Identification of Leishmania infantum chagasi proteins in urine of patients with visceral leishmaniasis: a promising antigen discovery approach of vaccine candidates

Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500 000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver and bone marrow lesions and excreted in the patients urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1) and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in Escherichia coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2+ BpMPLA-SE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to VL and opens novel possibilities for vaccine development to other serious infectious diseases.

Anti-Leishmania infantum IgG Antibody Avidity in Visceral Leishmaniasis

IgG avidity tests are used to discriminate acute from chronic infections. There are few reports on the IgG avidity profile of patients with visceral leishmaniasis (VL). This study investigated the anti-Leishmania IgG avidity in patients with classic VL (n = 10), patients showing clinical cure after treatment (n = 18), and asymptomatic subjects with at least one positive Leishmania test (n = 20). All subjects were from areas in Brazil where VL is endemic. Serum samples were collected from each subject on two different occasions. IgG avidity was evaluated by Western blotting. The proportion of high-avidity antibodies was higher in all samples from patients with classic VL. In contrast, low-avidity antibodies predominated in subjects with a history of VL, including 13 cases (72.2%) in the first assessment and 14 (77.8%) in the second. Fifteen (75%) of the asymptomatic subjects presented a predominance of low-avidity antibodies in the first assessment, and the frequency of high-avidity antibodies increased over time in seven subjects (35%) of this group. Antibodies against the 14- and/or 16-kDa antigen fraction were detected in the first assessment in all patients with classic VL, in 10 (55.5%) treated patients, and in 10 (50%) asymptomatic subjects. These were high-avidity antibodies in most cases. In the asymptomatic group, an increase in IgG avidity against the 14- and/or 16-kDa antigen fraction was observed in three cases (15%). The results indicate distinct responses in infected and asymptomatic subjects, probably associated with the length of time after infection. In this respect, IgG avidity tests represent a new approach to better characterize asymptomatic VL.

Estudio de la infección por Leishmania infantum en el perro: utilidad de las técnicas diagnósticas no invasivas y nuevas alternativas terapéuticas

La leishmaniosis canina (Lcan) causada por Leishmania infantum es una zoonosis de distribución mundial endémica en la cuenca mediterránea. Es transmitida a humanos y animales mediante la picadura de las hembras de flebotomos, siendo el perro tanto un hospedador natural como el principal reservorio. Las manifestaciones clínicas de la infección por L. infantum en el perro son muy variables, desde una infección subclínica crónica hasta una enfermedad grave, que puede ser fatal. Debido a su compleja patogénesis y al importante papel que juega la respuesta inmunitaria, tanto el diagnóstico como el tratamiento y prevención de esta enfermedad son un reto desde el punto de vista veterinario y de Salud Pública. El objetivo fundamental de esta Tesis Doctoral ha sido encontrar nuevas alternativas tanto diagnósticas como terapéuticas en la Lcan. En cuanto al diagnóstico se ha evaluado el uso de muestras obtenidas de forma menos invasiva y dolorosa, y mejor aceptadas por los perros y por ende por sus propietarios. Y con respecto al tratamiento, se han evaluado dos nuevas moléculas buscando que sea más económico, eficaz, bien tolerado y de fácil administración. Para ello se han realizado tres ensayos clínicos: los dos primeros con un modelo experimental canino y el tercero en perros con infección natural por L. infantum. Para los dos primeros ensayos se empleó el mismo modelo experimental canino, que consistió en ocho perras de raza Beagle infectadas experimentalmente con L. infantum, a razón de 5x107 promastigotes/ml por vía intravenosa...

The effect of climate change on the potential distribution of the European Phlebotomus species and the parasite Leishmania infantum in 2011-2070. A climate envelope modeling approach

Aims: In the Mediterranean areas of Europe, leishmanisasis is one of the most emerging vector-borne diseases. Members of genus Phlebotomus are the primary vectors of the genus Leishmania. To track the human health effect of climate change it is a very important interdisciplinary question to study whether the climatic requirements and geographical distribution of the vectors of human pathogen organisms correlate with each other. Our study intended to explore the potential effects of ongoing climate change, in particular through a potential upward altitudinal and latitudinal shift of the distribution of the parasite Leishmania infantum, its vectors Phlebotomus ariasi, P. neglectus, P. perfiliewi, P. perniciosus, and P. tobbi, and some other sandfly species: P. papatasi, P. sergenti, and P. similis. Methods: By using a climate envelope modelling (CEM) method we modelled the current and future (2011-2070) potential distribution of 8 European sandfly species and L. infantum based on the current distribution using the REMO regional climate model. Results: We found that by the end of the 2060’s most parts of Western Europe can be colonized by sandfly species, mostly by P. ariasi and P. pernicosus. P. ariasi showed the greatest potential northward expansion. For all the studied vectors of L. infantum the entire Mediterranean Basin and South-Eastern Europe seemed to be suitable. L. infantum can affect the Eastern Mediterranean, without notable northward expansion. Our model resulted 1 to 2 months prolongation of the potentially active period of P. neglectus P. papatasi and P. perniciosus for the 2060’s in Southern Hungary. Conclusion: Our findings confirm the concerns that leishmanisais can become a real hazard for the major part of the European population to the end of the 21th century and the Carpathian Basin is a particularly vulnerable area.

Perfil imunológico associado à susceptibilidade, resistência e cura na infecção por Leishmania ( Leishmania ) infantum

Visceral Leishmaniasis (VL) is endemic in Brazil and the northeast region had the highest incidence of the disease , despite, in the last 30 years, it has spread to all geographic regions of the country. Leishmania infantum is the m ain etiological agent of VL in Latin America, Europe and North Africa. However, not all infected individuals develop the disease; in fact, the majority present spontaneous re solution of infection without symptoms. The evaluation of the immunological profil e has been mostly conducted stimulating, with Leishmania spp. antigen, peripheral blood mononuclear cells isolated from subjects with VL. These studies showed that VL patients had an inhibition of both, lymphocyte proliferation and proinflammatory response to Leishmania spp. antigen. Our study aimed to evaluate the immune response in active LV, cured post treatment and asymptomatic infection. To reach this aim, we analyzed immunophenotypic features related to activation, Treg and memory lymphocytes, by flow cytometry, as well as, evaluation of cytokine production, in ex vivo or in whole blood culture. In active VL volunteers, a longitu dinal study was conducted with reassessment at 4 and 14 months after clinical cure. The control group included individuals th at live d in endemic region and were either Positive Control, consisting of individuals with positive anti - L eishmania spp. serology and/or positive PCR for Leishmania  spp. and Negative Control composed by individuals with negative anti - Leishmania antibodie s serology and negative PCR for Leishmania . During VL, CD4 lymphocytes showed greater activation and memory profile s and were the major source of cytokines in culture when compared to CD8 lymphocytes , and these were not Leishmania specific. There were act ivated lymphocytes during VL (CD4 + CD69 + :4.9%) when compared to control groups, Positive (CD4 + CD69 + :1.96%, p=0.0045) and Negative (CD4 + CD69 + :1.35%, p=0.006), on the other hand, this was non - specific activation. The lymphocyte activation profile remain ed el evated even 14 months post treatmen t. A fter clinical cure , the activation was Leishmania specific (CD4 + CD25 + absence of SLA: 8.4%, and presence of SLA: 10.7% p=0.0279). CD8 + CD25 + lymphocytes were able to produce Leishmania specific IFN - γ in both, Positive Controls (absence of SLA 5.2% and presence of SLA: 9.5%, p=0.0391) and Cured 4 month (absence of SLA: 3.9%; presence of SLA: 10.7% p=0.0098). Whole blood culture cells, of VL patients, were able to produce IFN - γ, by SLA stimulation (absence of SLA: 28.0 pg ∕mL, and presence: 44.3 pg∕mL p=0.0020) as well as recovered groups (absence of SLA 2.3 pg∕mL and presence of SLA 139.8 pg∕mL, p=0.0005). However, the high level of IL - 10 seem ed to inhibit pro - inflammatory activity of IFN - γ and TNF - α during symptomatic dis ease . Unlike other pro - inflammatory cytokines, active VL group d id not produce Leishmania specific IL - 2 (absence of SLA 2.4 pg∕mL and presence of SLA: 2.6 pg∕mL). Based on these data we conclude that the restoration of lymphocyte activation and decreased i n IL - 10 Leishmania specific production were related to a protective immune profile.

Estudio de la enfermedad renal y la respuesta al tratamiento (antimoniales "versus" miltefosina) en perros con infección natural por "Leishmania infantum"

La leishmaniosis canina (Lcan) es una zoonosis en toda la Cuenca Mediterránea y está producida por un protozoo intracelular, Leishmania infantum, transmitido por especies del género Phlebotomus. La enfermedad se caracteriza por una presentación clínica heterogénea en la cual la lesión renal es, con frecuencia, la causa principal de mortalidad. La nefropatía asociada a Lcan se atribuye fundamentalmente al depósito intraglomerular de inmunocomplejos circulantes. La combinación de antimoniato de n-meglumina (antimoniales) con alopurinol se considera el tratamiento de primera línea en esta enfermedad, aunque algunos estudios han demostrado que la combinación de miltefosina y alopurinol tiene una eficacia similar. A pesar de ser considerado el tratamiento más eficaz, el uso de antimoniato se asocia a efectos adversos sobre la funcionalidad renal lo cual puede ocasionar complicaciones importantes en el manejo de estos pacientes. A ello se suma la escasa sensibilidad de las pruebas diagnósticas disponibles hasta ahora en la clínica veterinaria para poder establecer un diagnóstico precoz de lesión renal. Es decir, el propio tratamiento podría agravar una posible alteración en la funcionalidad renal en pacientes que ya la tuvieran alterada debido a la leishmaniosis. Los perros infectados y tratados, en la mayoría de los casos, no alcanzan la curación parasitológica y sufren recidivas después del tratamiento. Las técnicas habituales para valorar la respuesta al tratamiento son la evolución de la tasa de anticuerpos y la electroforesis de proteínas plasmáticas. La normalización de las proteínas plasmáticas ocurre en paralelo con la mejoría clínica, pudiéndose emplear como marcadores de respuesta al tratamiento; sin embargo, la serología parece menos útil, a corto plazo, porque el descenso del título de anticuerpos es más lento. Se ha postulado que otros parámetros laboratoriales, como las proteínas de fase aguda, pueden ser una buena opción como marcadores precoces de la enfermedad y también para la monitorización del tratamiento...

Estudo sobre a participação de roedores na cadeia de transmissão de Leishmania infantum (Protozoa: Trypanosomatidae) no Rio Grande do Norte

American visceral leishmaniasis is a zoonosis caused by Leishmania infantum and transmitted by the bite of the sand flies Lutzomia longipalpis.The main domestic reservoir is the dog, while foxes and opposums are the known wild reservoirs. However, identification of natural infections with L. infantum in rodents appears for need of investigating the participation of these rodents how source of infection of the parasite. In the present work the Leishmania infantum infection was investigated in rodents captured in Rio Grande do Norte, aiming at to offer subsidies to the understanding of the epidemic chains of LVA in the State. Thirteen Galea spixii were distributed in four groups, being G1 the group control with four animals and the others, G2, G3 and G4, with three animals each. Those animals were intraperitoneally inoculated with 107 promastigotas of L. infantum and accompanied for, respectively, 30, 90 and 180 days. Weekly the animals were monitored as for the corporal weight and rectal temperature. At the end of each stipulated period the animals were killed. Blood were used for determination of the parameters biochemical and haematological, PCR, ELISA, microscopic examination and cultivation in NNN medium. Liver, spleen and lymph node were used in Giemsa-stained impression and cultivation in NNN medium. Liver and spleen fragments were still used in PCR and histopathological, respectively. At the same time 79 rodents of the species Rattus rattus, Bolomys lasiurus, Oligoryzomys nigripis, Oryzomys subflavus and Trichomys apereoides were captured in the Municipal districts of Brejinho, Campo Grande, Coronel Ezequiel, Passa e Fica and Vázea for identification of natural infection with L. infantum. Evidence of infection was checked by direct examination of Giemsa-stained impression of liver, spleen and blood and culture of these tissues in NNN medium. Antibodies were researched by ELISA. They were not found differences among the weigh corporal final, rectal temperature and biochemical and haematological parameters of the Galea spixii controls and infected. The rectal temperature of the animals varied from 36OC to 40OC. For the first time values of the haematocrit (33,6% to 42,8%), hemoglobin (10,2 to 14,5g/dl), erythrocyts number (4,67x106 to 6,90x106/mm3), total leukocytes (0,9x103 to 9,2x103/mm3), platelets (49x103 to 509x103/mm3) total proteins (1,56 to 6,06 g/dl), albumin (1,34 to 3,05 g/dl) and globulins (0,20 to 3,01 g/dl) of the Galea spixii were determined. The lymphocytes were the most abundant leucocytes. Infection for L. infantum was diagnosed in two animals euthanasied 180 days after the infection. In one of the animals was also identified antibodies anti-Leishmania. The parasite was not found in none of the five other species of rodents captured. Galea spixii are resistant to the infection for L. infantum and they are not good models for the study for visceral leishmaniose, although they can act as infection sources. More studies are necessary to determine the paper of the rodents in the epidemic chain of transmission of the visceral leishmaniose in the State of Rio Grande do Norte

Estado nutricional e efeito da vitamina A na resposta imune frente à infecção por Leishmania Infantum

Nutritional status is an important determinant to the response against Leishmania infection, although few studies have characterized the molecular basis for the association found between malnutrition and the disease. Vitamin A supplementation has long been used in developing countries to prevent mortality by diarrheal and respiratory diseases, but there are no studies on the role of vitamin A in Leishmania infection, although we and others have found vitamin A deficiency in visceral Leishmaniasis (VL). Regulatory T cells are induced in vitro by vitamin A metabolites and are considered important cells implicated T CD4+ cell suppression in human VL. This work aimed to examine the correlation of nutritional status and the effect of vitamin A in the response against Leishmania infantum infection. A total of 179 children were studied: 31 had active VL, 33 VL history, 44 were DTH+ and 71 were DTH- and had negative antibody to Leishmania (DTH-/Ac-). Peripheral blood monuclear cells were isolated in a subgroup of 10 active VL and 16 DTH-/Ac- children and cultivated for 20h under 5 different conditions: 1) Medium, 2) Soluble promastigote L. infantum antigens (SLA), 3) All-trans retinoic acid (ATRA), 4) SLA + ATRA and 5) Concanavalin A. T CD4+CD25highFoxp3+, T CD4+CD25-Foxp3- and CD14+ monocytes were stained and studied by flow cytometry for IL-10, TGF-β and IL-17 production. Nutritional status was compromised in VL children, which presented lower BMI/Age and retinol concentrations when compared to healthy controls. We found a negative correlation between nutritional status (measured by BMI/Age and serum retinol) and anti-Leishmania antibodies and acute phase proteins. There was no correlation between nutritional status and parasite load. ATRA presented a dual effect in Treg cells and monocytes: In healthy children (DTH-/Ac-), it induced a regulatory response, increasing IL-10 and TGF-β production; in VL children it modulated the immune response, preventing increased IL-10 production after SLA stimulation. Furthermore, we found a positive correlation between BMI/Age and IL-17 production and negative correlation between serum retinol and IL-10 and TGF-β production in T CD4+CD25highFoxp3+ cells after SLA stimulus. Our results show a potential dual role of vitamin A in the immune system: improvement of regulatory profile during homeostasis and down modulation of IL-10 in Treg cells and monocytes during symptomatic VL. Therefore, the use of vitamin A concomitant to VL therapy might improve recovery from disease status in Leishmania infantum infection

A infecção por Leishmania infantum chagasi altera o metabolismo lipídico do hospedeiro

American visceral leishmaniasis (AVL), caused by Leishmania infantum chagasi (L.i.chagasi), stands as a public health problem in Brazil, with human and canine cases related in all states..Lipid metabolism can be modified in several status of infection. For example, experimental studies show that the cholesterol is necessary to internalization and replication of L.i.chagasi in macrophages through caveolar domains. Patients with AVL present low levels of cholesterol and a visible triglycerides increase. This work aimed to evaluate the lipid metabolism in several post-infection status by L.i.chagasi, including individuals with symptomatic infection (AVL), and asymptomatic. The levels of cholesterol, triglycerides, HDL and reactive C protein, were measured. Individuals with AVL were compared with individuals with assymptomatic infection and presented low levels of total cholesterol (128 ± 6.180 mg/dL vs. 158 ±5.733 mg/dL, p=0.0001), HDL (29 ± 1.746 mg/dL vs. 37 ± 1.647 mg/dL, p=0.0001), increased levels of triglycerides (149.5 mg/dL ± 12.72 vs. 78.00 ± 10.43 mg/dL, p=0.0095) and higher levels of reactive C protein (1.750± 0.4939 mg/dL vs. 0.40 ± 0.1707 mg/dL; p=0.0001). The expression of genes related to lipid metabolism, such as LXR-a, LXR-b, PPAR-a, PPAR-d, PPAR-g and APOE was evaluated by real time PCR. A reduction in the expression of those genes was found in the group of AVL patients corroborating the serum levels of the metabolites earlier quantified. Our findings suggest a modulation of metabolism of lipids, in the chronic phase of AVL, this could facilitate the survival of leishmania, due to the known reduction on the ability of macrophages in presenting antigens efficiently to the T cells due to the reduction in the cholesterol available, it results in a subversion of the host immunity.

Anticorpos anti-trypanosoma cruzi como preditivos de infecção por leishmania infantum

Leishmania infantum and Trypanosoma cruzi are trypanosomatids of medical importance and are, respectively, the etiologic agents of visceral leishmaniasis (VL) and Chagas disease (CD) in Brazil. People infected with L. infantum or T. cruzi may develop asymptomatically, enabling the transmission of pathogens through blood transfusion and / or organs. The assessment of the infection by T. cruzi is included among the tests performed for screening blood donors in Brazil, however, there is no availability of tests for Leishmania. Serological tests for T. cruzi are very sensitive, but not specific, and may have cross-reactions with other microorganisms. Thus, the aim of this study was to determine the prevalence of Leishmania infection in blood donors and assess whether the serological test for T. cruzi detect L. infantum. Among the 300 blood samples from donors, discarded in 2011, 61 were T. cruzi positive, 203 were from donors with other infections and 36 were from handbags with low blood volume, but without infection. We also assessed 144 samples from donors without infections and able to donate blood, totaling 444 subjects. DNA was extracted from blood samples of all to perform quantitative PCR (qPCR) to detect Leishmania DNA. The buffy coat obtained from all samples was grown in Schneider medium supplemented and NNN. All samples were evaluated for the presence of anti-Leishmania antibody. The serological results indicate a percentage of 22% of Leishmania infection in blood samples obtained from discarded bags. A total of 60% of samples positive in ELISA for T. cruzi were negative by IFI, used as confirmatory test, ie 60% false positive for Chagas. Among these samples false positive for Chagas, 72% were positive by ELISA for Leishmania characterizing the occurrence of cross reaction between serologic assays. Of the 300 cultures performed, 18 grew parasites that were typed by qPCR and specific isoenzymes, found the species Leishmania infantum crops. Among the 18 cultures, 4 were purged from scholarships for low volume and all negative serology blood bank, thus demonstrating that there is a real risk of Leishmania transmission via transfusion. It is concluded that in an area endemic for leishmaniasis in Brazil, serological diagnosis performed to detect infection by T. cruzi among blood donors can identify infection by L. infantum and although cause false positive for Chagas, this cross-reactivity reduces the risk of Leishmania infection via blood transfusion, since tests are not applied specific detection of the parasite. In this way, there remains the need to discuss the implementation of a specific serological screening test for Leishmania in endemic countries such as Brazil

Estudio de la capacidad inmunoprofiláctica de proteínas recombinantes de Leishmania infantum en el modelo murino

En esta Tesis Doctoral, cuatro proteínas que están sobre-expresadas en las fases infectivas del ciclo celular de L. infantum (STPKA, STPK, PP2B and PABP3) fueron purificadas para inmunizar ratones BALB/c, con el interés de estudiar su posible capacidad protectora contra la VL. Tres de ellas indujeron una fuerte respuesta humoral en los ratones, rLiSTPKA, rLiSTPK y rLiPABP3, a diferencia de la proteína rLiPP2B. Además, la rLiPABP3 fue la única que indujo una disminución significativa de la carga parasitara tanto en bazo como en hígado 2 meses después de la infección experimental con el parásito. Con el objetivo de determinar los efectos que la inmunización con rLiPABP3 tenía sobre el sistema inmune murino, se determinó la expresión génica de 106 genes relacionados con el sistema inmune en ratones control, inmunizados e infectados. Los niveles de expresión génica fueron comparados con los obtenidos para los grupos control. Los resultados mostraron que durante todo el experimento la proteína rLiPABP3 promueve la inhibición de la respuesta inmune inflamatoria en el bazo de los ratones infectados. Además, no se observa una respuesta adaptiva claramente polarizada hacia un tipo concreto. Un mes después de la infeccion, en los animales previamente inmunizados se observa la sobre-expresion de Il2rb lo que nos hace pensar que rLiPABP3 podría estimular la presencia de linfocitos T memoria. En fases más avanzadas de la infección, a pesar de que no observamos una clara diferenciación de una población concreta de linfocitos T efectores, sí observamos la infra-expresión del gen codificante del TNF-alfa, lo que unido a la sobre-expresión del gen Cxcr4 y la ausencia de cambios de la expresión del gen Ccr7 nos hace pensar que la inmunización no sólo mantiene la micro-arquitectura del bazo, sino que también promueve la correcta migración de las DCs desde la MZ hasta el PALS.

Evidence of Leishmania infantum infection in rabbits (Oryctolagus cuniculus) in a natural area in Madrid, Spain

Leishmaniasis is one of most important neglected zoonosis and remains endemic in at least 88 developing countries in the world. In addition, anthropogenic environmental changes in urban areas are leading to its emergency world-wide. Zoonotic leishmaniasis control might only be achieved by an integrated approach targeting both the human host and the animal reservoirs, which in certain sylvatic cycles are yet to be identified. Recently, hares have been pointed out as competent reservoirs of Leishmania infantum in Spain, but the role of other lagomorphs has not been clarified. Here, 69 rabbits (Oryctolagus cuniculus) from a natural area in Madrid in which a high density was present were analyzed using indirect (immunofluorescence antibody test, IFAT) and direct (PCR, culture) techniques. Fifty-seven (82.6%) of the animals were positive to at least one technique, with IFAT yielding the highest proportion of positive samples. L. infantum was isolated in 13% animals demonstrating the occurrence of infection in this setting. Our results suggest that rabbits could play a role of competent reservoir of L. infantum and demonstrate that the prevalence of infection is high in the analyzed area.

Susceptibility of peritoneal macrophage from different species of neotropical primates to Ex vivo Leishmania (L.) infantum chagasi-infection

Este estudo examinou a susceptibilidade do macrófago peritoneal (PM) dos primatas neotropicais: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus e Callimico goeldii para a infecção ex vivo por Leishmania (L.) infantum chagasi, o agente etiológico da leishmaniose visceral americana (LVA), como método de triagem para avaliar o potencial desses primatas como modelo de estudo da LVA. A susceptibilidade do PM para a infecção foi investigada através do índice de infecção do PM (PMI) a intervalos de 24, 72 horas e, ainda, pela média dessas taxas (FPMI), assim como, pelas respostas do TNF-α, IL-2 (ELISA de captura) e óxido nítrico (NO) (método de Griess). Às 24hs da infecção experimental, o PMI do primata A. azarae infulatus (128) foi maior que aqueles de C. penicillata (83), C. goeldii (78), S. sciureus (77) e C. jacchus (55). Às 72hs, houve uma redução significativa do PMI de quatro primatas: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) e C. jacchus (55/12), com exceção de C. goeldii (78/54). O FPMI dos primatas A. azarae infulatus (82.5) e C. goeldii (66) foi maior que do primata C. jacchus (33.5), porém, não foi maior que dos primatas C. penicillata (60.5) e S. sciureus (57.5). A resposta do TNF-α foi mais regular nos quatro primatas que reduziram o PMI no intervalo de 24-72hs: C. jacchus (145/122 pg/µL), C. penicillata (154/130 pg/µL), S. sciureus (164/104 pg/µL) e A. azarae infulatus (154/104 pg/µL), com exceção de C. goeldii (38/83 pg/µL). A resposta de IL-12 foi, principalmente, marcante nos primatas A. azarae infulatus e C. goeldii, os quais apresentaram as maiores taxas do FPMI, e a resposta do NO foi maior no primata C. goeldii, em especial no intervalo de 72hs. Estes achados sugerem, fortemente, que estes primatas neotropicais parecem ter desenvolvido mecanismos resistentes de resposta imune inata capaz de controlar o crescimento intracelular da infecção por L. (L.) i. chagasi no macrófago, o que não encoraja o uso destes primatas como modelo de estudo da LVA.

Susceptibility of Cebus apella monkey (Primates: Cebidae) to experimental Leishmania (L.) infantum chagasi-infection

Na Amazônia Brasileira, o macaco Cebus apella (Primata: Cebidae) tem sido associado com o ciclo enzoótico da Leishmania (V.) shawi, um parasito dermotrópico causador da Leishmaniose Tegumentar Americana (LTA). Ele tem sido também empregado com sucesso como modelo experimental para estudo da leishmaniose tegumentar. Neste trabalho, foi investigada sua susceptibilidade à infecção experimental por Leishmania (L.) infantum chagasi, o agente etiológico da Leishmaniose Visceral Americana (LVA). Foram usados dez espécimes de C. apella oito adultos e dois jovens, quatro machos e seis fêmeas, todos nascidos e criados em cativeiro. Dois protocolos de infecção experimental foram feitos: i) seis macacos foram inoculados por via intradérmica (ID), na base da cauda com 2x106 formas promastigotas em fase estacionária de crescimento; ii) outros quatro macacos foram inoculados com 3x107 formas amastigotas de infecção visceral de hamsteres por duas vias diferentes: a) dois por via intravenosa (IV) e, b) outros dois pela via intraperitoneal (IP). A avaliação da infecção incluiu parâmetros: clínico: exame físico do abdômen, peso e temperatura corporal; b) parasitológico: aspirado de medula óssea por agulha para procura de amastigotas (esfregaço corado por Giemsa) e formas promastigotas (meio de cultura); c) imunológico: Reação de Imunofluorescência Indireta (RIFI) e, resposta de hipersensibilidade tardia (DTH). Nos seis macacos inoculados ID (formas promastigotas) todos os parâmetros de avaliação da infecção foram negativos durante o período de 12 meses. Entre os quatro macacos inoculados com formas amastigotas, dois inoculados IV mostraram parasitos na medula óssea do primeiro ao sexto mês p.i. e em seguida houve a resolução da infecção, no entanto os outros dois inoculados IP foram totalmente negativos. Esses quatro macacos apresentaram resposta específica de anticorpo IgG desde o terceiro mês p.i. (IP: 1/80 e IV: 1/320) até o décimo segundo mês (IP: 1/160 e IV: 1/5120). A conversão DTH ocorreu em apenas um macaco inoculado IV com uma forte reação na pele (30 mm). Considerando esses resultados, nós não recomendamos o uso do macaco C. apella como modelo animal para estudo da LVA.