5株鲆鲽鱼类病原菌多克隆抗体的制备与应用

本研究制备了鳗弧菌Vibrio anguillarum、杀鲑气单胞菌Aeromonas salmonicida、副溶血弧菌V. parahaemolyticus、哈维氏弧菌V. harveyi和腐败希瓦氏菌Shewanella putrefaciens 5株鲆鲽鱼类病原菌的兔抗血清,建立了5种菌的间接ELISA检测方法,并将该检测方法用于鱼类细菌分离物病原检测。人工感染结果显示,5株菌对大菱鲆的半数致死量(LD50)在102~107CFU/fish;制备的兔抗血清效价分别为1∶2 048 000、1∶16 000、1∶16 000、1∶1 024 000、1∶128 000;交叉反应结果显示,鳗弧菌、副溶血弧菌、哈维氏弧菌3株弧菌与抗血清相互之间存在交叉反应;抗血清特异检测灵敏度分别为104、108、107、105、106cell/ml;对13株海水鱼类细菌分离物进行检测,有1株腐败希瓦氏菌阳性,两株哈维氏弧菌阳性;1株副溶血弧菌和哈维氏弧菌均为阳性,该结果与16S rDNA序列的分子分析方法一致。     

大亚湾海洋异养细菌的初步研究

文章根据2004年3月对大亚湾6个站位表层水异养细菌的调查资料,分析研究了大亚湾表层水中异养细菌的数量分布和种类组成.结果表明,大亚湾表层水中异养细菌数量变化范围为7.15×102 ～91.0×102 cfu ·mL-1,异养细菌数量从湾顶到湾口依次减少;表层水中异养细菌的优势种有19种,利用Biolog微生物鉴定系统对优势菌进行鉴定,它们均为革兰氏阴性菌,隶属于10属14种;大亚湾表层水中异养细菌的种类组成以气单胞菌(Aeromonas sp.)、弧菌属(Vibrio sp.)和伯克霍尔德氏菌属(Burkholderia sp.)为优势属,舒氏气单胞菌[A.schubertii(DNA group 12)]、最小弧菌(V.mimicus)和荚壳伯克霍尔德氏菌(B.glumae)为优势种.     

水产动物6种主要病原菌与抗血清的免疫交叉反应

采用ELISA、试管凝集、Western-blot等方法,分析了鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(V. harveyi)、溶藻胶弧菌(V. alginolyticus)、副溶血弧菌(V. paraheamolyticus)、迟钝爱德华氏菌(Edwardsiella tarda)和荧光假单胞菌(Psedomonas fluorescens)等水产养殖中主要病原细菌与抗血清之间的免疫交叉反应.结果表明,弧菌属细菌之间的交叉反应程度比较大,而与其他两属的细菌之间存在的交叉反应程度小,或不存在交叉反应;Western-blot分析结果显示,哈维氏弧菌、溶藻胶弧菌和副溶血弧菌抗血清分别与其他3种弧菌在分子量为135.6 kD和121.5 kD;95.6 kD,48.4 kD,39.2 kD和34.9 kD;55.1 kD的蛋白带处存在交叉反应,而这些分子量的蛋白带与其他两属的抗血清均不发生反应.     

养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性分析

为了解引起养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性情况,针对2002-2010年由不同地区病样分离的27株细菌性病原进行了16S rDNA鉴定,并采用K-B法测定了27株细菌对22种抗生素的耐药性,分析了病原菌的耐药谱及耐药率变化.结果显示,大菱鲆腹水病病原菌主要有大菱鲆弧菌(Vibrio scophthalmi)、迟钝爱德华氏菌(Edwardsiella tarda)、鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(Vibrio harveyi)、假交替单胞菌(Pseudoalteromonas espejiana).山东青岛地区以大菱鲆弧菌为主,威海地区以迟钝爱德华氏菌为主,烟台地区菌株种类分布平均.5类细菌对青霉素类、头孢菌素类、大环内酯类、复方新诺明耐药率高于50％.只有1株迟钝爱德华氏菌对氟苯尼考产生了耐药,其余菌株对其均没有耐药性,且在长期使用中不易产生耐药性,证实氟苯尼考为当前防治腹水病的一种良好抗菌药物.27株病原菌的耐药谱数量为27个,每个菌株具备自己独特的耐药谱,74.1％的菌株对10种以上的抗菌药物产生了耐药性,均有多重耐药性.     

河蟹养殖主要疾病控制技术

据几年来的初步调查和研究,目前严重影响我国河蟹养殖业发展的病害有上岸症、黑壳病、黑鳃病、颤抖病、蟹奴病、肠炎病、烂爪病、性早熟等十多种。以上河蟹病害的发生,其病原大致为：细菌性、病毒性、原生动物性及生理性几大类,以细菌类的疾病危害最大。影响最深的细菌以弧菌属的霍乱弧菌、副溶血弧菌、溶藻弧菌、鳗弧菌、创伤弧菌、迟缓爱德华氏菌为主；气单胞菌属中以嗜水气单胞菌、气单胞菌为主；原生动物中以钟型虫、聚缩虫等纤毛虫类为主；生理性的以性早熟蟹为代表,加上投饵不科学,摄食量降低,导致营养奇缺,     

牙鲆腹水病病原免疫血清的制备和纯化试验研究

用迟钝爱德华氏菌免疫家兔,制备出高效价迟钝爱德华氏菌免疫血清.先制备迟钝爱德华氏菌抗原, 耳缘静脉注射免疫家兔.经微量反应板法检测血清效价, 效价达到1 ∶ 2560;用Millipore Montage抗体纯化试剂纯化抗体,纯化的抗体经SDS-PAGE电泳,杂带较少,条带主要集中于50 kD处;用斑点酶联免疫吸附试验检测时, 迟钝爱德华氏菌呈阳性, 温和气单胞菌、嗜水气单胞菌、河流弧菌、溶藻弧菌、鳗弧菌、腐败希瓦氏菌、产碱普罗威斯登菌、阪崎肠杆菌和大肠杆菌均呈阴性.试验结果表明,特异、高效价的迟钝爱德华氏菌免疫血清,为快速检测牙鲆腹水病病原奠定了基础.     

我国首个海水鱼活疫苗获评2019农业农村部十大新技术

正近日,从2019中国农业农村科技发展高峰论坛获悉,华东理工大学的"鲆鲽鱼类爱德华氏菌病活疫苗创制技术"被评为"2019年农业农村部十大新技术"。这种"鲆鲽鱼类爱德华氏菌病活疫苗创制技术"主要针对危害我国多宝鱼养殖业的重要病害——爱德华氏菌病,建立了从病原分离鉴     

养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性分析

为了解引起养殖大菱鲆(Scophthalmus maximus)腹水病的病原多样性及其耐药性情况,针对2002–2010年由不同地区病样分离的27株细菌性病原进行了16S r DNA鉴定,并采用K-B法测定了27株细菌对22种抗生素的耐药性,分析了病原菌的耐药谱及耐药率变化。结果显示,大菱鲆腹水病病原菌主要有大菱鲆弧菌(Vibrio scophthalmi)、迟钝爱德华氏菌(Edwardsiella tarda)、鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(Vibrio harveyi)、假交替单胞菌(Pseudoalteromonas espejiana)。山东青岛地区以大菱鲆弧菌为主,威海地区以迟钝爱德华氏菌为主,烟台地区菌株种类分布平均。5类细菌对青霉素类、头孢菌素类、大环内酯类、复方新诺明耐药率高于50%。只有1株迟钝爱德华氏菌对氟苯尼考产生了耐药,其余菌株对其均没有耐药性,且在长期使用中不易产生耐药性,证实氟苯尼考为当前防治腹水病的一种良好抗菌药物。27株病原菌的耐药谱数量为27个,每个菌株具备自己独特的耐药谱,74.1%的菌株对10种以上的抗菌药物产生了耐药性,均有多重耐药性。     

28株水产动物致病菌的编码鉴定

采用国产革兰氏阴性杆菌编码鉴定系列培养基对28株细菌进行鉴定。结果显示：除2株细菌未能得到鉴定外，其余26株细菌均鉴定到属或种水平，可鉴定率为92．86％．但各菌株的鉴定概率和模式频率有所不同。26株可鉴定菌分布于气单胞菌属(Aeromonas)、假单胞菌属(Pseudomonas)、不动杆菌属(Acinetobacter)、摩根氏菌属(Morganella)、弧菌属(Vibrio)、变形杆菌属(Proteus)、黄单胞菌属(Xanthomonas)、邻单胞菌属(Plesiomonas)和埃希氏菌属(Escherichia)9个菌属，其中13株为气单胞菌(Aeromonas)；3株为恶臭假单胞菌(P．putide)；2株为摩氏摩根氏菌(M.morganella)；2株为不动杆菌(Acinetobacter)；2株为类志贺邻单胞菌(P．shigelloides)；拟态弧菌(V．mimicus)、普通变形杆菌(P．vulgaris)、嗜麦芽黄单胞菌(X．maltophilia)和大肠埃希氏菌(E．coli)各l株。基本上摸清了安徽省水产养殖动物常见病原菌的种类。     

Surface-exposed expression of Edwardsiella tarda EseB in live attenuated Vibrio anguillarum based on novel surface display systems

Live, attenuated Vibrio anguillarum strains can serve as vectors for the delivery of heterologous antigens for development of multivalent recombinant vaccines. Based on the outer membrane anchoring elements of V. anguillarum , we have previously constructed several efficient surface display systems Lpp-Omporf1, Lpp-OmpU, Lpp-Omp26La, Wza-Omporf1, Wza-OmpU and Wza-Omp26La. In this study, with these constructed surface display systems, a putative antigen protein EseB from pathogenic Edwardsiella tarda was successfully expressed on the surface of an attenuated V. anguillarum strain to get multivalent vaccine candidates. Further immune protection evaluation in zebra fish ( Danio rerio ) demonstrated that the V. anguillarum EseB-display strain AV/pW-26La-B could trigger full protection against V. anguillarum infection and early protection against E. tarda infection in the immunized fish. These results suggest that surface display of heterologous protective antigens in attenuated V. anguillarum could be used as a tool to develop potential V. anguillarum vector vaccine.     

Characterization of Edwardsiella tarda strains isolated from turbot, Psetta maxima (L.)

The biochemical, serological and molecular characteristics of a group of 21 Edwardsiella tarda strains isolated from turbot, Psetta maxima, in two different areas of Europe were analysed and compared with a total of 13 strains of this bacterial species with different geographical and host origins. All the turbot isolates were biochemically identical to the E. tarda strains included as reference. The use of different techniques including microagglutination, dot blot and Western blot of lipopolysaccharides allowed us to determine that all the turbot isolates constitute an homogeneous and distinctive serological group. Genetic analysis by randomly amplified polymorphic DNA (RAPD) analysis demonstrated that although the E. tarda strains from turbot were compiled in a unique group using the primers P3 and P6, two clonal lineages could be detected when oligonucleotides P4 and P5 were employed.     

中华鳖爱德华菌病病原菌的分离鉴定及致病因子研究

采用API 20E系列生化鉴定及16S rDNA和gyrB基因序列同源性分析方法,对从患病中华鳖(Trionyx sinen-sis)肝脏中分离到的一株细菌TL5m进行了鉴定,并通过人工感染试验,对该菌株进行了毒力检测;此外分别提取该TL5m株的主要致病因子外膜蛋白、脂多糖和胞外产物,对中华鳖进行毒力和免疫保护率试验。结果显示:菌株TL5m的API 20E鉴定编码为4544000,99.9%为迟钝爱德华氏菌(Edwardsiella tarda);其16SrDNA序列和gyrB基因序列(GenBank登录号分别:EF121756和GU563803)与迟钝爱德华氏菌的同源性最高(分别为94%和98%);菌株TL5m对中华鳖的半数致死量LD50为2.45×106 CFU/ind。药敏感结果显示菌株对磷霉素、菌必治、头孢孟多、头孢噻吩、壮观霉素高度敏感。外膜蛋白攻毒剂量60μg/ind和脂多糖攻毒剂量400μg/ind时,对中华鳖的致死率都为33.3%,胞外产物对中华鳖的LD50为31.73μg/ind;全菌灭活苗、胞外产物、外膜蛋白和脂多糖的免疫保护率分别为75%、62.5%、25%和87.5%。结果表明,发病中华鳖的病原菌为迟钝爱德华氏菌,其分泌的胞外产物对中华鳖具有较高毒力;提取的脂多糖对中华鳖遭受迟钝爱德华氏菌攻击具有较高免疫保护率。     

Multiplex nested-polymerase chain reaction for the simultaneous detection of Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae, four important fish pathogens in subtropical Asia

A multiplex nested-polymerase chain reaction (PCR)-based (m-nested PCR) method was developed for simultaneous detection of four important freshwater/marine fish pathogens in subtropical Asia, including Aeromonas hydrophila, Edwardsiella tarda, Photobacterium damselae and Streptococcus iniae . The specificity of the oligonucleotide primers used for PCR detection was confirmed to generate specific amplicons for the corresponding pathogens. Moreover, non-specific amplicons were observed when the primers were tested using pure DNA extracted from 31 related bacterial strains belonging to 23 species or tissue homogenates of infected tilapia. This m-nested PCR approach could detect 19 colony forming unit (CFU) for A. hydrophila , 62 CFU for E. tarda , 280 CFU for P. damselae subsp. piscicida and 179 CFU for S. iniae in infected tilapia kidney homogenates, consistent with the results derived from bacteriological methods. The assay described in this paper is a sensitive and effective method for simultaneous detection of multiple fish pathogens.     

Isolation and identification of fish pathogen Edwardsiella tarda from mariculture in China

The causative agent was isolated from diseased turbots (Scophthalmus maximus) stricken by a high‐mortality outbreak of bacterial septicaemia occurring in a mariculture farm in Yantai, a northern coastal city of China. Seven pure isolates, namely EH‐15, EH‐103, EH‐107, EH‐202, EH‐203, EH‐305 and EH‐306, belonged to Edwardsiella tarda. The phenotypic features of the cultures were analysed extensively. Three of the isolates showed high 16S rDNA sequence similarities with E. tarda sequence (GenBank accession no. EF467289). However, unlike the E. tarda ATCC 15947, all the isolates, except EH‐15, contained a novel large plasmid sized about 23.7 kb. Furthermore, pathogenicity of the isolates was addressed by experimental challenges with fish models. The isolates exhibited strong virulence to swordtail fish with LD₅₀ ranging between 3.8 × 10³ and 3.8 × 10⁵ CFU g^?1, and EH‐202 displaying the lowest LD₅₀ value among them. Antibiotic susceptibilities of E. tarda isolates were assayed. Compared with E. tarda ATCC 15947, the isolates displayed strong resistance to chloramphenicol, and the probable dominant chloramphenicol resistance determinant was cat III. Depicting the main biological properties of turbot‐borne E. tarda strains in China, the study provided useful information for further unveiling their pathogenic mechanisms.     

利用mini-Tn10转座子文库筛选鳗弧菌M3表型发生变化的基因

为了寻找影响鳗弧菌(Vibrio anguillarum)表型变化的基因，本研究使用转座子mini-Tn10 (pLOF/Kana)构建了鳗弧菌M3突变株文库，筛选影响表型变化的菌株及相关基因，证明这些表型变化的突变子与毒力存在一定的相关性。对M3突变文库的1152突变子进行筛选，获得泳动能力改变的突变子1个(编号为6G_1)，酪蛋白酶活性发生改变的突变子3个(编号为5A_11、7B_12和7E_12)，明胶酶活性发生改变的突变子1个(编号为7H_1)，以及菌膜形成能力发生显著变化的突变子3个(编号为5E_2、6A_2和6E_12)。对转座子插入位点进一步分析显示，一个磷酸二酯酶相关基因突变引起泳动能力增强(P<0.05)，leuD、rseB和thiQ突变引起酪蛋白酶活性显著减弱(P<0.05)，potD突变引起明胶蛋白酶活性显著减弱(P<0.05)，leuO、ilvH和grpB的突变引起菌膜形成能力明显减弱(P<0.05)。对这些表型变化的突变子进行毒力感染，发现野生型M3是6G_1突变子的半数致死剂量(Lethal dose 50%，LD50)的2.04倍，该突变子毒力相对增强。5A_11、7B_12和7E_12的突变子LD50分别为野生型M3的2.96、3.25和3.36倍。7H_1的LD50是野生型M3的1.25倍，5E_2、6A_2和6E_12的LD50分别为野生型M3的3.34、4.08和1.84倍，这些突变子毒力相对减弱。本研究结果为进一步阐明鳗弧菌的发病机制提供了理论基础。     

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL⁻¹ of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g⁻¹ tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.     

Vibrio anguillarum serovars associated with vibriosis in fish

Abstract. A total of 517 Vibrio anguillarum strains isolated from discased fish together with 14 V. anguillarum serogroup O2 and V. ordalii type strains were serotyped using the European scrotyping system. Marked species differences were recorded. In isolates from salmonids serovar O1 (70.2%) and O2 (20.2%) were dominantwhilst a minor proportion belonged to other serogroups or were non-typeable. Figures for turbot were similar to those from salmonids. In 32 isolates from sea bass, sea bream and mullet, most strains belonged to serogroup O1. while one was O2a. one O7, and the rest non-typeable In cod serovar O2 was dominant while only a minor proportion belonged to other serogroups or were non-typeable. The ecl isolates belonged equally to serovars O2 and O3. All O2 strain were subtyped with absorbed O2a and O2b antisera. O2a was dominant in all fish species. but in cod. the relative number of O2b isolates was considerably higher than in other fish species. The applicability of the European serotyping system is discussed and compared with other serotyping systems.