Name: development of a larval zebrafish infection model for clostridioides difficile
Uploaded: 2020-02-14T13:30:00
Description: Watch this Scientific Journal Video about Development of a Larval Zebrafish Infection Model for Clostridioides difficile at JoVE.com

我们感谢蒂莫·弗里奇对动物的精心照料。我们感谢科斯特和施泰纳特实验室的成员的支持和有益的讨论。我们感谢韩丹丹博士对手稿的批判性阅读。我们感谢下萨克森州联邦州尼德塞奇斯·沃拉布（VWZN2889）的资助。

此处描述的所有动物工作均按照法律规定（欧盟指令 2010/63、许可证 AZ 325.1.53/56.1-TUBS 和许可证 AZ 33.9-42502-04-14/1418）进行。
1. 低熔甘蔗、凝胶板和微注射/微加注针的制备
<ol><li>在 10 mL 的 30% Daniau 的介质（0.12 mM MgSO4， 0.18 mM Ca [NO3+2]） 0.21 mM KCl） 中溶解 0.08 g 低熔融甘蔗 （材料表， 甘蔗 A2576）， 1.5 mM HEPES （pH = 7.2） 和 17.4 mM NaCl，在室?...

<ol>
	<li>Rupnik, M., Wilcox, M. H., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clostridium+difficile+infection:+new+developments+in+epidemiology+and+pathogenesis.">Clostridium difficile infection: new developments in epidemiology and pathogenesis.</a> Nature Reviews Microbiology. 7, 526-536 (2009).</li><li>Yang, Z., 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=Mechanisms+of+protection+against+Clostridium+difficile+infection+by+the+monoclonal+antitoxin+antibodies+actoxumab+and+bezlotoxumab.">Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab.</a> Infection and Immunity. 83, 822-831 (2015).</li><li>Kelly, C. P., Kyne, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+host+immune+response+to+Clostridium+difficile.">The host immune response to Clostridium difficile.</a> Journal of Medical Microbiology. 60, 1070-1079 (2011).</li><li>Britton, R. A., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interaction+between+the+intestinal+microbiota+and+host+in+Clostridium+difficile+colonization+resistance.">Interaction between the intestinal microbiota and host in Clostridium difficile colonization resistance.</a> Trends in Microbiology. 20, 313-319 (2012).</li><li>Goorhuis, 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=Emergence+of+Clostridium+difficile+Infection+Due+to+a+New+Hypervirulent+Strain,+Polymerase+Chain+Reaction+Ribotype+078.">Emergence of Clostridium difficile Infection Due to a New Hypervirulent Strain, Polymerase Chain Reaction Ribotype 078.</a> Clinical Infectious Diseases. 47, 1162-1170 (2008).</li><li>P&#233;pin, J., 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=Emergence+of+fluoroquinolones+as+the+predominant+risk+factor+for+Clostridium+difficile-associated+diarrhea:+a+cohort+study+during+an+epidemic+in+Quebec.">Emergence of fluoroquinolones as the predominant risk factor for Clostridium difficile-associated diarrhea: a cohort study during an epidemic in Quebec.</a> Clinical Infectious Diseases. 41, 1254-1260 (2005).</li><li>Merrigan, M. M., Sambol, S. P., Johnson, S., Gerding, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Fatal+Clostridium+difficile+-Associated+Disease+during+Continuous+Administration+of+Clindamycin+in+Hamsters.">Prevention of Fatal Clostridium difficile -Associated Disease during Continuous Administration of Clindamycin in Hamsters.</a> The Journal of Infectious Diseases. 188, 1922-1927 (2003).</li><li>Chen, X., 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=A+Mouse+Model+of+Clostridium+difficile-Associated+Disease.">A Mouse Model of Clostridium difficile-Associated Disease.</a> Gastroenterology. 135, 1984-1992 (2008).</li><li>Page, D. M., 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=An+evolutionarily+conserved+program+of+B-cell+development+and+activation+in+zebrafish.">An evolutionarily conserved program of B-cell development and activation in zebrafish.</a> Blood. 122, 1-12 (2014).</li><li>Benard, E. L., 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=Infection+of+Zebrafish+Embryos+with+Intracellular+Bacterial+Pathogens.">Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens.</a> Journal of Visualized Experiments. (61), e3781 (2012).</li><li>Theriot, C. M., Young, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+Between+the+Gastrointestinal+Microbiome+and+Clostridium+difficile.">Interactions Between the Gastrointestinal Microbiome and Clostridium difficile.</a> Annual Review of Microbiology. 69, 445-461 (2015).</li><li>Pham, L. N., Kanther, M., Semova, I., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+generating+and+colonizing+gnotobiotic+zebrafish.">Methods for generating and colonizing gnotobiotic zebrafish.</a> Nature Protocols. 3, 1862-1875 (2008).</li><li>Ransom, E. M., Ellermeier, C. D., Weiss, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+mCherry+red+fluorescent+protein+for+studies+of+protein+localization+and+gene+expression+in+Clostridium+difficile.">Use of mCherry red fluorescent protein for studies of protein localization and gene expression in Clostridium difficile.</a> Applied and Environmental Microbiology. 81 (5), 1652-1660 (2015).</li><li>Cocchiaro, J. L., Rawls, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microgavage+of+Zebrafish+Larvae.">Microgavage of Zebrafish Larvae.</a> Journal of Visualized Experiments. (72), e4434 (2013).</li><li>Chen, X., 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=A+Mouse+Model+of+Clostridium+difficile-Associated+Disease.">A Mouse Model of Clostridium difficile-Associated Disease.</a> Gastroenterology. 135 (6), 1984-1992 (2008).</li><li>Hutton, M. L., Mackin, K. E., Chakravorty, A., Lyras, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+animal+models+for+the+study+of+Clostridium+difficile+disease+pathogenesis.">Small animal models for the study of Clostridium difficile disease pathogenesis.</a> FEMS Microbiology Letters. 352, 140-149 (2014).</li><li>Brugman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+as+a+model+to+study+intestinal+inflammation.">The zebrafish as a model to study intestinal inflammation.</a> Developmental &amp; Comparative Immunology. 64, 82-92 (2016).</li><li>Goulding, D., 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=Distinctive+profiles+of+infection+and+pathology+in+hamsters+infected+with+Clostridium+difficile+strains+630+and+B1.">Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1.</a> Infection and Immunity. 77, 5478-5485 (2009).</li><li>Toh, M. 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=Colonizing+the+Embryonic+Zebrafish+Gut+with+Anaerobic+Bacteria+Derived+from+the+Human+Gastrointestinal+Tract.">Colonizing the Embryonic Zebrafish Gut with Anaerobic Bacteria Derived from the Human Gastrointestinal Tract.</a> Zebrafish. 10, 194-198 (2013).</li><li>Bloemberg, G. V., 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=Comparison+of+static+immersion+and+intravenous+injection+systems+for+exposure+of+zebrafish+embryos+to+the+natural+pathogen+Edwardsiella+tarda.">Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda.</a> BMC Immunology. 12, 58 (2011).</li><li>D&#237;az-Pascual, F., Ort&#237;z-Sever&#237;n, J., Varas, M. A., Allende, M. L., Ch&#225;vez, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+host-pathogen+interaction+as+revealed+by+global+proteomic+profiling+of+zebrafish+larvae.">In vivo host-pathogen interaction as revealed by global proteomic profiling of zebrafish larvae.</a> Frontiers in Cellular and Infection Microbiology. 7, 1-11 (2017).</li><li>Valenzuela, M. J., 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=Evaluating+the+capacity+of+human+gut+microorganisms+to+colonize+the+zebrafish+larvae+(Danio+rerio).">Evaluating the capacity of human gut microorganisms to colonize the zebrafish larvae (Danio rerio).</a> Frontiers in Microbiology. 9, (2018).</li></ol>

提出的方法修改和扩展了现有的方法，通过执行注射和微藻10，14感染斑马鱼幼虫。它还演示了一种研究斑马鱼幼虫22厌氧病原体的方法。此外，该议定书还有助于分析CDI和斑马鱼中C.困难的体内先天免疫细胞反应。该方法可重复，易于在常规实验室或临床环境中进行。
监测白细胞的C.困难的噬菌体， ?...

梭菌是一种克阳性、孢子成形、厌氧和毒素产生的杆菌，是胃肠道1严重感染的主要原因。CDI的典型症状包括腹泻，腹痛，和致命的假膜结肠炎，它有时可能导致死亡1，2。有证据表明，宿主免疫反应在这种疾病的进展和结果中都起着关键作用。除了免疫反应，本地肠道微生物群对CDI4的发病和发病机制至关重要。在过去十年中，由于出现C.困难性超毒菌株（BI/NAP1/027）5、6 ，CDI的病例数和死亡率均显著增加。更好地了解潜在的免疫机制和在CDI期间微生物群的作用将有助于导致新的治疗发展和进步，从而更好地控制这一流行病。
一些动物模型，如仓鼠和老鼠，已经开发出来，以提供洞察对C.困难7，8的免疫防御。然而，对先天免疫细胞的作用仍然知之甚少，特别是因为先天免疫细胞的行为主要来自组织学分析或体外培养细胞。因此，建立一个透明的斑马鱼模型，揭示活体脊椎动物有机体内部对C.困难的先天免疫反应，将有助于此类研究。斑马鱼幼虫具有功能性先天免疫系统，但它们缺乏适应性免疫系统，直到受精9周后4-6周。这一独特特征使斑马鱼幼虫成为研究CDI中先天免疫细胞的分离反应和功能的优秀模型。
本报告描述了使用斑马鱼幼虫研究梭菌和先天免疫细胞（如巨噬细胞和嗜中性粒细胞）之间的相互作用的新方法。首先，提出了一种局部的显微注射方案，包括C.困难接种和染色。利用体内共聚焦延移成像，记录中性粒细胞和巨噬细胞对感染部位的反应，并观察嗜中性粒细胞和巨噬细胞对细菌的噬菌体。然而，据报道，注射本身导致组织损伤，并导致独立于细菌10的白细胞的招募。因此，随后描述了一种非侵入性微藻方案，用于将C.困难输送到斑马鱼幼虫的肠道。先前的研究已经表明，本地胃肠道微生物群保护宿主免受C.困难11的殖民化。因此，诺生物斑马鱼幼虫也被用来使被感染的斑马鱼容易感染。之后，进行肠道解剖以恢复可行的C.困难，并验证它们在斑马鱼肠道中的持续时间。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A2576</td>
			<td>Ultra-low gelling agarose</td>
		</tr>
		<tr>
			<td>Agarose low-melting (LM)</td>
			<td>Pronadisa</td>
			<td>8050</td>
			<td>It is used in agarose plates</td>
		</tr>
		<tr>
			<td>BacLight Red Bacterial Stain</td>
			<td>Thermo Fisher Scientific</td>
			<td>B35001</td>
			<td>Fluorescent dye</td>
		</tr>
		<tr>
			<td>Brain-Heart-Infusion Broth</td>
			<td>Carl Roth GmbH</td>
			<td>X916.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Brass (wild-type)</td>
			<td></td>
			<td></td>
			<td>deficient in melanin synthesis, used to generate stable transgenic lines</td>
		</tr>
		<tr>
			<td>Calcium nitrate (Ca(NO3)2)</td>
			<td>Sigma-Aldrich</td>
			<td>C1396</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary Glass</td>
			<td>Harvard Apparatus</td>
			<td>30-0019</td>
			<td>Injection needles</td>
		</tr>
		<tr>
			<td>Clostridioides difficile</td>
			<td></td>
			<td></td>
			<td>R20291,, a ribotype 027 strain, TcdA+/TcdB+/CDT+ production</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Carl Roth GmbH</td>
			<td>A994</td>
			<td></td>
		</tr>
		<tr>
			<td>FIJI</td>
			<td>open-source platform</td>
			<td></td>
			<td>Image processing</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth GmbH</td>
			<td>6763</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal needle puller</td>
			<td>Sutter instrument Inc</td>
			<td>P-87</td>
			<td></td>
		</tr>
		<tr>
			<td>L-cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>168149</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Application Suite X (LAS X)</td>
			<td>Leica</td>
			<td></td>
			<td>Image processing</td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>Carl Roth GmbH</td>
			<td>P026</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro injector</td>
			<td>eppendorf</td>
			<td>5253000017</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection molds</td>
			<td>Adaptive Science Tools</td>
			<td>TU1</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica SP8 confocal microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol Red</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Carl Roth GmbH</td>
			<td>5346</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Carl Roth GmbH</td>
			<td>9265</td>
			<td></td>
		</tr>
		<tr>
			<td>Taurocholate</td>
			<td>Carl Roth GmbH</td>
			<td>8149</td>
			<td></td>
		</tr>
		<tr>
			<td>Tg(lyZ: KalTA4)bz17/Tg(4xUAS-E1b:EGFP)hzm3</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which in which the lyZ promoters drive the expression of EGFP fluorescent protein in neutrophils</td>
		</tr>
		<tr>
			<td>Tg(mpeg1.1: KalTA4)bz16/Tg(4xUAS-E1b:EGFP)hzm3</td>
			<td></td>
			<td></td>
			<td>stable transgenic line in which in which the mpeg1.1 drive the expression of EGFP fluorescent protein in macrophages</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Bacto</td>
			<td>212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of a larval zebrafish infection model for clostridioides difficile

梭状体困难感染 （CDI） 被认为是美国最常见的医疗保健相关胃肠道感染之一。已描述对C.困难的先天免疫反应，但中性粒细胞和巨噬细胞在CDI中的确切作用较少。在目前的研究中，达尼奥雷里奥（斑马鱼）幼虫被用来建立一个C.困难感染模型，用于成像这些先天免疫细胞在体内的行为和合作。为了监测梭菌，已经建立了一个使用荧光染料的标签协议。局部感染是通过标记C.困难的显微注射实现的，这种注射在斑马鱼肠道中积极生长，并模仿CDI中的肠道上皮损伤。然而，这种直接感染方案是侵入性的，导致显微伤口，这可能会影响实验结果。因此，这里描述了一种更无创的微藻协议。该方法包括通过张口插管将C.困难细胞直接输送到斑马鱼幼虫的肠道。这种感染方法与C.困难器的自然感染途径相近。

梭子是严格无氧的，但荧光蛋白的色度通常需要氧气才能成熟。为了克服这个问题，荧光染料被用来染色活的C.困难细胞，这些细胞正在积极生长（R20291，核糖核酸型027菌株;图 1A.使用Gal4/UAS系统，生成稳定的转基因斑马鱼线进行活成像，其中mpeg1.1或lyZ启动子以Gal4依赖性的方式驱动巨噬细胞和中性粒细胞中EGFP荧光蛋白的表达。
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Watch this Scientific Journal Video about Development of a Larval Zebrafish Infection Model for Clostridioides difficile at JoVE.com

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

这里介绍的是一种安全有效的方法，通过微注射和非侵入性微藻感染斑马鱼幼虫荧光标记厌氧C.困难。

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 ...

这种方法可以回答有关先天免疫系统在梭菌感染中的作用的关键问题，如巨噬细胞是否和如何识别或吞噬这种病原体。这种方法的主要优点是，它可以应用于许多不同的厌氧细菌，感染时间可以操纵，口服给药代表正常的人类感染更近。培养后，将培养的一毫升转移到分光光度计的库维特中，并测量OD600。将所需量转移到新鲜 1.5 毫升管中，以便在最终体积的一毫升内达到 1 到 1.2 的最终 OD。用1X PBS的1毫升清洗一次，在室温下以5000次g离心，3分钟。在 1X PBS 的一毫升中重新发送。将准备好的荧光染料的工作溶液的三微升加入一毫升细菌悬浮液中。在黑暗中室温下孵育样品15分钟。之后，用1X PBS清洗染色的梭三二胺困难一次，去除残留染料，并在1X PBS的一毫升中重新暂停，实现OD600的1.0。首先，将0.8%的低熔化阿加罗斯滴到宠物菜中的斑马鱼幼虫上盖上。轻轻地将幼虫调整到横向位置。将培养皿放在冰上30至60秒，使低熔化的红糖凝固。加入30%达尼欧的介质，含有0.02%至0.04%三分之一，以覆盖红糖。要准备注射溶液，在PBS溶液中加入0.5%酚红色微升，加入染色的梭三二胺困难二恶英的9微升中。使用微加载器使用喷射溶液加载校准的微注射针。将加载的针头安装到微操纵器中，并在立体显微镜下放置。调整 600 到 900 个下顶卡之间的喷射压力。将喷射时间设置为 0.1 至 0.3 秒，以获得 0.5 至 1.0 纳米光。将微操纵器中的针头设置为大约 45 度角，指向嵌入的幼虫。将针尖放在胃肠道上方，靠近泌尿生殖孔。穿过阿加罗斯，然后用针尖刺穿肌肉。然后将其插入肠道流明，并注射0.5至1.0纳米光的梭三二胺困难。使用荧光显微镜监测注射的幼虫，并使用灵活的微加载器尖端拾取正确注射的幼虫进行共合成像。在带凹槽的1.5%的加糖板块中，将0.8%的低熔加糖滴到斑马鱼幼虫上覆盖。轻轻调整幼虫，头部朝直，在凹槽中以 45 度角朝头，尾部靠槽壁。轻轻操作针头通过阿加罗斯，然后进入斑马鱼幼虫的嘴里，通过食道。一旦针头的尖端位于前肠道球内，按下注射踏板，释放0.5至1纳米细菌培养。填充肠道的流明。不要让它溢出食道或氯卡。轻轻地从斑马鱼的嘴里取针。在加夫格之后，用灵活的微装载机尖端从阿加罗斯中救出受感染的斑马鱼幼虫，先将幼虫切开，然后抬起幼虫。这些幼虫转移到无菌的30%达尼欧的培养，并冲洗两次。麻醉斑马鱼幼虫后，加入200至300微升1%的低熔化阿加罗斯，以覆盖麻醉幼虫。将幼虫的感染区域尽可能放在玻璃滑梯上。让加糖在冰上凝固30至60秒。将阿加罗斯淹没在含有0.02%至0.04%三分之三的介质中。继续用共和激光扫描显微镜对幼虫进行成像。对受感染的斑马鱼幼虫进行安乐死后，将针头插入靠近斑马鱼幼虫头部的后躯干，使斑马鱼固定。用长矛把刺后面的头部去掉。将第二根针插入后躯干中间。将第三根针插入斑马鱼的腹部，将肠子从体腔中拉出。使用微注射针将 10 至 15 肠道转移到含有 200 微升无菌 1X PBS 的 1.5 毫升管中。用害虫使肠道均质化，以扰乱组织并准备同质化。确保害虫到达管子底部，完全破坏所有肠道。加入同质，加入含有D-环素和西福克斯西丁的梭三二恶英困难养殖介质，带或不带陶罗胆碱，并在厌氧室中孵育。在这项研究中，嗜中性粒细胞和巨噬细胞都到达感染点。激活巨噬细胞噬菌体化两种细菌的例子显示了通过吞噬和消化标记的梭三二胺困难清除。在受精后五天将荧光标记的梭菌二恶英异位到巨噬细胞和嗜中性粒细胞的肠道流明中，这种微量作用模仿了梭三二恶虫难治感染的自然路径。然而，嗜中性粒细胞和巨噬细胞在微粒后长达12小时内未明显向胃肠道迁移。与此同时，标记的梭三二恶英的荧光在微重力后大约五个小时后消失。感染后24小时对肠道样本进行解剖，显示细菌生长。对照组没有细菌生长。在后来的时间点，孵育样本只生长在含有TCA的介质中，这表明肠道中总的梭三二恶英困难已经形成孢子。16S rDNA PCR 将生长的细菌确定为梭三二胺困难，它产生大约 800 个碱基对的可预测大小的特定 PCR 安普利素。生长在 CHROMID 板上的细菌培养物以典型的黑菌落出现，这进一步表明斑马鱼肠中的细菌是梭三二二恶英。与厌氧细菌一起工作要求有氧步骤保持短，因为这当然会影响细菌的生物学。斑马鱼中的先天免疫细胞可以纵。例如，他们可以破译先天免疫细胞对感染的反应机制。我们的方法表明，斑马鱼是研究人类肠道厌氧病原体的工具。使用多抗病原体需要采取适当的安全措施。

Research

JoVE Journal

Developmental Biology

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Imaging Subcellular Structures in the Living Zebrafish Embryo

在活斑马鱼胚胎成像的亚细胞结构

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

科研

发育生物学

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

分析<em&gt;在体内</em&gt;细胞迁移使用细胞移植和时间推移成像在斑马鱼胚胎

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

斑马鱼发展长期成像的多功能安装方法

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Dissection of Larval Zebrafish Gonadal Tissue

幼虫斑马鱼性腺组织的解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Electroretinogram Recording in Larval Zebrafish using A Novel Cone-Shaped Sponge-tip Electrode

一种新型锥形海绵尖电极在幼虫斑马鱼中的电录记录

The zebrafish (Danio rerio) is commonly used as a vertebrate model in developmental studies and is particularly suitable for visual neuroscience. For ...