Study On Preparation And Immune Efficacy Of A Bivalent Oral Vaccine Of Edwardsiella Ictaluri And Yersinia Ruckeri

Ictalurus punctatus is an important economic freshwater fish of China. In recent years, an increasing number of bacterial diseases plague the aquaculture of Ictalurus punctatus, which the Edwardsiella ictaluri disease and Yersinia ruckeri disease are particularly seriously endangering. Antibiotics are the main methods to prevent and control the bacterial diseases of fish, but this has been bringing some problems, such as bacterial resistance, drug residues, food safety and environmental pollution. Vaccines to control fish disease outbreaks are the most effective way. Currently, the use of natural polymer materials for carrier preparation of oral microsphere vaccine has become a hot topic. In the study, for the E.ictaluri disease and Y.ruckeri disease of I.ctalurus punctatus, sodium alginate was used as a vaccine carrier for preparing bivalent microspheres oral vaccine. Specific studies are as follows:1、Optimization of preparation conditions by response surface methodology and characterization of oral microsphere vaccineIn order to optimize the conditions of preparation in microsphere vaccine, sodium alginate and formalin-inactivated E. ictaluri were used as carrier material and antigen model respectively, and the response surface methodology was the way to optimization the preparation conditions. Then, the oral vaccines of E.ictaluri and Y.ruckeri were prepared through the optimized conditions. Single factors including sodium alginate concentration, water-oil ratio, emulsifier concentration and emulsification time which were investigated separately. On the basis of single-factor experiments, the relationships among main preparation conditions were modeled using a 4-factor,3-level Box-Behnken experimental design. The established model was analyzed by response surface methodology to obtain the optimal preparation conditions. All results showed that the optimal preparation conditions were as follows: sodium alginate concentration was 3.5%, water-oil ratio was 2.4:7.6, emulsifier concentration was 5.4%, and emulsification time was 13 min. Under these conditions, the encapsulation efficiency of microspheres were 96.37%(microspheres vaccine of E.ictaluri) and 94.51%(microspheres vaccine of Y.ruckeri). It matched with the predicted encapsulation efficiency. The microspheres had good form, homogeneous diameter particles distribution, and remarkable acid resistance, enteric and high security, which was prepared by response surface methodology. Its diameters were (8.88±1.26)μm, (8.76±1.73) μm, and span were 0.38,0.47. For the development of aquaculture vaccine provides a theoretical basis, the effectiveness of the vaccine will be continuing to study in subsequent trials.2、The immune efficacy of oral microspheres vaccineTo evaluate the immune effect of oral vaccine of E.ictaluri and Y.ruckeri, the oral microspheres vaccines were prepared by the optimal preparation conditions. After oral immunization with I.punctatus, the effect of the vaccine was evaluated by serum non-specific immune parameters, antibody titers and relative percent survival detection. Results showed that the total protein content, total superoxide dismutase activity (T-SOD) and serum lysozyme activity significantly increased in vaccinated I.punctatus. Serum agglutination titers of the microspheres vaccine of E.ictaluri peaked (1:16) at 5th week, and the specific antibodies could still be detected at 7th week. Serum agglutination titers of the microspheres vaccine of Y.ruckeri peaked (1:8) at 5th week and the specific antibodies could not be detected at the 8th week. Challenged by E.ictaluri and Y.ruckeri, the relative percent survivals of I.punctatus were 60.7%and 65.5%. In conclusion, the oral microspheres vaccine of E.ictaluri and Y.ruckeri played an important role in preventing E.ictaluri disease and Y.ruckeri disease.3、The immune efficacy of a bivalent oral microspheres vaccineTo evaluate the immune effect of bivalent oral vaccine of E.ictaluri and Y.ruckeri, the bivalent oral vaccine was prepared with the optimal preparation conditions. Three immunization doses were designed, in which bivalent microspheres vaccine as low-dose group, bivalent microspheres vaccine as medium-dose group and bivalent microspheres vaccine as high-dose group. After oral immunization with I.punctatus, the effect of the vaccine was evaluated by serum non-specific immune parameters, antibody titers and relative percent survival detection. To investigate the effects of oral vaccine on immune-related genes of I.punctatus, real-time PCR was used. Results showed that the bivalent oral vaccine can be more prolonged to boost non-specific immune function. Non-specific immune index of bivalent microsphere vaccines low, medium and high dose groups compared with control had significantly difference at 6th week (P<0.01). The serum agglutination titers of low dose bivalent microspheres vaccine group was higher than medium and high dose group in which the anti-E.ictaluri antibody and anti-Y.ruckeri antibody were 1:32 and 1:16, and they all peaked at 5th week. Challenged by E. ictaluri, the relative percent survivals of the three dose bivalent microspheres vaccine were 75.0%、67.9% and 60.7%, and challenged by Y.ruckeri, the relative percent survivals of the three doses bivalent microspheres vaccine were 65.5% 69.0% 65.5%. TNF-α, MHC-Ⅱβ and CD4L-2 expressed higher in low and medium dose groups of bivalent microsphere vaccines than others, and IL-1β1 of head kidney and spleen in low dose group expressed significantly higher than other groups (P<0.01) by real time PCR. In conclusion, the bivalent oral vaccine of E.ictaluri and Y.ruckeri can effectively boost immune function, which is a good vaccine to preventing E.ictaluri disease and Y.ruckeri disease of I.punctatus.