维氏气单胞菌菌蜕疫苗免疫后鲤免疫应答与hepcidin基因表达特征

【目的】为探讨接种维氏气单胞菌菌蜕疫苗后鲤的应答反应特征及与甲醛灭活苗的效果比较,在实验条件下研究了接种疫苗后鲤(Cyprinus carpio)的特异性、非特异性免疫指标及hepcidin基因表达量的变化。【方法】在开始实验的第1、15和29天使用菌蜕疫苗(CLGs)和甲醛灭活疫苗(FKC)对鲤进行注射3次免疫,并设一个注射磷酸缓冲液(PBS)的对照组,每个组设3个重复。从第一次免疫后每隔7 d从每个重复中随机取三尾鱼进行取样,对血清抗体滴度、相对免疫保护率(RPS)、溶菌酶(LZM)、呼吸暴发、补体C3、髓过氧化物酶(MPO)、丙二醛(MDA)及hepcidin基因表达量进行测定。【结果】二次免疫后,CLGs组抗体凝集效价达2~6-2~8,显著高于FKC和PBS组(P0.05),且其呼吸暴发、LZM、MPO含量和hepcidin基因表达量显著高于PBS组(P0.05),MDA含量显著低于PBS组(P0.05);CLGs组C3含量在35和42 d显著低于PBS组(P0.05);FKC组MPO含量和hepcidin基因表达量显著低于菌蜕组(P0.05)。CLGs组的RPS为57.70%,FKC组RPS为30.77%。【结论】接种一定强度维氏气单胞菌菌蜕疫苗,能够提高鲤血清抗体效价和呼吸暴发、LZM、MPO等非特异性免疫指标及hepcidin基因表达,而且整体免疫效果好于甲醛灭活苗。     

三种创伤弧菌免疫原的制备及其对黄姑鱼的免疫保护效果

从创伤弧菌(Vibrio vulnificus)中提取菌体脂多糖(lipopolysaccaride,LPS)和外膜蛋白(outer membrane protein,OMP)并制备福尔马林灭活全菌苗(FKC),腹腔注射接种黄姑鱼。分别在注射第0、7、14、21和28天后测定了受免鱼血清中凝集抗体效价、血清溶菌酶活性和血液白细胞吞噬活性,以及免疫28d后的相对免疫保护率。结果表明,3种抗原对黄姑鱼均有较强的免疫原性。免疫后,免疫组血清凝集抗体效价逐渐增高,第28天时最高;溶菌酶活性(LMZ)、白细胞吞噬活性(PP)和吞噬指数(PI)显著升高(P<0.01),第21天达到峰值,随后逐渐下降。各组之间比较表明,受免后7、14、21和28d,免疫组黄姑鱼凝集抗体效价、PP、PI和LMZ显著高于对照组(P<0.05),LPS和OMP组凝集抗体效价低于FKC组,LPS和OMP组的相对免疫保护率高于FKC组,各组间免疫保护率大小顺序为LPS组>OMP组>FKC组>对照组。     

鲁氏耶尔森氏菌口服微球疫苗特性分析及免疫效果研究

为制备并评价鲁氏耶尔森氏菌口服微球疫苗的免疫效果,实验采用天然高分子聚合物海藻酸钠为疫苗载体,鲁氏耶尔森氏菌灭活疫苗为抗原,制备鲁氏耶尔森氏菌口服疫苗。以拌料口服方式进行免疫,通过血清非特异免疫指标、抗体效价、免疫保护率等综合评价疫苗的免疫效果。结果表明,鲁氏耶尔森氏菌口服微球疫苗成球性好,粒径均匀,粒径(8.761.73) m,跨距0.47,包封效率94.51%,具备良好的抗酸性、肠溶性及高安全性的特点。将疫苗免疫斑点叉尾,能显著提高斑点叉尾血清中溶菌酶活力、总超氧化物歧化酶活力(T-SOD)以及补体替代途径活性(ACH50);血清凝集效价于第5周达到峰值为1:8,至免疫后第8周仍能检测到特异性抗体;口服鲁氏耶尔森氏菌微球疫苗的斑点叉尾获得的抗鲁氏耶尔森氏菌相对免疫保护率为65.52%。综上所述,实验制备的鲁氏耶尔森氏菌口服微球疫苗能够对鲁氏耶尔森氏菌病起到较好的预防作用。       

嗜水气单胞菌菌蜕的制备及其对银鲫的口服免疫

菌蜕系统是一个自身具有佐剂性质的新型疫苗体系,不含细胞质内容物但具有细菌的完整表面抗原结构,可诱导机体的体液、细胞免疫应答及增强黏膜免疫反应.本研究通过将带有裂解基因E的质粒pElysis转化至嗜水气单胞菌J-1株中,对Ah J-1(pElysis)进行温度诱导,温度从28℃升至42℃,每隔15min检测菌液的OD600值,测定其溶菌动力学,并做无菌检验,用扫描电镜观察裂解后的细菌形态,研究其作为口服疫苗对银鲫的效果.结果显示,通过温度诱导,嗜水气单胞菌J-1(pElysis)OD值在诱导30min后开始持续下降,75min时开始趋于平稳,到120min溶菌效率达99.99%,诱导16h后进行无菌检验,证实其无活菌.扫描电镜观察绝大部分菌体经诱导后形成菌蜕,细胞两端有溶菌通道.动物试验表明,用菌蜕口服免疫的银鲫,在第5周产生较高的凝集抗体,达到27,并能维持2周;而甲醛灭活苗组为26,维持时间仅一周;生理盐水对照组效价仅2.攻击试验表明,菌蜕疫苗组和甲醛灭活疫苗组对嗜水气单胞菌强毒株J.1的攻击均有保护作用,其相对保护率分别为16/20(78.95%)和12/20(57.9%),显示菌蜕疫苗比普通灭活疫苗能更有效地激活机体的免疫保护.     

重组大肠杆菌菌蜕与幽门螺杆菌蛋白质疫苗协同免疫BALB/c小鼠

目的:考察重组菌蜕和蛋白质疫苗混合后对BAB/c小鼠机体免疫反应的影响。方法:首先构建展示尿素酶B抗原表位的大肠杆菌菌蜕,同时通过融合PCR构建分子内佐剂蛋白质疫苗rLTBKAT,然后分别利用该菌蜕和蛋白质疫苗免疫BAB/c小鼠。结果:当rLTBKAT和重组菌蜕联合口服免疫BAB/c小鼠时,其抗尿素酶B抗体的效价由单独免疫时的1∶(239±23)提高到1∶(681±76),二者差异极显著(P=0.009);而其抗过氧化氢酶抗体的效价则由单独免疫时的1∶(2800±275)下降至1∶(1800±400),二者差异不显著(P=0.08)。抗体分型试验表明,单独和联合免疫时,其抗尿素酶B抗体类型均以IgG1为主,而抗过氧化氢酶抗体类型则由单独免疫时的IgG1为主转变为联合免疫时的IgG2a为主。联合免疫时,小鼠抗尿素酶B和抗过氧化氢酶IgA抗体水平均高于单独免疫时。结论:重组菌蜕和蛋白质疫苗联合免疫,可提高机体对某些抗原的免疫水平或改变其反应类型。     

迟缓爱德华菌抗独特型单克隆抗体的制备及鉴定

迟缓爱德华菌(Edwardsiella tarda)是肠杆菌科爱德华菌属的一种条件致病菌,可引起败血病,危害鲆、鲤等经济养殖鱼类。但是,经过20余年的研究,虽有各种灭活疫苗和组分疫苗的试验,却无针对该菌病的商品化疫苗的使用报道。抗独特型抗体疫苗技术已成功用于肿瘤、细菌、病毒、寄生虫的防治,具有安全、易大规模生产等优点。鉴于药物防治鱼菌病的局限性,作者在制备了抗迟缓爱德华菌单克隆抗体的基础上,制备抗该菌的抗独特型单克隆抗体,用该菌抗独特型抗体免疫牙鲆, 为预防该菌病提供一种安全有效的新方法。       

利用展示尿素酶B表位的大肠杆菌菌蜕递送幽门螺杆菌核酸疫苗

探讨和分析了重组大肠杆菌菌蜕递送系统能否进一步提高幽门螺杆菌核酸疫苗的免疫应答水平.首先构建了展示尿素酶B表位的重组大肠杆菌菌蜕,然后以此菌蜕包裹幽门螺杆菌核酸疫苗pcDNAKAT,经DNA凝胶电泳、荧光显微镜观察和FACS分析,表明该质粒DNA可以非特异地结合到大肠杆菌菌蜕内膜上.该核酸疫苗经重组菌蜕包裹后免疫小鼠,其血清抗过氧化氢酶抗体效价由单独免疫时的1∶(307±39)提高到1∶(520±54),二者差异极显著(P=0.005 8),结果表明利用菌蜕递送系统能较好地提高核酸疫苗的免疫应答水平.     

迟钝爱德华氏菌菌蜕系统的构建

菌蜕系统（Bacterial Ghost,BG）的形成是利用噬菌体PhiX174的裂解蛋白E在革兰阴性菌细胞膜形成一个跨膜孔道结构,使细菌胞内物质由孔道排出而引起死亡。这种基因灭活的过程不引起细菌表面结构的任何理化变性,因此生成的细菌空壳具有与活菌相同功能的膜抗原结构,可诱导机体的体液免疫和细胞免疫应答。检测和比较了在铁调控启动子PyncE和温度调控启动子PR/cI控制下的E基因对迟钝爱德华氏菌菌蜕系统（EBG）的生成效率。结果显示,2种启动子均能成功生成EBG,电镜下可观察到细菌两端有直径约为80～400 nm的孔洞。传统菌蜕系统所用的热启动子在诱导后3 h开始裂解,8 h后细菌停止死亡;而新型铁诱导启动子在诱导后2 h细菌即完全停止生长。本研究为将来开发菌蜕载体疫苗防治爱德华氏菌症奠定了基础。     

不同多糖对史氏鲟非特异性免疫反应的影响

本文旨在探讨注射不同多糖后史氏鲟非特异性免疫反应的差异。分别将4种不同来源的多糖（壳聚糖、水苏糖、酵母聚糖和米糠脂多糖）腹腔注射到史氏鲟体内,注射9d后,观测血液中淋巴细胞α－醋酸萘酯酶（α－naphthyl acetate esterase,ANAE）阳性率、血清溶菌活性（Bacteriolytic activity）和血清旁路补体途径溶血活性（ACP hemolytic activity）。结果显示壳聚糖（Chitosan）在几种多糖中免疫刺激作用最强。壳聚糖组与对照组相比,所有的免疫指标活性均有显著提高。壳聚糖组ANAE活性和溶菌活性与其他实验组相比也有显著提高。水苏糖（Stachyose）组、酵母多糖（Yeast polysaccharide）组和脂多糖（LPSR）组与对照相比血清旁路补体途径溶血活性增强,而对ANAE活性和溶菌活性没有显著影响。     

菌蜕系统作为新型渔用疫苗体系的研究进展

菌蜕系统作为一种新型疫苗体系,是通过对噬菌体PhiX174裂解基因E的精确表达调控建立的.细菌菌蜕兼顾了组合抗原免疫原性、佐剂效应、靶向性载体等作用,特别适合于黏膜免疫及口服免疫;由于缺乏内含物而更加安全;生产过程简单,适宜大规模生产.这些特性决定了细菌菌蜕是一种具有良好应用前景的候选疫苗及递送系统.近年来随着水产养殖业的发展,鱼类细菌性疾病大规模爆发并造成严重损失.鉴于化学药物和常规疫苗的种种缺陷,新型渔用疫苗的研究日渐受到人们重视.在阐述细菌菌蜕系统形成和调控机制的基础上,着重介绍菌蜕疫苗在几种鱼类细菌性病害防治中的研究进展,并对菌蜕系统作为新型渔用疫苗体系的可行性和优越性进行讨论,对其应用前景进行展望.相信随着研究的深入,渔用菌蜕疫苗将在水产养殖病害防治中发挥着越来越重要的作用.     

Protection of tilapia (Oreochromis mosambicus) from edwardsiellosis by vaccination with Edwardsiella tarda ghosts

The vaccine potential of Edwardsiella tarda ghosts produced by gene E mediated lysis was investigated using tilapia (Oreochromis mosambicus). Tilapia immunized with E. tarda ghosts (ETG) and formalin killed E. tarda (FKC) vaccines showed significantly higher serum agglutination titers than control fish. Fish immunized with ETG showed no significant differences with fish immunized with FKC in serum agglutination titers, but showed significantly higher bactericidal activity than fish immunized with FKC. Furthermore, fish immunized with ETG showed higher protection than fish immunized with FKC. As this promising type of a non-living whole cell envelope preparation seems to be favorable over conventional vaccines, we suggest E. tarda ghosts as a new vaccine candidate.     

Effect of oral immunization with Aeromonas hydrophila ghosts on protection against experimental fish infection

Aims:  To investigate whether oral immunization with Aeromonas hydrophila ghosts (AHG) vaccine can elicit mucosal and systemic immune responses of Carp ( Carassius auratus gibelio ) compared to conventional formalin-killed bacteria (FKC).
Methods and Results:  Fish were fed diets coated with AHG, FKC or phosphate buffered saline (PBS) alone, after immunization, more antigen-specific antibody was significantly detected in serum and intestinal mucus in AHG group than FKC group and PBS group. In addition, after challenged with the parent strain J-1, the survival of bacterial ghost-vaccinated fish was higher than PBS group and FKC group, the relative per cent survival (RPS) being 76·8%, 58·9%, respectively.
Conclusions:  Oral immunization with A. hydrophila ghosts can elicit systemic and mucosal adaptive immune responses and has higher potential to induce protective adaptive immunity than normal vaccine.
Significance and Impact of the Study:  Oral immunization with bacterial ghosts is a promising new solution with potential application to prevent diseases in fish.     

Evaluation of biofilm of Aeromonas hydrophila for oral vaccination of Clarias batrachus--a carnivore model

Biofilm of Aeromonas hydrophila was evaluated for oral vaccination of walking catfish (Clarias batrachus L.). Fish were fed with fish paste incorporating biofilm (BF) or free cells (FC) of A. hydrophila for 20 days and monitored for serum antibody production up to 60 days post-vaccination. Serum agglutinating antibody titre and relative percent survival (RPS) following challenge were found to be significantly higher in catfish fed with BF vaccine compared to that with FC.     

家蚕蛹表达的重组Vp28疫苗对克氏原螯虾的抗病毒保护效应

对虾白斑综合症病毒(WSSV)的致病性强、危害性大、地域分布和宿主范围广泛,目前还不能有效地控制疫情。将含有WSSV囊膜蛋白Vp28基因的重组杆状病毒HyNPV-Vp28感染家蚕(Bombyx mori)蛹,对发病蚕血淋巴进行SDS-PAGE和Western blotting分析,结果表明Vp28在家蚕体内得到了表达。将重组病毒囊膜蛋白rVp28疫苗配制成药饵,持续口服免疫75天,对克氏原螯虾进行预防WSSV,实验虾分为2%重组Vp28疫苗、2%普通蚕蛹组织匀浆(阳性对照)和普通饵料(阴性对照)3个处理组。免疫35天后进行口服攻毒,20天内rVp28疫苗组的累积存活率为63.33%,与阳性和阴性对照比差异显著(P<0.05),PRP分别达54.16%和59.26%;注射攻毒后20 天内rVp28疫苗组的累积存活率与阳性和阴性对照组比差异不显著(P>0.05),PRP分别为46.12% 和49.99%。第55天对存活虾再口服攻毒,20天内rVp28疫苗组与阳性和阴性对照组比累积存活率差异显著(P<0.05),PRP分别为55.80%和63.16%;二次注射攻毒后,rVp28疫苗组的PRP均为31.25%。对vVp28疫苗组存活虾的胃、肠和肝胰腺组织进行病毒的原位杂交检测均呈阴性反应,而对照组死亡虾组织都呈阳性反应。本研究表明,口服免疫家蚕蛹表达的病毒囊膜蛋白Vp28能诱导螯虾产生抗病毒保护作用,对应用疫苗预防对虾的病毒性疾病具有重要意义。     

Comparative immunogenecity of foot and mouth disease virus antigens in FMD-haemorrhagic septicaemia combined vaccine and FMD vaccine alone in buffalo calves

Humoral immune response was evaluated by monitoring the serum antibody titres and virus specific IgM titres against Foot and Mouth Disease (FMD) virus antigens in serum samples obtained from different groups of calves inoculated with combined vaccine or FMD vaccine alone, on 0, 7, 14, 21, 28, 42 and 56 days post-vaccination (DPV). The cellular immune response was monitored by MTT based lymphoproliferation in peripheral blood mononuclear cell cultures. Higher liquid phase blocking (LPB) ELISA antibody titres were observed in calves receiving combined vaccine as compared to calves immunized with FMD vaccine alone with the peak titres in both the groups obtained on 21 days post-vaccination. However, the virus specific IgM titres were significantly higher in group of calves inoculated with combined vaccine than FMD vaccine alone. The lymphoproliferative responses against FMDV types O, A22 and Asia 1 in the groups receiving combined vaccine and FMD vaccine alone started increasing gradually after day 14 and reached peak levels on 28 DPV followed by a gradual decline subsequently. The group receiving combined vaccine showed higher proliferative responses on in vitro stimulation with FMD virus type O, whereas, with FMD virus type Asia 1, the responses were significantly higher on 14 and 21 DPV as compared to the group immunized with FMD vaccine alone. However, in the group receiving combined vaccine, the responses on in vitro stimulation with FMD virus type A22 were significantly higher than FMD vaccine alone group on all DPV except on 42 DPV.     

Development of an effective Edwardsiella tarda vaccine for cultured turbot (Scophthalmus maximus)

Since 2004 Edwardsiella tarda has become one of the most important emerging pathogens in turbot aquaculture industry in Europe causing serious economic losses. Therefore, this study aimed to design an effective vaccination strategy to prevent edwardsiellosis in this fish species. Two vaccine formulations, an adjuvanted vaccine and an aqueous bacterin, and different routes of administration, bath and intraperitoneal injection (i.p.), were tested. The effectiveness of the different immunization strategies was evaluated in terms of relative percent survival (RPS) and antibody levels. On the basis of the results obtained we recommend the i.p. administration of a non-mineral oil adjuvanted vaccine via i.p., which confers RPS values over 90% at least 6months post-vaccination.     

Selenium nanoparticles enhance the efficacy of homologous vaccine against the highly pathogenic avian influenza H5N1 virus in chickens

A proper vaccination against avian influenza viruses in chicken can significantly reduce the risk of human infection. Egypt has the highest number of recorded humans highly pathogenic avian influenza (HPAI)-H5N1 infections worldwide despite the widespread use of homologous vaccines in poultry. Enhancing H5N1 vaccine efficacy is ultimately required to better control HPAI-H5N1. The aim of this study is to boost chicken immunity by combined with inactivated HPAI-H5N1 with selenium nanoparticles (SeNPs). The chickens groups 1–3 were fed diets supplemented with SeNPs concentrations (0.25, 0.5, and 1 mg/kg) for 3 weeks and then vaccinated (inactivated HPAI-H5N1). while groups 4,5 and 6 were fed with SeNPs free diets and administered with 0.5 ml of the vaccine combined with 0.02, 0.06, and 0.1 mg/dose of SeNPs and then all groups were challenged with homologous virus 3 weeks post-vaccination (WPV). Group 7, 8 were used as control positive and negative respectively. At 4, 5, and 6 WPV, antibody titer was considerably higher in the group fed a meal supplemented with 1 mg SeNPs/kg. In contrast, both methods of SeNPs supplementation significantly increased the Interleukin 2 (IL2), Interleukin 6 (IL6), and Interferon γ (IFNγ) expressions in the blood cells in a dose-dependent manner, with a higher expression observed in the group that was vaccinated with 0.1 mg/dose. After the challenge, all groups that received SeNPs via diet or vaccines dose showed significant reduction in viral shedding and milder inflammation in lung, trachea, spleen, and liver in addition to higher expression of IL2, IL6, and IFNγ, with the highest expression observed in the group that was vaccinated with 0.1 mg/dose compared the plain vaccinated group. The groups of 1 mg SeNPs/kg and combined vaccinated with 0.1 mg/dose showed the best vaccine efficacy. However, the group vaccinated with 0.1 mg/dose showed the earliest reduction in viral shedding. Overall, SeNPs supplementation in the diet and the administration of the vaccine formula with SeNPs could enhance vaccine efficacy and provide better protection against HPAI-H5N1 in chickens by enhancing cellular immunity and reducing inflammation. We recommend using SeNPs as a vaccine combination or feeding with diet to increase the immunity and vaccine efficacy against H5N1.     

Novel Expression System for Combined Vaccine Production in <Emphasis Type="Italic">Edwardsiella tarda</Emphasis> Ghost and Cadaver Cells

To develop combined vaccine systems, we have generated Edwardsiella tarda ghosts (ETG) displaying a foreign protein on the outer membrane and also Ed. tarda cadaver (ETC) expressing a heterologous protein in the cytoplasm. Green fluorescent protein (GFP) was used as a model foreign protein. A constitutive promoter (EtPR C28-1) cloned newly from Ed. tarda was used as a promoter for the expression of foreign protein. Comparison of the strength of the new promoter with a commercially available constitutive promoter (P_HCE) showed higher expression levels of the novel expression system. The N-terminal domain of ice nucleation protein (InaN), an outer membrane protein of Pseudomonas syringae, was used as an anchor motif for surface display of GFP. By transformation of Ed. tarda with the constructed vectors, GFP was successfully expressed on the surface of ETG and in the cytoplasm of ETC. When compared to P_HCE driven expression, approximately more than 2 times of GFP was expressed on ETG and in ETC by EtPR C28-1 promoter when judged by fluorescent spectrophotometry. Furthermore, significantly higher expression of GFP on the surface of ETG by EtPR C28-1 than by P_HCE was demonstrated by serum agglutination assay. These results suggest that the newly cloned Ed. tarda constitutive promoter is capable to express foreign proteins not only on the surface of Ed. tarda ghosts but also in the cytoplasm of Ed. tarda cadavers, and can be used as an efficient promoter for the expression of heterologous antigens of the ETG and ETC-based combined vaccines.