多亏了旋律尼利, 乔恩艾伦, 巴塞尔 Abuaita, 唐娜 Runft, 他们努力帮助开发斑马鱼模型。这项研究报告得到了国家卫生研究院的国家过敏和传染性疾病研究所的支持, R21AI095520 和 R01AI127390 (对杰弗里 h. Withey) 的奖励数字。内容完全是作者的责任, 不一定代表国家卫生研究院的官方意见。

这里描述的所有方法都得到了韦恩州立大学机构动物护理和使用委员会 (IACUC) 的批准。该方法首次在 Runft等中被描述。12
1. 肠道定植水平的测定
注: 肠道定植是斑马鱼模型中最有用的指标, 因为它可以用来比较各种v 型霍乱菌株的相对适用性或突变或基因挖空的影响。
<ol><li>利用浸泡法接种斑马鱼 注意: 这种接触方式类似于自然感染过程。<ol>
<li>霍乱弧菌的制备
		<ol>
<li>接种5毫升的溶源性汤 (磅) 与一个孤立的v 型霍乱菌群从 LB 板和孵化它在37°c 与震动 (180 rpm) 为 6–8 h (隔夜文化)。</li>
			<li>在250毫升锥形烧瓶中接种25毫升的新鲜蒸压 LB 培养基, 上面有50µL 的隔夜养殖。孵化它为 16–18 h 在37°c 与震动 (150 转每分钟)。</li>
			<li>阅读的光学密度在 600 nm (od600) 的一夜文化的1/10 稀释, 以估计细菌的数量每毫升 (OD600 = 1 是 ~ 109 CFU/毫升.)</li>
			<li>以6000克的离心方式收割细胞, 10 分钟. 用吸管取出介质或将其倒入消毒剂溶液中。并用重悬细胞在无菌 PBS 到期望集中, 通常在 1 x 107到 1 x 1010每毫升 PBS。 注: 在这里, 我们提到的蒸压反渗透水含有60毫克/升海盐作为无菌感染水。</li>
		</ol>
</li>
		<li>
接种和孵化
		<ol>
<li>将四或五斑马鱼放入400毫升烧杯中, 含200毫升无菌感染水。</li>
			<li>添加1毫升霍乱弧菌(从步骤 1.1.1.4) 获得所需的感染浓度 (通常为 5 x 104到 5 x 107 CFU/毫升) 在200毫升的感染水。</li>
			<li>用穿孔盖子盖上烧杯, 防止鱼跳出来;200µL 提示框的顶部工作正常。</li>
			<li>标签每个烧杯, 并把它们放到一个玻璃前孵化器设置在28摄氏度的试验期间。</li>
		</ol>
</li>
		<li>
暂时曝光程序 注: 暂时暴露于霍乱弧菌(通常为6小时) 之后, 除去接种细菌对研究传播和更准确地量化排泄细菌是有用的。<ol>
<li>经过6小时的曝光后, 通过渔网将烧杯中的水倒入收集鱼。将受感染的水丢弃到漂白容器中, 以杀死霍乱弧菌。</li>
			<li>将被去除的鱼放在200毫升无菌感染水的烧杯中。允许鱼在这干净的水中游泳5分钟, 以去除鱼表面的细菌。</li>
			<li>重复网络过程 (步骤 1.2.2), 并把鱼放在一个新的烧杯200毫升无菌感染水。在实验期间把鱼放在这个烧杯里。</li>
		</ol>
</li>
	</ol></li>
	<li>安乐 死
	<ol>
<li>当实验达到其预期的终点时, 取一个样本的感染水 (15 毫升通常是足够的), 以允许排泄细菌计数和测量腹泻 (如粘蛋白化验, 蛋白质含量和/或 OD), 如果需要 (2 节)。</li>
		<li>将剩余的水通过渔网 (收集鱼) 倒入漂白剂中, 以杀灭水中的霍乱弧菌。</li>
		<li>将鱼放入含有感染水的烧杯中, 再加336µg/毫升 3-氨基苯甲酸甲基磺酸乙酯 (tricaine), 并在该 tricaine 溶液中孵化出20分钟的鱼。 注: 罟用10% 漂白剂溶液消毒。</li>
	</ol></li>
	<li>夹层
	<ol>
<li>鱼的准备<ol>
<li>用一次性塑料勺子舀出 tricaine 溶液中的鱼, 将其放在解剖表面。</li>
			<li>将鱼与腹侧朝上, 将其钉在下颌上, 用针的钝端从身体中心处倾斜。把另一根针放在肛门后部, 也可以从身体角度离开。</li>
		</ol>
</li>
		<li>肠道暴露<ol>
<li>用70% 乙醇蘸上无绒布的擦拭, 拭去鱼腹面。</li>
			<li>用70% 乙醇蘸一把手术刀和 Vannas 剪刀, 然后将它们燃烧起来。</li>
			<li>通过穿透鳞片和使用手术刀, 在皮肤下面纵向切开一个小切口。小心不要切得太深, 因为肠道位于皮肤下面。</li>
			<li>使用剪刀, 沿着身体的长度仔细地伸展切口, 没有比皮肤更深的切割和避免肛门。</li>
			<li>用剪刀对鱼头进行两次横向切割, 使切口开口。</li>
			<li>将侧切口两侧的皮肤固定在解剖表面, 将针脚从身体上钓出来。 注意: 别针的尖端以前是通过酒精燃烧而消毒的。</li>
		</ol>
</li>
		<li>
肠道切除 注意: 肠道应该是可见的, 在其他器官之上的苍白, 非常薄的管。<ol>
<li>用火焰消毒镊子, 然后用它们去除整个肠道 (通常12–15毫米)。将肠道放入含有玻璃珠的均匀化管 (见步骤 1.4. 1–1.4 2) 和1毫升 1x PBS 或磅, 在冰上。</li>
		</ol>
</li>
	</ol></li>
	<li>均匀
	<ol>
<li>先将1.5 克1毫米玻璃珠倒入2毫升螺帽管 (大约半路), 然后用热处理消毒。</li>
		<li>添加1毫升不育 LB 或 1x PBS。</li>
		<li>一旦斑马鱼的肠道被添加 (步骤 1.3.3.1) 到管, 拧紧的上限, 然后确保在漩涡均质管。</li>
		<li>融汇1分钟的样品在最大的设置, 然后冷却他们在冰上的最小. 重复此均匀化循环一次, 以完全均匀化。 注意: 几种不同的均匀化方法也会奏效。</li>
	</ol></li>
	<li>定量肠道定植水平
	<ol>
<li>在每管中加入900µL 的 LB 或 1x PBS, 准备管子 (小玻璃试管或1.5 毫升离心管), 用于连续稀释匀浆。为每条鱼, 准备5–6管和做10倍的肠道匀浆稀释, 添加100µL 的匀浆到第一管, 涡流它混合, 然后增加100µL 从第一管到第二管。</li>
		<li>重复此过程, 直到所有稀释都已准备就绪。</li>
		<li>在含有100µg/毫升链霉素 (如果使用链霉素抗性菌株) 和40µg/毫升 X-加仑 (5-溴-4-氯-3-哚β D-galactopyranoside) 的 LB 琼脂板上的每稀释100–200µL。在30摄氏度孵化出 16–18 h 的盘子。</li>
		<li>在夜间孵化后, 在盘子上数诉霍乱菌落, 或者使用自动菌落计数器或手动计数菌落;用标记尖标记计数的殖民地是有用的。</li>
		<li>在考虑到所镀的体积的情况下, 用悬浮液的稀释系数乘以板数, 确定每条鱼肠的 CFU。</li>
	</ol></li>
</ol>2. 鱼腹泻的测量
注: 四种不同的指标用于评估鱼腹泻: 水中的黏蛋白水平、水中的总排泄蛋白水平、水的 OD600和霍乱弧菌在水中的 CFU13。如上文所述, 斑马鱼接触到霍乱弧菌后, 腹泻通常在视觉上明显接近6小时。
<ol><li>
确定黏蛋白水平 注: 粘液分泌在霍乱弧菌定植期间诱发, 是一种很好的排泄水平, 尤其是早期感染13。粘蛋白测定是微量滴定周期酸性席夫碱测定19的一种变异。<ol>
<li>准备以下材料: 50% (w/v) 周期性酸库存溶液和0.1% 周期酸工作溶液 (10 µL 50% 周期性酸库存添加到5毫升的 7% mL 乙酸-立即使用后), 黏蛋白标准, 96 井微量滴定板, 希夫的试剂。<ol>
<li>通过悬浮以下数量的黏蛋白 (从猪胃 III.) 在乙酸钠缓冲液 (100 毫米醋酸钠, 5mM EDTA, pH 值到5.5 与冰川乙酸) 制备粘蛋白标准: 400 µg/毫升, 300 µg/毫升, 200 µg/毫升, 150 µg/毫升, 100 µg/毫升, 75 µg/毫升, 50 µg/毫升, 25 µg/毫升, 10 µg/毫升。 将100µL 的感染水添加到每一个井中, 并将其用于化验, 如下所示: 空白 1x PBS;如上文所述的黏蛋白标准, 或从鱼类感染中抽取水样。每样样品三份。</li>
		</ol>
</li>
		<li>添加50µL 新鲜0.1% 周期酸溶液, 每井与多通道吸管, 并混合它由吹打上下几次。</li>
		<li>把盘子紧紧地裹在塑料包里, 在37摄氏度的 1–1.5 h 上孵化。</li>
		<li>把盘子冷却到室温, 5–10最小. 添加100µL 的品红溶液 (希夫的试剂) 给每个井, 并吸管上下混合。</li>
		<li>包装在塑料包装板, 并在室温下孵化它在一个摇摆平台, 直到颜色已经发展;颜色一般是在20分钟内开发的. 用平板阅读器读取 560 nm 的吸光度。</li>
		<li>绘制一个黏蛋白标准曲线 (OD560与µg/毫升粘蛋白标准)。通过确定每个未知的 OD560在标准曲线上的位置, 并读取该值的相应黏蛋白浓度, 确定未知的黏蛋白水平。</li>
	</ol>
</li>
	<li>
确定蛋白质水平 注: 除粘蛋白外, 水中的总蛋白质水平是鱼类产生的腹泻 (混浊、液体大便) 水平的另一个代表。本程序使用标准的布拉德福德化验来估计蛋白质水平。蛋白质水平估计根据牛血清白蛋白 (BSA) 标准曲线。<ol>
<li>混合100µL 的 BSA 标准之一 (见下文) 或100µL 感染水 (含受感染鱼类的烧杯中的水) 与900µL 的布拉德福德化验试剂 (皮尔斯蛋白化验试剂工作很好, 这) 在一次性试管。在室温下孵化所有样品2分钟。 注: 使用了以下 BSA 标准: 1800 µg/毫升, 1000 µg/毫升, 750 µg/毫升, 500 µg/毫升, 250 µg/毫升, 125 µg/毫升, 50 µg/毫升, 25 µg/毫升。在蒸压蒸馏水中, BSA 标准被稀释。</li>
		<li>阅读每个标准的 OD660或使用分光光度计取样。</li>
		<li>绘制 BSA 标准曲线 (OD660与µg/毫升)。确定未知的蛋白质水平, 通过确定每个未知的 OD660落在 BSA 标准曲线上, 并读取该值的相应粘蛋白浓度。</li>
	</ol>
</li>
	<li>
测量 OD600 注: 腹泻 (混浊, 液体大便) 的存在明显明显后, 几个 h 和一个简单的 OD600确定可以用来量化这一点。<ol>
<li>将含感染水的管倒置数次, 均匀分布。将1毫升的水样本添加到一次性试管。以1毫升无菌感染水为阴性对照。</li>
		<li>使用负控制样本, 使用 600 nm 的分光光度计执行 "空白" 测量。阅读每个水样600毫微米和记录的结果。</li>
	</ol>
</li>
	<li>
感染后 CFU霍乱弧菌排泄量的测定 注: 斑马鱼产生的腹泻的一个主要成分是排出霍乱弧菌。CFU/mL 的测定可用于评估鱼类肠道细菌复制, 以及作为传播实验中传染性剂量的量度。这项措施最好是在发生短暂感染时进行的。如果使用 24 h 感染, 则此检测将测量联合接种加排出的霍乱弧菌。<ol>
<li>在所需的时间点取一个水样, 并按前面所描述的顺序稀释它。</li>
		<li>在选择性培养基上印上系列稀释 (含有链霉素的 LB 琼脂或另一种适当的抗生素或 DCLS), 并将其孵化为24小时30摄氏度。</li>
		<li>数殖民地。计算步骤1.5 中描述的每毫升水的 CFU。</li>
	</ol>
</li>
</ol>

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+adult+rabbit+model+for+Vibrio+cholerae+and+enterotoxigenic+Escherichia+coli+diarrhea.">Simple adult rabbit model for Vibrio cholerae and enterotoxigenic Escherichia coli diarrhea.</a> Infection and Immunity. 32 (2), 739-747 (1981).</li><li>Ritchie, J. M., Rui, H., Bronson, R. T., Waldor, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Back+to+the+future:+studying+cholera+pathogenesis+using+infant+rabbits.">Back to the future: studying cholera pathogenesis using infant rabbits.</a> mBio. 1 (1), (2010).</li><li>Kilcoyne, M., Gerlach, J. Q., Farrell, M. P., Bhavanandan, V. P., Joshi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Periodic+acid-Schiff's+reagent+assay+for+carbohydrates+in+a+microtiter+plate+format.">Periodic acid-Schiff's reagent assay for carbohydrates in a microtiter plate format.</a> Analytical Biochemistry. 416 (1), 18-26 (2011).</li><li>Balaji, V., Sridharan, G., Jesudason, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytotoxicity+of+non+O1,+non+O139+Vibrios+isolated+from+fresh+water+bodies+in+Vellore,+south+India.">Cytotoxicity of non O1, non O139 Vibrios isolated from fresh water bodies in Vellore, south India.</a> Indian Journal of Medical Research. 110, 155-159 (1999).</li><li>Hasan, N. A., 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=Nontoxigenic+Vibrio+cholerae+non-O1/O139+isolate+from+a+case+of+human+gastroenteritis+in+the+U.S.+Gulf+Coast.">Nontoxigenic Vibrio cholerae non-O1/O139 isolate from a case of human gastroenteritis in the U.S. Gulf Coast.</a> Journal of Clinical Microbiology. 53 (1), 9-14 (2015).</li></ol>

作者没有什么可透露的。

斑马鱼是研究霍乱弧菌的一种比较新的模式, 但对于今后发现霍乱弧菌生物学和发病机制11、12、13等未知方面有很大希望。.成年斑马鱼模型具有天然v 型霍乱宿主的优势, 包括完整的、成熟的肠道微生物群和环境模型。该模型的缺点是, 两个主要的人类毒力因素, 霍乱毒素和毒素-coregulated pilus, 不需要为斑马?...

霍乱弧菌是一种水生, 革兰阴性菌, 导致人类疾病霍乱以及零星腹泻1,2。霍乱是在全球许多地区的环境中发现的, 通常与其他水生生物有关。这些关联有机体包括浮游生物, 昆虫蛋大量, 贝类和脊椎动物种类3,4,5,6,7。有几项研究从7、8、9、10不同地理区域的鱼类肠道中分离出霍乱弧菌。在鱼类中存在霍乱弧菌, 表明鱼可能充当环境水库。鱼类还可能牵涉到向人类传播这种疾病, 并在地理上传播霍乱6株。
为了更好地了解霍乱弧菌与鱼类的相互作用,斑马斑马, 更被称为斑马鱼, 是研究霍乱e11的模型系统。斑马鱼原产于南亚, 包括孟加拉湾地区, 被认为是霍乱弧菌最早的水库。在1817年开始的第一次霍乱大流行之前, 除了目前的印度和孟加拉国以外, 没有报告霍乱。因此, 斑马鱼和霍乱弧菌在进化时间尺度上几乎肯定是相互关联的, 这表明斑马鱼是12自然环境中的五个霍乱弧菌宿主。
斑马鱼模型为霍乱弧菌是简单的执行和可用于研究整个致病性霍乱弧菌生命周期。鱼通过沐浴在已接种了已知数量的霍乱弧菌的水中, 接触到霍乱弧菌。在几个小时内, 肠道定植发生, 其次是腹泻的产生。腹泻包括粘液、蛋白质、排泄细菌和其他肠道内容。腹泻的程度可以用一些简单的测量13来量化。被感染的鱼排出的霍乱弧菌可以继续感染天真的鱼, 完成感染周期。因此, 斑马鱼模型概括 v.12、14的霍乱弧菌。
最常用的v. 霍乱动物模型历来是老鼠和兔子14,15,16,17,18。这些模型有助于增加我们对霍乱病发病机制的认识。然而, 由于老鼠和兔子不是天然的v. 霍乱弧菌宿主, 因此, 对霍乱弧菌生命周期的哪些方面可以进行研究。霍乱弧菌对小鼠和家兔的定植通常要求缺乏肠道微生物群或用抗生素进行预处理, 以损害肠道微生物群。两种模型都要求饲将细菌引入消化道或手术操作, 直接将细菌注入肠道。斑马鱼有一个优势, 即成年鱼类与完整的肠道微生物群容易被殖民和感染过程自然发生, 没有任何操作需要。
目前的研究表明, 斑马鱼作为霍乱弧菌感染模型的应用。12、13, 对霍乱弧菌的感染、解剖、计数, 以及霍乱弧菌引起的腹泻的量化进行了说明。这种模式可能对那些对霍乱弧菌疾病过程和霍乱弧菌的环境生活方式感兴趣的科学家有用。

<table>
	<tbody>
		<tr>
			<td>Instrument</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker incubator</td>
			<td>New Brunswick Scientific, Edison, NJ</td>
			<td>Excella E25</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>NUAIRE, Plymouth, MN</td>
			<td>Auto Flow</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Thermo, Waltham, MA</td>
			<td>Geaesys 6</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex homogenizer</td>
			<td>Minibeadbeater24</td>
			<td>112011</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing Machine</td>
			<td>Ohaus, Columbia, MD</td>
			<td>Adventurer Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Stirer</td>
			<td>Corning, Corning, NY</td>
			<td>PC-420D</td>
			<td></td>
		</tr>
		<tr>
			<td>Burner</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>automated colony counter</td>
			<td>REVSCI</td>
			<td>120417B</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>400 ml glass beakers</td>
			<td>Pyrex</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>perforated lids</td>
			<td>Microtip holder with holes from tip box</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>disposable plastic spoons</td>
			<td>Office Depot, Boca Raton, FL</td>
			<td>D15-25-7008</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish Tank System</td>
			<td>Aquaneering, San Diego, CA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RO Water Purifier</td>
			<td>Aqua FX</td>
			<td>TK001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fish net</td>
			<td>Marina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>fish food</td>
			<td>Tetra fin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Brine Shrimp</td>
			<td>Red jungle brand</td>
			<td>O.S.I. pro 80</td>
			<td></td>
		</tr>
		<tr>
			<td>Styrofoam board</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pins</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpels</td>
			<td>Fine Scientific tools, Foster City, CA</td>
			<td>10000-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Scientific tools, Foster City, CA</td>
			<td>11223-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Vannas scissors</td>
			<td>Fine Scientific tools, Foster City, CA</td>
			<td>15000-11</td>
			<td></td>
		</tr>
		<tr>
			<td>2 ml screw cap tubes</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>02-681-375</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm glass beads</td>
			<td>Bio Spec</td>
			<td>11079110</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads for spreading</td>
			<td>Sigma, St. Louis, MO</td>
			<td>18406-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri plate</td>
			<td>Fisher Brand, Hampton, NH</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml centrifuge tube</td>
			<td>Midsci, Valley Park, MO</td>
			<td>AVSS1700</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml centrifuge tube</td>
			<td>Corning Falcon, Corning, NY</td>
			<td>352098</td>
			<td></td>
		</tr>
		<tr>
			<td>Test tubes</td>
			<td>Pyrex</td>
			<td>9820</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Pipette</td>
			<td>Fisher Brand, Hampton, NH</td>
			<td>13675K</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro pipettes</td>
			<td>Sartorius Biohit, G&#246;ttingen, Germany</td>
			<td>m1000/m200/m20</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips</td>
			<td>Genesee Scientific, San Diego, CA</td>
			<td>24-150RS/24-412</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instant Ocean salts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>phosphate buffered saline</td>
			<td>VWR Life Science, Radnor, PA</td>
			<td>K813-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine (ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td>Sigma, St. Louis, MO</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside</td>
			<td>Sigma, St. Louis, MO</td>
			<td>10651745001</td>
			<td></td>
		</tr>
		<tr>
			<td>Schiff&#8217;s reagent</td>
			<td>Sigma, St. Louis, MO</td>
			<td>84655-250 mL</td>
			<td></td>
		</tr>
		<tr>
			<td>periodic acid</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>10450-60-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin from porcine stomach</td>
			<td>Sigma, St. Louis, MO</td>
			<td>M2378-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>9046-46-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660nm Protein Assay Reagent</td>
			<td>Thermo, Waltham, MA</td>
			<td>22660</td>
			<td></td>
		</tr>
		<tr>
			<td>LB medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypton</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Teast Extract</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>NACL</td>
			<td>Fisher Scientific, Hampton, NH</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>TCBS Agar</td>
			<td>BD Biosciences, San Jose, CA</td>
			<td>265020</td>
			<td></td>
		</tr>
		<tr>
			<td>DCLS Agar</td>
			<td>Sigma, St. Louis, MO</td>
			<td>70135-500gm</td>
			<td></td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft office</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 5</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying vibrio cholerae colonization and diarrhea in the adult zebrafish model

霍乱弧菌是最有名的感染性药物, 引起了人类疾病。在人类宿主之外,霍乱弧菌主要存在于水生环境中, 在那里它与各种更高的水生物种相互作用。众所周知, 脊椎动物是一种环境宿主, 是一种潜在的霍乱弧菌储层。霍乱弧菌和硬骨鱼类鱼类种类斑马斑马, 俗称斑马鱼, 起源于印度次大陆, 表明在水生环境中存在长期的相互作用。斑马鱼是研究生物的许多方面的理想的模型有机体, 包括传染性疾病。在水中暴露后, 斑马鱼可以很容易和迅速地被霍乱弧菌所殖民。霍乱弧菌引起的肠道定植导致腹泻的产生和复制的霍乱弧菌的排泄。这些排泄出来的细菌可以继续殖民新的鱼类寄主。在这里, 我们示范如何评估v.斑马鱼的霍乱-肠道殖民化, 以及如何量化五、霍乱引起的斑马鱼腹泻。殖民模式应该是有用的研究人员谁正在研究是否有兴趣的基因可能是重要的宿主殖民和/或对环境的生存。对于研究任何有兴趣探索斑马鱼作为模型系统的肠道病原体的研究人员来说, 斑马鱼腹泻的量化应该是有用的。

五、霍乱斑马鱼肠道的定植
为了提供我们观察到的典型的殖民化水平的例子, 我们接种了 5 x 106 CFU 的大流行性流感病毒菌株 N16961 在装有几只斑马鱼的烧杯中的200毫升水中。经过6小时的感染, 这些鱼被淡水洗涤, 并转移到一个烧杯200毫升的蒸压感染水, 如议定书所述。18 h 在转移之后 (或 24 h 在主要传染以后),...

Watch this Scientific Journal Video about Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model at JoVE.com

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

斑马鱼是一种天然的霍乱弧菌宿主, 可以用来重述和研究整个感染周期从殖民化到传播。在这里, 我们演示如何评估霍乱弧菌的殖民水平和量化腹泻的斑马鱼。

成年斑马鱼模型中霍乱弧菌定植与腹泻的量化研究

Vibrio cholerae is best known as the infectious agent that causes the human disease cholera. Outside the human host, V. cholerae primarily exists in the ...

quantifying-vibrio-cholerae-colonization-diarrhea-adult-zebrafish

Research

JoVE Journal

Immunology and Infection

Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

8.3K 次观看.  Wayne State University School of Medicine. 斑马鱼是一种天然的霍乱弧菌宿主, 可以用来重述和研究整个感染周期从殖民化到传播。在这里, 我们演示如何评估霍乱弧菌的殖民水平和量化腹泻的斑马鱼。

视频: Quantifying Vibrio cholerae Colonization and Diarrhea in the Adult Zebrafish Model

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

造型粘膜念珠菌在斑马鱼幼虫通过鳔注射

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

科研

免疫与感染

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

为梭状体开发拉瓦尔斑马鱼感染模型

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

发育生物学

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

优化 GF 斑马鱼模型:生成、维护和微生物研究

Generation, Maintenance, and Identification of Germ-Free Zebrafish Models from Larvae to Juvenile Stages

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent embryos developed in a relatively clean chorion environment, and extremely rapid development of larvae compared to rodent models. Germ-free (GF) zebrafish (Danio rerio) are crucial for evaluating pollutant toxicity and establishing human-like disease models related to microbial functions. In comparison to conventionally raised (CR) models (fish in common husbandry), GF zebrafish allow for more accurate manipulation of the host microbiota, aiding in determining the causal relationship between microorganisms and hosts. Consequently, they play a critical role in advancing our understanding of these relationships. However, GF zebrafish models are typically generated and researched during the early life stages (from embryos to larvae) due to limitations in immune function and nutrient absorption. This study optimizes the generation, maintenance, and identification of early GF zebrafish models without feeding and with long-term feeding using GF food (such as Artemia sp., brine shrimp). Throughout the process, daily sampling and culture were performed and identified through multiple detections, including plates and 16S rRNA sequencing. The aseptic rate, survival, and developmental indexes of GF zebrafish were recorded to ensure the quality and quantity of the generated models. Importantly, this study provides details on bacterial isolation and infection techniques for GF fish, enabling the efficient creation of GF fish models from larvae to juvenile stages with GF food support. By applying these procedures in biomedical research, scientists can better understand the relationships between intestinal bacterial functions and host health.

作者聚焦:建立无菌斑马鱼用于微生物群-宿主关系研究

This protocol outlines the primary steps for obtaining germ-free (GF) fish embryos and maintaining them from larvae until the juvenile stage, including sampling and detecting their sterile status. The use of GF models with infection is important for understanding the role of microbes in host health.

从幼虫到幼虫阶段的无菌斑马鱼模型的生成、维持和鉴定

Zebrafish serve as valuable models for research on growth, immunity, and gut microbiota due to their genomic similarities with mammals, transparent ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

marinum 分枝杆菌感染成人斑马鱼结核的建模

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

Oral Intubation of Adult Zebrafish: A Model for Evaluating Intestinal Uptake of Bioactive Compounds

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water environment. Developing effective methods for oral delivery of immunostimulants or vaccines, which activate the immune system against infectious diseases, is highly desirable. In devising prophylactic tools, good experimental models are needed to test their performance. Here, we show a method for oral intubation of adult zebrafish and a set of procedures to dissect and prepare the intestine for cytometry, confocal microscopy and quantitative polymerase chain reaction (qPCR) analysis. With this protocol, we can precisely administer volumes up to 50 &#181;L to fish weighing approximately 1 g simply and quickly, without harming the animals. This method allows us to explore the direct in vivo uptake of fluorescently labelled compounds by the intestinal mucosa and the immunomodulatory capacity of such biologics at the local site after intubation. By combining downstream methods such as flow cytometry, histology, qPCR and confocal microscopy of the intestinal tissue, we can understand how immunostimulants or vaccines are able to cross the intestinal mucosal barriers, pass through the lamina propria, and reach the muscle, exerting an effect on the intestinal mucosal immune system. The model could be used to test candidate oral prophylactics and delivery systems or the local effect of any orally administered bioactive compound.

The protocol describes intubating adult zebrafish with a biologic; then dissecting and preparing the intestine for cytometry, confocal microscopy and qPCR. This method allows administration of bioactive compounds to monitor intestinal uptake and the local immune stimulus evoked. It&#160;is relevant for testing the intestinal dynamics of oral prophylactics.

成年斑马鱼的口服插管: 一种评价生物活性化合物肠道吸收的模型

Most pathogens invade organisms through their mucosa. This is particularly true in fish as they are continuously exposed to a microbial-rich water ...

Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected antigen or a combination of antigens into a plasmid, which enables a tailor-made and safe design. The administration of DNA vaccines into host cells leads to the expression of antigens that stimulate both humoral and cell-mediated immune responses. This report describes a protocol for the cloning of antigen sequences into the pCMV-EGFP plasmid, the immunization of adult zebrafish with the vaccine candidates by intramuscular microinjection, and the subsequent electroporation to improve intake. The vaccine antigens are expressed as green fluorescent protein (GFP)-fusion proteins, which allows the confirmation of the antigen expression under UV light from live fish and the quantification of expression levels of the fusion protein with ELISA, as well as their detection with a western blot analysis. The protective effect of the vaccine candidates is tested by infecting the fish with Mycobacterium marinum five weeks postvaccination, followed by the quantification of the bacteria with qPCR four weeks later. Compared to mammalian preclinical screening models, this method provides a cost-effective method for the preliminary screening of novel DNA-based vaccine candidates against a mycobacterial infection. The method can be further applied to screening DNA-based vaccines against various bacterial and viral diseases.

Here we describe a protocol for the immunization of the adult zebrafish (Danio rerio) with a DNA-based vaccine and demonstrate the validation of a successful vaccination event. This method is suitable for the preclinical screening of vaccine candidates in various infection models.

成人斑马鱼对 DNA 疫苗临床前筛选的免疫研究

The interest in DNA-based vaccination has increased during the past two decades. DNA vaccination is based on the cloning of a sequence of a selected ...

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Due to their transparency, genetic tractability, and ease of maintenance, zebrafish (Danio rerio) have become a widely-used vertebrate model for infectious diseases. Larval zebrafish naturally prey on the unicellular protozoan Paramecium caudatum. This protocol describes the use of P. caudatum as a vehicle for food-borne infection in larval zebrafish. P. caudatum internalize a wide range of bacteria and bacterial cells remain viable for several hours. Zebrafish then prey on P. caudatum, the bacterial load is released in the foregut upon digestion of the paramecium&#160;vehicle, and the bacteria colonize the intestinal tract. The protocol includes a detailed description of paramecia maintenance, loading with bacteria, determination of bacterial degradation and dose, as well as infection of zebrafish by feeding with paramecia. The advantage of using this method of food-borne infection is that it closely mimics the mode of infection observed in human disease, leads to more robust colonization compared to immersion protocols, and allows the study of a wide range of pathogens. Food-borne infection in the zebrafish model can be used to investigate bacterial gene expression within the host, host-pathogen interactions, and hallmarks of pathogenicity including bacterial burden, localization, dissemination and morbidity.

Using the Protozoan Paramecium caudatum as a Vehicle for Food-borne Infections in Zebrafish Larvae

Zebrafish (Danio rerio) are becoming a widely-used vertebrate animal model for microbial colonization and pathogenesis. This protocol describes the use of the protozoan Paramecium caudatum as a vehicle for food-borne infection in zebrafish larvae. P. caudatum readily internalizes bacteria and get taken up by larval zebrafish through natural preying behavior.