Ⅰ型鸭肝炎病毒的分离鉴定及其高免卵黄抗体的制备  

Isolation and identification of type I Duck hepatitis A virus and preparation of high-titer yolk antibody

作　　者：李娟 王岩 刘秋晨 于洪敏 徐福利 单春乔 刘艳 LI Juan;WANG Yan;LIU Quchen;YU Hongmin;XU Fuli;SHAN Chunqiao;LIU Yan(Dalian Sanyi Animal Medicine Co.,Ltd.,Dalian 116036,China;Jiangsu Sanyi Biological Engineering Co.,Ltd.,Pizhou 221300,China)

机构地区：[1]大连三仪动物药品有限公司,辽宁大连116036 [2]江苏三仪生物工程有限公司,江苏邛州221300

出　　处：《黑龙江畜牧兽医》2023年第18期72-77,136,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：辽宁省“兴辽英才计划”项目(XLYC1803014)。

摘　　要：为了确定山东某养殖场疑似患有鸭病毒性肝炎(duck viral hepatitis,DVH)病死鸭的病原,并针对该病原制备高效且安全的卵黄抗体(antibody of yolk,IgY),试验无菌采集病死鸭肝脏组织样品,通过细菌检测、病毒的分离与增殖、特异性基因的RT-PCR鉴定与序列分析及动物回归试验确定病原,采用Reed-Muench法计算半数致死量(LD_(50)),并针对病原制备油乳剂灭活苗,按照免疫程序将其免疫产蛋鸡,收集免疫鸡的鸡蛋,采用生理盐水法提取IgY,测定IgY的中和效价,最后进行动物被动免疫试验,探究IgY对攻毒雏鸭的免疫保护作用。结果表明:将病死鸭肝脏组织划线接种于营养琼脂培养基、麦康凯琼脂培养基,培养后均无细菌生长,病死鸭肝脏组织和增殖尿囊液均扩增出Ⅰ型鸭肝炎病毒(DHV-Ⅰ)特异性基因片段,目的基因与GenBank中的9株DHV-Ⅰ参考株的相应基因序列的相似性为99.1%~99.2%,确定分离出1株DHV-Ⅰ,命名为SD,分离株SD对9日龄鸭胚的LD_(50)为1×10^(-8.25)/0.2 mL。将分离株重新接种健康雏鸭,出现DVH典型的症状,死亡率为100%,提取死亡雏鸭肝脏组织中的病毒RNA,经RT-PCR后能扩增出目的条带,测序结果与分离株SD一致。制备的油乳剂灭活苗稳定性良好,30 min内不分层,雏鸭注射后14 d内生长无异常。制备的IgY抗体中和效价为1∶256,注射IgY后第1~5天攻毒,10 d后的保护率为87.5%~100%,注射IgY后第7天攻毒,10 d后的保护率为75%。说明从发病鸭群肝脏组织中分离出1株高致病性的DHV-Ⅰ,制备的油乳剂灭活苗免疫蛋鸡后获得高效价IgY,对攻毒雏鸭具有很好的免疫保护作用。In order to determine the causative agent of suspected duck viral hepatitis(DVH)infection in ducks in a breeding farm in Shandong Province,and to prepare an efficient and safe antibody of yolk(IgY)against the agent,in this experiment,the liver tissue samples of dead ducks were collected aseptically;the pathogen was determined by bacterial detection,virus isolation and proliferation,RT-PCR identification and sequence analysis of specific genes,and animal regression test.The median lethal dose(LDso)was calculated by Reed-Muench method,and the oil emulsion vaccine was prepared against the pathogen,which was immunized to priced laying hens according to immunization procedures.The immunized eggs were collected;IgY was extracted with normal saline method to determine the neutralization titer of IgY.Finally,passive immunization test was conducted to explore the immune protection effects of IgY on virus-challenged ducklings.The results showed that the liver tissue of dead ducks was inoculated with streaks on nutrient AGAR medium and MacConkey AGAR medium;no bacteria grew after culture.The specific gene segment of DHV-I was amplified from liver tissue and proliferating alllic fluid of dead ducks.The similarity between the target gene and the corresponding gene sequences of 9 DHV-I reference strains in GenBank was 99.1%-99.2%.A strain of DHV-I was isolated and named SD,whose LD_(50) was 1×10^(-8.25)/0.2 mL to the 9-day-old duck embryo.The strain was immunized to healthy ducklings again and the typical symptoms of duck viral hepatitis appeared,with a mortality rate of 100%.The virus RNA was extracted from the liver tissue of the dead ducklings,and the target band was amplified by RT-PCR.The sequencing results were consistent with SD.The prepared oil emulsion vaccine had good stability with no stratification within 30 min.No abnormal growth of ducklings appeared within 14 days after injection.The neutralization titer of the prepared IgY antibody was 1:256.Challenge was carried out 1-5 days post IgY injection,and the protect

关 键 词：Ⅰ型鸭肝炎病毒(DHV-Ⅰ) 分离 鉴定 半数致死量(LD50) 灭活疫苗 卵黄抗体(IgY) 被动免疫 

分 类 号：S834*.89[农业科学—畜牧学]