| The wild carnassial animals,such as wild wolves and dogs,red foxes and raccoons,are natural carriers or spreaders of rabies virus. To prevent irradiation of rabies virus from wild carnassial animals to human being and domestic animals is one of keys for rabies prevention and control. The oral vaccine against rabies using in the wild carnassial animals consists of three parts,i.e. antigen,adjuvant and bait,comparing the injecting vaccine for human being and domestic animals. The purposes in this study are 1) to select optimal immune adjuvants used in oral vaccine from bacterial lipopolysaccharide, zinc hydroxide, medlar polysaccharide and manna oligosaccharide, and to proof the effect of its immune enhancement, and 2) to prepare different baits used in oral vaccine by beef, chicken, fish, blood and milk powders, and to detect their foraging and embedding effect.Experimental methods The rabies virus SRV9 was cultured and proliferated in BHK-21 cells by synchronous inoculation method. The titer of antibody against rabies virus was detected by median fluorescence focus Dose(FFD50). The formulas of complex immunity adjuvant were selected and determined from bacterial lipopolysaccharide, zinc hydroxide, medlar polysaccharide and manna oligosaccharide by orthogonal test and test of immune effect was performed in Kunming mice. The mixture of optimal immune adjuvant and rabies virus SRV9 was given orally to mice in experimental groups. The groups injected intraperitoneally rabies vaccine for human beings for 3 times, and inoculated orally rabies virus SRV9 alone for 2 and 3 times, respectively, were used as control. The blood examples from the mice in experimental and control groups was collected for detecting serum IgG at, 14 days after inoculation(DAI14), DAI 21, DAI 28 and DAI 35, respectively. The antibody in serum samples at DAI 35 in experimental groups were detected by fluorescent antibody virus neutralization tests(FAVN). The formulas of different bait were optimized by orthogonal test, and the foraging and the embed effect of the baits was determined by physical and chemical approach.Experimental results The results were shown 1) the titer(FFD50) of collected rabies virus SRV9 was10-5.42/0.1 mL. 2) The optimal formulas of complex immunity adjuvants was bacterial lipopolysaccharide100 μg, zinc hydroxide 0.8 mg, medlar polysaccharide 50 mg and manna oligosaccharide 10 mg. The means values of serum IgG and SIgA in experimental groups were 3239 μg/mL and 1.56 ng/mL,respectively, at DAI 28. 3)At DAI 35, the effect of humoral immunity in the group induced by complex adjuvant plus the rabies virus SRV9 was batter than those received rabies virus SRV9 alone for 2 or 3 times, and the means titer of serum IgG were 0.63 IU/mL, reaching to the protection level. 4) The five formulas of baits with different flavor were prepared, i.e. fish, blood, beef, chicken, milk flavored baits. 5)The fish flavored bait was quickly eaten by the experimental cats, dogs, wolves and foxes in the ingestion test, next to the blood and milk flavored baits. 6)The five type of baits were well long-term preserved at80℃,-20℃. 4℃ for 7 days. The baits were not deformed,and leakage of embed vaccine plus dye was not seen in these baits. The fish and blood flavored baits were kept relative stability at 37℃ for 12 h, i.e.suitable texture without leakage of embed vaccine, while beef, chicken and milk flavored baits were deformed and leaked.Conclusion: The optimal formula of complex immunity adjuvants used in oral vaccine was selected.The complex immunity adjuvant plus rabies virus SRV9 could enhance the immune effect of rabies virus vaccine. The five formulas of baits with different flavor were prepared for oral vaccine against rabies, and the foraging and the embed effect of the baits was decided. The optimal formula of complex immunity adjuvant and baits could be the tools for the further study recombinant attenuation vaccine use in the wild carnassial animals. |
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