Isolation And Identification Of Group Ⅰ Fowl Adenovirus And Preparation And Application Of Trivalent Yolk Antibody

Group I fowl adenovirus（FAd V-Ⅰ）belongs to aviadenovirus,which is divided into 5species A-E and 12 serotypes 1-7,8a,8b,9-11,which mainly caused inclusion body hepatitis（IBH）and hydropericardium-hepatitis syndrome（HHS）.In recent years,the incidence of IBH and HHS has been increasing in China.IBH mainly affects broilers aged from 3-7 weeks,and serum type 4 can cause severe pericardial effusion with a mortality rate of 30%-80%.All serotypes can cause IBH,which is characterized by hepatic enlargement,hemorrhagic spots and bleeding spots of varying degrees,mortality of 10%-30%,and huge economic losses to the poultry industry.With the continuous expansion of intensive aquaculture production,especially viral infectious diseases have become an important factor restricting the further development of aquaculture.Yolk antibody is the specific antibody accumulated in yolk after poultry immunized with specific antigen,and yolk antibody has been widely used in the prevention and treatment of poultry viral diseases with the advantages of strong specificity,low cost,safety and high efficiency.In this study,group I fowl adenovirus was isolated and identified from clinical samples suspected to be infected,and sequences of the isolated strains were compared and analyzed.The dominant serotype strains were selected to prepare oil emulsion inactivated vaccine and yolk antibody,and SPF chickens were tested for prevention protection and challenge protection,aiming to provide application value for the prevention and control of groupⅠfowl adenovirus.1.Isolation and identification of groupⅠfowl adenovirusIn this study,suspected group I fowl adenovirus infected materials were collected from Shandong and surrounding provinces from 2020～2021.The virus was isolated from chicken embryonic liver cell and detected by PCR.Results a total of 9 strains of groupⅠfowl adenovirus were isolated,Hexon gene was sequenced,homology comparison and genetic evolution analysis were performed on the sequenced results,3 strains were serotype 4,4strains were serotype 8b and 2 strains were serotype 11.The dominant serotypes FAd V-4（SDTA）,FAd V-8b（SDLY）and FAd V-11（SDWF）were selected and amplified with chicken embryo liver cells,and the virulence was determined.The results showed that TCID₅₀were FAd V-4（SDTA）:10^-6.67/0.1 m L,FAd V-8b（SDLY）:10^-5.39/0.1 m L and FAd V-11（SDWF）:10^-6.5/0.1 m L.The results of animal regression test of 28-day-old SPF chickens by intramuscular injection of FAd V-4（SDTA）,FAd V-8b（SDLY）and FAd V-11（SDWF）showed that liver enlargement,hemorrhagic spots and bleeding spots on the surface,kidney and thymus enlargement,hemorrhage,and kidney enlargement.More than 90%of FAd V-4（SDTA）challenged chickens had pericardial effusion,which was light yellow and clear,some of which were gelatinous.2.Preparation of trivalent inactivated vaccine of groupⅠfowl adenovirusThe dominant serotypes FAd V-4（SDTA）,FAd V-8b（SDLY）,and FAd V-11（SDWF）were selected to prepare trivalent antibody stock in a ratio of 1:1:1.After formaldehyde inactivation,it was mixed with sterile Tween-80 in a ratio of 96:4 as the aqueous phase of vaccine preparation.Trivalent inactivated vaccine of groupⅠfowl adenovirus was prepared according to the ratio of oil to water 2:1.3.Preparation and application of trivalent yolk antibody against groupⅠfowl adenovirusThe prepared inactivated vaccine was immunized to laying hens for four times with an interval of 14 days.The eggs were collected 14 d after each immunization,and the egg yolk antibody was extracted by the caprylic acid method.The prevention protection test and challenge protection test of yolk antibody were carried out.In the prevention protection test,chickens in each group at 28 days of age were intramuscular injected with 0.5 m L and 1.0 m L yolk antibody,respectively.After 24 h,FAd V-4(TCID₅₀=10^-4.67/0.1 m L),FAd V-8b(TCID₅₀=10^-3.39/0.1 m L),FAd V-11(TCID₅₀=10^-4.50/0.1 m L)were challenged with 0.5 m L/bird.The results showed that the protection rate of FAd V-4,FAd V-8b and FAd V-11 by injection dose of 1.0 m L yolk antibody was 100%.The protective rate of FAd V-8b and FAd V-11 at 0.5 m L/bird was 100%.The protective rate of FAd V-4 at 0.5 m L/bird was 90%.It has good protective effect.In the challenge protection test,Chickens in each group were challenged at the age of 28 days,24 h later,0.5 ml and 1.0 m L yolk antibodies were injected intramuscularly.The results showed that the protective rates of yolk antibody injection doses of 0.5 m L/bird and 1.0 m L/bird against FAd V-8b and FAd V-11were 100%;The protective rates of 0.5 m L/bird and 1.0 m L/bird were 80%and 90%respectively.Anal swab detoxification test was carried out on chickens in prevention and protection test and challenge protection test groups 1,3,5,7 and 10 days after challenge.The results showed that the detoxification time of FAd V-4,FAd V-8b and FAd V-11 challenge control group was long and the positive rate was high.The injection amount of yolk antibody of 0.5 m L/bird and 1.0 m L/bird in the experimental group significantly reduced the detoxification rate of FAd V-4,FAd V-8b and FAd V-11.In conclusion,the groupⅠfowl adenovirus trivalent yolk antibody prepared in this study has a good protective effect against FAd V-4,FAd V-8b and FAd V-11,and has a good therapeutic effect on challenging SPF chickens,and can play a good preventive and therapeutic effect on groupⅠfowl adenovirus infection.To provide a safe and efficient new biological product for the prevention and control of the virus.