【作者】 刘忠伟；

【导师】 周碧君； 虞天德； 李永明； 王开功； 吴彤； 文明；

【作者基本信息】 贵州大学 ， 预防兽医学， 2006， 硕士

【摘要】 本文由四部分构成，一是制备了山羊痘正向间接血凝诊断试剂。二是采用自制的正向间接血凝诊断试剂（IHA）对贵州省镇远、德江2个地区及广西省柳州市兽防站送检的血清样品进行了山羊痘免疫效果监测。三是对山羊痘病变及山羊痘病毒进行了比较详细的透射电镜观察。四是应用PCR技术检测山羊痘病毒。主要试验和结果如下： 1．通过Vero-E6细胞盲传3代后观察到有规律的CPE；细胞培养物琼脂扩散试验阳性；反向间接血凝试验阳性；由此确定成功分离了一株贵州山羊痘流行毒株，命名为LD毒株。通过硫酸铵盐析、Sephadex G-200柱层析纯化山羊痘病毒，以病毒抗原致敏绵羊红细胞制备了山羊痘正向间接血凝诊断试剂，进行了最佳试验条件的摸索，并与琼脂扩散试验相比较，表明正向间接血凝试验比琼脂扩散试验敏感性高。本次试验制备的正向间接血凝试验诊断液可用于兽医临床上进行快速诊断。 2．应用自制的山羊痘正向间接血凝诊断试剂对贵州省和广西柳州市共604头份山羊血清进行了抗体检测。同时对三种山羊痘疫苗进行了免疫效果比较，测定了山羊痘疫苗的免疫抗体消长规律、免疫保护期，对皮内、皮下、肌肉接种疫苗的免疫效果进行了比较，试验结果显示山羊痘弱毒疫苗免疫后7～8d可检测出抗体，皮内、皮下注射接种方式的免疫效果优于肌肉注射。制定了对山羊痘病在贵州省的防疫工作具有一定的指导意义的免疫程序：使用B组疫苗，采用皮内注射方式对山羊进行免疫，产生抗体的时间较快，水平较高，维持时间可达196d以上。 3．对山羊痘病变及山羊痘病毒进行了比较详细的透射电镜观察。在感染的皮肤和肺上皮细胞的胞浆内发现有大量不同成熟阶段的典型的山羊痘病毒颗粒。病毒颗粒也可见于巨噬细胞、成纤维细胞、血管内皮细胞的胞浆内，甚至少量在血管腔内。有髓神经纤维也被病毒和被感染的巨噬细胞所波及。在山羊痘痘疹材料感染的鸡胚绒毛尿囊膜痘斑的上皮细胞和成纤维细胞以及感染的Vero-E₆细胞中也可见成熟和不成熟的病毒颗粒。 4．建立了山羊痘的PCR检测方法。用特异性引物扩增出山羊痘弱毒疫苗和皮肤痘疹病毒P32基因，得到约1kb条带，阴性产物及羊接触传染性脓疱皮炎病毒、口蹄疫病毒不产生此条带，表明该引物可准确扩增山羊痘病毒P32基因。此方法具有快速、准确、特异和灵敏的特点，加快了山羊痘的分子生物学诊断速度，缩短了实验室对其检测的时间。可用于临床上症状相类似的疾病如口蹄疫、接触传染性脓疱皮炎病毒的鉴别诊断。更多还原

【Abstract】 This paper is composed of four parts. The first part is preparation of the indirect hemagglutination diagnostic reagent. The second part is survallance of immunological effects of serum samples collected from Zhengyuan, Dejiang country of Guizhou province and Liuzhou city of Guangxi province. The third part is observation of goat poxvirus (GTPV) by transmission electronic microscopy. The fourth part is detection of GTPV by polymerase chain reaction (PCR).1. Isolation and identification of GTPV has been conducted using cell culture and serological test. One virus was isolated and designated as LD strain. The LD strain virus has been purified through ammonium sulfate sedimentation and Sephadex G-200 chromatography. The sheep erythrocyte was sensitized with purified virus and the indirect hemagglutination assay (IHA) was developed. Meanwhile, the optimal conditions for the experiment have been surveyed. The serum samples of affected goat were detected by IHA and agar gel precipitation (AGP) simultaneously. The result indicated that IHA was more sensitive than AGP and the reagent could be used for diagnosing goat pox quickly.2. A total of 604 goat sera form Guizhou Province and Liuzhou City of Guangxi province were detected by IHA test. The immunological effects of three goat pox vaccines have been compared. The antibody fluctuation curve, protective period and the effects of different inoculation routs, including introcutaneous, subcutaneous and muscle injection have been measured. The results showed that the antibody could be detected after 7~8 days vaccinated with attenuated goat poxvirus vaccines, the immundogical effects of introcutaneous and subcutaneous inoculation has advantageous to muscle inoculation. The immunological programme has been made to prevent goat pox in Guizhou province. With introcutaneous injection, the antibody level was higher and the period of higher antibody maintained over 196 days.3. The lesions of goat pox and goat pox virus were observed using transmission electronic microscopy. Numerous typical goat poxvirus in different mature stages were found in the cytoplasma of the affected epithelial cells in skin and lung , and some also could be found in the cytoplasma of macrophages ,更多还原