肉种鸡产蛋高峰期的马立克氏病鉴定及马立克氏病病毒标准抗原、标准血清的研制

发布时间：2018-01-21 20:37

本文关键词： 鸡马立克氏病病毒肉种鸡产蛋高峰期标准抗原标准血清　出处：《扬州大学》2016年硕士论文　论文类型：学位论文

【摘要】：鸡马立克氏病(Marek's disease, MD)由疱疹病毒科的马立克氏病病毒引起,1907年Josef Marek首次报道,主要引起各种脏器、皮肤的淋巴单核细胞浸润,通常于10-12周龄发病,可造成严重的经济损失。本研究以琼脂扩散试验,间接免疫荧光、病毒分离鉴定等方法,结合病理切片结果,在排除REV和ALV感染的基础上,对250日龄已免疫马立克疫苗肉种鸡鸡群中的肿瘤病原进行了鉴定。同时本研究参考OIE官方公布的MDV琼扩抗原制备方法,经改进制备出MDV标准抗原、标准血清,为MDV的诊断防治以及诊断试剂的标定提供了有效的方法和材料。1.肉种鸡产蛋高峰期马立克氏病的鉴定鸡马立克氏病通常于10-12周龄发病,由马立克氏病病毒引起。本研究以琼脂扩散试验,间接免疫荧光试验、病毒分离鉴定等方法,结合病理切片结果,在排除REV和ALV感染的基础上,诊断出250日龄已免疫马立克病疫苗肉种鸡鸡群的马立克氏病,并且成功分离到一株马立克氏病病毒,命名为MDV-AH1410。对其meq和gB基因进行PCR检测和序列分析,结果显示分离株与MDV Ⅰ型强毒及超强毒株的同源性为99.3%～100%,具有强毒特征。研究表明,鸡马立克氏病病毒不仅毒性在逐渐增强,而且鸡群发病的时间段也在延伸。2.马立克氏病病毒标准抗原、标准血清的研制选取MDV国际标准毒株RB1B作为候选毒株,用有限稀释法纯化病毒,将纯化好的病毒通过PCR的方法进行了纯异性分析,参考OIE公布的MDV琼扩抗原的制备方法经改进研制出MDV琼扩标准抗原,对其进行特异性分析以及琼扩效价测定。结果表明纯化的MDV-RBIB病毒无CAV、ALV、REV、EDS-76V、GPV、IBV、ILTV、IBDV等外源病毒,制得的琼扩抗原只与MDV阳性血清反应,与其他相关血清没有交叉反应,琼扩抗原效价为1：4。用1日龄SPF鸡腹腔注射MDV-RBIB毒株2000PFU/羽,每周采血血分离血清进行抗体检测,待琼扩试验阳性后无菌采血,通过间接免疫荧光和血凝抑制等方法进行纯异性分析,同时检测其荧光效价和琼扩效价。间接免疫荧光试验结果显示该制得的抗MDV血清不与ALV、GPV、REV、IBV、IBDV、NDV、AIV、EDS-76V等外源病毒反应,只与MDV反应,荧光效价为1：1600,琼扩抗体效价为1：4。制备的琼扩抗原加入1%的BSA,制备的多抗血清加入0.01%硫柳汞,分别冻干分装。按照中国兽药典进行无菌检验、支原体检验合格；检查分装差异系数CV值、物理性状、稳定性、均一性、保质期等均符合要求,最后获得了高质量的MDV标准抗原和标准血清,为MDV的诊断防治以及诊断试剂的标定提供了有效的方法和材料。
[Abstract]:Marek's disease (Marek's, disease, MD) caused by Marek's disease virus, herpes simplex virus, first reported in 1907, Josef Marek, is mainly caused by the various organs, skin, lymph and mononuclear cell infiltration, usually in the pathogenesis of 10-12 weeks of age, can cause severe economic loss. This study by agar diffusion test, indirect immunofluorescence virus isolation and identification, and other methods, combined with the pathological results, excluding the foundation of REV and ALV infection, 250 days of age has been immune vaccine tumor pathogenic Marek broiler breeder chickens were identified. At the same time this study reference OIE official MDV AGP antigen preparation method, improved the preparation of MDV the standard antigen and standard serum for the diagnosis of MDV control and calibration of diagnostic reagents provides effective methods and materials of.1. broiler breeder egg peak of Marek's disease and identification of chicken Marek's disease usually in 10-12 weeks. The disease, caused by Marek's disease virus. This study by agar diffusion test, indirect immunofluorescence test, virus isolation and identification methods, combined with the pathological results, excluding the foundation of REV and ALV infection, diagnosed at the age of 250 days has been immune to Marek's disease Marek's disease vaccine broiler chickens, and successfully isolated a strain of Marek's disease virus, named MDV-AH1410. was analyzed by PCR detection and sequence of the meq gene and gB gene showed 99.3% homology to 100% isolates with MDV type virulent and very virulent, with strong toxic characteristics. The results show that not only the toxicity of chicken Marek's disease virus in gradually increased. Time and the incidence of chickens is also an extension of Marek's disease virus.2. standard antigen and standard serum development selection of international standard MDV strain RB1B as candidate strains, purification of the virus by limited dilution, the purified virus The pure specific analysis by PCR method, preparation method of reference OIE released MDV AGP antigen and improved the development of MDV standard AGP antigen, carries on the analysis and determination of specific AGP titer. The results showed that the purified MDV-RBIB virus CAV, ALV, REV, EDS-76V, GPV, IBV, ILTV IBDV, exogenous virus, AGP antigen prepared only reacted with MDV positive serum, no cross reaction with other serum AGP antigen titer was 1:4. with 1 day old SPF chickens were intraperitoneally injected with MDV-RBIB strain 2000PFU/ plume, weekly blood separation serum antibody detection, the AGP test positive after sterile blood. Pure were analyzed by indirect immunofluorescence and hemagglutination inhibition method, and detecting the fluorescence titer and AGP titer. Indirect immunofluorescence assay showed that the prepared anti MDV serum and ALV, GPV, REV, IBV, IBDV, NDV, AIV, EDS-76V etc. The source of the virus, only reacted with MDV, fluorescent titer of 1:1600 antibody titer, AGP AGP antigen 1:4. were prepared by adding 1% BSA, preparation of polyclonal sera with 0.01% thimerosal, respectively. Aseptic freeze-dried packing inspection according to Chinese Veterinary Pharmacopoeia, Mycoplasma inspection; check the quantity difference coefficient CV, physical properties, stability, uniformity, shelf life and so on are in line with the requirements, finally get the high quality MDV standard antigen and standard serum for the diagnosis of MDV control and calibration of diagnostic reagent provides effective method and material.

【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S858.31

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