Morphological changes in carp epithelial cells infected with Aeromonas hydrophila

The in vitro interaction of Aeromonas hydrophila with epithehoma papillosum cells of carp (EPC) was studied. All the virulent and type strains invaded and multiplied inside EPC cells. Morphological changes were induced by the virulent and type strains during the processes of invasion and intracellular replication. Among the virulent group, strain PPD134/91 required the shortest time (30 min post-infection) to induce cytopathic effects in the EPC monolayers. The EPC cells infected with PPD 134/91 became retracted and condensed, lost their attachment abilities, and eventually disintegrated. Confocal microscopy revealed that tubular structures measuring 0.77 ± 0.19 μm appeared to connect the retracted EPC cells. The cytopathic effect was attributed to the growing and metabolically active bacteria. Avirulent strains such as L37 did not multiply nor induce cytopathic effects in the EPC monolayers.     

Lemon,Citrus aurantifolia,peel and Bacillus licheniformis protected common carp,Cyprinus carpio,from Aeromonas hydrophila infection by improving the humoral and skin mucosal immunity,and antioxidative responses

The role of dietary lemon peel (LM) and/or Bacillus licheniformis (BL) on the growth, immunity, and resistance against Aeromonas hydrophila in common carp, Cyprinus carpio was investigated in this study. LM and BL were included in diets at 0% (T0), 10⁸ CFU/g BL (T1), 1.5% LM and 10⁸ CFU/g BL (T2), and 3% LM and 10⁸ CFU/g BL (T3). Fish fed with T1, T2, or T3 had higher weight gain, specific growth rate, white blood cells count, and blood total protein with lower feed conversion ratio than T0 group (p < .05). The albumin increased significantly (p < .05) in fish fed both BL and LM (T3). The serum superoxide dismutase (SOD), catalase, lysozyme, and bactericidal activities were significantly increased in fish fed both BL and LM (T2 and T3), while serum glutathione peroxidase increased in fish fed BL (T2) (p < .05). Fish fed T1, T2, and T3 diets displayed higher SOD and lower malondialdehyde than fish fed T0 (p < .05). After the A. hydrophila challenge, the mortality rate was significantly lower in T1, T2, and T3 groups than the T0 group (p < .05). The obtained results revealed that LM and BL could be used to increase resistance against A. hydrophila infection in carp. However, further field studies should be performed to confirm the obtained results.     

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.     

Chemotaxis towards, adhesion to, and growth in carp gut mucus of two Aeromonas hydrophila strains with different pathogenicity for common carp, Cyprinus carpio L

Characteristics that promote bacterial colonization of the intestinal mucosal surface were examined in two strains of the common fish pathogen Aeromonas hydrophila, with different pathogenicity. The characteristics examined were chemotactic activity towards mucus, bacterial adherence to mucus and growth in mucus. Intestinal gut mucus of healthy common carp was used. The results indicate that chemotaxis is not necessary for a bacterium to become pathogenic, but it may be a necessary parameter for a bacterium to be an obligate pathogen. Adhesion also seems to be a factor influencing pathogenicity. The results suggest that higher adhesion to mucus and subsequent growth is associated with differences in pathogenicity.     

嗜水气单胞菌感染后牛蛙血清中抗菌物质的初步研究

用1×107 mL-1的嗜水气单胞菌腹腔注射感染牛蛙,注射后7 d内试验组牛蛙血清抗菌活性都高于对照组,其中在3 d、7 d时存在显著性差异,峰值出现在感染后第3天。收集第2批人工感染后3 d时牛蛙的血清。该血清经Sephadex G25凝胶柱分离后,并在波长280 nm处测定紫外吸收值,得到两个吸收峰。以嗜水气单胞菌检测分离物的抗菌活性,结果表明经诱导的血清抗菌物质主要集中在第1个吸收峰。通过对金黄色葡萄球菌等10株指示菌抗菌谱的测定,其对大肠杆菌、枯草芽孢杆菌、铜绿假单胞菌等具有明显的抗菌作用;SDSPAGE分析显示抗菌蛋白分子量较大。结果说明：牛蛙被嗜水气单胞菌感染后能很快产生多种抗菌物质,主要的抗菌物质是分子量比较大的物质,这些抗菌物质对部分革兰氏阴性菌和革兰氏阳性菌有抗菌作用。图3表1参24 关键词：牛蛙;嗜水气单胞菌;抗菌蛋白;抗菌活性     

Effect of using Spirulina and Chlorella as feed additives for elevating immunity status of Nile tilapia experimentally infected with Aeromonas hydrophila

The present trial was performed to evaluate the effect of dietary incorporation of dried Spirulina, Chlorella and their mixture on the immune status of Nile tilapia (Oreochromis niloticus) before and after artificial infection with pathogenic bacterium Aeromonas hydrophila (A. hydrophila). Two hundred and forty fish were divided into four groups: (a) a control group fed on a basal diet only, (b) a second group fed on a diet containing Spirulina 15%, (c) a third group fed on a diet containing Chlorella 15%, and (d) a fourth group fed on a diet containing a mixture of both Spirulina 15% and Chlorella 15%. At the end of the experiment (9 weeks), the four groups were experimentally infected with A. hydrophila for 7 days. Antioxidant enzymes, lysozyme and bactericidal activities and histopathological changes were determined just before the challenge test and 7 days post‐challenge. Significant (p ≤ 0.05) increases in fish body protein% before the challenge test and increases in serum antioxidant enzymes, lysozyme and bactericidal activity in the Chlorella and algal mixture groups before and after the challenge test were observed. Spirulina, Chlorella and their mixture groups significantly decreased serum malondialdehyde compared to the control group before and after the challenge test. Using Spirulina, Chlorella and their mixture mitigated the necrotic and degenerative changes induced by A. hydrophila and revealed well‐developed and multiple melanomacrophage centres. Thus, dietary Spirulina, Chlorella and their mixture inclusion in Nile tilapia fish proved to have a protective effect against A. hydrophila infection.     

Resistance of genetically different common carp, Cyprinus carpio L., families against experimental bacterial challenge with Aeromonas hydrophila

The objective of this study was to determine the differences in disease resistance against artificial infection with Aeromonas hydrophila between genetically different common carp families. Four strains differing in their origin and breeding history were selected from the live gene bank of common carp maintained at the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI, Szarvas, Hungary) to establish families with wide genetic background: Szarvas 15 (15), an inbred mirror line; Tata (T) scaly noble carp; Duna (D), a Hungarian wild carp and Amur (A), an East Asian wild carp. A diallele mating structure was used to allow the assessment of genetic variation within and between the tested 96 families for a variety of traits. The existing technologies of fertilization and incubation of carp eggs, as well as larval and fingerling rearing had been modified because of the large number of baseline populations. Two challenge trials of the 96 families of carp with Aeromonas hydrophila were done. The 10 most resistant and 10 most susceptible families to A. hydrophila were identified from these two challenges. The crosses that produced the most resistant families were mainly those having parents from Tata and Szarvas 15 domesticated strains, while the most susceptible families were from the wild strains Duna and Amur.     

鲫源嗜水气单胞菌毒力基因多重PCR检测及ERIC-PCR分子分型

为快速了解鲫源嗜水气单胞菌株毒力基因携带情况及与菌株基因型的相关性,建立多重PCR法和ERIC-PCR分子分型,为临床快速检测、菌株分型和菌株致病性分析提供依据。通过单重PCR法检测出标准菌株ATCC7966内5个毒力基因气溶素(aerolysin,aer)、溶血素(hemolysin,hly)、细胞毒性肠毒素(cytotoxic enterotoxin,alt)、胞外蛋白酶(extracellular protease,ahp)和细胞肠兴奋性肠毒素(intestinal cells of excitatory enterotoxin,act),其扩增产物长度依次为300 bp、592 bp、442 bp、856 bp和500 bp。在此基础上,优化并建立特异性高,敏感度达7.2×102cfu·m L-1多重PCR法,用于检测从江苏射阳地区患病水产动物体内分离的17株嗜水气单胞菌5个毒力基因携带率。结果显示,毒力基因act的携带率为100%,而80%的菌株5个毒力基因均有检出。采用ERIC-PCR分子分型技术,以标准菌株ATCC7966为对照,对17株鲫源致病性嗜水气单胞菌进行基因分型,获得两种基因型,分别描述为A型和B型,其中B型菌株14株,带型与ATCC7966一致,认为是当地的主要流行株。探究菌株基因型与毒力基因分布相关性,携带5个毒力基因的均为B型菌株,而所有A型菌株存在一或多个毒力基因缺失,有可能是此类菌株更易发生毒力基因漂变,但还需进一步研究。     

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。     

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。     

Enhancement of protective immunity in blue gourami,Trichogaster trichopterus (Pallas), against Aeromonas hydrophila and Vibrioanguillarum by A. hydrophila major adhesin

Blue gourami, Trichogaster trichopterus (Pallas), were intraperitoneally immunized with major adhesin, a 43 kDa OMP protein isolated from fish Aeromonas hydrophila, in the presence of Freund's complete adjuvant (FCA). Three weeks later, a booster injection of adhesin without FCA was administered. Control group fish were similarly treated with phosphate‐buffered saline (PBS) and FCA. Results showed that anti‐adhesin serum obtained from fish after booster immunization exhibited very strong ability in agglutinating bacterial cells. Although this antiserum had no bactericidal effect, it could significantly inhibit serologically different strains of A. hydrophila from invading EPC (Epithelioma papillosum of carp) cells in vitro. In addition, the proliferative response of head kidney leucocytes of these immune fish was significantly increased as compared to that of the control. The results also showed that the major adhesin could provide significant protective immunity to fish against the challenge by homologous and heterologous strains of A. hydrophila and one virulent strain of Vibrio anguillarum.     

Protection of crucian carp (Carassius auratus Gibelio) against septicaemia caused by Aeromonas hydrophila using specific egg yolk immunoglobulins

Egg yolk immunoglobulins (IgY) were obtained from laying hens immunized with inactivated Aeromonas hydrophila. The purified IgY was shown to inhibit the growth of A. hydrophila in vitro and the optimum concentration for inhibition of A. hydrophila‐specific IgY was 75 mg mL^?1. In a subsequent challenge trial, 100 carp (200~250 g) were assigned to one of ten tanks with ten carp per tank. The fish in one tank were unchallenged whereas the remaining 90 fish were injected intraperitoneally with 100 μL of A. hydrophila at a concentration of 10⁸ cfu mL^?1. For the next 21 days, all fish were moved in their respective groups to a clean tank for 20 min day^?1. The fish in four tanks (one unchallenged tank and three challenged tanks) received no treatment whereas the fish in the remaining six tanks were immersed in either 0.5 g L^?1 aqueous nonspecific IgY (N = 3) or 0.5 g L^?1 aqueous specific IgY (N = 3). Haemoglobin concentrations, white and red blood cell numbers as well as the mortality of specific IgY‐treated fish were significantly different from those of the control. These results suggest that passive immunization by immersion with pathogen‐specific IgY may provide a valuable treatment for A. hydrophila infection in carp.     

鲤嗜水气单胞菌感染症及其病原生物学特性

对一起养殖鲤(Cypr inus carp io )发生的病害进行了发病情况、临床特征、病理变化等方面的检验。以3尾病(死)鲤进行病变组织中的细菌检查及细菌分离, 对分离菌进行了形态特征、理化特性等较系统的表观分类学指征鉴定; 同时选择代表菌株进行了16S rRNA 基因的分子鉴定, 测定了16S rRNA 基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树。结果表明, 分离菌为气单胞菌属(Aeromonas K luyv er and Van N iel 1936) 的嗜水气单[A.hydrophila (Chester 1901) Stan ier 1943] , 代表菌株( HC060718- 1株) 16S rRNA基因序列长度( 不包括引物结合区)为1 454 bp( GenBank登录号: EF669478)。择代表菌株做对健康鲤的人工感染试验, 表明了分离鉴定的嗜水气单胞菌在被检鲤病例的相应原发病原学意义及较强的致病作用。药敏试验结果显示, 对供试37种抗菌药物中的头孢噻肟等23种药物高度敏感, 对氨苄青霉素等5种药物敏感, 对青霉素G等9种药物耐药。     

Immune protection in carp, Cyprinus carpio L., after immunization with Aeromonas hydrophila crude lipopolysaccharide

Abstract. Vaccination with crude lipopolysaccharide (LPS) induced better protection against infection with Aeromonas hydrophila in carp than vaccination with formalin killed vaccine. Dipping fish in vaccine for 2 h at 25°C was more effective than intraperitoneal injection of the vaccine in procedural simplicity, lower stress loading and the degree of protection acquired. In carp immunized with crude LPS by the dip method, antibodies were not detected by bacteriai agglutination, passive haemagglutination and the agar diffusion tests. The results indicate that the protection against A. hydrophila infection in carp is not dependent on humoral immunity.     

饲料VC 对草鱼生长、肌肉品质及非特异性免疫的影响

采用维生素C(Vc,以Vc磷酸酯为Vc源)添加量分别为50mg/kg、100mg/kg、150mg/kg、200mg/kg、300mg/kg饲料的配合饲料,喂养体质量(42.4±5.0)g的草鱼(Ctenopharyngodon idellus)60d,以不添加Vc的基础饲料饲喂组为对照,探讨vc对草鱼生长性能、肌肉品质和非特异性免疫的影响.结果表明,饲料中添加Vc能显著提高草鱼增重率,降低饲料系数,当Vc添加量为150～200mg/kg时,草鱼生长性能最佳:随Vc添加量增加,肌肉失水率、肌纤维直径和肌原纤维耐折力呈增加趋势;肌肉胶原蛋白含量和肝脏Vc含量随Vc添加量的增加而增加;Vc添加量为150mg/kg及以上水平时,各实验组草鱼血清碱性磷酸酶(AKP)活性比对照组显著提高(p＜0.05);Vc添加量达到300mg/kg后血清溶菌酶(LSZ)活性比对照组显著提高(P＜0.05);各组间血清超氧化物歧化酶(SOD)活性差异不显著(P＜0.05).上述研究表明,饲料中添加Vc能促进草鱼生长,改善肌肉品质,增强非特异性免疫力.适宜的Vc添加量为150mg/kg饲料.     

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.     

鲤嗜水气单胞菌感染症及其病原生物学特性

对一起养殖鲤(Cyprinus carpio)发生的病害进行了发病情况、临床特征、病理变化等方面的检验.以3尾病(死)鲤进行病变组织中的细菌检查及细菌分离,对分离菌进行了形态特征、理化特性等较系统的表观分类学指征鉴定;同时选择代表菌株进行了16S rRNA基因的分子鉴定,测定了16S rRNA基因序列、分析了相关细菌相应序列的同源性、构建了系统发生树.结果表明,分离菌为气单胞菌属(Aeromonas Kluyver and Van Niel 1936)的嗜水气单胞菌[A.hydrophila(Chester 1901)stanier 1943],代表菌株(HC060718-1株)16S rRNA基因序列长度(不包括引物结合区)为1 454 bp(GenBank登录号:EF669478).择代表菌株做对健康鲤的人工感染试验,表明了分离鉴定的嗜水气单胞菌在被检鲤病例的相应原发病原学意义及较强的致病作用.药敏试验结果显示,对供试37种抗菌药物中的头孢噻肟等23种药物高度敏感,对氨苄青霉素等5种药物敏感,对青霉素G等9种药物耐药.     

Effect of dietary willow herb,Epilobium hirsutum extract on growth performance,body composition,haematological parameters and Aeromonas hydrophila challenge on common carp,Cyprinus carpio

The present study was conducted to evaluate the use of Epilobium hirsutum extract in diet of common carp, Cyprinus carpio (20 ± 2 g). Different levels of plant extract (0%, 0.5%, 1, 3% and 3%+2‰ multivitamin (2 g of multivitamin per kg diet) were spread on commercial diet. The feed was offered by 8 weeks. Results showed that fish fed experimental diets had no significant difference (P>0.05) in specific growth rates, condition factor, feed conversion ratios and survival compared with control. There were no significant difference (P>0.05) in moisture, crude protein, crude lipid and ash content of common carp fed diets containing various levels of plant extract. The mortality of fish challenged by Aeromonas hydrophila have been recorded for 30 days after challenging, results showed that mortality decreased significantly (P<0.05) in fish fed diet containing plant extract compared with the control. The lowest mortality observed in group fed diet containing 3% plant extract. Haematological parameters showed that white blood cells had significantly (P<0.05) increased in infected and uninfected groups compared with the control. Red blood cells, haemoglobin and haematocrit had no significant change (P>0.05) in infected and uninfected groups compared with the control.