Effects of azadirachtin on the biology of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) adult female, the main vector of American visceral leishmaniasis.

The effects of azadirachtin A added to the sucrose diet of the adult females on the mortality, oviposition, and hatching of the sand fly vector of American visceral leishmaniasis Lutzomyia longipalpis (Lutz & Neiva, 1912) were investigated. Concentrations of 0.1, 1.0, and 10.0 microg/mg of azadirachtin significantly increased insect mortality in comparison with control insects. The same dose also significantly reduced oviposition but not hatching. After a long development period, significantly fewer adult insects were obtained from eggs hatching by azadirachtin-treated females in a dose-response manner. These results indicate that azadirachtin is a potent sterilizer that could be used against the development of Lu. longipalpis populations and as a tool for studying physiological and biochemical processes in phlebotomine species.

A prospective study on the dynamics of the clinical and immunological evolution of human Leishmania (L.) infantum chagasi infection in the Brazilian Amazon region.

This prospective study was carried out from October 2003 to December 2005 and involved a cohort of 946 individuals of both genders, aged 1-89 years, from an endemic area for American visceral leishmaniasis (AVL), in Pará State, Brazil. The aim of the study was to analyze the dynamics of the clinical and immunological evolution of human Leishmania (L.) infantum chagasi infection represented by the following clinical-immunological profiles: asymptomatic infection (AI); symptomatic infection (SI=AVL); subclinical oligosymptomatic infection (SOI); subclinical resistant infection (SRI); and indeterminate initial infection (III). Infection diagnosis was determined by the indirect fluorescent antibody test and leishmanin skin test. In total, 231 cases of infection were diagnosed: the AI profile was the most frequent (73.2%), followed by SRI (12.1%), III (9.9%), SI (2.6%) and SOI (2.2%). The major conclusion regarding evolution dynamics was that the III profile plays a pivotal role from which the cases evolve to either the resistant, SRI and AI, or susceptible, SOI and SI, profiles; only one of the 23 III cases evolved to SI, while most evolved to either SRI (nine cases) or SOI (five cases) and eight cases remained as III.

EST sequencing of blood-fed and Leishmania-infected midgut of Lutzomyia longipalpis, the principal visceral leishmaniasis vector in the Americas.

Leishmaniasis is an important worldwide public health problem. Visceral leishmaniasis caused by Leishmania infantum chagasi is mainly transmitted by Lutzomyia longipalpis in the Americas. Leishmania development within the sand fly vector is mostly restricted to the midgut. Thus, a comparative analysis of blood-fed versus infected midguts may provide an invaluable insight into various aspects of sand fly immunity, physiology of blood digestion, and, more importantly, of Leishmania development. To that end, we have engaged in a study to identify expressed sequenced tags (ESTs) from L. longipalpis cDNA libraries produced from midguts dissected at different times post blood meal and also after artificial infection with L. i. chagasi. A total of 2,520 ESTs were obtained and, according to the quality of the sequencing data obtained, assembled into 378 clusters and 1,526 individual sequences or singletons totalizing 1,904 sequences. Several sequences associated with defense, apoptosis, RNAi, and digestion processes were annotated. The data presented here increases current knowledge on the New World sand fly transcriptome, contributing to the understanding of various aspects of the molecular physiology of L. longipalpis, and mechanisms underlying the relationship of this sand fly species with L. i. chagasi.

A longitudinal study on the transmission dynamics of human Leishmania (Leishmania) infantum chagasi infection in Amazonian Brazil, with special reference to its prevalence and incidence.

This was a longitudinal study carried out during a period over 2 years with a cohort of 946 individuals of both sexes, aged 1 year and older, from an endemic area of American visceral leishmaniasis (AVL) in Pará State, Brazil. The object was to analyze the transmission dynamics of human Leishmania (Leishmania) infantum chagasi infection based principally on the prevalence and incidence. For diagnosis of the infection, the indirect fluorescent antibody test (IFAT) and leishmanin skin test (LST) were performed with amastigote and promastigote antigens of the parasite, respectively. The prevalence by LST (11.2%) was higher (p < 0.0001) than that (3.4%) by IFAT, and the combined prevalence by both tests was 12.6%. The incidences by LST were also higher (p < 0.05) than those by IFAT at 6 (4.7% x 0.6%), 12 (4.7% x 2.7%), and 24 months (2.9% x 0.3%). Moreover, there were no differences (p > 0.05) between the combined incidences by both tests on the same point surveys, 5.2%, 6.3%, and 3.6%. During the study, 12 infected persons showed high IFAT IgG titers with no LST reactions: five children and two adults developed AVL (2,560-10,120), and two children and three adults developed subclinical oligosymptomatic infection (1280-2560). The combined tests diagnosed a total of 231 cases of infection leading to an accumulated prevalence of 24.4%.

A phylogenetic lineage of closely related trypanosomes (Trypanosomatidae, Kinetoplastida) of anurans and sand flies (Psychodidae, Diptera) sharing the same ecotopes in brazilian amazonia.

Analysis of the phylogenetic relationships among trypanosomes from vertebrates and invertebrates disclosed a new lineage of trypanosomes circulating among anurans and sand flies that share the same ecotopes in Brazilian Amazonia. This assemblage of closely related trypanosomes was determined by comparing whole SSU rDNA sequences of anuran trypanosomes from the Brazilian biomes of Amazonia, the Pantanal, and the Atlantic Forest and from Europe, North America, and Africa, and from trypanosomes of sand flies from Amazonia. Phylogenetic trees based on maximum likelihood and parsimony corroborated the positioning of all new anuran trypanosomes in the aquatic clade but did not support the monophyly of anuran trypanosomes. However, all analyses always supported four major clades (An01-04) of anuran trypanosomes. Clade An04 is composed of trypanosomes from exotic anurans. Isolates in clades An01 and An02 were from Brazilian frogs and toads captured in the three biomes studied, Amazonia, the Pantanal and the Atlantic Forest. Clade An01 contains mostly isolates from Hylidae whereas clade An02 comprises mostly isolates from Bufonidae; and clade An03 contains trypanosomes from sand flies and anurans of Bufonidae, Leptodactylidae, and Leiuperidae exclusively from Amazonia. To our knowledge, this is the first study describing morphological and growth features, and molecular phylogenetic affiliation of trypanosomes from anurans and phlebotomines, incriminating these flies as invertebrate hosts and probably also as important vectors of Amazonian terrestrial anuran trypanosomes.

Revisão sobre a patogenia da leishmaniose tegumentar americana na Amazônia, com ênfase à doença causada por Leishmania (V. ) braziliensis e Leishmania (L. ) amazonensis / Revisiting the pathogenesis of the american tegumentary leishmaniasis in Amazonian, with emphasis to the disease due to Leishmania (V. ) braziliensis and Leishmania (L.) amazonensis

A patogenia da leishmaniose tegumentar americana (LTA) na Amazônia foi revisada à luz dos mais recentes aspectos associados ao espectro clínico, histopatológico e imunopatológico da doença causada por Leishmania (V.) braziliensis e Leishmania (L.) amazonensis. Esta revisão mostrou a existência de uma dicotomia entre as duas espécies de Leishmania e a resposta imune celular; enquanto a L. (V.) braziliensis mostra forte tendência em dirigir a infecção, a partir da forma central do espectro clínico-imunológico, a leishmaniose cutânea localizada (LCL), para o pólo imunológico hiperreativo, representado pela leishmaniose cutâneo-mucosa (LCM), com exacerbação da hipersensibilidade e perfil da resposta CD4 tipo-Thl, a L. (L.) amazonensis mostra o oposto, dirige a infecção para o pólo imunológico hiporreativo, representado pela leishmaniose cutânea anérgica difusa (LCAD), com forte inibição da hipersensibilidade e perfil da resposta CD4 tipo- Th2. Entre a forma central LCL e as formas polares LCM e LCAD a infecção passa por uma fase intermediária, a leishmaniose cutânea disseminada borderline (LCDB), com inibição parcial da hipersensibilidade e peifil da resposta CD4 Thl + Th2. Estes são, provavelmente, os principais mecanismos imunológicos que modulam a patogenia da LTA causada por L. (V.) braziliensis e L. (L.) amazonensis.

The pathogenesis of American tegumentary leishmaniasis (ATL) was reviewed ifl the light of more recent features of clinical, histopathological and immunopathological spectrum of disease caused by Leishmania (V.) braziliensis and Leishmania (L.) amazonensis. This review has shown a dichotomy in the interaction between these two species of Leishmania with the human .cellular immune response; while L. (V.) braziliensis shows a clear tendency to direct infection, from the localized cutaneous leishmaniasis (LCL) in the center of the clinical-immunological spectrum of disease, to the hyperactive immunologic pole represented by mucocutaneous leishmaniasis (MCL), which shows exacerbated hypersensitivity reaction and CD4 Thl-type immune response, L. (L.) amazonensis shows the opposite,. directing infection to the hypoactive immunologic pole consisted by anergic diffuse cutaneous leishmaniasis (ADCL), associated with a marked inhibition of hypersensitivity reaction and CD4 Th2type immune response. Between the central LCL and thetwo polar MCL and ADCL forms the infection may present an intermediary phase, borderline disseminated cutaneous leishmaniasis (BDCL), which shows partial inhibition of hypersensitivity reaction and a mixed CD4 Thl plus Th2 immune response. These are probably the main immunological mechanisms regarding the immune response dichotomy that modulates the pathogenesis of ATL caused by these Leishmania parasites.

Effect of Lutzomyia longipalpis salivary gland extracts on leukocyte migration induced by Leishmania major.

The mechanism by which the salivary gland lysate (SGL) of Lutzomyia longipalpis enables Leishmania infection remains under investigation. One possibility is that saliva promotes cellular recruitment leading to development of skin lesions. In this study, we investigated leukocyte recruitment induced by L. major, L. major + SGL, or SGL alone into the peritoneal cavity of BALB/c mice. The administration of L. major with or without SGL induced neutrophil migration six hours after infection. Interestingly, after seven days, the BALB/c mice still had eosinophils and mononuclear cells in their peritoneal cavities. Flow cytometric analysis showed an increase in the CD4(+) CD45RB(low) T cell subset (effector or memory cells) compared with the CD4(+) CD45RB(high) subset (naive cells). Moreover, the co-injection of L. major with SGL enhanced production of interleukin-10. These results suggest that SGL can facilitate Leishmania infection by modulating leukocyte recruitment and Th2 cytokine production at the inflammatory focus.

Effect of salivary gland extract of Leishmania vector, Lutzomyia longipalpis, on leukocyte migration in OVA-induced immune peritonitis.

Salivary gland extracts (SGE) from Lutzomyia longipalpis potentate L. major infection by inducing a Th2 immune response. However, the effect of SGE on the effector phase of immune response is not known. Herein, we demonstrate that SGE inhibited neutrophil migration in ovalbumin (OVA)-induced peritonitis in immunized mice. SGE pretreatment of mice inhibited OVA-induced CD4+ and CD8+ T lymphocyte migration. The OVA-induced production of TNF-alpha, IL-1beta and leukotriene B4 (LTB4), neutrophil chemotactic mediators in this model, were inhibited by SGE. On the other hand, SGE enhanced production of IL-10 and IL-4. In naive mice, SGE also blocked LTB4-induced neutrophil migration, but not that induced fMLP. Moreover, co-incubation of LTB4 (but not fMLP, TNF-alpha and MIP-1alpha) with SGE inhibited the ability of LTB4 to induce neutrophil migration in vivo and in vitro. Altogether, the results suggest that SGE has anti-inflammatory properties that are associated with inhibition of TNF-alpha and LTB4 production and/or with the neutrophil chemotactic activity of LTB4. The effectiveness of SGE in inhibiting neutrophil migration and inflammatory mediators release in a Th1 immune inflammatory response model reinforces the need for isolation of the compounds responsible for these activities, which could be used as prototypes for the development new anti-inflammatory drugs.

Recent observations on the sand fly (Diptera: Psychodidae) fauna of the State of Rondônia, Western Amazônia, Brazil: the importance of Psychdopygus davisi as a vector of zoonotic cutaneous leishmaniasis.

Sand flies were collected in the central region of the state of Rondônia (W 64 degrees 30' to 63 degrees 00' and S 10 degrees 00'to 11 degrees 00') using Shannon and CDC light traps from October 1997 to August 2000. A total of 85,850 specimens representing 78 named species were captured. Of these 14 were new records for Rondônia. The proportion of males/females was 1/1.131. Trypanosomatids, that are presently being identified, were detected in 11 species. Leishmania (Viannia) naiffi was recorded from Psychodopygus davisi and P. hirsutus. In the present study the dominant species was P. davisi (39.6%) followed by Lutzomyia whitmani (13.1%), P. carrerai (11.6%), and P. hirsutus (10.2%). The importance of P. davisi as a vector of zoonotic cutaneous leishmaniasis is discussed.

Recent observations on the sand fly (Diptera: Psychodidae) fauna of the State of Rondônia, Western Amazônia, Brazil: the importance of Psychdopygus davisi as a vector of zoonotic cutaneous leishmaniasis

Sand flies were collected in the central region of the state of Rondônia (W 64 30' to 63 00' and S 10 00'to 11 00') using Shannon and CDC light traps from October 1997 to August 2000. A total of 85,850 specimens representing 78 named species were captured. Of these 14 were new records for Rondônia. The proportion of males/females was 1/1.131. Trypanosomatids, that are presently being identified, were detected in 11 species. Leishmania (Viannia) naiffi was recorded from Psychodopygus davisi and P. hirsutus. In the present study the dominant species was P. davisi (39.6 percent) followed by Lutzomyia whitmani (13.1 percent), P. carrerai (11.6 percent), and P. hirsutus (10.2 percent). The importance of P. davisi as a vector of zoonotic cutaneous leishmaniasis is discussed

Comparative studies of populations of Lutzomyia umbratilis (Diptera: Psychodidae) in Brazil and Venezuela.

We analyzed 64 quantitative and qualitative morphological characters in different populations of Lutzomyia (Nyssomyia) umbratilis Ward & Fraiha from Venezuela (Gran Sabana, Bolívar/BO) and Brazil (Serra do Navio, Amapá/AP Manaus, Amazonas/AM, Monte Dourado, Pará/ PA, and Peixoto de Azevedo, Mato Grosso/MT). Based on an analysis of qualitative characters, no differences were observed that could distinguish between the populations. Parametric tests were used to verify differences between sample means and variance homogeneity. The analysis-of-variance (ANOVA) test showed the existence of differences between character means in the five populations. In the graphic representation of the Student-Newman-Keuls test (Student-Newman-Keuls), we observed that 77% of the analyzed characters displayed disjunctions between the Venezuelan and Brazilian populations. Considering only the Brazilian population averages, we concluded that they are statistically different. When the Student-Newman-Keuls test was applied, no pattern was found that could separate them. However, in part, the phenetic analysis showed some evidences of heterogeneity in MT population.

Variation between geographical populations of Lutzomyia (Nyssomyia) whitmani (Antunes & Coutinho, 1939) sensu lato (Diptera: Psychodidae: Phlebotominae) in Brazil

Phylogenetic analysis of morphometric and biological characteres indicated that there are two distinct forms of Lutzomyia whitmani in Brazil: one is present both north and south of the river Amazonas in the state of Pará while the other occurs in northeast Brazil, in the state of Ceará, and further south, including the type locality in state of Bahia. The Amazonian form is reportedly neither strongly anthropophilic nor synanthropic, and it is the vector of Leishmania shawi; whereas the southern form is often collected peridomestically, while biting man, and has been found infected with Le. (V.) braziliensis. The ratio of the length of the genital filaments to that the genital pump was found to be consistently smaller in males of the Amazonian populations. A middle repetitive DNA element was isolated by differentially screening a genomic library made using Amazonian material, and the sequence was diagnostic for this form of Lu. whitmani (being absent or occurring in low copy number in the southern form). The total evidence suggests there are at least two, geographically-isolated forms of Lu. whitmani, which may represent different cryptic species.

Doença de chagas na bacia amazônica: ocorrência de um novo caso autoctone do Estado do Pará / Chagas Disease in Amazon basin: occurrence of the new autochthon case in the Para state

Os autores registram a ocorrencia de mais de um caso autoctone de doenca de Chagas no Estado do Para, desta feita na localidade de Sao Francisco do Para, distante 95 km da capital. Esse caso constitui o 11ä registrado no Estado e o 29ä da bacia amazonica. O paciente, um menino de 7 anos de idade, teve diagnostico sorologico e parasitologico positivos para T. cruzi. O perfilenzimatico da amostra isolada foi caracterizado por eletroforese de enzimas como zimodema

Leishmaniose cutânea experimental: II. Aspectos evolutivos da infecçäo no primata Cebus apella (Cebidae) pela Leishmania (V.) braziliensis e L. (L.) amazonensis / Experimental skin leishmaniasis: II. Course of the infection in the Cebus apella primate (Cebidae) caused by Leishmania (V.) braziliensis and L. (L.) amazonensis

Objetivando avaliar o potencial do primata C. apella como modelo experimental da leishmaniose cutânea produzida pela L. (V.) brasiliensis e L. (L.) amazonensis, inocularam-se, via intradérmica, 3 X10**6 de promastigotas dessas leishmanias, em 8 sítios da cauda de 10 espécimens desse desse primata, 5 deles com a L. (V.) braziliensis e outros 5 com a L. (L.) amazonensis. Posteriormente, às inoculaçöes, o exame semanal dos animais e biópsias mensais, revelaram os seguintes resultados relativos a cada parasita: a) L. (V.) brasiliensis: o periodo de incubaçäo foi d e15-20 dias; aos 30 dias evidenciaram-se lesöes pápulo-eritematosas, que evoluiram para nódulo ao fim de 60 dias; no 3§ mês, notou-se espontânea destas lesöes e, no 4§ mês, deu-se o inicio da reparaçäo das lesöes ulceradas, culminando com a cura em um dos animais após 5 meses, em dois após 6 meses, noutro após 7 meses e, no ultimo, após 10 meses. Quanto ao parasitismo nas lesöes, foi demonstrado nos 5 animais, até 90 dias; depois disto, somente em 2 até 120 dias e, por fim, até 180 dias apenas naquele que curou depois de 10 meses. b) L. (L.) amazonensis: o período de incubaçäo foi de 20 dias; aos 30 dias notou-se lesöes pápulo-eritematosas, que também evoluiram para nódulos ao fim de 60 dias, porém, a partir do 3§ mês, estas lesöes regrediram rapidamente ao fim de 90 dias, quando näo mais detectou-se o parasita na pele dos animais. Em relaçäo aos testes de Montenegro, somente 2 dos 5 animais infectados com a L. (V.) braziliensis reagiram ao teste, 6 e 90 dias após as inoculaçöes. Os resultados observados permitiram confirmar a infectividade do C. apella a estas leishmanias e, também, reforçar a indicaçäo desse primata como modelo experimental da leishmaniose cutânea por estes parasitas

Leishmaniose cutanea experimental: I-sobre a susceptibilidade do primata cebus apella (Cebidae) a infecçäo pela Leishmania (Viannia) Lainsoni Silveira, Shaw, Braga e Ishikawa, 1987 / Experimental cutaneous leishmaniasis: I-on the susceptibility of the primate cebus apella (Cebidae) to the infection caused by Leishmania (Viannia) Lainsoni Silveira, Shaw, Braga and Ishikawa, 1987

Foi investigada a susceptibilidade do primata Cebus apella (Cebidae) à infecçäo experimentada pela Leishmania (Viannia) lainsoni, com o objetivo de estudar a patogenia desse parasita, ainda pouco conhecido para o homem. Desse forma, cinco espécimes jovens daquele primata, 2 machos e 3 fêmeas, foram inoculados, itraderme, em oito sítios diferentes da regiäo dorsal da cauda com 3 x 10***6 de promastigotas do parasita (MHOM/BR/81/M6426, Benevides, Pará), obtidas de cultura da fase estacionária. Em seguida às inoculaçöes, a infecçäo experimental no animais foi comprovada, näo só pela presença de amastigotas do parasita na pele dos animais inoculados, mas, também, pela concomitância desse achado associado ao desenvolvimento de lesäo cutânea nos pontos da pele onde o parasita foi inoculado. Diante desses resultados, ficou demonstrada a susceptibilidade do primata Cebus apella à infecçäo experimental pela leishmania lainsoni, cujo período de infecçäo durou quese quatro meses, suficiente para testar drogas antileishmanióticas e estudar a patogênese da doença causada por este parasita

Sobre a sensibilidade da cultura de leucócitos circulantes na detecçäo de Leishmania no sangue periférico de pacientes com leishmaniose tegumentar / Sensitivity of the culture of circulating leucocytes in the detection of Leishmania in the peripheral blood of patients with tegumentary leishmaniasis

Foi investigada a presença de Leishmani, através da cultura de leucócitos circulantes, no sangue periférico de 60 pacientes portadores de leishmaniose tegumentar americana, nas suas diferentes formas clínicas, assim como nas principais fases evolutivas da doença. Biópsias de lesöes cutâneas e/ou de mucosa desses pacientes foram obtidas com a finalidade de isolar e caracterizar os parasitas, através da técnica de anticorpos monoclonais. Dos 60 pacientes examinados, foram isoladas 40 amostras de Leishmania das lesöes biopsiadas, sendo 5 de Leishmania (V.) brasiliensis, 3 de L. (V.) guyanensis, 1 de L. (V.) lainsoni, 13 de L. (L.) amazonensis e 18 näo puderam ser caracterizados a nível específico, porém, reagiram com anticorpos monoclonais do grupo brazilensis. Quanto à pesquisa através das culturas de leucócitos circulantes, esta revelou resultados completamente negativos. Com base nesses achados, os autores concluíram ser pouco consciente atribuir valor à cultura de leucócitos para o diagnóstico da leishmaniose tegumentar

Epidemiologia de um caso de doença de Chagas na Ilha do Mosqueiro - Pará / Epidemiology of a case of Chagas' disease in the Mosqueiro Island - Para (Brazil)

Os autores apresentam os resultados do estudo epidemiológico de um caso autóctone da fase aguda da doença de Chagas na ilha do Mosqueiro. Estado do Pará, aproximadamente 75km da capital, Belém. O caso já havia sido objeto de uma publicaçäo anterior. Agora säo apresentadas informaçöes epidemiológicas. Nas proximidades da casa do paciente foram capturados em duas palmeiras de Inajá (Maximilian regia) e em uma da Mucajá (Acrocomia sclerocarpia) 114 triatomineos: Rhodnius pictipes, R. robustus, Panstrongylus lignarius, P. geniculatus e Microtriatoma trinidadensis, com tripanossomas em 31 deles. Na casa do paciente foram encontrados exemplares de Rhodnius pictipes, infectados com formas metacíclicas do Trypanosoma cruzi. Em 14 marsupiais, capturados na localidade, haviam 3 infectados com organismos semelhantes ao T. cruzi. A eletroforese dos isoenzimas nos tripanossomas isolados do paciente, de R. pictipes e de Didelphis marsupialis os classificou como zimodema 1. Os autores concluem que a doença de Chagas do paciente teve origem silvestre