【作者】 王泽霖； 张建武； 罗新霞； 黄娟； 刘志军；

【Author】 Wang Zelin Zhang Jianwu Luo Xinxia Huang Juan Liu Zhijun (Avian disease reseach institute,Henan agricultural university ZhengZhou 450002)

【机构】 河南农业大学禽病研究所；

【摘要】 应用(NH4)2SO4盐析法从A型魏氏梭菌培养滤液中粗提卵磷酯酶(PLC)(蛋白含量为2.798mg/ml).与等体积的M41或宜毒株的新鲜尿囊液混合,在37℃水浴作用2小时并不断振荡来制备HI抗原。在HI试验中.该抗原不与AIV H5,AIV H9.NDV.EDS-76,IBD单因子血清和SPF鸡血清.小牛血清、兔血清反应.仅与IBV阳性血清反应.有高度的特异性。通过对25份非免疫鸡血清抗体的测定与攻毒试验.确定了免疫临界线为4log2.HI滴度≥4log2,相当于ELIsA P/N值大于2.1.中和效价大于1:20.攻毒保护5/5;HI滴度<4log2.相当于ELISA P/N值小于2.1.中和效价小于1:20.攻毒保护3/9。HI试验与中和试验,ELISA试验有很高的相关性。用建立的HI试验进行了IBV的分型研究,通过与15个不同毒株间的交叉HI试验.表明不同毒株间有广泛交叉,可将10个毒株分为三大群.其中M类呼吸型仍占主要地位(50%).宜毒株类也占一定比例(30%).因此可选用M41+宜或M41+当地毒株来制备IB疫苗。IBV HI试验具有型特异性。初次免疫应用疫苗株同型毒株做抗原.再次免疫交叉反应增大,可采用混合毒株做抗原。用HI试验进行IB的血清流行病学调查,出壳雏鸡母源抗体在5-10天后降至临界值4log2水平。25日龄初免鸡.IB抗体在免疫后21天达到峰值6log2,维持一个月后降至临界值4log2;70日龄进行二免的青年鸡,IB抗体21天后升至峰值10log2,至实验结束时维持3个月无明显下降;成年鸡随鸡龄的增大而抗体滴度升高,维持时间则更长。实践表明用自制IBV HI抗原建立的HI试验简便、快速、准确,可为基层提供一种用于IBV的诊断.免疫效果检测和血清流行病学调查研究的新方法,还可用于IBV分型、疫苗株的选择、免疫程序的制订。更多还原

【Abstract】 Crude phospholipase C(PLC. protein density was 2. 798mg/ml) was extracted from the filtered cultivating liquid of clostricdium perfringens A using (NH4)2SO4. IBV HI antigen was prepared by mixing rude extract PLC with equal volume of fresh AAF of M41 and Y strains by incubating under 37 C for two hours with continuing shaking. This antigen did not react with sera of AIV H5.AIV H9.NDV. EDS -76,SPF chicken, habbit and cow. but only react with IBV serum .indicating a indicate high specifity.After the detection of 25 negative serum and challenge test threashold of HI litre was defined as 4log2. HI titre abover 4log2 is equal to ELISA(P/N) above 2. 1,1:20 of neutralization tilre and 10/11 of challege lesl(P/C); HI titre below 4log2 is equivalent to P/N of ELISA below 2. 1 ,neutualizalion lilre below 1:20, 3/9 of challege test (P/C) 3/9. These resulls were obviously inlerrelaled . Serotype was classified by established HI test. Eleven IBV isolates can be divided into three groups. Among which, serotype M41is dominant (50%); Y strain has a certain proportion(30%). We can select M+Y or M +field isolate strain to produce IB vaccine. IBV HI test has high specifity. We should select the same serotype with vaccine strain to produce antigen in primary vaccination but select the mixed serotype strains to produce antigen in the second vaccination. Epidemic investigation of IB serum is done by IBV HI test. Maternal antibody would drop to boundary line(4log2) between 5 to 10 days posthatchery. The HI titre of different age adult hens were low (41og2)before vaccination and raised to 8-12log2. HI titre of 25-day -old chickens reached the peak at 21s day poslvaccinalion and mailained one month and dropped to the boundary line. HI titre of 70-day vaccinated hen also reached the peak 21days poslvaccinalion . HI titre of adult hen will enhance with their age and mailains longer. Praclice proved that IBV HI test was simple, quick, accurate and fit for field application.It can be used in diagnosis,detecling the effect of vaccinalion and epidemic investigalion of IB . It can also be used in classificalion of IBV,selection of vaccine strains and formulalion of vaccinalion program.更多还原