Study On Preparation And Immune Efficacy Of Chitosan-coated Alginate Microspheres Incorporating Aeromonas Hydrophila

Aeromonas hydrophila is one of important pathogens of freshwater fish and has made substantial economic losses for fish industry and became a potential threat to human health. Possible consequences of A.hydrophila infection to fish are skin lesions,which can result in haemorrhagie septicemia,followed by high mortalities.Control actions against A. hydrophila should be strongly considered during intensive fish culture.Oral vaccination is a relevant alternative for fish immunization in intensive culture.However,its effectiveness is limited by possible vaccine degradation in the fish digestive system.The purpose of this study was to obtain stable biocompatible alginate-chitosan microspheres entrapping inactive Aeromonas hydrophila cells for fish oral immunization.The microspheres were prepared through modified spraying and ion cross-linking method,using chitosan and sodium alginate.The best preparation method was established by varying the main factors such as the initial A.hydrophila concentration,the concentration of sodium alginate,the spraying rate,the stirring rate,which could affect the mean diameter of the microspheres.The characteristics of the microspheres such as the average diameter, size distribution,morphology,the percentage of bacteria incorporation,the release characteristics in vitro,safety property and stability of storage etc.were evaluated in vitro. The optimization parameters for the preparation of the microspheres are as following,2% sodium alginate(w/v),the initial concentration of bacteria as 5×10⁹ cfu/mL,stirring at 800rpm,spraying at 7mL/min.The optimization microspheres stable under gastrointestinal conditions presenting mean diameters 11.010±0.145μm and good safety property are spherical and homogeneous with bacterium encapsulation efficiencies up to(96.52±0.17)%and bacterium loading capacity up to(2.41±0.32)×10⁸ cfu/mg;The antigenicity of the bacteria entrapped in microspheres was not significantly affected in the entrapment procedure.The microspheres vaccine was stable under difference temperature conditions(-20℃,4℃,25℃) for more than 9 months.The results showed that the several characteristics of the microsphere antigen delivery system developed are satisfying.To evaluate the immune response and protective efficacy of vaccination with the oral microspheres vaccine prepared according the optimized preparation methodology in mice, BALB/c mice were immunized with oral Ah-ACMS vaccines at the difference doses and medicaments.Several other vaccines were used as control.In the fourth week after the first immunization,the several immune parameters,such as the serum antibody titers, macrophage bactericidal activity,spleen lymphocyte proliferation activity,main cytokines relative expression and relative percent survival etc.were investigated.The immune response and protective efficacy of vaccination with oral Ah-ACMS vaccine in mice were evaluated.The immunological parameters of mice immunized with different dosage Ah-ACMS vaccines were significantly higher than those of the Ah directly oral vaccination group and control group(P＜0.05).The most immunological parameters between the high-dosage oral vaccine group(groupâ… ) and the medium-dosage oral vaccine group (groupâ…¡) were not significantly different(P＞0.05).The bactericidal activity of celiac macrophages and main cytokines expression of lymphocytes of mice immunized with the mixture of Ah,chitosan and sodium alginate were significantly different with those of the control mice.The RPS of immunized mice were groupâ… with 46.7%,groupâ…£with 53.3%, groupâ…§with 86.7%.100%of mice immunized orally with killed-Ah and the control mice died.The results showed the developed oral microspheres vaccine can provide protective immunity against Ah in mice.The oral adjuvant can strengthen the immunological effect of developed Ah-ACMS vaccine,making the RPS of the immunized mice higher 13.3%.To evaluate the immune response and protective efficacy of vaccination with the developed oral microspheres vaccine in aquatic animals,then carp(Carassius auratus gibelio) were immunized with oral Ah-ACMS vaccines at the difference doses and medicaments.Formalin-killed Ah vaccines(FKC) was injected intraperitoneally as positive control.In the fourth week after the first immunization,the several immune parameters, such as the serum antibody agglutination titers,macrophage bactericidal activity,relative percent survival post-challenge with 50LD₅₀ bacteria(strain Ah-J1),were investigated.The immune response and protective efficacy of vaccination with oral Ah-ACMS vaccine in carps were evaluated.The serum agglutination antibody titers and bactericidal activity of the macrophages in the carps orally immunized with low-dosage vaccine were significantly higher than those of negative control group.The oral immunization with Ah-ACMS induced cellular immune and antigen specific antibody responses.The oral adjuvant(MonTanide IMS 1312) could significantly enhance the immunogenicity of the developed Ah-ACMS vaccine.The RPS of carps immunized orally with the adjuvant-Ah-ACMS and intraperitoneally with FKC was 50.0%and 82.1%respectively,while 93.33%of the negative control carps died.These results showed that the oral Ah-ACMS vaccine was immunogenie,and could confer protective immunity in carps.The results of the study showed the microspheres delivery system can protect the antigenicity of the bacteria and keep the bacteria intact in gut.It could confer protective immunity in mice and fish.The study provides reference for the sequent oral microsphere vaccine study using the polymers.The delivery system could been applied to other pathogens of fish.