【作者】 张静；

【导师】 潘玲；

【作者基本信息】 安徽农业大学 ， 基础兽医学， 2008， 硕士

【摘要】 新城疫(Newcastle disease,ND)是鸡的一种高度接触性、毁灭性的传染病,给我国的养禽业造成巨大的经济损失,因此被国际兽医局(Office InternationaldesEpizooties,OIE)列为必须通报的传染病之一。在安徽地区随着疫苗的广泛应用,ND大规模爆发流行己受到明显控制,但由于疫苗质量、使用方法、免疫程序等问题,导致了非典型ND的广泛发生;而新城疫病毒(Newcastle disease vires,NDV)宿主范围的不断扩大和变异毒株的出现和流行,都使ND的防制和诊断工作更加复杂,引起社会各行对NDV检测和防治高度重视。首先,从临床上收集到的具有典型和非典型ND临床症状的病料组织20份,组织为脑。采用SPF鸡胚分离、纯化病毒,用HA与HI进行病毒尿囊液的初步鉴定。结果鉴定出三株新城疫病毒,分别命名为NDV-AH1、NDV-AH2、NDV-AH3。其次,利用NDV-AH1、NDV-AH2、NDV-AH3制备成免疫原,免疫7日龄的非免疫雏鸡,免疫3次后采血,分离血清,用微量法在同一个条件下用不同的分离毒株和Lasota疫苗株做为抗原做交互血凝抑制试验,测定免疫血清的HI抗体滴度,检测这三株分离毒分别与Lasota疫苗株抗原相关性,计算出R值比较其抗原相关性的差异。结果表明:三个毒株的R值为:NDV-AH1毒株与Lasota株两者间的抗原性差异R=0.90,NDV-AH2与Lasota两毒株间的抗原性差异R=0.95,NDV-AH3与Lasota两毒株间的抗原性差异R=0.91。当R值在0.67≤R≤1.5之间,说明分离毒株与Lasota毒株抗原性无明显差异。由此可知NDV-AH1、NDV-AH2、NDV-AH3与Lasota疫苗株之间抗原性无明显差异。为以后这些地区接种ND疫苗提供试验依据。再者,试验还建立了F48E9强毒株感染的PCEF细胞,观察细胞病变,用病变细胞进行免疫组化检测一抗和二抗最适试验浓度条件的优化,经过优化试验得出,一抗以1:10倍稀释,37℃孵化1h,酶标二抗以1:400稀释时,孵育时间为37℃,40min,染色效果达到最佳状态,具有染色效果好,背景清晰的优点。最后,进一步用所建立免疫组化的方法来检测F48E9强毒株和NDV-AH1毒株分别人工感染7日龄的非免疫雏鸡,取感染后3-4天的鸡全部扑杀,采集脑,肺,肝脏,脾脏,胰脏,十二指肠,气管来观察这些组织中的F48E9病毒和NDV-AH1毒株分布情况,以了解两株ND病毒感染动物时,病毒在体内各个组织中的分布情况。结果:F48E9感染组可以检测到病毒在肺、肝、脑组织中有分布,分离毒株NDV-AH1感染组也可以检测到病毒在肺、肝、脑脑组织中有分布。综上所述,试验从临床中收集到的病料,分离鉴定出三株NDV,利用交互免疫试验,说明分离毒株与Lasota毒株抗原性无明显差异。选取一株分离毒株和标准强毒株F48E9同时感染非免疫鸡,进一步用所建立免疫组化方法来检测NDV在机体的分布情况。更多还原

【Abstract】 Newcastle disease(ND) is one of the infectious diseases with high contiguity and truculence;which induces big economic loss of poultry production in our country,and as one of the official reported infectious diseases.listed by Office International desEpizooties. In Anhui province,along with the generally use of vaccine,the cosmically break out of ND has been obviously controlled.However,due to the problem of the quality of vaccine,the use method and the process of immunity,the non-typical ND was induced in a mass of occurrence.Along with the appearance and popularity of enlarge of animal species which are infected and virus variance,the prevention and diagnoses of ND become more difficulty,this is induced high attention to detect and prevent NDV in every walk of life.Firstly,twenty materials with typical and non-typical ND clinical symptom are collected,the brain tissue were sampled.The NDV are isolated and purified by SPF embryo of eggs.The primary identification of allantois of eggs is carded out by HA and HI. The results identify three kinds of NDV,named NDV-AH1,NDV-AH2 and NDV-AH3.Secondly,the NDV-AH1,NDV-AH2 and NDV-AH3 were made for immunogenic antigen,the non-immune chicken are injected vaccine for seven days.The blood is collected after injected for three times,and then serum is separated.The mutual test of HI were done on the same condition between different separated viruses and Lasota which are used for antigen by minimal method,the HI antibody of serum are measured to detect the relativity between different separated viruses and Lasota,and the R value is caculated to compare the difference of their relativity in antigen.The results show that:the R value of three different separated viruses is 0.90 between NDV-AH1 and Lasota,0.95 between NDV-AH2 and Lasota,0.91 between NDV-AH3 and Lasota.The immunity was no obvious differences when the value of R:0.67≤R≤1.5,so the differences between NDV-AH1 and Lasota,NDV-AH2 and Lasota,NDV-AH3 and Lasota were not obvious.It can provide theory of inoculating ND vaccine in these areas.Furthermore,F48E9 virus with strong virulence has built in the test to infect PCEF, and then the pathological changes cells are observed,the pathological changes cells are used as material to establish immunohistochemical method,the best test consistency in conditions of the first antibody and second antibody are built,the results indicate:the first antibody is diluted 10 times,the condition of the reaction is 37℃for one hour,the second antibody which is signed by enzyme is diluted 400 times,the condition of the reaction is 37℃for forty minute,under these conditions,the effect of test reach high-point,the slices of cell are fine dyed,the background is clear.Finally,the non-immune chicken which are infected by F48E9 and NDV-AH1 for seven days are used as material to establish immunohistochemical method,the chicken are killed after infected for three or four days,then the brain,lung,liver,pancreas,duodenum and trachea are collected to observe the distribution of F48E9 and NDV-AH1,to find out the distribution of virus in each tissues when the animal are infected by two kinds of ND. The results indicate:the viruses distribute in lung、liver and brain when infected by F48E9, the virus distribute in lung、liver and brain when infected by NDV-AH1.Above all,three kinds NDV were collected;separated and identified in twenty materials with typical and non-typical ND clinical symptom.The mutual test of immunity indicates that the differences between separate NDV and Lasota aren’t obvious.Chicken which are infected by F48E9 and NDV-AH1 are used as material to establish immunohistochemical method to find out the distribution of virus in each tissues.更多还原