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本研究采用核酸适配体的单链DNA浓度来表征其亲和力,通过测定核酸适配体与靶细菌哈维氏弧菌(Vibrio harveyi)结合后ssDNA的浓度,来研究该核酸适配体的亲和特异性和亲和常数,并通过荧光显微镜法对其亲和特异性进行验证。结果显示,采用单链DNA浓度法测得该核酸适配体对靶细菌哈维氏弧菌的亲和力是非目标菌的15.2倍以上;荧光显微镜直接观察发现只有靶细菌能较好结合有荧光标记的核酸适配体,并呈现明显荧光;荧光阻断法发现靶细菌和非靶细菌都未呈现明显荧光,证明了该适配体有较好的亲和特异性,也进一步验证了单链DNA法的测定结果。在亲和常数的测定方面,利用单链DNA浓度法测得该核酸适配体的亲和常数K_d=(33.70±7.83) nmol/L,相应的拟合系数为R~2=0.960,有较好的准确性和可靠性,说明采用单链DNA浓度法来测定适配体的亲和力和亲和常数是可行的。 相似文献
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于5L试验容器内,以副溶血弧菌和溶藻弧菌通过菌浴和口服法感染拟穴青蟹溞状幼体,比较2种弧菌对拟穴青蟹溞状幼体感染的致病力。海水水温25～26℃,盐度30.1,紫外线消毒。试验结果表明,副溶血弧菌菌浴感染拟穴青蟹幼体,24h对Z1至Z5幼体半致死密度分别为4.03×105、7.67×105、8.56×105、1.98×106、8.99×106cfu/mL;溶藻弧菌菌浴拟穴青蟹幼体,24h对Z1至Z5幼体半致死密度为5.76×106、7.64×106、7.92×106、1.02×107、6.02×107 cfu/mL;口服感染试验的幼体死亡率随投喂次数和时间增加而提高。在5×106cfu/mL密度下2种弧菌的混合感染组(密度比1∶1)24h幼体死亡率低于对照副溶血弧菌感染组,高于对照溶藻弧菌组。 相似文献
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流沙湾海区异养菌和弧菌动态变化规律研究 总被引：1，自引：0，他引：1
对流沙湾海区异养菌、弧菌动态变化规律及其优势菌群进行了初步探讨。采用平板稀释法对异养菌和弧菌数量进行了周年检测;对优势菌株进行分离纯化,并对代表性月份样品优势菌中的弧菌进行菌种鉴定。结果显示,流沙湾海区的异养菌和弧菌密度分别为8.8×103~9.60×105cfu/mL和1.0×103~2.45×104cfu/mL,其中异养菌数量高峰出现在6月,弧菌数量高峰出现在12月,异养菌、弧菌数量与水温变化的相关性不明显;流沙湾海区优势菌群中存在着溶藻弧菌等多种致病性弧菌。 相似文献
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《水产科学》2021,(4)
为提高罗非鱼链球菌病发前的早期预警,根据海豚链球菌的16S rRNA基因的部分序列和无乳链球菌的Sip基因特异性序列,分别合成特异性检测引物,经过对反应体系和反应条件的优化,建立检测较低含量的无乳链球菌和海豚链球菌的分子技术。试验结果显示,所建立的双重PCR可扩增出870 bp无乳链球菌和614 bp海豚链球菌的特异性片段,而迟钝爱德华氏菌、维氏气单胞菌、创伤弧菌、温和气单胞菌、溶藻弧菌、大肠杆菌均为阴性;无乳链球菌和海豚链球菌基因组DNA最低检测量分别为9.84×10~(-5) ng/μL和9.30×10~(-5) ng/μL,菌液最低检测量分别为2.76×10~3 cfu/mL和2.51×10~3 cfu/mL,远低于其半致死密度10~7 cfu/mL;对无乳链球菌和海豚链球菌混合感染的模拟临床样品的检出率为100%。研究结果表明,该分子技术的特异性较强、灵敏度较高、检出率较高,可用于较低含量的无乳链球菌和海豚链球菌的快速鉴别,为罗非鱼链球菌的早期预警分子检测提供技术支持。 相似文献
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对河蟹育苗场各期蚤状幼体(Z1～Z5)、大眼幼体至Ⅴ期幼蟹进行了病原菌的分离采样。采用"细菌生化鉴定编码"对育苗期的中华绒螯蟹的致病菌进行鉴定,主要为溶藻弧菌(V.alginolyticus)、霍乱弧菌(V.cholerae)、荧光假单胞菌(P.fluorescens)、豚鼠气单胞菌(A.caviae)和梅氏弧菌(V.metschnikovii)。采用扩散法(K-B法)对上述菌株进行了药物敏感性试验,结果表明:上述各菌株对庆大霉素、氟哌酸、头孢哌酮、丙氟哌酸、氟嗪酸、复方新诺明、环丙沙星具有较高的敏感性,抑菌直径>16mm。同时选择氟哌酸和环丙沙星对荧光假单胞菌、溶藻弧菌进行了最小抑菌浓度(MIC)与最小杀菌浓度(MBC)的测定。氟哌酸对荧光假单胞菌和溶藻弧菌的MIC分别为25μg/mL和0.78μg/mL;环丙沙星对荧光假单胞菌和溶藻弧菌的MIC分别为6.25μg/mL和0.025μg/mL。氟哌酸对荧光假单胞菌和溶藻弧菌的MBC均为6.25μg/mL;环丙沙星对荧光假单胞菌和溶藻弧菌的MBC分别为50μg/mL和0.1μg/mL。 相似文献
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一株短小芽孢杆菌B1的筛选鉴定及其抗菌特性研究 总被引：3，自引：0，他引：3
为筛选抗水产弧菌的益生菌株,自西沙海域采集的珊瑚和海水样品共分离到125株细菌,从中筛选出1株无溶血性且对多株病原菌具有拮抗作用的海洋细菌B1。基于生理生化及16SrDNA序列鉴定海洋细菌B1为短小芽孢杆菌。以副溶血弧菌13150为病原指示菌,用琼脂扩散法测定发酵液抑菌活性,并通过卤虫浸泡方式和药敏试验方法检测短小芽孢杆菌B1的安全性,同时进行温度、酸碱度、盐度的发酵条件优化研究。试验结果显示,短小芽孢杆菌B1对副溶血弧菌、溶藻弧菌、哈氏弧菌等有显著的拮抗作用;共培养试验显示,加入等量短小芽孢杆菌B1无菌上清液可显著抑制副溶血弧菌的生长;安全性试验发现,短小芽孢杆菌B1对卤虫的72h半致死密度为108cfu/mL,副溶血弧菌13150的72h半致死密度为105cfu/mL。此外,药敏试验表明,短小芽孢杆菌B1只对20种常规抗生素中的5种产生抗性;优化后的短小芽孢杆菌B1发酵条件为:温度30℃,pH 7.0,NaCl质量分数为2%。研究表明,短小芽孢杆菌B1具有防治水产养殖病原弧菌的应用潜力。 相似文献
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鱼肠道弧菌(Vibrio ichthyoenteri)可引起多种养殖鱼类发病死亡, 给鱼类养殖业带来严重经济损失。为解决养殖过程中鱼肠道弧菌的现场快速检测问题, 本研究研制了鱼肠道弧菌胶体金快速检测试纸。通过制备兔抗鱼肠道弧菌多克隆抗体, 间接ELISA分析发现其与鱼肠道弧菌的外膜蛋白、鞭毛蛋白、胞外产物及全菌破碎蛋白发生阳性免疫反应, 与副溶血弧菌(Vibrio parahaemolyticus)、鳗弧菌(Vibrio anguillarum)、溶藻弧菌(Vibrio alginolyticus)、哈维氏弧菌(Vibrio harveyi)的4种抗原蛋白发生程度不等的免疫交叉反应。制备胶体金标记鱼肠道弧菌抗体, 确定了鱼肠道弧菌4种抗原蛋白的划线浓度, 以4种抗原蛋白作为4条检测线, 羊抗兔IgG作为质控线, 基于竞争法免疫层析技术研制出鱼肠道弧菌快速检测试纸。对鱼肠道弧菌、副溶血弧菌、鳗弧菌、溶藻弧菌和哈维氏弧菌的检测结果显示, 该试纸可以准确鉴别出鱼肠道弧菌, 并能判别其他病原菌的交叉反应。该试纸对鱼肠道弧菌的最低检测限为5×105 CFU/mL, 检测耗时为10 min。对患病牙鲆组织的检测结果显示, 该试纸与ELISA结果一致, 表明具有较好的检测准确性。本研究为水产养殖现场的鱼肠道弧菌快速、准确检测提供了有力工具。 相似文献
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溶藻弧菌是中国南部水产养殖业中弧菌病的最主要病原菌,数量居海洋类弧菌之首,其快速检测具有重要意义。以溶藻弧菌国内标准株ATCC17749基因组DNA为模板,PCR扩增出外膜蛋白OmpK基因,将其克隆入表达载体pET-28a中,获得重组质粒pET-28a-OmpK,限制性内切酶结合琼脂糖凝胶电泳分析和序列测定结果表明,该序列开放读码框正确且与已发表的OmpK结构编码序列完全一致。将重组质粒pET-28a-OmpK转化大肠杆菌BL21 pLys E,高效表达重组蛋白,将其纯化后免疫小鼠所得的高免血清能外膜蛋白OmpK特异性结合,表明体外表达的重组蛋白OmpK具有良好的免疫原性和反应原性。利用该抗溶藻弧菌外膜蛋白OmpK的抗血清建立了间接ELISA检测方法,检测灵敏度为104 CFU/mL,能特异性检测出溶藻弧菌,与其他菌株无交叉反应。 相似文献
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金乌贼幼体弧菌性坏死病的病原、组织病理特征和药物敏感性 总被引：1，自引：0，他引：1
对青岛市黄岛区人工繁育金乌贼(Sepia esculenta)苗种大规模坏死病进行了病原、组织病理、传播途径和防控药物研究。发病金乌贼幼体不摄食,生长缓慢,体色发黑;体表、肌肉、口腕、触腕及内脏团大面积溃烂,死亡率高达90%;组织病理学显示:患病金乌贼皮肤色素细胞崩解,肌丝断裂,鳃丝崩解,肝细胞大量坏死脱落,肠道微绒毛脱落;从患病金乌贼分离出3株优势菌(SE-A、SE-B和SE-C),人工感染实验表明,SE-B、SE-C对金乌贼半数致死量分别为3.98×10~6 CFU/mL和8.91×10~5 CFU/mL,有较强致病力,为该病的致病菌;根据细菌形态、生理生化和16S rDNA序列分析将SE-B和SE-C鉴定为溶藻弧菌(Vibrio alginolyticus)和塔氏弧菌(Vibrio tubiashii)。分析金乌贼幼体、育苗用水和生物饵料糠虾的病原菌来源、分布和优势度,发现致病菌主要由糠虾携带进入育苗系统。分析了2株致病菌的药物敏感性,SE-B对强力霉素和氟苯尼考等敏感,对链霉素、庆大霉素等耐药;SE-C对氟苯尼考和罗美沙星等敏感,对新霉素和克拉霉素等耐药。 相似文献
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哈维氏弧菌(Vibrio harveyi)是引起海南省后水湾深水网箱养殖卵形鲳鲹(Trachinotus ovatus)"烂身病"的主要病原菌。为了能够快速诊断该病原菌,急需建立一种耗时短、准确以及便捷的检测方法。本研究利用分离到的致病菌株QT520的ToxR基因序列设计特异性引物和加入SYTO-9特异荧光染料,建立了一种可以实时、快速检测哈维氏弧菌的LAMP法(RT-LAMP)。该方法对哈维氏弧菌的基因组DNA及菌液灵敏度分别为100 fg/μL和10~3 cfu/mL,与Real-time PCR法检测灵敏度相当,比普通PCR的检测灵敏度要分别高1000倍和10倍,能有效区分哈维氏弧菌与坎氏弧菌(Vibrio campbellii);可以实时观察检测结果,且检测时间只需要40 min;具有耗时短、特异性和灵敏度高、仪器便携、操作简单且能实时观察检测结果等优点,非常适合在生产现场进行哈维氏弧菌的检测。 相似文献
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Qing Yu Mingzhu Liu Hehe Xiao Siting Wu Xianling Qin Ke Ke Siqiao Li Huizhi Mi Deqiang Shi Pengfei Li 《Journal of fish diseases》2019,42(11):1523-1529
As the major opportunistic pathogen to both marine animals and humans, Vibrio alginolyticus (V. alginolyticus) has caused heavy economic losses to mariculture. ssDNA aptamer VA2 targeting live V. alginolyticus was generated by systematic evolution of ligands by exponential enrichment (SELEX) technology in our previous study. In this study, we first developed aptamer (VA2)‐based enzyme‐linked apta‐sorbent assay (VA2‐ELASA) for rapid detection of mariculture pathogen V. alginolyticus. The VA2‐ELASA could achieve the rapid detection for V. alginolyticus infection with high specificity and sensitivity. The VA2‐ELASA could specifically identify V. alginolyticus, but not other non‐target bacterial strains. VA2‐ELASA could detect V. alginolyticus at the concentration of 5 × 104/ml, the incubation time short to 1 min and the incubation temperature as high as 45°C, which proved sensitivity and stability of the novel VA2‐ELASA in this study. It took less than one hour to accomplish the detection process by VA2‐ELASA. The characteristics of specificity, sensitivity and easy operation make VA2‐ELASA a novel useful technology for the rapid diagnosis of pathogen V. alginolyticus in mariculture. 相似文献
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鳗弧菌O1/O2二价灭活疫苗免疫大菱鲆的抗体持续期和免疫保护期 总被引：2，自引：0，他引：2
本研究分析了鳗弧菌(Vibrio anguillarum)O1/O2血清型二价灭活疫苗免疫大菱鲆后的抗体持续期和免疫保护期。以鳗弧菌O1血清型VAM003株和O2血清型VAM007株为抗原制备了福尔马林灭活二价疫苗,将疫苗按照三种剂量(10~7 cells/尾、10~8 cells/尾、10~9 cells/尾)以腹腔注射途径免疫大菱鲆,在免疫后3 d、7 d、14 d、30 d、60 d、90 d、120 d、150 d,用血清凝集实验检测了免疫鱼血清的VAM003和VAM007抗体效价,用攻毒实验检测了疫苗的免疫保护率(RPS)。结果显示,在免疫后7 d三个剂量组的大菱鲆均产生了特异抗体,并获得27%～60%的RPS。三个剂量组大菱鲆的O1血清型抗体持续期分别90 d (10~7 cells/尾组)、150 d (10~8 cells/尾组)、150 d (10~9cells/尾组),而三个剂量组大菱鲆的O2血清型抗体持续期均150 d。三个剂量组的大菱鲆获得的免疫保护持续期均150 d;以RPS75%为有效免疫保护,各剂量组大菱鲆抵抗O1血清型病原感染的有效免疫保护期为:14～120d(10~7 cells组)、14～120 d (10~8 cells/尾)、14～150 d (10~9 cells/尾),抵抗O2血清型病原感染的有效免疫保护期为:14～60 d (10~7 cells组)、14～120 d (10~8 cells/尾)、14～120 d (10~9 cells/尾)。研究结果表明鳗弧菌二价灭活疫苗可为大菱鲆提供有效而稳定的免疫保护,获得的抗体持续期和免疫保护期为该疫苗的临床中试研究提供了基础。 相似文献
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Identification and Detection of the Pathogenic Bacteria Responsible for Swollen Abdomen Disease in Cultured Turbot,Scophthalmus maximus,and Flounder,Paralichthys olivaceus 
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下载免费PDF全文 Swollen abdomen disease (SAD) seriously threatens the aquaculture of turbots and flounders. Two dominant bacterial strains, FS1 and FS2, were isolated from the livers and kidneys of fish with diagnosed SAD. Applications of biochemical analyses, sequence analyses of 16S ribosomal RNA gene and heat shock protein 60 gene revealed two distinct pathogenic bacterial species, Edwardsiella tarda and Vibrio alginolyticus. These two hypothesized SAD‐causing pathogens were validated by challenge trials on flounder, Paralichthys olivaceus. Challenges with E. tarda and V. alginolyticus demonstrated lethal dose 50 (LD50) values at 1.51 × 105 colony‐forming units (CFU) and 1.05 × 105 CFU, respectively. To develop a rapid SAD diagnosis method in flounders and turbots, a multiplex polymerase chain reaction (PCR) assay method was developed to simultaneously detect E. tarda and V. alginolyticus. Our multiplex PCR assay successfully detected as low as 105 CFU/mL E. tarda and V. alginolyticus in flounders and turbots. No other common fish pathogens were detected with the multiplex PCR, suggesting a high specificity of this assay. The multiplex PCR assay developed in this study showed a great reliability in detecting SAD‐causing bacterial pathogens. Further optimization of this assay may contribute to the development of a novel SAD diagnosis tool in aquaculture. 相似文献
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Vibrio alginolyticus as an important pathogen in aquaculture can encounter the oxidative stress produced by the immune system during infection. Previous studies showed that sRNAs have important functions in response to oxidative stress in bacteria; however, less of sRNAs related to oxidative stress response were identified in V. alginolyticus. In this study, a total of 749 novel sRNAs were identified by RNA sequencing; among them, 128 sRNAs were up‐ or downregulated in response to oxidative stress. In addition, 1,870 genes exhibited variation on mRNA levels in oxidative stress response. By analysing the target genes of the sRNAs, we concluded that these sRNAs could regulate expressions of genes responsible for iron transport, catalase, GSH‐dependent defence system, electron transferred and stress response. Moreover, the functions of the sRNAs are also seemed related to the pathogenicity in V. alginolyticus. Based on the results, we constructed the oxidative stress model in V. alginolyticus. This study provides us the first outlook of sRNAs function in oxidative stress response in V. alginolyticus. Furthermore, this study can help us to prevent and control this important opportunistic pathogen in aquaculture. 相似文献
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María-José Chapela Martiña Ferreira Asela Ruiz-Cruz Iris Martin-Varela Jacobo Fernández-Casal Alejandro Garrido-Maestu 《Journal Of Applied Aquaculture》2018,30(1):76-89
In the present study a multiplex real-time PCR method was developed for early detection of diseased fish infected by Aeromonas salmonicida, Vibrio anguillarum, and/or Tenacibaculum maritimum. The method consisted of the detection of three species-specific genes after DNA extraction with a commercial kit. Three types of samples were tested, and the results were compared with those of traditional diagnosis. The method obtained a limit of detection of 104 cfu/mL (2 x 102 cfu/tube). Additionally, 27 samples from fish showing signs of disease were correctly diagnosed by the developed methodology, demonstrating its suitability for implementation in aquaculture. 相似文献
18.
为探讨一株杀鱼假交替单胞菌 2515 Pseudoalteromonas piscicida (简称菌株 2515)在对虾养殖中的抗弧菌效果, 以鳗弧菌(Vibrio anguillarum)为指示菌, 以鳗弧菌的敏感抗生素新霉素为参比, 采用牛津杯打孔法, 比较了菌株 2515 与抗生素抗菌等效关系。设置浓度分别为 104 CFU/mL、105 CFU/mL、106 CFU/mL、107 CFU/mL 的菌株 2515 与鳗弧菌共培养, 评价了该菌对鳗弧菌的拮抗效果。将含量 103 CFU/g、105 CFU/g、107 CFU/g, 及 103 CFU/mL、 105 CFU/mL、107 CFU/mL 的菌株 2515 分别添加到对虾饲料和养殖水体中, 养殖凡纳滨对虾(Litopenaeus vannamei), 分析了菌株 2515 对水体和对虾肠道细菌总数、弧菌数及对抗副溶血弧菌效果的影响。结果显示, 相同抗鳗弧菌效果条件下菌株 2515 数量与新霉素质量存在对应关系, 与鳗弧菌混合培养时, 浓度 106 CFU/mL 及 107 CFU/mL 的菌株 2515 对鳗弧菌表现出强的抑菌作用。饲料中添加菌株 2515 浓度 105 CFU/g 及 107 CFU/g 时能够显著降低对虾肠道中的弧菌数量(P < 0.05), 提高对虾养殖的存活率。采用副溶血弧菌(V. parahaemolyticus)攻毒, 显示在饲料中添加菌株 2515 浓度为 105 CFU/g 一组对虾死亡率最低, 为(43.3±5.8)%, 对副溶血弧菌的相对保护率达(53.7±6.2)%。结果表明, 在饲料和养殖水体中添加适宜浓度的菌株 2515 均能够提高凡纳滨对虾抗副溶血弧菌的能力, 降低对虾肠道内的弧菌数量。本研究结果为对虾养殖弧菌病生物防控提供了一种技术支撑。 相似文献
19.
Lixing Huang Lina Guo Xiaojin Xu Yingxue Qin Lingmin Zhao Yongquan Su Qingpi Yan 《Journal of fish diseases》2019,42(5):703-712
Vibrio alginolyticus is a leading aquatic pathogen, causing huge losses to aquaculture. rpoS has been proven to play a variety of important roles in stress response and virulence in several bacteria. In our previous study, upon treatment with Cu2+, Pb2+, Hg2+ and low pH, the expression levels of rpoS were downregulated as assessed by RNA‐seq, while impaired adhesion ability was observed, indicating that rpoS might play roles in the regulation of adhesion. In the present study, the RNAi technology was used to knockdown rpoS in V. alginolyticus. In comparison with wild‐type V. alginolyticus, RNAi‐treated bacteria showed significantly impaired abilities of adhesion, growth, haemolytic, biofilm production, movement and virulence. Meanwhile, alterations of temperature, salinity, pH and starvation starkly affected rpoS expression. The present data suggested that rpoS is a critical regulator of virulence in V. alginolyticus; in addition, rpoS regulates bacterial adhesion in response to temperature, pH and nutrient content changes. These are helpful to explore its pathogenic mechanism and provide reference for disease control. 相似文献
20.
感染溶藻弧菌对日本蟳肝胰腺免疫功能的影响 总被引：1，自引：0，他引：1
摘 要：为探讨日本蟳感染溶藻弧菌的致病机理,采用溶藻弧菌人工感染健康日本蟳,分析了感染前后日本蟳肝胰腺 PO、SOD和LSZ活性变化及注射免疫多糖后的免疫保护率,并研究了细胞超微结构的病理组织学。结果表明：日本蟳感染溶藻弧菌后,肝胰腺中PO、SOD和 LSZ的活性均受到不同程度的影响,感染6~12 h,3种酶的活性均有一个上升的过程,但之后随时间的延长呈现下降趋势;免疫感染组因感染弧菌前注射了免疫多糖,日本蟳肝胰腺中PO、SOD和LSZ酶活性均显著高于感染组的酶活性,感染7 d后的免疫保护率高达72.73％,表明免疫多糖可提高酶的活性,使机体的抗病菌能力增强。超微结构显示：对照组日本蟳肝胰腺细胞结构形态正常,上皮细胞表面的微绒毛排列整齐,各细胞器结构完整;免疫组内质网、糖原颗粒和线粒体丰富,微绒毛排列致密;感染组肝胰腺组织受到明显的损伤,细胞和部分微绒毛脱落、受损;线粒体和粗面内质网变形、水肿或仅剩一空泡;核高度异染色质化,核膜破裂。与感染组相比,免疫后感染组具有形态结构较正常的细胞核、整齐致密的微绒毛和丰富的溶酶体,但依然可见水肿的内质网和狭长畸变的线粒体。 相似文献