19株海水鱼致病性弧菌OmpK基因序列及其抗原性分析

从哈维氏弧菌(Vibrio harveyi)、溶藻弧菌(V.alginolyticus)、副溶血弧菌(V.parahaemolyticus)克隆、测定了共19株海水鱼类致病性弧菌外膜蛋白OmpK基因序列,探讨其作为海水鱼类致病性弧菌共同抗原的分子基础.根据已知的弧菌外膜蛋白OmpK序列设计1对简并引物,利用聚合酶链式反应(PCR)方法从19株弧菌总DNA中分别扩增得到约800bp外膜蛋白OmpK的基因片段,将其克隆到pDM18-Tvector载体筛选阳性重组子进行序列测定.结果显示,OmpK基因分别含有786bp～849 bp的开放读码框,编码261～282个氨基酸,其核苷酸序列之间的相似性在72%～100%,推测氨基酸序列的相似性为71%～100%,且种内OmpK氨基酸序列的相似性比种间高.序列分析还表明,每一种弧菌OmpK基因都有一段特异性序列,可用于设计核酸探针或特异性引物来诊断、检测哈维氏弧菌等海水鱼致病性弧菌.本研究不仅从基因水平上证实了外膜蛋白OmpK广泛存在于海水鱼致病性弧菌中,而且证明了它们之间具有较高的相似性.由结果推测外膜蛋白OmpK是哈维氏弧菌、溶藻弧菌、副溶血弧菌等致病性弧菌的一种共同抗原,是较好的亚单位疫苗候选成分,为进一步研制广谱的海水鱼类致病性弧菌外膜蛋白基因工程亚单位疫苗提供了理论基础.     

同时检测两种对虾病毒和4种弧菌的同步PCR方法的建立

通过检索、多重比对、分析和筛选GenBank中对虾白斑综合征病毒(WSSV)、传染性皮下和造血器官坏死病毒(IHHNV)、副溶血孤菌、创伤弧菌、哈维氏弧菌和溶藻胶弧菌的基因序列,设计了10对特异性引物,以已知毒株和菌株的DNA为模板进行PCR,均能扩增出与实验设计相符合的DNA片段,对PCR扩增条件进行优化,建立了可同时检测鉴别WSSV、IHHNV、副溶血弧菌、创伤弧菌、哈维氏弧菌和溶藻胶弧菌,并且能同时区分WSSV不同地理毒株的同步PCR方法.研究结果表明,该方法检测特异性好,检测通量大,适合于对虾多种病原的同时检测.     

不同海洋弧菌中5类溶血素基因的分布及其与溶血活性和磷脂酶活性的相关性

用57株海洋弧菌,包括26株弧菌标准菌株、20株哈维氏弧菌、11株副溶血弧菌(从不同宿主和不同地理环境中分离得到),用PCR法合成5类相应的地高辛标记的溶血素基因探针,利用其进行Southern Blot,检测这5类溶血素基因在57株弧菌中的分布。结果显示,在57海洋株弧菌中,含有TDH、HlyA、TLH、δ-VPH和HLX溶血素基因的菌株分别为2株、2株、49株、3株和30株。另外,1株霍氏格里蒙菌(Grimontia hollisae)和1株副溶血弧菌(V.parahaemolyticus)中分别含有2个TDH溶血素基因。用鱼血平板和卵磷脂平板检测57株弧菌的溶血活性和磷脂酶活性,结果表明,弧菌溶血活性和磷脂酶活性与TLH溶血素基因具有显著相关性,与另外4类溶血素基因的关系不明显。     

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。     

哈维氏弧菌外膜蛋白OmpK基因的克隆及原核表达

根据哈维氏弧菌外膜蛋白OmpK的基因序列设计一对引物，应用聚合酶链式反应（PCR）方法，从分离自患病大黄鱼的哈维氏弧菌基因组中扩增获得一段约800bp的序列。将其克隆到pGEM-Teasy载体，测序结果证明该序列是哈维氏弧菌外膜蛋白OmpK基因。用PCR方法去除其信号肽序列，定向克隆到原核表达载体pGEX-4T-2构建重组表达质粒pGEX-4T-OmpK。IPTG诱导后能够在大肠杆菌BL21中高效表达分子量约为53kD的GST-OmpK融合蛋白。用纯化后的融合蛋白免疫新西兰兔获得了高效价的抗血清。Western-blotting分析表明，它与从哈维氏弧菌中提取的约27kD的外膜蛋白能够发生特异反应，提示外膜蛋白OmpK可能是哈维氏弧菌的重要保护性抗原之一。     

斜带石斑鱼3种致病性弧菌的分子生物学鉴定

从患病斜带石斑鱼分离到3株病原菌EcGS020802、EcGS021001、EcGS020801，经常规生理生化鉴定均属于弧菌属的种类。为了进一步确定其分类地位，测定了3株病原菌的16SrRNA和HSP60(heat shock protein，HsP60)基因部分序列。16SrRNA基因系统进化分析表明，3株病原菌与哈维氏弧菌、溶藻弧菌、副溶血弧菌亲缘关系较近，相互之间同源性均大于98．6％，差异不明显。HSP60基因序列分析表明，菌株EcGS020802、EcGS021001、EcGS020801 HSP60基因序列分别与哈维氏弧菌(AF230934)、溶藻弧菌(Ab230931)、副溶血弧菌(AF230951)HSP60基因的同源性最高，依次为95．7％、99．8％和99．8％，而与其它弧菌HSP60基因的同源性均低于90．6％，3株病原菌相互之间的同源性低于91．0％。HSP60基因构建的系统进化树表明，EcGS020802、EcGS021001、EcGS020801分别与Vibrio harveyi、Vibrio alginolyticus、Vibrio parahaemolyticus聚类。综合上述结果，菌株EcGS020802、EcGS021001、EcGS020801可分别鉴定为哈维氏弧菌、溶藻弧菌、副溶血弧菌。结果表明，HSP60基因比16SrRNA基因更适合用于海水鱼致病性弧菌种问的分类研究。     

大菱鲆肠道乳杆菌Lactobacillus sp.N5的筛选鉴定及其抑制水产病原菌的研究

从大菱鲆（Scophthalmus maximus）肠道内分离出8株乳酸菌，经16S r DNA鉴定为Lactobacillus sp.，以对水产病原菌的抑制和对抗生素的敏感性为指标，研究乳酸菌的特性。其中，菌株N5对鳗弧菌（Vibrio anguillarum）、哈维氏弧菌（V. harveyi）、副溶血弧菌（V. parahaemolyticus）和金黄色葡萄球菌（Staphylococcus aureus）均具有较强的抑制作用，对氨苄青霉素、庆大霉素和罗红霉素敏感。菌株N5发酵上清液经加热、排酸和过氧化氢酶处理后，仍有抑菌作用；经蛋白酶处理后失去抑菌能力，推测菌株N5分泌的抑菌物质为耐热的乳酸菌素，其可以抑制病原菌生物膜的形成。菌株N5在凡纳对虾（Litopenaeus vannamei）肠道定植5 d后，加入鳗弧菌和哈维氏弧菌，继续养殖7 d，死亡率显著降低。本研究结果可为菌株N5制备水产养殖益生菌剂提供实验依据。     

流水式养殖动物中弧菌和嗜水气单胞菌的PCR法监测

为了解流水式养殖场养殖动物中弧菌和气单胞菌的感染状况,分别针对霍乱孤菌肠毒素A亚单位(ctxA)基因、0139和01群群特异基因、毒力协同调节菌毛A亚单位基因(tcpA)、中心调节蛋白基因(toxR)、副溶血弧菌不耐热溶血素基因(tlh)、创伤弧菌Vibrio vulnificus溶细胞素基因(vvhA)和嗜水气单胞菌的气溶素基因(aerA)设计引物,建立了聚合酶链式反应(PCR)检测技术,PCR产物经电泳,根据扩增条带的大小和数目,检测和区分霍乱孤菌、嗜水气单胞菌和副溶血孤菌.对南昌沿岸霍乱流行水域及附近养殖场的养殖动物进行了18个月的连续监测,对1 440份水产品的增菌液进行PCR检测,17份检出副溶血弧菌,25份检出霍乱弧菌,创伤弧菌和嗜水气单胞菌均未检出.扩增结果与传统培养检测法的结果一致.监测结果表明,南昌附近地区霍乱弧菌和副溶血弧菌检出率分别为1.74%和1.18%,总体感染状况处于较低水平,但个别养殖品种弧菌检出率较高.     

苍术和番石榴的不同炮制方法及部位对水产病原菌抑制效果的影响

采用微量稀释法,以溶珊瑚弧菌（Vibrio coralliilyticus）、哈维弧菌（旷harveyi）、美人鱼发光杆菌杀鱼亚种（Photobacterium damselae subsp piscicida）、副溶血弧菌（V.parahaemolyticus）、摩氏摩根氏菌（Morganellafulton）、创伤弧菌（V.vulnificus）6种病原菌共7株为受试菌,研究苍术（Atractylodeslancea）不同炮制品和番石榴（Psidiumguajava）不同部位的抑菌作用,并进一步测定其最小抑菌浓度（MIC）和最小杀菌浓度（MBC）。结果显示,番石榴叶和枝果部位以及苍术不同炮制品对溶珊瑚弧菌、美人鱼发光杆菌杀鱼亚种和哈维弧菌抑菌效果较明显,MIC和MBC分别为10^-3g·mL^-1和10^-1g·mL^-1,抑菌效果由高至低依次为番石榴叶〉番石榴果〉番石榴枝,麸炒苍术〉盐炙苍术〉苍术〉醋炙苍术。     

凡纳滨对虾弧菌病及其防治方法研究进展

近年来,凡纳滨对虾(Litopenaeus vannamei)养殖生产中弧菌病的发病率越来越高,严重阻碍了我国海水养殖业的发展.文章介绍了凡纳滨对虾养殖过程中容易发生的几种弧菌病及其防治方法的相关研究,并对常见的哈维氏弧菌、溶藻弧菌、霍乱弧菌和副溶血弧菌这4种弧菌的研究进展进行了综述,以期为海水养殖中弧菌病的防治和控制...     

Molecular cloning and functional analysis of Photobacterium damselae subsp. piscicida haem receptor gene

A haem receptor gene from Photobacterium damselae subsp. piscicida (formerly known as Pasteurella piscicida) has been cloned, sequenced and analysed for its function. The gene, designated as pph, has an open reading frame consisting of 2154 bp, a predicted 718 amino acid residues and exists as a single copy. It is homologous with the haem receptors of Vibrio anguillarum hupA, V. cholerae hutA, V. mimicus mhuA and V. vulnificus hupA at 32.7, 32.7, 45.6 and 30.9%, respectively, and is highly conserved, consisting of a Phe-Arg-Ala-Pro sequence (FRAP), an iron transport related molecule (TonB) and a Asn-Pron-Asn-Leu sequence (NPNL), binding motifs associated with haem receptors. As a single gene knockout mutant P. damselae subsp. piscicida was able to bind haem in the absence of pph, suggesting that other receptors may be involved in its iron transport system. This study shows that the P. damselae subsp. piscicida pph belongs to the haem receptor family, is conserved and that its iron-binding system may involve more than one receptor.     

Identification of a marine antagonistic strain JG1 and establishment of a polymerase chain reaction detection technique based on the gyrB gene

A marine antagonistic bacterium, JG1, was isolated from rearing water of healthy turbot (Scophthalmus maximus) in Qingdao, China. Strain JG1 was Gram‐negative, straight rod and motile by polar flagella. The colony, when cultured for 24 h under room temperature, produced a yellow pigment. On the basis of morphological, physiological and biochemical characteristics, along with 16S rDNA sequence analysis, JG1 was identified as Pseudoalteromonas flavipulchra. JG1 had good inhibitory effects on several bacterial pathogens of aquaculture in the genus Vibrio (V. anguillarum, V. alginolyticus, V. campbellii, V. harveyi, V. mimicus, V. parahaemolyticus and V. tubiashii) and Aeromonas (A. hydrophila and A. salmonicida). No mortality occurred 14 days after zebra fish and 7 days after mantis shrimp were intraperitoneally injected with JG1 at 10⁶ CFU per animal, and 7 days after scallop and clam were immersed in JG1 at 10⁷ CFU mL^?1. A good antagonistic effect on several bacterial pathogens and nontoxicity to the above‐mentioned animals make JG1 a potential probiotic in aquaculture. In addition, a fast detection technique based on polymerase chain reaction amplification of the gyrB gene was established to allow us to determine the fate of JG1 when it is applied in aquaculture in the future.     

对虾养殖池中一株弧菌拮抗菌的分离鉴定

从对虾养殖池中分离出1株编号为2013082515(简称菌株15)的菌株,以鳗弧菌(Vibrio anguillarum)、哈维氏弧菌(Vibrio harveyi)和副溶血弧菌(Vibrio parahaemolyticus)为指示菌,分析了菌株15对指示菌的抑菌效果及最低抑菌浓度,同时,分析了菌株15对其他7株弧菌的抑菌效果.结果显示,菌株15对3株指示菌均具有抑菌效果,对鳗弧菌、哈维氏弧菌及副溶血弧菌的最低抑菌浓度分别为2.50×104、2.50×105、2.50×105 CFU/ml;对其他弧菌也具有一定的抑菌效果.采用注射及浸泡感染方法分析了菌株15对对虾的生物安全性,结果显示,在高浓度时,菌株15对对虾具有潜在毒性.分别用细菌全细胞脂肪酸气相色谱法和16S rDNA序列分析比对法对该菌进行分类鉴定,表明菌株15为一株假交替单胞菌(Pseudoalteromonas sp.).     

Characterization of Vibrio spp. Associated with Diseased Shrimp from Culture Ponds of Andhra Pradesh (India)

Abstract.— Surveys undertaken on diseases caused by Vibrio spp. in Penaeus monodon from culture ponds of coastal Andhra Pradesh recorded the occurrence of five types of diseases: tail necrosis, shell disease, red disease, loose shell syndrome (LSS), and white gut disease (WGD). Among these, LSS, WGD, and red disease caused mass mortalities in shrimp culture ponds. Six species of Vibrio—V. harveyi , V. parahaemolyticus, V. alginolyticus, V. anguillarum , V. vulnificus , and V. splendidus —are associated with the diseased shrimp. The number of Vibrio spp. associated with each disease ranged from two to five. Additionally, shrimp with red disease had concurrent infections with white spot syndrome virus. Vibrio harveyi in the case of LSS and WGD, V. parahaemolyticus for red disease, and V. alginolyticus for shell disease are the major etiologcal agents. Differences occur in the degree of virulence of different species of Vibrio and also different isolates of the same species. Vibrio harveyi isolated from LSS shrimp is the most virulent. In general, all the Vibrio isolates from LSS shrimp tend to be more virulent as compared to their counterparts from other diseased shrimp. It is apparent that the degree of virulence of various Vibrio isolates depends on its source and the pond environmental conditions. Most of the Vibrio isolates showed susceptibility to oxytetracycline, norfloxacin, and ciprofloxacin. The luminous V. harveyi exhibited resistance to many antibiotics and susceptibility to only three drugs. Considering the emergence of antimicrobial resistant strains of Vibrio , the need for using probiotics in place of antibiotics for disease control is stressed.     

哈维氏弧菌(Vibrio harveyi)ML01株胞外产物及分泌性蛋白的分离与特性分析

本研究以引起珍珠龙胆石斑鱼[Epinephelus fuscoguttatus(♀)×Epinephelus lanceolatus(♂)]幼鱼“皮肤溃疡病”的哈维氏弧菌(Vibrio harveyi) ML01株为研究对象,采用平板膜覆盖技术和柱层析技术,分离、纯化了ML01株的胞外产物及分泌性蛋白.应用毒性试验、质谱分析与分子克隆技术,对纯化的胞外产物和3种主要的分泌性蛋白进行了特性分析与鉴定.结果显示,哈维氏弧菌ML01株的胞外产物(Extracellular products,ECPs)具有酯酶、明胶酶、淀粉酶、酪蛋白酶活性,无脲酶活性.ECPs对羊红细胞无溶血性,对斑马鱼(Danio rerio)的半数致死剂量(LD50)为19.55μg/g鱼体重.从ML01株中分离到3种主要的分泌蛋白P42、P36、P31,其分子量分别为42、36、31 kDa.经质谱鉴定和分析,这3种蛋白分别为哈维氏弧菌的外膜蛋白OmpU和OmpN,以及一种功能未知的蛋白.利用同源克隆,成功地从ML01株基因组中扩增到了P42、P36、P31的基因.序列测定和比对结果显示,ML01株的这3个基因与哈维氏弧菌ATCC 33843(GenBank CP009467)的相应基因相比,其开放阅读框(Open reading frame,ORF)序列的相似性分别为97.08％、100％、99.67％,其编码的多肽序列的相似性分别为99.71％、100％和99.93％.本研究对进一步分析哈维氏弧菌ML01株的致病机理、研发该菌的亚单位疫苗具有重要的参考价值.     

鲍养殖水体中致病性弧菌的调查

在18份鲍鱼养殖厂的各种水样和不同生长期的鲍样品中,共分离出17株弧菌:溶藻性弧菌11株,副溶血弧菌3株,霍乱弧菌2株,拟态弧菌1株。其中,溶藻性弧菌分布比较广,存在于各种样品;副溶血弧菌主要分布于鲍的肠道内;而非O1 O139群霍乱弧菌和拟态弧菌则分布于源头水中。鲍养殖水体中细菌总数的计数可以作为监测细菌性病害的指标之一。副溶血弧菌在鲍肠道内的高检出率提示鲍与副溶血弧菌的关系值得深入研究。     

Isolation of a novel strain of Bacillus pumilus from penaeid shrimp that is inhibitory against marine pathogens.

A bacterium was isolated from the mid-gut of healthy black tiger shrimp, Penaeus monodon , based on a large zone of inhibition in mixed culture on solid medium. The isolate was a Gram-positive, motile spore former, with an optimum pH range for growth in tryptone soya broth containing 2% NaCl of between pH 6 and 9. The bacterium was highly salt tolerant with concentrations between 0% and 8% having no detrimental effect on growth. The isolate was identified as Bacillus pumilus based on physiological capabilities using the API50CHB and Biolog systems. Amplification and sequencing of the 16S rRNA gene followed by phylogenetic analysis confirmed its identity. The Bacillus pumilus isolate was strongly inhibitory against the marine bacterial pathogens Vibrio alginolyticus , V. mimicus and V. harveyi , and weakly inhibitory against V. parahaemolyticus in cross-streaking assays on solid medium. The organism was marginally self-inhibitory, and inhibited B. licheniformis and B. subtilis . The suitability of the B. pumilus isolate for use as a probiotic in farmed shrimp was further supported by the absence of any of the known B. cereus enterotoxin genes. Based on these in vitro results, in vivo safety and efficacy trials are underway to determine suitability of the novel strain as a commercial probiotic.