| Serotype 4 Fowl Adenovirus(FAdV-4)belongs to the genus Aviadenovirus.In recent years,the cases of inclusion body hepatitis(IBH)and hydropericardium syndrome(HPS)caused by FAdV-4 in domestic chicken flocks are increasing gradually.The outbreak of FAdV-4 occurs not only in 3-7 weeks old broiler flocks,but also in 10-20 weeks old layer flocks,which has caused serious economic losses to the poultry industry in China.Due to the development and application of inactivated vaccines,how to distinguish the chickens with natural infection from these vaccinated with the inactivated vaccine is crucial for effective and timely prevention and control for the disease caused by the infection of FAdV-4.In this study,two nonstructural proteins 22K and 52K were selected as targets for peptide synthesis,and the peptide-based ELISAs were developed for detection of antibodies against 22K and 52K respectively.The established ELISAs were evaluated for the potentials for distingushing the FAdV-4 naturally infected flocks from flocks vaccinated with the inactivated vaccine.1.Establishment of an ELISA for the detection of FAdV-4 non-structural proteins 22K and 52KIn order to distinguish the naturally infected chickens from chickens vaccinated with the inactivated vaccine,the antigenicity of two nonstructural protein 22K and 52K of FAdV-4 was firstly analyzed using DNAStar,and several peptides derived from 22K and 52K respectively were synthesized and screened.The peptide-based ELISA for the detection of antibodies against 22K and 52K were generated.Peptide screening revealed that 22K-4P and 52K-4P peptides showed strong reactivity with FAdV-4 positive sera.Therefore,22K-4P-ELISA and 52K-4P-ELISA were established for the detection of FAdV-4 antibodies by optimizing the conditions.The CUT-OFF value of 22K-4P-ELISA and 52K-4P-ELISA was 0.1668 and 0.182 respectively.Specificity analysis demonstrated that two ELISAs only reacted with the FAdV-4 sera,but not with sera against the other avian viruses including Chicken anemia virus(CAV),Serotype 8 fowl adenoviruses(FAdV-8),Reticuloendotheliosis virus(REV),Infectious bronchitis virus(IBV),Marek's disease virus(MDV),Newcastle disease virus(NDV),Avian influenza virus(AIV)tested.Reproducibility assay showed that both the intra-and inter-assay coefficients of variation of two ELISAs were less than 10%.The establishment of 22K-4P-ELISA and 52K-4P-ELISA for the detection of FAdV-4 antibodies provided technical possibilities for distinguishing the naturally infected chickens from the chickens vaccinated with the inactivated vaccine.2.Preliminary application of ELISA for detection of FAdV-4 non-structural protein 22K and 52K antibodiesTo evaluate whether 22K-4P-ELISA and 52K-4P-ELISA could be used to distinguish the chickens with natural infection from these vaccinated with the inactivated vaccine,sera from chickens experimentally infected with FAdV-4,clinical chickens with disease caused by FAdV-4,and chickens vaccinated with the inactivated vaccine respectively were tested by the two ELISA generated here,and also by the Fiber2-based ELISA and IFA method.Data showed that the positive rate of 22K-4P-ELISA,52K-4P-ELISA,Fiber2-ELISA and IFA was 87.5%,95.8%,100%and 100%respectively for 24 sera from chickens experimentally infected with FAdV-4,that for sera from clinical chickens with disease caused by FAdV-4 was 85.7%,88.9%,96.3%and 93.7%and that for sera from chickens vaccinated with the inactivated vaccine was 2.5%,0%,100%and 100%.Our data demonstrated that 22K-4P-ELISA and 52K-4P-ELISA developed here could be efficiently applied in differentiating the chicken flocks with the natural infection from the chicken flocks vaccinated with the inactivated vaccine,which proving novel tools for better controlling the disease caused by FAdV-4. |
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