Detection Of Four Immunosuppressive Pathogens In Poultry From Jiangsu Area,Establishment And Application Of Triplex Real-time QPCR For Oncoviruses

The pathogens for immunosuppressive disease not only cause the primary infection or even death of poultry flocks,but also impair the immune system,which leads to the decrease of the immunity and the secondary infection of other pathogens,resulting in decreased production function of the poultry flocks and higher morbidity and mortality.Chicken anemia virus(CIAV),Marek’s disease virus(MDV),Reticuloendotheliosis virus(REV)and Avian leukosis virus(ALV)are four common viruses that cause immunosuppression in poultry,the detected rations of them are high in chicken farms of China and they usually exist in the form of subclinical infection.At present,the epidemiological investigation on these immunosuppressive diseases mainly focus on the area of Guangxi,Anhui,Shandong and Henan,while there is little data on poultry in Jiangsu area.MDV,REV and ALV-J are difficult to diagnose because they can cause similar symptoms in chicken,and the conventional PCR is still used to detect these three pathogens in the lab.Therefore,it is necessary to establish a specific,sensitive,rapid method for clinical diagnosis and detection.In this study,four immunosuppressive pathogens of poultry flocks from Jiangsu area were detected by PCR,and a triplex real-time qPCR was developed for the simultaneous detection of MDV,REV and ALV-J,and the method had been preliminarily applied to the detection of clinical samples.We expected to understand the infection of immunosuppressive pathogens in poultry from Jiangsu area,and provided a rapid,specific and sensitive new technique for the diagnosis of oncovirus in poultry.1 Detection of four immunosuppressive pathogens in poultry from Jiangsu areaThe polymerase chain reaction(PCR)technique for the detection of CIAV,MDV,REV and ALV was developed and used for the epidemiology investigation of 507 chickens,80 ducks,129 geese and 42 pigeons collected from Jiangsu area.It was found that the detected rations of all samples was 66.07%,the detected rations for CIAV,MDV,REV and ALV were 36.09%,25.84%,10.85%and 32.94%,and the detected rations of single infection for these four virus were 13.61%,8.88%,1.18%and 12.03%,respectively.The most common types of co-infection of two virus and three virus were CIAV+ALV(detected ration was 7.69%)and CIAV+MDV+ALV(detected ration was 3.94%),respectively.The detected ration of co-infection of four virus was 0.99%.The test results of chickens at different ages showed that the detected rations for CIAV were declined when chickens older than 6 months.the detected rations for MDV and ALV were higher when chickens among 3 and 6 months;The test results of chickens with different species showed that the detected rations for MDV and REV in layer chickens were significantly higher than that in broilers,grass broilers and three-yellow broilers;The test results from 2016 to 2019 showed that immunosuppressive viruses were still prevalent in different degrees in chickens of Jiangsu Province,and there was difference in detection rate for some pathogens in each year.The comparison of interactions of these four immunosuppressive pathogens by statistical analysis indicated that there was a mutually reinforcing trend between the infection of CIAV and REV,CIAV and ALV(P<0.01).209 samples of waterfowls were negative for CIAV,the detected rations for MDV,REV and ALV were 0.96%,1.44%and 3.35%,respectively.None of the four immunosuppressive pathogens were detected in the samples of pigeons.The data indicate that the infection of immunosuppressive pathogens are prevalent in chickens.The detected rations for these four immunosuppressive pathogens in waterfowls were significantly lower than that in chickens.Pigeon is less likely to be infected with these four immunosuppressive pathogens.2 Establishment of triplex real-time qPCR for detection of oncoviruses in chickensTo develop an assay for simultaneous detection of MDV,REV and ALV-J,a triplex real-time qPCR method was established with three pairs of specific primers targeting the sequences of MDV meq gene,REV gp90 gene and ALV-J env gene,along with 3 kinds of TaqMan probes labeled with different,distinguishable reporter dye for each virus.After optimizing the reaction conditions,the triplex real-time qPCR assay was able to detect MDV,REV and ALV-J simultaneously.Three single real-time qPCR methods were also developed for the detection of MDV,REV and ALV-J.The results showed that three single real-time qPCR methods had same reaction system and conditions,it was easy to operate and rapid to detect.No genomic DNA or cDNA from other virus were amplified in these single and triplex real-time qPCR methods.The detection limits of three single real-time qPCR methods for MDV,REV and ALV-J were 3.23×101 copies/μL,3.23×100 copies/μL and 3.23×101 copies/μL,respectively.The detection limits of triplex real-time qPCR method for MDV,REV and ALV-J were 3.23×100 copies/μL,3.23×100 copies/μL and 3.23×101copies/μL,respectively.The variation coefficient of triplex real-time qPCR were less than 5%.Furthermore,120 samples were tested by the triplex real-time qPCR and conventional PCR,and the coincidence rate of two assays was 94.17%.The results indicated that the established method could provide a rapid,specific,and sensitive new technique for detection and epidemiological investigation of MDV,REV and ALV-J in clinical samples.3 Detection of oncoviruses in chickens by triplex real-time qPCR and identification and analysis of two Marek’s disease virus strainsIn order to further verify the accuracy of the established triplex real-time qPCR technique by clinical practice,the triplex real-time qPCR method was used to detect the sick chickens suspected to be infected with oncovirus from two farms in Jiangsu Province.The virus were isolated and identified at the same time.And then the sequences of meq and pp38 genes of the isolated MDV were compared and analyzed.The results of triplex real-time qPCR showed that the sick chickens from two farms were positive for MDV,and negative for REV and ALV-J.The results of isolation and identification of the virus from two farms showed that two strains of serotype 1 MDV were isolated.The results of nucleotide homology and genetic evolution showed that JS0316 was the closest relative to Jiangsu strain Js201801,while JS0424 was closest relative to the domestic epidemic strains.Comparison of the deduced amino acid sequence of the meq gene of MDV strains showed that the two MDV isolates in this study had no characteristics of the attenuated vaccine strain(A71S,P194-),and had the molecular characteristics of virulent MDV isolates from immune failure chickens in China(D80Y,V115A,T139A,P176R).There was the same mutation in meq gene between JS0316 and Js201801 isolate(Q93R)besides these characteristics of the virulent MDV,which may be the new molecular biological characteristics of MDV Jiangsu isolate.In addition,there were new mutations in meq gene of JS0316(V123A)and JS0424(A/P217T)isolates,and there were new mutations in pp38 gene of JS0316(L981,F240S)and JS0424(W67G,V210A)isolates.The results indicated that the test results of triplex real-time qPCR were consistent with that of isolation of virus,the chickens from two farms were infected by MDV which had molecular characteristics of virulent strain,and the triplex real-time qPCR method could be used for clinical detection.