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Detection of natural infection in Lutzomyia cruzi and Lutzomyia forattinii (Diptera: Psychodidae: Phlebotominae) by Leishmania infantum chagasi in an endemic area of visceral leishmaniasis in Brazil using a PCR multiplex assay

https://doi.org/10.1016/j.actatropica.2008.04.015Get rights and content

Abstract

In order to identify Lutzomyia spp. naturally infected by Leishmania parasites a PCR multiplex assay coupled to non-isotopic hybridization was used for the analysis of insect samples collected by CDC light traps in an endemic area of visceral leishmaniasis (VL) in the municipality of Corumbá, Mato Grosso do Sul State, Brazil in May/June 2006. Wild sand flies were identified and grouped into pools of 10 female specimens and 27 groups in total were collected. Positive results were obtained from Lutzomyia cruzi (2 out of 13 pools) and Lutzomyia forattinii (1 out of 14 pools). The positive pools were confirmed as being infected by Leishmania infantum chagasi after hybridizing the PCR products with a species-specific biotinylated probe derived from the kinetoplast minicircle conserved sequence. Given that we detected infection in 3 out of 27 groups and that there was at least 1 infected insect in each, it was possible to infer an infection rate of 1.5% for Lu. cruzi and 0.7% for Lu. forattinii in the analyzed samples. These results confirm the vectorial role of Lu. cruzi in transmitting L. infantum chagasi and suggest Lu. forattinii as a potential VL vector in the municipality of Corumbá, where notifications of the disease in humans and dogs have increased over the last two decades.

Introduction

Visceral leishmaniasis (VL) is endemic in several Latin American countries and the disease was classically observed in the northeast of Brazil (Deane and Deane, 1962). In the past two decades the re-emergence and urbanization of VL in Brazil has led the disease to become one of the major health problems in the country (Arias et al., 1996, Lainson and Rangel, 2005), where Leishmania infantum chagasi Cunha & Chagas, 1937 is the etiologic agent and Lutzomyia longipalpis (Lutz & Neiva, 1912) acts as primary vector (Deane and Deane, 1962, Lainson and Rangel, 2005).

In the State of Mato Grosso do Sul, VL was reported as early as 1911 with sporadic human cases. Since then, there has been an increase in the number of VL notifications, and spread to new areas so that it can actually be considered as being endemic in several regions within Mato Grosso do Sul (Arruda et al., 1949, Arias et al., 1996, Oliveira et al., 2006). The municipality of Corumbá in the northwest of the State represents one of these endemic areas with active transmission of canine and human visceral leishmaniasis. Although three males of Lu. longipalpis were found in 2001 in Corumbá (Santos et al., 2003), other entomological studies have not demonstrated its presence in the area (Galati et al., 1985, Galati et al., 1997, Brazil et al., 2002). The predominance of Lutzomyia cruzi (Mangabeira, 1938) and Lutzomyia forattinii (Galati et al., 1985) and their anthropophily suggested a possible participation of both sand fly species in the transmission of the disease in Corumbá (Galati et al., 1997). Moreover, the finding of natural Leishmania infection in Lu. cruzi strongly supported its identification as a VL vector in this region (Santos et al., 1998).

In this study, a PCR multiplex non-isotopic hybridization assay was used to evaluate natural infection rates of sand flies captured in a peri-domiciliary area in the municipality of Corumbá by Leishmania spp. The data presented here confirm previous findings regarding the participation of Lu. cruzi in the transmission of L. infantum chagasi, and describe for the first time the occurrence of natural infection by this parasite in Lu. forattinii, bringing a better understanding of the eco-epidemiology of leishmaniasis in the State of Mato Grosso do Sul, Brazil.

Section snippets

Sand fly capture and taxonomic identification

Sand flies were collected in May/June 2006 with CDC light traps (Sudia and Chamberlain, 1962) placed in a pigpen of a peridomicile (19°01′75″S, 57°08′33″W), in Cristo Redentor district of Corumbá, Mato Grosso do Sul State. Alive insects were transferred to labelled containers, taken to the laboratory and killed in the freezer. Using a stereoscopic microscope, pools of 11 individuals were usually performed based on their external coloration (dark in Lu. cruzi and completely pale in Lu. forattinii

Results and discussion

One important step towards the incrimination of Leishmania vectors is the report of the occurrence of naturally infected sand flies (Killick-Kendrick and Ward, 1981). In most epidemiological studies, accurate knowledge of the Leishmania parasite and its vector species can result in an appropriate intervention when transmission is taking place in a given area. Although sand fly digestive tract dissection is the method most commonly used to reveal the rate of natural infections in endemic areas,

Acknowledgements

This investigation was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico—CNPq (476380/2004-9) and FAPERJ. Daniela Pita-Pereira is a fellow PhD student from FIOCRUZ/CAPES; Dr. Constança Britto is a fellow of the CNPq Institution. We would like to thank Dr. J. Gordon Hamilton for the English revision.

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