Name: a model for experimental exposure of humans to larval ixodes scapularis ticks
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这项研究得到了美国国立卫生研究院（NIH）国家过敏和传染病研究所（National Institute of Allergy and Infectious Diseases）校内研究计划的支持。我们感谢 Linden T. 胡、Sam R. Telford III、Kenneth Dardick、Carla Williams、Erin Chung 和 Christina Brandeburg 参与制定程序。

让志愿者暴露于蜱虫叮咬是一种实验方法，必须根据相关监管机构批准的临床研究方案进行。临床研究（NCT01143558、NCT02446626 和 NCT05036707）由各自的机构审查委员会批准，在美国食品和药物管理局授予的研究性器械豁免下进行，并根据良好临床实践指南进行。此外，这些研究已在 ClinicalTrials.gov 注册，并获得了所有参与者的书面知情同意书。
1. 密闭敷料准备
<ol><li>要创建测量模板，请沿测量线的一侧复印 3“ x 3” 水胶体密封敷料（图 1A 和 材料表）。</li>
	<li>沿 1 英寸线切割复印模板。然后，沿着 2 英寸的线切割，创建一个 2 英寸的圆，中心为 1 英寸（图 1B）。</li>
	<li>使用此模板，切割非粘性泡沫敷料，创建一个 2 英寸的圆圈，中心为 1 英寸（图 2A、B 和 材料表）。</li>
	<li>使用相同的模板，在 4“ x 4” 超薄水胶体敷料的中心切一个 2 英寸的圆圈（图 2C，D）。</li>
	<li>在 11/2“ 线上切割 3” x 3“ 密闭敷料的水胶体层。</li>
	<li>拉回安全壳敷料的水胶体层的纸衬垫，露出粘合剂。将切割的泡沫敷料放入粘合剂的 2 英寸圆内（图 3A）。这将确保泡沫敷料延伸到安全壳敷料的开口之外。将纸衬垫更换到敷料的水胶体层上（图3B）。 注：这些测量值用于涉及多达 10 个 幼虫 I. scapularis 蜱的研究。对于涉及多达 30 只 肩胛蜱 幼虫的研究，泡沫中心的模板在 1 英寸和 11/2 英寸标记之间切割，以留出更大的放置区域。密闭敷料的测量值可以根据研究设计进行调整。</li>
</ol>2. 刻度放置
<ol><li>即时报价存储<ol><li>将 肩胛蜱 幼虫储存在医疗级冰箱中，温度设置为 9-10 °C。</li>
		<li>将蜱虫存放在覆盖有网帽的小瓶中，以允许足够的气流。用轻轻湿润的海绵将一小瓶蜱虫包装在可密封的塑料袋中，以保持湿度并防止干燥。</li>
	</ol></li>
	<li>放置刻度<ol><li>从冰箱中取出蜱虫，让它们加热到室温。蜱虫可以在冰箱外短短 15 分钟内变得活跃。</li>
		<li>在放置区域周围放置一张白纸，以便轻松看到任何无意中掉落的刻度。</li>
		<li>用清水或生理盐水弄湿 4 英寸 x 4 英寸的纱布垫，然后用它来清洁放置部位的皮肤。该区域应拍干或让其风干。 注意：在选择放置蜱虫的身体部位时，请考虑不限制志愿者日常活动的区域，并且参与者可以轻松监测敷料的完整性。不要将蜱虫放置在可能因直接压力而被压碎的区域（例如背部）。评估参与者的体毛，如有必要，使用剪刀在粘合剂部位小心修剪过多的体毛，避免对皮肤造成伤害。避免使用剃须刀脱毛，因为它可能会增加皮肤受伤和感染的风险。</li>
		<li>一旦皮肤干燥，从准备好的敷料的水胶体侧取下粘性衬垫，并将其牢固地固定在放置部位的皮肤上，确保网状层保持开放。</li>
		<li>将木棉签（见 材料表）对半掰开，露出锯齿状的末端。</li>
		<li>小心地打开一小瓶蜱虫。</li>
		<li>使用木棉签的锯齿状端，从小瓶中转移活性蜱虫，并将它们放在收容敷料内的皮肤上（图4A）。让额外的团队成员监控蜱虫放置部位，以确保蜱虫留在收容敷料内。在此过程中，使用胶带捕获从小瓶中逸出的蜱虫。 注意： 打破木签并使用锯齿状的一端可以有效地操作和转移蜱虫，而不会对蜱虫造成损坏。带有浅色柔软刷毛的小刷子也可用于将蜱虫从容器转移到皮肤部位。</li>
		<li>放置研究的指定数量的刻度后，从密闭敷料的网状层上取下粘性衬垫，并将其封闭在开口处。</li>
		<li>在确保粘合剂牢固附着后，将准备好的 4“ x 4” 超薄水胶体敷料涂在密闭敷料上以增加安全性（图 4B）。 注：有关无病原体肩胛胛蜱菌落和引用研究中使用的蜱暴露程序持续时间的更多信息，可在已发表研究19,22 的方法部分找到。</li>
	</ol></li>
</ol>3. 现场护理
<ol><li>为研究参与者提供防水罩和 2 英寸低过敏性胶带（参见 材料表）。</li>
	<li>提供护理蜱虫遏制部位的说明。<ol><li>定期检查敷料并监测边缘是否牢固地粘附在皮肤上。根据需要使用额外的胶带来加固敷料边缘。</li>
		<li>在研究期间，请勿刮擦敷料部位，并避免打开或取下敷料。</li>
		<li>避免洗澡或浸泡在水中。此外，避免热水淋浴并限制淋浴的时间。淋浴前，用提供的防水罩保护安全壳敷料，并在完成淋浴后将其取下。轻轻拍干敷料，并检查它以确保它保持牢固。</li>
		<li>避免可能导致大量出汗和损害敷料完整性的活动（例如，有氧运动、长时间徒步旅行）。</li>
	</ol></li>
</ol>4. 去除蜱虫
<ol><li>收集瓶制备<ol><li>在蜱虫收集瓶上贴上所需的标识符（例如，研究代码、日期）。</li>
		<li>如果需要保存活蜱虫，用20G针在小瓶盖上穿孔。使用一层尼龙或聚酯网以及瓶盖密封小瓶。将一小块湿海绵与一小瓶蜱虫一起放入可密封的袋子中以保持湿度。</li>
	</ol></li>
	<li>去除蜱虫<ol><li>在绷带部位周围铺上白色床单，并确保收集盆触手可及。</li>
		<li>使用酒精湿巾，小心地从皮肤上取下绷带。</li>
		<li>完全取下后，将绷带放入收集盆中，并检查是否存在脱落的蜱虫。</li>
		<li>评估放置地点是否有附着的蜱虫。已经补充的蜱虫将分离（图5A）。对于剩余的附着的蜱虫，使用细尖镊子去除（图5B）。</li>
		<li>用酒精湿巾或肥皂和水清洁皮肤。</li>
	</ol></li>
</ol>

人类接触幼虫蜱虫用于蜱传疾病研究

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Adriana Marques 博士拥有美国专利 8,926,989 B2;并且是全球莱姆病联盟和美国莱姆病基金会的无薪科学顾问。Siu Ping Turk 和 Aleah Eschman 没有可能构成利益冲突的关联。本出版物的内容并不一定反映美国卫生与公众服务部的观点或政策，提及商品名称、商业产品或组织也不意味着美国政府的认可。

虽然涉及暴露于蜱虫的动物研究 4,5,6,7,21 对于提高我们对宿主对蜱传疾病和蜱虫叮咬反应的理解非常宝贵，但这些模型在预测人类宿主反应的程度上存在局限性。该模型描述了以可控方式将人类暴露于蜱虫叮咬的方法，可以很容易地适应回答不同的研究问题，并扩展了在人?...

硬蜱（硬蜱科：Acari）是全球范围内发生的专性血液摄食性体外寄生虫，能够传播广泛的病原体，包括细菌、病毒和寄生虫，具有重要的医学和兽医意义。硬蜱蜱必须与宿主保持附着数天才能完成血粉，它们有能力保持附着在皮肤上，同时避免识别，防止局部血液凝固，并促进长期摄食1,2,3。动物研究表明，不宽容的宿主会通过反复接触蜱虫叮咬获得对蜱虫叮咬的抵抗力，这可能导致传播病原体的能力降低，而蜱虫可以反复寄生于宽容的宿主。获得性蜱耐药性取决于宿主免疫反应的性质 4,5,6,7。
蜱传疾病在美国是一个日益严重的威胁，2004 年至 2016 年间报告的病例数增加了一倍多 8,9。由于气候变化，不同蜱虫的地理范围继续扩大10,11。美国主要的蜱传疾病包括莱姆病、无形体病、埃立克体病、斑疹热立克次体病、巴贝虫病、土拉菌病和波瓦桑病毒病8。莱姆病由伯氏疏螺旋体感染引起，是美国和欧洲最常见的蜱传疾病12.美国每年约有 476,000 人被诊断患有莱姆病，这给个人和社会带来了公共卫生和经济负担 13,14,15。
肩胛硬蜱 （黑腿蜱或鹿蜱）是莱姆病的主要传播媒介，在美国也是无形体病、巴贝虫病、 宫本疏螺旋体 病和波瓦桑病毒病的主要传播媒介。在美国，其他具有医学重要性的蜱种包括 Amblyomma americanum （孤星蜱）、 Dermacentor variabilis （美国狗蜱）、 太平洋硬蜱 （西部黑腿蜱）、 Dermacentor andersni （落基山木蜱）、 Cookei硬蜱 （土拨鼠蜱）、 Dermacentor occidentalis （太平洋沿岸蜱）、 Rhipicephalus sanguineus （棕色狗蜱）和 Amblyomma maculatum （墨西哥湾沿岸蜱）16。
开发一种使研究志愿者暴露于蜱虫叮咬的方法支持使用自然载体寻找感染证据的研究，这一过程称为异种诊断 17,18,19,20,21，并更多地了解暴露于蜱虫引起的免疫力，这有助于发现抗蜱虫疫苗 5,6,7.本文描述的程序是在2014年发表的第一项人类研究中开发和使用的，该研究使用实验室饲养的肩胛硬蜱幼虫在抗生素治疗（NCT01143558）后对伯氏疏螺旋体感染进行异种诊断19。该系统已成功用于一项 2 期研究，该研究调查了阳性异种诊断是否与莱姆病 （NCT02446626） 抗生素治疗后症状的持续存在相关，以及一项探索宿主对蜱虫叮咬反应的研究 （NCT05036707）。
该程序协议描述了创建收容敷料、蜱虫放置程序和蜱虫清除程序的过程，以及维持收容敷料所需的现场护理。关于上述研究中使用的无病原体肩胛胛蜱菌落和蜱暴露程序的详细信息之前已经描述过19,22。这种方法提供了一种灵活的研究工具，可用于研究人类宿主对蜱虫叮咬的反应、蜱虫预防药物的有效性以及莱姆病和其他蜱传疾病的不同方面。

<table><tbody><tr><td>20 G needle</td><td>Any brand</td><td></td><td>For puncturing the vial cap.</td></tr><tr><td>3&quot; x 3&quot; containment dressing</td><td>Monarch Labs Names</td><td>LeFlap</td><td>https://www.monarchlabs.com/ordering</td></tr><tr><td>4&quot; x 4&quot; extra-thin hydrocolloid dressing</td><td>ConvaTec</td><td>DuoDerm</td><td>https://www.convatec.com/products/advanced-wound-care/brand-names/pc-wound-duoderm-granluflex/duoderm-extra-thin-dressing/</td></tr><tr><td>4&quot; x 4&quot; gauze</td><td>Monarch Labs Names</td><td></td><td>For cleaning skin</td></tr><tr><td>Clean water or saline</td><td></td><td></td><td>For cleaning skin</td></tr><tr><td>Moisture barrier (e.g. 7&quot; x 7&quot;)</td><td>AquaGuard</td><td>TIDI</td><td>For showering, ttps://www.tidiproducts.com/product-listing/aquaguard-shower-cover-sheets&amp;nbsp;</td></tr><tr><td>Non-adhesive foam dressing</td><td>Coloplast</td><td>Biatain</td><td>https://www.coloplast.us/biatain-non-adhesive-en-us.aspx</td></tr><tr><td>Roll of 2&quot; hypoallergenic tape</td><td>Monarch Labs Names</td><td>Durapore</td><td>For reinforcing containment dressing.</td></tr><tr><td>Roll of adhesive tape</td><td></td><td></td><td>For trapping ticks</td></tr><tr><td>Vials for collection (e.g. cryovials)</td><td>Ependorf</td><td>ECC200</td><td></td></tr></tbody></table>

a model for experimental exposure of humans to larval ixodes scapularis ticks

蜱传疾病是美国和全世界的一个重大公共卫生问题。蜱虫是专性吸血节肢动物;硬蜱必须保持附着在宿主的皮肤上，并完成其为期多天的摄食过程以获取其血粉。将动物暴露于蜱虫是研究宿主对蜱虫叮咬和蜱传疾病反应的常见做法。我们开发了该程序，进行了第一项人体研究，并发表了关于将人类志愿者暴露于未感染的 肩胛硬蜱 幼虫的研究结果。本文介绍了用于构建收容敷料的方法，如何将蜱虫应用并固定到宿主身上，如何维护敷料，以及如何从宿主中清除蜱虫。让志愿者暴露于蜱虫叮咬是一种实验程序，必须根据相关监管机构批准的临床研究方案进行。这种方法允许转化研究，以更好地了解人类对蜱虫叮咬的反应，并促进蜱传疾病的诊断、预防和治疗的发展。

该研究表明，该手术是安全且耐受性良好的，主要不良事件是叮咬部位的轻度瘙痒，在 58% 的手术中观察到。当使用干净的实验室饲养的肩 胛蜱幼 虫时，没有发生与该程序相关的严重不良事件19.在进行的 43 个程序中，与放置的蜱虫相比，附着的蜱虫的平均恢复百分比为 45% ± 27% （SD），中位数百分比为 40%（图 6）。
肩胛蜱</e...

Watch this Scientific Journal Video about A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks at JoVE.com

作者聚焦：用于蜱虫研究和莱姆病研究的受控人类暴露模型

本文介绍了将人类暴露于 肩胛硬蜱 幼虫以进行临床研究的方法。该技术比较简单，研究志愿者可以忍受，并且可以根据实验需要进行修改。此类涉及人类受试者的研究必须根据适当监管机构批准的临床研究方案进行。

人类接触 肩胛蜱 幼虫的实验暴露模型

Tickborne diseases are a significant public health problem in the United States and worldwide. Ticks are obligate blood-feeding arthropods; an ixodid tick ...

该协议为人类受控地暴露于蜱虫提供了一种有效的方法。对于研究，该协议可用于研究人类免疫反应蜱叮咬，以及开发针对蜱传疾病的诊断和预防措施。没有公布对人类研究志愿者进行蜱虫的程序。挑战在于开发一种系统，让蜱虫能够进食数天，同时又让参与者感到舒适。该模型包含喂养期间的蜱虫，并允许研究参与者继续进行日常活动，并对生活方式进行微小的调整。

a-model-for-experimental-exposure-humans-to-larval-ixodes-scapularis

Research

JoVE Journal

Immunology and Infection

A Model for Experimental Exposure of Humans to Larval Ixodes scapularis Ticks

449 次观看.  National Institutes of Health. 该协议为人类受控地暴露于蜱虫提供了一种有效的方法。对于研究，该协议可用于研究人类免疫反应蜱叮咬，以及开发针对蜱传疾病的诊断和预防措施。没有公布对人类研究志愿者进行蜱虫的程序。挑战在于开发一种系统，让蜱虫能够进食数天，同时又让参与者感到舒适。该模型包含喂养期间的蜱虫，并允许研究参与者继续进行日常活动，并对生活方式进行微小的调整?...

视频: 作者聚焦：用于蜱虫研究和莱姆病研究的受控人类暴露模型

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 ...

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

Transmission of the etiologic agent of Lyme disease, Borrelia burgdorferi, occurs by the attachment and blood feeding of Ixodes species ticks on mammalian hosts. In nature, this zoonotic bacterial pathogen may use a variety of reservoir hosts, but the white-footed mouse (Peromyscus leucopus) is the primary reservoir for larval and nymphal ticks in North America. Humans are incidental hosts most frequently infected with B. burgdorferi by the bite of ticks in the nymphal stage. B. burgdorferi adapts to its hosts throughout the enzootic cycle, so the ability to explore the functions of these spirochetes and their effects on mammalian hosts requires the use of tick feeding. In addition, the technique of xenodiagnosis (using the natural vector for detection and recovery of an infectious agent) has been useful in studies of cryptic infection. In order to obtain nymphal ticks that harbor B. burgdorferi, ticks are fed live spirochetes in culture through capillary tubes. Two animal models, mice and nonhuman primates, are most commonly used for Lyme disease studies involving tick feeding. We demonstrate the methods by which these ticks can be fed upon, and recovered from animals for either infection or xenodiagnosis.

Lyme disease is the most commonly-reported vector-borne disease in North America. The causative agent, Borrelia burgdorferi is a spirochete bacterium transmitted by Ixodid ticks. Transmission and detection of infection in animal models is optimized by the use of tick feeding, which we describe here.

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 ...

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a three host tick that requires a blood meal from a vertebrate host for each development stage, and the adult females require a blood meal for reproduction. Larval ticks attach to their host for 3 - 5 days for feeding and drop off the host when fully engorged. This dependency on several different hosts and the lengthy attachment time for engorgement complicates tick rearing in the laboratory setting. However, to understand tick biology and tick-pathogen interactions, the production of healthy, laboratory-reared ticks is essential. Here, we demonstrate a simple, cost-effective protocol for immature tick feeding on mice. We modified the existing protocols for decreased stress on mice and increased tick feeding success and survival by using disposable cages without mesh bottoms to avoid contact of ticks with water contaminated with mice urine and feces.

Rearing Ixodes scapularis, the Black-legged Tick: Feeding Immature Stages on Mice

The production of healthy laboratory-reared ticks is essential to studies on tick biology, and tick-pathogen interactions. Here we demonstrate a simple protocol for immature tick feeding that is cost-effective and less stressful to mice.

饲养<em&gt;肩突硬蜱，</em&gt;黑腿蜱:饲养对小鼠未成熟阶段

Ixodes scapularis, the vector of Lyme disease, is one of the most important disease vectors in the eastern and Midwestern United States. This species is a ...

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight loss, depreciation of the animals&#39; leather and also can act as a vector of several pathogens. Due to the exorbitant costs to control these parasites, damage to the environment caused by the inappropriate use of chemical acaricides, and the increased resistance against traditional parasiticides, alternative control of ticks, by the use of entomopathogenic fungi, for example, has been considered an interesting approach. Nevertheless, few studies have demonstrated how the tick&#39;s immune system acts to fight these entomopathogens. Therefore, this protocol demonstrates two methods used for entomopathogen inoculation into engorged females and two techniques used for hemolymph collection and hemocytes harvesting. Inoculation of pathogens at the leg insertion in the tick female&#39;s body allows evaluation of females biologic parameters unlike the inoculation between the scutum and capitulum, which frequently damages Gen&#233;&#39;s organ. Dorsal hemolymph collection yielded a higher volume recovery than collection through the legs. Some limitations of tick hemolymph collection and processing include i) high rates of hemocytes&#39; disruption, ii) hemolymph contamination with disrupted midgut, and iii) low hemolymph volume recovery. When hemolymph is collected through the leg cutting, the hemolymph takes time to accumulate at the leg opening, favoring the clotting process. In addition, fewer hemocytes are obtained in the collection through the leg compared to the dorsal collection, even though the first method is considered easier to be performed. Understanding the immune response in ticks mediated by entomopathogenic agents helps to unveil their pathogenesis and develop new targets for tick control. The inoculation processes described here require very low technological resources and can be used not only to expose ticks to pathogenic microorganisms. Similarly, the collection of tick hemolymph may represent the first step for many physiological studies.

Disclosing Hemolymph Collection and Inoculation of Metarhizium Blastospores into Rhipicephalus Microplus Ticks Towards Invertebrate Pathology Studies

Analysis of tick hemolymph represents an important source of information on how some pathogens cause disease and how ticks immunologically respond to this infection. The present study demonstrates how to inoculate fungal propagules and collect hemolymph from Rhipicephalus microplus engorged females.

在无脊椎动物病理学研究中发现血淋巴收集和接种转移到大黄微加分中

Ticks are obligate hematophagous ectoparasites and Rhipicephalus microplus has great importance in veterinary medicine because it causes anemia, weight ...

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

基于胶囊的模型，用于在实验鼠身上的不成熟硬滴答阶段侵扰

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...

生物学

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

从蜱 离体 唾液腺培养物和细胞外囊泡中分离microRNA

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

用于黑腿蜱、肩突硬蜱基因编辑的胚胎注射技术

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...

Tick Artificial Membrane Feeding for Ixodes scapularis

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ticks during both study and rearing can limit experimental questions or the ability of labs to research ticks and their associated pathogens. An artificial membrane feeding system can reduce these problems and open up new avenues of research that may not have been possible with traditional animal feeding systems. This study describes an artificial membrane feeding system that has been refined for feeding and engorgement success for all Ixodes scapularis life stages. Moreover, the artificial membrane feeding system described in this study can be modified for use with other tick species through simple refinement of the desired membrane thickness. The benefits of an artificial membrane feeding system are counterbalanced by the labor intensiveness of the system, the additional environmental factors that may impact feeding success, and the need to refine the technique for each new species and life stage of ticks.

Tick Artificial Membrane Feeding for Ixodes scapularis

Presented here is a method to blood feed ticks in vitro via an artificial membrane system to allow for partial or full engorgement of a variety of tick life stages.

肩突硬蜱人工膜喂养

Ticks and their associated diseases are an important topic of study due to their public health and veterinary burden. However, the feeding requirements of ...

人类接触肩胛蜱幼虫的实验暴露模型

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此视频中的章节

RNA干扰在蜱

莱姆病的病原体的蜱肠道内的快速传递和本地化的方法

唾液，唾液腺，淋巴收集肩突硬蜱蜱

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

饲养

在无脊椎动物病理学研究中发现血淋巴收集和接种转移到大黄微加分中

基于胶囊的模型，用于在实验鼠身上的不成熟硬滴答阶段侵扰

从蜱离体唾液腺培养物和细胞外囊泡中分离microRNA

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肩突硬蜱人工膜喂养

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副本

摘要

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饲养

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基于胶囊的模型，用于在实验鼠身上的不成熟硬滴答阶段侵扰

从蜱 离体 唾液腺培养物和细胞外囊泡中分离microRNA

用于黑腿蜱、肩突硬蜱基因编辑的胚胎注射技术

肩突硬蜱人工膜喂养

人类接触肩胛蜱幼虫的实验暴露模型

从蜱离体唾液腺培养物和细胞外囊泡中分离microRNA