The PEARL toolkit: Using sand flies to identify leishmaniasis animal reservoirs

In many leishmaniasis foci, reservoirs that maintain infection remain unknown. Here, we developed a field-applicable toolkit based on the analysis of individual blood fed sand flies (IBF) to identify reservoirs. Sand flies were given a Leishmania donovani -infected first blood meal (iBM1) by feeding artificially on a membrane or naturally on a clinically ill animal followed by two subsequent uninfected blood meals (BMS ⁺ ). Bulk-RNAseq was used to identify two target parasite genes, sherp and a novel hypothetical gene ( HPB ), which exhibited a significantly higher expression in BMS ⁺ compared to iBM1 sand flies. DNA and RNA were co-extracted from IBF. DNA was used to detect Leishmania infection and the blood meal source; RNA was used to assess expression of target genes by qRT-PCR. Linear discriminant analysis (LDA) of target gene expression classified sand fly specimens based on their iBM1 or BMS ⁺ status. Co-extraction yielded a mean of >800ng per IBF for DNA and RNA. We detected ≥1 parasite/s by kDNA qPCR and ssu rRNA RT-qPCR. LDA identified iBM1 parasites with a predictive accuracy of ∼87% and ∼82%, in membrane or naturally fed sand flies, respectively. This toolkit provides an innovative approach to identification of leishmaniasis reservoirs informing targeted control strategies.