Abstract

Natural infections with Leishmania were found in females of the phlebotomine sand flies Lutzomyia neivai (Pinto) (=Nyssomyia neivai) and Lutzomyia sallesi (Galvão & Coutinho) (=Evandromyia sallesi) (Diptera: Psychodidae) from Lassance, in the Brazilian state of Minas Gerais. Promastigotes were found in the pyloric region of the former species and in the abdominal midgut of the latter species. Insects found to be infected by microscopic examination were macerated in saline solution and inoculated into hamsters. Subsequent analysis by polymerase chain reaction-restriction fragment length polymorphism revealed both isolates to belong to the species Leishmania infantum chagasi Cunha & Chagas.

Hematophagous insects are the biological or mechanical vectors of various pathogens, among them members of the genus Leishmania Ross, etiological agents of the leishmaniasis. These parasites are transmitted by the bite of phlebotomine sand flies and may infect various species of mammals, including humans.

In recent years, the epidemiological profile of these diseases has changed. Formerly restricted to forested rural areas, they are now also present in periurban and urban habitats, including large cities such as the Brazilian state capitals of Campo Grande, Teresina, and Belo Horizonte (Costa et al. 1990, Silva et al. 2001, Oliveira-Pereira et al. 2006). However, rural outbreaks of leishmaniasis remain a problem, with cases reported from several Brazilian states, including Minas Gerais (Maywald et al. 1996, Gontijo et al. 2002, Nunes et al. 2006, Saraiva et al. 2006).

The subfamily Phlebotominae is composed of ≈500 species in the Americas, some of which feed on cold-blooded vertebrates and are unable to transmit Leishmania. Several species have been found naturally infected with these parasites, but only nine have proven vectorial capacity and another six are suspected of being vectors (Killick-Kendrick 1990, Santos et al. 1998). The latter include Lutzomyia neivai (Pinto) (=Nyssomyia neivai), suspected of transmitting Leishmania (Viannia) braziliensis Viannia, the principal etiological agent of American cutaneous leishmaniasis (ACL) in Brazil (Andrade Filho et al. 2007).

With regard to visceral leishmaniasis (VL), the principal vectors in Brazil are Lutzomyia longipalpis (Lutz & Neiva) and Lutzomyia cruzi (Mangabeira). Caused by Leishmania infantum chagasi Cunha & Chagas, this clinical form of the disease is almost 100% fatal in untreated cases.

The natural Leishmania infection rate in sand flies reported in most studies is very low, rarely reaching 1%. Detection methods include dissection under the microscope and molecular biology techniques such as the polymerase chain reaction (PCR), targeting both the conserved and variable regions of Leishmania kinetoplast (k)DNA minicircles. Recent studies have used PCR (Perez et al. 1994, Rodriguez et al. 1999, Jorquera et al. 2005, Kato et al. 2005), this being the most specific and sensitive tool for the detection and identification of Leishmania spp. (Schonian et al. 2003, Manna et al. 2004).

The objective of the current study was to determine the natural Leishmania infection rate in sand flies captured in the municipalities of Corinto and Lassance in the Brazilian state of Minas Gerais.

Materials and Methods

Both Corinto (18° 21′ S; 44° 27′ E) and Lassance (18° 01′ S; 44° 47′ E) are located in the upper São Francisco River region. Corinto has a population of 24,139 inhabitants distributed in an area of 2,552 km2, whereas Lassance has 6,491 inhabitants in 3,214 km2 (IBGE 2007). The principal economic activities of the region are agriculture, cattle ranching, and fishing, both municipalities being situated on the banks of the Velhas River. Mean annual temperature is ≈23°C, and the predominant vegetation type is cerrado (tropical savana). There were 18 cases of ACL reported in Corinto between 2001 and 2006 and a further seven in Lassance (SVS 2007), with no records of human VL in either municipality and no serological surveys in dogs having been conducted during these years.

Sand flies were sampled using illuminated Shannon traps and manual aspirators (Shannon 1939) from 1800 to 2200 hours at two sites. The first was located in a henhouse in Corinto on the left bank of the Velhas River (17° 58.115′ S, 44° 32.581′ E), where sand flies were collected from June 2003 through July 2004. The other site was seven km away, also on the left bank of the river but within the municipality of Lassance. Sampling was carried out from August 2004 through July 2005 in the peridomicile of a fishing camp (17° 54.739′ S, 44° 34.460′ E) within a highly degraded gallery forest habitat.

The insects collected were held in sand fly rearing pots inside styrofoam boxes. Humidity was maintained by placing pads of moistened cotton wool inside the boxes and the insects were supplied with sugar solution ad libitum and maintained alive for later dissection and specific identification. The authors have listed species names sensu Young and Duncan’s classification system (Young and Duncan 1994) followed by the corresponding nomenclature sensu Galati (2003) in brackets.

Dissection consisted of removing the sand fly gut in a drop of buffered saline on a microscope slide, using a pair of mounted entomological pins. A coverslip was then placed over the drop, and the entire length of the gut examined for promastigotes under an optical microscope. During the microscopic examination sand flies were identified to species, principally based on spermathecal morphology.

For positive slides, a macerate of sand fly guts was inoculated with a 1-ml syringe into the hind leg of an adult male hamster [Mesocricetus auratus (Waterhouse)], which was examined periodically thereafter. Seven or eight mo after inoculation, the hamsters were sacrificed, and samples of skin, liver, and spleen removed.

Fragments of each of these tissues were added to Novy-MacNeal-Nicolle medium to which liver infusion tryptose liquid medium had been added in an attempt to isolate the parasites. DNA also was extracted from the samples using the Genomic Prep Cells and Tissue DNA Isolation kits (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) and following the manufacturer’s protocol.

The DNA extracted was analyzed by to PCR to detect Leishmania, by using a pair of primers that amplified a 120-bp fragment within the conserved region of the kDNA minicircle: primer A: 5′-(C/G)(C/G)(G/C) CC(C/A) CTA T(T/A)T TAC ACC AAC CCC-3′ and primer B: 5′-GGG GAG GGG CGT TCT GCG AA-3′ (Degrave et al. 1994). The reactions were prepared to a final volume of 25 μl containing 10× buffer solution consisting of 1.5 mM MgCl2, 50 mM KCl, and 10 mM Tris-HCl, pH 8.0, 200 μM dNTPs, 10 pmol of each primer, 1.25 U of Taq DNA polymerase (GE Healthcare), and 2 μl of DNA. The PCR products were visualized by electrophoresis in 6% polyacrylamide gel stained with 0.2% silver nitrate.

Identification of the Leishmania species in the skin samples was performed using PCR-restriction fragment length polymorphism (RFLP) of the 120-bp fragment from the kDNA minicircles as described previously (Volpini et al. 2004). In brief, 5 μl of the PCR product obtained with primers A and B was digested by adding 1 U of the restriction enzyme HaeIII and its appropriate buffer (1×). The mix was incubated at 37°C for 3 h, and the restriction fragments were separated by electrophoresis in 10% polyacrylamide gel and visualized after silver staining.

The band profile obtained through amplicon digestion was compared with those from the DNA of the reference strains for each Leishmania species: Leishmania amazonensis Lainson & Shaw (IFLA/BR/67/PH8), Le. braziliensis (MHOM/BR/75/M2903), and Le. infantum chagasi (MHOM/BR/74/PP75).

Results

Collections at sites one and two numbered 342 females of six species and 133 specimens of seven species respectively (Table 1). The predominant species at both sites was Lu. neivai followed by Lutzomyia intermedia (Lutz & Neiva) (=Nyssomyia intermedia).

Table 1

Natural Leishmania infection rates of sand flies collected with illuminated Shannon traps in Lassance and Corinto, Minas Gerais, Brazil, 2003–2005

One specimen each of Lu. neivai and Lutzomyia sallesi (=Evandromyia sallesi) (Galvão & Coutinho) was found naturally infected with promastigotes at site 1. The former was captured in February and the latter in March 2004. No infected specimens of either species were found at the second site. The location of infections differed between the two species, promastigotes in Lu. neivai being present in the pyloric region and the parasites were bound together by a gel-like matrix. In Lu. sallesi, promastigotes were found in the abdominal midgut, were not to the gut wall and were not encumbered by a gel matrix. No host bloodmeal remnants were found with either infection.

Promastigotes could not be isolated due to fungal contamination in the culture tubes. Both hamsters presented no clinical signs compatible with Le. infantum chagasi infections or lesions characteristic of dermotropic species of the subgenera Viannia or Mexicana. Skin samples isolated from these animals were positive by conventional PCR for the genus Leishmania. PCR-RFLP allowed the species in these samples to be identified as Le. infantum chagasi (Fig. 1).

Fig. 1

Polyacrylamide 10% gel stained with silver nitrate showing specific diagnosis of samples positive for PCR-RFLP. PM, molecular weight marker 50 bp; 01, positive control (Le. amazonensis); 02, positive control (Le. braziliensis); 03, positive control (Le. infantum chagasi); 04, sample of Lu. neivai; 05, sample of Lu. sallesi.

Fig. 1

Polyacrylamide 10% gel stained with silver nitrate showing specific diagnosis of samples positive for PCR-RFLP. PM, molecular weight marker 50 bp; 01, positive control (Le. amazonensis); 02, positive control (Le. braziliensis); 03, positive control (Le. infantum chagasi); 04, sample of Lu. neivai; 05, sample of Lu. sallesi.

Discussion

It was not possible to carry out PCR directly after dissection from material on the slide holding the sand fly gut macerate. However, the positive results obtained using PCR for the genus Leishmania from the hamster skin, liver, and spleen fragments and PCR-RFLP from the skin samples demonstrated the practicality of this technique, principally in prolonged field studies where limited technology is available.

Although neither Corinto nor Lassance presented cases of VL, PCR-RFLP revealed the presence in the area of Le. infantum chagasi. The main Brazilian vector of this parasite is Lu. longipalpis, which was not found during the current study, despite being recorded infrequently from these municipalities (Saraiva et al. 2008).

None of the species sampled has been associated with VL to date. Only three, i.e., Lu. intermedia, Lu. neivai, and Lutzomyia whitmani (Antunes & Coutinho) (=Nyssomyia whitmani) are suspected vectors of the etiologic agent of ACL in other regions of Brazil (Queiroz et al. 1991, Luz et al. 2000, Andrade Filho et al. 2007) and are also found in VL-endemic areas from which Lu. longipalpis is absent (Oliveira et al. 1959, Souza et al. 2003).

The overall natural infection rate was 0.42%, close to that found in previous studies that used molecular techniques to detect natural infections in sand flies (Feliciangeli et al. 1994, Miranda et al. 2002, Pita-Pereira et al. 2005, Oliveira-Pereira et al. 2006). With respect to studies that used the same methodology as used in this study, Paiva et al. (2007) found 1.24% of sand flies collected in the Brazilian state of Mato Grosso do Sul to be infected, whereas Casanova et al. (1995) obtained a value of 0.24% for Lu. neivai (considered Lu. intermedia) in São Paulo.

This is the first report of natural infection of Lu. sallesi with Leishmania, although Mayrink et al. (1979) observed infections with trypanosomatids in specimens from eastern Minas Gerais. Although this sand fly is common in Minas Gerais (Martins et al. 1978), there are no records to date of it biting man. However, it may be involved in a sylvatic or rural cycle of Leishmania transmission among other vertebrate hosts. Two other species belonging to the same taxonomic group (migonei) (or series cortelezzii, sensu Galati) as Lu. sallesi have been found naturally infected, i.e., Lutzomyia cortelezzii (Brèthes) (=Evandromyia cortelezzii) and Lutzomyia edwardsi (Mangabeira) (=Evandromyia edwardsi), in Minas Gerais and São Paulo, respectively (Sucen 2005, Carvalho et al. 2008).

Lu. neivai has already been found naturally infected with Leishmania spp. in Argentina (Córdoba-Lanús et al. 2006). This species was considered to be a junior synonym of Lu. intermedia (Young and Duncan 1994), to which it is very similar morphologically (Marcondes 1996, Andrade Filho et al. 2003), there being considerable intraspecific polymorphism (Andrade Filho et al. 2006). Because of this erroneous synonymy, several studies of natural infection refer to Lu. intermedia as the probable vector of the etiologic agent responsible for ACL in southeastern and southern Brazil (Andrade Filho et al. 2007), when the species involved was actually Lu. neivai. This species is highly anthropophilic, including sand flies of the Lassance population. Several specimens have been collected inside houses by our group on previous occasions, sometimes biting the inhabitants (Andrade Filho et al. 2007).

Although the classification of Lainson and Shaw (1987) included Le. infantum chagasi among the Suprapylaria, in which the infection is initially established in the abdominal midgut, virtually nothing is known in relative to the development of this parasite in both Lu. neivai and Lu. sallesi. Experimental infections of Lu. neivai with several Leishmania species should be carried out to observe the distribution of parasites in the sand fly gut and the possibility of it acting as a vector.

A better understanding of Leishmania transmission requires that the relationship between the parasite, the vector and the reservoir to be determined. The ecology and epidemiology of the leishmaniases is such that they are considered to be among the most complex of all vector-borne diseases, the wide range of mammal and sand fly species involved in their transmission cycles producing considerable topogeographical variation of disease foci (Lainson and Shaw 1979, Ashford et al. 1998, Ashford 2000, Shaw 2003).

Given that in the study area Le. infantum chagasi presents a predominantly sylvatic cycle without human involvement, the presence of natural infections in these sand flies does not necessarily mean that these insects are responsible for transmission of the parasites. Further studies should be carried out in these municipalities to clarify the data presented here, as well as other aspects of this disease.

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