Vibrio mediterranei117-T6引发的坛紫菜黄斑病的初步研究

以一株黄斑病致病菌Vibrio mediterranei 117-T6感染坛紫菜的自由丝状体,研究了环境因子对该菌株生长的影响及其最易感条件,检测了该条件下V. mediterranei 117-T6引发的坛紫菜丝状体的抗氧化酶(SOD、POD)活性以及藻胆蛋白、叶绿素a(Chl. a)、可溶性蛋白、游离脯氨酸(Pro)和丙二醛(MDA)含量等生理指标的变化。结果显示,V. mediterranei117-T6的最优生长条件为30°C、pH 7.0、盐度20,最易感条件为30°C、pH 6.0、盐度20。在易感条件下培养12 h后,感染组坛紫菜丝状体的SOD和POD的活性,藻胆蛋白、可溶性蛋白以及游离Pro含量等指标均显著高于对照组;MDA含量峰值出现在6 h,显著高于对照组;在24 h后,感染组的Chl. a含量达到峰值,亦显著高于对照组。研究表明,短期内弧菌感染虽然可以刺激坛紫菜丝状体产生胁迫应激反应,但是随着感染时间的延长,致病菌感染引发的藻体内活性氧及渗透压胁迫加剧,最终导致紫菜死亡。高温和酸化等不良的环境会加剧V. mediterranei 117-T6引发的坛紫菜黄斑病的恶化,其作用程度依次为温度、pH和盐度。     

养殖环境及贝源溶藻弧菌MLST分型及其毒力基因、耐药性分析

溶藻弧菌(Vibrio alginolyticus)是近年来贝类病害中最常见的细菌性病原之一，对贝类养殖产业的健康发展构成严重威胁。本研究旨在分析不同养殖环境水体和贝类组织中，溶藻弧菌的基因变异、毒力基因、耐药性及其分布规律。对12株溶藻弧菌分离株开展多位点序列分型(multilocus sequence typing, MLST)、毒力因子以及菌株耐药性分析，结果显示，12株溶藻弧菌的序列型(sequence typing, ST)分型互不相同，7株为PubMLST数据库已经收录的ST型，5株因管家基因的等位基因位点变化而形成新的ST型，贝类养殖环境中的溶藻弧菌具有较高的遗传多样性。12株溶藻弧菌都携带tlh、fur和collagenase三种毒力基因，但均未检测到tdh、trh、toxR和tcpA毒力基因。溶藻弧菌携带毒力因子的种类和数量受地区分布等因素的影响。不同来源的溶藻弧菌均具有多重耐药特征，对青霉素和氨苄西林产生抗性。本研究表明，贝类养殖环境中的溶藻弧菌具有种群复杂、遗传多样性高的特点；不同来源菌株在毒力基因携带和耐药性方面存在较大差异。本研究通过探究不同区域内不同来源的溶藻弧菌遗传变异及耐药性差异，对贝源溶藻弧菌的有效防控提供一定理论参考。     

溶藻细菌A3的溶藻特性

本研究以锥状斯氏藻(Scrippsiella trochoidea)、蛋白核小球藻(Chlorella pyrenoidosa)、四尾栅藻(Scenedesmus quadricauda)、条纹小环藻(Cyclotella striata)作为实验藻种,将浓度为107 CFU/ml的菌株A3分别加入到4种微藻的单种藻液、2种藻混合藻液、3种藻混合藻液中,每48 h观察藻细胞形态并统计藻细胞数量,实验周期为10d,以探究菌株A3对4种微藻的溶藻效果.结果显示,在单种藻实验中,加菌组锥状斯氏藻细胞于第1天失去运动活性,细胞拉长变形,第5天细胞壁破裂溶解,第10天细胞密度为7.07× 102 cells/ml,显著低于对照组的2.90× 104 cells/ml (P＜0.05);实验期间,加菌组蛋白核小球藻细胞形态保持完整,第10天藻细胞密度为2.58× 107 cells/ml,显著高于对照组的2.09× 107 cells/ml (P＜0.05);加菌组四尾栅藻细胞形态保持完整,与对照组藻细胞密度无显著差异(P＞0.05);加菌组条纹小环藻细胞于第8天溶解,第10天对照组与加菌组藻细胞密度分别为4.38× 105 cells/ml、1.78×105 cells/ml,加菌组藻细胞密度显著低于对照组(P＜0.05).混合藻实验中,菌株A3对各种微藻的溶藻效果与单种藻实验结果类似,菌株A3对锥状斯氏藻生长具有显著的溶藻作用,对蛋白核小球藻与四尾栅藻无溶藻作用,对条纹小环藻生长具有较弱的溶藻作用.研究表明,菌株A3具有溶藻选择性,对锥状斯氏藻具有显著的溶藻作用,而对其他3种藻无溶藻作用或溶藻作用相对较弱.     

溶藻弧菌外膜蛋白（Va-OMP）的免疫原性及免疫保护性

利用初步制备的溶藻弧菌外膜蛋白（outer membrane protein of Vibrio alginolyticus, Va-OMP）、溶藻弧菌蛋白分子量58 kD外膜蛋白（Va-OMP58）和溶藻弧菌（Vibrio alginolyticus, Va）灭活菌苗、溶藻弧菌脂多糖（lipopolysaccharides of Vibrio alginolyticus, Va-LPS）菌苗等进行主动保护性和被动保护性的实验比较，VaOMP、Va-OMP58免疫组小鼠免疫后用活菌攻击，免疫保护率为62.5%～86.7%,而Va-LPS组和Va灭活菌苗组的免疫保护率为50%～62.5%，对照组的免疫保护率仅为6.7%。用不同抗血清免疫小鼠，活菌攻击后，Va-OMP组存活率达到53.3%和66.7%，VaOMP58组次之，存活率为40%和50%，Va灭活菌组的存活率为33.3%和46.7%，对照组为13.3%。与对照组相比较，Va-OMP组差异较显著，保护效果较好。溶藻弧菌外膜蛋白和蛋白分子量58 kD外膜蛋白的保护性效果高于溶藻弧菌灭活菌苗和溶藻弧菌脂多糖的保护性效果，证明外膜蛋白（outer membrane protein, OMP）具有较强的免疫原性。迟发性超敏反应试验也证明，OMP能诱发变态反应，间接证明OMP具有较强的引起细胞介导免疫反应的能力。因此实验证明，Va-OMP、Va-OMP⁵⁸是能在小鼠产生体液免疫和细胞介导免疫的保护性抗原。     

龙须菜苯丙烷类抑藻活性物质的分离纯化及其对6种赤潮微藻的抑藻活性

采用溶剂浸提、液液萃取、硅胶柱层析和硅胶薄层层析等分离方法以及红外光谱、质谱和核磁共振谱等光谱技术,初次从龙须菜中纯化得到4种苯丙烷类化合物:邻苯二丙酸、gossonorol、7,10-epoxy-ar-bisabol-11-ol和对羟基苯乙醇,并进一步分析了此4种苯丙烷类化合物对强壮前沟藻、赤潮异弯藻、米氏凯伦藻、球形棕囊藻、东海原甲藻和中肋骨条藻生长的影响。结果表明,它们对以上6种赤潮微藻的生长表现出明显的选择性抑制作用。其中,邻苯二丙酸、gossonorol和对羟基苯乙醇表现出更为广泛的抑藻活性。比较此4种苯丙烷类化合物和重铬酸钾对赤潮微藻生长的半抑制效应浓度EC50-96 h,发现邻苯二丙酸对强壮前沟藻、赤潮异弯藻和球形棕囊藻,gossonorol和羟基苯乙醇对赤潮异弯藻和球形棕囊藻,7,10-epoxy-ar-bisabol-11-ol对赤潮异弯藻和米氏凯伦藻在生长抑制方面比重铬酸钾更具有优势。     

南美白对虾致病性弧菌的鉴定及药敏分析

由丹东东港市某养殖场患病的南美白对虾(Penaeus vannamei)幼虾组织体内分离出一株优势菌,通过全自动细菌分析仪及16srDNA序列分析对该菌株进行鉴定,命名为DS180707。并进行人工感染试验和药敏实验。结果显示,本次患病幼虾致病菌为溶藻弧菌,且具有较强的致病性。该菌对氟苯尼考、氯霉素、恩诺杀星、诺氟沙星、氧氟沙星、左氧氟沙星等6种抗生素敏感。     

溶藻弧菌溶血素基因反向PCR克隆及其原核表达

溶藻弧菌的胞外产物(ECP)在其致病过程中发挥重要作用，已经观察到溶藻弧菌ECP所致的溶血现象，关于溶藻弧菌溶血素的种类和其对溶藻弧菌的致病性贡献的报道非常稀少。本文利用已经报道的多种弧菌溶血素基因序列设计通用引物，检测溶血素基因在溶藻弧菌中的分布，发现96个菌株中有74株扩增出溶血素基因(vah)片段。对致病株ZJ0451的 vah片段测序。根据已测得的片段序列设计引物，通过反向PCR扩增及后续克隆测序获得全长vah基因及部分侧翼序列。经比对证实溶藻弧菌vah与多种弧菌的TLH类溶血素高度相似，且其肽链N端有信号肽。利用pET32a载体构建了两种包含不同长度融合标签的vah表达载体，并均在大肠杆菌BL21(DE3)中获得可溶性表达。在26℃的诱导温度下，9h时vah表达量达到相对最大。在原核表达系统中，vah蛋白信号肽可以被切除。     

2株溶藻弧菌烈性噬菌体的分离鉴定及其生物学特性

为寻找并丰富溶藻弧菌噬菌体资源,实验以溶藻弧菌VAHN1为宿主菌,采用双层平板法从海南虾塘水样及福建海产品样本中分离溶藻弧菌噬菌体。通过透射电镜、限制性内切酶及构建发育树等方法对所获溶藻弧菌噬菌体进行分类鉴定;同时分析其生理生化性能。结果显示,本研究分离获得2株溶藻弧菌噬菌体VAP9与VAP21,其噬菌斑均清晰透亮,直径约1.5～2.0 mm。2株噬菌体核酸均为双链DNA,于透射电镜下可见其头部均呈正二十面体结构,2株噬菌体均属肌尾噬菌体科。噬菌体VAP9与VAP21对理化环境具有良好的耐受性;VAP9最适pH为6～8,VAP21最适pH为7～11;2株噬菌体可耐受通用杀菌浓度的过氧乙酸,且对氯仿与乙醚不敏感,同时对紫外线具有一定耐受性。2株噬菌体的最佳感染复数均为0.001,对供试溶藻弧菌的裂解率达95.2%,可裂解部分副溶血性弧菌,但无法裂解除溶藻弧菌与副溶血性弧菌外的弧菌属、葡萄球菌属、假单胞菌属等其他种属的供试细菌。噬菌体VAP9与VAP21可高效抑制溶藻弧菌VAHN1的生长,且2株噬菌体的混合制剂对溶藻弧菌的抑制效果优于单株噬菌体。将噬菌体VAP9及VAP21保守蛋白序列于N...     

养殖大黄鱼低温弧菌病病原的研究

浙江沿海深水网箱养殖大黄鱼冬天发生大量死亡，从病鱼体内分离出致病力较强的细菌GYC0227。人工感染试验证实这株菌为大黄鱼的病原菌。对细菌进行了形态、生理生化特性的测定，并进行了16S rRNA分子鉴定。PCR扩增获得了GYC0227菌株大小约1．5kb的16S rDNA部分基因片段，产物经回收纯化，克隆到pMD18-T载体，转化大肠杆菌TG1，对阳性克隆子进行酶切及PCR鉴定，并测序，将测定的序列递交NCBI进行BLAET同源序列比对，与溶藻弧菌的标准菌株的16S rDNA具有96％的同源性。结合菌株的生理生化特性，GYCD227菌株属于溶藻弧菌。该菌适应于低温生长，生长温度范围为7—25℃，并测定了其对14种药物的敏感性。     

溶藻弧菌双组分调控系统KdpDE减毒活疫苗的构建及其免疫效果评价

为了研究溶藻弧菌双组分调控系统KdpDE减毒活疫苗对鱼体的免疫保护作用,本研究采用Overlap PCR和同源重组技术,构建了kdpD/kdpE（kdpDE）基因无标记基因框内敲除突变株。对野生株和缺失突变株的生物学特性及致病性进行了比较研究,发现kdpDE的缺失对溶藻弧菌的生长速率和胞外蛋白酶活性的影响不明显,但是突变株的泳动能力、生物被膜形成能力较野生株下降。斑马鱼致病性实验结果显示,突变株毒力下降78.5倍,表明减毒株构建成功,可以作为减毒活疫苗的候选菌株。突变株在鱼体内存活能力实验结果显示,注射免疫7 d后,鱼体内不能检测到该菌。以突变株ΔkdpDE为抗原,分别用注射和浸泡的方式免疫斑马鱼,免疫后第28天用溶藻弧菌和哈维氏弧菌攻毒,评估该减毒活疫苗及不同免疫方式对斑马鱼的保护效果。实验结果表明,免疫后的斑马鱼能够抵抗溶藻弧菌的感染,其中注射免疫组的免疫保护率达83.3%,浸泡免疫组次之（66.7%）;同时,该减毒活疫苗能刺激斑马鱼对哈维氏弧菌产生交叉保护效应,其中注射免疫组的免疫保护率为65.5%,浸泡免疫组为55.2%。以上结果表明,kdpDE基因敲除突变株可能是抵抗溶藻弧菌感染的有效的候选减毒活疫苗。     

铜藻粉末的关键工艺研究

对铜藻进行组织化处理制成铜藻粉末,本试验研究了铜藻粉末的三个关键工艺点。研究表明:①铜藻粉末脱腥工艺:铜藻粉末脱腥效果较为理想的是添加乙醇量与铜藻液量比为1:1,微沸时间为30min。②铜藻粉末漂白工艺:漂白工艺的最佳条件为次氯酸钠浓度0.3%,pH值9,液固比4:1,漂白时间30min。③铜藻粉末粒度选择:选用铜藻粉末粒度为100目左右,它的持水力、膨胀力、溶解性较合适,可作为食品添加剂在膳食纤维制品中使用。     

In vitro effect of the red alga Hydropuntia cornea (J. Agardh) on the respiratory burst activity of sole (Solea senegalensis, Kaup 1858) phagocytes

The stimulatory effect of different extracts from the red alga Hydropuntia cornea (formerly Gracilaria cornea) on the respiratory burst activity of sole phagocytes was evaluated in vitro. Aqueous and ethanolic extracts and extracellular polysaccharidic fractions from algal cultures were assayed. Phagocytes, isolated from sole kidney, were incubated in vitro with different algal fractions at concentrations of 10, 5, 2 and 1 mg mL⁻¹. As a positive control, commercial β‐glucan from the alga Euglena gracilis was used. The results obtained indicate that an ethanolic extract from H. cornea, at 10 mg mL⁻¹, is capable of enhancing superoxide anion production in sole phagocytes when the cells are incubated for 30 min with the bacterium P. damselae ssp. piscicida and the extract. On the other hand, the commercial β‐glucan leads to a significant increase at highest concentration, 10 mg mL⁻¹, after 30 min of incubation.     

溶藻细菌控藻作用及其在对虾养殖池塘中的应用前景

浮游微藻与养殖池塘的水质及对虾的健康状况密切相关,控制有害微藻的过度繁殖,维持优良的藻相对于养殖水环境的稳定、减少对虾疾病的发生具有重要意义。文章对浮游微藻与对虾养殖的关系及溶藻细菌的研究进展进行了概述,分析了溶藻细菌在对虾养殖中的应用前景,提出了利用溶藻细菌控制对虾养殖池塘有害微藻的研发思路。     

中性柠檬酸菌对几种常见藻类生长的他感作用

在４５００～４８００ｌｕｘ光照,２６～２８℃的培养条件下,从绿裸藻（Ｅｕｇｌｅｎａｖｉｒｉｄｉｓ）水华中分离的优势菌种之一中性柠檬酸菌（Ｃｉｔｒｏｂａｃｔｅｒｉｎｔｅｒｍｅｄｉｕｓ）的分泌物对斜生栅藻（Ｓｃｅｎｅｄｅｓｍｕｓｏｂｌｉｑｕｕｓ）、粉核小球藻（Ｃｈｌｏｒｅｌａｐｙｒｅｎｏｉｄｏｓａ）、羊角月牙藻（Ｓｅｌｅｎａｓｔｒｕｍｃａｐｒｉ ｃｏｒｍｕｔｕｍ）、水华鱼腥藻（Ａｎａｂａｅｎａｆｌｏｓ－ａｑｕａｅ）和易变鱼腥藻（Ａｎａｂａｅｎａｖｑｒｉａｂｉｌｉｓ）的生长有促进作用;对银灰平裂藻（Ｍｅｒｉｓｍｏｐｅｄｉａｇｌａｕｃａ）、莱茵衣藻（Ｃｈｌａｍｙｄｏｎｏｎａｓｒｅｉｎｈａｒｄｉ）和铜绿微囊藻（Ｍｉｃｒｏｃｙｓｔｉｓａｅｒｕｇｉｕｏｓａ）的生长具有抑制作用。中性柠檬酸菌胞外分泌物能促进粉核小球藻细胞个体的长大,对其他藻类主要是影响细胞的大小。中性柠檬酸菌对不同藻类影响的差异对水华过程可能具有重要意义。     

Serratia marcescens: a potential pathogen for fish

Abstract. Characterization of a red pigmented enterobacterium isolated from natural population of white perch, Morone americanus (Gmelin), during the course of a bacteriological survey in the Back River (Baltimore, Maryland, USA) indicated that the bacterium belonged to the species Serratia marcescents. The virulence properties of this isolate (RB 469), studied in comparison with the reference strain of S. marcescens ATCC 1800 and S. plymuthica K1R, revealed that all the strains were highly pathogenic for fish with LD₅₀s raging from 5 × 10³ to 1 × 10⁵. Similarly, te extracellular products (ECP) from the three isolates were lethal for fish (LD₅₀ ranging from 0·22 to 4·8 μg protein g^-1 fish). However, only ECP from strains with strong proteolytic activity (the white perch isolate and S. plymuthica ) were cytotoxic for both in fish and homoiothermic cell cultures and both activities were completely destroyed by heating at 100°C for 10min. In contrast, only the two S. marcescens strains which possessed phospholipase active were pathogenic for mice and produced enterotoxins. None of the Serratia strains displayed dermonecrotic factor in rabbits. All these lindings indicate that a direct relationship between eytotoxicity and virulence cannot be established.     

球等鞭金藻生长抑制物对自身藻细胞的抑制效应

研究了生长抑制物1-羟基，丙二酸二乙酯-十二烯酸异丙酯（HDMA）对自身细胞生长、叶绿素和丙二醛（MDA）含量、胞外可溶性蛋白质和多糖含量、硝酸还原酶（NR）、超氧化物歧化酶（SOD）和过氧化物酶（POD）活性等生理指标的影响，分析HDMA对球等鞭金藻的抑制效应。结果表明，HDMA显著降低了细胞密度、叶绿素含量以及NR、SOD和POD活性，显著增大胞外可溶性蛋白质和多糖的比值，并且还明显提高MDA含量。因此，我们认为HDMA能够影响微藻细胞的某些生理生化过程。其中包括降低叶绿素含量，影响光合作用；改变胞外可溶性蛋白质和多糖的比值，增强细胞的疏水性，促使细胞絮凝；降低微藻硝酸还原酶和抗氧化体系酶的活性，影响细胞内营养平衡，并加速细胞体内活性氧的积累，导致细胞发生过氧化反应，从而影响微藻生长。     

一株溶藻细菌的分离、鉴定及溶藻特性研究

从三亚市大东海分离出1株有较强溶藻活性的细菌,命名为L-D1,并对该菌株进行形态和生理生化鉴定。L-D1的菌悬液对中肋骨条藻的生长有很强的抑制作用。试验发现,L-D1是通过直接和间接的方式共同作用影响中肋骨条藻的生长的;L-D1的溶藻效果在温度为25℃时优于其他温度,盐度为20‰时优于其他盐度,全黑暗条件下的溶藻效果优于其他光照条件下的。     

Nutritional Properties of the Marine Rotifer Brachionus plicatilis Fed the Freshwater Microalgae Selenastrum capricornutum

This study examined the effects of storage time on the fatty acid composition of freshwater Selenastrum capricornutum algal paste under 4 C refrigeration, the fatty acid composition of rotifers fed the fresh and stored algal paste, and the toxicological properties of the algal paste. Microalgae were produced in a hydraulically integrated serial turbidostat algal reactor (HIS-TAR), harvested as a paste, refrigerated and analyzed every 2 wk. Fresh Selenastrum capricornutum paste had almost three times greater concentration of unsaturated fatty acids than saturated fatty acids. Over 50% of the unsaturated fatty acids were made up of n-3 and n-6 fatty acids. Total unsaturated, n-3 group and n-6 group fatty acids decreased ( P 0.05) during storage, including the nutritionally important fatty acids (C20:4n6, C20:5n3, C22: 6n3). Rotifers fed 2– and 4-wk-old algal paste had a significantly lower ( P 0.05) total unsaturated fatty acid percentage and significantly greater ( P 0.05) total saturated fatty acids than those fed fresh algae. There were no significant changes in the n-6 group fatty acid level in the 4-wk-old paste feeding study or for the n-3 or n-6 groups in the 2-wk-old paste feeding study as compared to fresh algae. The levels of the three nutritionally important fatty acids (C20:4n6, C20:5n3, C22:6n3) did not differ between rotifers fed fresh and stored algae. Stored algae did not present toxicity to rotifers and Daphnia at the normal feeding concentration. These results indicated that the use of refrigerated freshwater algal paste for production of rotifers results in live feed with adequate nutritional properties for marine larviculture. This could eliminate the costs associated with production of marine algae, which could be replaced with freshwater algae, and may provide an alternative to cryopreservation.     

柱状黄杆菌强毒株和弱毒株胞外蛋白中差异蛋白的初步鉴定

柱状黄杆菌(Flavobacterium columnare)是一种世界范围的水产动物致病菌,是中国重要养殖鱼类草鱼(Ctenopharyngodon idellus)、鳜(Siniperca chuatsi)等烂鳃病的病原。本研究以1972年从患"烂鳃病"草鱼上分离的两株冻干柱状黄杆菌G4和G18菌株为研究对象,并将G4株再次分离纯化得纯化菌株,命名为G4R3。对草鱼鱼苗浸泡攻毒结果显示,G4R3的LD50至少比G18的高3个数量级,因此G4R3为"强毒株",G18为"弱毒株"。利用蛋白质组学方法分析柱状黄杆菌强毒株G4R3和弱毒株G18的胞外蛋白,经过双向电泳并结合图像分析,共发现了34个点是差异表达的蛋白。胶内酶解、肽质量指纹图谱和串联质谱分析后,鉴定出其中的7个蛋白点,代表滑动蛋白K、腺酐甲硫氨酸合成酶和一种可能的膜蛋白等3种蛋白,它们可能是柱状黄杆菌的毒力因子。     

The influence of salmon surface mucus on the growth of Flavobacterium columnare

Flavobacterium columnare is the causative agent of columnaris disease. The presence of lesions on the gills, skin and fins of diseased fish suggests that F. columnare is able to utilize fish skin mucus as a substrate for growth and that exposure to this material would alter the expression of genes involved in the colonization of the outer surfaces of the fish. Growth, biofilm formation, extracellular protease production and changes in protein expression of F. columnare strain C#2 cultured in media supplemented with juvenile Atlantic salmon skin mucus were compared with the same media without mucus. C#2 was able to grow by using mucus as the sole nutrient source. Growth in mucus-containing media induced cells to grow as a biofilm and extracellular protease activity increased in mucus-containing cultures. SDS-PAGE protein profiles showed that expression of six extracellular proteins increased in mucus-containing media. These results demonstrate that salmon surface mucus promotes the growth of F. columnare and that exposure to mucus alters the growth characteristics of this bacterium with regard to protease production and biofilm formation. Further characterization of mucus-induced physiological changes will increase our understanding of the basis of virulence of this economically important fish pathogen.