Sources of Blood Meals of Sylvatic Triatoma guasayana near Zurima, Bolivia, Assayed with qPCR and 12S Cloning

David E. Lucero mail,
Wilma Ribera,
Juan Carlos Pizarro,
Carlos Plaza,
Levi W. Gordon,
Reynaldo Peña Jr,
Leslie A. Morrissey,
Donna M. Rizzo,
Lori Stevens

Published: December 04, 2014
DOI: 10.1371/journal.pntd.0003365

Abstract

Background

In this study we compared the utility of two molecular biology techniques, cloning of the mitochondrial 12S ribosomal RNA gene and hydrolysis probe-based qPCR, to identify blood meal sources of sylvatic Chagas disease insect vectors collected with live-bait mouse traps (also known as Noireau traps). Fourteen T. guasayana were collected from six georeferenced trap locations in the Andean highlands of the department of Chuquisaca, Bolivia.

Methodology/Principal Findings

We detected four blood meals sources with the cloning assay: seven samples were positive for human (Homo sapiens), five for chicken (Gallus gallus) and unicolored blackbird (Agelasticus cyanopus), and one for opossum (Monodelphis domestica). Using the qPCR assay we detected chicken (13 vectors), and human (14 vectors) blood meals as well as an additional blood meal source, Canis sp. (4 vectors).

Conclusions/Significance

We show that cloning of 12S PCR products, which avoids bias associated with developing primers based on a priori knowledge, detected blood meal sources not previously considered and that species-specific qPCR is more sensitive. All samples identified as positive for a specific blood meal source by the cloning assay were also positive by qPCR. However, not all samples positive by qPCR were positive by cloning. We show the power of combining the cloning assay with the highly sensitive hydrolysis probe-based qPCR assay provides a more complete picture of blood meal sources for insect disease vectors.

Author Summary

The World Health Organization (WHO) estimates that 7 to 8 million people are currently infected with Trypanosoma cruzi, the parasite that causes Chagas disease. The WHO recommends insect vector control as the primary prevention method; and insecticide spraying is the most commonly used intervention technique. Sylvatic insect vectors are a special concern because they are a source of reinfestation after insecticides have been applied to living quarters (domestic) and immediate surroundings (peridomestic). To better understand sylvatic insect vector movement, we used two molecular biology techniques to detect the blood meal sources of sylvatic insect vectors. The first technique, cloning of 12S PCR products, allows us to cast a wide net and detect blood meal sources with no previous knowledge of vertebrates or mammals in the study site. After acquiring knowledge of vertebrates in the study site (either through the aforementioned cloning technique, literature review or survey of the area), the second technique, the species-specific hydrolysis probe-based qPCR provides a highly sensitive assay for particular taxa.