南方九孔鲍培苗过程中潜在致病菌胞外产物的分析

从广东汕尾一养殖场鲍苗掉板池中(包括水、藻膜和变白鲍苗)分离筛选到105株菌,并对之进行了致病毒力因子(胞外酶及溶血作用)的分析,同时应用PCR对溶血毒素的归属进行了初步的探讨,采用API对菌株进行了种类鉴定。结果表明,105株菌中,仅35株菌具有较强的分泌胞外蛋白酶、明胶酶和脂肪酶的能力,尤其以菌株1、2、3、5、9以及16相对强大。在此35株菌中,85.6%的菌株(3035株)表现出溶血现象,但仅16株菌的TlhPCR呈阳性。API鉴定表明,35株菌中弧菌约占50%,溶藻弧菌又占弧菌的70%。研究结果揭示,和其它菌株相比,分离自变白鲍苗的6株溶藻弧菌(菌株1、2、3、5,13和16)和2株副溶血弧菌(菌株9和21)具有较强的分泌胞外酶和或溶血的能力,意味着极有必要对其作进一步的研究,以观测它们对鲍苗的影响。     

南方九孔鲍鲍苗掉板病原菌的药敏测定

为提高鲍鱼培苗的成活率,对分离自广东汕尾一养殖场鲍苗掉板池中(包括水、藻膜和变白鲍苗)的、经回归感染试验证明为致病菌的菌株进行了鉴定和药物敏感性测定。API鉴定表明,这些致病菌株由Vibrio alginolyticus,Vibrio cholerae,Vibrio parahaemolyticus等组成,其中弧菌17株,约占总分离菌株的50％,而溶藻弧菌则为弧菌的优势菌株,有11株,约占弧菌总数的70％。药敏结果显示,绝大多数菌株对链霉素、红霉素和庆大霉素敏感;相反,四环素和新生霉素则对它们没有作用或不敏感。     

南方九孔鲍鲍苗掉板病原菌的药敏测定*

为提高鲍鱼培苗的成活率,对分离自广东汕尾一养殖场鲍苗掉板池中(包括水、藻膜和变白鲍苗)的、经回归感染试验证明为致病菌的菌株进行了鉴定和药物敏感性测定。API鉴定表明,这些致病菌株由Vibrio alginolyticus,Vibrio cholerae,Vibrio parahaem olyticus等组成,其中弧菌17株,约占总分离菌株的50%,而溶藻弧菌则为弧菌的优势菌株,有11株,约占弧菌总数的70%。药敏结果显示,绝大多数菌株对链霉素、红霉素和庆大霉素敏感;相反,四环素和新生霉素则对它们没有作用或     

溶藻弧菌的毒力相关基因及其对小鼠的致病力

【目的】通过多重PCR检测和小鼠动物实验,对溶藻弧菌环境分离株的毒力因子进行评估,以期获得较强致病菌株和弱致病菌株之间的差别,并初步探讨该菌毒力因子对小鼠的致病机理。【方法】采用多重PCR体系检测毒力相关基因,我妻氏血平板溶血实验和平板酶活实验检测溶藻弧菌株的溶血素和胞外酶;以昆明小白鼠为实验动物,攻毒方式为灌胃和腹腔注射,根据小鼠的致病症状和死亡情况来分析和对比溶藻弧菌的胞外分泌物以及菌体本身的毒性。【结果】10株溶藻弧菌产淀粉酶、卵磷脂酶的比例为100%,脂肪酶、明胶酶次之(为70%),脲酶均未被检出;神奈川现象阳性菌株率为60%。毒力基因检测的结果显示10株溶藻弧菌中toxR、Collagenase、tlh、FlaA、ompW、AspA、fur这些与毒力有关的基因均有分布,而toxS、trh、tdh、UreR并未检出。10株溶藻弧菌中的VA009对小鼠显示了较强的致病性,能造成腹腔积液,经腹腔注射感染此菌后7 d内死亡率高达80%。【结论】不同的溶藻弧菌对小鼠的致病性存在较大差异,溶藻弧菌菌体本身比胞外分泌物对其毒性的贡献要大,而副溶血弧菌的毒性则由其胞外分泌物起主要作用;比较我们筛选出的强致病菌株与弱致病菌株,其上述毒力基因的分布并没有差别,说明溶藻弧菌可能存在一套与副溶血弧菌不同的独立的毒力基因系统。     

VHH/TLH溶血素基因在海洋弧菌中分布的研究

哈维氏弧菌(V.harveyi)的VHH溶血素是对海水养殖鱼类的潜在致病因子。哈维氏弧菌的VHH溶血素基因与副溶血弧菌(V.parahaemolyticus)的TLH热不稳定性溶血素基因具有高度相似性,其氨基酸序列的相似性达到85.6 %。根据哈维氏弧菌vhhA溶血素基因序列,合成一个地高辛标记的VHH基因探针,利用其进行Southern Blot ,检测VHH溶血素基因在57株弧菌(包括26株国际标准菌株,20株哈维氏弧菌,11株副溶血弧菌)中的分布情况。结果显示,VHH基因探针与13株弧菌标准菌株有强杂交信号,包括2株溶藻胶弧菌(V.alginolyticus) ,2株哈维氏弧菌以及1株霍氏格里蒙菌(Grimontia hollisae) ,坎贝氏弧菌(V.campbellii) ,辛辛那提弧菌(V.cincinatiensis) ,费氏弧菌(V.fischeri) ,拟态弧菌(V.mimicus) ,飘浮弧菌(V.natriegens) ,副溶血弧菌,解蛋白弧菌(V.proteolyticus)和火神弧菌(V.logei)。与6株弧菌标准菌株有弱杂交信号,包括鳗弧菌(V.anguillarum) ,河口弧菌(V.aestuarianus) ,美人鱼发光杆菌(Photobacterium damselae subsp.damselae) ,河弧菌(V.fluvialis) ,弗尼斯弧菌(V.furnissii)和创伤弧菌(V.vulnificus) ,而另外7株弧菌标准菌株中无杂交信号。所有的哈维氏弧菌菌株至少含有一条杂交带,其中菌株VIB645 , VIB 648和SF-1分别含有2条杂交带。11株副溶血弧菌中均含有一条杂交带。上述数据表明,vhh/tlh溶血素基因广泛分布于弧菌中,尤其是哈维氏弧菌相关菌株和费氏弧菌相关菌株中。另外对鳗弧菌VIB 72 ,坎贝氏弧菌VIB 285 ,飘浮弧菌VIB 299和哈维氏弧菌VIB 647的vhh/tlh溶血素基因进行克隆并测序,其氨基酸序列与VHH溶血素和TLH溶血素氨基酸序列的同源性分别为67 %~99 %和69 %~91 %。对vhh/tlh溶血素基因在弧菌中的分布研究,将有助于进一步确定这类溶血素基因在病原弧菌致病性中的作用。     

副溶血弧菌Ⅲ型分泌系统(T3SS)效应蛋白及其对宿主细胞的操控北大核心CSCD

副溶血弧菌是典型的食源性病原菌,也是全球范围内引起肠胃炎的主要病原菌。针筒状的Ⅲ型分泌系统(T3SS)为该菌主要的毒力因子,细菌感染时可将其效应蛋白直接注射至宿主细胞中,通过效应蛋白操纵宿主细胞,介导毒力的发挥。多数临床分离的副溶血弧菌含有2套T3SSs,其中T3SS1分泌的效应蛋白主要通过诱导细胞自噬、变圆和裂解等过程来发挥其细胞毒性,而T3SS2分泌的效应蛋白则主要通过破坏细胞骨架和操控细胞信号传导来发挥肠毒性。本文主要对副溶血弧菌T3SSs的组成和目前已发现的效应蛋白及其对宿主细胞的操控进行介绍。该研究不仅对深入了解该菌的致病机制有重要意义,而且也为宿主细胞信号转导机制研究提供新视角。     

浙江沿海地区海产品及环境中副溶血弧菌的分离与主要毒力基因分析

2007~2008年间, 我们调查了浙江沿海地区海产品和养殖环境中副溶血弧菌的污染状况, 并分析了不同来源副溶血弧菌中主要毒力相关基因tdh、trh、ureC和T3SS2(vscC2、vcrD2)的分布特征及溶血表型与尿素酶表型。结果显示, 566份样品中共分离到395株副溶血弧菌, 检出率高达70%, 毒力相关基因分析结果发现, tdh基因阳性率为10.1%, trh与ureC基因阳性率分别为 20.0%与 11.1%, 40株tdh+菌中组成T3SS2的vscC2基因阳性率为32.5%, 其中38株tdh+菌的神奈川试验亦呈阳性; 但在44株trh+-ureC+菌株中, 尿素酶表型阳性只有6株。试验表明, 浙江沿海地区海产品及其养殖环境中副溶血弧菌污染状况比较严重, 且有相当比例的菌株携带毒力或疑似毒力基因。研究结果为深入探索副溶血弧菌的致病性、基因结构与功能(或表型)及其分子演化提供基础。     

副溶血弧菌Ⅲ型分泌系统（T3SS）效应蛋白及其对宿主细胞的操控

副溶血弧菌是典型的食源性病原菌,也是全球范围内引起肠胃炎的主要病原菌。针筒状的Ⅲ型分泌系统(T3SS)为该菌主要的毒力因子,细菌感染时可将其效应蛋白直接注射至宿主细胞中,通过效应蛋白操纵宿主细胞,介导毒力的发挥。多数临床分离的副溶血弧菌含有2套T3SSs,其中T3SS1分泌的效应蛋白主要通过诱导细胞自噬、变圆和裂解等过程来发挥其细胞毒性,而T3SS2分泌的效应蛋白则主要通过破坏细胞骨架和操控细胞信号传导来发挥肠毒性。本文主要对副溶血弧菌T3SSs的组成和目前已发现的效应蛋白及其对宿主细胞的操控进行介绍。该研究不仅对深入了解该菌的致病机制有重要意义,而且也为宿主细胞信号转导机制研究提供新视角。     

红鳍东方鲀病原鱼肠道弧菌的生物学特性研究

对引起红鳍东方鲀发病死亡的病原细菌进行了分离和主要生物学特性研究,包括病原性、形态特征、理化特性、16S rRNA基因序列及其系统发育学分析、胞外酶及溶血素活性、K抗原及耐药性等.结果表明,引起红鳍东方纯发病死亡的病原细菌为弧菌属(-Vibrio Pacini 1854)的鱼肠道弧菌(V.ichthyoenteri Ishimaru,et al.1996),2株代表菌株16S rRNA基凶序列(GenBank登录号分别为:EF611424和EF635304)与GenBank数据库中鱼肠道弧菌的同源性在98%-100%,且在构建的MP系统发生树中与鱼肠道弧菌聚为一个分支.分离菌不具有淀粉酶、蛋白酶、脂肪酶、DNA酶、脲酶、明胶酶和卵磷脂酶活性,且在含7%家兔脱纤血液营养琼脂培养基上不溶血.不具有K抗原.人工感染试验中分离菌对红鳍东方纯表现出明显的致病性.药敏试验结果显示,4株分离菌对供试37种抗菌药物中的苯唑青霉素和杆菌肽2种耐药.     

副溶血弧菌海产品和临床分离株的表型及溶血素相关基因分析

副溶血弧菌是(Vibrio parahaemolyticus)常见的食源性病原菌,可污染多种水产品,并引起人的食物中毒,其致病性与溶血素密切相关,如直接耐热溶血素(TDH)、TDH-相关溶血素(TRH)、不耐热溶血素(TLH)。用PCR方法对分离自浙江省部分地区的副溶血弧菌临床和海产品分离株的3种溶血素基因进行检测。结果表明,所有副溶血弧菌菌株均可检测到tlh基因;11株临床分离株均检测到tdh基因,而42株水产品分离株中只有1株检出tdh基因,携带tdh的分离株神奈川试验(KP)均为阳性。所有分离菌株中均未检测到trh基因以及其尿素酶试验呈阴性,由此可知trh基因可能与尿素酶基因连锁。副溶血弧菌分离株中致病性相关毒力因子TDH的阳性率极低,然而副溶血弧菌性食物中毒发生率较高,它们之间的关系及其发病机制还有待深入研究。     

Effect of eight benthic diatoms as feed on the growth of red abalone (Haliotis rufescens) postlarvae

The growth rate of abalone post larvae of Haliotis rufescens fed ad libitum with a benthic monoalgal diatom culture maintained as monocultures on a semi-commercial scale, was evaluated and correlated with the biochemical composition of the diatoms. The cell size (7.0 × 4.0 μm to 21.0 × 7.5 μm), protein percentage (7.42% to 13.66%), and ash content (49.03% to 59.61%) were different among diatom strains; lipid percentage, nitrogen free extract, and energy content (Kcal g⁻¹) were similar among diatom strains. The values of essential and non-essential amino and fatty acids composition differed among diatom strains. Differences in the abalone shell length and orthogonal analyses revealed postlarval growth was dependent on the quality of the food source. Postlarvae abalone displaying the longest shell lengths were fed Nitzschia thermalis var. minor and Amphiprora paludosa var. hyalina (1,712.0 ± 61 μm and 1,709 ± 67 μm, respectively), followed by Navicula incerta (1,413.3 ± 43 μm). The fatty acid content of benthic diatoms and abalone growth rate were not correlated.     

Characterization and identification of virulent Klebsiella oxytoca isolated from abalone (Haliotis diversicolor supertexta) postlarvae with mass mortality in Fujian, China

An epidemic of mass mortality of abalone (Haliotis diversicolor supertexta) postlarvae aged 40 days or less has existed across south coast of China since the second half of 2002. Among 20 bacterial strains isolated from diseased abalone postlarvae on 2216E marine agar plates during an outbreak of postlarval disease in August 2005, a predominant strain (designated strain 20) was demonstrated to be virulent to postlarvae with an LD(50) value of 1.0x10(5) colony forming units (CFUml(-1)) on day 4, while the other 19 strains were either avirulent (16 strains) or weakly virulent (3 strains). The same bacterium could be re-isolated from postlarvae after bacterial challenge using 2216E marine agar plates. Preliminary toxicity tests of ECPs of strain 20 revealed that at 2.77mgproteinml(-1), crude ECPs completely liquefied postlarvae within 24h, leaving only shells. API 20E analysis identified strain 20 as Klebsiella oxytoca. 16S and ITS rDNA sequencing and phylogenetic analyses further confirmed this identification. Antibiotic susceptibility tests showed that strain 20 exhibited 94% of susceptibility to 16 various antibiotics tested and only showed resistance to streptomycin. Results of this work demonstrated that K. oxytoca is also linked to this epidemic in Fujian, China. This is considered to be the first report regarding K. oxytoca involved in the mass mortality of postlarval abalone in south China and the world.     

七株外生菌根真菌与三种松苗菌根的形成能力

松树外生菌根菌资源丰富,但实际应用的种类不多。为筛选出与松苗形成菌根能力较强的菌种,采用播种接菌和芽苗截根移栽接菌两种方法,对7株外生菌根真菌与马尾松、湿地松和黑松3种松苗的菌根合成进行了研究。结果表明：形成的菌根以二叉分枝状为主,棒状菌根相对较少,多叉状菌根以马尾松较多。PC2形成的菌根表面菌丝厚且紧密,504、EG、Pt,形成的菌根表面菌丝紧密程度中等,而505、ZJ和HX形成的菌根其表面菌丝则比较稀疏;Pt1、Pt2、EG形成的菌根外延菌丝较长,而505、HX形成的菌根外延菌丝极短。截根接菌时的感染率和感染指数要高于播种接菌。504形成菌根的能力最强,在3种松苗上的菌根感染率都达100％,感染指数最高可达90,最低也达70;Pt2和EG与马尾松和黑松形成菌根的能力较强;而505和HX仅与马尾松形成菌根的能力较强;Pt1形成菌根的能力较差,在3种松苗上菌根感染率和感染指数都较低。在3种松苗中,马尾松的菌根化状况最好,其次为黑松,湿地松的菌根化状况较差。     

九孔鲍消化道及养殖水体中异养细菌胞外产物的分析

从汕尾健生鲍鱼场养殖水体及鲍鱼消化道中分离筛选到26株异养细菌,分析了其胞外产物,并采用API条带对其进行了种类鉴定。结果表明,就整体而言,成鲍消化道菌株产蛋白酶、淀粉酶、明胶酶和溶血能力均高于养殖水体的菌株,而产脂肪酶和卵磷脂酶菌株的比例则低于后者。无论是消化道还是养殖水体,均存在着分泌胞外产物能力极强的菌株,且大部分为鞘氨醇单胞菌。因此,在该养殖环境中,鞘氨醇单胞菌应视为一种主要的条件致病菌,同时消化道和养殖水体均应视为潜在致病菌的源泉。此外,在考虑细菌对水产经济动物的致病作用时,除了优势种类外,还应从菌群结构的角度出发,考虑整个细菌群落胞外产物的作用。     

Vibrio parahaemolyticus infectious for both humans and edible mollusk abalone

The aims of this study are to report evidence of the first laboratory-acquired infection of Vibrio parahaemolyticus associated with handling experimentally infected abalones and to describe the virulence of the two bacterial strains tested in these animals. Two strains of V. parahaemolyticus, one from the stool of a patient with acute gastroenteritis (strain 880713) and the other from the hemolymph of a diseased small abalone Haliotis diversicolor supertexta (strain 880915), were identified and characterized. Both strains were lethal to small abalone, with similar LD(50) values (8.36-8.41 x 10(4) colony-forming units/g abalone). Laboratory-acquired infection resulted in one individual experiencing two episodes of acute gastroenteritis due to handling virulence tests during a 1-week interval. Our present results suggest that a V. parahaemolyticus strain isolated from the stool of a patient with gastroenteritis was infectious for small abalone, a major species of edible mollusk abalone cultured in Taiwan, while a similar strain isolated from hemolymph of a diseased small abalone was infectious for humans. This is the first report of V. parahaemolyticus virulent to small abalone as a zoonotic pathogen.     

Responses of Nouelia insignis Franch (Asteraceae) and Terminalia franchetii Gagnep. (Combretaceae) seedlings to long-term water stress

The responses in terms of seedling growth, biomass allocation and photosynthesis of Nouelia insignis Franch. and Terminalia franchetii Gagnep. to long-term water stress were studied in a greenhouse experiment. Three-month-old seedlings were subjected to the following water supplies for about 79 days: 80, 60, 40 and 20% of field water capacity (FC). The results showed that, as water stress was increased, seedling height, leaf number, root length, specific leaf area (SLA), biomass production and stomatal limitation value (L_s) of both seedlings decreased, and water use efficiency (WUE) increased, and biomass allocation, the diurnal gas exchange and P_n-PAR response curves also altered. Water supply <40% FC was the threshold of drought-initiated negative effects on these parameters of both seedlings. Most growth and biomass parameters of T. franchetii seedlings were much higher than those of N. insignis seedlings. However, T. franchetii seedlings were more vulnerable to long-term water stress than N. insignis seedlings. Simultaneously, N. insignis seedlings had a higher ability to use strong light and a lower ability to use weak light than T. franchetii seedlings. Although water stress had a great impact on their growth, biomass allocation and photosynthesis, both of N. insignis and T. franchetii seedlings displayed considerable water stress resistance, and they tolerated even the most severe water stress (20% FC), and no seedlings died. However, the field conditions were worse; most of them might be hard to get though the long dry season.     

Cloning, characterization and tissue expression of disk abalone (Haliotis discus discus) catalase

Catalase is an antioxidant enzyme that plays a significant role in protection against oxidative stress by detoxification of hydrogen peroxide (H2O2). A gene coding for a putative catalase was isolated from the disk abalone (Haliotis discus discus) cDNA library and denoted as Ab-catalase. The full-length (2864 bp) Ab-catalase cDNA contained 1,503 bp open reading frame (ORF), encoding 501 amino acid residues with 56 kDa predicted molecular weight. The deduced amino acid sequence of Ab-catalase has characteristic features of catalase family such as catalytic site motif (61FNRERIPERVVHAKGAG77), heme-ligand signature motif (351RLYSYSDT358), NADPH and heme binding residues. Phylogenetic and pairwise identity results indicated that Ab-catalase is more similar to scallop (Chlamys farreri) catalase with 80% amino acid identity except for other reported disk abalone catalase sequences. Constitutive Ab-catalase expression was detected in gill, mantle, gonad, hemocytes, abductor muscle and digestive tract in tissue specific manner. Ab-catalase mRNA was up-regulated in gill and digestive tract tissues for the first 3h post injection of H2O2, showing the inducible ability of abalone catalase against oxidative stress generated by H2O2. The purified recombinant catalase showed 30,000 U/mg enzymatic activity against H2O2 and biochemical properties of higher thermal stability and broad spectrum of pH. Our results suggest that abalone catalase may play an important role in regulating oxidative stress by scavenging H2O2.     

Pathogenic Vibrio harveyi,in contrast to non‐pathogenic strains,intervenes with the p38 MAPK pathway to avoid an abalone haemocyte immune response

Vibrio harveyi is a marine bacterial pathogen responsible for episodic abalone epidemics associated with massive mortalities in France, Japan, and Australia. The aim of this study was the understanding of a possible role of the p38 MAPK in abalone haemocyte responses towards this bacterium. First, the pathogenicity of different V. harveyi strains was compared in both immersion and injection trials, and clear differences were detected. The three strains, ORM4, 04/092, and 05/053, all isolated from moribund abalone, induced up to 80% mortalities in immersion or injection challenges (LD₅₀ (ORM4) = 2.5 × 10² CFU animal^?1). The two strains, LMG 4044T and LMG 7890 were non‐pathogenic towards abalone in immersion trials, and needed very high numbers for killing by intramuscular injections (LD₅₀ = 8.9 × 10⁴ and 1.6 × 10⁵ CFU animal^?1, respectively). To start unraveling the mechanism explaining these differences, the p38‐MAPK, a keyplayer in antimicrobial immune response, was studied. The non‐pathogenic strain, LMG 7890 can be eliminated by abalone haemocytes and induces haemocyte phagocytosis and high ROS production. With different concentrations of a p38‐specific inhibitor, SB203580, p38 implication was shown. This inhibitor reduced phagocytosis and ROS induction leading to LMG 7890 proliferation. In the case of the pathogenic ORM4 which can not be eliminated by abalone haemocytes, no phagocytosis and ROS production was induced, and a retarded p38 activation was observed. Taken together, our results suggest that p38 MAPK modulation may be one of the ways of virulent V. harveyi to attack its host and escape abalone immune response. J. Cell. Biochem. 106: 152–160, 2009. © 2008 Wiley‐Liss, Inc.     

皱纹盘鲍的个体能量收支

对皱纹盘鲍的呼吸、摄食、生长及能量收支等实验研究表明, 鲍的耗氧率与壳长、体重、温度及昼夜变化有关, 耗氧率与壳长、体重均呈幂函数关系, 一天中16~4时(夜间)耗氧率高于4~16时(白天)且在18~20时达峰值.同温度下鲍日摄食率与体重呈幂指数关系, 日(相对)摄食率随温度升高而增加, 而日相对摄食率、日相对生长率均随壳长、体重增加呈下降趋势.鲍在14~20℃内对海带的总转化效率为53%.鲍软体部、海带及鲍粪便干品的比能值分别为19.2、8.57和7.23kJ·g^-1.14~20℃皱纹盘鲍摄入能量的34.6~48.6%为粪能, 22.0~38.2%的能量用于自身代谢, 5.6~28.2%用于贝体软体部的生长.