Contribution of AhyR to virulence of Aeromonas hydrophila J-1

Aeromonas hydrophila is a broad-host-range pathogen and its pathogenesis is multifactorial. A regulatory mechanism known as quorum sensing has been found to be involved in the regulation of virulence in many bacteria. In A. hydrophila the ahyR gene encodes LuxR-type response regulator. Here we describe the inactivation of the ahyR gene of A. hydrophila J-1 by the insertion of a DNA fragment containing a kanamycin resistance determinant and reintroduced by allelic exchange into the chromosome of A. hydrophila J-1 by means of the suicide plasmid pJP5603. Cytotoxic effects on EPC cells assay and LD(50) determinations in fish demonstrated that the ahyR mutant was highly attenuated relative to the wild-type strain. Compared with the parent strain, some characteristics, such as biochemical characters and outer membrane protein profiles, had changed. Some main virulent determinants could not be detected, including proteases, amylase, Dnase, hemolysin and S layer. This article confirmed the important function of AhyR in the pathogenesis of A. hydrophila J-1.     

鸭源致病性嗜水气单胞菌的分离鉴定与生物学特性分析

为了解嗜水气单胞菌的肉鸭健康带毒、致病与耐药情况,对贵州省三穗县某肉鸭屠宰场临床健康肉鸭随机取样,进行嗜水气单胞菌的分离鉴定、毒力基因检测及耐药性分析。结果显示,分离菌具有嗜水气单胞菌典型的培养特征,菌落形态、菌体形态和生化特性均与嗜水气单胞菌相符;16 S rRNA基因序列系统进化树显示,该分离菌与嗜水气单胞菌聚为一支,同源性均>99%;动物回归试验显示,该分离菌对小鼠有较强的致病性;毒力基因PCR检测显示,该分离菌携带aer、hly、epa、act、alt和ahp等6种毒力基因;药敏试验结果显示,该分离菌对复方新诺明、磺胺嘧啶、环丙沙星和诺氟沙星等15种药物耐药;耐药基因PCR检测显示,该分离菌携带qnrB、Sul1和IntI1等3种耐药基因,与药敏试验表型相符。研究结果为嗜水气单胞菌的生物学特性研究及防控提供参考依据。     

Blood changes in carp (Cyprinus carpio) induced by ulcerative Aeromonas salmonicida infections

Only recently Aeromonas salmonicida has been recognized as a significant bacterial pathogen in ulcerative disease of cyprinid fish. Our attempts to formulate a vaccine based on bacterial surface antigens were unsuccessful in conferring reliable protection against lethal challenge. This lead us to study pathological changes in the humoral defense system during ulcerative A. salmonicida infection in carp. High numbers of opportunist pathogens such as A. hydrophila and Pseudomonas sp. were frequently recovered from the internal organs of moribund fish, in addition to A. salmonicida. These findings together with leucopenia in moribund fish suggest that pathogenesis is characterized by a state of immune suppression. In addition, fish which had sustained a sublethal infection were not protected against a subsequent lethal challenge. However, fish previously injected with a concentrated and inactivated culture supernatant showed protection. Differential blood cell counts did not differ between experimental and control groups during sublethal infection in contrast to serum proteins. Furthermore infected non-immune carp showed a progressive decrease of immunoglobulin and total serum protein levels before the day of peak mortality whereas protected carp maintained the immunoglobulin concentration despite a decrease in protein. Our observations suggest the involvement of multiple pathogenic events, affecting different parts of the humoral defense system during ulcerative A. salmonicida infection. The immunosuppressive effects can be minimized by prior vaccination with culture supernatant.     

Correlation between production of acyl homoserine lactones and proteases in an Aeromonas hydrophila aroA live vaccine

Aeromonas hydrophila is a pathogen that causes disease in a wide range of homeothermic and poikilothermic hosts due to its multifactorial virulence. We have previously described the characterisation and use of an auxotrophic aroA mutant of the A. hydrophila AG2 strain as a live attenuated vaccine against A. hydrophila infections in rainbow trout (Oncorhynchus mykiss). In this study we report the expression of extracellular proteolytic activities and of quorum-sensing molecules by this mutant grown under different culture conditions, and in vaccine inocula. The aroA strain expresses extracellular proteases efficiently during in vitro growth and this ability is retained in vaccine inocula that were prepared by washing the bacterial cultures and resuspending the cells in phosphate-buffered saline. Since proteases are considered to be major bacterial antigens, the expression of these enzymes in the live attenuated vaccine may contribute to the superior protection afforded by these kind of vaccines. On the other hand, the production of serine- and metalloprotease activities in A. hydrophila has been described as controlled in a cell density-dependent fashion, through a mechanism known as quorum sensing. A microtiter method was developed that allowed correlation of the production of quorum-sensing molecules and of proteases produced by the aroA strain during in vitro growth and in the vaccine inocula. The production of both products was related to the type of culture medium and conditions used to grow the aroA mutant, whereas there was no correlation between the concentration of acyl homoserine lactones and protease production.     

企鹅源嗜水气单胞菌的分离鉴定

从上海某水族馆因血痢、厌食、消瘦而死亡的秘鲁帝企鹅的心脏、肝脏中分离到2株细菌SH051237和SH060104,其培养性状及生化特性基本一致,经生化试验、VITEK试验等鉴定为嗜水气单胞菌.动物试验表明,分离株对小鼠均具有较强的致病性,且从死亡鼠回收到相应细菌.结果表明,分离到的菌株为致病性嗜水气单胞菌.     

Protection against atypical Aeromonas salmonicida infection in carp (Cyprinus carpio L.) by oral administration of humus extract

Humic substances are formed during the decomposition of organic matter in humus, and are found in many natural environments in which organic materials and microorganisms have been present. In the present study, oral administration of humus extract to common carp (Cyprinus carpio L.) induced effective protection against experimental atypical Aeromonas salmonicida infection. Mortality of fish and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with 10%, 5% or 1% humus extract adsorbed on dry feeding pellets. The median surviving days was also greater in fish treated with 10% or 5% humus extract than in untreated fish. Atypical A. salmonicida was isolated from ulcerative lesions of part of dead fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying bacterial population changes during the progression of skin lesions. These results clearly show that treatment of fish with humus extract is effective in preventing A. salmonicida disease.     

嗜水气单胞菌Ⅲ型分泌系统AopN蛋白原核表达及鉴定

根据GenBank上登陆的嗜水气单胞菌AH-1株Ⅲ型分泌系统aopN基因序列,设计1对特异性引物,以J-1株的基因组为模板,PCR扩增得到aopN基因,连入pET-32a(+),转化至大肠杆菌表达,同时PCR检测aopN基因在66株嗜水气单胞菌中的分布情况。aopN基因测序分析发现,片段长度为874 bp,与嗜水气单胞菌AH-1、SSU、AH-3的同源性分别为97%、81%、82%,与杀鲑气单胞菌杀鲑亚种A449的同源性为81%,与温和气单胞菌的同源性为82%。PCR检测结果表明,57株能检测到aopN基因的目的片段。SDS-PAGE分析表明重组蛋白分子量为54.5ku,用兔抗J-1株的全菌抗血清进行Western blot分析表明,该蛋白具有较好的免疫反应性。由于多数菌株都含有aopN基因,提示AopN可能是嗜水气单胞菌的共同保护性抗原。     

Carp erythrodermatitis (CE) due to an Aeromonas hydrophila infection. Casuistic and experimental results

In November 1987 high losses of carp (Cyprinus carpio) with the main symptom of skin ulcera were observed in a farm in northern Greece. Sixty-six isolates of bacteria, characterized mainly as Aeromonas hydrophila or Pseudomonas spp. could be isolated from lesions of diseased fish. Transmission experiments with these isolates using mirror carp showed that Aeromonas hydrophila strains induced identical clinical and pathological pictures after intra- or subcutaneous injection. Extracts of these Aeromonas hydrophila isolates, as well as a supernatant of culture bouillon were toxic for carp and mice, indicating the presence of endo- and exotoxins. The results prove that carp erythrodermatitis (CE) may be caused by different bacteria, mainly including A. hydrophila.     

维氏气单胞菌研究进展

维氏气单胞菌（Aeromonas veronii）渐已成为重要的人-鱼共患致病菌。文章简单介绍了其分类地位及常规和特殊的生物学性质,并进一步综述了该菌的毒力因子、鉴定方法、流行病学和疫苗研究。作为气单胞菌属的成员,维氏气单胞菌具有该属典型的毒力因子,如气溶素、肠毒素、一系列粘附因子、磷脂酶、丝氨酸蛋白酶和核酸酶等。目前,维氏气单胞菌的鉴定主要依靠生理生化方法,也有人应用分子生物学和免疫学方法鉴定。维氏气单胞菌对水产动物有相当高的致死率,患有肝胆疾病的人感染后常常产生严重的后果,需要加强针对该菌所致疾病的诊断。在疫苗研究方面,有学者研究了口服疫苗和DNA疫苗,达到了一定的免疫效果。     

杀鲑气单胞菌的快速检测

杀蛙气单胞菌引起的鱼类疖疮病是进口动物检疫对象。该菌只能在疾病流行期从病鱼体内分离。在水和鱼卵中虽有此菌，也无法用常规细菌分离和培养法进行分离和鉴定。用ELISA技术快速检测水样中的该菌获得成功，为进口虹鳟鱼卵时检测该菌提供了方法。     

Contribution of food deprivation to the immune response in rainbow trout (Oncorhynchus mykiss) vaccinated against Cryptobia salmositica and Aeromonas salmonicida

The aims of the present study were to determine (a) the effectiveness of an attenuated live Cryptobia salmositica vaccine; (b) the effects of food deprivation on the immune response and its duration in rainbow trout (Oncorhynchus mykiss) immunised with a live C. salmositica vaccine or with a killed Aeromonas salmonicida vaccine. The fish were divided into three groups (I, II and III; 14 fish per group), those in Groups I and II were under food deprivation (0.40% of body weight), while Group III fish were fed to satiety. The study showed that the attenuated strain of C. salmositica did not cause anaemia and disease, and the fish were protected from clinical disease when they were challenged with virulent parasites. Parasitaemia in all fish vaccinated and challenged with virulent C. salmositica fluctuated and was relatively low; however, fish in Group III had higher parasitaemia than those in Groups I and II between weeks 8 and 14. The numbers of activated neutrophils increased [nitroblue tetrazolium (NBT) assay] after immunisation with both Cryptobia and Aeromonas vaccines and they remained high throughout the experiment. Antibody production (ELISA values) increased after vaccination and were slightly higher in Group III. ELISA titres against A. salmonicida increased after vaccination and decreased after 5 weeks. The titres increased again after the vaccinated fish were given booster, and they were higher than those in the first vaccinated fish.     

致病性嗜水气单胞菌PCR快速检测方法的建立

本试验根据GenBank已登录的致病性嗜水气单胞菌保守序列16S rDNA和Aero,设计2对引物,以嗜水气单胞菌纯培养物为起始材料,建立PCR检测方法。从12株分离物中均扩增到16S rDNA片段,从3株分离物中均扩增到Aero片段,经序列测定和分析,所扩增的片段均为嗜水气单胞菌的核苷酸序列。结果表明,建立的PCR方法可用于检测致病性嗜水气单胞菌。     

Development of a PCR protocol for the detection of Aeromonas salmonicida in fish by amplification of the fstA (ferric siderophore receptor) gene

The aims of the study were to evaluate a new PCR protocol designed to detect Aeromonas salmonicida in fish tissues and to develop a non-destructive method for the diagnosis of furunculosis. A set of primers (Fer3, Fer4), flanking a fragment of the fstA gene (coding for the ferric-siderophore receptor) was designed, showing to be sensitive and specific. When compared to PCR methods previously reported, the new protocol recognized all the 69 A. salmonicida strains evaluated, with no cross-reactions with the other bacterial species analysed. Sensitivity assays were performed in fish tissues seeded with serial dilutions of pure cultures of A. salmonicida and mixed cultures of this bacterium with Vibrio anguillarum and Aeromonas hydrophila. Detection limits obtained were of 60 and 450 bacterial cells 100 mg(-1) of tissue, respectively. Mucus and blood were evaluated in order to develop a non-destructive tool to detect the pathogen. The detection limits in seeded mucus and blood samples were 2.5 x 10(2) and 1 x 10(5) bacterial cells mL(-1), respectively. When the method was used to detect A. salmonicida in asymptomatic wild salmon, four samples of mucus and six of blood were positive, corresponding to 6 out of the 31 fish examined, whereas only one of the samples resulted positive by culture methods. It is concluded that the PCR protocol evaluated is fast, specific and sensitive to detect A. salmonicida in infected and asymptomatic fish, and will be helpful for the control of the disease through the prompt detection of carriers within fish populations.     

Isolation and identification of motile Aeromonas species from chicken.

In this study a total of 140 broiler carcasses and carcass parts purchased at different supermarkets in Ankara including 50 whole carcass, 30 wing, 30 leg and 30 breast samples were analysed for the presence of motile Aeromonas species. According to analysis findings, motile Aeromonas spp. were isolated from 116 (82.9%) of total 140 samples. The distribution of the isolates were 94%, 86.6%, 80%, 63.3% in broiler carcass, wing, leg and breast samples, respectively. Aeromonas hydrophila was isolated the most prevalent species with 56% the range followed by Aeromonas sobria with 29.3% and Aeromonas caviae with 14.7% from all of the carcass and carcass part samples, respectively. Consequently, it was supposed that, examined broiler carcass and carcass parts have been contaminated to important level with motile Aeromonas species and it has been risk for public health.     

Development of multiplex PCR assay for simultaneous detection of five bacterial fish pathogens

A multiplex polymerase chain reaction (PCR) method was designed for the simultaneous detection of the five major fish pathogens, Aeromonas hydrophila, Aeromonas salmonicida subsp. salmonicida, Flavobacterium columnare, Renibacterium salmoninarum, and Yersinia ruckeri. Each of the five pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The detection limits of the multiplex PCR was in the range of 2, 1, 1, 3, and 1CFU for A. hydrophila, A. salmonicida, F. columnare, R. salmoninarum, and Y. ruckeri, respectively. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria. The multiplex PCR assay was useful for the detection of the bacteria in naturally infected fish. This assay is a sensitive and specific and reproducible diagnostic tool for the simultaneous detection of five pathogenic bacteria that cause disease in fish. Therefore, it could be a useful alternative to the conventional culture based method.     

嗜水气单胞菌不同分离株生化特性及胞外蛋白酶的检测

嗜水气单胞菌可引起多种宿主患病,该菌致病性与其产生的多种毒力因子特别是胞外蛋白酶有关。本研究检测了26株嗜水气单胞菌不同分离株的生化指标,采用脱脂乳平板法检测了胞外蛋白酶的产生情况,并利用PCR技术检测了2种胞外蛋白酶基因ahp-A(编码丝氨酸胞外蛋白酶)和eprCAI(编码温敏胞外蛋白酶)。结果表明,大部分菌株生化指标表现为蔗糖(+)、阿拉伯糖(+)、葡萄糖(+)、甘露醇(+)、鸟氨酸脱羧酶(-)、肌醇(-)、七叶苷(+);脱脂乳平板法检测发现,91.7%(22/24)的致病菌株均能产生胞外蛋白酶,而2株无毒株则不能产生,对2个胞外蛋白酶基因的检测也得到相似的结果。本研究将为嗜水气单胞菌的流行病学调查和致病机制的深入研究奠定基础。     

Protection against atypical Aeromonas salmonicida infection in common carp, Cyprinus carpio L., by oral administration of a mixed microbial culture of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae

A microbial culture was prepared by co-cultivation of Lactobacillus paracasei, Pichia membranifaciens and Saccharomyces cereviciae for 48 hr at 30°C in rice bran extract medium, supplemented with dextrose. Oral administration of the resulting non-viable heat-inactivated microbial culture to common carp, Cyprinus carpio L., delivered in feed for four weeks, induced effective protection against experimental atypical Aeromonas salmonicida infection which causes "ulcer disease". After challenge of the carp by immersion, fish mortality and development of skin lesions such as hemorrhages and ulcers were significantly suppressed in carp treated with mixed microbial culture adsorbed on dry pellets relative to carp treated with medium or without extract. Atypical A. salmonicida was re-isolated from ulcerative lesions in parts of dead and surviving fish, but Aeromonas hydrophila and Flavobacterium sp. were also isolated from these fish, verifying microbial population changes during the progression of skin lesions. Among interleukin-1β (IL-1β), tumor necrosis factor-α, as well as CXC-α and CXC-β chemokines, gene expression of IL-1β was up regulated in the spleen and head kidney three weeks after administration of the mixed microbial culture. These results clearly show that this mixed microbial culture, delivered in feed, is effective in preventing A. salmonicida disease in carp.     

嗜水气单胞菌弹性蛋白酶基因的克隆表达

为了研究嗜水气单胞菌重组弹性蛋白酶的酶学性质,试验根据GenBank中的嗜水气单胞菌弹性蛋白酶基因ahyB设计1对含酶切位点的特异引物,以嗜水气单胞菌J-1(AhJ-1)株为模板,经PCR扩增得到不含信号肽的成熟弹性蛋白酶基因片段(787 bp),并与pMD18-T载体连接、测序,再用DNAStar软件分析。结果表明:该基因片段与豚鼠气单胞菌胞外蛋白酶同源性高达95%,与嗜水气单胞菌AG2株弹性蛋白酶ahyB基因同源性为92%,与铜绿假单胞菌LasB基因同源性为82%;将PCR产物连入表达载体pET-32a,转化至大肠杆菌BL21菌株中进行诱导表达,出现50 ku的融合表达蛋白,该表达产物纯化复性后表现出酶的活性。