两株气单胞菌的分离与鉴定

从福建省某牛蛙养殖场病死牛蛙和某鸭场病死鸭体内分离到两株细菌，检验结果表明，两株分离菌均为有运动力的革兰氏阴性杆菌。根据其临床症状、细菌培养特性、生化特性、动物回归试验以及溶血试验等一系列试验，研究证明牛蛙感染的病原为嗜水气单胞菌，鸭感染的病原为温和气单胞菌。     

Isolation and characterization of potentially human pathogenic, cytotoxin-producing aeromonas strains from retailed seafood in Berlin, Germany

The presence of potentially human pathogenic strains of Aeromonas was investigated in 84 samples of seafood which were purchased from retail traders in Berlin, Germany in spring 2000. A total of 134 Aeromonas strains were isolated on selective [GSP agar and Aeromonas (Ryan) agar] and unselective (standard count agar and enterohaemolysin agar) media from 27 (32.1%) of the samples and were classified as Aeromonas hydrophila (67.9%), A. caviae (26.1%) and A. sobria (6.0%) by biotyping. Thirteen (48.1%) of the 27 positive samples contained more than one species of Aeromonas. Production of haemolysins on enterohaemolysin agar was found with 132 (98.5%) of the strains at 28 degrees C and with 130 strains (97.0%) at 37 degrees C growth temperature. Vero cytotoxins were produced by 99 (73.9%) of the strains when grown at 28 degrees C but only by 24 of the strains (17.9%) at 37 degrees C. The latter strains were identified as A. hydrophila (n = 22) and A. sobria (n = 2) which came from 17 (20.2%) samples of raw seafood and from ready-to-eat salted herring 'Matjes' products. Cytotoxin-encoding genes for aerolysin (aer) and haemolysin A (hlyA) were investigated by PCR. Aer and hlyA genes were detected in both, strains which produced toxins only at 28 degrees C and strains which produced toxins at 37 degrees C. Our data indicate that raw seafood and ready-to-eat fish products can harbour potential human pathogenic, cytotoxin producing Aeromonas strains.     

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.     

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.     

中华鳖几种常见疾病病原的分离鉴定及药敏试验

从患“穿孔病”、“腐皮病”、“溶血性腹水病”等濒死鳖的肝、心血和腹水中分离到20株细菌，其中温和气单胞菌（Aeromonas sobria）5株，豚鼠气单胞菌（A.caviae）3株、凡隆气单胞菌（A.veronii）2株、奇异变形杆菌（Proteus mirabilis）2株、产碱假单胞菌（Pseudomonas alcaligene）2株、无色杆菌（Achro sp.）3株、不动杆菌（Acinetobacter sp.）2株、黄杆菌（Flavobacterium sp.）1株。人工感染试验证实，运动型气单胞菌的温和气单胞菌和豚鼠气单胞菌是其主要病原菌。药敏试验结果表明，分离菌株对新霉素、菌必治、丁胺卡那、庆大霉素、环丙沙星高度敏感。     

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。     

温和气单胞菌RC-07-KA株溶血素基因克隆、表达及活性检测

为研究温和气单胞菌(A.sobria)溶血素基因的特性,本研究根据GenBank登录的气单胞菌属溶血素基因序列设计一对引物,经PCR扩增得到大小约1.5kb的A.sobria RC-07-KA株溶血基因片段,将该片段克隆到pGEM-T载体,测序结果显示:溶血素基因片段大小为1 467 bp,编码487个氨基酸残基,遗传进化分析结果表明该基因与气单胞菌属中的A.hydrophila菌株Sb (AY611033)、NLEPA-1607 (AF410466)、AEF (HM853019),A.sobria菌株357 (AY157998)、人源分离株(EF620533)和A.salmonicida菌株17-2 (X65048)的溶血素基因亲缘关系较近,同源性大于95％,而与其他菌株的同源性较低.通过构建溶血素重组表达质粒pET-HIy,诱导表达并通过western blot鉴定表达蛋白,结果显示重组菌能高效表达重组溶血素,而且纯化的重组溶血素具有溶解鲤鱼红细胞的活性.为该菌进一步深入研究奠定了基础.     

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.     

鲫腹水症病原菌分离鉴定及药敏试验

从河南新乡某养殖场患腹水症的鲫鱼体内分离纯化获得1株细菌,编号为CA1618,通过细菌形态学观察、Biolog微生物鉴定系统,进一步采用PCR方法扩增其16SrDNA序列分析。结果表明CA1618菌株为革兰阴性杆菌,利用葡萄糖、麦芽糖、半乳糖、海藻糖等多种碳源,与Aeromonas sobria特征同源性为0.575、遗传距离为6.226;测序获得16SrDNA序列片段为1 363bp,与Aeromonas sobria序列同源性为99%~100%,构建系统发育树表明与模式菌株Aeromonas sobria ATCC43979T(GenBank登录号:X74683)亲缘关系最近,最终鉴定为温和气单胞菌(Aeromonas sobria)。CA1618菌株人工感染鲫鱼半数致死量(LD50)为1.55×10~7 CFU/mL,药敏试验显示对头孢哌酮、头孢噻肟、头孢克肟、萘啶酸、诺氟沙星、复方新诺明、四环素、氯霉素等抗生素敏感。     

嗜温气单胞菌的鉴定及其外膜蛋白型研究

从病鳖、鳗鱼体内分离到2株嗜温气单胞菌(TAh-3、EAs-1),经形态学观察、生理生化特性等鉴定,确定TAh-3为嗜水气单胞菌,EAs-1为温和气单胞菌。动物感染试验结果表明,TAh-3株为致病性嗜水气单胞菌,对鲫鱼的致死率100%,EAs-1株为非致病性温和气单胞菌。SDS-PAGE电泳结果显示,TAh-3株外膜蛋白(OMPs)型由7条蛋白带组成,其中1条主带大小为40.0kDa;EAs-1株OMPs型由8条蛋白带组成,其中2条主带大小为38.0kDa、45.0kDa。     

鱼源气单胞菌的主要特性研究

对分离于草鱼肠炎、白鲢鱼打印病、鲢鱼白皮病、草鱼烂鳃、鲤鱼体表出血感染及甲鱼败血症等病例的80株细胞进行了主要特性研究。结果表明均为气单胞菌属（Aeromonas)的细菌，其中嗜水气单胞菌（A.hydrophila)29株（占36．25％）、豚鼠气单胞菌（A.caviae)38株（占47．5％）、温和气单胞菌A.sobria）13株（占16．25％）。在被检验的病例中，或为其中一种气单胞菌的单独感染，或为两种、三种气单胞菌的混合感染。经做人工感染试验检查，表明这些分离菌均为相应感染症的致病菌。     

解淀粉芽孢杆菌的分离鉴定及其对嗜水气单胞菌抑制作用研究

本试验从鲫鱼肠道中分离到1株芽孢杆菌,经生物学特性测定及16S rRNA测序分析,鉴定该分离菌株为高产蛋白酶的解淀粉芽孢杆菌(命名为JX001).对该分离菌进行嗜水气单胞菌体外抑菌试验,结果发现有明显的抑菌圈和抑菌带,说明该分离菌对嗜水气单胞菌具有较强的抑制作用,在养殖水中加入分离菌JX001可防止嗜水气单胞菌的暴发;同时分离菌JX001在生长代谢过程中产生大量的蛋白酶,可有效地促进蛋白质水解,用于蛋白质饲料的降解.     

中华绒螯蟹细菌性病原的分离与鉴定

对杭州地区患病的中华绒螯蟹进行了病原的分离和鉴定 ,从病蟹体内分离到 2株革兰氏阴性杆菌 ,单极生鞭毛 ,运动 ,兼性厌氧 ,发酵葡萄糖产酸产气 ,氧化酶、过氧化氢酶阳性 ,七叶苷、蔗糖、水杨素阴性 ,还原硝酸盐 ,对氨苄青霉素和羧苄青霉素敏感等 ,同易损气单胞菌的形态特征、生理生化特性基本相同 ,故将这 2株病原菌鉴定为易损气单胞菌 (Aeromonas trota)。人工注射感染健康蟹后 ,均在 2 d内死亡 ,死亡率为 10 0 %,从而证实易损气单胞菌为此次患病中华绒螯蟹的致病菌。     

Isolation and haemolytic activity of Aeromonas species from domestic dogs and cats.

Rectal swabs from 120 domestic dogs and 15 domestic cats were examined for Aeromonas species using alkaline peptone water (pH 8.6) as the enrichment medium and blood agar containing 15 mg/l ampicillin as the plating medium. Aeromonads were isolated from 13 (10.8%) dogs and from 1 (6.7%) cat. Of the 14 aeromonads isolated in the present study only 9 were available for speciation and testing in the haemolysin assay. Of these 5 were A. sobria (including one from a cat), 2 were A. hydrophila and 2 were A. caviae. Six were positive in the haemolysin assay; 4 A. sobria (one from a cat) and 2 A. hydrophila. The presence of haemolysin producing-Aeromonas species in the faeces of domestic dogs and cats may pose a public health problem for humans who come into contact with such animals.     

鳜鱼嗜水气单胞菌的分离与鉴定

在建立和完善北方地区鱼类疫病的流行病学调查研究时,从丹东鸭绿江一网箱养殖场患病鳜鱼中分离到了一株优势菌株。对该菌株进行营养琼脂及绵羊血琼脂培养基培养、革兰氏染色镜检、Vitek细菌鉴定仪鉴定及16SrRNA序列分析比对。经细菌形态学特征分析、生理生化试验及16SrRNA核苷酸序列同源性分析和系统发育树的构建比对,结果表明该分离菌株为嗜水气单胞菌。本研究的开展为有效防控疫病及疫苗制备等方面提供参考和借鉴,同时为开展该疫病的监测、防治和预警,促进鱼类及其产品的进出口贸易,意义十分重大。     

Isolation and Identification of Aeromonas hydrophila from the Case of Channel Catfish Bowel Disease

To ensure the cause of suspected bacteria disease for channel catfish, the bacteria from clinical cases were isolated and cultured, then the biochemical and molecular biology assay, and animal regression test were used to identify the species of the isolated bacteria, bacteria medicine sensitive test was used to detect the bacterial drug resistance. The results showed that the bacteria were gram negative bacilli. The colony morphology and biochemical characteristics of the isolated bacteria were same as Aeromonas hydrophila. The sequencing test of the 16S rDNA also confirmed the isolated bacteria were Aeromonas hydrophila. The isolated bacteria could infect the healthy channel catfish and cause the same symptoms as the clinical case. The isolated bacteria were sensitive to floroquinolones and resistant to penicillin and cephalosporin drugs. The results showed that the pathogen of case catfish bowel disease was Aeromonas hydrophila, and the floroquinolones could use to prevent and treat the disease.     

斑点叉尾鮰套肠病病原嗜水气单胞菌的分离与鉴定

为确定疑似细菌感染的斑点叉尾鮰病原,本试验对临床病例进行细菌分离培养、生物化学鉴定和分子生物学鉴定、动物回归试验和病原菌药敏试验。结果显示,分离菌为革兰氏阴性杆菌,菌落形态和生化特性与嗜水气单胞菌相符;16S rDNA检测与测序分析结果进一步证实分离菌确为嗜水气单胞菌;动物回归试验显示,分离菌能引起与临床自然病例相似的症状;药敏试验显示,分离菌对喹诺酮类药物敏感,对青霉素类、头孢类药物产生耐药性。结果表明,疑似细菌感染斑点叉尾鮰病例为嗜水气单胞菌感染,可选择喹诺酮类药物进行病例的预防治疗。     

斑鳢细菌性败血症病原菌的分离与鉴定

由患细菌性败血症的斑鳢分离到１１株菌,根据直接荧光抗体法鉴定结果,对其中７株菌的菌落特性进行了观察,分析了菌株的生化特性,并测试了其对斑鳢、乌鳢和鲫的致病力。结果证明,斑鳢的细菌性败血症是由嗜水气单胞菌（Ａｅｒｏｍｏｎａｓｈｙｄｒｏｐｈｉｌａ）引起的     

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.     

从江县鲤爆发性死亡病例实验室诊断

2018年10月贵州省从江县某水产养殖专业合作社鲤爆发死亡,本研究对死亡病例进行了流行病学调查、病理学观察、寄生虫学检查、细菌分离鉴定和病毒PCR检测等诊断。结果显示:病鲤病变以鳃丝溃烂、体表出血、肛门红肿和眼球凹陷为主要特征,组织的主要病变是肾小球萎缩出血、肾小囊空腔、鳃丝肿胀粘连及上皮细胞脱落等;细菌分离培养可得到圆形凸起、表面光滑的灰白色菌落,经染色镜检、生理生化鉴定和16S rRNA基因测序分析,鉴定该分离菌为嗜水气单胞菌;PCR检测显示鲤浮肿病毒(Carp edema virus,CEV)为阳性,未检出鲤春病毒血症病毒(Spring viremia of carp virus,SVCV)和鲤疱疹病毒(Koi herpesvirus,KHV);显微镜观察亦未见到寄生虫虫体;分别取分离菌和鲤浮肿病毒阳性病例组织悬液进行人工感染试验,均可引起健康鲤发生死亡。上述结果表明,从江县鲤的死亡是嗜水气单胞菌和鲤浮肿病毒混合感染所致。