Natural infection of phlebotomines (Diptera: Psychodidae) by Leishmania (Leishmania) amazonensis in an area of ecotourism in Central-Western Brazil

Background Bonito municipality, known as an area of ecoturism, in Mato Grosso do Sul state, Brazil, is also a focus of visceral and cutaneous leishmaniases, with cases registered in both human and canine populations. This study sought to investigate natural infection by flagellate forms of Leishmania in phlebotomines of the urban area of Bonito. Findings Sand flies were collected fortnightly from October 2005 to July 2006 with modified automatic light traps installed in peridomiciles and animal shelters in the center and on the outskirts of the city. The females were dissected and their guts observed under an optical microscope. A total of 1977 specimens were captured, Lutzomyia longipalpis (88.4 %) and Bichromomyia flaviscutelata (3.0 %) being the most frequent species. Bi. flaviscutellata was found infected by flagellates that were identified as Leishmania (Leishmania) amazonensis by indirect immunofluorescence reaction, employing monoclonal antibodies and the biotin-avidin system. This is the first report of natural infection by L. amazonensis in Bi. flaviscutellata in a Brazilian urban area. Conclusions As Bi. flaviscutellata is only slightly attracted by humans, the transmission of L. amazonensis in the study area may have a zoonotic character; however, the sympatric occurrence of this parasite and Lu. longipalpis should be taken into consideration by the local health authorities since this sand fly has already been found with L. amazonensis DNA in a focus of canine visceral leishmaniasis in Bonito municipality.


Findings
Leishmaniases are a group of anthropozoonoses caused by protozoan parasites of the genus Leishmania (Kinetoplastida: Trypanosomatidae) and transmitted by the bite of several species of phlebotomine sand flies (Diptera: Psychodidae) [1]. The rate of naturally infected sand flies with Leishmania in endemic areas and the identification of the parasite in a particular phlebotomine species are important in vectorial and epidemiological studies of leishmaniases [2].
Bonito municipality, an important area of ecotourism due to the commercial exploitation of its great scenic beauty, is also a focus of visceral and cutaneous leishmaniases [3,4]. Studies of the phlebotomine fauna in its rural and urban environments have revealed several vectors or probable vectors of cutaneous and visceral leishmaniasis agents [3][4][5][6]. The aim of this study was to investigate natural infection by Leishmania spp. in phlebotomines of the urban area of Bonito.
The study was carried out in the urban area of Bonito (21°07'16"S and 56°28'55"W) located on the Bodoquena Range in the southwestern region of Mato Grosso do Sul state, Brazil. Phlebotomines were collected fortnightly from October 2005 to July 2006 with modified automatic light traps coupled to cages at six sites in nine ecotopes, one of them in the central district and the other five at various points on the outskirts, covering peridomestic areas and animal shelters.
The females were immobilized with ethyl ether and transferred to a slide containing a drop of saline solution. The dissection was performed under a stereoscope to expose their digestive tract and genitalia, which were covered by a cover glass and examined under a microscope to search for flagellates in the gut and identification of the phlebotomine species according to Galati's classification [7]. The males and undissected females were clarified before the identification. The abbreviations of the names of the phlebotomine species follow Marcondes [8].
When flagellates were observed more saline solution was added to the liquid containing the digestive tract; then liquid together with gut aspirated from the slide were inoculated into the hind paws of two hamsters (Mesocricetus auratus). As soon as the lesions developed, fine needle aspirations from these were inoculated into a blood agar base culture medium NNN (Neal, Novy, Nicolle) with the liquid phase consisting of BHI (bovine brain-heart infusion) plus penicillin (1000 IU/mL) and streptomycin sulfate (100 μg/mL). Once the stain was isolated, it was it was sent to the Tropical Medicine Nucleus, Pará Federal University, Belém, and subsequently analyzed by indirect immunofluorescence reaction, employing monoclonal antibodies and the biotinavidin system [9].
The number of sand fly species collected as well as their sex and ecotope are listed in Table 1. Of these 1977 phlebotomines (1515 males and 462 females), Lutzomyia longipalpis (88. 5 %)the known vector of Leishmania infantum chagasiand Bichromomyia flaviscutellata (3.0 %)the vector of Leishmania (L.) amazonensiswere the predominant species. Lu. longipalpis presented the highest frequencies in ecotopes close to animal shelters such as hen houses with wooden walls, roofs of palm leaves or plywood and the pigpens fenced with boards. This species was less common in forested areas and hillside savannah. Bi. flaviscutellata was captured in a hillside vestigial savannah area about 50 m away from the peridomiciles, which consisted of native plants and fruit trees such as banana, mango, papaya, guava and citrus. Nyssomyia whitmani, the recognized vector of Leishmania (Viannia) braziliensis, presented a low frequency (1.6 %) and was collected only on the urban perimeter on forested slopes and in peridomiciles surrounded by savannah.
Of 462 females collected, 430 (93.1 %) were dissected. Suprapylarian natural infection by flagellates was found in one female Bi. flaviscutellata (1.9 %) and inoculated into two hamsters. After five weeks, cutaneous lesions  Trap  T1  T2  T3  T4  T5  T6  T7  T8  T9 Subtotal Total Br. avellari Ev. corumbaensis Lu. longipalpis Mi. quinquefer were present in both the hind paws of the animals from which the strain was isolated and subsequently identified by immunofluorescence reaction, employing monoclonal antibodies and the biotin-avidin system, as L. (L.) amazonensis. Leishmania (L.) amazonensis causes cutaneous and occasionally anergic diffuse cutaneous leishmaniasis in individuals with faulty cell-mediated immunity and is widely distributed in Latin America [1]. However, in the Brazilian state of Bahia, it has previously been associated with human visceral leishmaniasis [10]. Although this parasite is predominantly associated with human and animal infection in rural environments [11,12], it has been found in dogs in Araçatuba municipality in São Paulo state and in domestic cats in urban environments in the municipalities of Campo Grande and Ribas do Rio Pardo in Mato Grosso do Sul state [13][14][15].
The slight attractiveness of humans to Bi. flaviscutellata [16] may perpetuate the transmission of L. amazonensis as a zoonotic affliction in the Bonito region. However, Lu. longipalpis has its capacity to transmit L. amazonensis to hamsters demonstrated experimentally and has already been associated with L. amazonensis in a focus of human cutaneous and canine visceral leishmaniases in the Bonito municipality [2,17].
In the opinion of these authors, the above scientific information should be taken into consideration by the authorities responsible for public health policies in Bonito in their establishment of prevention measures against leishmaniasis, which would include integrated control of vectors and reservoirs, precocious diagnosis of canine and human cases, treatment of human cases and relevant educational measures.

Ethics committee approval
The present study was not evaluated by the Animal Ethics Committee, because it was performed prior to the publication of the Brazilian law 11.794 of October 2008 laying down procedures for scientific use of animals.