Polymerase chain reaction-based assay for the detection and identification of sand fly gregarines inLutzomyia longipalpis, a vector of visceral leishmaniasis

Abstract

Gregarines that parasitise phlebotomine sand flies belong to the genus Psychodiella and, even though they are highly hostspecific, only five species have been described to date. Their most outstanding features include the unique localisation of the oocysts in the accessory glands of the female host, which ensures contamination of the egg surface during oviposition, and the fact that they naturally parasitise the vectors of Leishmania, causal agent of leishmaniasis. The type species, Ps. chagasi, was first described in Lutzomyia longipalpis, vector of visceral leishmaniasis (VL), from Brazil. We recently reported Ps. chagasi sequences in Lu. longipalpis from Posadas (Misiones, Argentina), an endemic VL location where this gregarine had not been previously recorded. In order to analyse the incidence of Ps. chagasi infections in Lu. longipalpis from this location, the aim of this study was to develop a diagnostic assay for sand fly gregarine parasites in Lu. longipalpis. For this, we designed primers using the Ps. chagasi sequences we previously identified and performed an in vitro validation by PCR amplification of the original sand fly samples. Their specificity and sensitivity as diagnostic primers were subsequently confirmed by PCR reactions using total DNA extracted from naturally infected Lu. longipalpis from the same location (Posadas, Argentina). Journal of Vector Ecology 39 (1): 83-93. 2014.

Optimization of DNA Extraction from Individual Sand Flies for PCR Amplification

“…In our studies we were interested in detecting parasite infection in Lutzomyia spp. sand flies by PCR amplification [11]. Nevertheless, as parasite DNA is not necessarily expected, we first needed to confirm that the DNA extraction had been successful, to ensure that negative results were not due to a poor extraction.…”

Optimisation of DNA Extraction from Individual Sand Flies for PCR Amplification

“…In our studies we were interested in analysing parasite infection in Lutzomyia spp. sand flies by PCR amplification [1]. For this, we previously compared two traditional DNA extraction methods using pools of 5 and 10 field-captured L. longipalpis sand flies: one that uses detergents (sodium dodecyl sulfate, SDS) and salt (potassium acetate) to eliminate DNA-associated proteins, and which was reported as an optimisation for the extraction of DNA from smaller sand flies [16]; and another, which we here refer to as pAC, that uses detergent (SDS), proteinase K, and phenol-chloroform extraction ( [2] and Acardi personal communication).…”