Application And Comparison Of Different Detection Methods Of P27 Antigen Of Avian Leukosis Virus In Eradication Program

Avian Leukemia(AL)is one of the most prevalent neoplastic diseases in Chinese flocks,which is caused by Avian Leukosis Virus(ALV).Since the first report of AL in 1908,the outbreak of the disease has been spreading all over the world.In present,there is a widespread infection of ALV in chicken flocks in China,especially in the local breed chickens,a serious threat has been raised to the safety of the local breed resources and huge economic losses have been brought to the poultry industry.Therefore,the monitoring and eradication of ALV is increasingly concerned.At present,the virus isolation or to determine and remove the positive of p27 antigen in different samples are the indispensable measures in eradication program of ALV.In the process of eradication,the core is to select the exactly samples and detection to determine the infection chickens.In this present study,we had conducted on the different detection between IFA performed by the monoclonal antibody against p27 protein and ELISA,the virus isolation and the detection for p27 antigen in samples of cloacal swabs or egg albumen from the same chicken were also compared,to provide technical guidance for ALV eradication.1.Correlation analysis of the p27 antigen of ALV in cloacal swabs or egg albumen with virus isolation in different types of chicken breedsTo investigate the correlation of the p27 antigen of ALV in cloacal swabs or egg albumen with virus isolation,the different types of chicken flocks,including the SPF chickens which were inoculated artificially with ALV-A in different route,the imported ancestral broilers which were completed ALV eradication,and green shells layers,Langya chickens,Xianju chickens,Luhua chickens.They were wingsbanded numbers in order,the samples of cloacal swab and egg albumen were collected according to the number and detected by ELISA kits.The sample of plasma was inoculated into DF1 cells for virus isolation,to observe and compare the ALV-p27 from cloacal swabs or egg albumen with virus isolation.The results showed that the cloacal swab ALV-p27 antigen and virus isolation of SPF chickens artificially infected subgroup ALV-A had higher anastomosis rate.Compared with virus isolation,there was one false-positive and three undetected cases in total 119 samples.The results showed that the samples were negative for p27 by cell culture in total 200 imported broilers by three times.While the positive rate of the cloacal swabs for ALV-p27 antigen were 4.0%(8/200),2.97%(5/168)and 2.61%(4/153),respectively.The average false-positive rate of the cloacal swab ALV-p27 antigen was 3.3%(17/521).Compared with virus isolation,the result of cloacal swab ALV-p27 antigen and virus isolation of local breed chickens of different genetic backgrounds demonstrated the false-positive rate of cloacal swab ALV-p27 antigen was as high as 93.2%~100%,and there was a certain proportion of false-negative rate(1/9,1/4,1/3,respectively).A large amount of data of this study suggested that the detection of cloacal swab ALV-p27 antigen in different types of chickens not only had a high false-positive rate,but also there was a certain proportion undetection rate compared with virus isolation.Considering the cost during the ALV eradication program,it is not recommended to use cloacal swabs for the test material.This study will provide reference for the eradication and clinical diagnosis of avian leukosis virus in china.2.Comparison of sensitivity between ELISA and IFA against p27 antigen in detection of different ALV subgroupsTo evaluate the sensitivity of IFA detection using 4F12 ascites and ELISA detection for p27 antigen,five strains of SDAU14A1,SDAU09C2,733,JS11C1 and JS11C3,which were classified into A,B,J,K subgroup respectively,were performed diluted with 10-fold cell culture solution(10-1~10-8),100 μL of each dilution was taken and inoculated into a line of DF-1 cells in the 96-well plate and maintained after 2 h.Each strain was subjected to 3 repeats,and the cell culture supernatant was detected at 3,6 and 9 d,respectively for p27 antigen with ELISA kits,and the cells were detected by IFA after immobilization.The viral TCID50 of the two methods was calculated with Reed-Muench method.The results showed that the specific green fluorescence signal was observed under the fluorescence microscope when IFA was used to detect the DF-1 cells infected by A,B,J,K subgroup of ALV,there was no fluorescence signal in the control group,however.Compared with the ELISA detection,IFA could detect higher virus dilution and more positive cell wells.The TCID50 were SDAU14A1 strain of 10-4.33 TCID50/0.1 mL and 10-4.80 TCID50/0.1 mL,SDAU09C2 strain of 10-4.50 TCID50/0.1 mL and 10-5.43 TCID50/0.1 m L,733 strain of 10-5.20 TCID50/0.1 m L and 10-5.43 TCID50/0.1 mL,JS11C1 strain of 10-4.80 TCID50/0.1 mL and 10-5.20 TCID50/0.1 mL,JS11C3 strain of 10-4.67 TCID50/0.1 m L and 10-5.33 TCID50/0.1 mL,respectively by the Reed-Muench for ELISA and IFA.It could be seen that the TCID50 of IFA was significantly higher than that of ELISA.This study indicates that the monoclonal antibody 4F12 against p27 protein of ALV can specifically bind to A,B,J,K subgroup of ALV;IFA has a higher sensitivity than ELISA of p27 antigen.In this study,the assessment method established provides more technical reference for the eradication of avian leukosis virus in breeders,particularly for the selection criteria for ALV test kits.