Kariu Toru, Coleman Adam S, Anderson John F, Pal Utpal
Department of Veterinary Medicine, University of Maryland, USA.
J Vis Exp. 2011 Feb 14(48):2544. doi: 10.3791/2544.
Lyme disease is caused by infection with the spirochete pathogen Borrelia burgdorferi, which is maintained in nature by a tick-rodent infection cycle. A tick-borne murine model has been developed to study Lyme disease in the laboratory. While naíve 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. 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. This technique can also be used to deliver a variety of other biological materials into ticks, for example, specific antibodies or double stranded RNA. 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.
莱姆病由螺旋体病原体伯氏疏螺旋体感染引起，该病原体在自然界通过蜱 - 啮齿动物感染循环得以维持。已开发出一种蜱传播的小鼠模型用于在实验室研究莱姆病。虽然未感染的蜱通过吸食感染小鼠可感染伯氏疏螺旋体，但蜕皮过程需要数周至数月才能完成。因此，开发更快速高效的蜱感染技术，如基于显微注射的方法，是研究莱姆病的重要工具。该方法只需数小时就能产生感染的蜱，并能控制在一组蜱中输送等量的螺旋体。这一点尤为重要，因为利用小鼠通过自然吸食过程产生感染伯氏疏螺旋体的蜱无法确保100%的感染率，并且可能导致吸食后的蜱之间病原体负荷存在差异。此外，在减毒株无法在小鼠中建立感染从而蜱无法自然获得的情况下，显微注射可用于用伯氏疏螺旋体分离株感染蜱。该技术还可用于将多种其他生物材料输送到蜱体内，例如特定抗体或双链RNA。在本文中,我们将演示用体外培养的伯氏疏螺旋体对若蜱进行显微注射。我们还将描述一种使用共聚焦免疫荧光显微镜在蜱肠道中定位莱姆病病原体的方法。
