Monitoring of the Parasite Load in the Digestive Tract of Rhodnius prolixus by Combined qPCR Analysis and Imaging Techniques Provides New Insights into the Trypanosome Life Cycle.

Felipe De Almeida Dias,Ana Caroline Paiva Gandara,Didier Salmon,Flavio Alves Lara,Raquel Juliana Vionette Do Amaral,Emiliano Medei,Pedro Lagerblad De Oliveira,Renata Estebanez Vollú,Marcos Henrique Ferreira Sorgine,Barbara Guerra,Hugo Diego Perdomo,Larissa Rezende Vieira,Suzete Araujo Oliveira Gomes,Alvaro Acosta-Serrano

doi:10.1371/journal.pntd.0004186

Abstract

BackgroundHere we report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI). These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection.Methodology/Principal FindingsqPCR analysis of the parasite load in the insect gut showed several limitations due mainly to the presence of digestive-derived products that are thought to degrade DNA and inhibit further the PCR reaction. We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut (anterior midgut, AM, posterior midgut, PM, and hindgut, H). Using combined fluorescence microscopy and BLI imaging as well as qPCR analysis, we showed that during their journey through the insect digestive tract, most of the parasites are lysed in the AM during the first 24 hours independently of the gut microbiota. During this short period, live parasites move through the PM to establish the onset of infection. At days 3–4 post-infection (p.i.), the parasite population begins to colonize the H to reach a climax at day 7 p.i., which is maintained during the next two weeks. Remarkably, the fluctuation of the parasite number in H remains relatively stable over the two weeks after refeeding, while the populations residing in the AM and PM increases slightly and probably constitutes the reservoirs of dividing epimastigotes.Conclusions/SignificanceThese data show that a tuned dynamic control of the population operates in the insect gut to maintain an equilibrium between non-dividing infective trypomastigote forms and dividing epimastigote forms of the parasite, which is crucial for vector competence.

Highlights

Chagas disease is considered to be a neglected major parasitic tropical illness in Latin America that affects 7–8 million people distributed in 21 endemic countries and is responsible for 12,000 deaths each year
We report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by quantitative real-time PCR (qPCR) coupled with fluorescence and bioluminescence imaging (BLI)
We developed a real-time PCR strategy targeting the T. cruzi repetitive satellite DNA sequence using as internal standard for normalization, an exogenous heterologous DNA spiked into insect samples extract, to precisely quantify the parasite load in each segment of the insect gut

Summary

Introduction

Chagas disease is considered to be a neglected major parasitic tropical illness in Latin America that affects 7–8 million people distributed in 21 endemic countries and is responsible for 12,000 deaths each year (http://www.who.int/mediacentre/factsheets/fs340/en/). This disease is an increasing worldwide public health problem due to the migration of infected subjects to non-endemic, more developed regions, mainly North America and Europe, where they could potentially transmit the disease by either hemotransfusion, organ donation, or pregnancy. We report the monitoring of the digestive tract colonization of Rhodnius prolixus by Trypanosoma cruzi using an accurate determination of the parasite load by qPCR coupled with fluorescence and bioluminescence imaging (BLI) These complementary methods revealed critical steps necessary for the parasite population to colonize the insect gut and establish vector infection

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