Name: environmental screening of aeromonas hydrophila, mycobacterium spp., and pseudocapillaria tomentosa in zebrafish systems
Uploaded: 2017-12-08T12:30:00.000+00:00
Duration: 9 min 58 s
Description: Watch this Scientific Journal Video about Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems at JoVE.com

作者想感谢弗朗西斯克里克研究所的 BRF 游泳队的技术帮助和关键投入。这项工作得到了弗朗西斯克里克研究所的支持, 它获得了癌症研究英国 (FC001999)、英国医学研究理事会 (FC001999) 和惠康信托基金 (FC001999) 的核心资助。

1. 在循环系统中暴露预过滤哨兵
<ol><li>
设置一个清洁的8升坦克从一个循环系统。用坑的水来填满它。从系统中添加2个生物介质的陶瓷珠或海绵立方体 (图 1)。添加 1-2鱼/L (即, 12 条鱼)。使用野生型鱼类的优势遗传背景的设施, 例如 AB。
	<ol>
<li>选择至少6条鱼如下: 至少一个女性和一个男性在6月的年龄以下, 一女性和一男性在6和18月之间, 并且一女性和一男性在18月年龄之上。</li>
	</ol>
</li>
	<li>
每天喂一次。改变饮食 (如: 如, 干性饮食, 盐水虾), 以确保哨兵是暴露在所有的饮食使用的斑马鱼设施。更改水在星期一, 星期三, 星期五,即, 每周三次, 为期4月。
	<ol>
<li>进行水的变化, 转移哨兵和生物媒体到一个临时的坦克。清空完全的哨兵坦克和清洁它。只用水池水重新填充哨兵舱。把哨兵和生物媒体放回去</li>
	</ol>
</li>
	<li>把哨兵4月的时间暴露在水池里安乐通过一种经批准的方法, 如浸泡在 2-乙醇 (3 毫升/升) 过量溶液中的鱼。</li>
	<li>
确认死亡, 在停止鳃运动后等待10分钟。
	<ol>
<li>用钳子抓住尸体, 将其完全冻结在-80 ° c 的容器中。这将用于 PCR12,15。</li>
		<li>另外, 切下 tailat 的尾梗, 尼克腹壁, 并设置在4% 福尔马林。 注意: 使用手套和通风柜进行组织病理学。标记示例容器。</li>
	</ol>
</li>
</ol>2. 水池拭子
<ol><li>使用塑料轴的无菌干拭子。戴上手套找到要擦拭的表面 (在水面上的水池壁), 并移除任何防止容易接触到表面的物品。选择低流量的水槽表面。</li>
	<li>
拔出通过除去外包装, 将无菌棉签暴露在空气中。避免接触拭子的交叉污染, 注意不要触摸未经测试的表面。
	<ol>
<li>用拭子在 5-10 厘米的水池壁上吸收水和生物膜在水池水面水平。</li>
		<li>将拭子套回或将笔尖折断成无菌离心管。给样品贴上标签, 在-80 ° c 时进行 PCR 检测或冷冻。</li>
	</ol>
</li>
</ol>3. 在罐底检测毛白杨卵
<ol><li>
污泥的显微分析
	<ol>
<li>使用60毫升注射器9来吸入污水坑底部的污泥或任何包括哨兵在内的鱼罐。将样品分成15毫升的管子。用螺丝顶盖上管子, 并将管子贴上标签。</li>
		<li>通过在177毫升的热水中掺入227克砂糖, 用磁性搅拌器14来制备糖饱和溶液 (比重 = 1.27)。</li>
		<li>离心机的15毫升管在 175-250 x g 为10分钟的离心与摆动桶。醒酒管, 并保持在他们的管沉淀物。</li>
		<li>用糖饱和溶液将管子填满一半。关闭管与他们的螺丝顶部和彻底混合的沉淀物与解决方案。</li>
		<li>把管子放在离心机的摆动桶里, 用糖饱和溶液填满它们的顶端。在每根管子的顶端轻轻地设置一个盖子玻璃, 并与糖饱和溶液接触。</li>
		<li>离心机在 175-250 x g 10 分钟注意到, 一些盖玻璃可能在离心过程中脱落和断裂, 因此每个60毫升样品有4管。提起盖子玻璃并将其放在玻璃滑梯上。用铅笔或记号笔标记幻灯片。<ol>
<li>以防太多的盖子玻璃破损发生, 填补了大部分的管与糖饱和溶液, 离心机在 175-250 x g 10 分钟, 填补了与糖饱和溶液的顶部, 然后轻轻地设置盖玻璃。等待30分钟。</li>
		</ol>
</li>
		<li>使用显微镜 13 (图 2和视频 1) 查找毛的毛鸡蛋。在放大倍数 400X6中识别双极性插头。 注: 卵的大小为 57-78 µm 长和 27-39 µm 直径16,17。请注意, 一个积极的幻灯片是足够的60毫升样本被宣布为正数。</li>
	</ol>
</li>
	<li>
在检疫中筛选进口动物为毛白杨卵
	<ol>
<li>在坦克中设置男性和女性的D. 鱼。添加到坦克通常用于收获和保存产卵的设备, 但在这里使用它收集粪便 (图 3)。 注: 例如, 一个完整的1升养殖罐 (外罐和带磨碎底部的内胆) 完全浸没在一个13升的水箱中, 允许鱼自由进出繁殖装置。</li>
		<li>在一周后取出繁殖装置, 并在3.1 步 "显微镜下的污泥分析" 中所描述的繁殖装置中收获收集的污泥。</li>
	</ol>
</li>
</ol>4. 污泥沉积物的 PCR
<ol><li>用60毫升注射器9将容器底部的污泥吸吸, 并将样品转移到60毫升管。关闭管与它的螺丝顶部和标签的管。把注射器处理掉</li>
	<li>摇动60毫升管和转移15毫升到15毫升管。关闭管与它的螺丝顶部和标签的管。</li>
	<li>离心机的15毫升管在 175-250 x g 为10分钟的离心与摆动桶。醒酒管, 并保持在管道中的沉淀物。</li>
	<li>
拔出用棉签除去外包装, 将无菌的棉签暴露在空气中。避免接触拭子的交叉污染, 注意不要触摸未经测试的表面。
	<ol>
<li>在试管中拭去十五年代的沉淀物。</li>
		<li>将拭子套回或将笔尖折断成无菌离心管。标签的样本, 冻结在-80 ° c, 并发送 PCR 测试。 注:45 毫升保持在60毫升管。如步骤3.1 所述, 可以通过显微镜下的污泥分析来检测出毛白杨卵, 以确认 PCR 结果。在步骤3.2 之后, 可以尝试在污泥中筛选出进口动物的 PCR 方法。</li>
	</ol>
</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+Assessment+of+the+Efficacy+of+Five+Veterinary+Broad-Spectrum+Anthelmintics+to+Control+the+Intestinal+Capillariasis+in+Zebrafish+(Danio+rerio).">Experimental Assessment of the Efficacy of Five Veterinary Broad-Spectrum Anthelmintics to Control the Intestinal Capillariasis in Zebrafish (Danio rerio).</a> Zebrafish. 12 (3), 255-267 (2015).</li><li>Collymore, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+Efficacy+of+Emamectin+Benzoate+and+Ivermectin+for+the+Treatment+of+Pseudocapillaria+tomentosa+in+Laboratory+Zebrafish+(Danio+rerio).">Tolerance and Efficacy of Emamectin Benzoate and Ivermectin for the Treatment of Pseudocapillaria tomentosa in Laboratory Zebrafish (Danio rerio).</a> Zebrafish. 11 (5), 490-497 (2014).</li><li>Chang, C. T., Whipps, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+of+Antibiotics+against+Mycobacterium+Species+Commonly+Found+in+Laboratory+Zebrafish.">Activity of Antibiotics against Mycobacterium Species Commonly Found in Laboratory Zebrafish.</a> Journal of Aquatic Animal Health. 27 (2), 88-95 (2015).</li><li>Chang, C. T., Doerr, K. M., Whipps, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibiotic+treatment+of+zebrafish+mycobacteriosis:+tolerance+and+efficacy+of+treatments+with+tigecycline+and+clarithromycin.">Antibiotic treatment of zebrafish mycobacteriosis: tolerance and efficacy of treatments with tigecycline and clarithromycin.</a> J Fish Dis. , (2017).</li><li>Peterson, T. S., Ferguson, J. A., Watral, V. G., Mutoji, K. N., Ennis, D. G., Kent, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paramecium+caudatum+enhances+transmission+and+infectivity+of+Mycobacterium+marinum+and+M.+chelonae+in+zebrafish+Danio+rerio.">Paramecium caudatum enhances transmission and infectivity of Mycobacterium marinum and M. chelonae in zebrafish Danio rerio.</a> Dis Aquat Organ. 106 (3), 229-239 (2013).</li><li>Watts, S. A., Lawrence, C., Powell, M., D'Abramo, L. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Vital+Relationship+Between+Nutrition+and+Health+in+Zebrafish.">The Vital Relationship Between Nutrition and Health in Zebrafish.</a> Zebrafish. 13, 72-76 (2016).</li></ol>

作者没有什么可透露的。

技术、关键步骤和疑难解答的限制:
哨兵的年龄、性别、应变和暴露时间不规范。这在表 1中显示。在6月龄以下的鱼类或老鱼的筛查很少。可能有一些病原体影响幼鱼, 因为有一些病原体是较常见的在老年人口10,18,19,20。同样, 在某些哨兵组的选择中也没有考虑到性别问题, 尽管有些报告说某些病原体存在性别偏见21。建议的技术试图解决这些问题, 虽然应变的选择可以根据特定的病原体进行监测。例如, TU 可以帮助检测分枝杆菌,12,22, 但有一个风险, 哨兵将作为一个水库或显示临床症状。关于曝光时间的长短, 斑马鱼国际资源中心的做法10增加了检测病原体的几率, 而这些病菌在污染期内可能会被遗漏。长期暴露的需要意味着哨兵不容易获得。添加的环境样品允许一些灵活性和增殖的筛选事件。例如, 取样可以每隔一个月进行一次, 每次筛选方法间隔4月。这可能会减少在新引入的病原体被检测之前的时间的流逝。
环境筛选技术依赖于对环境中病原体的检测。病原体由鱼流下, 因此在系统水中稀释。没有探讨通过水过滤来捕获病原体的可能性23 。我们所描述的方法只有当病原体有足够的时间在鱼和生物膜上繁殖以达到允许检测的污染阈值时才有效。通过对取样点的关键选择, 将这种技术的限制降到最低限度: 对罐内的污泥进行取样, 而不是在废油坑污泥中取样, 而水和生物膜则是在污水坑的表面采样, 而不是在罐内或后过滤。尽管如此, 同一系统中的所有样本都不可能提供相同的结果。使用另一种方法 (组织病理学、PCR 或污泥分析) 可以证实对毛白杨的阳性结果。杆菌 PCR 阳性结果可以通过培养或其他诊断实验室确认。然而, 在建立健康状况时, 建议进一步的样本, 以确认任何环境筛选技术的负面结果。
技术在现有/替代方法方面的重要性:
分枝杆菌在环境中是常见的, 它们在污水坑中的存在并不能预测其致病性12。马可9表明, 监测死亡率是调查健康问题发展的关键。动物标本的使用对任何兽医调查都是必不可少的。健康监测意味着在一个设施中检测所有流行的病原体, 而这不能仅仅通过环境筛选技术来实现。然而, 缺乏灵敏度的诊断工具可以延缓或防止准确描述健康状况。虽然哨兵的使用减少了检测在人群中流行的微生物所需的鱼的数量, 但是缺乏敏感性增加了使用多种方法的重量, 包括环境筛选5,23。事实上, 特定的病原体自由状态通常被定义为在设施中缺少一个物种, 这样环境和动物样本必须测试负的24,25。
该水池拭子的结果, 以确定分枝杆菌, 表明, 依赖于鱼类样本可能会导致一个虚假的负面健康状况。6试验过的杆菌种被描述为斑马鱼的致病或潜在致病性15 , 有些不会被鸡蛋表面消毒, 氯26在检疫中例行执行。因此, 伪负可能会对导入行的协作者产生一定的影响。例如, m 杆菌在鱼样本上被 pcr 遗漏, 但超过一半的废油坑棉签 pcr 检测到它。考虑到这些分枝杆菌对氯的耐药性比其他人多, 而且它们在水系统中的生长能力27, 这是不受污染的进口设施的风险。为了允许导入行, 经理需要信任和比较出口设施的健康报告与他们的。委员会性能评估程序28是啮齿类动物过程中的关键。RESAMA 网络报告法语D. 鱼11中的m gordonae和m mucogenicum的检测。这些分枝杆菌不是在我们使用的商业实验室的面板中提出的。这将是有用的扩展委员会程序和统一的诊断化验以及致病物种名单29。
水气也是一种病原体, 在导入动物时有可能被引入, 尽管它对氯气的敏感度30使其在常规的蛋表面消毒过程中更容易消除。水池, 棉签和污泥的结果表明, 环境筛选可以用来检测这种病原体。其他细菌如分枝杆菌在污泥中被检测到 PCR23。这种类型的样品是特别相关的, 因为它可以检测棚病原体。例如, 另一个新的应用是污泥分析筛检进口鱼在检疫为P. 毛白杨。寄生虫是威胁动物的健康13和肿瘤模型16。此外, 在常规斑马鱼蛋表面消毒中使用的氯浓度不是有效的31。因此, 以一周的营业额和没有任何鱼类安乐死来筛选进口动物的能力似乎很有吸引力。这种技术可以通过允许进口的分流来影响检疫和生物安全规则。然后根据出口设施中流行的病原体、进口鱼样本中检测到的病原体以及危害进口设施的健康状况的风险, 设计了一个决策过程10。
掌握这些技术后的未来应用或方向:
即使是常规的检疫治疗是选择的选择, 这种药物的功效32,33,34,35,36可以用育种装置的污泥分析评估。更普遍地, 环境筛选可以被用于测试化合物反对细菌和寄生虫根除, 包括在鱼群落生境。环境筛选的另一个利基应用是监测活饲料中的病原体的数量37,38。虽然这些技术的主要应用是作为一个宝贵的除了诊断工具箱的健康监测在斑马鱼设施。由于更准确, 成本和时间的有效定义的健康状况, 废油坑棉签和污泥分析是互补的哨兵监测和例行检疫的做法。事实上, 这些技术的未来将成为任何水生实验室健康报告的例行部分。

为了保护研究和动物福利1,2, 在动物设施内监测病原体的存在。在斑马鱼, 健康监测3,4,5,6,7,8,9,10,11通常依赖于分析的动物尸检后的经病理组织学, 细菌培养, 或分子方法。仅检测菌落动物不是推荐的方法, 因为鱼的数量和相关的费用将被要求检测低发病率的病原体。因此, 首选的方法是将一小群动物暴露在更高的污染物负荷中。这些鱼被称为预过滤哨兵。这种暴露持续了几个月, 它涉及增加动物看护人的工作量和/或一些专门建造的工程解决方案。另一项挑战是, 在检疫中筛选出可育的动物要保持活力的进口线, 这与对胴体的常规化验不相容。
我们在这里描述了一些方法来检测某些斑马鱼病原体 (水气, 分枝杆菌, 和毛白杨) 通过筛选水生系统的环境。目的是减少用于健康监测的鱼的数量, 并优化检测的营业额、成本和灵敏度。这种方法是一种替代动物的使用, 一些技术可以用于甄别进口的检疫。例如, 马可9能够通过在水池拭子上进行 PCR 而不是斑马鱼 (包括哨兵) 来识别更多的致病杆菌物种, 这是以较少的样本获得的。在同一研究中, 利用浮选和显微显微镜对坦克污泥进行筛选, 而不是通过 PCR 和组织病理学检测鱼, 来检测出更敏感的毛白杨卵。
表 1概述了哨兵程序的各种特征345,6789用于许多斑马鱼设施。后过滤哨兵接收水的方式与任何殖民地的鱼一样, 而预过滤哨兵接收水一旦它已经通过殖民地的鱼缸首先。例如, 可以在循环系统上设置预过滤哨兵, 不断接收污水坑水。这可能不是一个选项, 当有许多独立的系统在一个房间。在这种情况下, 一罐预过滤哨兵可以用来屏蔽整个房间。哨兵是在一个静态的坦克, 出循环系统, 他们的水是定期改变, 只使用预过滤水即, 从所有的系统在房间的污水。该技术被描述为与环境筛选效果比较的基线。该建议的设置是为了控制水质问题, 如降低 pH 值或氮污染。
细菌环境筛选的概念依赖于这样的假说, 即细菌在生物膜中被发现, 如在水表面的污水坑壁上或在罐底的污泥中找到。在循环养殖系统中, 水池似乎是一个理想的取样点, 因为它从所有的罐前过滤器收集废物 (水、粪便、饲料和其他有机物)。水池的表面经常容易地是可到达的, 抽汲是快速的, 并且它可以执行菌避免样品的交叉污秽 (从手套例如)。该概念用于鉴别斑马鱼系统中常见的致病性分枝杆菌9,12。下面介绍了该技术, 我们还报告了斑马鱼水槽表面拭子和污泥中的水气检测。
对寄生虫卵进行环境筛选是基于默里et al.的检测13和浮选技术是常规用于寄生虫和粪便中寄生虫卵的显微筛选14。马可9提出了一种替代取样过程的方法, 并表明该技术可用于检测鱼类群落生境的其他种类。感染的D. 鱼通过P. 毛白杨将它们的粪便和虫卵留在池底的污泥中。它们可以在那里收集, 因为它们的密度大于水。鸡蛋的密度也用来处理环境样品。第一次浮选与离心分离水和轻的残骸从重的物质。第二次离心依赖于饱和糖溶液 (密度大于密度的毛白杨卵), 允许寄生虫卵出现在试管的表面。
通过对第一次离心后获得的污泥样品进行 PCR, 对生物膜中的细菌和罐底的毛白杨进行筛选。这优化了取样时间。该方法如下所述。我们还建议在隔离环境中使用这些技术。为了屏蔽进口的成年斑马鱼 , 需要保持活着 , 一个育种装置入到检疫坦克。一周后, 在育种装置中的粪便和其他废弃物的收集和筛选通过显微镜或 PCR。下面介绍了该技术, 并在这方面显微镜检测到一些毛白杨卵。

<table><tbody><tr><td>Aqua-Sed 250 mL</td><td>Vetark</td><td></td><td>2-phenoxyethanol</td></tr><tr><td>Tubed Sterile Dryswab Tip</td><td>mwe</td><td>MW100</td><td>Sump surface</td></tr><tr><td>BD Plastipak Disposable Syringe 50mL Eccentric</td><td>Becton&lt;br/&gt; Dickinson</td><td>300866</td><td>They are actually&lt;br/&gt; graduated to 60 ml</td></tr><tr><td>Centrifuge tube 15 mL Corning</td><td>Corning</td><td>430766</td><td></td></tr><tr><td>Centaur 2 benchtop centrifuge with 4 x 200 mL Swing&amp;ndash;Out Rotor (unsealed)</td><td>Sanyo</td><td>MSB020.CX1.5</td><td></td></tr><tr><td>Cover Glass 22 mm x22 mm</td><td>Menzel-Glaser</td><td>MNJ-350-020H</td><td></td></tr><tr><td>Plain Swab Sterile Plastic Applicator Rayon Tipped White Cap</td><td>Sterilin Ltd Thermo Fisher Scientific</td><td>F155CA</td><td>Swab sediment from sludge</td></tr><tr><td>50 mL Self-Standing Centrifuge Tube CentriStar Cap</td><td>Corning</td><td>430921</td><td></td></tr><tr><td>In-Tank Spawning Tray Set</td><td>MBK Installations Ltd</td><td></td><td></td></tr></tbody></table>

environmental screening of aeromonas hydrophila, mycobacterium spp., and pseudocapillaria tomentosa in zebrafish systems

健康监测系统是开发和使用在斑马鱼的研究设施, 因为病原体的斑马鱼, 如单胞菌水气, 分枝杆菌, 和Pseudocapillaria 毛白杨有潜力损害动物福利和研究。鱼通常被分析为检测微生物的宰后。使用哨兵是一个建议的方式, 以提高灵敏度的监测和减少动物数量的样本。介绍了一种用于循环系统的预过滤哨兵箱的设置。该技术的发展是为了防止水污染, 并通过仔细选择年龄、性别和菌株来代表鱼类种群。为了使用最小的动物数量, 屏幕环境的技术也详细。聚合酶链反应 (PCR) 在表面废油坑拭子是用来显着改善检测一些流行和致病杆菌物种, 如分枝杆菌杆菌, 分枝杆菌 haemophilum, 和龟分枝杆菌。另一种环境方法是处理在贮水池底部的污泥, 以寻找毛白杨卵。这是一种廉价和快速的技术, 可以应用在检疫的饲养设备被淹没在进口动物的控股罐。最后, 将 PCR 技术应用于污泥样品, 并在底池和表面检测到水气。一般来说, 这些特定的病原体的环境筛选技术, 比起前过滤哨兵的测试, 更敏感。

在图 4中的结果支持与鱼样相比, 废油坑拭子的优势, 以确定流行的分枝杆菌。在测试的115条鱼中, 分别在5% 和3% 中检测到了m 龟和m haemophilum 。没有发现其他致病的杆菌种。从同一系统中, 49 个水池拭子发现有5杆菌的物种存在。赔率比是计算的假设, m 龟和m. 杆菌是检测更频繁的 PCR 在表面水池拭子比在鱼样本。这是统计学上的显著性与各自的赔率比率 11 (95% CI: 4 至 29;p < 0.0001) 和 306 (95% CI:18 至 5208;p = 0.0001)。结果表明, 表面底壳拭子技术是一个有价值的替代方法的唯一使用哨兵筛选斑马鱼设施的分枝杆菌。环境示例还用于筛选水气。这些细菌是在鱼、污泥和表面样品中检测到的 (图 4)。这也支持建议的技术的能力筛选鱼群落生境。
关于污泥分析检测到的毛白杨卵, 马可9通过 PCR 和组织病理学检测了27% 条鱼样本中的寄生虫, 而在93% 的同一系统中检测出的污泥中发现了虫卵。在这里, 该技术被挑战, 以重现检疫检查。在这种情况下, 不能对进口的动物进行取样, 及时评估其健康状况有助于生物安全管理。从P. 毛白杨阳性装置中的未知健康状态的鱼在8罐中设置了繁殖装置: 最大 16 fish/13 L 罐, 7 转基因和1野生型线, 混合性别, 年龄 4-24 月。一周后从装置中收获污泥, 用显微镜进行分析。在 7 (88%) 样品中观察到了毛的卵. 最后, 对该寄生虫的 PCR 检测试用在底泥和污泥沉积物上。4 6 污泥样品中均为 PCR 阳性, 表面拭子均为阴性。这并不奇怪, 因为污泥筛选依赖于鸡蛋的能力下降到底部的坦克。这表明, 污泥分析技术可用于筛选鱼水族馆的毛白杨害虫, 这些方法可用于筛选进口动物。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55306/55306fig1.jpg"> 图 1: 从循环系统中取出预过滤哨兵箱.一个8升坦克充满了水和生物介质从系统的坑的屏幕。两个白色陶瓷生物媒体珠坐在底部的坦克 (在图片中间)。根据年龄、品系和性别选择12条鱼, 并将其添加到哨兵池中。<a href="//cloudflare.jove.com/files/ftp_upload/55306/55306fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55306/55306fig2.jpg"> 图 2: 在污泥分析过程中通过显微镜检测到的毛. 使用的放大倍数是400X。箭头表示双极性插头。缩放栏 = 50 µm.<a href="//cloudflare.jove.com/files/ftp_upload/55306/55306fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55306/55306fig3.jpg"> 图 3: 在鱼缸中浸泡的养殖装置收集污泥进行分析.这个坦克是设置在一个长凳上的图片的目的;它在循环系统否则被设置。<a href="//cloudflare.jove.com/files/ftp_upload/55306/55306fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55306/55306fig4.jpg"> 图 4: 在鱼、表面水池拭子和废油坑污泥中, 用 PCR 方法对分枝杆菌、水气和毛白杨进行鉴定的百分比.百分比是通过将每种病原体的阳性结果除以被试样品的数量来获得的。阳性结果的百分比由样本类型作为鱼、面或污泥, 并在细菌的名称后表示。用 PCR 法检测了115条鱼和49种表面底池拭子, 鉴定了分枝杆菌。数据是用马可的9结果编译的, 因为它是本研究的扩展。根据协议1条, 鱼类主要是预过滤哨兵。很少, 当哨兵不是可利用的, 逃脱者和老殖民地鱼 (> 18 月) 被抽样了。对分枝杆菌的所有测试系统都在鱼和油槽拭子上进行了测试。赔率比是计算的假设, m 龟和m. 杆菌是检测更频繁的 PCR 在表面水池拭子比在鱼样本。这是统计学上的显著性与各自的赔率比率 11 (95% CI: 4 至 29;p < 0.0001) 和 306 (95% CI:18 至 5208;p = 0.0001)。marinum 分枝杆菌PCR 是阴性的所有样品, 因此它被认为没有从这些设施和不包括在分析。在水气的存在下, 用 PCR 方法检测了12条鱼、14表面水池拭子和 6个废油坑污泥。对6坑在表面和污泥中的存在进行了测试. 聚合酶链反应。CI = 置信区间。* 和 ** 表示统计学意义;其他比较没有统计学意义。<a href="//cloudflare.jove.com/files/ftp_upload/55306/55306fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Video 1" src="/files/ftp_upload/55306/55306vid1.jpg"> 视频 1: 扫描一个鸡蛋的毛白杨与显微镜.滑块是从污泥分析中获得的, 如步骤3.1 所述。将扫描该字段以检测P. 毛白杨的卵。一旦结构被识别, 它将被放大以在更高的分辨率下确认识别。<a href="http://cloudflare.jove.com/files/ftp_upload/55306/video_scan_for_egg.avi" target="_blank">请单击此处查看此视频。(右键单击可下载.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>作者</td>
			<td>接触开始时的年龄</td>
			<td>曝光长度</td>
			<td>取样年龄</td>
			<td>前或后 过滤</td>
			<td>性别</td>
			<td>应变 (s)</td>
		</tr>
<tr>
<td>巴顿et al.3
</td>
			<td>4月</td>
			<td>6月</td>
			<td>10月</td>
			<td>预过滤</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>博尔et al.4
</td>
			<td>6月</td>
			<td>6月</td>
			<td>12月</td>
			<td>预过滤</td>
			<td>N/A</td>
			<td>AB 野生型</td>
		</tr>
<tr>
<td>弗兰克·科利莫尔et al.5
</td>
			<td>尽可能年轻</td>
			<td>3月</td>
			<td>< 6 月</td>
			<td>预过滤</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>格斯勒et al.6
</td>
			<td>4月</td>
			<td>4月</td>
			<td>8月</td>
			<td>前和后 过滤</td>
			<td>N/A</td>
			<td>AB 野生型</td>
		</tr>
<tr>
<td>刘et al.7
</td>
			<td>3月</td>
			<td>1-6 月</td>
			<td>4-9 月</td>
			<td>前和后 过滤</td>
			<td>N/A</td>
			<td>野生型</td>
		</tr>
<tr>
<td>马丁斯et al.8
</td>
			<td>3月</td>
			<td>3月</td>
			<td>6月</td>
			<td>预过滤</td>
			<td>N/A</td>
			<td>野生型</td>
		</tr>
<tr>
<td>马可9
</td>
			<td>< 6 月, 6-12 月, > 18 月</td>
			<td>4月</td>
			<td>7-24 月</td>
			<td>预过滤</td>
			<td>1女性和 1男性 每个年龄组</td>
			<td>AB 野生型</td>
		</tr>
<tr>
<td>默里et al.10
</td>
			<td>3-4 月</td>
			<td>3月, 6 月, 1 年</td>
			<td>7、10和16 月</td>
			<td>前和后 过滤</td>
			<td>N/A</td>
			<td>AB 野生型</td>
		</tr>
</tbody></table>表 1: 斑马鱼设施中哨兵设置的比较.哨兵鱼可根据年龄、性别或应变选择。他们被暴露在一个明确的时期, 他们接收预先或后过滤系统的水。这些数据是从2016的斑马鱼杂志健康特刊上编译的3,4,5,678910。

Watch this Scientific Journal Video about Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems at JoVE.com

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

本议定书描述了使用水槽拭子和对斑马鱼系统的污泥分析, 这导致检测比唯一使用哨兵检测病原体, 如单胞菌水气, 分枝杆菌, 和Pseudocapillaria 毛白杨。此外, 还提出了一种在检疫中监测毛白杨卵的系统。

斑马鱼系统中水气单胞菌、分枝杆菌和Pseudocapillaria 毛白杨的环境筛选

Health monitoring systems are developed and used in zebrafish research facilities because pathogens of Danio rerio such as Aeromonas hydrophila, ...

environmental-screening-aeromonas-hydrophila-mycobacterium-spp

Nanyang Technological University

Research

JoVE Journal

Biology

Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

9.7K 次观看.  The Francis Crick Institute. 本议定书描述了使用水槽拭子和对斑马鱼系统的污泥分析, 这导致检测比唯一使用哨兵检测病原体, 如单胞菌水气, 分枝杆菌, 和Pseudocapillaria 毛白杨。此外, 还提出了一种在检疫中监测毛白杨卵的系统。

视频: Environmental Screening of Aeromonas hydrophila, Mycobacterium spp., and Pseudocapillaria tomentosa in Zebrafish Systems

Experimental Protocol for Examining Behavioral Response Profiles in Larval Fish: Application to the Neuro-stimulant Caffeine

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and toxicological studies. Using automated digital tracking platforms, recent efforts in neuropharmacology are leveraging larval fish locomotor behaviors to identify potential therapeutic targets for novel small molecules. Similar to these efforts, research in the environmental sciences and comparative pharmacology and toxicology is examining various behaviors of fish models as diagnostic tools in tiered evaluation of contaminants and real-time monitoring of surface waters for contaminant threats. Whereas the zebrafish is a popular larval fish model in the biomedical sciences, the fathead minnow is a common larval fish model in ecotoxicology. Unfortunately, fathead minnow larvae have received considerably less attention in behavioral studies. Here, we develop and demonstrate a behavioral profile protocol using caffeine as a model neurostimulant. Though photomotor responses of fathead minnows were occasionally affected by caffeine, zebrafish&#160;were markedly more sensitive for photomotor and locomotor endpoints, which responded at environmentally relevant levels. Future studies are needed to understand comparative behavioral sensitivity differences among fish with age and time of day, and to determine whether similar behavioral effects would occur in nature and be indicative of adverse outcomes at the individual or population levels of biological organization.

Here, we present a protocol to examine larval zebrafish and fathead minnow locomotor activities and photomotor responses (PMR) using an automated tracking software. When incorporated in common toxicology bioassays, analyses of these behaviors provide a diagnostic tool to examine chemical bioactivity. This protocol is described using caffeine, a model neurostimulant.

检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

Fish models and behaviors are increasingly used in the biomedical sciences; however, fish have long been the subject of ecological, physiological and ...

科研

环境科学

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

播散性念珠菌在斑马鱼幼虫的非侵入性成像

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

免疫与感染

Studying Neurobehavioral Effects of Environmental Pollutants on Zebrafish Larvae

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae are a preeminent model for the neurobehavioral study of environmental pollutants. Here, a detailed experimental protocol is provided for the evaluation of the neurotoxicity of environmental pollutants using zebrafish larvae, including the collection of the embryos, the exposure process, neurobehavioral indicators, the test process, and data analysis. Also, the culture environment, exposure process, and experimental conditions are discussed to ensure the success of the assay. The protocol has been used in the development of psychopathic drugs, research on environmental neurotoxic pollutants, and can be optimized to make corresponding studies or be helpful for mechanistic studies. The protocol demonstrates a clear operation process for studying neurobehavioral effects on zebrafish larvae and can reveal the effects of various neurotoxic substances or pollutants.

A detailed experimental protocol is presented in this paper for the evaluation of neurobehavioral toxicity of environmental pollutants using a zebrafish larvae model, including the exposure process and tests for neurobehavioral indicators.

环境污染物对斑马鱼幼虫的神经行为影响研究

Recent years more and more environmental pollutants have been proved neurotoxic, especially at the early development stages of organisms. Zebrafish larvae ...

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions.&#160;The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model.&#160;In this respect, the present manuscript describes the use of zebrafish as an&#160;in vivo&#160;model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate.

Deciphering and Imaging Pathogenesis and Cording of Mycobacterium abscessus in Zebrafish Embryos

Optically transparent zebrafish embryos are widely used to study and visualize in real time the interactions between pathogenic microorganisms and the innate immune cells. Micro-injection of Mycobacterium abscessus, combined with fluorescence imaging, is used to scrutinize essential pathogenic features such as cord formation in zebrafish embryos.

破译和成像发病机制与盘带<em&gt;脓肿分枝杆菌</em&gt;斑马鱼胚胎

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen ...

Modeling Fetal Alcohol Spectrum Disorders in Zebrafish to Characterize the Impact of an Adverse Embryonic Environment on Adult Social Behavior

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, and cognitive abnormalities are associated with FASD. Social difficulties are common behavioral abnormalities associated with FASD and often result in serious health issues. Animal models are critical to understanding the mechanisms responsible for ethanol-induced social defects. Zebrafish are social vertebrates that produce externally fertilized transparent eggs; these characteristics provide researchers with a precise yet simple procedure for creating the FASD phenotype and an innate behavior that can be leveraged to model the social deficits associated with FASD. Thus, zebrafish are ideal for characterizing the social deficits of FASD. The goal of the current protocol is to provide the user with a simple behavioral assay that can be used to characterize the consequences of a negative environment early during development and the effects it can have on social behavior in adulthood. The protocol can be used to characterize the effect mutations or teratogens have on adult social behavior. The protocol outlined here demonstrates how to characterize the social behavior of individual fish during a 20-min social assay. Furthermore, the data obtained using the current protocol provides evidence that the protocol can be used to characterize the effects of embryonic ethanol-induced social defects in adult zebrafish.

<meta charset="utf8"></head><body>对斑马鱼的胎儿酒精谱系障碍进行建模，以表征不良胚胎环境对成人社交行为的影响

The goal of the current protocol is to outline the steps necessary to establish and use a social preference assay for adult zebrafish and demonstrate that it can be used to characterize ethanol-induced social defects.

对斑马鱼的胎儿酒精谱系障碍进行建模，以表征不良胚胎环境对成人社交行为的影响

Fetal alcohol spectrum disorders (FASD) describe all alcohol-induced birth defects. Birth defects such as growth deficiencies, craniofacial, behavioral, ...

行为学

Boldness, Aggression, and Shoaling Assays for Zebrafish Behavioral Syndromes

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is important to standardize protocols to ensure proper analyses and interpretations. In our previous studies, we have measured boldness by monitoring a series of behaviors (time near surface, latency in transitions, number of transitions, and darts) in a 1.5 L trapezoidal tank. Likewise, we quantified aggression by observing bites, lateral displays, darts, and time near an inclined mirror in a rectangular 19 L tank. By dividing a 76 L tank into thirds, we also examined shoaling preferences. The shoaling assay is a highly customizable assay and can be tailored for specific hypotheses. However, protocols for this assay also must be standardized, yet flexible enough for customization. In previous studies, end chambers were either empty, contained 5 or 10 zebrafish, or 5 pearl danios (D. albolineatus). In the following manuscript, we present a detailed protocol and representative data that accompany successful applications of the protocol, which will allow for replication of behavioral syndrome experiments.

This manuscript describes the setup, implementation, and analysis of boldness, aggression, and shoaling in zebrafish and testing for the presence of a behavioral syndrome. A standardized approach for behavioral quantification will allow for easier comparison across studies. Modifications to this protocol are possible as each assay can be run individually.

气魄，侵略和浅水测定斑马鱼行为综合征

A behavioral syndrome exists when specific behaviors interact under different contexts. Zebrafish have been test subjects in recent studies and it is ...

医学

Rapid Evaluation of Toxicity of Chemical Compounds Using Zebrafish Embryos

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has transparent embryos and rapid external development. Zebrafish embryos can, therefore, also be used for the rapid evaluation of toxicity of the drugs that are precious and available in small quantities. In the present article, a method for the efficient screening of the toxicity of chemical compounds using 1-5-day post fertilization embryos is described. The embryos are monitored by stereomicroscope to investigate the phenotypic defects caused by the exposure to different concentrations of compounds. Half-maximal lethal concentrations (LC50) of the compounds are also determined. The present study required 3-6 mg of an inhibitor compound, and the whole experiment takes about 8-10 h to be completed by an individual in a laboratory having basic facilities. The current protocol is suitable for testing any compound to identify intolerable toxic or off-target effects of the compound in the early phase of drug discovery and to detect subtle toxic effects that may be missed in the cell culture or other animal models. The method reduces procedural delays and costs of drug development.

Zebrafish embryos are used for evaluating the toxicity of chemical compounds. They develop externally and are sensitive to chemicals, allowing detection of subtle phenotypic changes. The experiment only requires a small amount of compound, which is directly added to the plate containing embryos, making the testing system efficient and cost-effective.

利用斑马鱼胚胎对化合物毒性的快速评价

The zebrafish is a widely used vertebrate model organism for the disease and phenotype-based drug discovery. The zebrafish generates many offspring, has ...

行为的：评估成年斑马鱼神经毒性作用的测试电池

Neurotoxicity Assessment in Adult Danio rerio using a Battery of Behavioral Tests in a Single Tank

The presence of neuropathological effects proved to be, for many years, the main endpoint for assessing the neurotoxicity of a chemical substance. However, in the last 50 years, the effects of chemicals on the behavior of model species have been actively investigated. Progressively, behavioral endpoints were incorporated into neurotoxicological screening protocols, and these functional outcomes are now routinely used to identify and determine the potential neurotoxicity of chemicals. Behavioral assays in adult zebrafish provide a standardized and reliable means to study a wide range of behaviors, including anxiety, social interaction, learning, memory, and addiction. Behavioral assays in adult zebrafish typically involve placing the fish in an experimental arena and recording and analyzing their behavior using video tracking software. Fish can be exposed to various stimuli, and their behavior can be quantified using a variety of metrics. The novel tank test is one of the most accepted and widely used tests to study anxiety-like behavior in fish. The shoaling and social preference tests are useful in studying the social behavior of zebrafish. This assay is particularly interesting since the behavior of the entire shoal is studied. These assays have proven to be highly reproducible and sensitive to pharmacological and genetic manipulations, making them valuable tools for studying the neural circuits and molecular mechanisms underlying behavior. Additionally, these assays can be used in drug screening to identify compounds that may be potential modulators of behavior.
We will show in this work how to apply behavioral tools in fish neurotoxicology, analyzing the effect of methamphetamine, a recreational drug, and glyphosate, an environmental pollutant. The results demonstrate the significant contribution of behavioral assays in adult zebrafish to the understanding of the neurotoxicological effects of environmental pollutants and drugs, in addition to providing insights into the molecular mechanisms that may alter neuronal function.

作者聚焦：成年斑马鱼环境和药物诱导的行为变化评估 

Here, we present a comprehensive behavioral test battery, including the novel tank, Shoaling, and social preference tests, to effectively determine the potential neurotoxic effects of chemicals (e.g., methamphetamine and glyphosate) on adult zebrafish using a single tank. This method is relevant to neurotoxicity and environmental research.

斑马鱼系统中水气单胞菌、分枝杆菌和Pseudocapillaria 毛白杨的环境筛选

摘要

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检测幼虫行为反应剖面的实验协议: 在神经刺激咖啡因中的应用

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破译和成像发病机制与盘带

对斑马鱼的胎儿酒精谱系障碍进行建模，以表征不良胚胎环境对成人社交行为的影响

对斑马鱼的胎儿酒精谱系障碍进行建模，以表征不良胚胎环境对成人社交行为的影响

气魄，侵略和浅水测定斑马鱼行为综合征

利用斑马鱼胚胎对化合物毒性的快速评价

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估

在单个水箱中使用一系列行为测试对成年 Danio rerio 进行神经毒性评估