Preparation And Application Of Yolk Antibody Against Fowl Adenovirus Type 4

Fowl Adenovirus type 4（FAdV-4）is a highly contagious infectious agent that infects poultry and mainly causes hepatitis and hydropericardial syndrome in clinical infection of chickens.In recent years,the disease is widespread in various regions of China,mainly affecting 3～7 weeks of age chickens,rapid onset,mortality up to 80%,bringing great economic losses to the poultry industry.Domestic commercial vaccines for FAdV-4 prevention and control cannot fully meet the needs of prevention and control.Yolk antibody is a specific antibody that gathers in the yolk of poultry after immunization with specific antigens.With advantages of strong specificity,low cost and safety,it is applied to the prevention and treatment of poultry diseases.In this study,the FAdV-4 DQ15 strain was inoculated into suspension-cultured LMH cells.The cell culture was harvested 72 h later,and the virus-containing supernatant was collected after centrifugation.The virulence and specificity of the cultured virus were tested.The 100-fold diluted virus preparation caused 70%morbidity and mortality in SPF chickens of 42-day-old.The virus was neutralized by the FAdV-4-specific serum.To prepare the virus,the optimal cell density was determined to be 1.4×10⁶～1.6×10⁶ cells/mL,the optimal cell density was 8.0×10⁶ cells/mL and the optimal inoculation dose was 0.1%.Consequently,the virus titer was reached to10^9.23TCID₅₀/0.1mL.Formaldehyde solution was added into the virus solution to the final concentration of 0.1%and was kept at 37℃for 16 h.The immunogen of virus was prepared by emulsification with white oil adjuvant.The safety,formulation,physical properties,sterility and immune efficacy of the immunogen were tested.The immunogen formulation is oil-in-water,physical properties are stable,safety is good,no bacteria and other exogenous microorganisms were detected.The SPF chickens of 21-day-old were immunized with the immunogen,and the immune efficacy test was conducted.The SPF chickens were challenged with the FAdV-4 at 21days post immunization.All chickens were alive in 10 days post infection,and no pathological changes were observed by autopsy.The protection of disease was 100%.Laying hens were immunized with three doses of the immunogen with the interval of 21 days.Sera of immunized chickens were collected each week after the first immunization,and yolk antibodies were collected from the eggs for the detection of neutralization titer.Results showed that the level of specific yolk antibody increased continuously after immunization and decreased gradually after reaching the plateau at the 10th week post immunization.Optimum conditions for purification of the yolk antibody were measured.The optimum dilution of acidified water was 1:8at the pH of 5.2,and the optimum concentration of caprylic acid was 1%.The yolk antibody against FAdV-4 was prepared according to the optimized technical procedure,and the yolk antibody against FAdV-4 was prepared by collecting eggs from hyper-immunized hens through eggshell disinfection,yolk separation,acetic acid degrease,caprylic acid extraction,filtrations and sterilization,and then inactivated with formaldehyde.The physical properties,caprylic acid residue and the safety of purified yolk antibody were tested.In order to exam the emergency preventive effect of the FAdV-4 yolk antibody,SPF chickens of 21-day-old were challenged by FAdV-4 after 7 days being injected with different neutralizing titers of yolk antibody.The preventive effect of different neutralizing titers of yolk antibody against FAdV-4 infection was evaluated.Results showed that all SPF chickens that injected with the yolk antibody with a neutralizing titer of 1:64 were died within 6 days after challenge.Among chickens infected with the yolk antibody with neutralizing titer of 1:128,two survived 10 days after challenge,and one was detected shedding FAdV-4.When the injected yolk antibody with neutralization titer of 1:256,all chickens resisted to the FAdV-4 challenge.No side effects of the antibody were observed within 10 days after injection,and no pathological changes were observed at autopsy.Only one chicken was detected shedding virus on the third day after challenge.Therefore,the protection of disease by the antibody of 1:256 was 100%.In the post-infection treatment experiment,21-day-old SPF chickens infected with the FAdV-4 through chest muscle were given the treatment with yolk antibody with neutralizing titer of1:256 at 24 h and 36 h after the infection,respectively.The results showed that within 10 days after the injection of yolk antibody,all the chickens subjected the antibody treatment were alive and there was no pathological change in autopsy.All the chickens in the no antibody-treatment group died.In the group received the antibody-treatment 36 h post infection,viral shedding was detected from 2 and 4 chickens,respectively,within 3～5 days post infection.Most chickens were negative in viral shedding within 10 days,and the passive immunization protected 100%of experimental chickens infected with FAdV-4.In conclusion,the inactivated immunogen and the yolk antibody against FAdV-4 prepared in this study effectively protected SPF chickens against the infection of FAdV-4.The production process of FAdV-4 yolk antibody is simple and in low cost.The product is safe,and the protective effect is solid with a long duration.The FAdV-4 yolk antibody can be effectively used in the emergency prevention and the treatment of FAdV-4 infection,and therefore has potential applications in disease control.