An experimental vaccine against Aeromonas hydrophila can induce protection in rainbow trout,Oncorhynchus mykiss (Walbaum)

A candidate vaccine against Aeromonas hydrophila in rainbow trout, Oncorhynchus mykiss, was developed using a bacterial lysate. To test the strength of protection, A. hydrophila challenge models were compared using injection into both the intraperitoneal (IP) cavity and the dorsal sinus (DS) with selected doses of live bacteria washed in saline or left untreated. Unlike the IP route, injection into the DS with either saline washed or unwashed cells resulted in consistent cumulative mortality and a dose response that could be used to establish a standard challenge having an LD₅₀ of approximately 3 × 10⁷ colony forming units per fish. Survivors of the challenge suffered significantly lower mortality upon re‐challenge than naïve fish, suggesting a high level of acquired resistance was elicited by infection. Passive immunization using serum from hyper‐immunized fish also resulted in significantly reduced mortality indicating protection can be transferred and that some portion of resistance may be antibody mediated. Vaccination of groups of rainbow trout with A. hydrophila lysate resulted in significant protection against a high challenge dose but only when injected along with Freund’s complete adjuvant. At a low challenge dose, mortality in all groups was low, but the bacterial lysate alone appeared to offer some protection.     

鳜浸泡嗜水气单胞菌全菌疫苗后皮肤黏液抗体的变化

应用间接ELISA方法,研究经嗜水气单胞菌(Aeromonas hydrophila)疫苗浸泡免疫后鳜(Siniperca chuatsi)皮肤黏液中抗体消长规律,以揭示鳜局部黏膜免疫在免疫保护中的作用及浸泡免疫的保护效果。结果表明,鳜皮肤黏液中抗体滴度在免疫后第7天达到峰值211,抗体从开始形成到消失持续21 d;添加佐剂(IMS1312、葡聚糖、莨菪碱、食盐)可以提高鳜皮肤黏液及血清中抗体滴度和相对免疫保护率,其中添加IMS1312组免疫保护率最高,达77.8%,免疫保护与抗体滴度成正相关(R2=0.79,P<0.05);以溶菌酶为代表的非特异性免疫在免疫后1周内即抗体未形成时,对鱼体起主要保护作用。此外,通过比较鳜血清与皮肤黏液中的抗体消长规律,发现血清中抗体滴度峰值出现时间较迟(第14天),抗体持续时间较长(42 d),初步推断鳜皮肤黏膜中可能存在相对独立于全身免疫系统的局部黏膜免疫系统。     

不同溶氧条件下黄颡鱼免疫机能及抗病力的研究

研究了水温(28.4±1.0)℃时不同溶氧条件下(A组:(2.28±0.56)mg/L;B组:(4.04±0.38)mg/L;C组:(6.51±0.64)mg/L;D组:(9.11±1.24)mg/L)饲养8周后,黄颡鱼(Pelteobagrus fulvidraco)的免疫机能及对嗜水气单胞菌(Aerom onas hydrophila)的抗病力。结果显示:A组脾脏系数、吞噬百分率极显著低于其他3组(P<0.01),淋巴细胞转化率显著低于其他3组(P<0.05),红细胞数(RBC)显著高于其他3组(P<0.05),白细胞数(WBC)显著高于C、D两组(P<0.05);B组脾脏系数极显著低于C、D两组(P<0.01),吞噬百分率和淋巴细胞转化率显著低于C、D两组(P<0.05),攻毒1周后,A组受试鱼累积死亡率显著高于B组(P<0.05),极显著高于C、D两组(P<0.01)。表明慢性低氧胁迫抑制了黄颡鱼免疫机能。饲养8周后各组黄颡鱼血清皮质醇水平和溶菌酶活力差异均不显著(P>0.05),表明这两个指标不宜作为鱼类慢性胁迫中的应激信号。     

Analysis of virulence and immunogenic factors in Aeromonas hydrophila: Towards the development of live vaccines

Aeromonas hydrophila, a bacterium that is widespread in aquatic environments, is responsible for causing haemorrhagic disease in both aquatic and terrestrial species. With the purpose of developing a live vaccine, herein we have investigated nine strains of A. hydrophila (Ah-01 to Ah-09) isolated from diseased fish. A study of virulence factors that contribute to pathogenicity and immunogenicity in the host Cyprinus carpio suggests that the presence of β-hly, act and fla genes contribute to pathogenesis: strains Ah-01, Ah-02 and Ah-03 (β-hly⁺/act⁺/fla⁺ genotype) were highly pathogenic to C. carpio, whereas Ah-05 and Ah-06 (β-hly⁻/act⁻/fla⁻ genotype) showed weak pathogenicity. Accordingly, Ah-02 and Ah-03 were selected to prepare inactivated vaccines, whereas Ah-05 and Ah-06 were chosen as live vaccines. Ah-06 live vaccine was found to have the best protective efficacy, with a protective rate of about 85%, whereas rates of other vaccines were significantly lower, in the range 37%–59%. In addition, DNA vaccines based on genes altA, aha and omp showed immune protection rates of 25%, 37.5% and 75%, respectively. Our data demonstrate that the β-hly⁻/act⁻/fla⁻/altA⁺/aha⁺/omp⁺ genotype has weak pathogenicity and high immunogenicity, and provide a simple and effective way to screen for live A. hydrophila vaccines.     

灭活菌苗免疫的南方鲇外周血液细胞免疫指标的变化

以嗜水气单胞菌(Aeromonashydrophila)灭活菌苗为免疫原,以平均体重为86 20g人工饲养的健康南方(Silu rusmeridionalis)为实验对象,通过腹腔注射免疫原(浓度为1 0×108/mL)免疫,剂量为0 2mL/尾,并分别在免疫后的第1、2、4、7、14、21、28天对实验鱼尾静脉采血,进行血液生理指标和血液中吞噬细胞活性的测定。结果显示,嗜水气单胞菌灭活菌苗(FKC)可以增加南方外周血液白细胞数量并引起不同种类白细胞组成比例的变化,提高吞噬细胞的吞噬活性。在免疫后第1、2、4、7、14、21、28天,南方外周血中白细胞数量呈明显上升趋势,其中嗜中性粒细胞、单核细胞、淋巴细胞变化趋势更为明显;而且吞噬细胞活性大为提高,吞噬百分比较对照组平均升高了17 66%,吞噬指数比对照组平均升高了1 16,在免疫后第4天吞噬百分比和吞噬指数均达到最高值,吞噬百分比为48 40%,吞噬指数为4 82;免疫后第21天,吞噬百分比和吞噬指数开始下降,但仍高于对照组。由此表明,使用疫苗后,鱼类非特异细胞免疫在初期(尤其是在第2～7天)明显增强,这是鱼用疫苗在免疫早期保护作用的主要机制之一。     

Microbial levan in the diet of Labeo rohita Hamilton juveniles: effect on non-specific immunity and histopathological changes after challenge with Aeromonas hydrophila

A 60-day feeding trial was conducted to study the immuno-protective effect of microbial levan on Labeo rohita juveniles challenged with Aeromonas hydrophila. Six purified diets were prepared with different levels of microbial levan: control (no levan), T1 (Basal + 0.25%), T2 (Basal + 0.50%), T3 (Basal + 0.75%), T4 (Basal + 1%) and T5 (Basal + 1.25%), fed to six groups of fish in triplicate. Among the treatment groups the haemoglobin content and total leucocyte count were increased with a dietary supplementation of levan at 1% or more. An increasing trend for total erythrocyte count was observed with increasing level of dietary levan. Lower levan-supplemented groups showed a higher albumin/globulin ratio. As the levan supplementation was increased, there was a gradual increase in serum lysozyme activity and respiratory burst activity [nitroblue tetrazolium (NBT) assay] reduction values. The highest lysozyme activity and NBT were observed in the T5 group although this was similar to the T4 group ( P  > 0.05). No significant histo-architectural changes were associated with dietary levan levels. After challenge with A. hydrophila , moderately degenerated hepatocytes, oedema and leucocytic infiltration in parenchymatous tissues, and extensive haemorrhage and haemosiderosis in the kidney were observed in the control group. However, the T5 group supplemented with 1.25% levan showed infiltrating leucocytes in the liver while the kidney showed only moderate degeneration of renal tubules. The relative survival per cent of juveniles after challenge with A. hydrophila was the highest in the T5 group followed by T4. This suggests that microbial levan at 1.25% can be used as dietary immunostimulant for L. rohita juveniles.     

嗜水气单胞菌温敏胞外蛋白酶基因eprJ的克隆与检测

根据已发表的人源嗜水气单胞菌(Aeromonas hydrophila,Ah)温敏胞外蛋白酶基因eprA1的核甘酸序列,设计合成一对引物,以嗜水气单胞菌鱼源株Ah J-1基因组DNA为模板,通过PCR扩增得到1 005 bp的胞外蛋白酶基因eprJ1,将该基因片段连接到pMD18-T载体中,构建克隆载体pMD-eprJ1。序列分析发现,其与发表的Ah AH1的胞外蛋白酶eprA1基因的同源性有91%。根据测序结果在保守区重新设计一对引物以增强特异性,扩增片段大小为387 bp。对1990~2004年15年间从浙江、福建、湖北及江苏等地不同患病水产动物肝肾等脏器分离到的23株Ah检测结果显示,从中国东南地区分离的21株Ah水生动物致病性菌株扩增均为阳性,2株Ah非致病性菌株为阴性,推测该基因普遍存在于致病性Ah中。检测模板DNA的灵敏度可达1.5×10-3ng/μL。     

Development and characterization of cell lines derived from rohu, Labeo rohita (Hamilton), and catla, Catla catla (Hamilton)

Two new cell lines, designated RE and CB, were derived from the eye of rohu, Labeo rohita , and the brain of catla, Catla catla , respectively. The cell lines were maintained in Leibovitz's L-15 supplemented with 20% foetal bovine serum. The RE cell line was sub-cultured for more than 70 passages and the CB cell line for more than 35 passages. The RE cells are rounded and consist predominantly of epithelial cells. The CB cell line consists of predominantly fibroblastic-like cells. Both cell lines are able to grow at temperatures between 25 and 32 °C with an optimum of 28 °C. The growth rate of the cells increased as the foetal bovine serum concentration increased from 2% to 20% at 28 °C, with optimum growth at concentrations of 15% or 20% foetal bovine serum. The cells were successfully cryopreserved and revived at different passage levels. The cell lines were not susceptible to four marine fish viruses. Extracellular products from Aeromonas sp . were toxic to the cell lines. When the cells were transfected with plasmid eukaryotic green fluorescent protein (pEGFP [Clontech, Carlsbad, CA, USA]) vector DNA, a significant fluorescent signal was observed suggesting that these cell lines could be a useful tool for transgenic and genetic manipulation studies. Polymerase chain reaction amplification of mitochondrial 12S rRNA from rohu and catla confirmed that the cell lines originated from these fish species. The cell lines were further characterized by immunocytochemistry using confocal laser scanning microscopy.     

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。     

褐牙鲆腹水症病原菌的分离鉴定及其灭活疫苗的研制

从患腹水病褐牙鲆体内分离致病菌,通过生理生化和分子生物学方法进行鉴定;该菌的16S rDNA序列与标准株同源性达99.8%,证明该菌为嗜水气单胞菌.制备灭活疫苗后分别注射小鼠和牙鲆,测定其免疫效果.试验结果表明,用所制备的灭活疫苗免疫小鼠,其保护率达到90%,免疫牙鲆的保护率达到80%;采用渗透压差法浸泡免疫牙鲆在攻毒后发病率仅为6%,攻毒后保护率为85%;通过间接ELISA检测表明受免动物体内产生了较高水平的抗体;剖检及病理切片结果表明免疫过的动物内脏健康无异样,而未免动物肝脏充血,肠腔有积水.说明该疫苗安全有效.     

Experimental challenge studies in Vietnamese catfish,Pangasianodon hypophthalmus (Sauvage), exposed to Edwardsiella ictaluri and Aeromonas hydrophila

The two main diseases in the pangasius catfish industry are bacillary necrosis of Pangasianodon (BNP) and motile aeromonas septicaemia (MAS), where the aetiological agents have been identified as Edwardsiella ictaluri and Aeromonas hydrophila, respectively. In this study, apparently healthy Pangasianodon hypophthalmus were exposed to E. ictaluri, A. hydrophila or both bacterial species by intraperitoneal injection or immersion. There were 20 fish per treatment group, and the bacterial isolates used for the study were recovered from natural infections of BNP or MAS in farmed Vietnamese P. hypophthalmus. The results of the experimental infections mimicked the natural disease outbreaks reported from these pathogens in P. hypophthalmus. Furthermore, it was clearly demonstrated that E. ictaluri was only recovered from the fish exposed to the bacterium and not recovered from the animals receiving A. hydrophila.     

Use of garlic, Allium sativum, to control Aeromonas hydrophila infection in rainbow trout, Oncorhynchus mykiss (Walbaum)

Garlic, Allium sativum , which was fed at 0, 0.05, 0.1, 0.5 and 1.0 g per 100 g of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), led to control of experimental infection with Aeromonas hydrophila. At doses of 0.5 and 1.0 g garlic per 100 g of feed, there was a reduction in mortalities to 4% compared with the controls (88%). Moreover, there was a significant increase in growth, feed conversion and protein efficiency. There was stimulation of the number of erythrocytes and leucocytes, a significantly higher haematocrit, enhancement of phagocytic activity, respiratory burst, lysozyme, anti-protease and bactericidal activities following feeding with garlic.     

大黄鱼致病嗜水气单胞菌的分离鉴定及其多克隆抗体制备

为探究引起网箱养殖大黄鱼肠炎病的病原及其特性,自患病大黄鱼肝脏组织分离到1株优势菌NDLc-P,经回归感染试验确认该菌为大黄鱼致病菌.人工感染7 d后,该菌对体质量(46.4±3.5)g的健康大黄鱼的半数致死密度为3.98×107 cf u/m L,表明该菌株对大黄鱼有一定的致病性.利用梅里埃微量多项鉴定系统对病原菌N...     

香鱼嗜水气单胞菌人工染病后血液生理生化指标变化

从患病香鱼的体腔液中分离获得嗜水气单胞菌病原,经培养稀释的菌液,腹腔注射人工感染健康香鱼,出现典型的出血症后,进行血液生理生化指标的测定。结果发现,病鱼红细胞数量为(1.21±0.08)×1012个/L,血栓细胞数量(189.00±5.19)×1012个/L,较健康香鱼红细胞[(2.41±0.16)×1012个/L]、血栓细胞[(380.0±20.00)×1012个/L]呈极显著降低(P0.01),血红蛋白含量呈显著减少(P0.05),而白细胞数量却显著增加(P0.05)。染病香鱼总蛋白为(47.56±2.63)g/L,健康鱼为(49.23±3.06)g/L,经检验无显著差异;而碱性磷酸酶、Cl-、Ca2+等3项血液生化指标值均呈极显著减少(P0.01),乳酸脱氢酶呈极显著增加(P0.01)。     

The garlic component,allicin, prevents disease caused by Aeromonas hydrophila in rainbow trout,Oncorhynchus mykiss (Walbaum)

Allicin was fed at 0 (= control), 0.5 and 1.0 mL of Allimed^® liquid 100 g^?1 of feed for 14 days to rainbow trout, Oncorhynchus mykiss (Walbaum), fingerlings before infection with Aeromonas hydrophila with a resultant reduction in mortalities from 80% in the controls to 8% [relative percentage survival (RPS) = 90%] and 0% (RPS = 100%) among the treated fish. Allicin was strongly antibacterial compared to the control, with a minimum inhibitory concentration (MIC) of >400 μL mL^?1 of Allimed^® liquid. Use of allicin led to a lower number of white blood cells (132.0 ± 0.4 × 10³) compared to 175.0 ± 0.1 × 10³ in the controls, but elicited increased phagocytic activity, i.e. a phagocytic value of 39.2% compared to 13.6% in the controls, and serum lysozyme activity, which showed significant (P > 0.05) differences compared to the control at 15 and 30 min after the first reading at 0 min of incubation.     

鳜嗜水气单胞菌GYK1株胞外产物提取、纯化及生物学活性分析

以鳜分离的嗜水气单胞菌GYK1株为代表,采用玻璃纸覆盖技术提取了8株嗜水气单胞菌和1株温和气单胞菌的胞外产物,SDS-PAGE电泳分析显示,8株嗜水气单胞菌的胞外产物均有35kDa的共同蛋白带。酶活性和溶血性分析表明,GYK1株胞外产物具有酪蛋白酶、淀粉酶、脂肪酶、明胶酶活性,但不具备脲酶活性;溶血性较强,蛋白浓度为365μg·mL-1的胞外产物对鳜血细胞的溶血价为213,对加州鲈、银鲫、小白鼠、兔、绵羊、人O型血的红细胞的溶血价在211~214之间。应用聚丙烯酰胺葡聚糖凝胶柱层析,对GYK1株的胞外产物进行了初步纯化,得到分子量为35kDa的蛋白带,该纯化产物具溶血性,但溶血性比粗胞外产物降低,对鳜和小鼠血细胞的溶血价均为25。     

The Use of American Ginseng (Panax quinquefolium) in Practical Diets for Nile Tilapia (Oreochromis niloticus): Growth Performance and Challenge with Aeromonas hydrophila

This study was carried out to evaluate the effect of American ginseng (AG), Panax quinquefolium, on growth and resistance to Aeromonas hydrophila in Nile tilapia, Oreochromis niloticus. Ginseng was included in practical test diets at rates of 0.0 (control), 0.50, 1.0, 2.0, or 5.0 g/kg diet. Fish (9.1 ± 0.3 g) were distributed into quadricated 100-L aquaria at a density of 20 fish per aquarium. Fish in all treatments were fed up to satiation twice daily for 8 weeks. After the feeding trial, fish of each treatment were intraperitoneally injected with pathogenic A. hydrophila and kept under observation for 10 days. Highest growth was obtained at 1.0 – 5.0 g AG/kg diet. The survival of fish challenged by A. hydrophila increased with increasing AG levels in fish diets. Cost-benefit analysis indicated that ginseng supplementation could reduce per kg costs by 15% with an optimum inclusion level of 2.0 g/kg.