Protective Efficacy Of Microencapsulated Enterotoxin Oral Vaccine Against Enterotoxigenic Escherichia Coli Infection In Weaned Rats

Diarrhea in weaned piglets is one of the most common diseases in intensive pig farms around the world,affecting the growth of piglets and causing huge economic losses to the pig industry.Enterotoxigenic Escherichia coli(ETEC)is the main pathogen of post-weaning diarrhea,and fimbriae and enterotoxins are the major pathogenic factors of ETEC.Currently,vaccination is an effective way to prevent diarrhea in weaned piglets.In this study,the micro-encapsulation technology was used to prepare an oral microencapsulated vaccine with recombinant enterotoxin as the main antigen component,and then the protective efficacy of the novel oral vaccine was evaluated in a rat model.The experimental content of this research mainly includes the following:The recombinant SLS enterotoxin was used as the core material of vaccine antigen,with natural polymer materials sodium alginate and chitosan as coating wall materials to prepare sodium alginate-chitosan SLS-protein microcapsules.Firstly,the effects of calcium chloride concentration,sodium alginate concentration,chitosan concentration and gelation pH value on the encapsulation rate of microcapsules were studied by single factor experiment.Orthogonal experiments were designed to optimize the preparation process of micro-encapsulated vaccine.The results showed that the best preparation condition is:the sodium concentration was 2%,the calcium chloride concentration was 3%,the chitosan concentration was 0.8%,and the gelation pH was 5.The sodium alginate-chitosan SLS-protein microcapsule antigen prepared under the optimal preparation condition showed an encapsulation rate of 83.43%,with a round shape.The release rate,swelling property and the effects of ionic strength on the swelling property of microcapsules were investigated in order to test whether the microcapsules could reach the intestinal tract and the release effect of microcapsules.The concentration of Na+ and Cl-could influence the space structure and the swelling rate of the micro-capsulated antigen.The release of antigen from microcapsules in gastric juice was about 16%and the microcapsules did not swell significantly.The microcapsules showed a good release effect in intestinal fluid,with the release amount and swelling rate were 87%and 2%,respectively,indicating that the microcapsules could protect the stability of antigen in gastric juice and maintain its biological activity,and it had a good release effect in the intestinal fluid and promoted the immune effect of the antigen.The experiment evaluated the immune effect of micro-capsulated vaccine on weanling pups.Three-week-old rats were divided into five groups,namely as follows:control group,uncoated group,coated group,propolis coated group and aluminum adjuvant coated group.The levels of specific antibodies and cytokines in the serum of rats after immunization were tested.The intestinal damage of young mice after ETEC challenge was also evaluated.Oral immunization with the micro-encapsulated vaccine induced an increase of specific antibody against ETEC enterotoxin three week post vaccination,and the IgG titer of the three coated groups were significant higher than control and the uncoated group(P<0.01).The level of IFN-γ,IL-2 and IL-6 cytokines of the coated groups were higher than those of the control and uncoated group.Two of the adjuvant coated groups maintained higher levels,and the propolis coated group had the highest cytokine levels.The increase of antibodies and cytokines suggested the microencapsulation could promote the antigen protein stimulate the body continuously and enhance the immune responses.The addition of adjuvant could further enhance the immune effect.The challenge experiment showed that oral with the coated vaccines could provide protection to rats and the survival rate were all above 80%.By contrast,the survival rate of control group was 30%.The results of histological observation were also indicated that the immunization with the microcapsules vaccines effectively protect the small intestines against ETEC invasion.In summary,the sodium alginate-chitosan SLS-protein microcapsules prepared in this study was able to elicit strong immune responses and provide protection to post-weaning rats against ETEC infection.