Preparation And Oral Immunization Of Microencapsulated Killed Vaccine Against Grass Carp Hemorrhage

Grass carp hemorrhage is the most serious disease of grass carp cultured inChina, the causative pathogen is the grass carp reovirus (GCRV). Grass carphemorrhage has been spreading in a wide range of areas in China with long durationand high mortality, and caused huge economic losses. Grass carp hemorrhage hasbecome a great threat to the healthy development of the industry of grass carp culture.Therefore, it is of importance to explore the effective measures for preventing andcontrolling grass carp hemorrhage. At present, immunization with vaccine againstgrass carp hemorrhage is reported the most effective method through intraperitonealinjection and immersion routines. Because of easy delivery, non-limited of fish sizeand farming scale, more attentions has been paid on the oral vaccine development forfish. However, due to the antigenic protein might be destructed easily by digestiveenzyme in vivo, the development and application of oral vaccine for fish is limitedcurrently. The development microencapsulated technology and the application ofmicroencapsulated vaccine reduced the loss of vaccine antigenicity, and enhanced theimmune effect, therefore, study of the microcapsule vaccine has been given more andmore attention.A complex co-acervation method was applied to microencapsulate killed vaccineagainst grass carp hemorrhage by using Sodium alginate and Chitosan as the wallmaterials, and the killed vaccine of grass carp hemorrhage as microcapsule corematerial. Firstly, by single factor experiment, five factors which affectmicroencapsulated vaccine entrapment efficiency were studied, including theconcentration of sodium alginate, chitosan concentration, calcium chlorideconcentration, pH of chitosan solution and adding vaccine volume. Throughorthogonal test, the optimal preparation technology of oral SA-CS microencapsulated vaccine was determined. Finally, morphology observation and releasing test ofmicroencapsulated vaccine in vitro, and the properties of resistance to acid and basewere studied. The results of single factor test showed that, in the preparation ofSA-CS microencapsulated vaccine, sodium alginate concentration should bemaintained at around1.5%, chitosan concentration should be maintained at about1%,and pH of chitosan solution should be maintained at4-5, calcium chlorideconcentration should be maintained at4%, and the optimal dose of vaccine is2mL;the affecting degree of the factors for the microencapsulated vaccine entrapment ratefrom large to small is: sodium alginate concentration> vaccine dosage> chitosanconcentration＞calcium chloride concentration. The optimum preparation ofmicroencapsulated vaccine is: sodium alginate concentration1.5%, chitosanconcentration1%, chloride calcium concentration4%, vaccines volume2mL. Theresults of performance in vitro of the microencapsulated vaccine showed that, themicroencapsulated vaccine presented in homogeneous round under light microscope,withan average particle size of (7.023.95) μm. The average encapsulation rate was60.53%, the average carrier protein was8.12mg/g, and the microencapsulated vaccinehad a good releasing performance in PBS solution (pH7.4) and physiological salinesolution.The grass carp were immunized with SA-CS microencapsulated vaccine storedwith different time. By the serum neutralizing antibody titer detection, the stability ofthe microencapsulated vaccine was studied. After immunizing the grass carp, it wasconfirmed that the vaccine was uptook in grass carp intestinal by indirectimmunofluorescence technique. The blood and splanchnic tissues (liver, spleen andkidney) of grass carp were taken to compare the differences between microcapsulesimmune group, cell vaccine oral group and control group. By real-time fluorescentquantitative PCR method, the expression of the immune gene IgM and Mx of grasscarp were analyzed to determine the immune effect of microcapsule vaccine. Throughthe GCRV104challenge test, the immune protection of the microencapsulated vaccinewas determined. Results of the stability study of SA-CS microencapsulated vaccineshowed that, grass carp immunized with SA-CS microencapsulated vaccine stored at4℃for1month,3months,6months of were no significant difference between the3groups after detection of serum antibody titer(P＞0.05), which illustrated themicrocapsule vaccine can be stored at4℃at least6months; After immunization,results of serum antibody titers detection showed that, at7d, the antibody level of cell vaccine oral group was significantly higher than that of SA-CS microencapsulatedvaccine group (P <0.05); at14d, the immunized groups were maintained at a highlevel, the difference was not significant (P>0.05); at21d, SA-CS microencapsulatedvaccine group was significantly higher than that of cell vaccine group (P <0.05).Results of immune gene expression detection showed that, at7d, IgM of theimmunized group was significantly higher than that of the control group (P <0.05),and at14d, was significant difference (P <0.01); the expression level of Mx in theimmunized group is significantly higher than that in control group at3d (P <0.05),the difference is significant (P <0.01) at7d. Results of challenge test showed that, themortality rate of SA-CS microencapsulated vaccine group was lower than that ofcontrol group, and relative percent survival (RPS) of the microencapsulated vaccinewas62.5%.