用琼脂扩散法和间接ELISA法测定IgY效价以及体外中和病毒效价。在动物实验中,以纯化的CPV毒株实验室人工攻毒感染2～3月龄幼犬5只,待出现明显临床症状时,用特异卵黄抗体进行治疗,考察其疗效。进而在石河子兽医门诊临床治疗发病犬,从较大样本中统计治愈率。研究结果显示,分离CPV的接种F81细胞产生明显细胞病变,凝集猪红细胞效价达2~9,并能被犬细小病毒阳性血清所抑制;免疫电镜观察外观呈圆形、直径约20～24nm病毒粒子;根据犬细小病毒的基因组设计特异引物进行PCR扩增,得到犬细小病毒VP2基因组全长,提交GenBank(EU170352),经序列分析,确定分离毒株为CPV-2a型。暂命名为CPV-SHZ株。改良PEG6000法提取IgY的蛋白浓度高,而水提法IgY纯度高。琼脂扩散法与间接ELISA法测定IgY最后一次免疫后10天的效价分别为1:32和1:24000。实验室小规模动物实验和较大规模临床实验分别获得100%和79.4%的治愈率和明显的动物保护效果。本研究在新疆石河子地区成功分离到犬细小病毒CPV-SHZ株,为我国该病流行病学提供一定的参考资料,并且针对该病原体制备了高效价的IgY抗体,应用抗体的中和特性,临床治疗效果显著。所得抗体经济、方便、可大规模生产,研究结果显示,CPV卵黄抗体可进一步开发为抗体制剂,用于该病的预防和治疗。更多还原

【Abstract】 Canine parvovirus(CPV), one of the smallest and the simplest single stranded DNA virus, is a kind of lethal virus which can cause acute infectious canine diseases with clinical symptoms such as bloody enteritis and cardiac muscle inflammation. According to the statistical data of veterinary office in Shihezi region, CPV is the primar cause of morbidity and mortality of various infectious diseases in dogs. At present, there is not effect CPV treatment method .Development of CPV antibodies is considered as an important role for the diseases prevention and treatment.CPV samples were collected from infected dogs according to the clinical symptoms. The embryonic feline kidney cell line F81 was used to multiplicate the viruses. The virus was than purified by Sepharose-4B column chromatography and Fractogel EMD TMAE(M) Anion exchange chromatography. HA/HI, immune-electron microscope (IEM) and PCR methods were used to identify the CPV. Laying hens were immunised by purified CPV samples and the egg yolk antibodies (IgY) were extracted by modified PEG6000 method and water dilution method. The titres of IgY was tested with agar spread method and indirect ELISA. The anti- CPV IgY antibodies were than used to treat 2~3 months old dogs which were challenged with CPV perversely. Furthermore, field investigation using 34 CPV infected dogs was done in order to get the enlarged statistical data.The present research show that the cell supernatant fluid can be detected by HA, the diluter of HA range is 2~9. The purified virus can reacted with serum CPV antibody in immune-electron microscope observation (IEM) and the virus diameter is 20-24nm. The VP2 gene of the virus was amplified by PCR reaction, The sequences data of the gene were than submitted to GenBank(EU170352). The gene sequences were classified as a CPV-2a type with a few diversities. It is named as CPV-SHZ in the present paper. Different IgY antibody extraction methods were compaired. Higher protein concentration is observed with the use of modified PEG6000. However, there is higher IgY consentration by the use of water dilute method. The titer of IgY was tested as 1:32 by agar spread method and which is equaled to 1:24000 by indirect ELISA. The clinical results showed that four dogs in the IgY group were cured and one in the control group died, and the protection rate reached to 100%. The enlarged animal test with 34 dogs shows protection rate of 79.4%.The above showed results indicate that the CPV-SHZ strains have been successfully isolated. Furthermore, highly titred and neutralized IgY antibodies can be purified,and show good virus protection effects in both in vitro and in vivo. The present research suggests that anti-CPV IgY have the potential for further development and use of CPV prevention and treatment.更多还原