| One main reason that causes diarrhea of young animals is different serotypes' s pathogenic E. coli and it would generate great economical loss to livestock. Currently, normal prevention and treatment of this disease is using relevant medicine or vaccine injecting. Although the prevention effect of vaccine injecting is fine, it would cause animal stress as well as wasting time and energy. On the other hand, oral vaccine's advantage is its convenience and safety. It could stimulate the body to generate animal mucosal immune system response and humoral immune response. Therefore, the application value as well as development value of oral vaccine is high. This research plan to use microcapsules coated technology to prepare sheep pathogenic E. coli oral vaccine inactivated in order to establish a foundation of the researching the preparing method of lamb escherichia coli in a new oral vaccine formulations.The contents of the research are as following:1. The improvement of the preparing process of E. microencapsulated oral killed vaccine In this research, sheep pathogenic E. coli propolis-adjuvant and natural alginate polymer materialwas respectively selected by screening using adjuvants to be the core material and wall material of inactivated vaccine. Three often-used coated ways including spray drying, vacuum freeze-drying method, orificecoagulation bath were used to electing the best coating method. By testing the income tax rate of coated products,the preparing process of this oral vaccine could be improved.2. The research of how different E. microencapsulated adjuvant oral killed vaccine could affect the immune effect of mice This research took mice(male) as experimental subject, established freund's adjuvant injection group, aluminum gel adjuvant injection group, propolis adjuvant injection group and the control group. Serum antibody titer was tested by indirect ELISA test taking micro-agglutination test and established to evaluate the effect of different immune adjuvant vaccine. The best microencapsulated oral vaccine adjuvant vaccine was chosen to do the evaluation experiment of the efficiency of immune effect of mice and attack drug test and the evaluation of the Immunity protection efficiency is based on different volume.3.The research of the Immune effect of propolis E. Lamb of microencapsulated adjuvant oral inactivated vaccine 60 Hu lambs aged 7 days were used as experiment subjects in this experiment. Vaccine protection experiment was took by separating the subjects into blank control group(oral saline), injected group(1 mL/only) hand oral group. Among the oral group, there were low dose group(0.5 g oral powder vaccine/mouse) and high-dose group(1 g oral vaccine powder/only). The research also adopted micro-agglutination, indirect ELISA, lymp Hocyte transformation and tested the lamb mucosal secretoryimmunoglobulin A to measure the different index of humoral immunity, cellular immunity and mucosal immunity of lamb, using of poison attack experiment to validate the oral vaccine immune protection effect on the lamb.The result of the research is as following:The coating rate of microencapsulated product which is made by a spray drying method is 75.23%which is the highest, and the average value of the particle size is less than 10 ?m. The bacterial content of the microcapsules made after optimizing is 7.52 × 1011 bacterias/g of dry powder which accompanied by fine release as well as acid resistance. Three kinds of adjuvant screening discover that the immune protection rate of propolis adjuvant is the best which could be 100%. Besides, by using propolis as an adjuvant oral killed vaccine encapsulated E. coli oral vaccine to evaluate the effect of immunity mice, then testing serum antibody titers using micro-agglutination test and the established indirect ELISA, the result has showed that both oral group and injection group could produce antibody titers after 7 days and the OD492/630 of the injection group is notably higher than the oral test group(P < 0.05) after 28,35 days. After immunization, the 20-fold dose basis OD 492/630 of the oral test group is higher than 5 times the basal dose and the differences of 21 days, 28 days and 35 days is remarkable(P > 0.05). The 5-fold basal dose group could be recommend doses if the antibody titer level is not affected.In the experiment of using animal body 7-day-old Hu sheep to test the efficiency of the immune microencapsulated coli oral vaccine, it has been discovered that better cellular immunity could be attained when the amount of antigen dose oral high-powered group is 75.2 times higher than injection group. The content of the intestinal mucosa secretory immunoglobulin A(sIgA) of every test group lambs were all increased after the 28 th day of immunization and the sIgA of high-dose oral immunization of lamb is the highest which is 15.88 ?g/mL. Attack drug experiments, an injection group and oral group in the high dose group only for the number of lambs diarrhea 0, lamb immune protection.The result of this research shows:Microencapsulated sheep escherichia coli oral killed vaccine could stimulate lamb to effectively generate specific immune protection. Both mucosal immunity and humoral immune responses have fine.Additionally, this microencapsulated propolis adjuvant inactivated vaccine against E. coli have the possibility to be used in the prevention of diarrheal diseases lamb in production practice if the immunization dose and immunization time is optimized. |
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