作者要感谢妮可Hasenkampf和Amanda TARDO技术支持。我们也感谢博士。林登胡锦涛和阿德里安娜马尔克斯的LeFlap防护装置的建议，并厉色格恩博士指导毛细管喂养方法。这项工作是由美国国立卫生研究院/ NCRR格兰特8 P20 GM103458-09（MEE）和由国家研究资源中心和研究基础设施计划生透过出让P51OD011104/P51RR000164全国学院办公室（ORIP）的支持。

接种蜱和饲养动物时莱姆病研究的实验大纲如图1所示。 1。接种若虫硬蜱蜱与B螺旋体采用毛细管喂养当执行操作与蜱，白色的实验室大衣与弹性套筒，手套，一次性保护帽磨损。 <ol><li>我们的技术是所报道的布罗德沃特等人 49的修改版本。用移液管拉拔器加热和拉动巴斯德吸管打破薄准备毛细管。使用镊子和解剖范围，打破提示以最优的直径（约0.2​​mm）。标准化蜱口器大小的管子被用作上浆指导。面罩应该准备的移液管时佩戴。 </li><li> B.成长疏螺旋体 ，以BSK-H培养基（Sigma）浓度之间的2-8×10 7个/ ml的（对数中期）泰宁6％兔血清。 </li><li>使用已保存在23℃的4-6周后幼虫蜕皮蜱若虫。这里蜱上用双面胶带小的60×15mm陪替氏培养皿上的菜的外底表面上。将蜱腹面面朝上。 </li><li>浸毛细管尖端插入B。混合后螺旋体培养管。将毛细管过的蜱口器使用解剖范围的hypostome。使用成型粘土来固定管到位， 如图2A所示 。 </li><li>将培养皿里面一个大的透明塑料桶贴蜱围堵的添加水平。湿纸巾被添加到提供水分。将蜱在30分钟-2小时37°C的热孵化，直到排便是显而易见的。这表明，含有螺旋体传媒透剔已经过去了。 </li><li>休息蜱为2-4周，在23℃下，使适应环境打勾喂食前他们在动物身上。 </li></ol> 2。感染小鼠B。由蜱螺旋体 <ol><li>稀释氯胺酮股票在1:10无菌水。麻醉每只小鼠用腹腔内注射100毫克/千克氯胺酮用结核菌素注射器</li><li>当鼠标被完全麻醉，背部用细（雷明顿润滑，如丝般）电动修边刮胡子鼠标从耳朵到中间。 </li><li>在一个白色的锅，没有其他物体附近，由湿的画笔转移蜱若虫（即海氏螺旋体 ）鼠标的无毛区。可替换地，未感染的蜱可以放置在老鼠的小鼠具有可疑感染xenodiagnosis。蜱放置使用干净的白色表面，有助于确保任何独立的蜱将很容易看到。 </li><li>将鼠标放置在专门的隔离罩（阿伦敦囚禁，宾夕法尼亚州Allentown）。该关进笼子由一个不锈钢格栅从笼子的底部升高的。 Ŧ他笼顶部是由我们的内部机加工车间改装提升，足以让下方的鼠标自由移动的水壶架。泛充满了约半英寸的水陷阱，脱落小鼠（ 图3A）任何蜱。为了尽量减少体温过低的危险，可重复使用加热垫，微波在使用前，被放置在笼子底下，直到小鼠从麻醉中苏醒完全。动物通常共济失调，因为它们从麻醉和抗擦的食物和水的托盘中恢复，所以这些必须去除。水位足够低，以防止小鼠的四肢从浸没。 </li><li>将笼子已经内衬纠结陷阱糊（CONTECH，维多利亚，不列颠哥伦比亚省，加拿大）和带托盘内，以确保节肢动物的压迫。小鼠单笼，并在麻醉期间连续观察。 </li><li>在2小时内，当小鼠从麻醉中完全清醒，食品托盘和水瓶被替换到笼子里。 2后4小时，住房富集由塑料小屋和nylabone被替换。 </li><li>经过3，4，5天，点击鼠标，笼和笼水喂蜱。笼中的水通过一个白色的金属锅过筛（ 即 "淘金"）。冲洗馈蜱在清水中并存储在塑料罐（ 图3B）。在天3和4，在笼子里用清水代替水。第5天，不仅要检查笼子，但鼠标彻底蜱。通常通过这一点，所有蜱喂养和小鼠可以返回到正规的笼舍。 </li><li>将所有的废物从鼠笼，包括液体，在生物危害容器和高压灭菌处理。保留日志放置在老鼠和那些恢复在任何时候都蜱的数量。 </li></ol> 3。在非人灵长类动物的饲养为蜱感染B。螺旋体或xenodiagnosis <ol><li>准备剔防护装置:切在3英寸×1¾英寸直径的圆3英寸LeFlap（瓣）用干净的手术刀和测量指南。使用切出作为模板切割相同大小的圈中Biatane泡沫和Duoderm。该泡沫用于提升皮瓣在皮肤的表面上，防止蜱的可能破碎。的Duoderm添加缓冲的另一层和覆盖在遏制装置用于从蜱逃逸附加的安全的边缘。遏制装置的示意图示于图4。 </li><li>兽医工作人员将通过肌肉注射麻醉动物用5-8毫克/公斤Telazol。 </li><li>用夹子电动修边机（奥斯特）配备了大小40片动物的毛发。将要覆盖的外套所有区域都剪短背面，正面，上臂。使用剃须膏和双刀片一次性剃须刀，刮脸密切大约25厘米面积的垂直×20厘米水平。用干净湿纸巾和低热量干燥的皮肤吹干。 </li><li>将皮瓣在T他动物的背部，正下方的肩胛骨，对脊柱的两侧。使用一个标记，以跟踪在这个位置的圆圈。通过与SkinPrep擦拭准备周围一圈的皮肤区域。这消除了油的皮肤可能会影响胶水和密封装置的附着力。留下约的空间绕了一圈1厘米的圆周，涂上一层胶水皮肤（SkinBond）与〜4cm的宽度。 </li><li>拆下Biatane泡沫背胶，并加盖在适当的位置的皮肤。动物再次麻醉，由兽医人员以5毫克/公斤Telazol。轴封采用皮胶和Hypafix磁带的边缘。取下皮瓣背胶，并加盖在Biatane的顶部。放置Hypafix磁带绕LeFlap的边缘，然后用胶带向下折翼的网状瓣，然后将护套上的动物。磁带和昆布陷阱膏施加到地板在围绕非人灵长类动物笼舍为增加安全性的周边。 </li><li>为了尽量减少CHEMIC的影响在蜱喂养步骤3.4使用ALS，蜱添加24小时后防护装置到位。在这一点上，该装置的安全性也进行检查，如果需要加强。典型地，20不喂食若虫（4-8周后幼虫蜕皮）加入到用画笔装置内的皮肤。 </li><li>除去从襟翼的网状粘合剂衬垫和密封就位。最后，除去Duoderm膜露出胶，并将其放置在密封装置的顶部。加入跨开目圈了一块Hypafix磁带，并更换外衣。已完成的遏制装置示于图5A。 </li><li>后5天，麻醉动物如上述和外套被除去。先取出磁带通过网格（ 图5B）检查蜱喂养。仔细剥离Duoderm离瓣。 </li><li>回力网眼部分的边缘，以提供对蜱。美联储蜱常发现接近或下泡沫圈（ 图5C）和被除去并放置在干净的水用画笔。一旦移除所有可见美联储蜱收集到的设备（ 图5D）。 </li></ol>注意:通常情况下，该容器装置可以简单地被剥离远离皮肤。如果附着力强，并有可能损伤皮肤，Unisolve溶剂应用于为温和去除的区域。皮肤擦拭用异丙醇和蜱被存储在23℃下如果用于感染的蜱可以粉碎以确认含有B的数螺旋体，如果用于xenodiagnosis，蜱是前中肠内容分析保存1-3周。

作者宣称，他们有没有竞争的财务权益。

为了获得该蜱B.海港螺旋体对下游的研究，蜱可以是:（1）在幼虫期饲喂感染小鼠;（2）浸在乙螺旋体文化无论是在幼虫或若虫期48;（3）显微注射与B疏螺旋体 47，或（4）毛细管喂养B.螺旋体49。虽然每种方法都有其目的，确保用于港口感染B的蜱一大截螺旋体 ，我们看好毛细管喂养。如果不要求接种与已知数量的螺旋体，所述?...

2011年，莱姆病是在北美（第6最常见的在全国范围内通报的疾病<a href="http://www.cdc.gov/lyme/stats/index.html" target="_blank">http://www.cdc.gov/lyme/stats/index.html</a> ）B.螺旋体是一种多用途的微生物，无论是基因和抗原（1审阅）。其遗传组成包括一个大型（&gt; 900 KB）染色体和高达21质粒（12线，9圆），用分离株中质粒的内容不同。多是要了解该螺旋体，因为质粒的开放阅读框的90％以上是与任何已知的细菌序列2,3，B。螺旋体呈现多种抗原作为宿主免疫的潜在目标。然而，一个未经治疗的感染往往持续存在。螺旋体的蜱环境和脊椎动物宿主环境的相互作用就必须适应由B.螺旋体在整个感染过程。几个质粒编码的基因是已知的差异表达以响应变化的温度，pH，细胞密度和虱子的生命周期4-8的偶数级。 B的研究感染后的自然路线螺旋体适应整个流行性周期，宿主反应依赖于饲料蜱合适的动物模型的能力。这些研究都见了产生的滴答窝藏B的技术挑战螺旋体 ，并确保模型主机上的刻度高效传输和/或喂食。此外，受感染的蜱的遏制和恢复是至关重要的。其中所使用的模型是小鼠和非人类灵长类动物，其中每个作为莱姆病研究的重要工具。由于与白足鼠，这是一个自然储存宿主为B。螺旋体 ，实验室鼠标是支持持续感染的B.一个高度易感宿主螺旋体 9。折伞仰角调整疾病易感小鼠，如C3H品系的感染，螺旋体传播到多个组织，包括皮肤，膀胱，肌肉，关节和心脏。炎症反应的感染导致患病的心脏和关节组织。而螺旋体坚持这种主机和保持感染性，炎症性病变可能成为间歇性的，没有什么不同的进程中人类。在小鼠模型也因此提供了B.多的信息螺旋体引起的病变，包括关节炎和心脏炎和宿主免疫反应10-12。来自病原体的角度来看，哺乳动物感染过程中差异表达的某些基因已被鉴定，因为有一些必要的用于从蜱矢量13-21的传输。 虽然几种动物物种已被用于研究莱姆病22，猕猴最密切模仿人类疾病23的多器官字符。不像其他的动物模型中，疾病表现如游走性红斑，心脏炎，关节炎，以及外周和中枢神经系统的神经病变的广度在猕猴观察。在小鼠体内，储存宿主为B。螺旋体，病小鼠品系和24年龄而异，而早期和晚期弥漫性表现是罕见的9。此外，其它啮齿动物，兔类动物，犬科动物和所有不能从B.表现出神经系统疾病螺旋体感染25。重要的是，猕猴表现出的迹象表明，在所有三个阶段莱姆疏螺旋体病，即早期的本地化，早期传播，晚期莱姆病26-28的特点。游走性红斑（EM）被认为是发生在人类病例29 70-80％，并且也出现在恒河猴28,30。感染后，螺旋体从接种部位传播到多个器官。螺旋体DNA已在骨骼亩被发现scles，心脏，膀胱，外周神经和神经丛，以及存在于中枢神经系统（大脑，脑干和小脑，脊髓和硬膜）31。 勾选喂养小鼠已动用我们和其他研究小组对蜱菌落繁殖，在水库竞争力研究32-36和B的研究螺旋体病机37-40。这种技术也被用于xenodiagnosis和小鼠41-44疫苗效力的测试。我们已经喂硬蜱蜱的非人灵长类动物模型开发28，疫苗效力45的研究，并为xenodiagnosis在持久性后抗生素治疗46的评估。蜱的港口B.疏螺旋体可以被保持在一个天然的地方性动物病周期由在感染的小鼠喂养幼虫和使用若虫进行研究，作为螺旋体通过生命阶段发送。在这份报告中，我们指示如何生成感染了野生型和突变体B蜱螺旋体 ，利用毛细管喂养。这也可以通过显微注射47和通过浸入48来完成的。人工引入B的目的螺旋体进入蜱可学习的突变株，其传输率是未知的，生成一组蜱具有高感染率， ​​并保持清洁，否则未感染的壁虱的殖民地，以减少潜在的错误。此外，我们证明对小鼠和非人灵长类动物剔喂养，这样才能确保遏制和充满蜱的恢复。使用蜱喂养是对免疫反应B.今后的研究中必不可少的螺旋体感染，潜在的莱姆疫苗效力，并xenodiagnosis检测隐匿性感染。

<table border="1">
		<tbody>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Reagent</td>
		 </tr>
		 <tr>
			<td>BSK-H</td>
			<td>Sigma</td>
			<td>B-8291</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Ketamine HCl</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Tangle Trap coating Paste</td>
			<td>Ladd research</td>
			<td>T-131</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SkinPrep</td>
			<td>Allegro Medical Supplies</td>
			<td>177364</td>
			<td></td>
		 </tr>
		 <tr>
			<td>LeFlap, 3" x 3"</td>
			<td>Monarch Labs</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hypafix tape</td>
			<td>Allegro Medical Supplies</td>
			<td>191523</td>
			<td></td>
		 </tr>
		 <tr>
			<td>SkinBond</td>
			<td>Allegro Medical Supplies</td>
			<td>554536</td>
			<td></td>
		 </tr>
		 <tr>
			<td>UniSolve</td>
			<td>Allegro Medical Supplies</td>
			<td>176640</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Biatane Foam, adhesive 4"x4"</td>
			<td>Coloplast</td>
			<td>3420</td>
			<td></td>
		 </tr>
		 <tr>
			<td>DuoDerm CGF Dressing - 4" x 4", (3/4)" adhesive border </td>
			<td>Convatec</td>
			<td>187971 </td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nonhuman primate jackets with flexible 2" back panels; add drawstrings at top and bottom</td>
			<td>Lomir Biomedical Inc.</td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT </td>
		 </tr>
		 <tr>
			<td>Pipet puller</td>
			<td>David Kopf Instruments</td>
			<td>Model 700C</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dark field microscope</td>
			<td>Leitz Wetzlar</td>
			<td>Dialux</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dissecting microscope</td>
			<td>Leica</td>
			<td>Zoom 2000</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Mouse caging</td>
			<td>Allentown caging</td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

feeding of ticks on animals for transmission and xenodiagnosis in lyme disease research

莱姆病， 莱姆病螺旋体的病原体的传播，就会发生由硬蜱种的附着和吸血虱对哺乳动物宿主。在自然界中，这种人畜共患病原菌可使用多种储存宿主，但白足鼠（Peromyscus leucopus）是主要水库在北美幼虫和若虫蜱。人类是最常见的感染B.偶然的主机由蜱的若虫阶段。B.咬螺旋体螺旋体适应它的主机在整个地方性动物病周期，所以要探讨哺乳动物宿主这些螺旋体及其效果的功能的能力需要使用蜱喂养。此外，xenodiagnosis的技术（使用天然矢量检测和传染性病原体的恢复）已在神秘的感染研究是有用的。为了获得蜱若虫该港口B。螺旋体 ，蜱是通过毛细管喂活螺旋体的文化。两种动物模型，小鼠和非人灵长类动物，最常用于涉及蜱喂养莱姆病的研究。我们证明了这些蜱可在喂食，并从动物无论是感染或xenodiagnosis恢复的方法。

继完成毛细管喂养，蜱通常在休息23℃，2〜3周，他们被送到动物进行传输之前。利用毛细管喂养技术，我们发现美联储的90％以上蜱港口B.疏螺旋体。的阳性蜱的百分比是通过洗涤来确定蜱中过氧化物和乙醇，然后粉碎它们在无菌PBS用微量离心管形杵。肠内容物溢入PBS固定在玻片上，并用抗- 疏种抗体是FITC标记的。通过荧光显微镜观察代表蜱中肠涂片在图2B-C中所描绘<...

Watch this Scientific Journal Video about Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research at JoVE.com

Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

莱姆病是最常见报告的媒介传播疾病在北美。病原体， 莱姆病螺旋体是由蜱传播螺旋体细菌。传输感染的动物模型，并检测是通过使用刻度加料，其中我们在这里描述了优化。

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the  attachment and blood feeding of Ixodes species ticks on ...

feeding-ticks-on-animals-for-transmission-xenodiagnosis-lyme-disease

Research

JoVE Journal

Immunology and Infection

13.1K 次观看.  Tulane University Health Sciences Center. 莱姆病是最常见报告的媒介传播疾病在北美。病原体， 莱姆病螺旋体是由蜱传播螺旋体细菌。传输感染的动物模型，并检测是通过使用刻度加料，其中我们在这里描述了优化。 

视频: Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research

RNA Interference in Ticks

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors of human diseases1 and the most important vectors affecting cattle industry worldwide2. Ticks are classified in the subclass Acari, order Parasitiformes, suborder Ixodida and are distributed worldwide from Arctic to tropical regions3. Despite efforts to control tick infestations, these ectoparasites remain a serious problem for human and animal health4,5.
RNA interference (RNAi)6 is a nucleic acid-based reverse genetic approach that involves disruption of gene expression in order to determine gene function or its effect on a metabolic pathway. Small interfering RNAs (siRNAs) are the effector molecules of the RNAi pathway that is initiated by double-stranded RNA (dsRNA) and results in a potent sequence-specific degradation of cytoplasmic mRNAs containing the same sequence as the dsRNA trigger7-9. Post-transcriptional gene silencing mechanisms initiated by dsRNA have been discovered in all eukaryotes studied thus far, and RNAi has been rapidly developed in a variety of organisms as a tool for functional genomics studies and other applications10.
RNAi has become the most widely used gene-silencing technique in ticks and other organisms where alternative approaches for genetic manipulation are not available or are unreliable5,11. The genetic characterization of ticks has been limited until the recent application of RNAi12,13. In the short time that RNAi has been available, it has proved to be a valuable tool for studying tick gene function, the characterization of the tick-pathogen interface and the screening and characterization of tick protective antigens14. Herein, a method for RNAi through injection of dsRNA into unfed ticks is described. It is likely that the knowledge gained from this experimental approach will contribute markedly to the understanding of basic biological systems and the development of vaccines to control tick infestations and prevent transmission of tick-borne pathogens15-19.

A method for RNA interference (RNAi) by injection of dsRNA into unfed ticks is described. RNAi is the most widely used gene-silencing technique in ticks where the use of other methods of genetic manipulation has been limited.

RNA干扰在蜱

Ticks are obligate hematophagous ectoparasites of wild and domestic animals and humans, and are considered to be second worldwide to mosquitoes as vectors ...

科研

免疫与感染

Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1. A tick-borne murine model 2 has been developed to study Lyme disease in the laboratory. While na&iacute;ve ticks can be infected with B. burgdorferi by feeding them on infected mice, the molting process takes several weeks to months to complete. Therefore, development of more rapid and efficient tick infection techniques, such as a microinjection-based procedure, is an important tool for the study of Lyme disease 3,4. The procedure requires only hours to generate infected ticks and allows control over the delivery of equal quantities of spirochetes in a cohort of ticks. This is particularly important as the generation of B. burgdorferi infected ticks by the natural feeding process using mice fails to ensure 100% infection rate and potentially results in variation of pathogen burden amongst fed ticks. Furthermore, microinjection can be used to infect ticks with B. burgdorferi isolates in cases where an attenuated strain is unable to establish infection in mice and thus can not be naturally acquired by ticks 5. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA 6. In this article, we will demonstrate the microinjection of nymphal ticks with in vitro-grown B. burgdorferi. We will also describe a method for localization of Lyme disease pathogens in the tick gut using confocal immunofluorescence microscopy.

Lyme disease research studies often require generation of ticks infected with the pathogen Borrelia burgdorferi, a process that typically takes several weeks. Here we demonstrate a microinjection-based tick infection procedure that can be accomplished within hours. We also demonstrate an immunofluorescence method for in situ localization of B. burgdorferi within ticks.

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle 1.  ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

唾液，唾液腺，淋巴收集肩突硬蜱蜱

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

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

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B 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 ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

人精切肺切片中物质的细胞毒性及免疫调节作用的评价

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

大动物淋巴结的采集与处理 RNA 分析: 大动物 Transcriptomic 淋巴结的制备研究

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...

Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in hospitals and research laboratories. In addition, the Trypanosoma cruzi infection is transmitted congenitally from a&#160;chagasic mother to her offspring, but the male partner&#39;s contribution to in utero contamination is unknown. The findings of nests and clumps of amastigotes and of trypomastigotes in the theca cells of the ovary, in the goniablasts and in the lumen of seminiferous tubules suggest that T. cruzi infections are sexually transmitted. The research protocol herein presents the results of a family study population showing parasite nuclear DNA in the diploid blood mononuclear cells and in the haploid gametes of human subjects. Thus, three independent biological samples collected one year apart confirmed that T. cruzi infections were sexually transmitted to progeny. Interestingly, the specific T. cruzi antibody was absent in the majority of family progeny that bore immune tolerance to the parasite antigen. Immune tolerance was demonstrated in chicken refractory to T. cruzi after the first week of embryonic growth, and chicks hatched from the flagellate-inoculated eggs were unable to produce the specific antibody.&#160;Moreover, the instillation of the human semen ejaculates intraperitoneally or into the vagina of naive mice yielded T. cruzi amastigotes in the epididymis, seminiferous tubule, vas deferens and uterine tube with an absence of inflammatory reactions in the immune privileged organs of reproduction. The breeding of T. cruzi-infected male and female mice with naive mates resulted in acquisition of the infections, which were later transmitted to the progeny. Therefore, a robust education, information and communication program that involves the population and social organizations is deemed necessary to prevent Chagas disease.

The Trypanosoma cruzi agent of Chagas disease produces long-lasting asymptomatic infections that abruptly develop into clinically recognized pathology. The following research protocol describes a short-run family-based epidemiological study to unravel the T. cruzi infection transmitted sexually from parent to progeny.

美国锥虫从男性和女性到天真的命运的性传播

American trypanosomiasis is transmitted to humans by triatomine bugs through the ingestion of contaminated food, by blood transfusions or accidently in ...

Highly Efficient Transfection of Primary Macrophages with In Vitro Transcribed mRNA

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern recognition receptors (PRRs). Unfortunately, this also makes macrophages particularly challenging to transfect as the transfection reagent and the transfected nucleic acids are often recognized by the PRRs as non-self. Therefore, transfection often results in macrophage activation and degradation of the transfected nucleic acids or even in suicide of the macrophages. Here, we describe a protocol that allows highly efficient transfection of murine primary macrophages such as peritoneal macrophages (PM) and bone marrow-derived macrophages (BMDM) with mRNA in vitro transcribed from DNA templates such as plasmids. With this simple protocol, transfection rates of about 50-65% for PM and about 85% for BMDM are achieved without cytotoxicity or immunogenicity observed. We describe in detail the generation of mRNA for transfection from DNA constructs such as plasmids and the transfection procedure.

Macrophages, especially primary macrophages, are challenging to transfect as they specialize in detecting molecules of non-self origin. We describe a protocol that allows highly efficient transfection of primary macrophages with mRNA generated from DNA templates such as plasmids.

具有体外转录mRNA的初级巨噬细胞的高效转染

Macrophages are phagocytic cells specialized in detecting molecules of non-self origin. To this end, they are equipped with a large array of pattern ...

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of UTI in male and female mice have illustrated the procedures for bacterial inoculation and enumeration in urine and tissues. During an initial bladder infection in C57BL/6 mice, UPEC establish latent reservoirs inside bladder epithelial cells that persist following clearance of UPEC bacteriuria. This model builds on these studies to examine rUTI caused by the emergence of UPEC from within latent bladder reservoirs. The urogenital bacterium Gardnerella vaginalis is used as the trigger of rUTI in this model because it is frequently present in the urogenital tracts of women, especially in the context of vaginal dysbiosis that has been associated with UTI. In addition, a method for in situ bladder fixation followed by scanning electron microscopy (SEM) analysis of bladder tissue is also described, with potential application to other studies involving the bladder.

Recurrent Escherichia coli Urinary Tract Infection Triggered by Gardnerella vaginalis Bladder Exposure in Mice

A mouse model of uropathogenic E. coli (UPEC) transurethral inoculation to establish latent intracellular bladder reservoirs and subsequent bladder exposure to G. vaginalis to induce recurrent UPEC UTI is demonstrated. Also demonstrated are the enumeration of bacteria, urine cytology, and in situ bladder fixation and processing for scanning electron microscopy.

由加德纳菌阴道膀胱暴露在小鼠中引发的复发性大肠杆菌尿路感染

Recurrent urinary tract infections (rUTI) caused by uropathogenic Escherichia coli (UPEC) are common and costly. Previous articles describing models of ...