Development Of Preparation Technology And Potency Tests Of Propolis Adjuvant Inactivated Vaccine Against Duck Adenovirus

The research was used duck adenovirus which was propagated in embryonated duck eggs inoculated via the allantoic cavity. After 24-120 h, chilled at 4 ℃, separate under aseptic conditions, then the allantoic and amniotic fluids were harvested. The duck adenovirus continue propagating till to be stability. The allanto-amniotic fluid (AAF) having duck adenovirus suspension gave high HA titer 1:216 with the duck erythrocytes. The EID50 of the virus was 10-7.51/0.2ml. The formaldehyde, when admixed in the duck adenovirus suspension at the rate of 0.3%, inactivated the virus with 48 hours incubation at 37 ℃. The confirmed Duck adenovirus was processed for preparation of propolis, which propolis was 10 mg/ml. The vaccine was safety.The research developed an indirect ELISA method to study the ducks immunity response of propolis adjuvant inactivated vaccine against duck adenovirus.The optimum reaction conditions for ELISA were determined:The protein quantity of purified antigen was 2.45 μg/ml; the best dilution of serum was 1:100; the best reaction time for serum was 90 min. The best dilution of second antibody was 1:2000; The threshold of 32 negative serum was 0.2310. The coefficient of variation in the plate was 1.09%-2.67%; the coefficient of variation among plates was 2.79%-4.06%. Neither of them were more than 10%. The positive serum of Escherichia coli, Pasteurella multocida, Duck plague virus, Duck viral hepatitis, Salmonella and duck adenovirus were detected by ELISA which coating with duck adenovirus and the results showed the positive serum of Escherichia coli, Pasteurella multocida, Duck plague virus duck viral hepatitis and Salmonella were negative except duck adenovirus.The breeder ducks were vaccinated with propolis adjuvant inactivated vaccine against duck adenovirus. The serum antibody was detected by ELISA and HI methods. ELISA results showed that the antibody could be tested by ELISA at 3 d post inoculation (PI), it reached peak (OD450=0.628) at 14 d PI in immune once group and decreased slowly afterwards, but it is still kept positive at 56 d. Immune twice group reached the highest titer (OD450=0.759) at 21 d. Comparing with the control group, the OD450 value of immune once group was significantly higher (P<0.05). At the same time, The OD450 value immune twice group was significantly higher than immune once group (P<0.05) and the control group (P<0.05). The HI test showed that immune once group can detect antibody 2 at 3 d. Then the level of antibody increased gradually at 3-14 d. At 14 d after vaccination reached the highest HI titer 211.52, then decline gradually and slowly. At 56 d, the HI titer was 29.38. Immune twice group reached the highest titer 216.00 at 21 d, then decline slowly. The HI titer was 213.30 at 56 d after vaccination. The research showed that the HI titer of immune once group was very significantly higher than control group (P<0.05) and the HI titer of immune twice group was very significantly higher than immune once group (P<0.05) and the saline control group (P<0.05). The sensitivity tests between ELISA and HI test were performed and showed the ELISA had much higher sensitivity than HI test.The breeder ducks were vaccinated with propolis adjuvant inactivated vaccine against duck adenovirus. The cellular immunity was conduced by MTT method and Leukophago-cytic function test. The MTT result showed that the transformation ability of T-lymphocyte immune once group is enhanced at 3 d. There was a significantly difference between the vaccination group and the saline control group (P<0.05). Then OD value decreased rapidly after reaching highest point at 10 d. The transformation ability of T-lymphocyte of immune twice group maintained high level at 10-14 d, then decreased gradually. Immune twice group was dramatically higher than immune once group (P<0.05) and the saline control group (P<0.05) at 10 d、14 d、21 d、28 d post inoculation. There was no significant difference among immune once group, immune twice group and saline control group at 42 d. Leukophagocytic function test results showed that phagocytic index (PI) reached peak at 21 d post inoculation which is 3.03, then decreased rapidly. Immune twice group was higher than immune once group and control group by contrast leukocyte phagocytic index. Immune twice group reached the highest index at 21 d post inoculation.