Optimization Of The Multivalent Inactivated Vaccine Of Avian Adenovirus In The Chicken Subgroup ? And The Study Of Its Immune Efficacy

Group I Fowl Adenovirus(FAdV-I)belong to the genus Aviadenovirus under the family Adenoviridae,it have been subgrouped into five species(A to E)based on their molecular structure and have been further divided into 12 serotypes(FAdV-1?7?FAdV-8a?FAdV-8b?FAdV-9?11)based on cross-neutralization tests.Inclusion body hepatitis(IBH)and Hydropericardium hepatitis syndrome(HPS)are the two most important disease caused by Group I Fowl Adenovirus in the world,including China.All 12 serotypes have been incriminated in IBH,whereas serotype 4 has been implicated in HPS.In recent five years,poultry farms in many provinces of China have outbreaks of HPS and IBH,and there is no commercial vaccine to prevent and control the infection of FAdV-I in China.However,there are many serotypes of FAdV-I,and there is no or only weak cross protection between different serotypes,which brings great difficulties to clinical prevention and control.Thus this study selected four dominant serotypes(FAdV-2,FAdV-4,FAdV-5,FAdV-8b),belonging to D,C,B and E four species respectively to prepare and optimize the multivalent oil emulsion inactivated vaccine of Group I Fowl Adenovirus,which has important application value in the prevention and control of FAdV-I and the healthy development of chicken industry.1.Screening of methods for serotype 2,4,5 and 8b Group I Fowl Adenovirus proliferation To rejuvenate and proliferate the FAdV-2,FAdV-5 and FAdV-8b epidemic serotype strains which preserved in the laboratory and the FAdV-4 strain which Isolated and identificated after collectioning by laboratory.After comparing the effects of virus proliferation,choosing the most suitable way among chicken embryos,293A cells,chicken embryo hepatocytes and LMH cells.Finally,the LMH cells were selected,which are convenient and stable,can produce obvious pathological changes of Fowl Adenovirus,and can effectively increase virus titer.Testing the egg 50%infective dose and tissue 50%culture infective dose of the stable proliferating virus to determine the virulence level of the virus.The EID50 of s seed batch Fowl Adenovirus FAdV-2?FAdV-4,FAdV-5 and FAdV-8b were 10-6.7/0.2 mL,10-7.38/0.2 mL,10-7.83/0.2 mL,10-7.62/0.2mLrespectively,TCID50were 10-6.77/0.1mL,10-7.4/0.1mL,10-7.63/0.1 mL,1 0-7 6/0· 1 mL respectively.2.Preparation of Fowl adenovirus tetravalent inactivated vaccine and optimization of oil-water ratioThe experiment continued to use the traditional formalin inactivation method to inactivate antigen.The serotype 2,4,5 and 8b FAdVs were mixed according to the volume ratio of 1:2:1:1,and the final concentration of formaldehyde was 0.15%.After 24h inactivated at 37?,antigenic solution was made.Three oil-water ratios were set up to prepare three batches of oil emulsion inactivated vaccine for contrastive experiment.The viscosity,stability,asepsis,safety and other quality of vaccine were examined.The results showed that when the oil and water ratio of the vaccine was 2:1 and 3:1,the characters were stable,and a small amount of liquid precipitated after the centrifugation in the 4:1 group,it showed the stability of 4:1 group was slightly worse.The preservation period of 2:1 group was longer than the 3:1 group and the 4:1 group.The three groups were all qualified for sterility test and safety test.3.Study on Immune Effect of Group I Fowl Adenovirus Tetravalent Inactivated VaccineEach minimum immunization doses of three kinds of oil-water ratio(group 2:1,3:1 group and 4:1 group)oil emulsion inactivated vaccine,which immunized 7 day old SPF chickens is 0.3mL.After two weeks of immunization,the antibody level of the immune group was monitored by agar gel diffusion test and neutralization test every week.The results of neutralization test was clearer and more sensitive.The antibody level began to increase two weeks after vaccination,and reached the peak at the fourth week,then decreased.Because the stability of the oil-water ratio group 2:1 is the highest and its immune effect is the best,this group is the best oil-water ratio.Then carry it out to do the immunization challenge test.The immunization challenge test showed that the protective rate of the inactivated vaccine against the four serotypes virus was 100%,while the attack group had death and clinical symptoms,and the vaccine could effectively resist the infection of the chicken avian adenovirus.