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

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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.