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 flies (Lainson and Rangel 2005). Two sand fly 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). Thus, 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 L. infantum infection was identified in two of those dogs (unpublished data from the Health Department of Santa Catarina). More detailed investigation showed that all four animals never left 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 fly fauna and examined the captured specimens for the presence of Leishmania spp. DNA.

Florianópolis is located on an island of about 672 km2 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 (27o33′38″S, 48o27′13″W) where the present study was developed. The Conceição Lagoon is an urban district that offers ecological trails, extreme sports options, and wonderful sightseeing. It has a few brick houses surrounded by fruit trees and modified 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. The 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. This 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 different areas located at the highest part of the Conceição Lagoon district at altitudes between 25 m and 55 m. Those areas were chosen according to two criteria: the occurrence of canine cases of VL and/or ecological conditions favoring the presence of phlebotomine sand flies 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. The captured specimens were sent to the Leishmaniases Laboratory at the Centro de Pesquisas René Rachou (CPqRR) for identification using taxonomic keys (Young and Duncan 1994), comparison with specimens deposited at the Reference Collection of the CPqRR, and specific published descriptions. Lu. neivai was identified 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 amplification with specific primers for a cacophony gene (Lins et al. 2002).

The 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). The first amplification step was performed with primers R221 and R332, which are specific for the order Kinetoplastida but not exclusively for Leishmania. The resulting PCR product was then amplified in the presence of Leishmania-specific primers (R223 and R333) (Cruz et al. 2002). Amplifications 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. The amplified products were analyzed by electrophoresis in 2% agarose gels.

The amplified 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 softwares. Multiple alignments were performed against the known sequences of the SSUrRNA genes for L. braziliensis and L. amazonensis, both etiological agents of ACL, and for L. infantum, the etiological agent of VL in Brazil.

A total of 815 males and 658 females of phlebotomine sand flies was captured during our study. The species and respective specimen numbers are listed in Table 1. Concerning phlebotomine females, the main species were Lutzomyia fischeri (69.0%), Lu. migonei (19.3%), and Lu. neivai (9.1%), totaling 94.4% of all the specimens captured.

Table 1.  Phlebotomine sand flies captured in the Conceição Lagoon in Florianópolis (Santa Catarina state, Brazil) between August and December, 2010.
SpeciesSpecimens captured
 Male%Female%Male + female
Brumptomyia mangabeirai 60.700.06
B. nitzulescui 70.900.07
Lu. edwardsi 60.791.415
Lu. fischeri 35843.945469.0812
Lu. firmatoi 40.510.25
Lu. lanei 10412.800.0104
Lu. migonei 31839.012719.3445
Lu. neivai 91.1609.169
Lu. pascalei 30.400.03
Lutzomyia sp.00.030.53
Lu. tupynambai 00.040.64

To investigate the natural infection by Leishmania, 136 pooled samples of Lutzomyia spp. females (66 of Lu. fisheri, 25 of Lu. neivai, 34 of Lu. migonei, six of Lu. edwardsi, four of Lu. tupynambai, and one of Lu. firmatoi) were submitted to total DNA extraction. The presence of a cacophony gene for phlebotomine sand flies was confirmed by the presence of the characteristic 220 bp amplicon on agarose gels (data not shown). After LnPCR, nine among those 136 pooled samples displayed the characteristic 335 bp fragment of leishmanial DNA that confirms the presence of the parasite in those samples (Figure 1). Leishmania spp. DNA was present in 4/25 pooled samples of Lu. neivai, 4/66 pooled samples of Lu. fischeri, and 1/34 pooled samples of Lu. migonei. Considering at least one infected insect in each pooled sample, it is possible to infer minimal Leishmania infection rates of 1.6% for Lu. neivai, 0.6% for Lu. fischeri, and 0.3% for Lu. migonei, respectively.


Figure 1. Electrophoresis on 2% agarose gels of the products obtained after amplification of phlebotomine sand fly total DNA by LnPCR. The 335bp amplicon is Leishmania-specific (Cruz et al 2002). The numbers on top of the gel identify the pooled sample. The number of specimens in the pooled sample was ten in 69 and 72; eight in 117; six in 75; three in 125; two in 36, 79 and 88, and one in 136. The pool samples that did not show any amplification product on the gel are not shown. M- 100bp DNA ladder; PC- positive control (L. infantum DNA); NC- negative control (no DNA).

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Molecular typing of the Leishmania species was accomplished for a single pooled sample of Lu. neivai (numbered 125). Multiple DNA alignment returned 100% identity with L. infantum (Figure 2). These data indicate the natural infection of Lu. neivai, a putative vector of the American cutaneous leishmaniasis (ACL), with the etiological agent of VL. Unfortunately, we were unable to identify the Leishmania at a specific level in the other positive samples for leishmanial DNA.


Figure 2. Nucleotide (nt) alignment of the 335bp fragment of Leishmania spp. DNA detected in Lu. neivai (this study) with Leishmania (Viannia) braziliensis (M80292.1), L. (V.) amazonensis (M80293.1) and L. (L.) infantum (M81430.1) (syn L. chagasi). Identical nucleotides are represented by dots and nucleotide deletion by a hyphen.

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The state of Santa Catarina is considered a vulnerable, but not a transmission area of VL, and it is a transmission area of the American cutaneous leishmaniasis (ACL). Between 2007 and 2010, 236 cases of ACL have been registered by the Brazilian Health System, 20 of which were in Florianópolis (Brazil 2012). Thus, suspected or putative vectors of ACL would be expected to occur there. In fact, the three major phlebotomine sand fly females (Lu. migonei, Lu. fischeri, and Lu. neivai) captured in the present study belong to that group (Peterson and Shaw 2003, Rangel and Lainson 2009, Salomón et al. 2010). All of them were found to be infected with etiological agents of ACL in Brazil (Pitta-Pereira et al. 2005, 2009, Rocha et al. 2010, Oliveira et al. 2011).

Concerning the etiological agent of VL, L. infantum, it was associated with ACL vectors in a few previous studies. One specimen of Lu. neivai was found to be infected by L. infantum in an urban area in the Brazilian state of Minas Gerais with no records of human VL and no data available for canine VL (Saraiva et al. 2009). The presence of L. infantum was also described in females of Lu. migonei in Brazil and in Argentina (Carvalho et al. 2010, Salomón et al. 2010). Association of those data and ours with epidemiological evidence suggests that other Lutzomyia species may be acting as vectors of VL, mainly in areas where Lu. longipalpis is absent (Souza et al. 2003, Carvalho et al. 2010, Salomón et al. 2010). Unfortunately, the data set available so far do not support more than a suggestion. According to Killick-Kendrick (1990), four criteria must be fulfilled before incriminating a given species as a vector of a zoonotic disease: to feed on humans and in the animal reservoir, to support the parasites after ingestion and expulsion of the infected blood meal, to display indistinguishable parasites from those isolated from patients, and to transmit the parasite by biting. Although it seems plausible to hypothesize that Lu. neivai and perhaps Lu. migonei might be participating in the transmission cycle of canine VL in Florianópolis, further studies will undoubtedly be demanded before this possibility might be confirmed.


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  2. Acknowledgments

We are grateful to Bento Pedro Simão, Elisa Filter, and João Goulart Filho from the Secretaria Estadual de Saúde of Florianópolis, for helping in the entomological captures. This work was financially supported by the Brazilian Ministry of Health through the Leishmaniases Web of Fundação Oswaldo Cruz (Fiocruz), CNPq, and FAPEMIG.


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