【作者】 张训海；

【导师】 陈溥言；

【作者基本信息】 南京农业大学 ， 预防兽医学， 2003， 博士

【摘要】 通过对商用CVI988疫苗细胞培养上清液浓缩与检测，鉴别出了其分泌性抗原成分，且琼扩滴度可达到1：4，而用该疫苗分别免疫SPF鸡和罗曼鸡后，在免疫后第3～8周内均未检测到特异性羽囊病毒沉淀抗原，但该羽囊浸提液经15～20倍浓缩后再测，则在免疫后第3～4周。SPF鸡和罗曼鸡都呈阳性，其后则迅速减小。在对商品CVI988疫苗和HVT(Fc-126)疫苗免疫及其免疫强毒感染的SPF鸡，分别进行羽囊病毒沉淀抗原和血清病毒沉淀抗体的检测。结果显示，单纯免疫鸡群均呈阴性；CVI988免疫接毒后的第一周即可检出羽囊病毒沉淀抗原，较HVT免疫接毒组早2周；而循环沉淀抗体的出现，却较HVT免疫接毒组迟2周，与同期攻毒对照组沉淀抗体的检出时间相当。此外，接毒后的第7～18d，尤其是9～18d，在接毒对照组的血清中发现有MDV沉淀抗原。我们对上述的检出现象分别进行了初步分析和较合理的解释。应用该MDV抗原和抗体同步琼扩检测法，对以安徽省为主的29个的县市部分饲养鸡群随机进行了MD污染状况的调查。结果表明，在调查的8个品种(系)、日龄幅度为31～434d的93个不同免疫状况饲养群中，呈检测阳性的为76个群，总阳性率为81.7％(76／93)。在有效检测报告为阳性的1386只(总计2071)鸡中，则MDV琼扩抗原和抗体的检出阳性率分别为34.1％(472／1386)和64.7％(866／1386)，剔除其中的抗原和抗体阳性重叠部分(＞115只)，MD污染鸡群琼扩法总检出阳性率达88.3％(1223／1386)。此结果与已报道的MDV分子生物学检测方法在其它地区的调查结果有较好的符合率。 由此并综合我们以往的研究结果，进而得出以下结论：疫苗CVI988和HVT的免疫鸡，虽然都含有不能为常规AGP法直接检测到的阶段性且低水平的羽囊MDV抗原，但并不影响羽囊病毒抗原经典AGP法对鸡群MDV强毒感染的特异性诊断；单纯疫苗CVI988和HVT的免疫抗体可能是低水平的，故亦不能被敏感性较低的经典AGP法所检出；在感染鸡的羽毛囊中所发现的MDV抗体，且其消长与血液中的病毒抗体消长呈正相关；而与羽囊病毒抗原消长呈负相关。MDV琼扩抗体与琼扩抗原一样，都是鸡只MDV强毒感染的特异性指征；该抗原和抗体的同步琼扩检测法对MDV强毒污染的检测，不仅简便、快捷、特异，而且重复性好、准确率高，可作为MDV强毒感染诊断的标准技术与方法。更多还原

【Abstract】 Soluble antigen of Marek’s disease virus(MDV) was concentrated about 100 folds with50% saturated ammonium sulfate cell culture fluids. The antigen were identified for thepresence of MDV vaccine CVI988/Rispens strain examined by agar-gel precipitation (AGP)test and its titre to 1:4. Some specific pathogen free(SPF) and commercial Lohmann chicksvaccinated respectively, with the commercial vaccine CVI988/Rispens and the viral antigen ofthe feather tips were always negative at 3 to 8 weeks post-vaccination(PV) by AGP test.However, when 10 to 30 feather-tips were pooled and cut into pieces in 2ml phosphatebuffered saline(PBS), the suspension after incubation was centrifuged and the buffer decreasedto about 1/15~ 1/20 times in volume was detected by AGP for viral antigens.lt showed that theviral antigens in the feather was recovered and always positive at 3 to 4 weeks PV, whereas itwas positive partly for the antigen at 5 or 6 week and none at 6 to 8 or 7 to 8 weeks PV in theSPF and Lohmann chicks, respectively. Both the feather tips and serum, obtained fromchickens vaccinated with the commercial vaccine CVI988/Rispens or turkeyherpesvirus(HVT), its chickens vaccinated infected and unvaccinated infected with a virulentMDV strain Beijing-1(BJ-1) at 14 days of age, were detected by agar-gel precipitation (AGP)test for the viral antigen and either viral antigen and/or viral antibody. Results showed thegroups of birds vaccinated alone were rarely positive for the presence either precipitatingantigen or precipitating antibody at 3 to 8 weeks of age. The precipitating antigens wereidentified at 1 week post-infected(PI) in the feather tip of birds CVI988-vaccinated infectedand were about 2 weeks earlier than the presence of HVT-vaccinated infected. However, theprecipitating antibodies in the serum of birds CVI988-vaccinated infected were identified later2 weeks than that of HVT-vaccinated infected, similar to birds infected alone with BJ-1 at thesame time. In addition, the precipitating antigens could be requently found in the serum ofbirds infected alone with BJ-1 range about 9~18 days PI. The reasons of all above result andapplications are discussed in our article. The two immunoassay was developed to identifychickens infected with pathogenic MDV. The assay for Marek’s disease (MD) of chicken wascarried out in 29 counties ( or cities ) distributed mainly in Anhui Province. 93 flocks ofopening or half-opening breeding chicken were surveyed, including 8 varieties of breeding fordifferent purpose and scope of age from 31 to 434 days with a total number of 2071 chickensunimmunized or immunized with different serotye vaccines. The results showed that 76(81.7%) of 93 flocks tested were positive for MDV-infection. Viral antigen positive rates and viral antibody positive rates were 24.2%(472/1947, cut out 124 not tested) and 42.9%(879/2071), while in the positive flocks of MDV-infection tested, the viral antigen positive rates and viral antibody positive rates were 34.1%(472/1386) and 64.7%(866/1386), respectively. However, the total identified positive rates of MDV-infection were increased significantly to 88.3%(1223/1386, removed over 115 overlapping identification) by the AGP test using both the precipitating antigen and the precipitating antibodies. The results obtained in this survey are in general agreement with the results of survey obtained in Guangxi Province by Wang guijun et al.detected using a PCR technique who found that MD was 84.91% positive of the flocks and 73.33% positive of the birds.Our results above and involved published suggest confirmly that the viral antigens could be found at low levels in feather tips of chickens vaccinated with either the CVI988/Rispens vaccine or HVT(Fc-126) vaccine, but couldn’ t be covered directly by the conventional AGP test. The precipitating antibodies as the same as the precipitating antigens was specific for specific diagnosis of virulent MDV-infection of chickens. And vaccination of chicks with ei更多还原