Application of parallel PCR and DNA sequencing for Ixodes scapularis host blood meal detection

Understanding which hosts are responsible for maintaining populations of Ixodes scapularis ticks and pathogens is biased by an historical focus on white-tailed deer (Odocoileus virginianus) and white-footed mice (Peromyscus leucopus), with considerably less attention paid to other species that may affect human exposure risk. The objective of this research is to apply parallel PCR and amplicon sequencing to determine the identity of the nymphal- and larval-stage blood meal of questing blacklegged ticks (Ixodes scapularis). We tested DNA extracts from flat nymphal stage I. scapularis ticks for which the larval blood meal host was known (positive controls) as well as on wild caught I. scapularis nymphs and adults from southwestern Vermont. We successfully identified 6/8 mammal species for which positive controls were available. Positive controls obtained from all tested bird species returned an accurate match to Passeriformes in our custom database. The best results were from the squirrel species, for which 6/10 ticks matched the family Sciuridae. The success rate of detection of wild caught ticks was lower in comparison to positive controls. Among the species identified as the larval-stage blood meal for Ixodes scapularis were white-tailed deer (Odocoileus virginianus) and Bobcat (Lynx rufus). The detection of juvenile feeding on white-tailed deer DNA was further supported by visual confirmation of feeding on road-killed O. virginianus and by the presence of Babesia odocoile in black legged nymphs. We expect this ongoing research to continue to provide new insights into the feeding behavior of I. scapularis in Vermont.