Effects of different samples on avian leukosis virus eradication and detection in yellow feather breeders
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摘要:目的
评估黄羽种鸡禽白血病的净化工作，比较源于同一鸡群不同样品对禽白血病病毒(ALV)检测结果的影响.
方法采用ALV抗原ELISA方法对广东一黄羽种鸡场采集的2 691枚种蛋进行检测，根据检测结果采集其中70份不同S/P区间所对应的母鸡抗凝血，分离血浆后分别同时接种于CEF和DF-1细胞，用病毒分离方法进行检测，并用PCR方法进行亚群鉴定.
结果和结论从送检种蛋蛋清共检出ALV p27阳性样品243份，阳性率为9.03%（243/2 691）；蛋清不同ELISA S/P区间所对应的病毒分离情况分别为：蛋清ELISA S/P ≥2.0的鸡只其血样CEF和DF-1细胞病毒分离率均为100%；1.5≤S/P < 2.0的鸡只病毒分离率均为88.9%；1.0≤S/P < 1.5的鸡只病毒分离率均为85.7%；0.2≤S/P < 1.0的鸡只病毒分离率分别为60%~75%(CEF)和40%~50%(DF-1)；0.1≤S/P < 0.2的鸡只病毒分离率为62.5%(CEF)和50%(DF-1)；S/P < 0.1的鸡只分别为27.2%(CEF)和9.1% (DF-1)的病毒分离率.PCR结果显示，所有7份CEF+DF-1-的CEF培养物中有6份为内源性ALV-E.研究表明，在蛋清ELISA检测时，S/P越高的阳性蛋清样品其对应母鸡越可能是外源性ALV病毒血症阳性；有一定比例S/P较低的阳性蛋清样品是由对应母鸡体内的内源性ALV排毒所致；而净化初期少数蛋清S/P为阴性的蛋清，其对应母鸡仍可能检出外源性ALV，说明在黄羽种鸡外源性ALV净化方案中单独使用1次蛋清ELISA检测可能会给禽白血病的净化检测造成一定的“误诊”和“漏检”.
Abstract:ObjectiveThe avian leukosis (AL) eradication protocol in yellow feather breeders was evaluated and the effects of different albumen samples from the same breeder chickens on the results of avian leukosis virus (ALV) detection were compared.
MethodA total of 2 691 albumen samples collected from a yellow feather breeders of Guangdong Province were tested using ALV p27 antigen ELISA.Seventy plasma samples were collected from corresponding hens based on the different S/P intervals results for virus isolation in CEF and DF-1 cells respectively, and the results were analyzed.PCR was used to identify subgroup.
Result and conclusionA total of 243 positive samples were detected in this local breeder flock, and the ALV p27 positive rate was 9.03%(243/2 691).The virus isolation ratios (VIR) at different S/P intervals were found as follows: both 100% VIR in CEF and DF-1 cells of hens' plasma samples with albumen S/P ≥2.0; both 88.9%VIR with 1.5≤S/P < 2.0; both 85.7% VIR with 1.0≤S/P < 1.5; 60%-75%(CEF)and 40%-50%(DF-1) with 0.2≤S/P < 1.0;62.5%(CEF)and 50% (DF-1) with 0.1≤S/P < 0.2; 27.2%(CEF)and 9.1% (DF-1) with S/P < 0.1. The PCR detection showed that six of seven CEF+DF-1- proviral DNA were positive with endogenous ALV-E and one positive with ALV-J.The results indicated that in albumen ELISA detection, the higher S/P value of positive albumen samples, the more likely exogenous ALV viremia positive in their corresponding hens. The corresponding hens of low S/P value of positive albumen samples were caused by a certain proportion of endogenous virus; it is noteworthy that exogenous ALV can still be isolated from some ELISA negative chickens at the beginning of eradication. This study suggests some "misdiagnosed" and "missed" chickens may be caused by using only one method of albumen p27 antigen ELISA.
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Keywords:
- yellow feather breeder /
- avian leukosis virus /
- virus detection /
- eradication
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图 1 CEF和DF-1细胞病毒分离相关性分析
Figure 1. Correlation analysis of virus isolation in CEF and DF-1 cells
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图 2 CEF细胞培养物的ALV PCR检测
M：DNA marker DL2000（从上到下依次为：2 000，1 000，750，500，250，100 bp）；1 ~ 8：检测样品；+：阳性对照；-：阴性对照.
Figure 2. ALV PCR detection of CEF cell cultures
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图 3 DF-1细胞培养物的ALV PCR检测
M：DNA marker DL2000（从上到下依次为：2 000，1 000，750，500，250，100 bp）；9 ~ 21：检测样品；+：阳性对照；-：阴性对照.
Figure 3. ALV PCR detection of DF-1 cell cultures
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图 4 gp85基因的PCR扩增
M：DNA marker DL2000；1 ~ 3：GD1401J、GD1402J、GD1403J.
Figure 4. PCR amplification of the gp85 gene
表 1 血浆样品细胞培养物的ALV抗原ELISA检测结果1）
Table 1 The results of ALV antigen-ELISA test of plasma cell cultures
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