Identification of phlebotomine sand fly blood meals by real-time PCR

Background: Phlebotomine sand flies are blood-feeding insects of great medical and veterinary significance acting as vectors of Leishmania parasites. Studying the blood-feeding pattern of these insects may help in the understanding of their interactions with potential reservoir hosts of Leishmania parasites. In this study, we developed real time PCR assays for the identification of sand fly blood meal. Methods: Six pairs of primers were designed based on cytochrome b gene sequences available in GenBank of the following potential hosts: dog, cat, horse, chicken, black rat, and human.Firstly, SYBR Green-based real time PCR assays were conducted using a standard curve with eight different concentrations (i.e., 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg and 1 fg per 2 μl) of DNA samples extracted from EDTA blood samples from each target animal. Then, DNA samples extracted from field-collected engorged female sand flies belonging to three species (i.e., Lutzomyia longipalpis, L. migonei and L. lenti) were tested by the protocols standardized herein. Additionally, female sand flies were experimentally fed on a black rat (Rattus rattus) and used for evaluating the time course of the detection of the protocol targeting this species. Results: The protocols performed well with detection limits of 10 pg to 100 fg. Field-collected female sand flies were fed on blood from humans (73%), chickens (23%), dogs (22%), horses (15%), black rats (11%) and cats (2%). Interestingly, 76.1% of the L. longipalpis females were positive for human blood. In total, 48% of the tested females were fed on single sources, 31% on two and 12% on three. The analysis of the time course showed that the real time PCR protocol targeting the black rat DNA was able to detect small amounts of the host DNA up to 5 days after the blood meal. Conclusions: The real time PCR assays standardized herein successfully detected small amounts of host DNA in female sand flies fed on different vertebrate species and, specifically for the black rats, up to 5 days after the blood meal. These assays represent promising tools for the identification of blood meal in field-collected female sand flies.