Cell-mediated protection in carp, Cyprinus carpio L., against Aeromonas hydrophila

Abstract. The mechanism of protection against Aeromonas hydrophila infection in carp was studied. Recipient fish, into which pronephric cells from carp previously immunized by dipping in bacterial crude lipopolysaccharide (LPS) at 25°C for 2h were transferred, demonstrated almost the same level of protective ability as an immunized control group. The protective ability was transmitted by non-adherent (to nylon fibre) immune pronephric cells. These non-adherent cells were damaged by anti-carp thymocyte serum and were, thus, considered to be T-like cells. The protective ability was depressed in immunized carp treated with anti-carp thymocyte serum in vivo and was also remarkably reduced in immunized carp whose macrophage function was impaired by Dextran sulphate-500 treatment. These results indicate that the protection shown by carp immunized by dipping in crude LPS is dependent on cellular immunity regulated by a T-like cell-macrophage system.     

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.     

鲤竖鳞病病原及其疫苗的研究

对发生在山东境内的鲤竖鳞病进行了病原学研究,经过病原分离,人工感染实验和细菌学鉴定,确定豚鼠气单胞菌（Ａｅｒｏｍｏｎａｓｃａｕｉａｅ）是鲤竖鳞病的病原菌,在此基础上,得到了免疫原性较高和生产工艺简单的鲤竖鳞病疫苗,同时,对该疫功的安全性,免疫保护力和效价等进行了系统研究,并进行了一定范围的口服免疫应用试验。     

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.     

Vaccine-induced protective immunity against Aeromonas salmonicida tested in experimental carp erythrodermatitis

Abstract The development and applicability of a dose-controlled experimental infection with atypical Aeromonas salmonicida in carp is described. The proliferation and clinical manifestations of experimentally induced carp erythrodermatitis mimicked a natural infection. An in-vivo assay was used to evaluate the lethal properties of cell-free culture supernatants and a simple serum-free growth medium was devised for maintaining the virulence of the challenge strain. Depending on the inoculation dose, a sublethal (chronic) to a lethal (acute) infection could be induced, and a dose-response relation was observed between A. salmonicida inoculum size and carp mortality. The dose-controlled experimental infection was used as a challenge test for laboratory evaluation of the efficacy of potential vaccine candidates. The vaccine candidates tested, a cell envelope preparation, purified lipopolysaccharide and purified A-layer (ACE) protein showed no protection or only a feeble one at the best, while formalinized whole cells showed a consistent but only moderate protection. In contrast, when concentrated, detoxified culture supernatant was used, the carp were protected against a subsequent lethal challenge. These observations indicate that immunity against A. salmonicida extracellular products is of prime importance.     

Immunization of rainbow trout Oncorhynchus mykiss (Walbaum) with a crude lipopolysaccharide extract from Flavobacterium psychrophilum

Control methods for Flavobacterium psychrophilum are limited and oftentimes ineffective; hence, research efforts have focused on vaccine development. This study tested the hypothesis that a crude lipopolysaccharide (LPS) extract from F. psychrophilum will elicit a protective immune response in rainbow trout Oncorhynchus mykiss (Walbaum) against F. psychrophilum challenge. Rainbow trout (mean weight, 3 g) were immunized intraperitoneally with the following treatment and control preparations: 10 μg of crude LPS with or without Freund's complete adjuvant (FCA), 25 μg of crude LPS with or without FCA and saline with or without FCA. Immunization of fish with 10 or 25 μg of crude LPS/FCA resulted in significant antibody responses against F. psychrophilum using ELISA with a whole‐cell lysate as the coating antigen, but only minimal levels of protection were conferred following F. psychrophilum challenge at 14 weeks post immunization. Western blot analyses demonstrated that fish exhibited antibodies specific for low‐molecular mass proteins present in the crude LPS extract, but did not exhibit antibodies specific for F. psychrophilum LPS. The results indicate that higher immunization doses and/or the use of an alternative extraction method that yields larger LPS molecules (23–70 kDa) may be necessary to elicit specific antibody responses against F. psychrophilum LPS.     

Immunological response of the Indian major carps to Aeromonas hydrophila vaccine

Abstract. Fingerlings of the three major types of Indian carp, i.e. Catla catla (Hamilton), Labeo rohita (Hamilton) and Cirrhunas mrigala (Hamilton), were immunized using a haemolysin-negative mutant of Aeromonas hydrophila . Very high titres of antibodies were induced in C. catla , followed by C. mrigala and L. rohita . Immunized fish showed good protection against homologous challenge. Moderate protection against heterologous challenge was observed in C. mrigala and L. rohita .     

Optimization by orthogonal array design and humoral immunity of the bivalent vaccine against Aeromonas hydrophila and Vibrio fluvialis infection in crucian carp (Carassius auratus L.)

Aeromonas hydrophila and Vibrio fluvialis are the causative agents of a serious haemorrhagic septicaemia that affects a wide range of freshwater fish in China. In order to develop a bivalent anti‐A. hydrophila and anti‐V. fluvialis formalin‐killed vaccine to prevent this disease, an orthogonal array design (OAD) method was used to optimize the production conditions, using three factors, each having three levels. The effects of these factors and levels on the relative per cent survival for crucian carp were quantitatively evaluated by analysis of variance. The final optimized formulation was established. The data showed that inactivation temperature had a significant effect on the potency of vaccine, but formalin concentration did not. The bivalent vaccine could elicit a strong humoral response in crucian carp (Carassius auratus L.) against both A. hydrophila and V. fluvialis simultaneously, which peaked at 3 or 5 weeks respectively. Antibody titres remained high until week 12, the end of the experiment, after a single intraperitoneal injection. The verification experiment confirmed that an optimized preparation could provide protection for fish at least against A. hydrophila infection, and did perform better than the non‐optimized vaccine judged by the antibody levels and protection rate, suggesting that OAD is of value in the development of improved vaccine formulations.     

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。     

被动转移免疫在鲫感染温和气单胞菌中的作用

实验用鲫平均体重100g，采用免疫物质被动转移试验，探讨体液免疫在鲫感染温和气单胞菌（Aeromonas sobria)中的保护作用。结果表明，无论免疫物质是来自异源还是自源，是由相关的嗜水气单胞菌（A.hydrophia)诱导产生还是由攻毒的温和气单胞直接刺激，均具有良好的免疫保护作用。实验还发现，再次攻毒时，在所有抗体组的鲫存活率为100%时，全血被动转移组的死亡率高达55.6%；免疫前后免疫复合物发生明显变化。说明抗体在保护鲫免疫受温和气单胞菌的致死性感染中发挥决定性的作用。     

Polysaccharide-induced protection of carp, Cyprinus carpio L., against bacterial infection

Abstract. The efficacy of 10 polysaccharides (curdlan, inulin, krestin, laminaran, lentinan, levan, schizophylian, selerogiucan, yeast glucan and zymosan) to enhance protection of carp, Cyprinus carpio L., against bacterial infection was investigated. Carp were intraperitoneally injected with the polysaceharides (2–l0 mgkg^-1) on days 1 and 4, and challenged with Edwardsiella tarda on day 7. Among the polysaccharides tested, lentinan, schizophyllan and scleroglucan, which are l,6-branchcd-β-l,3-glucans, significantly increased the survival rate. They also induced a protective effect against Aeromonas hydrophila at a dose of 5 mg kg^-1. The ability of the polysaccharides to activate the alternative complement pathway (ACP) was examined by incubating the polysaccharides with carp serum and measuring the residual ACP activity. At a final concentration of 0.l mgml^-1, l,6-branched-β-1,3-glucans greatly reduced (76–77%) the ACP activity. Therefore, it is suggested that the protective effect of the l,6-branched-β-1,3-glucans may be associated with the activation of ACP.     

两种鲤鱼对侵袭性病原体及嗜水气单胞菌抗病力的比较

调查同塘饲养的高寒鲤,散鳞镜鲤当年鱼对侵袭性病原体及人工感染嗜水气单胞菌耐受力进行比较,侵袭性病原体检出8种,其中绦虫只有散鳞镜鲤感染,感染率达到29％,其余种类二种鲤鱼体表,鳃都有寄生,但寄生的程度有所不同,高寒鲤的抵抗力要比散鳞镜鲤强,对人工感染的嗜水气单胞菌的耐受力高寒鲤比散镜鲤高,这可能与品种对病原菌耐性不同有关。     

银鲫肠道内抑制嗜水气单胞菌的潜在益生菌筛选及其特性研究

从养殖的银鲫(Carassius auratus gibelio)肠道中分离纯化得到60株具有不同特征的菌株,以嗜水气单胞菌(Aeromonas hydrophila)为指示菌,点种法初筛得到12株菌株,再通过牛津杯实验筛选出4株对嗜水气单胞菌有明显抑制作用(抑菌圈直径在14 mm以上)的益生菌疑似株,并进行了耐热试验、耐酸试验和人工肠液耐受试验,筛选出最符合益生菌功能特性的菌株YJ-9。该菌株在90℃高温水浴30 min后相对存活率为1.51%,在pH3的液体培养基中4 h内相对存活率为8.37%,活菌数都保持在106 CFU/mL以上,并且可以在人工模拟肠液中缓慢生长。在急性毒力实验中,10倍于实验剂量的菌株YJ-9对银鲫无毒,动物保护实验证明菌株YJ-9能有效增强银鲫对嗜水气单胞菌的免疫抵抗力,相对保护率达到80%。研究结果表明,菌株YJ-9可以作为一个新的饲用微生态制剂候选菌种。     

鱼对气单胞菌抗原接种的免疫效果观察

通过用分离于草鱼肠炎病例的嗜水气单胞菌制备成免疫原，经腹腔接种健康1龄鲤、50d后，进行免疫抗体检验，以确定鱼类对抗原刺激的应答强度，同时在免疫接种后60d,70d,90d，做相应气单胞菌的人工感染保护试验，结果表明鱼体对嗜水气单胞菌抗原免疫可以产生良好的免疫抗体应答，经免疫后的鱼对相应菌株的感染具有一定的免疫保护作用，保护效果与抗体水平有一定的相关性。     

鳗源嗜水气单胞菌β—溶血素基因的克隆及表达

应用PCR技术,从1株鳗源嗜水气单胞菌ML316中扩增得到β 溶血素基因AHL316HEM,将AHL316HEM克隆到pGEM TEasyVector,经分析验证后重组到pcDNA3.0中,构建了重组质粒PDLH。转化重组质粒PDLH的大肠杆菌(Escherichiacoli)DH5α能在血琼脂培养皿中形成明显的β 溶血斑,重组菌纯化的胞外产物溶血价为1.28×104HU·mg-1,同时重组菌的胞外产物能被原始菌株的高免血清特异性地识别。结果证实,克隆到鳗源嗜水气单胞菌β 溶血素基因,重组质粒PDLH能够表达具有天然生物学活性的β 溶血素,为构建嗜水气单胞菌核酸疫苗奠定了基础。     

鳗鲡外周血白细胞体外吞噬嗜水气单胞菌数量模型的建立

为建立鳗鲡外周血白细胞体外吞噬嗜水气单胞菌的数量模型,研究了感染时间一定、感染复数变化的情况下鳗鲡外周血白细胞吞噬的细菌数量和白细胞的存活率,同时还研究了感染复数一定、感染时间变化的情况下鳗鲡外周血白细胞吞噬的细菌数量和白细胞的存活率。结果发现,鳗鲡外周血白细胞对嗜水气单胞茵有较强的吞噬作用,在感染复数为100：1、感染时间为30min时细胞吞噬的细菌量达到饱和,而细胞本身仍可维持较高活性。表明100：1的感染复数及30rain的感染时间,是研究鳗鲡外周血白细体外吞噬嗜水气单胞菌数量模型的最佳参数。本研究还通过检测吞噬速率和杀菌速率评估建立起来的数量模型发现,鳗鲡外周血白细胞吞噬嗜水气单胞菌的速率较吞噬大肠杆菌的速率快,但杀灭该茵的速率却均显著低于杀灭大肠杆菌和金黄色葡萄球菌的速率,进一步证明建立的数量模型具有良好的可行性。     

多种接种途径对翘嘴鳜细菌性烂鳃和败血症的免疫预防效果

多种接种途径对翘嘴鳜细菌性烂鳃和败血症的免疫预防效果陈昌福，周文豪（华中农业大学水产学院，武汉４３００７０）陈昌福等（１９９５）先后用从患细菌性烂鳃病的翘嘴鳜（ＳｉｎｉｐｅｒｃａｃｈｕａｔｓｉＢ．）上分离的柱状嗜纤维菌（Ｃｙｔｏｐｈａｇａｃｏｌｕｍｎ...     

鲤鱼细菌性败血症病原菌的研究

从具有典型细菌性败血症症状的病鱼或濒死鱼中分离到2 株优势菌,经分离培养、人工感染试验、毒力因子、16S rRNA基因序列的测定及药敏试验,研究分离菌的生理生化特征、致病性及药物敏感性.根据2 株分离菌的菌落形态、生化特性和16S rRNA基因序列的测定结果(同源性为99%),确定为嗜水气单胞菌.人工感染试验的症状与自然发病相似,且再次感染后又分离到该菌株.2 株菌对阿米卡星、氨曲南、头孢噻肟等15 种药物均敏感,对氨苄西林均耐药,对复方磺胺表现出菌株差异.对其敏感的15 种药物可作为防治鲤鱼细菌性败血症的首选药物.     

嗜水气单胞菌3种疫苗对斑点叉尾免疫原性比较研究

将经福尔马林灭活的嗜水气单胞菌(Aeromonas hydrophila)、菌体脂多糖(LPS)和菌体外膜蛋白(OMP)作为免疫原,分别接种斑点叉尾(Ictalurus punctatus Rafinsque)后,通过测定受免鱼的凝集抗体效价,头肾和血液中吞噬细胞的吞噬活性和用A.hydrophila活菌攻毒的方法,探讨了斑点叉尾对A.hydrophila的3种疫苗的免疫应答状况和对活菌攻毒的免疫保护效果。试验结果显示:对斑点叉尾经腹腔注射接种3种疫苗均能刺激受免鱼产生较强的免疫应答,3种疫苗的受免鱼均产生了特异性凝集抗体,接种F-Ah灭活菌苗的试验鱼最高,接种OMP疫苗的试验鱼其次,而接种LPS疫苗的试验鱼血清中凝集抗体效价最低;与未接种疫苗的对照鱼相比,受免鱼头肾和血液中吞噬细胞的吞噬活性明显上升,头肾和血液中吞噬细胞活性由高到低依次是LPS、OMP和F-Ah免疫接种的斑点叉尾;活菌攻毒的结果证明接种3种疫苗的受免斑点叉尾A.hydrophila的感染产生了不同程度的相对免疫保护力(RPS),以OMP免疫接种后的斑点叉尾RPS最高,达到72.5%,接种LPS的斑点叉尾稍差,RPS为62.5%,而RPS最低的是接种F-Ah的免疫组,RPS只有24.2%。     

Effects of dietary Bacillus licheniformis on growth performance,intestinal morphology,intestinal microbiome,and disease resistance in common carp (Cyprinus carpio L.)

Aquaculture International - This study was to evaluate the growth, intestinal morphology, intestinal flora, and anti-infection activity against Aeromonas hydrophila in common carp upon adding...