Active Transport of Glutamate in Leishmania (Leishmania) amazonensis

Leishmania spp. are the causative agents of leishmaniasis, a complex of diseases with a broad spectrum of clinical manifestations. Leishmania (Leishmania) amazonensis is a main etiological agent of diffuse cutaneous leishmaniasis. Leishmania spp., as other trypanosomatids, possess a metabolism based significantly on the consumption of amino acids. However, the transport of amino acids in these organisms remains poorly understood with few exceptions. Glutamate transport is an important biological process in many organisms. In the present work, the transport of glutamate is characterized. This process is performed by a single kinetic system (K-m=0.59 +/- 0.04 mM, V-max=0.123 +/- 0.003 nmol/min per 20 x 10(6) cells) showing an energy of activation of 52.38 +/- 4.7 kJ/mol and was shown to be partially inhibited by analogues, such as glutamine, aspartate, alpha-ketoglutarate and oxaloacetate, methionine, and alanine. The transport activity was sensitive to the extracellular concentration of H+ but not to Na+ or K+. However, unlike other amino acid transporters presently characterized, the treatment with specific ionophores confirmed the participation of a K+, and not H+ membrane gradient in the transport process.

Leishmania spp. in Didelphis albiventris and Micoureus paraguayanus (Didelphimorphia: Didelphidae) of Brazil

Leishmaniasis is kept in nature by the participation of several animal species. This study evaluated the presence of Leishmania spp. in skin samples of free-ranging marsupials Micoureus paraguayanus (n = 95) and Didelphis albiventris (n = 191), captured in Morro do Diabo State Park and in sections of its surrounding forest, in the region of Pontal do Paranapanema, Sao Paulo State, Brazil. The samples were tested for the presence of kDNA of Leishmania spp. by polymerase chain reaction (PCR) and by real time PCR (qPCR). All samples from D. albiventris tested by PCR were negative for the presence of kDNA of Leishmania spp. However, when tested by qPCR, the positivity was 1.6%. A positivity of 7.4% by PCR and 11.6% by qPCR was observed for M. paraguayanus. Sixty-four per cent (9/14) of positive animals were limited to the same forest fragment. Presence of Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis was detected in M. paraguayanus samples. While D. albiventris is the most studied marsupial species due to its urban habits, other marsupial species such as M. paraguayanus can be potential reservoirs of Leishmania spp. and should also be studied. (C) 2010 Elsevier B.V. All rights reserved.

Seroprevalence of anti-Leishmania spp. antibodies in rural dogs from the city of Monte Negro, State of Rondonia, Brazil

The present study assessed the prevalence of anti-Leishmania spp antibodies in dogs from the city of Monte Negro, State of Rondonia, Brazil ELISA (NE >= 3) and IFAT (>= 1 40) were used to evaluate 161 serum samples collected from rural dogs from Monte Negro Forty-five (27 9%) dogs were positive by ELISA tests and five (3 1%) were positive by IFAT The present study showed for the first time the frequency of exposure to Leishmania spp in dogs in the State of Rondonia, Amazon Region

In vitro sensitivity of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonensis Brazilian isolates to meglumine antimoniate and amphotericin B

Resistance of Leishmania parasites to specific chemotherapy has become a well-documented problem in the Indian subcontinent in recent years but only a few studies have focused on the susceptibility of American Leishmania isolates. Our susceptibility assays to meglumine antimoniate were performed against intracellular amastigotes after standardizing an in vitro model of macrophage infection appropriate for Leishmania (Viannia) braziliensis isolates. For the determination of promastigote susceptibility to amphotericin B, we developed a simplified MTT-test. The sensitivity in vitro to meglumine antimoniate and amphotericin B of 13 isolates obtained from Brazilian patients was determined. L. (V.) braziliensis isolates were more susceptible to meglumine antimoniate than Leishmania (Leishmania) amazonensis. EC(50), EC(90) and activity indexes (calculated over the sensitivity of reference strains), suggested that all isolates tested were susceptible in vitro to meglumine antimoniate, and did not show association with the clinical outcomes. Isolates were also uniformly susceptible in vitro to amphotericin B.

A real-time polymerase chain reaction assay for the identification and quantification of American Leishmania species on the basis of glucose-6-phosphate dehydrogenase

A real-time polymerase chain reaction (PCR) test was developed on the basis of the Leishmania glucose-6-phosphate dehydrogenase locus that enables identification and quantification of parasites. Using two independent pairs of primers in SYBR-Green assays, the test identified etiologic agents of cutaneous leishmaniasis belonging to both subgenera, Leishmania (Viannia) and Leishmania (Leishmania) in the Americas. Furthermore, use of TaqMan probes enables distinction between L. (V.) braziliensis or L. (V.) peruviania from the other L. (Viannia) species. All assays were negative with DNA of related trypanosomatids, humans, and mice. The parasite burden was estimated by normalizing the number of organisms per total amount of DNA in the sample or per host glyceraldehyde-3-phosphate dehydrogenase copies. The real-time PCR assay for L. (Leishmania) subgenus showed a good linear correlation with quantification on the basis of a limiting dilution assay in experimentally infected mice. The test successfully identifies and quantifies Leishmania in human biopsy specimens and represents a new tool to study leishmaniasis.

False-positive results of a rapid K39-based strip test and Chagas disease

Background: The definitive diagnosis of visceral. leishmaniasis (VL) requires invasive procedures with demonstration of amastigotes in tissue or promastigotes in culture. Unfortunately, these approaches require laboratory materials not available in poor countries where the disease is endemic. The correct diagnosis of VL is important, and made more difficult by the fact that several common tropical diseases such as malaria, disseminated tuberculosis, and enteric fever share the same clinical presentation. Serological tests have been developed to replace parasitological diagnosis in the field. A commercially available K39-based strip test for VL has been developed for this purpose. The endemic area of leishmaniasis in Brazil overlaps the endemic area of Chagas disease, a disease that can cause false-positive serological test results. The aim of this study was to evaluate the incidence of false-positive exams using a rapid test for VL in patients with Chagas disease. Methods: A rapid test based on the recombinant K39 antigen of Leishmania was used in: (1) 30 patients with confirmed Chagas disease, (2) 30 patients with a serological diagnosis of Chagas disease by ELISA, indirect immunofluorescence, indirect hemagglutination, and chemiluminescence, (3) 30 healthy patients from a non-endemic area as the control group, (4) 30 patients with confirmed VL, and (5) 20 patients with proved cutaneous leishmaniasis. Results: The sensitivity and specificity of the rapid strip test were 100% when compared with healthy volunteers and those with confirmed Chagas disease. One false-positive result occurred in the group with Chagas disease diagnosed by serological tests (specificity of 96%). Conclusion: The rapid test based on recombinant K39 is a useful diagnostic assay, and a false-positive result rarely occurs in patients with a serological diagnosis of Chagas disease. (C) 2008 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

The effect of phospholipase A2 from Crotalus durissus collilineatus on Leishmania (Leishmania) amazonensis infection

In this study, the effect of phospholipase A2 (PLA2) derived from Crotalus durissus collilineatus was evaluated in vitro and in vivo on experimental cutaneous leishmaniasis. The promastigote and amastigote forms treated with PLA2 presented increased growth rate. In vivo studies showed that PLA2-treated Leishmania (Leishmania) amazonensis promastigotes increased the size of lesions in BALB/c mice, and histopathological analysis showed numerous necrotic regions presenting a higher density of polymorphonuclear, mononuclear, and amastigote cells. Additionally, infected macrophages treated with PLA2 were able to generate prostaglandin E2 (PGE2). Cytokine quantification showed that the supernatant from infected macrophages presented moderate and high amounts of IL-2 and IL-10, respectively. However, in PLA2-treated infected macrophages, suppression of IL-2 levels occurred, but not of IL-10 levels. Observation also revealed that both the supernatant and lysate of L. (L.) amazonensis promastigotes exhibited PLA2 activity, which, in the presence of dexamethasone, showed no reduction in their activities; while glucocorticoid maintained the ability of promastigote forms to infect macrophages, which presented values similar to controls. In conclusion, the results indicate that PLA2 may be a progression factor for cutaneous leishmaniasis, since the PLA2 effect suppressed IL-2 levels and generated PGE2, an inflammatory lipid mediator.

Surto de leishmaniose tegumentar americana em Itaporanga, São Paulo (Brasil)

O presente trabalho relata os aspectos epidemiológicos e clínicos de surto de leishmaniose tegumentar americana na região Sudoeste do estado de São Paulo, área de colonização antiga, não associada a derrubadas de matas. Foram examinados 231 indivíduos, observando-se: a) Sessenta e sete indivíduos (29%) apresentavam leishmaniose confirmada pela histopatologia das lesões cutâneas e intradermorreação de Montenegro. Destes casos, 40 (59,7%) eram homens; b) a idade variou entre 2 (5 casos) e 86 anos (1 caso); c) de acordo com o tipo de lesão, observou-se: 54 (80, 6%) pacientes apresentavam úlceras, 13 (19,4%) nódulos, 4 (5,9%) lesões úlcero-vegetantes e 3 (4,4%) cicatrizes típicas; d) a intradermorreção de Montenegro apresentou 94,7% de positividade nos casos de leishmaniose-doença. Este estudo reforça observações prévias de que, além da forma clássica de transmissão, a leishmaniose pode ser transmitida no intra e peri-domicílio.

Avaliação do efeito antiparasitário do omeprazol na prevenção do desenvolvimento de lesões cutâneas em hamsters infectados por Leishmania brasiliensis

FUNDAMENTOS: A leishmaniose tegumentar americana permanece doença endêmica em diversas regiões brasileiras. A sobrevivência do parasita no interior dos macrófagos se deve, em parte, pela atividade de uma K+/H+-ATPase de membrana que pode ser inibida pelo omeprazol. OBJETIVOS: Avaliar a eficácia do omeprazol na prevenção do desenvolvimento de lesões de leishmaniose em hamsters. MÉTODOS: Empregaram-se 18 hamsters, divididos em três grupos: o grupo L recebeu apenas a inoculação de L. brasiliensis na pata anterior direita, o grupo O recebeu apenas doses diárias de 0,4mg de omeprazol subcutâneo, e o grupo L+O recebeu o inóculo de leishmanias e o tratamento com omeprazol desde o dia da inoculação. O estudo foi conduzido por 42 dias, realizaram-se medidas dos diâmetros das patas semanalmente, e, ao final do estudo, foram realizados esfregaços das lesões para verificação dos parasitas. RESULTADOS: Os hamsters dos grupos L e L+O desenvolveram lesões de leishmaniose tegumentar havendo ulceração em duas patas do grupo L e uma do grupo L+O. Ao final do estudo, a mobilidade e vitalidade no grupo L foram menores que em L+O, e estas menores que no grupo O. Os diâmetros das patas inoculadas nos grupos L e L+O foram significativamente maiores que no início do estudo (p<0.05). Não houve diferença significativa entre os diâmetros das patas dos grupos L e L+O ao final do estudo (p0,05), sendo detectados parasitas no esfregaço das lesões dos dois grupos. CONCLUSÕES: Omeprazol, no protocolo utilizado, não evitou o desenvolvimento de lesões de leishmaniose tegumentar em hamsters.

Leishmaniose cutânea acidental

Os autores relatam o 3º caso de Leishmaniose acidental em laboratório, ocorrido em aluna do curso de Biologia, que contaminou-se por ocasião da passagem de formas amastigotas de Leishmania (Viannia) braziliensis de hamster infectado para hamster são. Chamam atenção de que, apesar de usar toda proteção exigida, a aluna foi mordida pelo hamster são, havendo como conseqüência, ruptura da luva e contágio através do inóculo.

Comparative studies of the anti-leishmanial activity of three Crotalus durissus ssp venoms

In this study, we compared the anti-leishmanial activity of three crotalic venoms (Crotalus durissus terrificus-Cdt, Crotalus durissus cascavella-Cdca, and Crotalus durissus collilineatus-Cdcol). Different concentrations of each venom incubated with Leishmania (Leishmania) amazonensis promastigotes were used. Cdt venom exhibited a higher anti-leishmanial activity (Inhibitory concentration-IC50-value of 4.70 +/- 1.72 mu g/ml) in comparison with that of Cdca venom (IC50 value of 9.41 +/- 1.21 mu g/ml), while Cdcol venom increased parasite numbers in 50% at a concentration of 44.30 +/- 2.18 mu g/ml. In addition, this venom showed a low anti-leishmanial activity in higher concentrations (IC50 value of 281.00 +/- 9.50 mu g/ml). The main fractions of Cdca venom were isolated and assayed under similar conditions used for assessing crude venom. The most active fractions were gyroxin and crotamine that had IC50 values of 3.80 +/- 0.52 mu g/ml and 19.95 +/- 4.21 mu g/ml, respectively. Convulxin also inhibited parasite growth rate, although this effect was not dose-dependent. Crotoxin was the least effective fraction with an IC50 value of 99.80 +/- 2.21 mu g/ml. None of the protein fractions presented cytotoxic effects against J774 cells in culture. In vivo assays using BALB/c mice revealed that crotoxin and crotamine were the main toxic fractions. In conclusion, C. durissus cascavella venom has three main fractions with anti-leishmanial activity. These results open new possibilities to find proteins that might be used as possible agents against cutaneous leishmaniasis.

Tratamento da leishmaniose cutanea difusa (LCD) com a associacao antimoniato-N-metilglucamina (Glucantime®) e sulfato de paramomicina (Gabbrox®)

Diffuse cutaneous leishmaniasis (DCL) is a rare form of tegumentary leishmaniasis (TL) and 350 cases have been reported world wide. Presently in Brazil, there were 31 cases reported corresponding to 8.9% of the world cases. The state of Maranhao contributed with ten cases (32.3%) of national reports. Recently the authors had opportunity to follow two new cases of DCL, both from the countryside of that state totaling 12 cases. The parasite Leishmania L. amazonensis was incriminated in the disease, confirming the available data as being the unique species to produce DCL in this state. The therapy associating meglumine anthimoniate with paramomicine sulphate (Gabbrox®) during 90 days, showed good tolerance and favorable response, being another option for fills form of DCL that constituted a challenge to science so far.

Flebotomíneos (Diptera, Psychodidae) de área de cerrado no Município de Corumbataí, Centro-leste do Estado de São Paulo, Brasil.

Distribution and ecology knowledge of sandfly species is essential for epidemiology vigilance and risk determination for transmission of leishmaniasis. Sandfly trapping was carried out in a cerrado strictu sensu vegetation fragment in a rural area of Corumbataí Municipality, São Paulo State, Brazil, during July to November 2004. Two CDC light traps were used from 18h to 8h, once a month, resulting in 112 total hours of capture. During this period, 60 sandfly specimens of ten different species were sampled. The most abundant species and the one found in all captures was Pintomyia monticola totalizing 15 (25.0%) specimens, the second more abundant one was P. pessoai with 14 (23.3%) and the third one was Brumptomyia guimaraesi with 12 (20.0%) individuals collected. Other captured species were Psathyromyia aragaoi, B. avellari, B. brumpti, B. cunhai, P. bianchigalatiae, Evandromyia termitophila and Nyssomyia whitmani which corresponded to 19 (31.2%) specimens captured. Pintomyia pessoai and N. whitmani presence indicates transmission risk of American Cutaneous Leishmaniasis in the area.

Ocorrência de anticorpos contra Leishmania spp., Neospora caninum e Toxoplasma gondii em soros de cães atendidos no Hospital Veterinário da Universidade Estadual de Londrina-Pr

The aim of this study was to detect the presence of IgG antibodies anti-Leishmania spp., Toxoplasma gondii and Neospora caninum in dogs from a Veterinary Hospital from Universidade Estadual de Londrina. Blood samples from 112 animals were obtained by jugular venipuncture to obtain sera. The samples were tested by indirect immunofluorescence to detect antibodies anti-Leishmania spp., anti-N. caninum and anti-T. gondii. Thirteen (11.61%), 25 (22.32%), and 57 (50.89%) samples were positive for Leishmania spp., N. caninum, and T. gondii, respectively. The co-presence of anti-Leishmania spp. and N. caninum was observed in 6 (5.36%), anti-Leishmania spp. and anti-T. gondii in 8 (14.7%), and anti-N. caninum and anti-T. gondii in 18 (16.07%) samples. The co-presence of anti-Leishmania spp., anti-N. caninum and anti-T. gondii was observed in 5 (4.46%) dogs. There was a higher prevalence of Leishmania in Toxoplasma and Neospora positive animals, however, these results were not statistically significant (range p = 0.052 p = 0.06). The dogs have an important role in the epidemiological cycle of these diseases, which are important in animal and public health. The northern state of Paraná is an endemic area for human cutaneous leishmaniasis, therefore, studies should be conducted to uncover the real role of dogs as reservoirs of Leishmania to humans in the state.

Estudo soroepidemiológico de leishmaniose em equinos na região de Araçatuba-SP, Brasil, área endêmica para leishmaniose visceral

The aim of the present study was to perform a leishmaniasis survey in horses from Araçatuba, São Paulo, an endemic area of Brazil. Of the 466 horses tested for the presence of anti-Leishmania chagasi titers by ELISA, 68 (14.59%) were seropositive, with titers varying between 0.324 and 0.813. ELISA positive samples were also tested by immunocromatography and 19/466 (4.08%) were positive. The results of the present study indicated that equines are in contact and can attract phlebotomines, and highlight the necessity of a more accurate investigation on the role played by the horses living in endemic areas, in order to help to control the spread of the illness.