| Summary: | We report the study of sandfly <i>Leishmania</i> infection in an area of low incidence of visceral leishmaniasis in Tunisia. Sandflies were collected monthly using CDC light-traps set in houses and animal shelters during May–November 2016 and 2017. All males were identified at the species level. A sample of 878 females including all gravid specimens was subjected to kDNA qPCR for <i>Leishmania</i> detection and parasite load estimation. <i>Leishmania</i> species were determined by ITS1 PCR sequencing, and species identification of infected sandflies was performed by DNA barcoding. <i>Phlebotomus perfiliewi</i> and <i>P. perniciosus</i> were the dominant species during the two-year period. However, comparison of their relative abundances showed that <i>P. perniciosus</i> was more abundant during peaks of 2017 with longer activity duration. Real-time kDNA PCR did not detect <i>Leishmania</i> infection in 2016, although it identified four positive specimens (0.7%) in 2017. All four infected specimens were identified as <i>P. perniciosus</i>. ITS1 PCR sequencing allowed <i>L. infantum</i> identification in one kDNA qPCR-positive specimen. This was a <i>P. perniciosus</i> gravid female with a high parasite load caught during the long-lasting peak of 2017. This work highlights the usefulness of multi-seasonal studies of sandfly dynamics and kDNA qPCR in screening <i>Leishmania</i> infection and determining <i>L. infantum</i> vectors in hypo-endemic foci of human leishmaniasis.
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