Development And Application Of A Method For The Detection Of FadV And FadV Contamination In Attenuated Vaccines

Fowl adenovirus（FAdV）is a DNA virus belonging to the family Adenoviridae and adenoviruses.Group I fowl adenovirus can be divided 5 species with 12 serotype（FAdV1-8a,8b-FAdV11）based on a common group antigen.Fowl adenovirus serotype 4 is commonly known as"Ankara"disease,which can cause severe pericardial effusion hepatitis syndrome and present a worldwide distribution.Since July 2015,IBH-HPS caused by FAdV has been occurred in many chicken flocks of different provinces in China,such as Henan,Hebei,Liaoning,Jilin,Heilongjiang,Xinjiang,Anhui,Shandong,Jiangxi,Hubei,Jiangsu and other provinces,which caused huge economic loss in poultry farming.Many live poultry attenuated vaccines have been confirmed to be contaminated with exogenous viruses such as ALV and REV.Pakistan has also reported that the large-scale spread of fowl adenovirus may be caused by potential virus contamination in the attenuated vaccine.In 2016,the avian adenovirus type-4 was isolated and identified from the live attenuated vaccine of Newcastle disease by PCR assay in China.With the rapid development of molecular biology techniques,virus detection methods are constantly being updated.Exogenous virus contamination in vaccines is usually low-dose,which puts higher requirements for the detection of fowl adenovirus type I in vaccines.Therefore,in this study,fowl adenovirus dot blot hybridization,TaqMan quantitative PCR,and drop digital PCR assays were established and provide rapid detection techniques for fowl adenovirus-infected flocks.At the same time,it will help vaccine companies to quickly and accurately detect the infection of poultry adenovirus 4 in live attenuated avian attenuated vaccines,which is of great significance in promoting the healthy development of the poultry industry.First,the FAdV-N22 strain contaminated with avian adenovirus type 4 vaccine was inoculated to chicken embryos,the virus titers were determined as 50%egg infectious doses（EID50）using the Reed-muench method.Based on the sequence of Hexon gene from FAdV-N22 isolate published on GenBank,the sequence was analyzed using Lasergene 7.0software and the conserved sequence was edited.Two pairs of primers including Dot-F1/Dot were synthesized using Primer 5.0 software.The-R1 amplification primers were used to prepare labeled probes,Hexon-F2/Hexon-R2 primers,and Taqman probes.The DNA fragments was amplified using Dot primers by polymerase chain reaction（PCR）,labeling this PCR recovered product with Digoxigenin as a probe to establish a dot blot hybridization method.Hexon-F2/R2 primers and Taqman probes were used to establish Taqman quantitative PCR and drop digital PCR.The reaction conditions for the three methods were optimized for sensitivity,specificity,and repeatability tests.The results showed that the method of PCR combined with dot blotting can detect the minimum concentration of the plasmid standard as 1,000 copies/μL.The Taqman fluorescence quantitative PCR method can detect the lowest concentration of the plasmid standard as 100 copies/μL.Droplet digital PCR can detect the lowest concentration of the plasmid standard as 10 copies/μL.The avian adenovirus contamination in live vaccine was artificially simulated experiment in this study,Conventional PCR can only detect the contamination of 100 EID₅₀/1,000 dosage adenoviruses in the vaccine.PCR and dot blot hybridization can detect the contamination of5 EID₅₀/1000 dosages in the vaccine.The qPCR method could detect FAdV contamination at a dose of 1 EID₅₀/1,000 while ddPCR method could detect FAdV contamination at a dose of0.1 EID₅₀/1,000,which was 1,000-fold more sensitive than the regular PCR detection(100 EID₅₀/1,000).In summary,the three molecular methods for detection of fowl adenovirus established in this study can quickly and effectively detect low-dose FAdV contamination in attenuated vaccines,and micro-droplet digital PCR can be used for low-dose contamination of attenuated vaccines,this assay shows the more sensitivity compare to other two assays.In conclusion,the three molecular detection methods of avian adenovirus established can effectively test the low dose of avian adenovirus in the attenuated vaccine.TaqMan qPCR and ddPCR can detect the fowl adenovirus type,and provide a new quantitative detection method for clinical ddPCR,which can more accurately monitor the changes in viral load,and further reveal the relationship between the low-dose contamination of attenuated vaccines and the occurrence of adenovirus and outbreak of the viral epidemics.