Application of parallel PCR and DNA sequencing for detecting remnant host DNA from the prior blood meal of Ixodes scapularis

项目总结/摘要 了解维持肩突硬蜱（莱姆病媒介）种群的宿主物种， 和莱姆病病原体伯氏疏螺旋体，对于改善预防策略至关重要， 这种使人衰弱的疾病在美国每年影响大约30万人。然而，在这方面， 大多数关于宿主物种的研究都集中在两种宿主物种上--白尾鹿 （Odocoileus virginianus）和白足鼠（Peromyscus leucopus），尽管有文献记载， 19种哺乳动物和60种鸟类是I.美国东部的肩胛肌。 本研究的具体目的是测试一种新的扩增子测序方案，用于确定 肩突硬蜱幼虫期的血粉。我们将专注于这一点，因为 是美国东部大多数莱姆病病例的生命阶段。 这种新的宿主检测方案利用了针对18 S、12 S和16 S rDNA的引物对的平行PCR 从幼虫阶段血粉中扩增相对较短的DNA基因区（115 - 294 nt）， 以及用于检测常见人类病原体的其它引物组（B.伯氏无形体 嗜吞噬细胞菌和田鼠巴贝斯虫，莱姆病、无形体病和人巴贝斯虫病的病原体， 分别）。随后将对扩增子进行高通量测序，并对病原体和 分类学分类方案将在唯一的阳性对照- DNA分离株文库中进行检测 从我。已知其幼虫期宿主的肩胛骨。此外，将对方案进行测试 在数百个I。在佛蒙特州南部收集的肩胛骨DNA分离物-一个最高的地区之一 莱姆病在美国的发病率-这是从9项长期研究中收集的 2015 - 2017年的网站。这些数据将提供必要的见解I。肩胛肌主机使用以及 储库容量，或储库宿主对病原体感染流行的绝对贡献 在蜱虫种群中。这些信息可用于预防莱姆病传播， 通过更好地了解野生动物宿主对环境的贡献， 病原体传播，并通过告知基于减轻 关键宿主物种的贡献。

PROJECT SUMMARY/ABSTRACT Knowledge of the host species that maintain populations of Ixodes scapularis, the Lyme disease vector, and Borrelia burgdorferi, the Lyme disease pathogen, is essential to improving preventative strategies for this debilitating disease that affects an estimated 300,000 people annually in the United States. However, the majority of research on host species is focused on only two host species - the white-tailed deer (Odocoileus virginianus) and the white-footed mouse (Peromyscus leucopus), despite documentation of 19 mammals and 60 bird species that are permissive hosts for I. scapularis in the eastern United States. The specific aim of this study is to test a new amplicon sequencing protocol for determining the identity of the larval stage blood meal of questing Ixodes scapularis nymphs. We will focus on nymphs because this is the life stage responsible for most Lyme disease cases in the eastern United States. This new host detection protocol utilizes parallel PCR with primer pairs targeting 18S, 12S and 16S rDNA to amplify relatively short DNA gene regions (115 - 294 nt) remnant from the larval stage blood meal, as well as additional primer sets for detection of common human pathogens (B. burgdorferi, Anaplasma phagocytophilum and Babesia microti, agents of Lyme disease, Anaplasmosis and human Babesiosis, respectively). This will be followed by high-throughput sequencing of the amplicons, and pathogen and taxonomic classification. The protocol will be tested on a unique library of positive controls - DNA isolates from I. scapularis nymphs for which the larval stage host is known. Additionally, the protocol will be tested on hundreds of I. scapularis DNA isolates collected in southern Vermont - a region with one of the highest rates of Lyme disease incidence in the United States - that were collected across nine long-term study sites from 2015 - 2017. These data will provide needed insights into I. scapularis host use as well as reservoir capacity, or the absolute contribution of a reservoir host to the prevalence of pathogen infection in a tick population. This information can be used to prevent Lyme disease transmission by improving estimates of risk over time and space through improved knowledge of wildlife host contributions to pathogen transmission, and also by informing management strategies based on mitigating the contributions of key host species.