Molecular Detection of Leishmania DNA in Wild-Caught Phlebotomine Sand Flies (Diptera: Psychodidae) From a Cave in the State of Minas Gerais, Brazil.

Leishmania spp. are distributed throughout the world, and different species are associated with varying degrees of disease severity. In Brazil, Leishmania transmission involves several species of phlebotomine sand flies that are closely associated with different parasites and reservoirs, and thereby giving rise to different transmission cycles. Infection occurs during the bloodmeals of sand flies obtained from a variety of wild and domestic animals, and sometimes from humans. The present study focused on detection of Leishmania DNA in phlebotomine sand flies from a cave in the state of Minas Gerais. Detection of Leishmania in female sand flies was performed with ITS1 PCR-RFLP (internal transcribed spacer 1) using HaeIII enzyme and genetic sequencing for SSUrRNA target. The survey of Leishmania DNA was carried out on 232 pools and the parasite DNA was detected in four: one pool of Lutzomyia cavernicola (Costa Lima, 1932), infected with Le. infantum (ITS1 PCR-RFLP), two pools of Evandromyia sallesi (Galvão & Coutinho, 1939), both infected with Leishmania braziliensis complex (SSUrRNA genetic sequencing analysis), and one pool of Sciopemyia sordellii (Shannon & Del Ponte, 1927), infected with subgenus Leishmania (SSUrRNA genetic sequencing analysis). The present study identified the species for Leishmania DNA detected in four pools of sand flies, all of which were captured inside the cave. These results represent the first molecular detection of Lu cavernicola with Le infantum DNA, Sc sordellii with subgenus Leishmania DNA, and Ev sallesi with Leishmania braziliensis complex DNA. The infection rate in females captured for this study was 0.17%.

Behavioral aspects of Lutzomyia longipalpis (Diptera: Psychodidae) in urban area endemic for visceral leishmaniasis.

The study of some of the behavioral aspects of the main vector of Leishmania infantum chagasi Cunha & Chagas in the Americas, Lutzomyia longipalpis (Lutz & Neiva), such as dispersion, population size, and vector survival rates, is important for the elucidation of the mechanisms of visceral leishmaniasis transmission. These parameters were studied by means of capture-mark-release-recapture experiments in an urban area of Campo Grande municipality, an endemic area of visceral leishmaniasis, situated in Mato Grosso do Sul state, Brazil. Six capture-mark-release-recapture experiments were undertaken between November 2009 and November 2010 and once in January 2012 with a view to assessing the population size and survival rate of Lu. longipalpis. The insects were released in a peridomicile surrounded by 13 residences. The recaptures were undertaken with automatic light traps for four consecutive weeks after release in the surrounding area. In total, 3,354 sand flies were captured, marked, and released. The overall recapture rate during the capture-mark-release-recapture experiments was 4.23%, of which 92.45% were recaptured at the release site, indicating limited dispersal. The greatest distance recorded from the release site was 165 m for males and 241 m for females. The male daily survival rate, calculated on the basis of regressions from the numbers of marked recaptured insects during the 15 successive days after release was 0.897. The estimated male population size measured by the Lincoln Index was 10,947.127. Though Lu. longipalpis presented a limited dispersion the physical barriers typical of urban environments did not prevent the sand flies from flying long distances.

Relationship between digestive enzymes and food habit of Lutzomyia longipalpis (Diptera: Psychodidae) larvae: Characterization of carbohydrases and digestion of microorganisms.

The sandfly Lutzomyia longipalpis (Lutz and Neiva, 1912) is the main vector of American Visceral Leishmaniasis. In spite of its medical importance and several studies concerning adult digestive physiology, biochemistry and molecular biology, very few studies have been carried out to elucidate the digestion in sandfly larvae. Even the breeding sites and food sources of these animals in the field are largely uncharacterized. In this paper, we describe and characterize several carbohydrases from the gut of L. longipalpis larvae, and show that they are probably not acquired from food. The enzyme profile of this insect is consistent with the digestion of fungal and bacterial cells, which were proved to be ingested by larvae under laboratory conditions. In this respect, sandfly larvae might have a detritivore habit in nature, being able to exploit microorganisms usually encountered in the detritus as a food source.

Synthetic sex pheromone attracts the leishmaniasis vector Lutzomyia longipalpis (Diptera: Psychodidae) to traps in the field.

Improving vector control remains a key goal in reducing the world's burden of infectious diseases. More cost-effective approaches to vector control are urgently needed, particularly because vaccines are unavailable and treatment is prohibitively expensive. The causative agent of American visceral leishmaniasis (AVL), Leishmania chagasi, Cunha and Chagas (Kinetoplastida: Trypanosomatidae), is transmitted between animal and human hosts by blood-feeding female sand flies attracted to mating aggregations formed on or above host animals by male-produced sex pheromones. Our results show the potential of using synthetic pheromones to control populations of Lutzomyia longipalpis Lutz and Neiva (Diptera: Psychodidae), the sand fly vector of one of the world's most important neglected diseases, AVL. We showed that a synthetic pheromone, (+/-)-9-methylgermacrene-B, produced from a low-cost plant intermediate, attracted females in the laboratory. By formulating dispensers that released this pheromone at a rate similar to that released by aggregating males, we were able to attract flies of both sexes to traps in the field. These dispensers worked equally well when deployed with mechanical light traps and inexpensive sticky traps. If deployed effectively, pheromone-based traps could be used to decrease AVL transmission rates through specific targeting and reduction of L. longipalpis populations. This is the first study to show attraction of a human disease-transmitting insect to a synthetic pheromone in the field, showing the general applicability of this novel approach for developing new tools for use in vector control.

Heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes.

We have examined the heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes (HBP-Lb) by chromatography assays. The proposed strategy to isolate an enriched fraction of the HBP-Lb consisted of an association of the Triton X-114 method with affinity chromatography in heparin-Sepharose 4B column. SDS-PAGE analysis of the eluted proteins showed two main protein bands (65.0 and 54.5 kDa), while a single protein band was observed in native electrophoresis gel. The hemagglutination property of HBP-Lb over rabbit erythrocytes was confirmed up to 6.3+/-0.5 microg of protein mL(-1). Additionally, we have assayed the potential of HBP-Lb labeled with sulfo-NHS-LC-biotin in binding to nitrocellulose-immobilized gut proteins extracted of Lutzomyia intermedia and Lutzomyia whitmani. The results indicated a similar profile of five ligands (67.0, 62.1, 59.5, 56.0 and 47.5 kDa) in both studied Lutzomyia species. This is the first direct description of this class of protein in L. (V.) braziliensis with a suggestion of its biological activity in the interaction of Leishmania with Lutzomyia gut cells, which maybe a crucial step during this parasite's life cycle.

Diagnosis of canine leishmaniasis in the endemic area of Belo Horizonte, Minas Gerais, Brazil by parasite, antibody and DNA detection assays.

Canine leishmaniasis caused by Leishmania chagasi (L. infantum) is found throughout the South American continent, including Brazil, and dogs are considered to be the main reservoir host for this parasite. To support the implementation of a diagnostic protocol for surveillance of the disease in the region of Belo Horizonte (Minas Gerais, Brazil) we have compared the sensitivity and specificity of two serological tests, indirect immunofluorescent antibody test (IFAT) and direct agglutination test (DAT), with the combination of direct microscopy-culture-PCR as the gold standard, using samples obtained from 103 dogs in the city of Belo Horizonte, Minas Gerais. The currently used standard serodiagnostic test, IFAT, had a sensitivity of 100% and its specificity was 74% compared to the gold standard of the study. The sensitivity and specificity of the DAT were 100% and 91%, respectively. On the basis of this study it is recommended to change from the IFAT to DAT for the serodiagnosis of canine leishmaniasis because of the superior specificity of the test combined with its user-friendliness.

Analysis of the sex pheromone extract of individual male Lutzomyia longipalpis sandflies from six regions in Brazil.

Although the phlebotomine sandfly Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) is generally accepted to be a species complex, it is unclear how many members there are, how they are related and which are the main vectors of leishmaniasis. The vectorial capacity of each sibling species is likely to differ, thus a means of identifying the most important vector species is of critical importance to the epidemiology and control of this debilitating disease in South and Central America. In Brazil four chemotypes have been distinguished by sex pheromone analysis. In this study the sex pheromone extracts of L. longipalpis from six regions of Brazil were analysed in detail. Samples included the sympatric 1-spot, 2-spot and intermediate spot morphotypes from Sobral, Ceará State. The results strongly suggest that members of the complex that produce different sex pheromones are reproductively isolated, thus strengthening the argument that the different chemotypes represent true sibling species. The study also found significant differences in morphology and the amounts of sex pheromone produced by members of each chemotype from different parts of Brazil, which suggests population substructuring that has not previously been recognized. Evidence of a fifth chemotype in Brazil is also presented.

A fourth chemotype of Lutzomyia longipalpis (Diptera: Psychodidae) from Jaibas, Minas Gerais State, Brazil.

Lutzomyia longipalpis (Lutz and Neiva) is a species complex of Lutzomyia pseudolongipalpis (Arrivillaga and Feliciangeli) and at least three other as yet undefined siblings. Isozyme and mitochondrial studies of allopatric populations across Central and South America have suggested the presence of four "clades" that have been hypothesized to have arisen mainly because of geographical isolation mechanisms. Parallel studies of sexual behavior as well as cross-mating and genetic analysis, of both allopatric and sympatric populations, suggest at least four sibling species that do not seem to correspond to the defined four "clades." In an effort to understand this apparent discrepancy, sympatric populations of L. longipalpis from a single South American country, Brazil, are being studied. In Brazil, three putative species can be identified by their male-produced sex pheromones: (S)-9-methylgermacrene-B, 3-methyl-a-himachalene, and a cembrene. We report here that analysis by coupled gas chromatography-mass spectrometry shows that L. longipalpis from Jaibas, Minas Gerais State, Brazil, occurs as two sympatric sex pheromone chemotypes. One chemotype is the cembrene type previously recorded in a L. longipalpis population from Sobral, Ceará State, Brazil, and the other is a new cembrene isomer not previously observed in L. longipalpis. The finding of this new chemotype strongly suggests that the L. longipalpis species complex in Brazil consists of four members rather than the three previously recognized and confirms previous analysis of genetic variation that had suggested the presence of a complex in Brazil.

Canine visceral leishmaniosis in Anastácio, Mato Grosso do Sul state, Brazil.

Canine visceral leishmaniosis (CVL) may be an important factor preceding human outbreaks of the disease. We report that the prevalence of canine visceral leishmaniosis infection has been increasing in recent years in Anastácio town, located in the central western region of Brazil. Serological investigations showed that 75.3% of dogs presented antibody titres ranging from 1/40 to 1/160 in the indirect immunofluorescence antibody test (IFAT). Bone marrow and lymph node aspirates provided positive cultures and furnished parasites for enzymological and serological typing in 42.5% and 41.1% of the cases, respectively. All the strains were typed as Leishmania (L.) chagasi. This is primarily a canine disease that spills over into the human population as a zoonosis. The study showed the epidemiological features of the infection in a region in which the problem of visceral leishmaniosis has been underestimated.

A new phlebotomine fossil species Trichopygomyia killickorum sp. n. (Diptera: Psychodidae) found in the Dominican Republic amber.

A description is given of male sandfly found in a piece of amber from Dominican Republic that dates from the Miocene period (20-15 m.y.a). It is assigned to the genus Trichopygomyia Barreto. The position of the spines on the style and the shape of the dorsal lobes of the paramere separate this fly from other species of the genus: it is here named Trichopygomyia killickorum sp. n.

Distribution of putative male sex pheromones among Lutzomyia sandflies (Diptera: Psychodidae).

Male Lutzomyia longipalpis produce terpene sex pheromones in glandular tissue underlying the cuticle. The pheromones are transmitted to the surface via cuticle-lined ducts (measuring 0.25 microm in diameter), each of which reaches the surface in the centre of a papule (measuring 3-3.5 microm in diameter). Similar papules, in a range of shapes but all characterized by the presence of a central pore and absence of macroserae, occur in some other species of sandfly. The aim of the present study was to determine the distribution of sex pheromones in sandflies of the genus Lutzomyia that do and do not have the papules. The results indicate that sex pheromones are not widely distributed amongst male Lutzomyia spp. Male members of the genus can be subdivided into three groups: those that produce terpenes and have cuticular papules; those that do not produce terpenes but still have the associated papules; and those that have neither terpenes nor papules. The papules seen in the species that do not synthesise sex pheromones are presumably vestigial, non-functional structures. Such species may have stopped producing pheromone as the result of changes in the way in which the females found and selected mates or changing feeding preferences. A similar event has occurred in the Lepidoptera, where vestigial pheromone-secreting structures remain in some species which no longer produce pheromone. Lutzomyia lenti collected in southern Brazil produced a novel diterpene whereas male L. lenti from north-eastern Brazil did not, supporting suggestions by others that L. lenti is, like L. longipalpis, a species complex.

Epidemiological studies of an outbreak of cutaneous leishmaniasis in the Rio Jequitinhonha Valley, Minas Gerais, Brazil.

We detected an outbreak of American cutaneous leishmaniasis in the Jequitinhonha River Valley, Minas Gerais, Brazil. Clinical and epidemiological aspects were studied for a period of two years. Data include results of physical examinations, Montenegro skin test and serology. In total 72 of the 299 individuals evaluated presented active lesions. Only one case out of these 72 patients showed the mucosal form of the disease. The precarious sanitary conditions, low educational level and low income found in the population studied demonstrated that, as with the other parasitic diseases, cutaneous leishmaniasis occurs with greater frequency in needy populations. A canine serological survey detected 20.3% (30/148) of dogs reactive to the Leishmania antigen. Lutzomyia intermedia was the predominant phlebotomine species and the majority of the specimens (84.9%) were captured in the peridomicile. Four samples from human and three from canine cases were isolated and characterised by PCR and isoenzymes as being Leishmania (Viannia) braziliensis. The peridomiciliary nature of the disease is discussed.

[Phlebotomine sand flies in the State of Tocantins, Brazil (Diptera: Psychodidae)]. / Flebotomíneos do Estado de Tocantins, Brasil (Diptera: Psychodidae).

Between 1997-1998, the authors carried out sporadic collection of sand flies in the municipalities of Paraíso de Tocantins, Monte do Carmo, Porto Nacional and Monte Santo all in the Tocantins State of northern Brazil. Human bait was used in Monte Santo and a battery operated light trap in other municipalities. The ecotypes chosen for the traps were in the peridomiciles, inside the houses, in the forest and the orchard. We identified 2,677 sand flies, belonging to 32 species. The most abundant species of sand flies were Lutzomyia whitmani, Lutzomyia longipalpis, Lutzomyia carmelinoi, Lutzomyia evandroi, Lutzomyia longipennis and Lutzomyia antunesi. Collections from the forest showed greater diversity of species, while the largest number of sand flies were caught around the houses. Several species known or suspected to be vectors of Leishmania in other regions of Brazil were captured.

Flebotomíneos do Estado de Tocantins, Brasil (Diptera: Psychodidae) / Phlebotomine sand flies in the State of Tocantins, Brazil (Diptera: Psychodidae)

Between 1997-1998, the authors carried out sporadic collection of sand flies in the municipalities of Paraíso de Tocantins, Monte do Carmo, Porto Nacional and Monte Santo all in the Tocantins State of northern Brazil. Human bait was used in Monte Santo and a battery operated light trap in other municipalities. The ecotypes chosen for the traps were in the peridomiciles, inside the houses, in the forest and the orchard. We identified 2,677 sand flies, belonging to 32 species. The most abundant species of sand flies were Lutzomyia whitmani, Lutzomyia longipalpis, Lutzomyia carmelinoi, Lutzomyia evandroi, Lutzomyia longipennis and Lutzomyia antunesi. Collections from the forest showed greater diversity of species, while the largest number of sand flies were caught around the houses. Several species known or suspected to be vectors of Leishmania in other regions of Brazil were captured.

Nos anos de 1997 e 1998 realizaram-se capturas esporádicas de flebotomíneos nos municípios de Paraíso de Tocantins, Monte do Carmo, Porto Nacional e Monte Santo, todos localizados no estado de Tocantins, região norte do Brasil, com o intuito de conhecer a fauna flebotomínica da região. No município de Monte Santo utilizou-se isca humana e nos demais armadilhas luminosas CDC. Os ecótopos utilizados foram: peridomicílio, intradomicílio, mata e pomar. Foram capturados 2.677 flebotomíneos, pertencentes a 32 espécies. As mais freqüentes foram Lutzomyia whitmani, Lutzomyia longipalpis, Lutzomyia carmelinoi, Lutzomyia evandroi, Lutzomyia longipennis e Lutzomyia antunesi. As capturas na mata apresentaram maior diversidade de espécies, enquanto no peridomicílio houve maior número de exemplares capturados. Foram capturadas neste estado várias espécies vetoras em outras regiões do Brasil.

Visceral leishmaniasis in the Metropolitan Region of Belo Horizonte, State of Minas Gerais, Brazil.

In the last few years the number of human cases of American visceral leishmaniasis in the Metropolitan Region of Belo Horizonte (MRBH), Minas Gerais, Brazil has increased, indicating an elevation in the transmission rate of the disease. The total number of notified human cases in the MRBH since 1994, when the first case was identified, up to 1999 was 345 of which 223 (65%) were from the city itself, indicating an urbanization of the disease in this region of Minas Gerais. The age distribution of visceral leishmaniasis cases in the MRBH shows a higher prevalence in children from 0-4 years old, responsible for 28.9% of the notifications. Clinical and immunological findings from dogs infected with Leishmania chagasi are described. The majority of these animals showed no sign of the disease. Sera from all infected dogs showed detectable Leishmania-induced high titles of antibodies based on the results of an indirect fluorescent antibody test. Samples of isolated Leishmania from human and dogs were characterized as L. (L.) chagasi by biochemical and molecular techniques.

A new sand fly, Lutzomyia campograndensis sp. n. (Diptera: Psychodidae: Phlebotominae) from the State of Mato Grosso do Sul, Brazil.

During studies of the phlebotomine sand fly fauna of Campo Grande, State of Mato Grosso do Sul, Brazil, a new species was captured with CDC light trap and is described here. The new species resembles Lutzomyia lutziana (Costa Lima) and is named Lutzomyia campograndensis sp. n. after the type locality.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis.

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative.

[Digestive tract microbiota in female Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) from colonies feeding on blood meal and sucrose plus blood meal]. / Microbiota do trato digestivo de fêmeas de Lutzomyia longipalpis (Lutz & Neiva, 1912) (Diptera: Psychodidae) provenientes de colônia alimentadas com sangue e com sangue e sacarose.

There are very few reports on the microbiota of the digestive tract of sand flies, an important omission considering that blood is not the only meal ingested. Male and female sand flies obtain sugar meals from several sources, thereby increasing their chance of infection with microorganisms. Chances of contamination are higher when insects are bred in the laboratory, and this may affect the development of Leishmania spp. From the digestive tract of 300 sand fly females separated in two groups we isolated 10 species of bacteria in group 1 and 8 species in group 2. In group 1, Enterobacteriaceae of the following genera were identified: Serratia, Enterobacter, and Yokenella and the non-fermenters: Pseudomonas, Acinetobacter, and Stenotrophomonas. In group 2, the Enterobacteriaceae Enterobacter and Serratia were identified as well as the non-fermenters Acinetobacter, Stenotrophomonas, Burkolderia, and Pseudomonas.

Characterization of constitutive and putative differentially expressed mRNAs by means of expressed sequence tags, differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR from the sand fly vector Lutzomyia longipalpis

Molecular studies of insect disease vectors are of paramount importance for understanding parasite-vector relationship. Advances in this area have led to important findings regarding changes in vectors' physiology upon blood feeding and parasite infection. Mechanisms for interfering with the vectorial capacity of insects responsible for the transmission of diseases such as malaria, Chagas disease and dengue fever are being devised with the ultimate goal of developing transgenic insects. A primary necessity for this goal is information on gene expression and control in the target insect. Our group is investigating molecular aspects of the interaction between Leishmania parasites and Lutzomyia sand flies. As an initial step in our studies we have used random sequencing of cDNA clones from two expression libraries made from head/thorax and abdomen of sugar fed L. longipalpis for the identification of expressed sequence tags (EST). We applied differential display reverse transcriptase-PCR and randomly amplified polymorphic DNA-PCR to characterize differentially expressed mRNA from sugar and blood fed insects, and, in one case, from a L. (V.) braziliensis-infected L. longipalpis. We identified 37 cDNAs that have shown homology to known sequences from GeneBank. Of these, 32 cDNAs code for constitutive proteins such as zinc finger protein, glutamine synthetase, G binding protein, ubiquitin conjugating enzyme. Three are putative differentially expressed cDNAs from blood fed and Leishmania-infected midgut, a chitinase, a V-ATPase and a MAP kinase. Finally, two sequences are homologous to Drosophila melanogaster gene products recently discovered through the Drosophila genome initiative

Short report: detection of Leishmania DNA by polymerase chain reaction on blood samples from dogs with visceral leishmaniasis.

Immunological, parasitological, and molecular techniques were applied to blood samples of dogs to diagnose Leishmania infections. In 1997, 644 domestic dogs were studied. Peripheral blood samples were collected for serological diagnosis and detection of Leishmania parasite by polymerase chain reaction (PCR). The indirect immunofluorescence test was positive in 139 (21.6%) of 644 dogs examined. The PCR was performed in 70 blood samples and 3 bone marrow aspirates. A 120-bp fragment specific for Leishmania was present in PCR hybridization analysis of all seropositive samples in the molecular assays. The PCR hybridization test, which used a minicircle of Leishmania chagasi as a probe, was negative in 20 seronegative dogs. These results suggest that a combined PCR-Southern hybridization technique is a highly sensitive approach to diagnose leishmaniasis in dogs, which are a zoonotic reservoir of leishmaniasis for humans.