Studies on Vibriosis of Pseudosciaene crocea—Pathogen Biology and Immunology

1、将副溶血弧菌培养至对数生长期，用天然海水洗脱，调整菌浓度至107cells/mL左右后置于28℃恒温培养箱进行饥饿试验。结果表明：在饥饿初期细菌总数、CFU和活菌数都有较大幅度上升；在饥饿中、后期，细菌总数、CFU和活菌数都呈下降趋势，其中总菌数与活菌数缓慢下降，而CFU数降低速度较快。结晶紫染色观察的结果表明：饥饿前副溶血弧菌呈短杆状，染色均匀；饥饿后许多细胞中央出现染色较浅的区域，而且细胞形态也变为椭圆形。以对数生长期的副溶血弧菌为对照，对饥饿60d的副溶血弧菌进行热处理和紫外线处理。结果表明饥饿60d的副溶血弧菌对热和紫外线更敏感。用间接ELISA测定饥饿前后副溶血弧菌的最低检测限，...1、 Pathogenic Vibrio parahaemolyticus in log-phase were collected and the concentration was adjusted to 107cells/ mL in natural sea water for starvation studies. The results showed that: all of the total bacteria number 、viable bacteria number and CFU number of V. parahaemolyticus increased remarkably at the initial starvation stages; all of the total bacteria number 、viable bacteria number and CF...学位：博士后院系专业：化学化工学院化学系_化学学号：BH1700003

绿色荧光蛋白基因在大黄鱼体内的表达

以绿色荧光蛋白基因为报告基因 ,以pIRESneo为载体质粒 ,制备含有报告基因的质粒 ,以肌肉注射的方式导入大黄鱼体内 ,每隔一周用荧光显微镜观察绿色荧光蛋白基因在鱼体不同部位的表达情况 .结果表明 ,绿色荧光蛋白基因可以在大黄鱼体内得到表达 ,表达时间高达 2 8d以上 .绿色荧光蛋白基因不仅可以在肌肉注射部位组织细胞得到表达 ,而且在其它部位的肌肉、肝脏、肾脏和心脏等组织中也有表达 ,但表达水平低于肌肉注射部位组

Establishment of Single Tube Quantitative Detection Technology for Escherichia coli Assay Based on Fluorescent Method

通过研究培养温度、培养基pH值对大肠杆菌生长和培养液荧光强度的影响,确定44℃,培养液pH值7.0～7.5为大肠杆菌MUG酶荧光检测的最佳条件。通过研究培养时间和接种菌浓度与培养液荧光强度的关系,建立起基于荧光方法检测大肠杆菌的单管定量检测技术。通过与平板菌落计数法和最大可能数法比较发现单管定量检测法的相对标准偏差小于这两种常用的方法,而且更为快速、经济,适合用于大肠杆菌的定量检测。Influence of incubation temperatures and pH values of culture liquid on growth of Escherichia coli and the fluorescent intensity of culture liquid had been investigated.E.coli incubated at 44℃ in the culture liquid with a pH value in the range of 7.0～7.5 was found to be the best condition for fluorescent assay.Single tube technology to detect E.coli was established through the studies on the relationship between incubation time,inoculation quantity of E.coli and the fluorescent intensity of culture liquid.Relatively standard diversity of single tube detection technology was lower than those of colony-counting method and the most probably number method.The results indicated that the single tube quantitative detection method was fit for E.coli quantitative detection because of the several advantages such as less time-consuming and more economical compared with traditional methods.国家高技术研究发展计划项目(863计划项目)(No2002AA639600);; 福建省自然科学基金(NoB0410022

发光细菌监测沿岸水质应用研究

采用发光细菌测试毒性法(MICrOTOX)检测了取自厦门大学海滨浴场、厦门大学医院排污口、厦门大学东大沟排污口的海水水样毒性;比较了四种稀释液:30‰nACl溶液、陈海水、人工海水Ⅰ及人工海水Ⅱ对发光菌发光强度的影响和在不同盐度条件下发光强度的变化。结果表明:(1)前两个站住水体无毒性反应,后一个站住水体存在毒性物质;(2)除人工海水Ⅱ对发光菌有较强的抑制作用外,其它两种稀释液的活化作用与常用稀释液(30‰nACl溶液)相近;(3)当盐度变化在25‰~40‰范围时,其对发光强度影响极小。国家自然科学基金;福建省自然科学基

Establishment of in vitro biofilm model and characteristics of biofilm formation of Vibrio harveyi

采用改良微孔板法建立哈维氏弧菌VIbrIO HArVEyI TS-628菌株生物被膜体外模型,并进一步研究环境因子对哈维氏弧菌体外形成生物被膜的影响。结果发现,哈维氏弧菌TS-628菌株可以在聚苯乙烯板中形成生物被膜,并且该菌生物被膜的形成受起始菌液浓度、温度、PH等理化因子的显著影响。温度为30℃、nACl质量分数为3%—6%、PH为7、孵育时间为36H是哈维氏弧菌形成生物被膜的最佳条件。此外哈维氏弧菌在分别经表皮黏液、鳃黏液、肠道黏液、肝脏提取液、脾脏提取液、肌肉提取液包被的基质上成膜效果显著,其中在经肝脏提取液包被的基质上形成的生物被膜量显著高于在其他生物基质上形成的生物被膜量(P<0.01)。Modified microtiter-plate test was used to establish an in vitro biofilm model of Vibrio harveyi TS-628 strain.Effect of environmental factors on bacterial biofilm formation in vitro was also studied.The results show that V.harveyi TS-628 strains could form biofilm in polystyrene plates,and biofilm formation of this bacterium was significantly affected by different environmental factors,such as temperature,initial bacterial concentration,pH,NaCl concentration,and so on.The optimum conditions for biofilm formation of V.harveyi TS-628 strain are at temperature of 30℃,pH=7,NaCl concentration of 3%?6%,and using 36h incubation time.Furthermore,V.harveyi TS-628 strain could form biofilm on the walls coated with skin mucus,gill mucus,intestinal mucus,liver extract,spleen extract,and muscle extract.The biofilm formation of V.harveyi on the walls coated with liver extract exhibited a remarkably higher value of D(590)(p<0.01)than that on the walls coated with other tissue extracts.国家高技术研究发展计划项目(2007AA09Z115

Approaches to fish DNA vaccine study

福建省自然科学基金(B0410022);; 福建省青年创新基金(2002J037

Influence of environmental factors on the adhesion of Vibrio alginolyticus to the intestinal mucus of Pseudosciaene crocea

研究了不同环境条件下溶藻弧菌对大黄鱼肠粘液的粘附作用。采用细菌计数法测定溶藻弧菌的粘附作用。粘附的细菌经SYBR GreenⅠ染色后在荧光显微镜下观察,用数码相机拍照后在电脑上计数。试验结果表明,溶藻弧菌对大黄鱼肠粘液的粘附量随菌浓度的升高而升高,并在1~1.5h内趋于饱和;粘附作用在15~30℃、pH偏酸时较强;盐度在5~35范围内对前肠粘液的粘附作用影响不明显,后肠粘液的粘附作用在此范围内随盐度增大而加强,在盐度为0时,溶藻弧菌对前、后肠粘液都无粘附作用;56℃热处理5min及60℃处理1h均能大幅减弱溶藻弧菌对两种肠粘液的粘附作用,表明溶藻弧菌表面的某些热敏结构在粘附作用中起着重要作用。根据以上结果,可以认为溶藻弧菌能够很好地粘附于大黄鱼肠粘液层,其粘附作用受温度、盐度、pH值等环境因子影响很大,溶藻弧菌表面的某些热敏结构可能在粘附过程中起着重要作用。研究结果表明,溶藻弧菌对大黄鱼肠粘液的粘附作用是可控制的,这对于鱼类养殖疾病的控制有一定的参考价值。The mucus layer is the most outer defence barrier of fish.The bacterial attachment to mucus layer is a critical step in the host colonization and subsequent occurrence of infection.Adhesion of Vibrio alginolyticus to the intestinal mucus of Pseudosciaene crocea has been investigated in order to understand the characteristics and mechanisms involved in the adherence of bacteria onto fish mucus so that preventive measures can be devised to reduce infection.In vitro adhesion assay was carried out by bacterial counting.The adhesive bacteria were stained with SYBR Green Ⅰ,observed under fluorescent microscope,counted on computer display after taking pictures.The results showed that the adhesive quantity of V.alginolyticus increases with bacterial concentrations and reached equilibrium after being incubated 1-1.5 h;the higher adhesive quantity was achieved at 15-30 ℃ and acid condition;adhesion of V.alginolyticus to the mucus of foregut was not obvious variety with salinity(5-35),but adhesive quantity to the hindgut mucus enhances while salinity increases;V.alginolyticus could not adhere to intestinal mucus without salinity;adhesive quantity reduced remarkably after heat treatment of the germ(56 ℃ 5 min,60 ℃ 1 h),which indicated that heat sensitive structure of V.alginolyticus might play an important role in adhesion.The conclusions of this paper showed V.alginolyticus could adhere to intestinal mucus of P.crocea facilely in seawater;this adhesion was remarkably affected by environmental factors,such as temperature,pH and salinity;V.alginolyticus adhered to the intestinal mucus of P.crocea by specific adhesion,some heat-sensitive structures on V.alginolyticus surface played a crucial role in adherence of the bacteria to gill mucus.The results indicated that the bacterial adhesion of V.alginolyticus to intestinal mucus of P.crocea was governable.The results obtained are useful for the epidemic control in fish culture.国家863计划项目(2002AA639600);; 福建省自然科学基金(B0410022);; 福建省青年创新基金(2002J037

口服免疫添加剂对养殖大黄鱼免疫机能影响的初步研究

对福建省连江县厦宫乡新辉村的养殖大黄鱼病害进行免疫防治试验 ,通过定期口服免疫添加剂后 ,检测大黄鱼血清中溶菌酶活力、抗菌活力和酚氧化酶活力 ,测定大黄鱼体长和体重 .研究结果表明 :大黄鱼在服用免疫添加剂后血清中的溶菌酶活力平均值提高了 2 6% ,抗菌活力增加了 18% ,酚氧化酶活力提高幅度高达 4 6% .与此同时 ,实验组大黄鱼的生长速度也比对照组要快 ,其中实验组平均体长比对照组高 3.8% ,平均体重增加 13.5%

Effect of experimental infection with Vibrio alginolyticus on immune parameters of Pseudosciaena crocea

为了解大黄鱼在抗溶藻弧菌感染时免疫功能的变化规律,将160尾健康大黄鱼分为感染组和对照组,通过腹腔分别注射0.2mL浓度为2×107CFU.mL-1的溶藻弧菌和灭菌生理盐水,在注射0,1,3,7,11,15,19d后从两组各取6尾大黄鱼,尾静脉取血,进行外周血的血相、NBT阳性细胞数、血清抗菌活力、抗体效价等免疫学指标的测定。结果显示:在人工感染的初期,感染组大黄鱼外周血的红细胞、白细胞、淋巴细胞和NBT阳性细胞的数量及血清抗菌活力等指标均较对照组有显著提高;在注射后1d外周血粒细胞的数量显著低于对照组;抗体效价在7d开始增加,15d达到峰值,且用间接ELISA和试管凝集两种方法所得结果具有非常高的同步性。结果表明:在感染溶藻弧菌后,大黄鱼能通过红细胞和白细胞增殖、释放抗菌物质、产生特异性抗体等方式提高其对溶藻弧菌的免疫力;在感染的早期阶段非特异性免疫因子起主要作用,在感染后期阶段特异性免疫因子起重要作用;NBT阳性细胞数可以作为细菌感染的指标。To investigate the effect of experimental infection with Vibrio alginolyticus on immune parameters of Pseudosciaena crocea,one hundred and sixty healthy fish were categorized into infected and control groups.Each fish in infected group was injected intraperitoneally with 0.2 mL of bacteria suspension with a concentration of 2×107 CFU·mL-1,while each fish in control group was injected i.p.with 0.2 mL of sterile saline solution. P.crocea in both groups were sacrificed at 0,1,3,7,11,15 and 19 d post-injection to evaluate immune parameters such as haematocyte counting,NBT-positive cells,serum antibacterial activity,and serum antibody titer.The results showed that: experimental infection with V.alginolyticus would increase the numbers of erythrocytes,leucocytes,lymphocytes in peripheral blood as well as peripheral serum antibacterial activity and antibody titer of P.crocea,and reduce peripheral blood granulocytes number significantly,in comparison with those in control group.These results suggested that after infected by V.alginolyticus,P.crocea would enhance its resistance to the invaded bacteria by means such as erythrocyte and leucocyte proliferating,antibacterial substance excreting,antibodies synthesizing;non-specific immune factors including antibacterial substance in peripheral blood played an important role at the initial stages of infection,and then specific immune factors such as antibody played an important role at the later stages;the number of NBT positive cell was a good indicator of bacterial infection.国家“八六三”高新技术发展计划(2002AA639600);; 福建省自然科学基金项目(B0410022);; 福建省青年创新基金项目(2002J037