种公鸡精液禽白血病病毒检测方法研究及初步应用

Comparison and Preliminary Application of Detection Methods for Avian Leukosis Virus in Breeder Cocks′ Semen

研究旨在探讨种公鸡精液禽白血病病毒（ALV）检测的可行胜与实用性，为种禽场禽白血病净化材料的选择与方案制定提供参考依据。对绿壳蛋鸡种公鸡精液不同处理和检测方法效果，以及不同精液稀释度对病毒分离效果的影响进行了研究，并将确定的检测方法应用于绿壳蛋鸡、矮脚鸡和芦花鸡3个地方鸡种合计551只种公鸡的检测净化中，同时与血浆病毒分离或血浆斑点杂交检测结果进行比较。结果显示，以血浆病毒分离为标准，精液样品经稀释离心后，取上清接种DF-1细胞，确保在培养基中最终稀释度为1：28-1：56时病毒分离效果最佳。初步应用结果显示，不同鸡种精液p27抗原ELISA检测阳性率均最高，但并不能将血浆和精液病毒分离检测为阳性的鸡均检出，存在“假阴性”；同一鸡种血浆和精液病毒分离阳性率高低不同，在绿壳蛋鸡和矮脚鸡种公鸡中，二者阳性符合率均低于50％，两种方法不可互相取代；芦花种公鸡精液病毒分离阳性率显著高于血浆病毒分离，亦高于血浆斑点杂交，二者阳性符合率分别为100％-36．4％，精液病毒分离效果优于血浆病毒分离。结果表明，对种公鸡精液进行ALV检测具有可行性和必要性，由于不同鸡种净化进程不同、带毒排毒状态不一，检测方法也应灵活运用。

The objective of this study was to evaluate the feasibility and practicality of detection methods for avian leukosis virus （ALV） in breeder cocks＇ semen, and provide the basis for material selection and development of ALV eradication program of breeder farm. 23 semen samples collected form Blue-shell breeder cocks were detected by 6 kinds of methods, the effect of semen dilution was taken into account. Then the selected methods were applied to detect ALV in 551 breeder cocks which were from Blue-shell, Bantam and Barred Plymouth rock breeds. As a result, the positive coincidence rate of isolation of exogenous ALV was the highest when semen samples were centrifuged and diluted at 1：28 to 1：56 in DF-1 cell culture compared with plasma samples. The preliminary application results showed that the positive rates of p27 antigen by ELISA of semen samples from different breeds were all the highest, but could not select out all the positive cocks by isolation of exogenous ALV in semen and plasma samples,there were false negative individuals. The isolation rates of exogenous ALV in plasma and semen samples collected from the same breed were different,their positive coincidence rates were both lower than 50% in Blue-shell and Bantam breeds,the two methods could not replace each other. The isolation rate of exogenous ALV in semen samples from Barred Plymouth rock was significantly higher than that of plasma samples and also higher than the positive rate of exogenous ALV in plasma samples detected by Dot-blot,their positive coincidence rates were 100% and 36.4%. The results indicted that it was feasible and necessary to detect ALV in breeder cocks＇ semen. As eradication process and infection status in different breeds were different,detection methods should be selected flexibly. Key words： avian leukosis; semen; plasma; isolation of virus ; p27 antigen