Name: sexual transmission of american trypanosomes from males and females to naive mates
Uploaded: 2019-01-27T13:00:00
Description: Watch this Scientific Journal Video about Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates at JoVE.com

我们感谢伊扎贝拉·杜拉多、卡拉·阿劳霍和聪明的戈梅斯的实验室设施和批评意见, 以及布鲁诺·达拉戈和拉斐尔·安德拉德的技术援助。我们感谢科学促进基金会、国家研究理事会、科技部和巴西教育部人力资源培训机构对这些机构的支持。调查。

巴西利亚大学医学院人类和动物研究委员会分别在 2011年2500.167567 和 1041111/研究议定书中批准了与人体和实验动物有关的所有程序。公共基金会医院 gaspar vianna (2009年和 conep 1116ne2009 号议定书) 的道德委员会批准了实地研究的自由同意书, 并扩大到卫生部国家人类研究委员会 (conep 2585/04)。对该程序进行了调整, 以评估二倍体血液单个核细胞和精液单倍体配子中的t. cruzi dna 。实验动物得到了人?...

<ol>
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在此, 我们讨论了一个基于家庭的研究协议, 该协议回答了人类恰加斯病是否源于性传播种内感染的问题。早期的研究无法提供t. cruzi感染的性传播证据, 可能是因为关于查加斯病的现有数据和信息是与个人3,4分开获得的, 5,6,7,8, 9,</su...

原生动物寄生虫三叶草属植物在哺乳动物寄主中经历了锥虫和三甲草的生命周期阶段, 并作为异位体存在于昆虫媒介的肠道和无菌培养。近几十年来, 几项研究表明, 在被认为三胺素无菌的四大洲国家, 有查加斯病的存在, 有 1、2、3、4、5 ,6,7.,8,9,10,11,12,13;美国锥虫体的分散最初归因于拉丁美洲移民到北半球, 但有些是本土的恰加斯病的可能性不能再被否认 3,4,5,6,7.,8,9,10,11,12,13,14. 唯一可识别的内源 t. cruzi传播的原因是, 在大约10% 的怀孕中, 长崎母亲将寄生虫转移到后代身上,15男性伴侣对精液射精在子宫内感染的贡献仍未得到承认。
一个多世纪前, 研究人员在卵巢的卵细胞和急性恰加斯病患者睾丸的生殖细胞中观察到细胞内的 t . cruzi。在致命急性恰加斯病病例的卵巢癌细胞、性腺和精原管腔 (图 1) 中, t. cruzi 色样体和阿玛菌的巢穴和团块在卵巢的器官中产生免疫特权。繁殖在没有炎症渗透 18,19。近几十年来, 一些实验研究表明, 在急性感染 小鼠的精子管、附属物、输便于和子宫、管和卵巢细胞中, 出现了圆形的 "1,20,21,22。此外, 在记录父母查加斯患者原生动物线粒体 dna 转移到后代的家庭研究过程中, t. cruzi核 dna (ndna) 在人类单倍体生殖细胞23中得到了验证,在马达加斯加小鼠24的射精中观察到寄生虫的生命周期阶段.这些发现与关于受 t . cruzi-感染的宿主在没有特定抗体1、25、26的情况下获得免疫耐受的报告一致。此外, 表明恰加斯病流行向其他大陆蔓延的流行病报告3、4、5、6、7、8 9、10、11、12、13现在得到实验研究的支持, 这些实验研究表明, 恰加斯病可以通过性行为传播.本研究提出了一个流行病学的家庭研究程序 , 并表明t . cruzi感染是通过传播的。

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			<td height="20" style="height:20px;width:336px;">BCIP and NBT redox system</td>
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			<td style="width:65px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Blood DNA Purification columns</td>
			<td style="width:183px;">Amersham Biosciences</td>
			<td style="width:102px;">27-9603-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">d-ATP, [&#945;-32P], 250 &#181;Ci.</td>
			<td style="width:183px;">&#160; Perkin Elmer&#160;&#160;</td>
			<td style="width:102px;">BLU012H</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">DNA, Solution Salt Fish Sperm</td>
			<td style="width:183px;">AMRESCO</td>
			<td style="width:102px;">064-10G</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:336px;">dNTP Set, 100 mM Solutions</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">28-4065-51</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Eco&#160;RI</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">15202-021</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Goat anti-human IgG- alkaline phosphatase conjugated</td>
			<td style="width:183px;">Southern Biotech</td>
			<td style="width:102px;">&#160;&#160;&#160;&#160;&#160;&#160; 2040-04</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Goat anti-human IgG- FITC conjugated</td>
			<td style="width:183px;">Biocompare</td>
			<td style="width:102px;">MB5198020</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Hybond &#8211; N+ nylon membrane</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">RPN303B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Hybridization oven</td>
			<td style="width:183px;">Thomas Scientific</td>
			<td style="width:102px;">95-0031-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Micro imaging software cell Sens software</td>
			<td style="width:183px;">Olympus, Japan</td>
			<td style="width:102px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Molecular probes labeling System</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">700-0030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Nsi I</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">R5584 1KU</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Plasmid Prep Mini Spin Kit</td>
			<td style="width:183px;">GE Healthcare</td>
			<td style="width:102px;">28-9042-70</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Plate reader&#160;</td>
			<td style="width:183px;">Bio-Tek GmBH</td>
			<td style="width:102px;">2015</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-chicken IgG-alkaline phosphatase conjugated</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">A9171</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-chicken IgG-FITC conjugated</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">F8888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-mouse IgG- alkaline phosphatase conjugated</td>
			<td style="width:183px;">Sigma Aldrich</td>
			<td style="width:102px;">A2418&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Rabbit anti-mouse IgG-FITC conjugated</td>
			<td style="width:183px;">Biorad</td>
			<td style="width:102px;">MCA5787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Spin Columns for radio labeled DNA purification, Sephadex G-25, fine</td>
			<td style="width:183px;">Sigma-Aldrich</td>
			<td style="width:102px;">G25DNA-RO&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Taq DNA Polymerase Recombinant</td>
			<td style="width:183px;">Invitrogen</td>
			<td style="width:102px;">11615-010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Thermal cycler system</td>
			<td style="width:183px;">Biorad, USA</td>
			<td style="width:102px;">1709703</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:336px;">Vector Systems</td>
			<td style="width:183px;">Promega</td>
			<td style="width:102px;">A1380</td>
			<td></td>
		</tr>
	</tbody>
</table>

sexual transmission of american trypanosomes from males and females to naive mates

美国锥虫病是通过摄入受污染的食物、输血或在医院和研究实验室意外传播给人类的。此外, 克鲁兹锥虫病感染是由长崎母亲自然传播给后代的, 但男性伴侣在子宫污染方面的贡献尚不清楚。在卵巢的细胞、性腺和精原管腔内发现的阿玛菌和色母细胞的巢穴和团块表明, t .克鲁兹感染是性传播的。本文的研究方案介绍了一个家庭研究群体的结果, 该群体在人体二倍体血单个核细胞和单倍体配子中显示了寄生虫核 dna。因此, 时隔一年收集的三个独立生物样本证实, t. cruzi感染是通过性传播到后代的。有趣的是, 大多数对寄生虫抗原具有免疫耐受能力的家族后代中都存在特异性 t. cruzi 抗体。在胚胎生长的第一周后, 鸡的难熔对t. cruzi 的免疫耐受性得到了证实, 而从鞭毛接种的卵中孵化出来的小鸡则不能产生特定的抗体。此外, 人精液射精射精射精腹腔内或阴道中的天真小鼠产生 t. cruzi amastigotes 在附属物, 精原体小管, 输精管和子宫管没有炎症 反应在免疫特权器官再生产。受 t. cruzi感染的雄性和雌性老鼠与天真的配偶的繁殖导致了感染的获取, 这些感染后来被传播到后代身上。因此, 有民众和社会组织参与的强有力的教育、信息和通信方案被认为是预防查加斯病的必要条件。

这项根据协议进行的研究, 旨在通过临床和寄生虫学检查检测急性恰加斯病病例。对静脉血液样本进行了直接显微镜检查和体外培养, 以促进寄生虫的生长。21例急性恰加斯病在血液中显示t. 克鲁齐。研究协议确保了 t. cruzi eci1-至 eci21 与急性恰加斯病的分离, dna 样本在其余研究人群中显示出阳性 dna 足迹: dna-pcr 检测产生了典型的端粒重复序列与188nt 乐队存在, ?...

Watch this Scientific Journal Video about Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates at JoVE.com

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

恰加斯病的锥虫瘤壳体产生长期的无症状感染, 突然发展成为临床公认的病理。下面的研究协议描述了一项以家庭为基础的短期流行病学研究, 以揭示从父母到后代性传播的t. cruzi感染。

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 ...

查加斯病的锥虫科鲁西剂可能会产生持久的症状感染，可能进入临床公认的病理学。在一名死于恰加斯病的男孩的半尼弗管中发现锥虫瘤的克鲁西巢，表明研究方案可以解开查加斯寄生虫可能的性传播。提供五年的医疗保健，并招募家庭，显示患有急性恰加斯病的临床症状的参与者，ACG。相隔一年三次获得15毫升家庭参与者的静脉血，分为三、五密耳，储存在四摄氏度。从成人志愿者那里收集的两毫升血清射精显示ACG病例血液中的T.cruzi，以验证分子检测。从ACG血液中分离T.cruzi，用抗凝剂储存在无菌的10ml管中。将5毫升血液接种到50毫升管倾斜，加上5毫升肝脏输液尝试性介质，在27摄氏度的摇床中孵育三个月。在玻璃幻灯片上 100 微升的超量镜中搜索显微镜。从离心的介质中收获旗帜，以 1，000 x G 进行 10 分钟，用 PBS pH 7.4 洗涤。接种 75 ml L6 肌肉细胞培养瓶每个与 10 至第六 T.cruzi 分离。用 15 ml 的 Dulbecco 最小基本介质喂养培养细胞，在 37 摄氏度时提供 DMEM。生长寄生虫分离物和正对贝雷尼斯T.克鲁西。在 DMEM 中将 T. cruzi 相对 Leishmania 巴西病作为负对照。使用 10 x 10 到第六组织培养物衍生出 T.cruzi 试虫感染小鼠。在一个月后，在肌肉和生殖器官的各部分搜索T.cruzi的巢。使用EDTA康宁无菌10ml管中的血液等分，在3000 x G的密度梯度离心中收集白细胞45分钟。用 PBS 清洗两次细胞，在 1，500 x G 下离心机，在不同的管下清洗 10 分钟。从从109个家庭受试者和21名参与者的精子中提取从109个家族受试者的贝雷尼斯T.cruzi的分离物中提取DNA，如在协议的第3.3步。测量0.8%的加糖凝胶上的DNA浓度，并在206纳米时吸收。在 PCR 分析中，使用 T.cruzi TCZ 一破折号两个底片退火到 188 核苷酸探针。使PCR与10纳米图模板DNA、每对底塑剂的0.4微摩尔、两个单位的DNA聚合酶、0.2微摩尔DNA-TPs和5微摩尔氯化镁在25微升最终体积中混合。执行步骤 3.5.2 中的 DNA 扩增，并在凝胶中显示 DNA 波段。使用 T.cruzi 188 核苷酸探针，将 α 32P dATP 的底注标签与 alpha 32P dATP 进行分离，并在 1.3% 的 agarose 凝胶上分离 PCR 产品，并转移到带正向的尼龙膜上。清洗膜，现在暴露在不同周期的X射线场。使用 TCZ 一破折号两个底转器，对从 X 射线场选择的克隆进行商业序列。在109名研究参与者中进行三个独立的免疫荧光测定，并在PBS中进行1至100个血清稀释1至100次稀释。将封闭血液中的5毫升等分在1000 x G下离心30分钟，将血清储存在零下20摄氏度。运行独立的免疫荧光血清稀释从研究样品获得三次一年分开。使用5微升正式孵育的T.cruzi母体或L.巴西西普罗斯龙，10个寄生虫每微升在PBS悬浮液玻璃幻灯片上，让它干燥过夜，如步骤5.3.3。用100微升的1至100微升的血清稀释剂孵育玻璃上的寄生虫，并在37摄氏度的湿润室中用滑液覆盖一小时，然后用PBS洗涤两次。用1到1000个荧光素的荧光素，将标有兔的抗体用于人类IgG，洗涤和干燥，孵育风干玻璃滑梯。用盖滑将玻璃滑梯安装，并在紫外光显微镜下检查。在100微升碳酸盐缓冲液pH9.6中检测T.cruzi和L.巴西易溶性抗原1微克。在三分四分之一井中用100微升的1至100微升血清稀释。用 PBS-Tween 溶液清洗板两次，然后等待干燥。孵育用100微升的1至1000稀释碱性磷酸酶结合，山羊抗兔IgG。用PBS-Tween清洗板两次，加入底材，加入亚硝基苯丙酸，等待颜色发展。在多模板读卡器中读取 630 纳米的光学密度。将肥沃的鸡蛋转干，在它们的气泡下在壳上打个洞。在模拟控制鸡蛋的每个壳培养基上接种100个试药。用胶带密封孔。在37摄氏度孵育卵子，65%湿度21天。从活对照蛋A组孵化的小鸡，从模拟对照组，B组和T.cruzi接种的卵子，C组孵化，直到他们的成年生活。挑战B组和C组的鸡两次与10×10到第七正式孵化的试杀剂每周。挑战五周后，从 A、B 和 C 鸡的翼静脉获得两毫升静脉血。分离血清并运行免疫荧光分析仪以检测T.cruzi抗体。通过出色的住房、食物和供水，确保动物福利。使用来自成人的精液射精和 APCR 阳性的 Chagas 病病例，以及从协议步骤 1.5 中的对照对象。将100微升的精液注入内皮内和小鼠的阴道。父母和后代小鼠的T.cruzi感染的性传播。急性恰加斯病的锥形瘤的足迹由与无线电标记探头提前形成的 nDNA-PCR 产品显示。T.cruzi的表型通过免疫荧光测定而揭示出来。反 T 。克鲁西抗体不能识别L.巴西西。ELISA 和 nDNA-PCR 测定的图形表示。组一和二分别负和正控制。第三组，阳性nDNA-PCR和特异性抗体测定。第四组与NDNA-PCR检测到的T.cruzi感染，但在没有特异性抗体的情况下。第五组，无感染参与者。家庭人口的地谱图和制图显示，反T的比例存在差异。克鲁兹抗体和nDNA-PCR测定的抗体。开放是方形和圆形，负男和女。红色与防 T 。克鲁西抗体和nDNA-PCR阳性。黑色， 正 ndna - pcr 单独。从T.cruzi接种的鸡蛋中孵化的鸡的免疫耐受性。A， 模拟控制， B， 活对控鸡挑战与 T. cruzi 抗原.C， 鸡孵化从 T. cruzi 接种鸡蛋后， 挑战与特定的抗原.T.cruzi的感染性在精液注入腹腔和阴道时表现出来。这些静止物中牺牲了三周的老鼠，显示出心脏、骨骼肌、血管和子宫管中的阿玛斯蒂奥特巢。小鼠模型系统中T.cruzi感染的性传播。T.cruzi感染的男性和女性，将感染传播给天真的伴侣。T.cruzi感染垂直从父母转移到后代小鼠。南方的印迹揭示了大多数F1垃圾中的寄生虫角带。免疫过氧化物酶污渍T.cruzi amastigotes显示，在表皮炎的间质和进入后代小鼠的半硝基管的流明中。在21例恰加斯病急性病例中，原生动物的寄生虫学证明对于验证所有受感染受试者的T.cruzi足迹至关重要。寄生虫抗体测定与核酸检测比例的差异表明，无抗体患者的大部分足迹。在胚胎发育的第一周后，在鸡模型中，向T.cruzi反映了广泛的差异。免疫耐受性成年鸡显示寄生虫的脚印，但没有特定的抗体。查加斯患者解热中至关重要的抗克鲁西在注入小鼠阴道以及腹腔后引起广泛感染。病理学显示，T.cruzi amastigote巢在心脏和骨骼肌肉，在血管延迟和子宫管。查加斯小鼠与天真伴侣的繁殖表明，受感染的雌性小鼠和雄性将T.cruzi传播给未受感染的幼鼠，它们的大部分垃圾获得感染垂直转移到后代。基于家庭的议定书涉及核酸检测，以便进行可靠的输血和流行病学调查，并阐明潜在的持续性传播恰加斯病。

Research

JoVE Journal

Immunology and Infection

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

科研

免疫与感染

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

Oral Transmission of Listeria monocytogenes in Mice via Ingestion of Contaminated Food

口头传播的<em&gt;单核细胞增生李斯特氏菌</em小鼠通过食入被污染的食物

L. monocytogenes are facultative intracellular bacterial pathogens that cause food borne infections in humans. Very little is known about the ...

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

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

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 ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

实验模型后的自然传播到研究结核病，疟疾混合感染结核分枝杆菌和伯氏疟原虫</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Investigating Mast Cell Secretory Granules; from Biosynthesis to Exocytosis

调查肥大细胞分泌颗粒;从合成到胞吐作用

Mast Cells (MC) are secretory cells of the immune system that accomplish their physiological and pathological functions by releasing pre-formed and newly ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

协议侦办主机组织分布，传播模式和效果上浓核病毒的主机健身的棉铃虫

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Phenotypic Analysis of Rodent Malaria Parasite Asexual and Sexual Blood Stages and Mosquito Stages

啮齿动物疟原虫无性、性血期和蚊子阶段的类状分析

Recent advances in genetics and systems biology technologies have promoted our understanding of the biology of malaria parasites on the molecular level. ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

人类的过表达和纯化<em&gt;顺</emβ-肾上腺素转移酶<em&gt;大肠杆菌</em

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...