Preparation And Evaluation Of Immune Efficacy Of Inactivated Flow Adenovirus 8a Serotype Oil-emulsion Vaccine

Since 2015,Fowl adenovirus has been reported to cause outbreaks in many countries worldwide.In May 2015,FAd V broke out in northern China,causing significant economic losses to the poultry industry.The FAd V strains detected in China are mainly FAd V-4,FAd V-8a,and FAd V-8b.FAd V-4 causes HPS in poultry,with mortality rates as high as 80%in broilers but only up to 40%in layer chickens.FAd V-8a and FAd V-8b cause IBH,leading to immunosuppression,decreased productivity,and even death.In recent years,FAd V-8a and FAd V-8b have become increasingly prevalent in China,with mortality rates of up to 30%in broilers aged 3-5 weeks.Currently,there are several commercial FAd V-4 oil-emulsion inactivated vaccines available in the Chinese market,and a commercial oil-emulsion inactivated vaccine for FAd V-8b is under review.However,there is no commercial oil-emulsion vaccine for FAd V-8a,and due to the large number of subgroups of FAd V serotypes in subgroup I,cross-protection between different serotypes is weak or absent.Therefore,the development of a commercial vaccine for FAd V-8a is of significant economic value to the domestic poultry industry.This experiment obtained a suspected inclusion body hepatitis（IBH）outbreak sample from a commercial broiler chicken farm in Weifang,Shandong province.A strain of FAd V-8a was successfully isolated through chicken embryo inoculation,and its Hexon gene was sequenced.A phylogenetic tree and homology comparison table were generated,and the strain was named FAd V-8a-CY21.A fluorescent quantitative PCR detection method for FAd V-8a was established using 8a-CY21 as the standard,with a detection limit of 4 copies/μL,which is 100 times more sensitive than the conventional PCR method.The intra-and inter-group reproducibility was good,with a coefficient of variation not exceeding 2%.There was no cross-reactivity with nucleic acids from other serotypes of avian adenovirus,avian influenza,Newcastle disease,infectious bursal disease,and other common diseases.The strain was then passaged in chicken liver cancer cells（LMH cells）and evaluated for its pathogenicity in chickens through artificial infection using specific-pathogen-free（SPF）chickens via intramuscular injection.The distribution of FAd V-8a in chickens was detected using fluorescent quantitative PCR,which showed that the virus was widely distributed in various organs.The liver of infected chickens was swollen,with a dull round edge and a yellow-brown color.There were also hemorrhagic spots on the surface,and the tissue was fragile.The viral load results showed that the virus was most abundant in the duodenum,peaking on the 6th day after infection.No chickens died during the experiment.The antigen was inactivated with 2‰formalin for 24 hours at 37℃,and the water phase and oil phase were mixed at a ratio of 1:3 to prepare an oil emulsion inactivated vaccine.The finished product was subjected to inactivation testing,safety testing,stability testing,and sterile testing in accordance with the《Chinese Veterinary Pharmacopoeia》and the results showed that all indicators of the vaccine met the standards.SPF chickens immunized with the inactivated vaccine via intramuscular injection reached peak antibody levels on the 21st day,with an average antibody titer of 7000.The protective efficacy of the inactivated vaccine against FAd V-8a was tested by intramuscular injection of the virus in chickens.The results showed that there were no obvious abnormalities in any organs of the immunized group,and no viral load was detected.In the control group,depression was observed,and liver lesions were present.Cloacal swabs were collected from each group to detect shedding,and the immunized group had significantly lower levels than the virus-challenged control group.When the viral titer reached 10^6.5 TCID₅₀/0.1m L,complete inhibition of shedding was achieved in the experiment chickens.The FAd V-8a oil emulsion inactivated vaccine prepared in this study is expected to become a candidate means for preventing the spread of IBH in Chinese poultry.This experiment isolated a strain of FAd V-8a in the Weifang region of Shandong province,China,and evaluated its pathogenicity.A protective model against the virus was established,and a fluorescent quantitative PCR detection method was developed.The protective efficacy of the inactivated FAd V-8a emulsion vaccine was measured,providing valuable data for the preparation of a domestic FAd V-8a vaccine.This vaccine has the potential to become a promising candidate drug for the prevention and reduction of inclusion body hepatitis transmission in China.