嗜水气单胞菌灭活疫苗免疫施氏鲟后的免疫应答反应与保护效果

为了研究嗜水气单胞菌灭活疫苗免疫施氏鲟(Acipenser schrenckii Brandt,1869)后的免疫应答反应与保护效果,本研究采用福尔马林灭活法制备嗜水气单胞菌灭活疫苗。通过腹腔注射途径免疫健康施氏鲟,对施氏鲟外周血的红细胞和白细胞数量、白细胞组成、吞噬活性、吞噬指数、血清酸性磷酸酶活性、血清溶菌酶活性、血清中和抗体效价等免疫指标及疫苗保护效果进行测定。结果显示,免疫组红、白细胞数量迅速上升,于免疫后第4天达峰值,分别为(8.50±0.17)×10~8个/mL和(8.96±0.44)×10~6个/mL;单核细胞和嗜中性粒细胞分类百分比于第7天达峰值,分别为10.50%和15.53%,极显著高于对照组(P0.01),淋巴细胞分类百分比在第21天才达峰值73.51%;吞噬指数和吞噬百分比均于第4天达到最高值,分别为4.53和30%;血清中溶菌酶活性和酸性磷酸酶活性分别在免疫后第7天和第14天达到峰值,极显著高于对照组(P0.01);血清抗体效价于免疫后第21天达到峰值1:203,极显著高于对照组(P0.01)。攻毒感染实验结果表明,免疫组的相对免疫保护率为76.84%。由此可见,嗜水气单胞菌灭活疫苗免疫施氏鲟后,能够显著诱导施氏鲟体内的免疫应答反应,增强鱼体的免疫保护能力。本研究为养殖鲟因嗜水气单胞菌感染引起疾病的免疫预防技术研究奠定了前期基础。     

嗜水气单胞菌灭活疫苗浸泡后鳜IgM基因表达量和抗体效价的变化

应用荧光定量PCR技术,研究了嗜水气单胞菌疫苗浸泡免疫后,鳜(Siniperca chuatsi)鳃、皮肤、脾脏和头肾中IgM基因表达量变化,同时应用ELISA检测皮肤黏液和血清中抗体滴度变化。结果显示:最早检测到IgMmRNA转录水平上调的是皮肤和鳃(第4天),而脾脏和头肾在第7天才达到高峰。IgM基因在头肾和脾脏中表达量较高(高峰值分别达到16.3和23.8),皮肤和鳃中的表达量较小(高峰值分别为4.3和8.6)。抗体效价方面,皮肤黏液中抗体滴度峰值出现时间较早(第7天),但抗体从开始形成到消失持续时间较短(28 d);血清中抗体滴度峰值出现时间较迟(第21天),但持续较长(42 d)。结果表明:浸泡免疫能够引发鳜机体和局部均出现免疫应答。从抗体滴度和IgM基因表达量方面考虑,系统免疫组织均高于黏膜免疫组织,表明前者是合成IgM的主要场所。从时间上比较,对抗原刺激最早做出应答的是皮肤黏膜和鳃,其后系统免疫才表现应答作用,这表明了局部黏膜能够在抗原入侵的早期起到抵制作用。     

甘露寡糖对草鱼感染嗜水气单胞菌后细胞因子表达的调节

在饲料中添加0.2%甘露寡糖(Mannan oligosaccharide,(MOS)),饲养草鱼(Ctenopharyngodon idellus)28 d后,草鱼经腹腔注射3.2×106cell/mL的嗜水气单胞菌(Aeromonas hydrophila,Ah)0.1 mL,在注射嗜水气单胞菌48 h、72 h和96 h后,分别取草鱼头肾、脾脏和肝脏,利用荧光定量PCR分析甘露寡糖对草鱼感染嗜水气单胞菌后各组织中IL-1β和IL-10的表达影响。结果显示,在草鱼头肾中,MOS/I(0.2%甘露寡糖+注射Ah)组IL-1β的表达在注射嗜水气单胞菌后48 h和72 h显著高于CON/I(基础饲料+注射Ah)组;MOS/I组中1L-10的表达在注射嗜水气单胞菌72 h显著高于CON/I组,在96h时显著低于CON/I组。在草鱼脾脏中,MOS/I组IL-1β的表达在注射后72 h和96 h显著低于CON/I组;IL-10的表达在注射嗜水气单胞菌后48 h和96 h显著低于CON/I组。在草鱼肝脏中,MOS/I组IL-1β和IL-10的表达在注射嗜水气单胞菌后48 h显著高于CON/I组,但在注射后72 h时该2种基因的表达显著低于CON/I组。表明甘露寡糖在鱼体感染嗜水气单胞菌时能快速调节头肾、脾脏和肝脏中细胞因子的表达,增强鱼体抵抗细菌性病原的免疫力。     

3月龄草鱼种免疫器官组织学及其免疫后免疫基因的转录差异

为研究处于同一时期但养殖条件不同的草鱼(Ctenopharyngodon idellus)鱼体规格与发育状态间是否具有一致性,观察压塘3月龄草鱼与普通3月龄草鱼头肾、中肾、肠道和脾脏的显微结构,并对压塘3月龄草鱼与普通3月龄草鱼进行腹腔注射疫苗处理,实时荧光定量PCR检测免疫7 d后、14 d后各期草鱼头肾、中肾、肠道和脾脏中IgM与MHCⅠ的表达量。结果显示,压塘3月龄草鱼各组织发育较普通3月龄草鱼迟缓。压塘3月龄草鱼与普通3月龄草鱼在免疫后,头肾、中肾、肠道与脾脏中IgM和MHCⅠ的表达量均有上升,且IgM与MHCⅠ的相对表达量峰值均出现在普通3月龄草鱼免疫14 d后的中肾中(IgM为74.51倍;MHCⅠ为56.02倍。结果表明,草鱼出血病活疫苗可以增强草鱼头肾、中肾、肠道和脾脏中IgM和MHCⅠ的表达,且在3月龄时期进行注射免疫效果最佳。     

投喂阿维拉霉素对鲤免疫应答的影响

将阿维拉霉素按照一定的浓度添加到饲料中,投喂经注射接种福尔马林灭活的嗜水气单胞菌(Aerom onashydrophila)菌苗的鲤,于免疫后的第7 d、14 d、21 d、28 d和35 d检测供试鲤血液中的白细胞吞噬活性、血清中溶菌酶活性、抗体效价、补体C3含量及免疫保护率(RPS),探讨了阿维拉霉素对受免鲤免疫应答的影响。结果表明,饲料中添加阿维拉霉素(4 mg/kg和8 mg/kg),投喂较长时间(≥28 d)会降低受免鲤的血液白细胞吞噬活性、血清中的溶菌酶活性和抗体效价,减少受免鲤血清中补体C3的含量,减弱鲤抵抗嗜水气单胞菌人工感染的能力,每公斤饲料中添加8 mg的阿维拉霉素其免疫抑制作用更强。     

嗜水气单胞菌疫苗免疫效果研究

选取对鲤鱼毒力强的嗜水气单胞菌菌株制成灭活疫苗,研究疫苗的免疫保护效应.采用甲醛灭活制备疫苗,分别对鲤鱼进行注射和浸泡试验,测定白细胞吞噬活性、淋巴细胞转化能力、血清、皮肤黏液的抗体效价及活菌攻毒后的免疫保护率.结果表明,受免鱼各项指标均明显高于对照组,注射组免疫保护率达90%,其中浸泡组各项略低于注射组.嗜水气单胞菌灭活疫苗对鲤鱼有显著免疫保护效应,可作为预防细菌性败血症感染的疫苗.     

山莨菪碱提高嗜水气单胞菌灭活疫苗浸泡免疫鲫的效果

为研究山莨菪碱作为佐剂在细菌灭活疫苗浸泡免疫中的作用,将山莨菪碱和嗜水气单胞菌全菌灭活疫苗联合浸泡免疫异育银鲫,首次浸泡免疫7d后加强免疫1次.通过实时荧光定量PCR检测第2、4、7、11、14和21天脾脏中IgM、IL-1β、C3、C-凝集素以及溶菌酶的mRNA表达量,并在第21天进行同源菌株活菌攻击实验.结果显示,山莨菪碱组第4天IgM和IL-1 β的表达量达到最高值,分别为28.3和332.7;而无佐剂疫苗组第11天IgM和IL-1β达到最高值,分别为56.1和791.8.山莨菪碱组的补体C3、C-凝集素以及溶菌酶的表达量都显著高于无佐剂组和对照组,且表达持续时间长.活菌攻击实验表明山莨菪碱组的相对免疫保护率可达80.0％,显著高于无佐剂组的56.0％.结果表明,山莨菪碱与嗜水气单胞菌灭活疫苗共同浸泡免疫银鲫,可以增强脾脏中IgM、IL-1β、C3、C-凝集素以及溶菌酶基因表达,提高银鲫相对免疫保护率.     

酵母免疫多糖对受免斑点叉尾(鮰)免疫应答的增强作用

在饲料中添加定量的酵母免疫多糖,投喂注射接种了福尔马林灭活的嗜水气单胞菌(Aeromonas hydrophila)菌苗的斑点叉尾(鮰)(Ictalurus punctatus),28 d后通过测定供试鱼的增重量、血清中凝集抗体效价、溶菌酶活性、谷丙转氨酶活性(SGPT)、血清总蛋白含量、白细胞吞噬活性以及活菌攻毒后的免疫保护率(RPS),探讨了酵母免疫多糖对受免斑点叉尾(鮰) 免疫应答的增强作用.结果表明,用添加200.0 mg/(kg·d)酵母免疫多糖的饲料投喂受免斑点叉尾(鮰),不仅可以使受免斑点叉尾(鮰) 对灭活嗜水气单胞菌的免疫应答水平提高,增强抵抗嗜水气单胞菌人工感染的能力,而且还具有一定的促进生长和改善肝功能的作用.     

酵母免疫多糖对受免斑点叉尾免疫应答的增强作用

在饲料中添加定量的酵母免疫多糖,投喂注射接种了福尔马林灭活的嗜水气单胞菌(Aeromonas hydrophila)菌苗的斑点叉尾(Ictalurus punctatus),28 d后通过测定供试鱼的增重量、血清中凝集抗体效价、溶菌酶活性、谷丙转氨酶活性(SGPT)、血清总蛋白含量、白细胞吞噬活性以及活菌攻毒后的免疫保护率(RPS),探讨了酵母免疫多糖对受免斑点叉尾免疫应答的增强作用。结果表明,用添加200.0 mg/(kg.d)酵母免疫多糖的饲料投喂受免斑点叉尾,不仅可以使受免斑点叉尾对灭活嗜水气单胞菌的免疫应答水平提高,增强抵抗嗜水气单胞菌人工感染的能力,而且还具有一定的促进生长和改善肝功能的作用。     

传染性造血器官坏死病灭活疫苗的制备及免疫保护效果

为了研发用于预防传染性造血器官坏死病(infectious hematopoietic necrosis,IHN)灭活疫苗,实验以不同感染复数(multiplicity of infection,MOI)在胖头鱥上皮细胞(Epithelioma papulosum cyprinid,EPC)上对传染性造血器官坏死病毒(infectious hematopoietic necrosis virus,IHNV)进行连续传代培养,通过测定各代病毒滴度,结合病毒收获时间确定最佳增殖方案;采用不同浓度的β-丙内酯(β-propanolactone,BPL)在24℃下灭活,经安全性实验验证后确定最佳灭活条件。以不同剂量腹腔注射免疫虹鳟,以磷酸盐缓冲溶液(PBS)为阴性对照组,通过检测攻毒后相对免疫保护率(relative percent survival,RPS)、免疫相关因子表达量及血清中和抗体效价来分析该疫苗的保护效果。结果显示,最佳增殖方案为以MOI为0.000 1接种,15℃培养3 d;最佳灭活条件为以终浓度3.0 mmol/L的BPL将IHNV在24℃下灭活24 h;以最佳免疫剂量10μL/尾腹腔注射免疫虹鳟,免疫后7、21、45和60 d的相对免疫保护率分别为91.37%、84.28%、84.15%和47.5%,免疫后60 d时RPS显著低于其他时间点免疫组;免疫相关基因的实时荧光定量PCR(realtime quantitative PCR,RT-qPCR)结果显示,与阴性对照组相比,Mx-1和IFN-γ表达量在免疫后7、15和30 d脾脏和头肾中均显著上调,在第7天时达到最大值(5倍);CD4和IgM表达量在免疫后15 d脾脏和头肾中均显著上调;在免疫后第30、45和60天虹鳟血清IHNV中和抗体效价依次为67.25、43.40和29.78,呈下降趋势且各组间差异显著。研究表明,该灭活疫苗可诱导虹鳟产生特异性免疫和非特异性免疫反应,对虹鳟具有显著的免疫保护作用。本研究为IHNV灭活疫苗研发提供参考。     

拥挤胁迫对已接种疫苗的大菱鲆部分免疫和应激指标的影响

通过对3个养殖初始密度(低、中、高密度组分别为5、10、20 kg/m~2)的大菱鲆(Scophthalmus maximus)接种减毒迟缓爱德华氏菌疫苗,观察其血液、脾脏、头肾、鳃和皮肤中部分免疫和应激指标的变化规律,研究经接种疫苗操作后大菱鲆应对拥挤胁迫的应激响应。结果显示,低密度养殖的白细胞介素(1L-1β)、补体(C3)、免疫球蛋白M(IgM)、溶菌酶(LZM)和肿瘤坏死因子(TNF-α)等免疫指标显著高于中、高密度组(P0.05),高密度组血液中皮质醇的含量高于中、低密度组(P0.05);通过基因表达分析发现,高密度组的热休克蛋白(HSP70)表达量显著高于中、低密度组;低密度组的免疫球蛋白M(IGM)表达量高于中、高密度组。研究表明,为提高大菱鲆的免疫效果、减少应激反应,可以采用低密度(5 kg/m~2)养殖。     

壳聚糖和益生菌对暗纹东方鲀抗病力和免疫功能的影响

为了解壳聚糖和益生菌刺激和增强免疫应答的可能作用,本试验在基础饲料中添加0.2%、0.5%、1.0%的壳聚糖,0.1%、0.2%、0.4%的益生菌,甘露聚糖与益生菌混合物,壳聚糖与益生菌的混合物,在室内水泥池中喂养暗纹东方鲀(3.15±0.05)g 2个月,根据抗菌活力筛选出免疫增强饲料组(0.2%壳聚糖,0.1%、益生菌,甘露聚糖与益生菌混合物,壳聚糖与益生菌混合物)测定免疫功能。结果表明,甘露聚糖与益生菌混合物仅提高头肾T淋巴细胞转化,壳聚糖与益生菌混合物显著增强头肾T、B淋巴细胞转化和脾脏B淋巴细胞体外培养时分泌的IgM。筛选出的四种免疫增强饲料均能使头肾B淋巴细胞体外培养时分泌的IgM显著增加。无论是脾脏B淋巴细胞还是头肾B淋巴细胞,体外培养时所分泌的IFN-α都不同程度地受抑制。     

初探母源免疫处理草鱼母本及其子代5种免疫因子含量的周年变化规律

为探究草鱼母源免疫因子传递特性,本实验借助ELISA和qPCR技术分别检测分析了GCRV弱毒疫苗免疫草鱼母本及其子代（即母源免疫子代）早期胚胎阶段至一龄期间5种免疫因子（IgM、C3、LZM、MBL和Bf）蛋白活性和基因表达水平的周年变化规律。结果显示,草鱼母本经GCRV弱毒疫苗免疫后,血液中5种免疫因子蛋白活性及基因表达水平在产卵期均显著高于对照组1.5倍以上,至免疫后第11个月与对照组无显著差异。母源免疫子代发育至水花（4 d）时,IgM、C3、MBL和Bf的基因表达水平均高于对照组;发育至乌仔（15 d）时,IgM、C3和Bf的蛋白活性水平均略高于对照组;发育至5~11月龄时,5种免疫因子在肝脏、脾脏、肾脏和头肾等免疫器官中仍存在高表达。研究表明,GCRV弱毒疫苗促使母源免疫因子在亲本和子代体内富集表达具有一定的时效性,最长可维持至11个月,为实际生产中草鱼母本疫苗免疫程序的制定提供参考依据。     

Study on the distribution and expression of a DNA vaccine against lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

The distribution and expression of lymphocystis disease virus (LCDV) vaccine, on the basis of DNA vaccine (pEGFP-N2-LCDV0.6 kb) construction, were analyzed in tissues of the Japanese flounder by PCR, RT-PCR and fluorescent microscopy. Results from PCR studies indicated that the vaccine-containing plasmids were distributed in injected muscle, muscle located opposite the injection site, hind intestine, gill, spleen, head kidney, liver and gonad 7 days after vaccination. However, these vaccine-containing plasmids disappeared by 90 days following vaccination. Fluorescent microscopy observations revealed that green fluorescence appeared in muscle, muscle located at the opposite side of the injection site, hind intestine, gill, spleen, head kidney and liver of fish 36 h after vaccination, and that green fluorescence did not appear in control tissue. The green fluorescence became weaker at 60 days post-vaccination, however, it remained detectable in the spleen 90 days post-vaccination. Results from RT-PCR studies indicated that the Mcp gene is expressed in all tissues of vaccinated fish 7-20 days after vaccination. These results demonstrate that the DNA vaccine is distributed and expressed in different tissues of vaccinated fish, and therefore, may have provided an antigen producing specific immune response.     

Changes in the spleen of the channel catfish Ictalurus punetatus Rafinesque indueed by infection with Aeromonas hydrophila

Abstract. A virulent, 2,3-butanediol positive culture of Aeromonas hydrophila , subspecies hydrophila , was fed or injected intraperitoneally into channel catfish. The positive infection and tissue involvement were monitored by the 2,3-butanediol test with tissue homogenate enriched with a buffered nutrient broth containing dextrose. The results revealed that the fish infected orally showed very little or no spleen involvement. Fish infected intraperitoneally, however, showed spleen involvement in a very short time, accompanied by the rapid onset of an acute systemic infection.
Light and electron microscope studies of the spleen revealed that the pathogenic bacteria were mainly located within the reticular sheath of the ellipsoids and only sparsely in the surrounding regions, and that intense phagocytic activity of macro-phages had taken place in the ellipsoids. It was also observed that the phagocytized bacteria divide extracellularly, and probably intracellularly, resulting in the destruction of the endothelial and reticular cells of the ellipsoids. This finding substantiates the cytolytic and fibrolytic capacities of A. hydrophila for destroying host defence systems and may contribute to its invasive powers. Other cytological changes in cells in spleen after infection are described, and the resistance of A. hydrophila to intracellular enzymatic digestion by the macrophages is discussed.     

Adjuvant effect of mushroom glucan and bovine lactoferrin upon Aeromonas hydrophila vaccination in catla, Catla catla (Hamilton)

Mushroom glucan and bovine lactoferrin (Lf), known for their immunostimulatory potential, were used as adjuvant in conjunction with a formalin-killed Aeromonas hydrophila vaccine in catla, Catla catla. In vitro antigen-specific responsiveness of catla leucocytes and protective responses against experimental challenge with homologous antigen were monitored following immunization. Antigen-specific proliferation, 'macrophage activating factor' (MAF) production and antibody production were significantly higher in fish injected with glucan adjuvanted vaccine. Lf adjuvanted preparations showed a weak proliferative response and MAF production, although the antibody production was significantly higher than the controls. A good degree of protection was achieved with the glucan adjuvanted vaccine. However, in spite of producing significant anti-A. hydrophila antibody, Lf adjuvanted vaccine did not confer any protection following challenge with A. hydrophila. The potential of adjuvanticity of mushroom glucan and bovine Lf in intraperitoneal vaccination is discussed.