DISTINCT CELLULAR MIGRATION INDUCED BY Leishmania infantum chagasi AND SALIVA FROM Lutzomyia longipalpis IN A HEMORRHAGIC POOL MODEL

Recruitment of a specific cell population after Leishmania infection can influence the outcome of the disease. Cellular migration in response to Leishmania or vector saliva has been reported in air pouch model, however, cellular migration induced by Leishmania associated with host's blood and vector saliva in this model has not been described. Herein we investigated cellular migration into air pouch of hamster after stimulation with combination of L. chagasi and host's blood and Lutzomyia longipalpis saliva. Migration induced by saliva was 3-fold more than those induced by L. chagasi alone. Additionally, L. chagasi associated with blood and saliva induced significantly even more leukocytes into air pouch than Leishmania alone. L. chagasi recruited a diverse cell population; however, most of these cells seem to have not migrated to the inflammatory exudate, remaining in the pouch lining tissue. These results indicate that L. chagasi can reduce leukocyte accumulation to the initial site of infection, and when associated with vector saliva in the presence of blood components, increase the influx of more neutrophils than macrophages, suggesting that the parasite has developed a strategy to minimize the initial inflammatory response, allowing an unlimited progression within the host. This work reinforces the importance of studies on the salivary components of sand fly vectors of leishmaniasis in the transmission process and the establishment of the infection.

FIRST REPORT OF CUTANEOUS LEISHMANIASIS CAUSED BY Leishmania (Leishmania) infantum chagasi IN AN URBAN AREA OF RIO DE JANEIRO, BRAZIL

SUMMARY American tegumentary leishmaniasis (ATL) is an infectious disease caused by protozoa of the genus Leishmania, and transmitted by sandflies. In the state of Rio de Janeiro, almost all of the cases of American tegumentary leishmaniasis (ATL) are caused by Leishmania (Viannia) braziliensis, while cases of visceral leishmaniasis (VL) are caused by Leishmania (Leishmania) infantum chagasi. The resurgence of autochthonous VL cases in Rio de Janeiro is related to the geographic expansion of the vector Lutzomyia longipalpis and its ability to adapt to urban areas. We report the first case of leishmaniasis with exclusively cutaneous manifestations caused by L. (L.) infantum chagasi in an urban area of Rio de Janeiro. An eighty-one-year-old woman presented three pleomorphic skin lesions that were not associated with systemic symptoms or visceromegalies. Multilocus enzyme electrophoresis identified L. (L.) infantum chagasi, but direct smear and PCR of bone narrow were negative for Leishmania sp. (suggesting exclusively cutaneous involvement). We discuss the different dermatological presentations of viscerotropic leishmaniasis of the New and Old World, and the clinical and epidemiological importance of the case. Etiologic diagnosis of ATL based upon exclusive clinical criteria may lead to incorrect conclusions. We should be aware of the constant changes in epidemiological patterns related to leishmaniases.

Leishmania (Leishmania) infantum chagasi em canídeos silvestres mantidos em cativeiro, no Estado de Mato Grosso

INTRODUÇÃO: Leishmaniose visceral é uma zoonose que acomete diversos mamíferos tendo os canídeos domésticos como principais reservatórios em ambiente urbano. A presente nota descreve a infecção de canídeos silvestres por Leishmania (Leishmania) infantum chagasi mantidos em cativeiro no Estado de Mato Grosso, Brasil. MÉTODOS: De seis raposas (Cerdocyon thous) e um cachorro vinagre (Spheotos venaticus), foram coletadas amostras de pele, medula óssea e linfonodo para detecção e caracterização de Leishmania sp pela técnica de PCR-RFLP. RESULTADOS: Todos as animais pesquisados apresentaram-se positivos para Leishmania (L.) infantum chagasi. CONCLUSÕES: Destaca-se a importância de monitoramento adequado dos mesmos, além do maior controle desta enfermidade já que estes animais estão em ambientes de recreação pública.

Serological cross-reactivity of Trypanosoma cruzi, Ehrlichia canis, Toxoplasma gondii, Neospora caninum and Babesia canis to Leishmania infantum chagasi tests in dogs

Introduction: The aim of this study was to evaluate the serological cross-reactivity between Leishmania sp. and other canine pathogens. Methods: Positive serum samples for Ehrlichia canis, Babesia canis, Toxoplasma gondii, Neospora caninum and Trypanosoma cruzi were tested using three serological methods enzyme linked immunosorbent assay (ELISA), indirect immunofluorescent antibody test (IFAT) and Kalazar Detect™, for canine visceral leishmaniasis. Results: Of the 57 dog samples tested, 24 (42.1%) tested positive using one of the three serological methods: 10/57 (17.5%) for ELISA, 11/57 (19.3%) for IFAT and 3/57 (5.3%) for Kalazar Detect™. Conclusions: Our results demonstrated that the presence of other infectious agents may lead to cross-reactivity on leishmaniasis serological tests.

Reproductive isolation between different forms of Lutzomyia longipalpis (Lutz & Neiva), (Diptera: Psychodidae), the vector of Leishmania donovani chagasi Cunha & Chagas and its significance to kala-azar distribution in South America

The males of the sandfly Lutzomyia longipalpis occur in two forms, one which bears a single pair of pale spots on tergite 4 and another in which an additional pair of spots characterizes tergite 3. In crosses between laboratory reared stocks of the two forms originating from allopatric and sympatric sites in Brazil nearly all males of one form fail to inseminate females of the other. In addition, insemination failure between some allopatric populaytions of Lu. longipalpis with similar tergal spot patterns is recorded, indicating the existence of additional forms in an apparent species complex. The possibility that Lu. longipalpis sensu latu represents more than a single taxon is discussed and the relevance of these findings to future epidemiological studies on kala-azar is considered.

Active cutaneous leishmaniasis in Brazil, induced by Leishmania donovani chagasi

L.d. chagasi was isolated from active cutaneous leishmaniasis in both human and canine infections in an endemic area in Rio de Janeiro, Brazil. Both isolates were identified by molecular and immunological characterization of the parasite using three different methods: electrophoretic mobility of isoenzymes; restriction endonuclease fragment analysis of kDNA and serodeme analysis using monoclonal antibodies. This seems to be the first well documented case in the New World of a "viscerotropic" Leishmania inducing a case of cutaneous leishmaniasis. This observation emphasizes that the diagnosis of the etiologic agent of human or canine visceral leishmaniasis based solely upon clinical and epidemiological critwria may lead to erroneous conclusions.

In vitro and in vivo activity of meglumine antimoniate produced at Farmanguinhos-Fiocruz, Brazil, against Leishmania (Leishmania) amazonensis, L (L.) chagasi and L (Viannia) braziliensis

The leishmanicidal activity of four batches of meglumine antimoniate, produced in Farmanguinhos-Fiocruz, Brazil (TAMs), was assessed and compared to Glucantime®-Aventis Pharma Ltda. Using the amastigote-like in vitro model, the active concentrations of Sb v varied from 10µg/ml to 300 µg/ml for L. (L.) chagasi and from 50µg/ml to 300µg/ml for L. (L.) amazonensis, with no statistically significant differences among the four batches of TAMs and Glucantime®. The inhibitory concentrations (IC50) determined by the amastigote-infected macrophage model for TAM01/03 and Glucantime® were, respectively: 26.3µg/ml and 127.6µg/ml for L. chagasi, 15.4µg /ml and 22.9µg/ml for L. amazonensis, and 12.1µg/ml and 24.2µg/ml for L. (V.) braziliensis. The activities of the four batches of TAMs were confirmed in an in vivo model by assessing, during eight weeks skin lesions caused by L. braziliensis in hamster that were treated with 20mg Sb v/Kg/day for 30 consecutive days. The meglumine antimoniate produced by Farmanguinhos was as effective as the reference drug, Glucantime®-Aventis, against three species of Leishmania that are of medical importance in Brazil.

Experimental infection parameters in Galea spixii (Rodentia: Caviidae) with Leishmania infantum chagasi

In order to better understand the epidemiological transmission network of leishmaniasis, an endemic disease in Northeast Brazil, we investigated the susceptibility of Spix yellow-toothed cavies (Galea spixii) to the Leishmania infantum chagasi parasite. Nine cavies were experimentally infected, separated into three groups and monitored at 30, 90 and 180 days, respectively. Amastigotes were identified in the spleen slides of two cavies killed 180 days after infection. Antibodies against the L. i. chagasi were identified in one of the cavies. This demonstrates that G. spixii is in fact capable of maintaining a stable infection by L. i. chagasi without alterations in biochemical and hematological parameters of the host and without perceivable micro and macroscopic lesions.

Biodistribution of meglumine antimoniate in healthy and Leishmania (Leishmania) infantum chagasi-infected BALB/c mice

Pentavalent antimonials such as meglumine antimoniate (MA) are the primary treatments for leishmaniasis, a complex disease caused by protozoan parasites of the genus Leishmania . Despite over 70 years of clinical use, their mechanisms of action, toxicity and pharmacokinetics have not been fully elucidated. Radiotracer studies performed on animals have the potential to play a major role in pharmaceutical development. The aims of this study were to prepare an antimony radiotracer by neutron irradiation of MA and to determine the biodistribution of MA in healthy and Leishmania (Leishmania) infantum chagasi-infected mice. MA (Glucantime(r)) was neutron irradiated inside the IEA-R1 nuclear reactor, producing two radioisotopes, 122Sb and 124Sb, with high radionuclidic purity and good specific activity. This irradiated compound presented anti-leishmanial activity similar to that of non-irradiated MA in both in vitro and in vivo evaluations. In the biodistribution studies, healthy mice showed higher uptake of antimony in the liver than infected mice and elimination occurred primarily through biliary excretion, with a small proportion of the drug excreted by the kidneys. The serum kinetic curve was bi-exponential, with two compartments: the central compartment and another compartment associated with drug excretion. Radiotracers, which can be easily produced by neutron irradiation, were demonstrated to be an interesting tool for answering several questions regarding antimonial pharmacokinetics and chemotherapy.

Diagnóstico molecular e frequência de anticorpos anti-Leishmania infantum chagasi em cães do município de Belém, Pará

A leishmaniose visceral é uma enfermidade cujo agente etiológico no Brasil é o protozoário Leishmania infantum chagasi. Os cães são considerados reservatórios urbanos da doença, sendo indicadores da ocorrência de casos humanos. O presente trabalho teve como objetivo diagnosticar a infecção por L. infantum chagasi em cães domiciliados e errantes do município de Belém, estado do Pará, através da reação em cadeia da polimerase (PCR) e da reação de imunofluorescência indireta (RIFI), empregando dois antígenos distintos. Amostras de sangue venoso de cães adultos, sem distinção de sexo ou raça, de diferentes bairros e épocas do ano da cidade de Belém-PA, foram colhidas em tubos sem e com anticoagulante para obtenção do soro e do DNA, respectivamente. Esses animais foram divididos em dois grupos: cães errantes capturados pelo Centro de Controle de Zoonoses (Grupo A) e cães domiciliados (Grupo B). Os soros foram analisados através do teste de RIFI para pesquisa de IgG utilizando-se dois antígenos distintos: 1) antígeno do kit Bio-Manguinhos/FIOCRUZ (Ag-PRO) contendo formas promastigotas de Leishmania sp. (complexo major-like); 2) Antígeno do Instituto Evandro Chagas (Ag-AMA) constituído por formas amastigotas de L. infantum chagasi. A avaliação dos dois antígenos foi realizada com as amostras reagentes a partir da titulação 1:80. Já a PCR foi realizada a partir do DNA extraído do sangue total dos animais e amplificado utilizando-se os iniciadores RV1e RV2. Das 335 amostras analisadas, 10,4% (35/335) foram reagentes na RIFI (Ag-PRO) e 0,9% (3/335) reagiram com o Ag-AMA. A distribuição das amostras positivas se deu da seguinte forma: Grupo A 14,8% (25/169) com Ag-PRO e 1,2% (2/169) com Ag-AMA; Grupo B 6% (10/166) com Ag-PRO e 0,6% (1/166) com Ag-AMA; sendo que todas as amostras positivas pelo teste de RIFI com o Ag-AMA também reagiram com o Ag-PRO e em nenhuma das amostras foi detectado o DNA de L. infantum chagasi. Os achados do presente estudo indicam que Belém ainda pode ser considerada área não endêmica para leishmaniose visceral canina e que a natureza do antígeno influencia no resultado da RIFI para a pesquisa de anticorpos anti-L. infantum chagasi em cães, sendo que a RIFI que utiliza formas promastigotas de Leishmania major-like como antígeno deve ser utilizada com cautela como método diagnóstico confirmatório em estudos epidemiológicos em áreas não endêmicas para LVC.

Phlebotomine sandfly fauna and natural Leishmania infection rates in a rural area of Cerrado (tropical savannah) in Nova Mutum, State of Mato Grosso in Brazil

Introduction American cutaneous leishmaniasis (ACL) has been reported in every municipality of the State of Mato Grosso, Brazil, but the transmission epidemiology remains poorly understood. Our study was developed in a rural area of the Nova Mutum municipality where four autochthonous cases of ACL were reported in 2009. Our aims were to describe the local phlebotomine sandfly fauna and to investigate the infection rates and infecting Leishmania species in the captured sandflies. Methods Entomological captures were performed bimonthly at 10 fixed sites close to the edge of a forested area between June 2011 and April 2012. Results A total of 3,743 phlebotomine sandflies belonging to 31 distinct species were captured. Approximately 75% of the specimens were females. The most abundant species (45.4%) was Lutzomyia antunesi, which was consistently captured at every site. Species that are epidemiologically important for ACL, such as L. flaviscutellata, L. whitmani and L. umbratilis, were also captured. L. antunesi and L. ubiquitalis were naturally infected by Leishmania braziliensis or Le. guyanensis, with minimum infection rates of 0.88% and 6.67%, respectively. Surprisingly, L. antunesi was infected by Le. infantum (synonym chagasi). Conclusions The natural infection of L. antunesi and L. ubiquitalis by Leishmania sp. suggests that these species might play a role in the zoonotic cycle of ACL in Nova Mutum. The presence of Le. infantum in L. antunesi suggests that there may be a risk of an outbreak of visceral leishmaniasis (VL) in Nova Mutum.

Susceptibility of laboratory-reared female Lutzomyia longipalpis (Lutz & Neiva, 1912) to infection by different species and strains of Leishmania Ross, 1903

A study was undertaken to compare the susceptibility of laboratory-reared female Lutzomyia longipalpis to infection by different species or strains of New World Leishmania. The sand flies proved to be highly susceptible to infection by a strain of Le. guyanensis, with flagellates developing in all (18/18) of the specimens examined. A lower infection rate of 37 per cents (10/27) was recorded in flies exposed to infection by a strain of Le. amazonensis. Flagellates developed in 13 per cents (6/46) of the sand flies that glood fed on dogs in the earlly stage of experimental infection with an old laboratory strain of Le. chagasi. In contrast, promastigotes did not develop in sand flies that blood fed on dogs with naturally acquired Le. chagasi. The naturally infected dogas were in an advanced stage of disease. Flagellates developed in 9// (3/32) of the sand flies that blood fed on lesions of hamsters infected with a strain of Le. braziliensis and in 9 per cents (3/34) of those that fed on hamsters with lesions due to a parasite fo the mexicana complex (strain MHOM/BR/73/BH121). Sand flies did not develop flagellate infections after blood feeding on hamsters bearing lesions induced by strain MHOM/BR/71/BR49. Factors influencing the susceptibility of Lu. longipalpis to infection by New World species of Leishmania are discussed.