Isolation And Identification Of Group Ⅰ Fowl Adenovirus And Development Of The Trivalent Oil-emulsion Inactivated Vaccine

Thenumberofclinicalcasesofinclusionbodyhepatitis（IBH）and hydropericardium-hepatitis syndrome（HHS）has been increasing,resulting in considerable economic losses in many countries.The HHS has been observed from chickens and geese with clinical signals of sudden increase in mortality,hydropericardium,hepatitis and nephritis.HHS was first reported in Angara Goth,Pakistan,and was subsequently observed in the broiler flocks of South Asia,the Arabian Peninsula,Eastern Europe,North and South America,Japan,South Korea and China.Previous studies revealed that the pathogenesis of fowl adenovirus infection is affected by the serotype or genotype involved.All 12 serotypes of fowl adenovirus have been associated with outbreaks of IBH.IBH is usually observed in chickens between 3 and 4 weeks of age and is characterized by mortality approaching10%²0%.The main lesions of IBH are a congested and enlarged liver with necrosis and petechial hemorrhage.On the other hand,HHS is caused by only fowl adenovirus serotype 4,and the characteristic features of the disease include accumulation of transparent or straw-colored fluid in the pericardial sac with pulmonary edema,nephritis,and hepatitis with extremely high mortality rate.The outbreak of inclusion body hepatitis（IBH）and hydropericardium-hepatitis syndrome（HHS）has caused enormous economic loss to the poultry industry.Previous study revealed that the group I fowl adenovirus was the causative agent of these diseases.To prevent and control the epidemic of the disease,the wild strains of the fowl adenovirus were isolated and identified.The sequence analysis and reproduction of experimental model of infection have proved that the isolates all belong to the group I fowl adenovirus,3 of serotype1,9 of serotype 4 and 3 of serotype 8a.The broilers were used to carry out the development of experimental infection of the isolates.During the period of observation,the challenged birds all showed the clinical signs of depression and anorexia,and the acute death would ensue.The results of autopsy showed that all broilers died in the challenged group show the obvious lesions of the swollen liver,spleen and kidney,the necrosis of liver,as well as hemorrhages of the kidney.What’s more,the accumulation of transparent or straw-colored fluid in the pericardial sac can be observed in birds challenged with the FAdV-4.All those above lesions are coincident with the cases reported previously.As for the histopathology examination,the inclusion body can be observed in the liver cell,the necrosis of spleen and liver,as well as inflammatory infiltrate in liver is typical.The virus strains with great antigenicity,SDJN,SDJX and SDRZ,were selected to develop the inactivated triplex vaccine.Different serotype viruses were culture by the SPF chicken embryo,inoculated via yolk-sac route,respectively.Eggs those died within 24h were abandoned,and then collected allantoic fluid of the remaining eggs at 120h post inoculation.In order to improve the antigen content,the allantoic fluid was concentrated by PEG20000.The allantoic fluid of each serotype was inactivated by adding 1‰formalin（v/v）and kept at37°C for 24 h,shaken every 6 hours.294.4mL sterilized mineral,19.2mL span-80（Sorbitan（Z）-mono-9-octadecenoate）and 6.4mL stearic acid aluminum were mixed uniformly as the oil phase.Then the oil phase and 200mL inactivated virus fluid（100mL of SDJX strain,50mL of SDJN strain and 50mL of SDRZ strain）,which contains sterilized 4%tween-80（Polysorbate-80）were churned by 3000 rounds per minute（r/min）for 30 minutes with homogenizer.The purity examination including sterilization examination,mycoplasma examination and exotic virus examination demonstrated that the all components of the vaccine were sterile.After the development of trivalent inactivated vaccine in the study,the evaluation of the vaccine was performed.Results showed that the vaccine can effectively protect broilers against the attack of fowl adenovirus serotype 1,serotype 4 and serotype 8a.And the specific antibody to different serotype of the fowl adenovirus was detected by the i ELISA assay coated by the whole virus.The blood samples were collected from vaccinated broiler chicks at1、4、7、10、13、17、20、25、42、50、60、64、67 days post vaccination.Results showed that when the OD450 value comes to the 0.7452,the vaccine can provide good protection to the serotype 1 attacking.What’s more,the value of serotype 4 and serotype 8 is 0.6215 and0.692,respectively.The antibody level of serotype 4 and serotype 8 began to rise at the 4 days post vaccination,while serotype 1 was at the 7 days post vaccination,and they all reached the peak of the level at the 25 days post vaccination.The ELISA OD₄₅₀50 value of antibody to all serotype was still higher than the value described previous at the 67 days post vaccination,indicating that the inactivated vaccine can provide good protection against the attack of the fowl adenovirus for a long period.What’s more the challengeing test revealed that the inactivated vaccine can provide the broilers with protection against not only the FAdV-1,FAdV-4,FAdV-8a but also the cross-serotype protection against FAdV-2 and FAdV-6.Therefore,the inactivated vaccine developed in the study can be effective in preventing the havoc caused by adenovirus.