The Development Of Inactivated Seedlings Of Avian Subfamily Avian Adenovirus Tetravalent Oil Emulsion In China

Inclusion body hepatitis(IBS)is an acute infectious disease,which caused by Fowl adenovirus I(FAV I).Recently,in parts of China,a large number of deaths occurred.After dissecting corpse,the main features of hydrocardia and hepatitis were found,so it was called hydrocardia-hepatitis syndrome(HHS).The virus of this disease has similar genetic evolutionary relationship with the serotype 4 of fowl adenovirus I.In recent years,the epidemic situation of inclusion body hepatitis and hydrocardia-hepatitis syndrome in China has been an upward trend which caused serious economic losses to Chinese poultry industry.In that case,3 standard strains of FAV I and HHSV were choosed to invent a polyvalent oil emulsion inactivated vaccine,which based on the prevalence of inclusion body hepatitis and hydrocardia-hepatitis syndrome in China.It will be of great application value for the prevention and treatment of the two diseases and the development of poultry industry.In this research,the heart and liver of the diseased chickens,which suspected to be infected with HHS,were collected from the animal hospital of Yangzhou University,and after processing,the samples were inoculated into the yolk sac of 7-day-old chick embryos.Virus was subcultured by collecting allantoic fluid of chicken embryo.Through the identification of PCR technology,4 strains were identified.The Hexon gene of these strains were amplified and compared with all serotypes of FAV I published in GenBank.The results showed that all four strains belonged to the same branch as the serotype 4 of fowl adenovirus I,and which had very high homology.The incidence and anatomy of SPF chickens,inoculated with four strains,were consistent with those of natural infection of HHSV.Choosing three standard strains of FAV I,which were preserved in our laboratory,producted the polyvalent oil emulsion inactivated vaccine by 1:1:1:2 ratio with HHSV.The changes in the level of antibody were detected after vaccination,in order to provide a scientific basis for preventing the occurrence and epidemicity of the two diseases.Three serotypes of FAV I(FAV-2,FAV-5,FAV-8)and HHSV were selected and subcultured by SPF chicken embryo yolk sac and CEL to amplify more viruses.The egg 50%infective dose,tissue 50%culture infective dose,optimal inactivation concentration and optimal inactivation time of each stain were tested.The polyvalent oil emulsion inactivated vaccine was manufactured after inactivation of formaldehyde.The physical properties of the vaccine,aseptic test,immune safety,minimum immune dose,immune effect and antibody levels were examined.The results showed that each EID50 of FAV-2,FAV-5,FAV-8,JS201501 was 10-5.21/0.2 mL,10-6 67/0.2 mL,10-6 48/0.2 mL,10-5.61/0.2 mL.each TCID50 of virus was 10-5.75/0.1 mL,10-7.61/0.1 mL,10-7.25/0.1 mL,10-6.37/0.1 mL,each concentrations of virus was 105 086 copies/?L,106.304 copies/?L,105.371 copies/?L,104 560 copies/?L.It is suggested that the final concentration of formaldehyde inactivation is 0.15%and the best inactivation time is 24 hours.It is determined that the vaccine is water-in-oil,the viscosity and stability are in line with the standard,and the sterility test is qualified.7-day-old chicken from the hatching of the SPF chick embryo were vaccinated.After vaccination there were no adverse reactions to be found,the security was good and the minimal immunization dose of each one was 0.4 mL.The antibody level was detected after two weeks of vaccination,and it reached a peak at week 4.After that,the antibody level began to decline rapidly.The results of immunization challenge test showed that the clinical protection rate of the vaccine was 100%,which could prevent the infection of the corresponding virus.