Needle in a haystack: A droplet digital polymerase chain reaction assay to detect rare helminth parasites infecting natural host populations

Abstract

Helminths infect humans, livestock, and wildlife, yet remain understudied despite their significant impact on public health and agriculture. Because many of the most prevalent helminth-borne diseases are zoonotic, the health of diverse host species are closely interconnected. Therefore, understanding helminth transmission among wildlife could improve predictions and management of infection risks across species. A key challenge to understanding helminth transmission dynamics in wildlife is accurately and quantitatively tracking infection levels across hosts and environments. Traditional methods, such as visual parasite identification from environmental samples or infected hosts, are time-consuming, while standard molecular techniques (e.g., PCR and qPCR) often lack the sensitivity to reliably detect lower parasit burdens. These limitations often underestimate the prevalence and severity of infection, hindering efforts to manage infectious diseases. Here, we developed a multiplexed droplet digital PCR (ddPCR) assay to quantify helminth levels in aquatic habitats using 18S rRNA target genes. Using Schistocephalus solidus and their copepod hosts as a case study, we demonstrate ddPCRs sensitivity and precision. By establishing a 1:1 infection standard in the lab, we contextualize ddPCR gene concentration data to quantify both host and parasite numbers in field samples. The assay is highly reproducible, reliably detecting target genes at concentrations as low as 1 picogram of DNA in lab standards and field samples (multi-species and eDNA). Thus, we provide a toolkit for quantifying infection loads in intermediate hosts and monitoring infection dynamics across spatio-temporal scales in multiple helminth systems of concern for public health, agriculture, and conservation biology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/634533v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1bc2e40org.highwire.dtl.DTLVardef@180822eorg.highwire.dtl.DTLVardef@1e2c2c8org.highwire.dtl.DTLVardef@621909_HPS_FORMAT_FIGEXP M_FIG Applications of ddPCR probe-primer design to parallel systems. Cyclopoid copepods serve as initial hosts for diverse helminthic diseases distributed globally. The primers designed in this assay are suitable for other systems, with minimal work required for probe design specific to each helminth species. C_FIG