嗜水气单胞菌是淡水鱼类的重要病原菌，给淡水养殖业带来了巨大的经济损失，同时也给人类健康带来潜在威胁。嗜水气单胞菌可引起淡水鱼类的出血性败血症，具有高死亡率。因此，规模化养殖场嗜水气单胞菌免疫与防控尤为重要。口服免疫是规模化鱼类养殖中具有良好应用前景的免疫方式。但是由于胃肠复杂环境对疫苗抗原成分的降解，其免疫效果往往不佳。本研究的目的是利用生物相容性好的高分子聚合物制备包裹有细菌的微囊传递系统，开发嗜水气单胞菌口服疫苗。 以海藻酸钠 (Sodium alginate，SA)和壳聚糖(Chitosan)为材料，利用改良喷雾-离子交联技术制备嗜水气单胞菌微囊（Microsphere，MS）口服疫苗。通过对影响微囊粒径的细菌浓度、海藻酸钠浓度、喷雾速度及搅拌速度等主要技术参数进行正交筛选，确定最优的制备技术路线，并对该口服疫苗微囊的形态、直径、包被效率、载药量、体外模拟释放性、疫苗抗原稳定性、疫苗安全性、储存稳定性等特性进行检测。所确定的最优微囊制备参数为：SA浓度2%（W/V），细菌浓度5×109 cfu/mL，搅拌速度800rpm，喷雾速度7mL/min；微囊圆整，平均粒径为11.010±0.145μm，粒径分布较均匀，83%的微囊直径小于15μm；包被率约(96.52±0.17)%；载菌量为（2.41±0.32）×108cfu/mg；微囊在模拟胃肠条件下稳定，表现出较好的突释性与缓释性；微囊中细菌抗原性保持良好，疫苗安全性与储存稳定性较好。结果表明所开发的嗜水气单胞菌微囊传递系统各项特性较理想，有进一步开发的前景。 为了评价所研制的嗜水气单胞菌海藻酸钠-壳聚糖微囊（Ah-ACMS）口服疫苗在动物体内的免疫效果，采用不同剂量、不同剂型（有无佐剂）的Ah-ACMS疫苗分别口服免疫BALB/C小鼠，对免疫小鼠的血清抗体水平、巨噬细胞吞噬活性、淋巴细胞主要细胞因子相对表达量及淋巴细胞转化率等多个免疫指标进行了检测分析，并通过攻毒试验对疫苗的保护效率进行了评价。初次免疫四周后，高、中、低口服疫苗免疫组的各项免疫学参数与细菌口服对照组及空白对照组均差异显著（P＜0.05），高、中剂量口服疫苗组间大部分免疫参数差异不显著（P＜0.05）。Ah+SA+Ch口服组与空白对照组的脾淋巴细胞主要细胞因子表达及巨噬细胞吞噬活性等免疫指标差异显著。各免疫组小鼠的相对保护效率分别为：ACMS高剂量组46.7%；ACMS佐剂组53.3%；Ah腹腔注射组86.7%；而空白对照组与细菌口服对照组的小鼠100%死亡。结果表明所制备微囊口服疫苗对Ah感染具有一定的免疫抵抗力，所用口服佐剂(MonTanide IMS 1312)能显著增强微囊口服疫苗的免疫效果。 由于哺乳动物与鱼类的免疫系统和胃肠环境有较大差异，为了科学评价所开发微囊口服疫苗的对水生动物的免疫效果，分别采用不同剂量，不同剂型（佐剂有无）的Ah-ACMS疫苗分别口服免疫银鲫，以腹腔注射福尔马林灭活的Ah（FKC）为阳性对照。初次免疫四周后，对免疫银鲫的血清抗体水平、巨噬细胞吞噬活性等免疫指标进行了检测分析，并通过攻毒试验对疫苗的保护效率进行了评价。低剂量的口服Ah-ACMS组的各项免疫指标与空白对照组之间有显著差异（P﹤0.05）。佐剂可显著增强所开发微囊口服疫苗的免疫原性。佐剂Ah-ACMS组及腹腔注射组的相对保护率分别为：50.00% 、82.14%、,而FKC口服对照组与空白对照组鲫鱼死亡率分别为96.67%和93.33%。结果表明所开发的Ah-ACMS疫苗对鲫鱼具有一定的免疫效应，可保护鲫鱼抵抗嗜水气单胞菌的感染。 综上所述，本研究所开发的聚合物微囊传递系统可有效保护抗原的免疫原性，对复杂的胃肠环境耐受，可诱导小鼠及鲫鱼产生免疫应答。该研究以期能为后继的聚合物微囊口服疫苗的开发提供参考。有望进一步推广应用于其他鱼类病原菌口服疫苗的开发。

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 haemorrhagic 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）×108cfu/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 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(group Ⅰ) and the medium-dosage oral vaccine group (group Ⅱ) were not significantly different. 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 group I with 46.7%, group Ⅳ with 53.3%, group Ⅷ 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. 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 50 LD50 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 50.00% and 82.14 % respectively, while 93.33% of the negative control carps died. These results showed that the oral Ah-ACMS vaccine was immunogenic, 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 vaccine study using the polymers. The delivery system could been applied to other pathogens of fish.