Name: the galleria mellonella waxworm infection model for disseminated candidiasis
Uploaded: 2018-11-17T15:00:00
Description: Watch this Scientific Journal Video about The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis at JoVE.com

作者希望感谢帕米拉·华盛顿和利亚·安德森在获得用于本研究的大肠杆菌方面的帮助。

所述的所有方法都依赖于无脊椎动物宿主的使用, 不需要机构动物护理和使用委员会 (iacuc) 的批准。
1.麦地那龙线虫幼虫
<ol><li>从批发商和供应商订购不向幼虫引入激素、抗生素或其他治疗方法并能够运送和运送活体标本的幼虫。<ol>
<li>在实验过程中, 一定要从同一供应商处购买所有幼虫。夏季订购幼虫时要小心, 因为温度超过30°c 会降低幼虫的生存能力。</li>
		<li>监?...

<ol>
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Y., Whiteway, M., Kavanagh, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correlation+between+virulence+of+Candida+albicans+mutants+in+mice+and+Galleria+mellonella+larvae.">Correlation between virulence of Candida albicans mutants in mice and Galleria mellonella larvae.</a> FEMS Immunology and Medical Microbiology. 34 (2), 153-157 (2002).</li><li>Fuchs, B. B., O'Brien, E., Khoury, J. B., Mylonakis, E. <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+using+Galleria+mellonella+as+a+model+host+to+study+fungal+pathogenesis.">Methods for using Galleria mellonella as a model host to study fungal pathogenesis.</a> Virulence. 1 (6), 475-482 (2010).</li><li>Kavanagh, K., Fallon, J. 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g. mellonella蜡虫模型是快速、可重复分析白色念珠菌毒力的有效工具。这一详细的协议依赖于在一批幼虫中一致地将确定的传染性剂量传递给同一地点。传染性剂量对 g. mellonella 的死亡率有深远的影响, 而从最初到达到收到后10天使用幼虫产生了类似的结果。失去c. albicansmtl 导致毒力下降, 与以前的实验一致的小鼠, 尽管中断 mtlα等位基因并不能改变幼?...

念珠菌是常见的人类共生体, 能够成为严重免疫功能低下和生物功能障碍患者的机会性病原体。虽然许多念珠菌类可以引起疾病,白色念珠菌是传播念珠菌病 1,2最普遍的原因。系统性疾病的原因是白色念珠菌通过直接渗透以前限制性的宿主屏障或在手术部位引入和其他身体接触 3.念珠菌利用一系列致病过程在宿主内引起全身性疾病, 包括丝状、生物膜形成、免疫细胞逃避和逃逸以及铁清除4。体外方法存在, 以调查个人的致病机制, 但动物模型继续提供了最好的选择, 以调查整个疾病结局 5,6。以前的研究详细介绍了许多有希望的体外研究毒力未能在体内繁殖7,8。因此, 仍然需要动物模型来评估体内的毒力.尽管白色念珠菌无法作为例长9自然地殖民小鼠系统, 但大多数疾病模型都依靠老鼠作为人类感染的代名词.无脊椎动物中的传播念珠菌模型包括线虫、果蝇果蝇和蜡虫的线虫, 尽管担心根本的区别在基本生理上, 宿主体温和暴露途径阻碍了它们的广泛接受 10,11 。
最近, g . mellonella蜡虫感染模型已被采用来模拟致病性的细菌和真菌病原体12,13,14。这种模式的优点包括, 与小鼠模型相比, 它的成本相对较低, 吞吐量增加, 易用性, 以及减少对动物慈善的伦理担忧。对于研究人员来说, 这转化为测试多个变量的能力增强、置信区间更强、实验速度更快、动物协议的旁路。在11、15例临床分离株的生物膜形成、丝状化和基因调控所需的基因受到干扰后, 美洛内拉已经成为快速评估白色念珠菌毒力的平台 ,16。最近的研究纳入了使用g. mellonella 进行抗真菌效果的研究, 以评估体内环境下药物活性和耐药性的药代动力学, 否则这些活性和耐药力具有挑战性和耗时17,18。然而, 据报道, 由于实验中的差异很大, 研究小组之间的重复也不一致, 从而产生不同的毒力表型, 这使得对白色念珠菌中白色念珠菌毒力的研究变得更加复杂老鼠和蜡虫之间 11,13,19, 20,21。在这里, 我们概述了一种g. mellonella 协议,以规范白色念珠菌感染, 提高毒力实验的重现性, 并证明与前面描述的小鼠毒力研究的一致性模型。
先前的研究表明, 5号染色体上的白色念珠菌交配样位点 (mtl) 调节细胞身份和交配能力, 类似于酿酒酵母和其他 ascomycete 真菌 22.白色念珠菌分离株的大多数在mtl位点是杂合, 编码每个mtla 和 mtlα等位基因 (mtla/α) 的一个, 因此是无菌的15,23,24. 由于杂合性 (loh) 丢失或突变而失去其中一个mtl等位基因, 会导致纯合mtla或mtlα菌株, 这些菌株可以经历从无菌 "白色" 状态到无菌 "白色" 状态到无菌 "白色" 状态的表型开关。配合主管 "不透明" 状态25。先前的研究表明, mtl杂合性的丧失也会降低不同应变背景的小鼠全身感染模型的毒力26。在这里, 我们详细介绍 g . mellonella模型的传播念珠菌病使用基因相似的实验集, 以描绘mtl杂合性对毒性的贡献。我们表明, mtl 构型影响白色念珠菌的致病性, 其中mtlα菌株的毒性较低, 无论是mtla/α和mtla 细胞, 类似于研究结果在小鼠感染模型26。

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			<td height="21" style="height:21px;width:427px;">Galleria mellonella</td>
			<td style="width:140px;">Snackworms.com</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Buy twice as many worms as expected to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 uL, Model 1701 N SYR Cemented needle, 26G, type 2 syringe</td>
			<td>Hamilton</td>
			<td>80000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish, 100X15 mm, 500 pack</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microcentrifuge tube, 1.7 mL, 500 pack</td>
			<td style="width:140px;">VWR</td>
			<td>87003-294</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:427px;">Phosphate Buffered Saline (Biotechnology grade), 500 mL</td>
			<td style="width:140px;">VWR</td>
			<td style="width:119px;">97062-818</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;width:427px;">Ethanol absolute, &#8805;99.5% pure, 500 mL</td>
			<td style="width:140px;">Millipore Sigma</td>
			<td>EM-EX0276-1S</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:427px;">autoclaved ddH2O</td>
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			<td></td>
		</tr>
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</table>

the galleria mellonella waxworm infection model for disseminated candidiasis

念珠菌是人类在皮肤、粘膜表面和胃肠道中的常见真菌共生体。在某些条件下,念珠菌可以过度生长他们的自然定位, 导致衰弱的黏膜感染以及危及生命的全身感染, 这是一个主要的研究焦点, 因为他们相关的高死亡率。为研究疾病进展和剖析念珠菌致病性特征, 存在传播感染的动物模型。其中,麦地那龙菌蜡虫感染模型为高通量系统毒力调查提供了一个具有成本效益的实验工具。许多其他细菌和真核感染因子已在g. mellonella中得到有效研究, 以了解致病性, 使其成为一个被广泛接受的模型系统。然而, 用于感染g. mellonella 的方法的变化可以改变表型结果, 并使结果的解释复杂化。在这里, 我们概述了蜡虫模型的好处和缺点, 以研究系统念珠菌的发病机制, 并详细介绍了提高重现性的方法。我们的研究结果突出了g. mellonella 的死亡率动力学范围, 并描述了可以调节这些动力学的变量。最终, 这种方法是一个合乎道德的, 快速的, 具有成本效益的方法来研究在一个模型的传播念珠菌病的毒力。

在这里, 我们演示了一种可重复的方法, 用于使用g. mellonella蜡虫, 以调查传播念珠菌感染模型使用白色念珠菌。适当储存、维护和选择用于感染的幼虫是确保g. mellonella 死亡率重现性的关键组成部分 (图 1a)。健康的幼虫是活跃的, 有浅黄色/颜色, 身体上没有黑色斑块应用于这个系统, 通常是可行的感染后, 抵达后两周。?...

Watch this Scientific Journal Video about The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis at JoVE.com

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

凝膜状杆菌是一种播散念珠菌病的无脊椎动物模型。在这里, 我们详细介绍了感染协议, 并为模型的有效性提供了支持数据。

弥散性念珠菌感染模型

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

此方法可以帮助回答在念珠菌领域有关菌株毒性和特定基因对这些过程的贡献的关键问题。这种技术的主要优点是，它避免了对动物使用的担忧，坚固、快速、便宜，并且每个实验提供大量的动物。一般来说，对这种方法有新的个体可能会有点困难，因为他们在实验开始之前很难评估蠕虫的可行性，或者他们可能有一些问题与蠕虫注射不一致。从不向幼虫提供激素、抗生素或其他治疗的批发商和供应商订购幼虫，这些批发商和供应商能够运送和运送活标本。当幼虫到达时，检查每个容器，确认幼虫的健康和生存能力。健康的幼虫将完全淹没在床上用品下，移动，并拥有黄色，浅褐色与少数含有黑色痕迹或变色的幼虫沿身体。幼虫可以在环境温度下储存在通风空间中的容器中长达两周。为了准备酵母细胞的画廊梅洛内拉感染，种植C.albicans菌株在三毫升新鲜酵母-肽-葡萄糖汤每株在标准培养管在中速和30摄氏度的旋转管。第二天早上，通过离心收集细胞，在五毫升的新鲜PBS离心中清洗酵母三次。最后一次洗涤后，在一毫升新鲜PBS中重新给细胞，将酵母转移到微离心管中。连续稀释其他PBS中的细胞，浓度为1至1000，1至10万浓度，并计算1至100和1000稀释的细胞。在新型微离心管中，将细胞调整为每毫升PBS浓度的2.5倍至第7细胞，通过电镀1至10万稀释的适当体积，在一个酵母-肽-脱糖质板上实现100个菌落形成单位，用于感染。要用念珠菌细胞感染幼虫，首先将少量蜡虫床上用品传播到一个空的、100至15厘米的无菌培养皿中，每次生物复制，然后对装有26米针的10微升注射器进行消毒，三次用10微升70%乙醇和3次每洗10微升PBS。接下来，涡旋接种稀释C.albicans，将10微升细胞加载到注射器中。打开一个G.mellonella幼虫的容器，用手指小心地把床上用品翻过来发现幼虫，选择体型相似、身体缺乏黑色色素沉着的黄色、棕褐色幼虫，用中间和食指和拇指以类似于抓铅笔的方式拾起一只幼虫。将幼虫卷到其背上，使腿朝上，在手指和拇指之间保持身体全长，使幼虫无法卷曲或离开注射。用另一只手旋转注射器，使针斜面朝上，并缓慢地将斜面的整个长度插入与最后左腿交界处的身体，确保针头穿透身体，而不是简单地向内推幼虫的身体。在这个程序中，最关键的步骤是注射蜡虫的方式，以尽量减少对幼虫身体的损害，并接种完整的脂肪。然后注射全10微升的念珠菌，并提取针头。接种后，每100至15毫米培养皿中注射多达10个幼虫，并在37摄氏度下孵育幼虫8天。每24小时检查一次餐具，以监测死亡情况。死亡率最初可以通过变暗色素沉着、黑斑或身体的形成以及缺乏运动来评估。为了确认死亡率，用钳子轻轻地将垂死的幼虫卷到背上，用钳子轻轻戳幼虫的底面。开始幼崽进入飞蛾的幼虫可以包含在分析中，但如果它们开始剥落，就应该将其移除。幼虫感染零或抵达后10天之间，G.mellonella死亡的动力学没有显著差异。幼虫的特征症状出现在两个年龄的幼虫中，这些年龄都死于感染，从变色开始，在完全失去活力并最终死亡之前，这种变色让位给黑斑。感染C.albicans加快了这种变色和发病的过程，但与未感染或PBS注射的幼虫相比，并没有显著改变导致死亡的表型标记的级联。值得注意的是，与PBS注射的动物相比，G.mellonella感染任何经测试的临床分离的C.albicans菌株的交配型样同性或异质酶，可产生高死亡率，而原生衍生菌株的注射则减弱了该模型中的致命效应。重要的是要记住选择健康的幼虫，并温柔地处理幼虫。如果多个菌株将被接种，注意不要让念珠菌细胞在PBS中坐得太久。按照这个程序，其他方法，如免疫学分析和在幼虫中的念珠菌定位，可以回答有关宿主和真菌病原体在感染期间相互作用的其他问题。开发后，该技术为研究人员使用高通量格式探索念珠菌发病机制铺平了道路。不要忘记，与 Candida albicans 合作需要小心，执行此程序时应始终采取标准预防措施，包括个人防护设备的磨损。

Research

JoVE Journal

Immunology and Infection

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Introducing Shear Stress in the Study of Bacterial Adhesion

在细菌粘附的研究剪应力

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

科研

免疫与感染

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

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 ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

昆虫<em&gt;大蜡螟</em作为一个强大的的感染模型探讨细菌致病

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

Use of Galleria mellonella as a Model Organism to Study Legionella pneumophila Infection

使用<em&gt;大蜡螟</em&gt;作为模式生物来研究<em&gt;嗜肺军团菌</em&gt;感染

Legionella pneumophila, the causative agent of a severe pneumonia named Legionnaires&#39; disease, is an important human pathogen that infects and ...

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

Analysis of the Epithelial Damage Produced by Entamoeba histolytica Infection

上皮损伤制作分析<em&gt;阿米巴</em&gt;感染

Entamoeba histolytica is the causative agent of human amoebiasis, a major cause of diarrhea and hepatic abscess in tropical countries. Infection is ...

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

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 ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

A Galleria mellonella Oral Administration Model to Study Commensal-Induced Innate Immune Responses

一种研究食细胞诱导免疫应答的 mellonella 口服液模型

The investigation of the immunogenic potential of commensal bacteria on the host immune system is one essential component when studying intestinal ...

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

Use of the Invertebrate Galleria mellonella as an Infection Model to Study the Mycobacterium tuberculosis Complex

利用无脊椎杆菌作为感染模型研究结核分枝杆菌复合物

Tuberculosis is the leading global cause of infectious disease mortality and roughly a quarter of the world&#8217;s population is believed to be infected ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

伪多莫纳斯航空感染模型实时评估工具

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...