A novel eDNA-based surveillance strategy targeting quiescent ticks in soil

Ticks are obligate hematophagous ectoparasites with worldwide distribution and the ability to transmit tick-borne pathogens to humans and animals. Their rapid geographic expansion and abundance of multiple vector species emphasize the need for systematic tick surveillance. Current tick surveillance efforts target the host-seeking phase, even though ticks spend most of their life in the upper soil in a quiescent state. In an effort to expand the existing tick surveillance strategies, a novel molecular approach was developed for the detection, identification, and relative quantification of tick DNA in soil samples. Amblyomma americanum Linnaeus, Ixodes scapularis Say, Rhipicephalus annulatus Say, and R. sanguineus Latreille (Ixodida: Ixodidae), all species of medical and veterinary health importance, were employed for protocol optimization. Ticks (individual or pooled) from all life stages were spiked in soil, and DNA was extracted using a verified protocol adapted for arthropods. Tick DNA was detected with multiplex Polymerase Chain Reaction (PCR) using novel species-specific primers designed based on sequence dissimilarities at the ribosomal RNA gene cluster (5.8S-ITS2-28S rRNA genes) so that the PCR products for each species would be distinguishable by size in gel electrophoresis. Individual target species were successfully detected and identified from soil, while in pooled samples, all 4 species were clearly distinguishable. Detection sensitivity was as low as 5 eggs/40 ml of soil. Specificity tests revealed the absence of cross-reaction between species-specific primers and nontarget species. This approach could provide a robust, standardized, and cost-efficient technique for tick detection in locations of putative tick abundance, complementing existing surveillance methods and facilitating control interventions.