JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Resultados
Discussion
Declarações
Acknowledgements
Materials
Referências
Please note that all translations are automatically generated. Click here for the English version.
Imunologia e Infecção

美国锥虫从男性和女性到天真的命运的性传播

Published: January 27, 2019
doi:
10.3791/57985
, , , , ,
1Chagas Disease Multidisciplinary Research Laboratory, Faculty of Medicine,University of Brasilia, 2Instituto Evandro Chagas

Summary

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。

Abstract

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。

Introduction

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。

Protocol

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?…

Representative Results

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, ?…

Discussion

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…

Declarações

The authors have nothing to disclose.

Acknowledgements

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。

Materials

BCIP and NBT redox system Sigma-Aldrich 681 451 001
Blood DNA Purification columns Amersham Biosciences 27-9603-01
d-ATP, [α-32P], 250 µCi.   Perkin Elmer   BLU012H
DNA, Solution Salt Fish Sperm AMRESCO 064-10G
dNTP Set, 100 mM Solutions GE Healthcare 28-4065-51
Eco RI Invitrogen 15202-021
Goat anti-human IgG- alkaline phosphatase conjugated Southern Biotech        2040-04
Goat anti-human IgG- FITC conjugated Biocompare MB5198020
Hybond – N+ nylon membrane GE Healthcare RPN303B
Hybridization oven Thomas Scientific 95-0031-02
Micro imaging software cell Sens software Olympus, Japan
Molecular probes labeling System Invitrogen 700-0030
Nsi I Sigma-Aldrich R5584 1KU
Plasmid Prep Mini Spin Kit GE Healthcare 28-9042-70
Plate reader  Bio-Tek GmBH 2015
Rabbit anti-chicken IgG-alkaline phosphatase conjugated Sigma-Aldrich A9171
Rabbit anti-chicken IgG-FITC conjugated Sigma-Aldrich F8888
Rabbit anti-mouse IgG- alkaline phosphatase conjugated Sigma Aldrich A2418 
Rabbit anti-mouse IgG-FITC conjugated Biorad MCA5787
Spin Columns for radio labeled DNA purification, Sephadex G-25, fine Sigma-Aldrich G25DNA-RO 
Taq DNA Polymerase Recombinant Invitrogen 11615-010
Thermal cycler system Biorad, USA 1709703
Vector Systems Promega A1380
DOWNLOAD MATERIALS LIST

Referências

  1. Araujo, P. F., et al. Sexual transmission of American trypanosomiasis in humans: a new potential pandemic route for Chagas parasites. Memórias do Instituto Oswaldo Cruz. 112 (6), 437-446 (2017).
  2. Teixeira, A. R. L., Nitz, N., Bernal, F. M., Hetch, M. M. Parasite Induced Genetically Driven Autoimmune Chagas Heart Disease in the Chicken Model. Journal of Visualized Experiments. (65), 3716 (2012).
  3. Kalil-Filho, R. Globalization of Chagas Disease Burden and New Treatment Perspectives. Journal of the American College of Cardiology. 66 (10), 1190-1192 (2015).
  4. Nunes, M. C. P., Dones, W., Morillo, A., Encina, J. J., Ribeiro, A. L. Chagas Disease: An Overview of Clinical and Epidemiological Aspects. Journal of the American College of Cardiology. 62 (9), 767-776 (2013).
  5. Pinazo, M. J., Gascon, J. Chagas disease: from Latin America to the world. Reports in Parasitology. 2015 (4), 7-14 (2015).
  6. Kessler, D. A., Shi, P. A., Avecilla, S. T., Shaz, B. H. Results of lookback for Chagas disease since the inception of donor screening at New York Blood Center. Transfusion. 53 (5), 1083-1087 (2013).
  7. Klein, N., Hurwitz, I. R. Globalization of Chagas Disease: A Growing Concern in Nonendemic Countries. Epidemiology Research International. , (2012).
  8. Pérez-Molina, J. A., Norman, F., López-Vélez, R. Chagas disease in non-endemic countries: epidemiology, clinical presentation and treatment. Current Infectious Diseases Report. 14 (3), 263-274 (2012).
  9. Hotez, P. J., et al. Chagas disease: "the new HIV/AIDS of the Americas". PLoS Neglected Tropical Diseases. 6, e1498 (2012).
  10. Schmunis, G. A., Yadon, Z. E. Chagas disease: a Latin American health problem becoming a world health problem. Acta Tropica. 115 (1-2), 14-21 (2010).
  11. Franco-Paredes, C., Bottazzi, M. E., Hotez, P. J. The Unfinished Public Health Agenda of Chagas Disease in the Era of Globalization. PLoS Neglected Tropical Diseases. 3 (7), e470 (2009).
  12. Teixeira, A. R. L., Vinaud, M., Castro, A. M. . Emerging Chagas Disease. In: Chagas Disease: – A Global Health Problem. 3, 18-39 (2009).
  13. Lee, B. Y., Bacon, K. M., Bottazzi, M. E., Hotez, P. J. Global economic burden of Chagas disease: a computational simulation model. Lancet Infectious Diseases. 13 (4), 342-348 (2013).
  14. Teixeira, A. R. L., Hecht, M. M., Guimaro, M. C., Sousa, A. O., Nitz, N. Pathogenesis of Chagas Disease: Parasite Persistence and Autoimmunity. Clinical Microbiology Review. 24 (3), 592-630 (2011).
  15. Murcia, L., et al. Risk factors and primary prevention of congenital Chagas disease in a nonendemic country. Clinical Infectious Diseases. 56 (4), 496-502 (2013).
  16. Chagas, C. New human trypanosomiasis. Morphology and lifecycle of Schizotrypanum cruzi, the cause of a new human disease. Memórias do Instituto Oswaldo Cruz. 1, 159 (1909).
  17. Vianna, G. Contribution to the study of the Pathology of Chagas disease. Memórias do Instituto Oswaldo Cruz. 3, 276 (1911).
  18. Teixeira, A. R. L., Roters, F., Mott, K. E. Acute Chagas disease. Gazeta Médica da Bahia. 3, 176-186 (1970).
  19. Teixeira, A. R. L., et al. Prevention and Control of Chagas Disease – An Overview. International STD Research, Reviews. 7 (2), 1-15 (2018).
  20. Rios, A., et al. Can sexual transmission support the enzootic cycle of Trypanosoma cruzi?. Memórias do Instituto Oswaldo Cruz. 113 (1), 3-8 (2018).
  21. Lenzi, H. L., et al. Trypanosoma cruzi: compromise of reproductive system in acute murine infection. Acta Tropica. 71 (2), 117-129 (1998).
  22. Carvalho, L. O. P., et al. Trypanosoma cruzi and myoid cells from seminiferous tubules: Interaction and relation with fibrous components of extra cellular matrix in experimental Chagas’ disease. International Journal of Experimental Pathology. 90 (1), 52-57 (2009).
  23. Hecht, M. M., et al. Inheritance of DNA transferred from American trypanosomes to human hosts. PLoS One. 12, e9181 (2010).
  24. Alarcon, M., et al. Presencia de epimastigotes de Trypanosoma cruzi en el plasma seminal de ratones con infección aguda. Boletín Malariología y Salud Ambiental. 51, 237 (2011).
  25. Teixeira, A. R. L., et al. Trypanosoma cruzi in the Chicken Model: Chagas-Like Heart Disease in the Absence of Parasitism. PLoS Neglected Tropical Diseases. 5 (3), e1000 (2011).
  26. Guimaro, M. C., et al. Inhibition of Autoimmune Chagas-Like Heart Disease by Bone Marrow Transplantation. PLoS Neglected Tropical Diseases. 8 (12), e3384 (2014).
  27. Oliveira, C. I., et al. Leishmania braziliensis isolates differing at the genome level display distinctive features in BALB/c mice. Microbes and Infection. 6 (11), 977-984 (2004).
  28. Mendes, D. G., et al. Exposure to mixed asymptomatic infections with Trypanosoma cruzi, Leishmania braziliensis and Leishmania chagasi in the human population of the greater Amazon. Tropical Medicine, International Health. 12, 629 (2007).
  29. Moser, D. R., Kirchhoff, L. V., Donelson, J. E. Detection of Trypanosoma cruzi by DNA amplification using the polymerase chain reaction. Journal of Clinical Microbiology. 27 (7), 1477-1482 (1989).
  30. Da Silva, A. R. . Sexual transmission of Trypanosoma cruzi in mus musculus [MsD thesis]. , (2013).
  31. Ribeiro, M., et al. Can sexual transmission support the enzootic cycle of Trypanosoma cruzi?. Memórias do Instituto Oswaldo Cruz. 113 (1), 3-8 (2018).
  32. Billingham, R. E., Brent, L., Medawar, P. B. Actively acquired tolerance of foreign cells. Nature. 172 (4379), 603-606 (1953).
  33. Burnet, F., Fenner, F. . The production of Antibodies. , (1949).
  34. Hasek, M. Parabiosis of birds during their embryonic development. Chekhoslovatskaia Biology. 2 (1), 29-31 (1953).
  35. Coura, J. R., Junqueira, A. C. V., Fernades, O., Valente, S. A., Miles, M. A. Emerging Chagas Disease in Amazonian Brazil. Trends Parasitology. Trends Parasitology. 18 (4), 171-176 (2002).
  36. Teixeira, A. R. L., et al. Emerging Chagas disease: Trophic network and cycle of transmission of Trypanosoma cruzi from palm trees in the Amazon. Emerging Infectious Diseases. 7 (1), 100112 (2001).
  37. Hazebroek, M., Dennert, R., Heymans, S. Idiopathic dilated cardiomyopathy: possible triggers and treatment strategies. Netherland Heart Journal. 20 (7-8), 332-335 (2012).
  38. Arimura, T., Hayashi, T., Kimura, A. Molecular etiology of idiopathic cardiomyopathy. Acta Myologica. 26 (3), 153-158 (2007).
  39. Dec, W. G., Fuster, V. Idiopathic Dilated Cardiomyopathy. New England Journal of Medicine. 33, 1564-1575 (1994).
  40. Niederkorn, J. Y. See no evil, hear no evil, do no evil: the lessons of immune privilege. Nature Immunology. 7 (4), 354-359 (2006).
  41. Smith, B. E., Braun, R. E. Germ cell migration across Sertoli cell tight junctions. Science. 338 (6118), 798-802 (2012).
  42. Garth, A., Wilbanks, J., Streilein, W. Fluids from immune privileged sites endow macrophages with the capacity to induce antigen-specific immune deviation via a mechanism involving transforming growth factor-β. European Journal of Immunology. 22 (4), 1031-1036 (1992).
  43. Meng, J., Anne, R., Greenlee, C., Taub, J., Braun, R. E. Sertoli Cell-Specific Deletion of the Androgen Receptor Compromises Testicular Immune Privilege in Mice. Biology of Reproduction. 85 (2), 254-260 (2011).
  44. Fujisaki, J., et al. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature. 474 (7350), 216-219 (2011).
  45. Wood, K. J., Sakaguchi, S. Regulatory T cells in transplantation tolerance. Nature Review Immunology. 3 (3), 199-210 (2003).

Tags

Trypanosoma CruziChagas DiseaseSexual TransmissionBlood SamplesCell CulturePCR AnalysisDNA Extraction

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Almeida, A. B., Araújo, P. F., Bernal, F. M., Rosa, A. d. C., Valente, S. A., Teixeira, A. R. Sexual Transmission of American Trypanosomes from Males and Females to Naive Mates. J. Vis. Exp. (143), e57985, doi:10.3791/57985 (2019).
Baixar citação
Reimpressões e Permissões

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image