Research Article: A Reverse Transcriptase-PCR Assay for Detecting Filarial Infective Larvae in Mosquitoes

Date Published: June 18, 2008

Publisher: Public Library of Science

Author(s): Sandra J. Laney, Caitlin J. Buttaro, Sabato Visconti, Nils Pilotte, Reda M. R. Ramzy, Gary J. Weil, Steven A. Williams, Thomas Raymond Unnasch

Abstract: BackgroundExisting molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript.Methodology/Principal FindingsCandidate genes identified by bioinformatics analysis of EST datasets across the B. malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen) was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes.Conclusions/SignificanceThis L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971) that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens) may also be useful for detecting infective stages of other vector-borne parasites.

Partial Text: Lymphatic filariasis (LF) is a disabling tropical disease that is caused by filarial nematode parasites that are transmitted by mosquitoes. Brugia malayi and B. timori account for approximately 10% of the global LF burden of 120 million infected individuals [1]. The Global Programme to Eliminate Lymphatic Filariasis (GPELF) has the ambitious goal of eliminating this disease by the year 2020 [2]. The program is largely based on a strategy of mass drug administration (MDA) of antifilarial medications to endemic populations with the aim of reducing human infection rates to levels that cannot sustain transmission by mosquitoes.

Several technical advances were required for a molecular L3 detection assay including a simple method for preserving parasite RNA in mosquitoes, a method for efficiently extracting RNA from parasites in pools of mosquitoes, identification of an L3-activated gene, and a method to sensitively detect that gene’s transcript. We found that parasite RNA could be efficiently extracted from infected mosquitoes preserved in RNAlater (Ambion, Inc.) using a simple BB-grinding technique and a phenol-based extraction procedure. We then identified TC8100 as a L3-activated transcript and developed methods for its detection by conventional RT-PCR and qRT-PCR.



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