Molecular Etiology Detection Of Subgroup J Avian Leukosis Virus In Wild Birds And Poultry In Live Poultry Market In 2016 In Jiangsu Yancheng

Avian Leukosis(AL)groups refer to avian infectious benign and malignant tumors,caused by avian leukosis virus(Avian Leukosis Virus,ALV).ALV can cause a variety of clinical and pathological changes,lead to performance descends and immunosuppression.Avian leukosis can be divided into endogenous avian leukosis(ALV-E)and exogenous avian leukosis.According to the genome structure,host range,serum neutralization and interference experiments,exogenous avian leukosis were divided into five subgroups(A,B,C,D,and J).The common subgroups in clinical practice are subgroup A and J.The ALV-J subgroup appeared in the 20 th century,and then spread and broke out worldwide.It caused great economic losses and attracted people's attention.Now there are many reports about the domestic epidemiology of avian leukosis,and the reports mainly concentrated on the poultry.There are less reports about the wild birds infected with avian leukosis.The natural host of avian leukosis virus is less,in which chicken is the main natural host of the disease.In order to understand the situation of avian leukosis virus in wild birds and live poultry market and the difference of infection among different animals,we selected Yancheng Wetlands and the live poultry market along the East Asia-Australia Flyway as the research bases.We investigated the epidemic situation of subgroup J avian leukosis virus in wild birds and live poultry market in Yancheng of Jiangsu province.In this study,we collected the fecal samples of wild birds and poultry markets in Yancheng,Jiangsu,China twice from 2015 to 2016.A total of 2427 samples were collected,of which,1091 fecal samples were from live poultry market,and 1336 fecal samples were from wild bird.The molecular pathogenicity and gene sequence analysis of subgroup J avian leukemia were performed.We isolated RNA of the fecal samples,and detected ALV-J by Real-Time PCR.Thepositive samples were amplified the gp85 gene fragment by RT-PCR,cloned and analyzed genen sequence.The RT-PCR positive fecal samples and some of the negative fecal samples were inoculated into 9-day-old SPF chicken embryos and CEF for virus proliferation.CEF was passed 3 times continuously,after 10 days,the cells and supernatant were harvested.Chick embryos were inoculated by allantoic cavity and passaged 3 times,chicken intestine and allantoic fluid were harvested.The DNA of cells and chicken embryo were extracted,and detected by regular PCR.The supernatant of the cells was detected P27 antigen by ELISA.The results of real-time PCR showed that there were 22 positive samples of 1091 samples in live poultry market,and the positive rate was 2.02%.There were 3 positive samples among the 1336 wild bird samples,and the positive rate was 0.22%.In the positive samples detected by real-time PCR,the results of RT-PCR showed that there were three positive samples in live birds and no positive samples in wild birds.Three924 bp ALV-J gp85 gene fragments in live poultry market samples(native chicken)were obtained by cloning sequencing.The homology of the three gene sequences was 93.7%-94.7%,and the homology between the three gene sequences and the thirteen ALV reference strains at home and abroad was 87.7%-95.7%.The results of regular PCR showed that the three ALV-J positive samples in live poultry market were positive by both real-time PCR and RT-PCR.The homology of the two positive samples was 98.3% and the homology between the two positive samples and international reference strain HPRS-103 was 97.8% and 98.1%by cloning sequencing.The supernatant of cells was negative by ELISA.In this study,the detection rate of ALV-J by real-time PCR was higher than the regular PCR amplification,and the results of regular PCR amplification showed that the detection accuracy of ALV-J was high.We investigated the prevalence of ALV-J in live birds market and wild birds in Yancheng,Jiangsu Province in 2016,The results indicated that ALV-J infection was present in avian market birds.However,ALV-J infection was not detected in wild birds by multiple retest,and that can't exclude the absence of ALV-J infection in wild birds.We investigated the the epidemic situation of ALV-J along East Asia-Australia wild bird migration route,which provided important monitoring data for ALV prevention,and provided some reference meaning for ALV prevention and control in Yancheng area.