| Porcine Epidemic Diarrhea (PED) is characterized as one important Viral intestinal disease which causes early death of the piglets and result in severe economic loss for swine industry. At present, conventional PED vaccines are not satisfactory in controlling PED’s regional prevalence process considering that they cannot effectively stimulate the body to produce enough sIgA, which plays an important role in anti-PEDV infection process. Therefore novel vaccines which can induce strong mucosal immunity, and require cellular and humoral immune responses are needed to effectively prevent epidemic of PED in China.In our study, we successfully prepared recombinant microencapsulated attenuated Salmonella by utilizing the constructed recombinant attenuated Salmonella(S.C 500/pVAX1-SAB、S.C500/pIRES-M-N) and sodium alginate with emulsion method. The process optimization and property analysis was performed and then piglets were orally immunized to evaluate the immune protect effect. The experiment was divided into three parts as follow:1.Preparation of recombinant attenuated Salmonella microcapsulesOrthogonal experiment based on single factor experiment was designed to optimize the preparation of microcapsules and the optimal preparation condition is: Sodium alginate concentration 1.5%, bacteria microcapsule ratio 1:6, Emulsifier content 0.4%, CaCl2 concentration 0.1%, mixing rate:600rpm, emulsification time: 10 min. under this condition, the embedding rate and bacterial content are 34.1%,4.31 ×109CFU/g respectively.2.Property examination of recombinant attenuated Salmonella microcapsulesPrepared microcapsules were treated in artificial gastric juice, artificial bile, artificial intestinal fluid and simulated human digestive tract environment respectively to examine microcapsule’s tolerance of acid, bile and bacteria-releasing efficacy. The results revealed that 4h after treatments in artificial gastric juice, the survival rate was 73.21%; while in artificial bile salt solution, the survival rate was 74.07%, the latter is significantly higher than that of recombinant attenuated Salmonella without microencapsulation (p<0.05); Microcapsule disintegration occurred after treatments in simulated human digestive tract environment for 120 min, suggesting successful release of bacteria body and satisfactory enteric solubility. The survival rate was 55.17%for Microencapsulated Salmonella after treatment in artificial digestive environment, while for the Salmonella without encapsulation the survival rate was only 5.42%. This proves that the microencapsulation can help reduce Gastric juice and bile’s destruction effects on the bacteria body and the body can successfully reach the intestine.3.Effect of oral immunization of piglets with Recombinant attenuated Salmonella microcapsulesIn order to evaluate and compare the effect of our prepared attenuated Salmonella microcapsules. We collected the PEDV antibody level, Salmonella antibody level, cell factor IL-4, IFN-γ level from serum and the dynamic changes of IgA from piglets stools in different immune periods by using ELISA kit and by immunizing piglets with the microencapsulated recombinant attenuated Salmonella, recombinant attenuated Salmonella without encapsulation, microencapsulated bacteria without vector, bacteria without vector nor encapsulation with 8.62×109CFU for each, and PEDV inactivated vaccine with 1.5ml for each. Every two weeks, we collected serum and stools from immured piglets to detect proliferation of T lymphocyte. Peripheral blood lymphocyte proliferation results show that 42 days after immunization, there was no significant difference between recombinant attenuated Salmonella microcapsule group, recombinant Salmonella without encapsulation group, microencapsulated bacteria without vector group, bacteria without vector nor encapsulation group as well as inactivated vaccine group(p>0.05).The blood serum examination result shows that the PEDV antibody level from inactivated vaccine group was significantly higher than the microencapsulated Salmonella group throughout the process (p<0.05), and 42 days after immunization, PEDV antibody level of the latter was significantly higher than that of recombinant Salmonella group without encapsulation, microencapsulated bacteria without vector group, bacteria without vector nor encapsulation group as well as the control group (p<0.05). The Salmonella antibody level result detected in immunized piglets serum shows that after 42 days, the microencapsulated Salmonella group’s antibody level was significantly higher than the inactivated vaccine group, recombinant Salmonella group without encapsulation, bacteria without vector nor encapsulation group as well as the control group (p<0.05). The IgA antibody level result from stools of the immune piglets shows that from the 42th day, the IgA antibody level of the microencapsulated Salmonella group was consistently and significantly higher than that of the inactivated vaccine group, microencapsulated bacteria without vector group, bacteria without vector nor encapsulation group as well as the control group (p<0.05). The Cell factor IL-4, IFN-Y from the blood serum result show that IL-4 level in serum from Microencapsulated Salmonella group rise during the immune process and was significantly higher than the recombinant Salmonella group without encapsulation, bacteria without vector nor encapsulation group as well as the control group(p<0.05), and there was no significant difference between the microencapsulated Salmonella group and the inactivated vaccine group as well as microencapsulated bacteria without vector group(p>0.05). IFN-γ level in serum from Microencapsulated Salmonella group was significantly higher than the control group (p<0.05), no significant difference was detected with recombinant Salmonella group without encapsulation, bacteria without vector nor encapsulation group, microencapsulated bacteria without vector group as well as the inactivated vaccine group (p>0.05). The challenge experiment to the piglets results show that the immunized piglets received satisfactory protection.Our results show that Microencapsulated recombinant attenuated Salmonella can help the bacteria body avoid the destruction from the Gastric acid and bile so that the plasmid can be delivered into the immune system of the piglets after the bacteria body reaches Intestines, this process can help stimulate humoral immunity, cellular immunity and mucosal immunity. Thus, our study can offer a foundational guidance for developing a safe and effective oral vaccine for PED. |
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