| In April2010, there was a disease outbreak which was characterized by rapid-egg-drop in the southern part of China, caused by Duck Tembusu Virus (DTMUV). DTMUV is a member of the Ntaya virus group within the genus Flavivirus, family Flaviviridae. This newly emerged disease spread rapidly with serious damage and caused huge economic losses to the duck farming industry. Thus an urgent need for a safe and effective vaccine to prevent and control the disease incidence and prevalence appears.FXV2010-180P strain, an attenuated virus of FXV2010, obtained by serial passage in chicken embryo fibroblaats cells (CEFs). In this study, the research of inactivated vaccine was developed by using FXV2010-180P as the seed virus. Firstly, we carried out the manufacturing process of inactive vaccine. The best inoculation amount and harvest time was optimized by using asynchronous inoculation method. The virus titer can be stabilized at106.0TCID50/0.1ml, when T75culture flasks that grown with monolayers DF-1cells inoculated with105.5TCID50at60h. Under the optimal conditions for culture expansion, virus was inactivated by using two inactivating agent formaldehyde and BPL, the result showed that both of them can inactivate virus completely at the concentration of2%o. After using the adjuvant Montanide ISA50V2to pre-emulsify at a low speed of100r/min, mixed with equal volume inactivated virus fluid and emulsified6000r/min5minutes to preparation inactivated vaccine. The inactivated vaccine received was a white water-in-oil emulsion fluid and was quite stable, it wasn’t break when centrifuged3000r/min for15minutes. Subsequent studies conducted in accordance with the preparation of the production process DTMUV inactivated virus vaccine.In order to investigate the effects of different inactivate agents and virus amounts in DTMUV inactivated vaccine. After inactivated with formaldehyde and BPL, the virus fluid was concentrated by using centrifugal filters and virus fluid with different antigen mount were prepared (equivalent to1times,2.5times,5times and10times of virus stock amount). Use these concentrated virus fluid to develop inactivated vaccines and immunized female ducks. The sera were collected after immunized1,2and3week and used Blocking-ELISA to detect the antibody titers. The results showed that the vaccine with high antigen content induced antibody earlier with higher titers. At the third week post immunized, ducks challenged with103.5TCID50of virulent. The immune efficacy of inactivated vaccine was evaluated through clinical symptoms, pathological changes and the virus isolation results of spleen, lung, kidney, brain, ovary and other samples. The results show that the protective efficacy of the vaccine has a significant correlation with antigen amount. In addition, compared with formaldehyde, use BPL as the inactivator provided a better protection from virulent attacked.The researches above showed that increase the antigen mount will help to improve the immune potency of inactivated vaccine, but use concentration technique to raise the antigen content will also increase the cost of vaccine’s production significantly. What’s more, the antibody titers produced by single immunization accompanied by higher dispersion. In view of these, this study further evaluated the efficacy of inactivated vaccine by using BPL as the inactivator and immunized two times. The results showed that the antibody titer produced by the original times was equivalent to2-fold concentrated vaccine. After virulent challenge, both of them can provide100%immune protection. |
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