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

Chicken infectious anemia(CIV)is caused by the chicken infectious anemia virus(CIAV)that leads to the aplastic anemia in chicks,which is characterized as immunosuppressive infectious diseases with systemic lymphoid atrophy.Fowl adenovirus(FAdV)can produce diseases such as inclusion body hepatitis(IBH),pericardial effusion syndrome(HPS)and gizzard erosion(GE)in chickens.Both CIAV and FAdV can cause the chickens to be immunosuppressed,increasing the susceptibility of chicken flocks to other pathogens,and reduce the vaccine protection efficacy against MDV,IBDV,REV,leading to increased high mortality rates for chickens in other infections,such as co-infection or secondary infection,which causes greatest economic losses to broiler and SPF egg production.In addition,CIAV and FAdV were infected by vertical and horizontal transmission,it also can be transmitted through the vaccine contamination.Therefore,in this study,we established a rapid detection for for monitoring CIAV and FAdV in attenuated vaccines,which will help enterprises to accurately and quickly to detect CIAV and FAdV contamination in the attenuated vaccines1.Establishment and application of SYBR Green quantitative PCR for the detection of CIAV contamination in attenuated vaccines.According to the partial VP2 gene of CIAV genome conserved region,a specific primer with a size of 154 bp was designed and synthesized.The SYBR Green I fluorescence quantitative PCR assay for rapid detection of CIAV was established by optimizing the reaction conditions for sensitivity,specificity and reproducibility tests.The results showed that there was a good linear relationship between Ct threshold of CIAV and standard concentration from 6.36 × 107 copies/?L to 6.36 copies/?L,the correlation co-efficient was R2= 0.999,the slope was-3.186,and the sensitivity was approximately 160-fold higher than that of the conventional PCR assay.This detection method has good specificity,cannot be used for carrying out cross-reaction with other REV,ALV,and MDV genes,the CV(coefficient ofvariation)of intra-assay and inter-assay were less than 2.5%,and also shows that the method has the characteristics of better repeatability.In this experiment,a low dosage of 1EID50/1000 plumes and 2 positive of 14 samples were detected for CIAV detection from commercial poultry vaccines by qPCR method.CIAV-positive samples were further subjected to classical SPF chicken test method,which gave positive results for exogenous virus detection..The results suggest that this developed SYBR Green quantitative PCR system has strong specificity,highly sensitivity and better repeatability,and can be easily used in diagnostic laboratories for detection of a low dosage CIAV contamination in attenuated vaccines.2.Establishment and application of PCR for the detection of FAdV contamination in attenuated vaccines.A specific primers with a size of 1310 bp were designed and synthesized according to the hexon gene of the JSJ13 strain from Genebank,the PCR detection method was established by optimizing the reaction conditions,and 32 batches of attenuated live vaccines were detected by this method.The results showed that one of the La Sota strain of Newcastle disease vaccine(Clone 30)was positive for FAdV detection.FAdV in vaccine by chicken embryo inoculation was isolated and identified by PCR amplication.The results showed that the hexon gene was the purpose band as same as the vaccine,the fragment length was 1310 bp,which indicated that there was FAdV contamination in the live vaccine of Newcastle disease,and completely isolated and identified in the vaccine.The isolated strain(FAdV-N22)were compared with the hexon gene of other different reference strains published in GenBank.The phylogenetic tree found that the nucleotide sequence homology of FAdV-N22 strain and other strains of FAdV hexon gene was from 98.3% to 99.8%,the highest homology was 99.8% with the Chinese isolate JSJ13(FAdV-4 type)in China from 2015.the homology was lower than that of A,B,C,D and E,the homology was lowest in the population of II and III which were only 56.9% and 40.9%,respectively.The phylogenetic tree found that all the isolates were divided into two groups,group I contained five species of A,B,C,D and E,and group II only contained hemorrhagic enteritis virus(HEV)belonging to the FAdV-II subgroup and the eggdropping syndrome virus(EDSV)belonging to the FAdV-III subgroup,it was confirmed that the isolated strain,FAdV-N22,belonged to FAdV-4 type of avian adenovirus C species.Chickens were infected with FADV N22,the results showed that the mortality rate was 100%after infection,chickens in the control group were all alive.One-day old chickens were all dead within one day after infection without any asymptomatic.no obvious symptoms in the first three days in chickens with 3 weeks old after infection,a large number of deaths chickens occurred on the 4th day,the chickens were sick,apathetic and feather fluffy,all of the cloacal swabs were positive for FAdV by PCR test,there were the typical clinical symptoms such as pericardial effusion after anatomy,the pathology histology showed that liver steatosis and bleeding,basophilic inclusion bodies could be seen in liver cells.In this study,a PCR method for specific detection of FAdV was established,which could be used to detect the contamination of FAdV in attenuated vaccines quickly and effectively,is conducive to monitor the live attenuated vaccine FAdV and other poultry exogenous virus contamination.FAdV-4 type was firstly isolated and identified in China by this method combined with virus isolation and identification,these results showed that FAdV contamination in live vaccines may be one of the important pathways for FAdV infection in chickens.