SUMMARYDNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei).

In order to characterize the epidemiology of American Cutaneous Leishmaniasis (ACL) in a periurban area of the municipality of Sabará in the metropolitan region of Belo Horizonte (MRBH), an area until then considered free of the disease, a cross sectional survey was undertaken in 1990. The survey of the population consisted of 1119 interviews and 881 clinical examinations using Montenegro's skin test (MST). A low prevalence (3.7%) of positive MST was encountered. The disease had been occurring in the area for about 20 years in the form of sporadic cases. The predominant species of sandfly both in domestic areas and nearby areas of secondary vegetation was Lutzomyia whitmani. A canine survey of delayed hypersensitivity to the antigen P10,000 identified only one dog with a positive reaction out of 113 examined. The transmission of ACL in MRBH was confirmed. The occurrence of the disease in women, children and individuals with no contact with forest areas as well as the presence of potential vector species in the domiciliar environment, suggests the transmission of the disease in this environment.

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Visceral leishmaniasis (VL)

A study of Lutzomyia longipalpis (Lutz and Neiva, 1912) (Diptera: Psychodidae), the primary vector of American visceral leishmaniasis (AVL), and the canine form of the disease, was carried out in Porteirinha. The city is situated in the northern part of the Brazilian State of Minas Gerais and is an endemic area of AVL. Systematic phlebotomine captures were performed in seven districts with previously reported cases of canine visceral leishmaniasis, during 2 years (January 2000--December 2001). A total of 2328 specimens of L. longipalpis were captured. The association between the local climate variables and the population density of L. longipalpis was evaluated and rainfall was determined to be a major factor, with increased populations during the rainy season (October--March). At the same time period, blood samples from every dog domiciled in the same seven districts, in total 14,077 animals, were analyzed for infection by viscerotropic Leishmania using indirect immunofluorescence assay (IFA). Accumulated incidence rates of canine VL per district varied from 3.40 to 14.34 for the 2-year period. A positive correlation between the population density of L. longipalpis and the canine cases of visceral leishmaniasis in Porteirinha was observed.

Haematophagous dipterans of the Nematocera and Brachycera suborders have been objects of study for a better understanding of the behavioral mechanisms used by insects to find their blood meal. This is usually elicited by chemical stimuli (semiochemicals) called kairomones that are often released by the vertebrate hosts (Gibson & Torr 1999).Among the more than 500 species of the subfamily Phlebotominae that are known in the Americas, over 400 belong to the genus Lutzomyia (Young & Duncan 1994, Galati 2003. Lutzomyia longipalpis (Lutz and Neiva) and L. intermedia (Lutz and Neiva) play an important role in the transmission of leishmaniases (Forattini 1973, Lainson 1989, Lemos & Lima 2005. Host-preference studies of Lutzomyia spp. frequently use animals, such as chicken, dogs, horses (Quinnell et al. 1992, Ximenes et al. 1999, Bongiorno et al. 2003, and occasionally humans as bait (Quinnell et al. 1992, Aguiar et al. 1996, Rebollar-Telez et al. 1999, Pinto et al. 2001. Laboratory assays (e.g., precipitin test) have been used for the identification of hosts that provide the blood meal for the phlebotomine sand flies (Christensen et al. 1982, Afonso et al. 2005). CDC light traps (Sudia & Chamberlain 1962) and Shannon traps (Shannon 1939) have been used as tools for monitoring phlebotomine sand flies, thus eliminating the ethical problems associates with using human bait. However, some studies have shown that certain phlebotomine sand fly species are highly anthropophilic but rarely phototropic (Alexander et al. 1995, Travi et al. 1998, while other species seem to be more phototropic than antropophilic, such as Lutzomyia verrucarum Townsend (Davies et al. 1995). It is possible that these species are responsive to non-specific kairomones such as CO 2 (Quinnell et al. 1992, Campbell-Lendrum et al. 1999a, and can be attracted by different species of hosts. CO 2 is the main volatile kairomone released by vertebrates and it is used by most haematophagous insects to find their hosts (Eiras 2001). Knudsen et al. (1979) demonstrated for the first time the attractiveness of CO 2 (released from dry ice) to phlebotomine sand flies. Light traps (LT) baited with CO 2 enhanced the capture of phlebotomine sand flies in the field (Chaniots 1983), despite the fact that attraction to CO 2 was not observed in the laboratory (Nigam & Ward 1991).The chemical 1-octen-3-ol (octenol) is an important kairomone used by zoophilic dipteran species of the families Muscidae, Tabanidae, Ceratopogonidae and Culicidae to seek their hosts (Kline at al. 1990, Gibson & Torr 1999, Bernier et al. 2000, Eiras 2001). To date, there is no evidence supporting the trapping of phlebotomine sand flies with this kairomone. Lactic acid was the first kairomone identified from human skin (Acree et al. 1968), and its effect in attracting mosquitoes has been evaluated, especially in combination with CO 2 (Kline et al. 1990, Eiras & Jepson 1991, ammonia and some fatty acids (Geier et al. 1999). Ammonia was identified from vertebrate odors in the 1970s (Larson et al. 19...

Among neglected tropical diseases, visceral leishmaniasis (VL) shows great relevance in global terms and is a serious public health concern due to the possibility of severe and lethal forms in humans. In this study, we evaluate entomological factors such as diversity and abundance of phlebotomine sand flies (Diptera:Psychodidae) and the Leishmania species circulating in these species in possible association with VL transmission in the Brazilian town Itaúna. The entomological collections were performed during three consecutive nights, always in the third week of each month, within a period of 12 mo. A total of 1,786 sand fly specimens were collected, from which 20% were collected inside houses. The influence of three local climatic variables (temperature, rainfall, relative humidity) on the population sizes of these insects was evaluated. Temperature was the most influential factor, with a significant positive correlation with the local population size of phlebotomine sand flies collected per month. Lutzomyia longipalpis (Lutz & Neiva, 1912) was the predominant species in the study area. Leishmania DNA was detected in nine out of 133 pools of sand fly females, using nested/PCR, which resulted in a minimal natural infection rate of 2.91%. DNA from Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatida), was detected in Evandromyia cortelezzii (Bréthes, 1923), Ev. evandroi (Costa, Lima & Antunes, 1936), Ev. lenti (Mangabeira, 1938), and Ev. termitophila (Martins, Falcão & Silva, 1964), besides Lu. longipalpis. Our study indicates favorable conditions for VL spreading in Itaúna due to the presence of Lu. longipalpis and Le. infantum-infected phlebotomine sand flies.

The phlebotomine sand fly Lutzomyia longipalpis has been incriminated as a vector of American visceral leishmaniasis, caused by Leishmania chagasi. However, some evidence has been accumulated suggesting that it may exist in nature not as a single but as a species complex. Our goal was to compare four laboratory reference populations of L. longipalpis from distinct geographic regions at the molecular level by RAPD-PCR. We screened genomic DNA for polymorphic sites by PCR amplification with decamer single primers of arbitrary nucleotide sequences. One primer distinguished one population (Marajó Island, Pará State, Brazil) from the other three (Lapinha Cave, Minas Gerais State, Brazil; Melgar, Tolima Department, Colombia and Liberia, Guanacaste Province, Costa Rica). The population-specific and the conserved RAPD-PCR amplified fragments were cloned and shown to differ only in number of internal repeats.