Identification of blood meals in field captured sand flies by a PCR-RFLP approach based on cytochrome b gene

Abstract

Leishmaniasis is a vector-borne disease transmitted by phlebotomine sand flies. Information about blood meal preferences in sand flies is essential to understand the epidemiology of the disease to adopt control measures. In previous studies, a polymerase chain reaction (PCR) of 359bp fragment of the conserved gene cytochrome b (cyt b) and further sequencing were applied in the study of blood meal sources in sand flies collected in the area of a leishmaniasis outbreak in southwest Madrid, Spain, providing significant information about blood meal preferences in the focus. In this work, a PCR-restriction fragment length polymorphism (RFLP) targeting a fragment of 359bp of vertebrate cyt b gene was developed. Restriction endonucleases HaeIII and HinfI generated specific patterns consistent with the blood meal sources found in sand flies. The protocol has been validated with twenty six engorged females collected in the field with CDC traps. Blood meals from nine vertebrates were identified based on PCR-cyt b and sequencing-human, dog, cat, horse, hare, rabbit, sheep, goat and chicken - and mixed blood meals (sheep/human; sheep/goat) - and successfully distinguished by PCR-RFLP. Therefore, this approach is an efficient and reliable alternative method to be applied in entomological surveys.

Keywords: PCR-RFLP; blood meal; cytochrome b; sand fly.