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

应用平行PCR和DNA测序检测肩胛硬蜱前期血粉中残留宿主DNA

• 批准号: 10193631
• 负责人: William Landesman
• 金额: $ 1.88万
• 依托单位: UNIVERSITY OF BRIDGEPORT
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-08 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10193631
• 关键词: Adoption; Affect; Anaplasma phagocytophilum; Anaplasmosis; Attention; Babesia microti; Babesiosis; Birds; Black-legged Tick; Blood; Borrelia burgdorferi; Classification; Communities; Competence; County; DNA; DNA sequencing; Data; Data Collection; Deer; Detection; Disease; Disease Vectors; Documentation; Ecology; Ecosystem; Exposure to; General Practitioners; Genes; Habitats; Health; High-Throughput Nucleotide Sequencing; Human; Incidence; Infection; Knowledge; Laboratories; Lead; Libraries; Life; Longitudinal Studies; Lyme Disease; Lyme Disease Vaccines; Mammals; Methods; Mitochondria; Nymph; Peromyscus; Polymerase Chain Reaction; Population; Prevalence; Prevention strategy; Preventive measure; Protein Analysis; Protocols documentation; Public Health; Recombinant DNA; Reporting; Research; Resolution; Risk; Risk Estimate; Risk Reduction; Role; Sampling; Site; Tail; Taxonomy; Testing; Tick-Borne Diseases; Ticks; Time; United States; Vaccines; Vermont; White-Footed Mouse; base; disease transmission; exposed human population; feeding; human pathogen; improved; insight; pathogen; permissiveness; prevent; stable isotope; tick feeding; tick population; transmission process; vector

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.

项目摘要/摘要 了解维持ixodes肩capularis的宿主物种，莱姆病载体， 莱姆病病原体Borrelia Burgdorferi对于改善预防策略至关重要 这种使人衰弱的疾病每年在美国影响约30万人。然而， 关于宿主物种的大多数研究仅集中在两种宿主物种上 - 白尾鹿 （Odocoileus virginianus）和白脚的老鼠（peromyscus leucopus），尽管有记录 美国东部的19种哺乳动物和60种鸟类是宽阔的scapularis宿主。 这项研究的具体目的是测试一种新的扩增子测序方案，以确定 幼虫阶段的血液粉ixodes肩ben若虫。我们将专注于若虫，因为这 是美国东部大多数莱姆病病例的生命阶段。 该新的主机检测协议利用靶向18s，12s和16s rDNA的引物对的平行PCR 从幼体阶段血粉中扩增相对较短的DNA基因区域（115-294 nt）， 以及用于检测普通人病原体的其他底漆集（B. burgdorferi，Anaplasma 吞噬细胞和贝贝西亚微阶级，莱姆病的药物，无肿瘤和人类Babesiosis， 分别）。随后将进行扩增子的高通量测序，病原体和 分类分类。该协议将在唯一的阳性对照库-DNA分离株上进行测试 来自肩cap骨若虫，幼虫阶段宿主已知。此外，该协议将进行测试 在佛蒙特州南部收集的数百个肩cap骨DNA分离株上 - 一个地区之一 在美国九个长期研究中收集的莱姆病发病率 2015年至2017年的网站。这些数据将为I. scapularis主机使用以及 储层容量，或储层宿主对病原体感染的绝对贡献 在tick人群中。该信息可用于通过改善莱姆病的传播 通过提高野生动植物寄主的贡献的知识来估计随时间和空间的风险估计 病原体的传播，以及通过缓解管理策略的通知 关键宿主物种的贡献。