这个项目是由美国凯斯西储大学启动资金提供给大肠杆菌乔利。感染性钉螺，血吸虫资源中心，国立卫生研究院NIAID的（NIAID的合同号HHSN272201000009I）。我们也感谢克里斯国王和罗纳德布兰顿确保感染血吸虫钉螺的支持和有益的讨论。

安全措施： 血吸虫尾蚴可直接穿透皮肤没有削减或毛孔需要，造成慢性疾病血吸虫病。S。血吸虫是一个生物安全2级的有机体，因此，适当的个人防护设备和感染尾蚴的工作时，必须遵循适当的安全措施。大多数必要的设备，成长和改造血吸虫是标准的，表中所指出的，该协议的结束与几个关键例外。所有的cercarial工作应该做在一个孤立的房间。 Schistosomules应培养在无菌的，经批准的生物安全罩。应佩戴两层腈或乳胶手套，实验室外套，一双防水的鞋子，裤子没有洞或泪水，并为您的手腕和手臂防水袖保护。在一个偶然泄漏的情况下，立即喷用70％乙醇或10％漂白粉的受污染的地区，揉大力如果你的人。这将是足以禁用的寄生虫，但所有可能感染的，应当报和重视。虽然schistosomules是不是天生感染，机构准则各不相同，应征询，以确定是否BSL2的预防措施，在非感染性的人生阶段所需的。来与任何潜在的感染性物质接触后，实验是在经批准的生物危害容器中进行丢弃废物的所有表面消毒。 注：盖蜗牛主机箱1〜2天前脱落和保护由轻 1。收获尾蚴从蜗牛主机<ol><li>填写一个干净，洗涤剂中途平衡至室温，用蒸馏水500ml烧杯。如果在不同的日期与毛蚴感染主机蜗牛的正常工作，使用感染日期为每一个单独的烧杯中（如果蜗牛将会为未来的实验中重用，这一点尤为重要）。烧杯的大小也可根据用户体验。 </li><li>收集你想摆脱仔细存款，他们使用一个标准的观赏鱼的网和通用钳需要烧杯中的受感染的蜗牛主机。 </li><li>泄漏应急放在烧杯托盘白炽灯光源下约8至12英寸。让我们站在为1-2小时，在灯光下。尾蚴离开蜗牛主机，当暴露在明亮的光线。 </li><li>仔细筛选出从尾蚴混合物蜗牛。采取额外的安全防范措施，作为混合物具有高度传染性。记住，尾蚴可直接穿透人体的皮肤！ </li><li>更换光源下约15分钟的混合物，使任何微粒废物问题解决。 </li><li>转移到一个干净的锥形烧瓶中，用吸管的尾蚴，避免在烧杯底部的碎片。烧瓶放置在约45 ° 45在黑暗中的冰角 - 60分钟，允许尾蚴融入一个角落。 </li><li>使用50 mL的吸管，去除75％至90％的上清液和存款中含10％漂白粉或70％乙醇的烧杯中，小心不要让水滴含有尾蚴从吸管滴。 </li><li>摇动三角瓶中含有尾蚴轻轻悬浮在溶液中的尾蚴。使用吸管样品转移到无菌50 ml锥形管。放在冰管，再次为在黑暗中20分钟。尾蚴将解决对试管底部，在黑暗中。 </li></ol> 2。手册转型的尾蚴schistosomules 开始之前：RPMI 1640完全“（5％胎牛血清的RPMI，1X笔/链球菌）和温暖至37 ° C。 （这是至关重要的，已孵化灭活胎牛血清使用56℃，1小时可以作为血吸虫敏感的补充）。 <ol><li>在生物危害罩，取出上清下降到5至10毫升每50 ml锥形解决方案，使用移液器或真空设备。 </li><li> 22计双端连接，露儿乐乳化针，以适当大小的注射器。 </li><li>慢慢地拉入注射器的混合物。收集来自所有50毫升注射器管的尾蚴。滴水周到。 </li><li>小心地翻过来，这样，独立的针尾注射器。将另一注射器乳化针的这一边。 </li><li>穿过针的混合物的40倍（20倍来回），改造尾蚴。 </li><li>位置垂直等混合物包含在底部注射器和顶部的注射器是空的注射器。卸下顶部的注射器和70％乙醇消毒。 </li><li>存款下降混合物放入一个干净的高壁百毫米x 50毫米耐热盘或结晶盘。 70％乙醇消毒针头和注射器。 </li><li>添加37℃的RPMI 1640完整的媒体，如培养皿的底部完全覆盖，仿佛一个卢里亚肉汤板浇筑。 </li><li>涡流轻轻收集在培养皿的中心schistosomules。避免飞溅。 注意：它可能是有用的这一步关闭罩灯，以更充分的可视化中心schistosomules的。 </li><li>立即存入一个新的12孔细胞培养板使用无菌滴管集中schistosomules。 </li><li>直到schistosomules菜（约10倍）的中心不再重复2.9和2.10。定金随后每一个新鲜以及的集合。 </li><li>填写半途而废（〜1.5毫升）预热的RPMI完整的媒体。 </li><li>倒置显微镜，可视化，以验证schistosomules和尾蚴的情况下是否存在。 </li><li>成长中的CO 2培养箱schistosomules设置为37 ° C和5％的CO 2。 </li></ol> 3。代表性的成果： 待转让schistosomules转化成文化传媒，下一个倒置的明场显微镜（见设备表为例）可​​视化应该产生一个类似于图1，其中只有转化schistosomules以及在目前的图像。潜在的污染物可能包括完整的尾蚴，切断cercarial尾巴，或其他杂物。如果没有倒置显微镜，一个标准的显微镜，甚至清扫范围，将有足够的schistosomules可视化。注意，此过程并不总是变换尾蚴的100％。 <img src="/files/ftp_upload/3191/3191fig1.jpg" alt="图1"/> 图1：转化Schistosomulum <img src="/files/ftp_upload/3191/3191fig2.jpg" alt="图2"/> 图2：尾蚴和痛苦的尾巴

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> N Engl J Med. 346, 1212-1220 (2002).</li><li>Steinmann, P., Keiser, J., Bos, R., Tanner, M., Utzinger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis+and+water+resources+development:+systematic+review,+meta-analysis,+and+estimates+of+people+at+risk.">Schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk.</a> Lancet Infect Dis. 6, 411-425 (2006).</li><li>Savioli, L., Albonico, M., Engels, D., Montresor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+the+prevention+and+control+of+schistosomiasis+and+soil-transmitted+helminthiasis.">Progress in the prevention and control of schistosomiasis and soil-transmitted helminthiasis.</a> Parasitol Int. 53, 103-113 (2004).</li><li>King, C. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Schistosoma+mansoni+daughter+sporocysts+from+mother+sporocysts+maintained+in+synxenic+culture+with+Biomphalaria+glabrata+embryonic+(Bge)+cells.">Production of Schistosoma mansoni daughter sporocysts from mother sporocysts maintained in synxenic culture with Biomphalaria glabrata embryonic (Bge) cells.</a> J Parasitol. 81, 714-722 (1995).</li><li>Colley, D. G., Wikel, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+simplified+method+for+the+production+of+schistosomules.">Schistosoma mansoni: simplified method for the production of schistosomules.</a> Exp Parasitol. 35, 44-51 (1974).</li><li>Lazdins, J. K., Stein, M. J., David, J. R., Sher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+rapid+isolation+and+purification+of+schistosomula+of+different+developmental+stages+by+centrifugation+on+discontinuous+density+gradients+of+Percoll.">Schistosoma mansoni: rapid isolation and purification of schistosomula of different developmental stages by centrifugation on discontinuous density gradients of Percoll.</a> Exp Parasitol. 53, 39-44 (1982).</li><li>Cousin, C. E., Stirewalt, M. A., Dorsey, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosoma+mansoni:+ultrastructure+of+early+transformation+of+skin-+and+shear-pressure-derived+schistosomules.">Schistosoma mansoni: ultrastructure of early transformation of skin- and shear-pressure-derived schistosomules.</a> Exp Parasitol. 51, 341-365 (1981).</li><li>Lewis, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schistosomiasis.">Schistosomiasis.</a> Curr Protoc Immunol. Chapter 19, 1-1 (2001).</li></ol>

没有利益冲突的声明。

在改造过程中可能出现的几个潜在的问题;然而，这个协议是为了避免这些问题大部分。一个潜在的危险是乳化针，这通常会出现由于在步骤1.3中的收获遗留下来的碎片堵塞。一定要允许所有的颗粒物质，解决在步骤1.5和转让步骤1.6尾蚴仔细，以避免这个问题。此外，如前所述，内生物安全柜工作，个人防护装备的使用（甚至面罩），避免过度的压力注射器最好。 分离cercarial尾巴可以在媒体上，这是贫困分离在步骤2.9可视化。尾巴表现出痉挛般的议案，并有不同的形状（见图2），而正是如此很容易分辨的schistosomules。可去除清洗多余的RPMI schistosomules完成schistosomules解决的尾巴。尾巴将保持浮动，并可以清理。 如果完整的尾蚴是目前在媒体上，在改造步骤2.5无法被正确执行。一个完整的尾蚴的形象描绘在图3b中，以供参考。如果发生此问题，确保正在使用的乳化针是适当的大小。此外，可以增加cercarial混合物是通过针通过倍，40倍，虽然通常比足够接近100％剪切cercarial尾巴。 尾蚴转化成schistosomules科利和Wikel（13）于1974年首次描述。从那时起，schistosomules准备已经完成在本议定书中，或使用一个振荡的方法和percoll梯度分离（14，15）或纷飞在这纸证明，使用针头和注射器剪切战略。这两种技术准备schistosomules精美的弗雷德刘易斯（16）和进一步培养长期增长schistosomules的信息，可在NIAID的血吸虫资源<a href="http://www.schisto-resource.org/">中心（</a> www.schisto resource.org ）。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th></tr><tr><td> 22克x 2-7/8“乳化针</td><td> Fisher Scientific则</td><td> 14 - 825 - 17G </td></tr><tr><td> RPMI 1640培养液1X（500毫升） </td><td> Invitrogen公司</td><td> 11835030 </td></tr><tr><td>胎牛血清（FBS），热灭活</td><td> Invitrogen公司</td><td> 10082139 </td></tr><tr><td>青霉素/链霉素（100X） </td><td> Invitrogen公司</td><td> 15070063 </td></tr><tr><td>结晶菜（100毫米） </td><td> Fisher Scientific则</td><td> 08 - 741D </td></tr><tr><td>通用镊子</td><td> Fisher Scientific则</td><td> 10 - 316C </td></tr><tr><td> PETCO水族馆净盐水虾</td><td> PETCO </td><td>商品编号：1190954 </td></tr></tbody></table>

cercarial transformation and in vitro cultivation of schistosoma mansoni schistosomules

血吸虫寄生虫病，血吸虫病，长期衰弱的疾病，影响全球超过200万人次，跻身在公众健康和社会经济的影响（1-4）方面寄生虫病的第二个疟疾的病原体。血吸虫寄生虫吸虫蠕虫，软体动物和哺乳动物的主机之间的寄生生活，一个复杂的生命周期转乘干预自由游动阶段。简言之，自由游动的尾蚴感染哺乳动物的主机，通过穿透皮肤分泌的蛋白酶的援助，在这段时间内尾蚴失去它们的尾巴，schistosomules改造。现在schistosomules必须逃避宿主的免疫系统，开发出红血细胞的消化肠道，并迁移虽然肺部，他们在肝门系统的最终目的地途中门静脉循环和最终肠系膜静脉（曼氏血吸虫）其中男性和女性对蠕虫和队友，每天生产数百个鸡蛋。有些是从体内排出的鸡蛋，鸡蛋孵化成自由游动的毛蚴（5-10）到淡水。毛蚴感染特定的蜗牛种，并转化为母亲和女儿sporocysts，这反过来，产生感染尾蚴，完成生命周期。不幸的是，整个生命周期血吸虫不能在体外培养，但可以转化成schistosomules感染尾蚴，schistosomules可周的培养，在体外或微阵列分析分析血吸虫发展。在这个协议中，我们提供了一个可视化描述cercarial改造， 并在体外培养的schistosomules 。我们从蜗牛主机Biomphalaria光滑棚感染尾蚴和手动变换成schistosomules使用乳化针双端分离它们​​的尾巴。 体外 cercarial的改造和schistosomules养殖技术的描述，避免使用哺乳动物的主机，从而简化了血吸虫的可视化和便于实验分析收集的寄生虫。体外转化和血吸虫培养技术多年来一直做的（11， 12），但没有视觉的协议已经开发了整个社会。

Watch this Scientific Journal Video about Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules at JoVE.com

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

我们描述了一个<em&gt;在体外</em&gt;扁虫的寄生虫血吸虫童虫培养方法<em&gt;曼氏血吸虫</em&gt;，通过收获和改造感染尾蚴的淡水蜗牛中间宿主Biomphalaria光滑。

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

Schistosome parasites are the causative agents of schistosomiasis, a chronically debilitating disease that affects over 200 million people globally and ...

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Immunology and Infection

Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

18.0K 次观看.  Case Western Reserve University. 我们描述了一个在体外扁虫的寄生虫血吸虫童虫培养方法曼氏血吸虫，通过收获和改造感染尾蚴的淡水蜗牛中间宿主Biomphalaria光滑。

视频: Cercarial Transformation and in vitro Cultivation of Schistosoma mansoni Schistosomules

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation. 
Following intraperitoneal sensitization and intravenous challenge, S. mansoni eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni eggs from infected livers (modified from 7), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis. 
Overall, this method provides an in vivo model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.

Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation

Schistosoma mansoni eggs are potent stimulators of the T helper type 2 (Th2) immune response, characteristic of parasite infection, asthma and allergic inflammation. This protocol utilizes S. mansoni egg injection to generate a CD4 Th2 cytokine-induced inflammatory response in the lung, characterized by lung granuloma formation around the egg, eosinophilia and macrophage alternative activation.

从使用鸡蛋<em&gt;血吸虫</em&gt;作为<em&gt;在体内</em&gt;蠕虫引起的肺部炎症模型

Schistosoma parasites are blood flukes that infect an estimated 200 million people worldwide 1. In chronic infection with Schistosoma, the severe ...

科研

免疫与感染

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and mammalian final host, respectively. The ability to isolate and sustain the different life cycle stages under in vitro culture conditions has greatly facilitated investigations of the cellular, biochemical and molecular mechanisms regulating parasite growth, development and host interactions. Transmission of schistosomiasis requires asexual reproduction and development of multiple larval stages within the snail host; from the infective miracidium, through primary and secondary sporocysts, to the final cercarial stage that is infective to humans. In this paper we present a step-by-step protocol for mass hatching and isolation of Schistosoma mansoni miracidia from eggs obtained from livers of infected mice, and their subsequent introduction into in vitro culture. It is anticipated that the detailed protocol will encourage new researchers to engage in and broaden this important field of schistosome research.

Mass Isolation and In Vitro Cultivation of Intramolluscan Stages of the Human Blood Fluke Schistosoma Mansoni

This article describes a protocol for the large-scale axenic isolation and collection of free-swimming miracidia of the human blood fluke Schistosoma mansoni and their subsequent processing for introduction into in vitro culture.

人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏

Human blood flukes, Schistosoma spp., have a complex life cycle that involves asexual and sexual developmental phases within a snail intermediate and ...

发育生物学

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm harbored in the intestine of canines. There is an urgent need for applied genetic research to understand the mechanisms of pathogenesis and disease control and prevention. However, the lack of an effective gene evaluation system impedes direct interpretation of the functional genetics of cestode parasites, including the Echinococcus species. The present study demonstrates the potential of lentiviral gene transient transduction in the metacestode and strobilated forms of E. granulosus. Protoscoleces (PSCs) were isolated from hydatid cysts and transferred to specific biphasic culture media to develop into strobilated worms. The worms were transfected with harvested third-generation lentivirus, along with HEK293T cells as a transduction process control. A pronounced fluorescence was detected in the strobilated worms over 24 h and 48 h, indicating transient lentiviral transduction in E. granulosus. This work presents the first attempt at lentivirus-based transient transduction in tapeworms and demonstrates the promising outcomes with potential implications in experimental studies on flatworm biology.

Transient Transduction of the Strobilated Forms of Echinococcus granulosus

We describe a rapid transient transduction technique in different developmental stages of Echinococcus granulosus using third-generation lentiviral vectors.

粒状棘球绦虫的短暂转导

Cystic echinococcosis or hydatid disease is one of the most important zoonotic parasitic diseases caused by Echinococcus granulosus, a small tapeworm ...

遗传学

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote its motility and dissemination. New work has shown that proteins involved in actin-based motility are also linked to autophagy, an intracellular degradation process crucial for cell autonomous immunity. Strikingly, host cells may prevent actin-based motility of S. flexneri by compartmentalizing bacteria inside &#8216;septin cages&#8217; and targeting them to autophagy. These observations indicate that a more complete understanding of septins, a family of filamentous GTP-binding proteins, will provide new insights into the process of autophagy. This report describes protocols to monitor autophagy-cytoskeleton interactions caused by S. flexneri in vitro using tissue culture cells and in vivo using zebrafish larvae. These protocols enable investigation of intracellular mechanisms that control bacterial dissemination at the molecular, cellular, and whole organism level.

Use of Shigella flexneri to Study Autophagy-Cytoskeleton Interactions

To counteract pathogen dissemination, host cells reorganize their cytoskeleton to compartmentalize bacteria and induce autophagy. Using Shigella infection of tissue culture cells, host and pathogen determinants underlying this process are identified and characterized. Using zebrafish models of Shigella infection, the role of discovered molecules and mechanisms are investigated in vivo.

使用<em&gt;福氏痢疾杆菌</em&gt;研究自噬，细胞骨架相互作用

Shigella flexneri is an intracellular pathogen that can escape from phagosomes to reach the cytosol, and polymerize the host actin cytoskeleton to promote ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of heterogeneous vesicles, whose size range between 40 and 1,000 nm. Accumulated evidence indicated that EVs play important regulatory roles in pathogen-host interactions. A deep understanding of schistosome EVs should provide insights into the mechanisms underlying schistosome-host interactions, enabling development of novel strategies against schistosomiasis. Here, we aim to further study EVs functions in schistosomes by presenting a protocol for the isolation and characterization of EVs from adult Schistosoma japonicum (S. japonicum). EVs were isolated from in vitro culture medium using centrifugation combined with a commercial exosome isolation kit. The isolated S. japonicum EVs (SjEVs) typically possess a diameter of 100 - 400 nm, and are characterized by transmission electronic microscopy and western blotting. The usage of PKH67 dye-labeled SjEVs has demonstrated that SjEVs are internalized by the recipient cells. Overall, our protocol provides an alternative method for isolating EVs from adult schistosomes; the isolated SjEVs may be suitable for functional analysis.

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Here, we present a protocol to describe extracellular vesicles (EVs) isolation in in vitro cultured medium from adult Schistosoma japonicum.

成年日本血吸虫细胞外囊泡的分离与鉴定

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and Strongyloides ratti. S.&#160;stercoralis is a human-parasitic, skin-penetrating nematode that infects approximately 610 million people, while the rat parasite S.&#160;ratti is closely related to S.&#160;stercoralis and is often used as a laboratory model for S.&#160;stercoralis. Both S.&#160;stercoralis and S.&#160;ratti are easily amenable to the generation of transgenics and knockouts through the exogenous nucleic acid delivery technique of intragonadal microinjection, and as such, have emerged as model systems for other parasitic helminths that are not yet amenable to this technique.
Parasitic Strongyloides adults inhabit the small intestine of their host and release progeny into the environment via the feces. Once in the environment, the larvae develop into free-living adults, which live in feces and produce progeny that must find and invade a new host. This environmental generation is unique to the Strongyloides species and similar enough in morphology to the model free-living nematode Caenorhabditis elegans that techniques developed for C. elegans can be adapted for use with these parasitic nematodes, including intragonadal microinjection. Using intragonadal microinjection, a wide variety of transgenes can be introduced into Strongyloides. CRISPR/Cas9 components can also be microinjected to create mutant Strongyloides larvae. Here, the technique of intragonadal microinjection into Strongyloides, including the preparation of free-living adults, the injection procedure, and the selection of transgenic progeny, is described. Images of transgenic Strongyloides larvae created using CRISPR/Cas9 mutagenesis are included. The aim of this paper is to enable other researchers to use microinjection to create transgenic and mutant Strongyloides.

Generating Transgenics and Knockouts in Strongyloides Species by Microinjection

The functional genomic toolkit for the parasitic nematodes Strongyloides stercoralis and Strongyloides ratti includes transgenesis, CRISPR/Cas9-mediated mutagenesis, and RNAi. This protocol will demonstrate how to use intragonadal microinjection to introduce transgenes and CRISPR components into S.&#160;stercoralis and S.&#160;ratti.

通过显微注射在类 圆线虫 物种中产生转基因和敲除

The genus Strongyloides consists of multiple species of skin-penetrating nematodes with different host ranges, including Strongyloides stercoralis and ...

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of Plasmodium deposited by mosquitoes in the skin of vertebrate hosts undergoes a phase of mandatory development in the liver before initiating clinical malaria. We know little about the biology of Plasmodium development in the liver; access to the sporozoite stage and the ability to genetically modify such sporozoites are critical tools for studying the nature of Plasmodium infection and the resulting immune response in the liver. Here, we present a comprehensive protocol for the generation of transgenic Plasmodium berghei sporozoites. We genetically modify blood-stage P. berghei and use this form to infect Anopheles mosquitoes when they take a blood meal. After the transgenic parasites undergo development in the mosquitoes, we isolate the sporozoite stage of the parasite from the mosquito salivary glands for in vivo and in vitro experimentation. We demonstrate the validity of the protocol by generating sporozoites of a novel strain of P. berghei expressing the green fluorescent protein (GFP) subunit 11 (GFP11), and show how it could be used to investigate the biology of liver-stage malaria.

Generating Genetically Modified Plasmodium berghei Sporozoites

Malaria is transmitted through inoculation of the sporozoite stage of Plasmodium by infected mosquitoes. Transgenic Plasmodium has allowed us to understand the biology of malaria better and has contributed directly to malaria vaccine development efforts. Here, we describe a streamlined methodology to generate transgenic Plasmodium berghei sporozoites.

生成转基因 伯氏疟原 虫孢子

Malaria is a deadly disease caused by the parasite Plasmodium and is transmitted through the bite of female Anopheles mosquitoes. The sporozoite stage of ...

Isolation And Dendritic Cell-Uptake of Small Extracellular Vesicles from Echinococcus granulosus

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In particular, small extracellular vesicles (sEVs) act as nano-carriers transferring natural antigens, which are critical in host immunomodulation and parasite survival. This article presents a step-by-step protocol to isolate sEVs from larval stage cultures of Echinococcus granulosus and analyzes their uptake by dendritic cells obtained from murine bone marrow, which acquire adhesion and antigen presentation capacity during their maturation after one week of in vitro culture. This article provides comprehensive information for generating, purifying, and quantifying sEVs using ultracentrifugation alongside parallel analyses of dynamic light scattering and transmission electron microscopy. Additionally, a detailed experimental protocol is outlined for isolating and cultivating mouse bone marrow cells and driving their differentiation into dendritic cells using Flt3L. These dendritic cells can present antigens to na&#239;ve T cells, thereby modulating the type of immune response in vivo. Thus, alternative protocols, including confocal microscopy and flow cytometry analysis, are proposed to check the acquired maturational phenotype of dendritic cells previously exposed to parasitic sEVs. Finally, it is worth noting that the described protocol can be applied as a whole or in individual parts to carry out parasite in vitro culture, isolate extracellular vesicles, generate bone marrow-derived dendritic cell cultures, and perform uptake assays with these cells.

Here, we describe in vitro culture conditions, isolation, and increased generation of extracellular vesicles (EVs) from Echinococcus granulosus. The small EVs were characterized by dynamic light scattering and transmission electron microscopy. The uptake by bone marrow-derived dendritic cells and their phenotypic modulation were studied using confocal microscopy and flow cytometry.

细粒棘球蚴小细胞外囊泡的分离和树突状细胞摄取

The secretion of extracellular vesicles by cestodes is crucial for enabling cellular communication not only among parasites but also with host tissues. In ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

鸡寄生虫的成核和活体繁殖

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...

生物学

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Malaria remains one of the most important public health problems, causing significant morbidity and mortality. Malaria is a mosquito borne disease transmitted through an infectious bite from the female Anopheles mosquito. Malaria control will eventually rely on a multitude of approaches, which includes ways to block transmission to, through and from mosquitoes. To study mosquito stages of malaria parasites in the laboratory, we have optimized a protocol to culture highly infectious Plasmodium falciparum gametocytes, a parasite stage required for transmission from the human host to the mosquito vector. P. falciparum gametocytes mature through five morphologically distinct steps, which takes approximately 1-2 weeks. Gametocyte culture described in this protocol is completed in 15 days and are infectious to mosquitoes from days 15-18. These protocols were developed to maintain a continuous cycle of infection competent gametocytes and to maintain uninterrupted supply of mosquito stages of the parasite. Here, we describe the methodology of gametocyte culture and how to infect mosquitoes with these parasites using glass membrane feeders.

Plasmodium falciparum Gametocyte Culture and Mosquito Infection Through Artificial Membrane Feeding

Detailed investigations on mosquito stages of malaria parasites are critical to design effective transmission blocking strategies. This protocol demonstrates how to effectively culture infectious gametocytes and then feed these gametocytes to mosquitoes to generate mosquito stages of P. falciparum.

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

摘要

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此视频中的章节

从使用鸡蛋

人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏

粒状棘球绦虫的短暂转导

使用

成年日本血吸虫细胞外囊泡的分离与鉴定

通过显微注射在类圆线虫物种中产生转基因和敲除

生成转基因伯氏疟原虫孢子

细粒棘球蚴小细胞外囊泡的分离和树突状细胞摄取

鸡寄生虫的成核和活体繁殖

恶性疟原虫通过人工膜喂养进行配子体培养和蚊子感染

Cercarial转型和在体外栽培曼氏血吸虫 Schistosomules

摘要

探索更多视频

此视频中的章节

从使用鸡蛋

人血 Intramolluscan 期的体外分离与培养的研究血吸虫氏

粒状棘球绦虫的短暂转导

使用

成年日本血吸虫细胞外囊泡的分离与鉴定

通过显微注射在类 圆线虫 物种中产生转基因和敲除

生成转基因 伯氏疟原 虫孢子

细粒棘球蚴小细胞外囊泡的分离和树突状细胞摄取

鸡寄生虫的成核和活体繁殖

恶性疟原虫 通过人工膜喂养进行配子体培养和蚊子感染

通过显微注射在类圆线虫物种中产生转基因和敲除

生成转基因伯氏疟原虫孢子

恶性疟原虫通过人工膜喂养进行配子体培养和蚊子感染