Development of a novel multipurpose model to propagate and study the tick transmission cycle of relapsing fever spirochetes from Eurasia.

项目摘要 回归热（RF）螺旋体是一组被忽视的媒介传播的病原体， 儿童发病率和死亡率。目前培养和评估RF螺旋体蜱传播的方法 在它们普遍存在的资源有限的环境中是不切实际的。例如，将螺旋体繁殖到 培养基昂贵且劳动密集。此外，在培养基中长时间的体外繁殖导致 它们已经非常复杂的基因组的DNA重排，这导致减少载体 定植和发病机制。有一个具有成本效益的实用方法来增长和研究的迫切需要 来自地球仪被忽视地区的RF螺旋体。该项目建立在我们之前的出版物报告的基础上， 第一次分离出高加索疏螺旋体，它是高加索种复合体的一部分，分布于 欧亚和中东。此外，我们最近的RF螺旋体基因组学进展解决了一个主要问题， 本发明克服了该领域的局限性，并产生了完整的染色体和质粒组装和注释的基因组。 我们开发的基因组学工具现在可以用来描述一个复杂的物种的模式。 有趣的是，历史文献表明含胚鸡蛋（ECEs）可以感染RF 螺旋体通过蜱叮咬和细菌生长在这个系统中。然而，在分子和基因组学 没有资源来描述这种模式，关于病原体的知识差距仍然存在 ECEs中的基因组稳定性利用RF螺旋体基因组学的最新进展，ECE模型现在可以 已经完全发育到可以研究紫茎泽兰复合种的程度。在这项研究中，提出了两个目标。第一个目标是利用 RF螺旋体的基因组学资源，评估质粒多样性，并确定RF螺旋体的独特质粒标记物。 B。高加索分离物通过比较基因组分析。第一个目标是完成参考质量 B的基因组组装。caucasica分离株，并进行比较基因组学分析，以确定质粒 标记。第二个目标是开发一组特异性探针，用于评估质粒保留和 在B.高加索生活史在第二个目标中，我们将评估B的基因组变化。 caucasica使用tick-ECE模型。第一个目标是发展B。高加索模式， 评估螺旋体的生长、蜱的获得以及随后蜱将螺旋体传播给未感染卵。的 第二个目的是评估质粒稳定性和B的能力。caucasica在传代后仍具有传染性 通过ECES。建立欧洲经委会模型将导致一个具有成本效益的方法，以放大射频 螺旋体在没有培养基的情况下。此外，该模型的开发还提供了一个实用的系统 研究RF螺旋体在资源有限环境中的传播周期和媒介能力。与 低收入国家RF螺旋体的出现和健康负担，重要的是要开发系统， 传播和研究这些被忽视的病原体。

Project Summary Relapsing fever (RF) spirochetes are a group of neglected vector-borne pathogens that are a significant cause of child morbidity and mortality. The current approaches to grow and assess tick transmission of RF spirochetes are impractical in resource limited settings where they are prevalent. For example, propagating spirochetes in culture medium is expensive and labor intensive. Moreover, prolonged in vitro propagation in medium causes DNA rearrangements of their already exceedingly complex genomes, and this results in diminished vector colonization and pathogenesis. There is a critical need for cost effective practical approaches to grow and study RF spirochetes from neglected regions of the globe. This project builds on our prior our publication reporting the first isolation of Borrelia caucasica, which is part of the Persica species complex and is distributed through Eurasia and the Middle East. Moreover, our recent advances RF spirochete genomics addressed a major limitation in the field and resulted in complete chromosome and plasmid assembled and annotated genomes. The genomics tools we developed can now be utilized to characterize a model for the Persica complex species. Interestingly, historical literature suggested that embryonated chicken eggs (ECEs) could be infected with RF spirochetes by tick bite and the bacteria grew in this system. However, at the time molecular and genomics resources were not available to characterize this model and knowledge gaps remain about the pathogens genomic stability in ECEs. Leveraging recent advances in RF spirochete genomics, the ECE model can now be fully developed to study Persica complex species. In this study, two aims are proposed. The first aim is to utilize genomics resources for RF spirochetes and evaluate plasmid diversity and identify unique plasmid markers of B. caucasica isolates through a comparative genomic analysis. The first objective is to complete reference quality genomes assemblies of B. caucasica isolates and perform a comparative genomics analysis to identify plasmid markers. The second objective is to develop a panel of specific probes needed to evaluate plasmid retention and rearrangements during the B. caucasica life cycle. In the second aim, we will assess genomic changes of B. caucasica using the tick – ECE model. The first objective is to develop the B. caucasica model in ECEs and evaluate spirochete growth, tick acquisition, and subsequent tick transmission of spirochetes to naïve eggs. The second objective is to assess plasmid stability and the ability of B. caucasica to remain infectious after passage through ECEs. The establishment of the ECE model will result in a cost-effective approach to amplify RF spirochetes in the absence of culture medium. Moreover, the development of this model offers a practical system to study the transmission cycle and vector competence of RF spirochetes in resource limited settings. With the emergence and health burden of RF spirochetes in low-income countries, it is important to develop systems to propagate and study these neglected pathogens.