Vol. 38, no. 1 Journal of Vector Ecology 193 Scientiic Note Detection of Leishmania infantum, the etiological agent of visceral leishmaniasis, in Lutzomyia neivai, a putative vector of cutaneous leishmaniasis Edelberto Santos Dias 1* , Érika Monteiro Michalsky 1 , João Cezar do Nascimento 2 , Eduardo de Castro Ferreira 1 , Josiane Valadão Lopes 1 , and Consuelo Latorre Fortes-Dias 3 1 Laboratório de Leishmanioses, Centro de Pesquisas René Rachou/FIOCRUZ, Belo Horizonte, MG, Brazil, edel@cpqrr.fiocruz.br 2 Laboratório de Entomologia, GEZOO/DIVE, Secretaria Estadual de Saúde, Florianópolis, SC, Brazil 3 Laboratório de Enzimologia Aplicada, Centro de Pesquisa e Desenvolvimento, Fundação Ezequiel Dias, Belo Horizonte, MG, Brazil Leishmania (Leishmania) infantum (syn. L. chagasi) (Kuhls et al. 2011) is the etiological agent of the American visceral leishmaniasis (VL) in Asia, Europe, and the Americas. VL transmission occurs through the bite of infected female phlebotomine sand lies (Lainson and Rangel 2005). Two sand ly species are vectors of VL in Brazil: Lutzomyia longipalpis (Lutz & Neivai, 1912) as a primary vector and Lu. cruzi (Mangabeira, 1938) as a secondary one (Brazil 2006). In general, VL is more prevalent among dogs than humans and human cases are preceded by canine cases in certain areas (Bevilacqua et al. 2001). hus, canine VL might be considered a risk factor to human VL. Ninety percent of the VL cases in Latin America occur in Brazil. Between 2007 and 2010, the Brazilian Health System (Brazil 2012) registered a total of 14,763 cases of VL. In the Brazilian state of Santa Catarina, a single case of human VL was registered in 2008. In 2010, however, four suspicious cases of canine VL were reported in the Conceição Lagoon district in Florianópolis, the capital of Santa Catarina state. L. infantum infection was identiied in two of those dogs (unpublished data from the Health Department of Santa Catarina). More detailed investigation showed that all four animals never let the region. Due to the absence of previous reports on the presence of Lu. longipalpis and/or Lu. cruzi in Florianópolis, we screened the local phlebotomine sand ly fauna and examined the captured specimens for the presence of Leishmania spp. DNA. Florianópolis is located on an island of about 672 km 2 with 421,240 inhabitants according to the last census performed by the Brazilian Institute of Geography and Statistics (IBGE) in 2010. It is one of the main destinations in Brazil for national and international tourists, with several places to be visited, including the Conceição Lagoon (27 o 33’38”S, 48 o 27’13”W) where the present study was developed. he Conceição Lagoon is an urban district that ofers ecological trails, extreme sports options, and wonderful sightseeing. It has a few brick houses surrounded by fruit trees and modiied Atlantic rainforest, that stretches from the northeastern to the southern regions of Brazil, northern Argentina, and southeastern Paraguay. In northeast Brazil, it occupies a thin coastal strip not exceeding 65 km in width, while in the south it extends from the coast to as far as 320 km inland. he Atlantic rainforest was reduced to mostly discontinuous fragments, mainly due to deforestation, but still holds considerable biodiversity. It contains multiple canopies that support an extremely rich vegetation mix. his includes an astonishing diversity of ferns, mosses, and epiphytes (‘air plants’ or plants that attach to other plants) including lianas, orchids, and bromeliads. Aiming to survey the local entomological fauna, captures were performed at seven diferent areas located at the highest part of the Conceição Lagoon district at altitudes between 25 m and 55 m. hose areas were chosen according to two criteria: the occurrence of canine cases of VL and/or ecological conditions favoring the presence of phlebotomine sand lies in the neighborhood. CDC light traps were mounted in the peridomiciles from 18:00 to 07:00 for three consecutive nights in the third week of each month between August and December, 2010. he captured specimens were sent to the Leishmaniases Laboratory at the Centro de Pesquisas René Rachou (CPqRR) for identiication using taxonomic keys (Young and Duncan 1994), comparison with specimens deposited at the Reference Collection of the CPqRR, and speciic published descriptions. Lu. neivai was identiied according to Marcondes (1996). A variable number of female specimens (one to ten), belonging to the same species and captured in the same spot, were pooled for total DNA extraction using the Tissue and Cell DNA Extraction Kit (GE HealthCare), independently of the date of capture. Reliability of the extracted DNA was checked by a previous ampliication with speciic primers for a cacophony gene (Lins et al. 2002). he presence of Leishmania DNA was investigated by means of Leishmania-nested PCR (LnPCR) targeting the gene SSUrRNA (small subunit ribosomal ribonucleic acid) (Van Eys et al. 1992, Cruz et al. 2002). he irst ampliication step was performed with primers R221 and R332, which are speciic for the order Kinetoplastida but not exclusively for Leishmania. he resulting PCR product was then ampliied in the presence of Leishmania-speciic primers (R223 and R333) (Cruz et al. 2002). Ampliications were performed with the Illustra PuReTaq Ready- To-Go PCR Beads kit (GE HealthCare). Negative (no DNA) and positive (20 ng of L. infantum DNA) controls were run with every reaction. he ampliied products were analyzed by electrophoresis in 2% agarose gels. he ampliied bands from the previous step were extracted from the gel with the QIAquick gel extraction kit (QIAGEN) and submitted to DNA sequencing with the DYEnamic ET dye Terminator kit (MegaBACE GE HealthCare). DNA editing and alignment were performed with the LaserGene sequence analysis package (DNAStar Inc.) and the BioEdit sotwares. Multiple alignments were performed against the known sequences of the SSUrRNA genes for L. braziliensis and L. amazonensis, both