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

蜱对动物饲养的传输和Xenodiagnosis在莱姆病研究

Published: August 31, 2013
doi:
10.3791/50617
, , ,
1Division of Bacteriology & Parasitology, Tulane National Primate Research Center,Tulane University Health Sciences Center

Summary

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。

Abstract

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。

Introduction

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http://www.cdc.gov/lyme/stats/index.html ）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（> 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。

Protocol

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。 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…

Representative Results

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<…

Discussion

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?…

Declarações

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

Referências

  1. Porcella, S. F., Schwan, T. G. Borrelia burgdorferi and Treponema pallidum: a comparison of functional genomics, environmental adaptations, and pathogenic mechanisms. Journal of Clinical Investigation. 107, 651-656 (2001).
  2. Fraser, C. M., et al. Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature. 390, 580-586 (1997).
  3. Casjens, S., et al. A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi. Molecular Microbiology. 35, 490-516 (2000).
  4. Carroll, J. A., Garon, C. F., Schwan, T. G. Effects of environmental pH on membrane proteins in Borrelia burgdorferi. Infection & Immunity. 67, 3181-3187 (1999).
  5. Gilmore, R. D., Mbow, M. L., Stevenson, B. Analysis of Borrelia burgdorferi gene expression during life cycle phases of the tick vector Ixodes scapularis. Microbes & Infection. 3, 799-808 (2001).
  6. Ramamoorthy, R., Philipp, M. T. Differential expression of Borrelia burgdorferi proteins during growth in vitro. Infection & Immunity. 66, 5119-5124 (1998).
  7. Ramamoorthy, R., Scholl-Meeker, D. Borrelia burgdorferi proteins whose expression is similarly affected by culture temperature and pH. Infection & Immunity. 69, 2739-2742 (2001).
  8. Schwan, T. G., Piesman, J. Temporal Changes in Outer Surface Proteins A and C of the Lyme Disease-Associated Spirochete, Borrelia burgdorferi, during the Chain of Infection in Ticks and Mice. J. Clin. Microbiol. 38, 382-388 (2000).
  9. Barthold, S. W., de Souza, M. S., Janotka, J. L., Smith, A. L., Persing, D. H. Chronic Lyme borreliosis in the laboratory mouse. Am. J. Pathol. 143, 959-971 (1993).
  10. Barthold, S. W., de Souza, M. Exacerbation of Lyme arthritis in beige mice. Journal of Infectious Diseases. 172, 778-784 (1995).
  11. Barthold, S. W., Feng, S., Bockenstedt, L. K., Fikrig, E., Feen, K. Protective and arthritis-resolving activity in sera of mice infected with Borrelia burgdorferi. Clin. Infect. Dis. 25, S9-S17 (1997).
  12. Miller, J. C., Ma, Y., Crandall, H., Wang, X., Weis, J. J. Gene expression profiling provides insights into the pathways involved in inflammatory arthritis development: Murine model of Lyme disease. Experimental and Molecular Pathology. 85, 20-27 (2008).
  13. Purser, J. E., Norris, S. J. Correlation between plasmid content and infectivity in Borrelia burgdorferi. Proceedings of the National Academy of Sciences of the United States of America. 97, 13865-13870 (2000).
  14. Grimm, D., et al. Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals. Proceedings of the National Academy of Sciences of the United States of America. 101, 3142-3147 (2004).
  15. Zhang, J. R., Norris, S. J. Kinetics and in vivo induction of genetic variation of vlsE in Borrelia burgdorferi. Infection & Immunity. 66 (1), 3689-3697 (1999).
  16. Hodzic, E., Feng, S., Freet, K. J., Borjesson, D. L., Barthold, S. W. Borrelia burgdorferi population kinetics and selected gene expression at the host-vector interface. Infection & Immunity. 70, 3382-3388 (2002).
  17. Hodzic, E., Feng, S., Freet, K. J., Barthold, S. W. Borrelia burgdorferi population dynamics and prototype gene expression during infection of immunocompetent and immunodeficient mice. Infection & Immunity. 71, 5042-5055 (2003).
  18. Liang, F. T., Nelson, F. K., Fikrig, E. Molecular adaptation of Borrelia burgdorferi in the murine host. Journal of Experimental Medicine. 196, 275-280 (2002).
  19. Samuels, D. S. Gene Regulation in Borrelia burgdorferi. Annual Review of Microbiology. 65, 479-499 (1146).
  20. Gilmore, R. D., et al. The bba64 gene of Borrelia burgdorferi, the Lyme disease agent, is critical for mammalian infection via tick bite transmission. Proceedings of the National Academy of Sciences. 107, 7515-7520 (2010).
  21. Fisher, M. A., et al. Borrelia burgdorferi σ54 is required for mammalian infection and vector transmission but not for tick colonization. Proceedings of the National Academy of Sciences of the United States of America. 102, 5162-5167 (2005).
  22. Barthold, S. W. Animal models for Lyme disease. Laboratory Investigation. 72, 127-130 (1995).
  23. Pachner, A. R. Early disseminated Lyme disease: Lyme meningitis. American Journal of Medicine. 98, 30S-37S (1995).
  24. Barthold, S. W., Beck, D. S., Hansen, G. M., Terwilliger, G. A., Moody, K. D. Lyme Borreliosis in Selected Strains and Ages of Laboratory Mice. Journal of Infectious Diseases. 162, 133-138 (1990).
  25. Philipp, M. T., Johnson, B. J. Animal models of Lyme disease: pathogenesis and immunoprophylaxis. Trends in Microbiology. 2, 431-437 (1994).
  26. Roberts, E. D., et al. Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system. Journal of Infectious Diseases. 178, 722-732 (1998).
  27. Roberts, E. D., et al. Chronic lyme disease in the rhesus monkey. Laboratory Investigation. 72, 146-160 (1995).
  28. Philipp, M. T., et al. Early and early disseminated phases of Lyme disease in the rhesus monkey: a model for infection in humans. Infection & Immunity. 61, 3047-3059 (1993).
  29. Steere, A. C., Sikand, V. K., 348, T. r. e. a. t. m. e. n. t. .. N. .. E. n. g. l. .. J. .. M. e. d. .. The Presenting Manifestations of Lyme Disease and the Outcomes of Treatment. N. Engl. J. Med. 348, 2472-2474 (2003).
  30. Pachner, A. R., Delaney, E., O’Neill, T., Major, E. Inoculation of nonhuman primates with the N40 strain of Borrelia burgdorferi leads to a model of Lyme neuroborreliosis faithful to the human disease. Neurology. 45, 165-172 (1995).
  31. Cadavid, D., O’Neill, T., Schaefer, H., Pachner, A. R. Localization of Borrelia burgdorferi in the nervous system and other organs in a nonhuman primate model of lyme disease. Laboratory Investigation. 80, 1043-1054 (2000).
  32. Mather, T. N., Wilson, M. L., Moore, S. I., Ribiero, J. M. C., Spielman, A. Comparing the Relative Potential of Rodents as Reservoirs of the Lyme Disease Spirochete (Borrelia Burgdorferi).. American Journal of Epidemiology. 130, 143-150 (1989).
  33. Mather, T. N., Telford, S. R., Moore, S. I., Spielman, A. Borrelia burgdorferi and Babesia microti: Efficiency of transmission from reservoirs to vector ticks (Ixodes dammini). Experimental Parasitology. 70 (90), 55-61 (1990).
  34. Telford, S. R., Mather, T. N., Adler, G. H., Spielman, A. Short-tailed shrews as reservoirs of the agents of Lyme disease and human babesiosis. Journal of Parasitology. 76, 681-683 (1990).
  35. Mather, T. N., Fish, D., Coughlin, R. T. Competence of dogs as reservoirs for Lyme disease spirochetes (Borrelia burgdorferi). J. Am. Vet. Med. Assoc. 205, 186-188 (1994).
  36. Telford, S. R., Mather, T. N., Moore, S. I., Wilson, M. L., Spielman, A. Incompetence of deer as reservoirs of the Lyme disease spirochete. Am. J. Trop. Med. Hyg. 39, 105-109 (1988).
  37. Lin, T., et al. Analysis of an Ordered, Comprehensive STM Mutant Library in Infectious Borrelia burgdorferi: Insights into the Genes Required for Mouse Infectivity. PLoS ONE. 7, e47532 (2012).
  38. Lin, T., et al. Central Role of the Holliday Junction Helicase RuvAB in vlsE Recombination and Infectivity of Borrelia burgdorferi. PLoS Pathog. 5, e1000679 (2009).
  39. Jacobs, M. B., Norris, S. J., Phillippi-Falkenstein, K. M., Philipp, M. T. Infectivity of the Highly Transformable BBE02- lp56- Mutant of Borrelia burgdorferi, the Lyme Disease Spirochete, via Ticks. Infection and Immunity. 74, 3678-3681 (2006).
  40. Jacobs, M. B., Purcell, J. E., Philipp, M. T. Ixodes scapularis ticks (Acari: Ixodidae) from Louisiana are competent to transmit Borrelia burgdorferi, the agent of Lyme borreliosis. J. Med. Entomol. 40, 964-967 (2003).
  41. Bockenstedt, L., Mao, J., Hodzic, E., Barthold, S., Fish, D. Detection of Attenuated, Noninfectious Spirochetes in Borrelia burgdorferi-Infected Mice after Antibiotic Treatment. The Journal of Infectious Diseases. 186, 1430-1437 (2002).
  42. Barthold, S. W., et al. Ineffectiveness of tigecycline against persistent Borrelia burgdorferi. Antimicrobial Agents & Chemotherapy. 54, 643-651 (2010).
  43. de Silva, A. M., Telford, S. R., Brunet, L. R., Barthold, S. W., Fikrig, E. Borrelia burgdorferi OspA is an arthropod-specific transmission-blocking Lyme disease vaccine. Journal of Experimental Medicine. 183, 271-275 (1996).
  44. Fikrig, E., et al. Vaccination against Lyme disease caused by diverse Borrelia burgdorferi. Journal of Experimental Medicine. 181, 215-221 (1995).
  45. Philipp, M. T., et al. The outer surface protein A (OspA) vaccine against Lyme disease: efficacy in the rhesus monkey. Vaccine. 15, 1872-1887 (1997).
  46. Embers, M. E., et al. Persistence of Borrelia burgdorferi in Rhesus Macaques following Antibiotic Treatment of Disseminated Infection. PLoS ONE. 7, e29914 (2012).
  47. Kariu, T., Coleman, A. S., Anderson, J. F., Pal, U. Methods for Rapid Transfer and Localization of Lyme Disease Pathogens Within the Tick Gut. J. Vis. Exp. , e2544 (2011).
  48. Policastro, P. F., Schwan, T. G. Experimental infection of Ixodes scapularis larvae (Acari: Ixodidae) by immersion in low passage cultures of Borrelia burgdorferi. J. Med. Entomol. 40, 364-370 (2003).
  49. Broadwater, A. H., Sonenshine, D. E., Hynes, W. L., Ceraul, S., de Silva, A. M. Glass Capillary Tube Feeding: A Method for Infecting Nymphal Ixodes scapularis (Acari: Ixodidae) with The Lyme Disease Spirochete Borrelia burgdorferi. Journal of Medical Entomology. 39, 285-292 (2002).
  50. Hodzic, E., Feng, S., Holden, K., Freet, K. J., Barthold, S. W. Persistence of Borrelia burgdorferi following antibiotic treatment in mice. Antimicrob Agents Chemother. 52, 1728-1736 (2008).
  51. Bockenstedt, L. K., Mao, J., Hodzic, E., Barthold, S. W., Fish, D. Detection of attenuated, noninfectious spirochetes in Borrelia burgdorferi-infected mice after antibiotic treatment. Journal of Infectious Diseases. 186, 1430-1437 (2002).
  52. Schwan, T. G., Burgdorfer, W., Garon, C. F. Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infection and Immunity. 56, 1831-1836 (1988).

Tags

Keywords: Tick FeedingLyme DiseaseBorrelia BurgdorferiIxodes TicksZoonotic PathogenReservoir HostsWhite-footed MousePeromyscus LeucopusNymphal TicksXenodiagnosisMammalian HostsSpirochetesAnimal ModelsMiceNonhuman Primates
check_url/pt/50617?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citar este artigo
Embers, M. E., Grasperge, B. J., Jacobs, M. B., Philipp, M. T. Feeding of Ticks on Animals for Transmission and Xenodiagnosis in Lyme Disease Research. J. Vis. Exp. (78), e50617, doi:10.3791/50617 (2013).
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