鳗弧菌和副溶血弧菌对短蛸感染的病理学研究

为了今后养殖病害的防治,了解病原微生物在养殖过程中对头足类动物产生的影响,选择在海水养殖中发病频率较高的弧菌,包括鳗弧菌(Vibrio anguillarum)和副溶血弧菌(Vibrio Parahemolyticus),研究它们对短蛸的毒力水平。通过将稀释菌液直接注射短蛸腕部的方式,观察感染后短蛸的致死率、半致死时间,以及各菌对短蛸肝脏、鳃、鳃心、白体等器官结构的影响。结果显示:鳗弧菌和副溶血弧菌均对短蛸有较强的致病性,分别在60、48 h时达到半致死,且发病的短蛸活性减退,虚弱无力,腹部肿大,解剖可见部分内脏肿胀明显,如肝脏;通过对病变组织和健康组织的石蜡切片和HE染色,以及超显微结构的观察,发现患病短蛸的肝脏、鳃、鳃心和白体均出现不同程度组织、细胞结构上的破坏。以上结果表明这两种弧菌对短蛸均具有明显的致病性,副溶血弧菌更强一些,合适条件下将会成为短蛸规模化养殖的重要威胁之一。     

对虾病原体鳗弧菌抗血清制备及其血清反应

对鳗弧菌抗血清学制备及其血清反应的研究结果表明，应用免疫注射法，先去除鞭毛抗原，可以获得对虾“黄鳃病”鳗弧菌的高效价的抗血清，其血清效价可以达到５１２０，这为对虾病原菌制备抗血清，提供了一个很好的技术和方法．     

大黄炮制前后体外抗菌活性比较

为了对大黄及其炮制品(酒大黄)的体外抗菌活性进行初步研究,采用超声法提取,利用滤纸片法,二倍稀释法分别检测MIC和MBC,对大肠杆菌、金黄色葡萄球菌、白色葡萄球菌、枯草杆菌及白色念珠菌五种细菌进行体外抗菌活性研究.结果发现,大黄、酒大黄乙醇提取物对实验菌种都有不同程度的抑菌及杀菌作用,且大黄炮制品(酒大黄)的抗菌效果略好于大黄,并在一定浓度范围中均具有杀菌作用.     

鳗弧菌质粒编码的铁吸收系统的分子调控

鳗弧菌是鱼类弧菌病的主要病原菌,该菌中质粒编码的铁吸收系统是重要的毒力因子．在铁吸收系统中,铁转运体系以及弧菌素合成基因受到AngR、TAF、Fur、反义RNAs、铁离子以及弧菌素等调控因子的调节,这些调控因子分别在转录或后转录水平参与铁吸收系统的正负调控、     

抗菌药物对布鲁氏菌体外最低抑菌浓度研究

目的:测定临床常用抗菌药物及新型抗菌药物对布鲁氏菌的最低抑菌浓度(MIC),以确定布鲁氏菌对各种抗菌药物的敏感性,为后期布鲁氏菌耐药性研究及布鲁氏菌病临床用药研究奠定基础.方法:采用美国临床实验室标准化协会(CLSI)规定的肉汤稀释法对布氏杆菌活疫苗S2株进行抗菌药物的MIC检测.结果各种药物的MIC值分别为:硫酸庆大霉素0.470μg/mL,硫酸卡那霉素1.362μg/mL,利福平1.821μg/mL,硫酸链霉素1.700μg/mL,四环素2.710μg/mL,莫西沙星0.298μg/mL.结论与其余5种药物相比,第4代喹诺酮类药物莫西沙星的MIC值最低,抑制布鲁氏菌生长能力最强.氨基糖苷类抗菌药物中,硫酸庆大霉素的MIC值最低,硫酸链霉素的MIC值最高.     

翘嘴红鲌对几种常用渔药的敏感性试验

报道了翘嘴红鲌对硫酸铜硫酸亚铁合剂、高锰酸钾、生石灰、三氯异氰尿酸、甲酸溶液、呋喃西林、孔雀石绿、食盐等九种常用渔药的敏感性，其安全浓度分别为0.088mg/L、0.314mg/L、9.018mg/L、0.136mg/L、5.561μL／L、2.394mg/L、0.031mg/L、和895.4mg/L，亚甲基兰对翘嘴红鲌的安全浓度大于3mg/L。     

基于斑马鱼模型的鳗弧菌减毒活疫苗的生物安全性评价

利用疫苗对养殖鱼类进行疾病预防正得到逐步应用,而减毒活疫苗通过浸泡方式也能使鱼体获得较高的免疫保护力。利用模式动物斑马鱼对鳗弧菌减毒活疫苗MVAV6203的生物安全性进行了评价。首先进行了疫苗对斑马鱼毒性实验,每条鱼注射免疫的半致死量为9．26×10^4CFU;其次分析了鳗弧茵在水体中的存活情况,菌体浓度在1．0×10...     

喙尾琵琶甲提取物体外抗菌作用研究

目的:研究喙尾琵琶甲不同极性组分的抗菌活性,筛选出抗菌活性部位。方法:通过硅胶柱层析得到不同极性的组分,采用试管稀释法测定不同组分对6种临床常见的病原菌的MIC及MBC。结果:乙酸乙酯、丙酮洗脱组分有较好的体外抗菌作用,乙酸乙酯组分活性强于丙酮组分;石油醚、氯仿、甲醇组分对所测菌株没有体外抗菌作用。结论:喙尾琵琶甲提取物具有良好的体外抗菌作用,乙酸乙酯组分为体外抗菌活性部位。     

铜绿体外抗菌作用的实验研究

目的 探讨中药铜绿对11种细菌的体外抑菌作用．方法 应用平板稀释法测定最低抑菌质量浓度(MIC)。结果铜绿对金葡菌、表皮葡萄球菌、乙型溶血铁球菌、白假丝酵母菌的MIC为0．00258g/L，，对大肠埃希菌、流感嗜血杆菌、乙型副伤寒杆菌、肺炎克雷伯杆菌、福氏志贺菌、铜绿假单胞菌的MIC为0．0125g/L。结论 铜绿在体外对G^ 球菌有较强的抑菌作用，对G^-杆菌抑菌作用较弱，对变形杆菌无抑制作用。     

花椒挥发油的提取、分离和抗菌实验

研究花椒(Zanthaxylum Bungearum Maxim)挥发油的抗菌作用．超临界CO2萃取法(CO2-SFE)和乙醇回流法提取花椒挥发油及硅胶柱分离乙醇回流法所得挥发油，适当处理后共得到8个样品，用滤纸片和试管法分别测定其抗菌活性和最低抑菌浓度(MIC)．结果表明，试验中8个样品分别对8种受试菌种有不同的抑制作用．说明花椒挥发油有明显的抑菌作用；挥发油中极性相对较小的成分主要对真菌起抑制作用，而极性相对较大的样品主要对细菌起抑制作用．     

Preparation and Identification of Monoclonal Antibodies Against Vibrio anguillarum

Monoclonal antibodies (Mabs) against V. anguillarum strain M3 are prepared, and their isotypes are also characterized. Among them, C1C5 is the only Mab which does not cross-react with other eleven non-V, anguillarum strains. The proteinase K digestion test shows that the epitopes recognized by C1C5, C6C3 and C6C32 Mabs contained protein. The periodate oxidation test showed that the epitopes recognized by Mabs except C1 C5 are glycosylated. In addition, results of additivity test indicate that the epitopes recognized by C6C3 and C6C32 Mabs are similar, and quite different from those recognized by MabC1C5.     

海洋鳗弧菌铁载体合成的调控

以一株鳗弧菌pJM1质粒缺陷为研究对象，考察了儿茶酚类铁载体Anguibactin的合成代谢调控。研究发现：在由染色体和质粒共同编码介导的铁载体Anguibactin的合成途径中，其分界点位于2，3－二羟基苯甲酸合成，2，3－二羟基苯甲酸合成之前代谢途径受染色体编码调节,后由质粒编码介导；编码2，3－二羟基苯甲酸的染色体相关基因的表达受环境铁离子浓度的诱导调节。     

Detection of Vibrio anguillarum and Its Virulent Metalloprotease Using the Method of ELISA

A method detecting pathogenic Vibrio anguillarum and its virulent metalloprotease is reported in this paper. The metalloprotease is isolated from extracellular product (ECP) of V.anguillarum by the cellophane plate technique and purified by gel filtration and ion-exchange chromatography. Anti-sera are prepared by injecting V.anguillarum cells and metalloprotease into the rabbits. Slide Agglutination Assay is used to detect V.anguillarum in the infection experiment and Enzyme Linked Immunosorbent Assay (ELISA) is carried out to detect concentration of metalloprotease. The result shows that bacterium strain M3 is able to diffuse into the viscera of infected fish through the blood circulating system 10 hours after intramuscular infection, and ELASA is a sensitive method to detect the metalloprotease with detectable amount of 7.8ng. The aim of this study is to establish a sensitive and specific method to observe the infection of V.anguillarum in the host.     

Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coil

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, hetemlogous expression of the empA gene encoding metallopmtease and export of the recombinant metallopmtease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide （611 amino acids） consisting of four domains： a signal peptide, an Nterminal pmpoptide, a mature region and a C-terminal pmpoptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metallopmtease as the mature pmtease was further confirmed by SDS-PAGE and im- munoblotting. It was found that recombinant metallopmtease with the EmpA activity and antigenicity was exported into the poriplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.     

Overexpression and export of Vibrio anguillarum metalloprotease in Escherichia coli

Vibrio anguillarum metalloprotease, an extracellular zinc metalloprotease involved in the virulence mechanism of Vibrio anguillarum, is synthesized from the empA gene as a 611-residue precursor and naturally secreted via Sec secretion pathway in Vibrio anguillarum. In this study, heterologous expression of the empA gene encoding metalloprotease and export of the recombinant metalloprotease in Escherichia coli were examined. The empA gene was subcloned into pBAD24 with arabinose promoter and sequenced. The sequence encoded a polypeptide (611 amino acids) consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. The empA gene inserted in plasmid pBAD24 was overexpressed in TOP10 strain of E. coli after arabinose induction. The 36kDa polypeptide of the recombinant metalloprotease as the mature protease was further confirmed by SDS-PAGE and immunoblotting. It was found that recombinant metalloprotease with the EmpA activity and antigenicity was exported into the periplasm of Escherichia coli cells via Sec translocation pathway, whereas it was secreted into extracellular environments in V. anguillarum. The results imply that the expression, export and processing mechanism of the protein in E. coli are similar to those in V. anguillarum.     

仿刺参硫氧还蛋白基因在灿烂弧菌和鳗弧菌刺激下的应激表达特征

分别用灿烂弧菌(Vibrio splendidus)和鳗弧菌(V.anguillarum)对仿刺参(Apostichopus japonicus)进行注射感染试验,取注射后0、3、6、9、12、24 h的仿刺参体壁、肠道、呼吸树、触手和体腔细胞5种组织或细胞进行了实时荧光定量PCR检测,分析硫氧还蛋白(Thioredoxin,Trx)基因在这5种组织或细胞中的表达特征,探讨了硫氧还蛋白在仿刺参先天免疫中的作用。结果显示,Trx基因在仿刺参体壁、肠道、触手、呼吸树及体腔细胞中均表达;在灿烂弧菌刺激下各组织或细胞中Trx基因变化趋势相似,呈现一种"突增-下降-再突增"的双峰型模式;鳗弧菌刺激下Trx基因表达量变化同样呈现先升高后降低的趋势,但与灿烂弧菌组相比反应时间与上调幅度都不同。总而言之,Trx基因在仿刺参体内有组成型和诱导型两种表达模式,并存在表达的组织特异性和病原特异性;Trx基因参与了仿刺参对病原感染的免疫应答,使机体免受氧化胁迫,在抵御外界病原入侵、维持胞内氧化还原状态方面起到重要作用。     

四种海洋致病弧菌对抗生素敏感性的测定

测定了4种海洋致病弧菌对8种常用抗生素药物的敏感性．测试结果表明，鳗弧菌对氯霉素、庆大霉素具有敏感性，但对其他所测药物均表现出耐药性；副溶血弧菌对四环素、卡那霉素、青霉素表现出耐药性，而对氯霉素、头孢娄新、庆大霉素、红霉素和链霉素则表现出敏感性；河流弧菌对四环素、链霉素表现出耐药性，对氯霉素、卡那霉素、头孢娄新、庆大霉素、青霉素和红霉素则表现出敏感性；溶藻弧菌仅对四环素表现出耐药性，对其他7种抗生素均表现出敏感性．同时针对表现出抗药性的药物，相应地确定了它们的最小抑菌质量浓度值．     

12种中草药粗提液抑真菌效果研究

选取了山豆根、麻黄草、穿山龙、茜草、穿心莲等12种中草药,采用水提和醇提两种方法制备药液,以番茄灰霉病菌、白菜灰斑病菌、柑橘炭疽病菌、辣椒炭疽病菌、小麦全蚀病菌、棉花枯萎病菌6种常见真菌为供试菌,采用室内抑制菌丝生长速率法进行抑菌实验,结果表明山豆根水提药液在5 000 mg·L-1浓度下对辣椒炭疽病菌的抑制率最高达到了52.84％,穿心莲醇提药液在400 mg·L-1浓度下对白菜灰斑病菌的抑制率最高达到了65.26％,进一步测定了茵陈醇提液、山豆根水提液的最小抑菌浓度.