禽腺病毒4型的分离鉴定及毒种免疫原性研究

发布时间：2018-02-10 13:41

本文关键词： 鸡禽腺病毒病毒分离鉴定免疫原性　出处：《内蒙古农业大学》2017年硕士论文　论文类型：学位论文

【摘要】："心包积液肝炎综合征"(hydropericardium-hepatitis syndrome,HHS)是由禽腺病毒4型引起的一种以鸡的心包积液和包涵体肝炎为特征的疾病。自2015年7月我国多个省相继暴发了该病,且多个品种鸡都易感。本研究应用ELISA检测病鸡血清抗体、PCR扩增病毒的hexon基因、病料接种SPF鸡胚卵黄囊分离病毒、动物回归实验、灭活病毒免疫试验等方法对该病进行临床调查、病毒的分离鉴定、毒株免疫原性研究,结果表明:(1)5个发病鸡场均有禽腺病毒感染,PCR扩增出了 4个大小为2819bp左右的目的基因,4个毒株同源性较高,均属于禽腺病毒4型;(2)从一个鸡场的病料接种鸡胚分离到一株无血凝性的病毒毒株,该毒株回归动物复制出了与该病相同的临床症状,在电镜下观察到肝细胞中有典型的腺病毒粒子。分离到的毒株通过卵黄囊、绒毛尿囊膜和尿囊腔三个途径感染鸡胚,发现通过绒毛尿囊膜感染鸡胚最适合病毒的增值,用该途径分离的毒株ELD50为105.1/0.1mL,该毒株能够使LMH细胞产生明显病变:(3)毒株感染34dSPF鸡第3d开始出现临床症状,4d～6d为死亡高峰期,第7d后病鸡开始恢复,发病率为75%,死亡率为67.86%。鸡感染病毒后第6d开始出现血清抗体,第10d抗体水平达到峰值。发病鸡眼观病变主要出现在肝脏,尤其是感染后第5d病变最严重,病理组织学检查见肝细胞核内有嗜酸性和嗜碱性的包涵体。免疫组织化学染色显示,病毒抗原主要分布在肝细胞、脾脏的网状细胞和肾脏的肾小管上皮细胞中;(4)分离毒株灭活后分0.2mL/只、0.3mL/只、0.5mL/只和1mL/只四个不同剂量免疫21d SPF鸡,32d后攻毒,攻毒后对照鸡80%死亡,免疫组100%存活。结论:调查的5个发病鸡场均感染了 FAdV,用其中一个发病鸡场的病料分离到一株FAdV 4型的强毒力毒株,该毒株灭活后免疫鸡群,对鸡有良好的保护效果。
[Abstract]:Hydropericardium-hepatitis syndrome (HHSS) is a disease caused by avian adenovirus type 4, characterized by pericardial effusion and inclusion body hepatitis in chickens. In this study, the hexon gene of the virus was amplified by ELISA in serum of infected chicken. The virus was isolated from yolk sac of chicken embryo inoculated with SPF, the animal regression test and the immunoassay of inactivated virus were used to investigate the disease. The isolation and identification of the virus and the study of the immunogenicity of the virus strains showed that the four target genes of about 2819 BP in size were amplified by PCR from all the 5 infected chicken farms, and the four strains had high homology. Both belong to Avian adenovirus type 4 (AAV4) A virus strain without hemagglutination was isolated from chicken embryo inoculated in a chicken farm, and the virus returned to animal to replicate the same clinical symptoms as the disease. Typical adenovirus particles were observed in hepatocytes under electron microscope. The virus strains were infected by yolk sac, chorioallantoic membrane and allantoic cavity. The virus strain ELD50 isolated by this route was 105.1 / 0.1 mL. The strain could cause obvious pathological changes in LMH cells. The virus strain infected with LMH strain on the third day of infection began to appear clinical symptoms on the 3rd day. The peak of death was 4 days after infection, and the sick chicken began to recover after 7 days. The morbidity was 75 and the mortality was 67.86.The antibody level of serum began to appear at the 6th day after infection, and the antibody level reached the peak on the 10th day. The pathological changes in the eyes of the affected chicken mainly occurred in the liver, especially on the 5th day after infection. Histopathological examination showed that there were eosinophilic and basophilic inclusion bodies in the nucleus of the liver. Spleen reticular cells and renal tubular epithelial cells were inactivated. After inactivation, the virus strains were divided into 0. 2 mL / 0. 3 mL / L and 1 mL / 4 different doses immunized with different doses for 21 d SPF chickens for 32 days, and then the control chickens died after attack. Conclusion: five infected chicken farms were infected with FAdV. A virulent strain of FAdV 4 was isolated from one of the infected chicken farms. After inactivation of the virus strain, the chickens were immunized with a good protective effect.
【学位授予单位】：内蒙古农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.4

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