荧光定量PCR方法在鸡马立克氏病疫苗免疫监测中的应用分析

Application analysis of fluorescence quantitative PCR in monitoring immunization of MD vaccine

鸡马立克氏病(MD)的防控主要依靠疫苗免疫,MD疫苗株在鸡体内的有效复制与最终的免疫效果直接相关,可通过这种相关性对MD疫苗进行免疫监测。为有效监测MD疫苗的免疫效果,本研究共采用两种血清1型MD疫苗免疫不同的鸡:(1)实验鸡分为两组:一组在1日龄时免疫血清1型MD疫苗814株,另一组为不免疫的对照组,选用双重荧光定量PCR方法分别对两组鸡免疫后7 d和14 d的脾脏和羽髓中的马立克氏病病毒(MDV)载量(log10 MDV拷贝数/106个细胞)进行检测;(2)商品鸡:选用上述双重荧光定量PCR方法分别对经血清1型MD疫苗CVI988/Rispens株免疫后7 d和14 d的商品蛋鸡脾脏和羽髓中的MDV载量进行检测。并针对荧光定量PCR的检测结果结合常规PCR检测和meq基因的序列比对分析各组鸡脾脏和羽髓样品是否被野毒感染;同时监测正常免疫商品鸡的产蛋率和死淘率等至8个月鸡龄。结果显示,实验鸡免疫后7 d,免疫组鸡脾脏和羽髓的MDV载量为3.39~4.69,对照组鸡相应样品的MDV载量为3.36~4.90;免疫后14 d,免疫组鸡MDV载量为3.77~4.86,对照组鸡相应样品的MDV载量为5.06~6.22,对照组显著高于免疫组(p<0.01)。PCR鉴定和meq基因序列分析证实,对照组鸡被MDV野毒感染,导致其体内MDV载量的显著上升。商品鸡免疫后7 d,脾脏和羽髓的MDV载量为3.75~4.59;免疫后14 d,MDV载量为3.71~4.92,并且在监测期内没有发生MD。以上结果表明,利用双重荧光定量PCR方法对血清1型MD疫苗进行免疫监测时,免疫后7 d~14 d鸡体内的病毒载量为3.39~4.86,载量过高则可能发生了免疫失败或者野毒感染。本研究结果表明双重荧光定量PCR方法可以用于MD疫苗免疫效果的监测,为临床鸡MD疫苗的免疫监测提供了参考数据。。

The prevention and control of chicken Marek’s disease(MD) mainly rely on vaccine immunization. The effective replication of the MD vaccine strain in chickens is directly related to the final immune effect, and the immune effect of MD vaccine can be monitored through this correlation. In order to effectively detect the immune effect of the MD vaccine, two serotype1 MD vaccines were used in this study to immunize different chickens as follows:(1) Experimental chickens: one group is immunized with serotype 1 MD vaccine 814 strains at 1 day-old, and the unimmunized group was as the control group. MDV load(log10 MDV copies/106 cells) in spleen and feather pulp of chickens of the two groups were detected by using the double fluorescent quantitative PCR at 7 and 14 days post vaccination(p.v.).(2) Commercial chickens: MDV load in the spleen and feather pulp of commercial chickens immunized with the serotype 1 MD vaccine CVI988/Rispens strain was detected by double fluorescent quantitative PCR at 7 and 14 days p.v.. According to the detection results by using fluorescence quantitative PCR, in combination with conventional PCR detection and sequence analysis of meq gene, wild virus infection was identified in each group. At the same time, the laying rate and death rate of normal immunized commercial chickens were monitored until 8 months old. The results showed that the MDV load of spleen and feather pulp in the immunized group and the control group was 3.39-4.69 and 3.36-4.90 at7 days p.v., respectively. At 14 days p.v., the MDV load in immunized group was 3.77-4.86, and the MDV load in the control group was 5.06-6.22, which was significantly higher than that of the immunized group(p<0.01). PCR identification and sequence analysis of meq gene confirmed that chickens in the control group were infected with the wild MDV, which resulted in a significant increase in MDV load. The MDV load of spleen and feather pulp in normal immunized chickens was 3.75-4.59 and 3.71-4.92 at 7 days and14 days p.v., respectively. Besides, there was no MD occurred during the monitoring period. The above results showed that the viral load in chickens between 7 days and 14 days p.v. was 3.39-4.86 by using dual fluorescence quantitative PCR to monitor serotype 1 MD vaccines. If the MDV load in chickens was too high, immune failure may occur, and chickens may be infected by wild viruses.This study shows that the dual fluorescence quantitative PCR can be effectively used for monitoring the immune effect of MD vaccines, and these results provides a reference data for monitoring the immune effect of MD vaccines in clinical flocks.