Development of new screening tools to evaluate dog exposure to Phlebotomus tobbi and Phlebotomus papatasi sand flies

Abstract Background Leishmaniases are a group of medically and veterinary important diseases caused by protozoan parasites of the genus Leishmania (Kinetoplastida) and transmitted by blood-feeding female sand flies (Diptera: Phlebotominae). To assess the risk of Leishmania transmission, host exposure to sand fly bites can be measured through the detection of host antibodies to vector salivary proteins. Anti-sand fly saliva antibodies are elicited by repeated exposure of the mammalian host to sand fly salivary proteins deposited into the host skin during blood feeding. These antibodies are species-specific and correlate with the intensity of host exposure to sand fly bites. The aim of our study was to develop enzyme-linked immunosorbent assays (ELISAs) on the basis of recombinant sand fly salivary antigens as tools to measure exposure to sand fly bites, hence serving as risk markers for Leishmania transmission. We focused on two Old World vector sand fly species: Phlebotomus tobbi as a vector of Leishmania infantum , and Phlebotomus papatasi as a vector of Leishmania major . Methods Dog sera from endemic areas in Türkiye were used to characterise the main salivary antigens of P. papatasi and P. tobbi in immunoprecipitation and immunoblot assays, followed by proteomic analysis. Four candidate salivary proteins from each species were expressed in Escherichia coli and subsequently evaluated and validated in an ELISA as potential risk markers of dog exposure to sand flies. Results Among the eight tested recombinant candidates, P. tobbi rSP38 (a yellow-related protein), P. papatasi rSP36 (an apyrase) and P. papatasi rSP42 (a yellow-related protein) were identified as the most reliable antigens to replace salivary gland homogenate (SGH) in serological assays. They demonstrated high correlation with SGH and exhibited high sensitivity and specificity. Conclusions These recombinant antigens can be developed into a standardized assay to measure dog exposure to sand flies, which can serve as a complementing tool for leishmaniasis surveillance and control. Graphical Abstract