Combined Inactivated Vaccine Against Duck Tembusu Virus And Avian Influenza Virus(H9 Subtype)

In the Southern China,April 2010,A disease which was characterized by laying egg rate in duck,caused by a new member of genus Flavivirus,family Flaviviridae,it is Duck Tembusu Virus(TMUV).It spread raidly and can caused huge economic losses to the duck farming industry since this Newly emerged disease outbreak,So thus an urgent realistic need to develop a effective vaccine to prevent and control the disease,but the TMUV is often mixed infections with H9 subtype avian influenza virus in actual production activities.Therefore,it is more significance to develop a combined inactivate vaccine.The conditions of TMUV proliferation were optimized firstly in this study,and then decided the optimum cell for virus proliferation was BHK-21-JS cell strain,the best inculation amount was MOI=0.001,and the harvest time was 60 hours after infection.That provided sufficient antigens for the preparation of inactived vaccine.To identify the best adjuvant for the combined vaccine of Tembusu virus(TMUV)and H9 avian influenza virus(H9 subtype),vaccines were prepared with inactivated TMUV JS804and H9N2 AIV NJ01 using oil,Carbomer,Aluninum hydroxide or propolis adjuvant in this study.Each vaccine formulation was evaluated for immunogenicity in ducks by comparing antibody levels of TMUV and AIV(H9 subtype)in serum samples collected at different times points of post immunization.The results showed that oil adjuvant vaccine could induce a faster antibody-producing process and higher antibody levels than the others.And the HI titers of AIV(H9 subtype)in 60%(6/10)serum samples was above 1:8,and the TMUV seroconversion rate was 80%(8/10)by 2 weeks post vaccination.This study support that oil is the optimum adjuvant for the preparation of TMUV-AIV(H9 subtype)combined vaccine.Study the effect of different infection ways of TMUV on ducks,In order to establish artificial infection model.60 35-day-old Cherry Valley ducks were randomly divided into 6groups.One to five groups were inoculated with TMUV-JS804 strain by intracerebralinjection,intramuscular injection,subcutaneous injection,intravenous injection and oral administration respectively,with dose of 5×10~4 PFU.The clinical symptoms were observedevery day and the body weight of each group was measured and analyzed at 0,3,6 and 10 day post infection.Serum samples were collected at 1,2,3,4 and 5 day post infection.Viral load in blood of infected ducks were detected by phage forming test.Gross andmicroscopic lesions of spleen of infected ducks were examined at 3 day post infection.The results showed that clinical symptoms such as depression and inappetence,and diarrhea were observed on some of the infected ducks,and the body weight showed a downward trend.Each infective route could induce obvious viremia in ducks,especially intramuscular injection.Spleen of infected ducks showed destruction of structure and necrosis of cells.Intramuscular injection is the best way to establish artificial infection model.To study the effect of different antigen content on inactivation,20-day-old Cherry valley duck was immunized with different doses of antigen,separate serum collection after immunization,and the blocking ELISA method was used to of serum antibody titers.The results showed that the ideal effective antibody level could be obtained when the minimum immune dose of inactivated vaccine was 0.5 mL,To further develop reasonable immunization procedures,The 20-day-old cherry valley duck was respectively immunized with the same dose after primary immunization and secondary enhancement,and then to detect antibody production levels and antibody duration,The results showed that the secondary immunization of inactivated vaccine could achieve better immune effect,the effective antibodies last only three weeks at a time of first immunization,After the second immunization,the antibody level rapidly increased to a higher level and can lasted for more than 18 weeks.