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

项目摘要/摘要 对寄主物种的知识，这些寄主物种维持着肩部硬蜱的种群，莱姆病媒介， 而莱姆病病原体伯氏疏螺旋体对于改善预防策略是必不可少的 这种使人衰弱的疾病每年在美国影响大约30万人。然而， 大多数关于宿主物种的研究只集中在两个宿主物种上--白尾鹿 (Odocoileus Virginianus)和白足小鼠(Permyscus Leucopus)，尽管有文献记载 19种哺乳动物和60种鸟类，它们是美国东部肩周炎的允许宿主。 这项研究的具体目的是测试一种新的扩增子测序方法，以确定其身份 寻找肩部硬体若虫幼虫阶段的血食。我们将重点关注若虫，因为这 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法采用并行聚合酶链式反应，并使用针对18S、12S和16S rDNA的引物对 从幼虫期血粉中扩增相对较短的DNA基因区(115-294个核苷酸)，如 以及用于检测常见人类病原体(伯氏杆菌、无浆体)的额外引物组 吞噬细胞和微小巴贝斯虫，莱姆病、无浆体病和人巴贝斯虫病的病原体， )。随后将对扩增产物、病原体和 分类分类。该方案将在一个独特的阳性对照文库--DNA分离物上进行测试 从已知幼虫期寄主的肩部若虫中分离出来的。此外，该协议还将进行测试 在佛蒙特州南部收集的数百个肩袖单胞菌DNA分离物上-一个地区是世界上最高的 莱姆病在美国的发病率--通过九项长期研究收集 2015-2017年间的网站。这些数据将提供所需的对肩胛虫寄主的使用情况以及 水库容量，或水库宿主对病原体感染流行的绝对贡献 在扁虱种群中。这些信息可以用来通过改善 通过改进对野生动物的了解来估计时间和空间上的风险 病原体传播，也通过告知基于减轻 关键寄主物种的贡献。

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.