【作者】 沈志强；

【导师】 汪明；

【作者基本信息】 中国农业大学 ， 预防兽医， 2004， 硕士

【摘要】 正文：本研究从山东流行烈性传染病的鸡群中分离出7株具有血凝活性的病毒，该7株病毒的血凝活性均能被新城疫LaSota株标准阳性血清所抑制，但病毒不能被中和，仍能致死鸡胚。经分离鉴定，分离毒均为新城疫病毒株，通过鸡胚半数致死量(ELD₅₀)、最小致死量致死鸡胚的平均时间（MDT）、1日龄雏鸡脑内致病指数（ICPI）、6周龄雏鸡静脉内致病指数（IVPI）、血凝解脱及血凝素稳定性试验等表明：7株分离毒株均为新城疫病毒强毒株，其毒力与标准强毒株F₄₈E₉株相似。 对4株具有一定代表性的NDV分离株的F基因进行RT-PCR扩增和序列分析，根据基因裂解位点的氨基酸序列推测，其中1株属于弱毒株，3株属于强毒株；核苷酸序列及其推导的氨基酸序列比较结果表明：3株强毒株与Clone30基因核苷酸序列的同源性在83.6％-84.0％之间；与F₄₈E₉典型NDV强毒株同源性在86.5.6％-88.3％之间，推导的氨基酸序列同源性与Clone-30株在85.9％-87.0％之间；与F₄₈E₉典型NDV强毒株在89.1％-91.3％之间；利用MegAlign软件绘制了NDV的系统发育进化树，其结果表明，3株分离强毒株为Ⅶ基因型，弱毒株为基因Ⅱ型。 通过鸡胚接种的方法研究了蜂胶乙醇浸出物（EEP）对NDV Clone-30株的灭活作用，结果表明，0.1-10mg／ml的蜂胶乙醇浸出物（EEP）对NDV Clone-30株的灭活作用，并且这种灭活作用与温度、乙醇浓度成正比，在3-30min内与作用时间无关。 用NDVClone-30株和分离毒株分别接种非免疫鸡胚，制备抗原液，经甲醛灭活后与纯化的天然免疫增强剂蜂胶乳化，研制成功复合新城疫蜂胶灭活疫苗，对制品的安全性和免疫原性进行了测定，对3-6周龄易感雏鸡以2倍使用剂量肌肉注射，安全性良好，对1日龄雏鸡接种1个使用剂量，进行急性毒性试验观测，15天生长曲线表明，对雏鸡无影响，对3-6周龄雏鸡注射1个使用剂量，每批疫苗免疫10只鸡，免疫5-32天鸡血清中ND血凝抑制（HI）抗体的几何平均滴度（GMT）分别为4.8-9.8 log2，对鸡新城疫免疫攻毒试验表明，3批疫苗，每羽份含有的半数保护量(PD₅₀)分别为≥131和≥127。注射部位的动态病理组织学比较观察研究结果表明，蜂胶疫苗免疫后对局部组织学影响明显小于油乳剂苗。蜂胶疫苗的免疫期暂定为6个月；疫苗的保存期为-10℃不结冰，可保存18个月以上，4-8℃为12个月，15-20℃为6个月。田间试验与2亿羽份现场应用结果表明，疫苗安全可靠，效果良好。更多还原

【Abstract】 Content: Seven viral strains which have heagglutination activity were isolated from 7 chicken flocks with high morbidity and mortality contagious disease. These strains were able to agglutinate chicken red blood cell which could be inhibited by Newcastle disease virus LaSota strain standard antisera, but could not be neutralized by the antisera, and were still able to kill chicken embryos. These strains were confirmed to be Newcastle disease virus based on the test of 50% embryo lethal dose, mean death time of chicken embryos, intracerebral pathogenicity index in 1-day-old chicken, intravenous pathogenicity index in 6-week-old chicken, dehaemagglutination and haemagglutinin heat-stability. The results demonstrated that these strains isolated were high virulent Newcastle disease virus, which were virulent as similar as F48E9 strain.Gene fragment with F protein splitting site of 4 typical NDV strains isolated was amplified by RT-PCR and analyzed by sequences. Maybe one strain was low mortality virus and three strains were high mortality virus according to amino acid sequence of splitting site of gene. The result of the sequencing and comparison of sequence demonstrated that the homogeneity of nucleoside sequence among the 3 high mortality virus strains and Clone30 was 83.6%-84.0%, among typical high mortality F48E9 strain was 86.5-88.3%.The homogeneity of amino acid sequence among the 3 high mortality virus strains and Clone30 was 85.9%-87.0%, among typical high mortality F48E9 strain was 89.1-91.3%. The NDV systemic evolution tree was depicted by MegAlign software and showed that the three high mortality viral strains were gene type VII and the low mortality one was gene type II.The inactivation effect of ethanol extract of propolis on Newcastle Disease virus Clone-30 strain was studied through chicken embryo inoculation. The result demonstrated that, 0.1-10mg/ml ethanol extract of propolis could inactivate NDV clone-30 and the inactivation effect directed ratio the temperature inactivated and concentrate of ethanol and had correlation with the reaction time within 3-30min.Egg embryo from hen which hadn’t been immunized were inoculated by NDV Clone-30 strain and virus strain isolated and the antigen was gathered and inactivated with formaldehyde solution and mixed with natural immunity intensifier propolis,so the compositive NDV proplis inactivated vaccine was made successfully. The safety and immunity of vaccines were detected and the result demonstrated that the safety was good when 3-6 week old chicken were immunized through muscle with twice dose; the 15 days growth curve demonstrated the vaccine hadn’t bad effect to chicken when they were immunized with one dose and studied the actuate toxicity test. The 3-6 week old chicken were immunized through muscle with one dose used and 10 chicken every batch vaccine, the GMT of HI antibodies of ND in the 5-32 days old chicken sera were 4.8 log2 and 9.81og2,respectively.The result of ND immunity and challenge to chicken demonstrated that every dose of three batches vaccine contain PD50 more than or equal to 131 and 127,respectively. The result of dynamic pathology tissue comparison of muscle injected demonstrated that the effect on the injection tissue of propolis vaccines was apparently less than that of oil vaccines. The immunity period of proplis vaccine was temporary as 6 months, the stored period of this vaccine was more than 18 months in -10℃ and didn’t ice up in this temperature, 12 months in 4-8 ℃ and 6 months in 15- 20℃. The result of field test and the effect of immunity 2 billions fowls demonstrated that the vaccine was safe and reliable, meanwhile had good immunity effect.更多还原