Infantum Chagasi Infection Research Articles

Leishmania (L.) infantum chagasi isolated from skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis lead to visceral lesion in hamsters.

In Central America, Leishmania (L.) infantum chagasi infection causes visceral leishmaniasis (VL) and non-ulcerated cutaneous leishmaniasis (NUCL). The aim of the present study was to evaluate the course of an experimental infection in hamsters caused by L. (L.) infantum chagasi isolated from patients affected by NUCL compared with a strain isolated from a patient with VL. Stationary phase parasites in culture were inoculated through subcutaneous and intraperitoneal routes in hamsters. Following the post-infection times, a histopathological study, parasite load and cytokine determination in skin from the cutaneous inoculation site and viscera were performed. Animals subcutaneously infected with the different strains did not develop macroscopic lesions at the inoculation site, and the histopathological changes in the dermis were very slight. Regarding the histopathological study of the viscera, we observed the portal mononuclear inflammatory infiltrate, the presence of nodules in the hepatic parenchyma and the proliferation of macrophages in the spleen, which increased over the infection course. Overall, the parasite load in the liver and spleen and in the total IgG titres in the sera of infected hamster showed an increase with the time of infection, regardless of the route of inoculation. Regarding cellular immunity, we did not observe an increase or decrease in pro- and anti-inflammatory cytokines compared to the healthy control, except for IL-10, which was evident in the infected animals. The data showed that strains isolated from NUCL cause visceral lesions in the hamsters regardless of the route of inoculation, and they were similar to parasites isolated from VL humans.

Visceral Leishmaniasis Urbanization in the Brazilian Amazon Is Supported by Significantly Higher Infection Transmission Rates Than in Rural Area.

This was an open cohort prospective study (2016-2018) that analyzed the prevalence and incidence rates of human Leishmania (L.) infantum chagasi-infection and the evolution of their clinical-immunological profiles in distinct urban and rural scenarios of American visceral leishmaniasis (AVL) in Pará State, in the Brazilian Amazon. These infection profiles were based on species-specific DTH/IFAT-IgG assays and clinical evaluation of infected individuals, comprising five profiles: three asymptomatic, Asymptomatic Infection [AI], Subclinical Resistant Infection [SRI], and Indeterminate Initial Infection [III]; and two symptomatic, Subclinical Oligosymptomatic Infection [SOI] and Symptomatic Infection [SI = AVL]. The two distinct scenarios (900 km away) were the urban area of Conceição do Araguaia municipality and the rural area of Bujaru municipality in the southeast and northeast of Pará State. Human populations were chosen based on a simple convenience sampling design (5-10% in each setting), with 1723 individuals (5.3%) of the population (32,464) in the urban area and 1568 individuals (8.9%) of the population (17,596) in the rural one. A serological survey (IFAT-IgG) of canine infection was also performed in both scenarios: 195 dogs in the urban area and 381 in the rural one. Prevalence and incidence rates of human infection were higher in the urban area (20.3% and 13.6/100 person-years [py]) than in the rural setting (14.1% and 6.8/100-py). The AI profile was the most prevalent and incident in both urban (13.4% and 8.1/100-py) and rural (8.3% and 4.2/100-py) scenarios, but with higher rates in the former. An III profile case evolved to SOI profile after four weeks of incubation and another to SI (=AVL) after six. The prevalence of canine infection in an urban setting (39.2%) was also higher (p < 0.05) than that (32%) in the rural zone. AVL urbanization in Pará State, in the Brazilian Amazon, has led to infection rates significantly higher than those in rural sites, requiring more intense control measures.

Evaluation of systemic immunity in atypical cutaneous leishmaniasis caused by Leishmania (L.) infantum chagasi.

In some central-American countries, Leishmania (L.) infantum chagasi infection can cause non-ulcerated or atypical cutaneous leishmaniasis (NUCL) in addition to the classic clinical form, visceral leishmaniasis (VL). Little is known about the host-parasite relationship that can contribute to the determination of one or another clinical form. The present study had the objective to evaluate the humoral and cellular immunity in the sera of individuals affected by NUCL to improve the comprehension of this atypical host-parasite interaction. Based on clinical and laboratory diagnosis, serum of 80 individuals was collected to evaluate the cytokines and immunoglobulins profile of NUCL (n=47), VL patients (n=5), and negative controls (n=28). Cytokines were detected using Cytokine Bead Array (CBA) Human Th1/Th2/Th17kit according to the manufacturer's instructions; class (IgG and IgM), and subclass of (IgG1 and IgG2) immunoglobulins was evaluated by ELISA using specific antigens. The concentration of TNF-α, IFN-γ, IL-2 and IL-4 cytokines in NUCL, VL and control was present below the detection threshold of CBA kit. IL-6, IL-10 and IL-17A cytokines was lower in NUCL compared to LV patients. Regarding to immunoglobulins, NUCL patients produced 4.0 times more IgG than the control, while VL patients produced 6.6 times more; and IgM level was 1.6 times higher in NUCL and 2.6 times in VL patients compared to the control. Concerning the immunoglobulins subclass, only VL patients showed positive reaction for IgG1, and IgG2 did not show positive reaction among the groups. The results showed a weak cellular and humoral systemic immune response in NUCL patients.

New record of preclinical diagnosis of American visceral leishmaniasis in Amazonian Brazil encourages optimizing disease control

The clinical-immunological spectrum of human Leishmania (L.) infantum chagasi-infection in Amazonian Brazil has recently been reviewed based on the combined use of the delayed-type hypersensitivity (DTH) and indirect fluorescence antibody test (IFAT-IgG/IgM), both with homologous L. (L.) infantum chagasi-antigens, and associated with the clinical evaluation of infected individuals. This diagnostic approach has allowed to identify the broadest clinical-immunological spectrum of human L. (L.) infantum chagasi-infection composed by five clinical-immunological profiles of infection: three asymptomatic, 1) Asymptomatic Infection (AI) [DTH+/++++, IFAT−], 2) Subclinical Resistant Infection (SRI) [DTH+/++++, IFAT+/++], and 3) Indeterminate Initial Infection (III) [DTH−, IFAT+/++], and two symptomatic ones, 4) Symptomatic Infection (SI) [=American visceral leishmaniasis - AVL] and, 5) Subclinical Oligosymptomatic Infection (SOI), both with the same immune profile [DTH−, IFAT+++/++++]. Herein, we confirm for the third time the preclinical diagnosis of AVL through IgM-antibody response in an early asymptomatic case of infection (profile III), a 17-year-old boy who evolved to AVL (=profile SI) six weeks after the initial infection diagnosis, confirming that the combined use of DTH and IFAT-(IgG/IgM) assays associated with the clinical evaluation of infected individuals is potentially useful for monitoring human L. (L.) infantum chagasi-infection in endemic areas as well as optimizing AVL control.

Infection by Leishmania (Leishmania) infantum chagasi causes intestinal changes B-1 cells dependent.

Evaluating the histopathological and morphometric changes caused by Leishmania (Leishmania) infantum chagasi infection either in the presence or absence of B-1 cells. Wild-type Balb/c and XID mice were used. Half of XID mice received B-1 cells adoptive transfer (XID+B1). Five animals from each group were infected (Balb/c I, XID I and XID+B1 I), totalizing six groups (n=5). After 45days of infection, the ileum was collected for histological processing and analysis. After infection, the XID animals showed an increase in the thickness of the intestinal layers, in the depth and width of the crypt and in the villi width. However, the Balb/c I group showed a reduction in almost all these parameters, whereas the villi width was increased. The villi height decreased in the infected XID animals; however, it was increased in the XID+B1 I group. Leishmania (L) infantum chagasiinfection caused a decrease in the number of Paneth cells; however, their area was increased. Finally, goblet cells and enterocytes presented different change profiles among groups. This study showed that the parasite infection causes structural and histopathological alterations in the intestine. These changes might be influenced by the absence of B-1 cells.

The F1F3 Recombinant Chimera of Leishmania donovani-Nucleoside Hydrolase (NH36) and Its Epitopes Induce Cross-Protection Against Leishmania (V.) braziliensis Infection in Mice.

Leishmania (V.) braziliensis is the etiological agent of Cutaneous (CL) and Mucocutaneous leishmaniasis (ML) in the New World. CL can be more benign but ML can be severe and disfiguring. Immunity to these diseases include hypersensitivity, an enhanced inflammatory response with strong IFN-γ and TNF-α secretion. Additionally, the production of IL-10 which down modulates the immune response is reduced. The Nucleoside hydrolase (NH36) of Leishmania (L.) donovani is the main antigen of the Leishmune veterinary vaccine and its F3 domain induces a CD4+ T cell-mediated protection against L. (L.) infantum chagasi infection. Prevention of L. (L.) amazonensis infection requires in contrast an additional CD8+ T cell mediated response induced by the F1 domain. Consequently, the F1F3 recombinant chimera, which contains both domains cloned in tandem, optimized the vaccine efficacy against L. (L.) amazonensis mouse infection. We compared the efficacies of NH36, F1, F3, and the FIF3 chimera against L. (V.) braziliensis mouse infection. The F1F3 chimera increased the NH36 specific IgA and response before and after infection and the IgG and IgG3 levels after challenge. It also induced a 49% stronger intradermal response to leishmanial antigen (IDR) than NH36 that was positively correlated to the levels of IFN-γ and TNF-α, IgG, IgG2a, IgG2b, and IgG3 anti-NH36 antibodies. However, stronger Th1 responses with elevated IFN-γ/IL-10 and TNF-α/IL-10 ratios were promoted by the F3 and F1 vaccines and detected in infected controls while the F1F3 chimera promoted the highest IL-10 secretion, which reduced the pathological Th1 response, and characterized the induction of a mixed and/or T-cell regulatory response. We identified the epitopes responsible for these immune responses. The F3 vaccine induced the earliest immunity and after challenge, the F1F3 chimera promoted the highest CD4+ and CD8+ cytokine-secreting T cell responses, and the predominant frequencies of multifunctional CD4+ and CD8+IL-2+TNF-α+IFN-γ+ T cells. Also as observed against L. (L.) amazonensis infection, the F1F3 chimera showed the strongest reduction of the ear lesions sizes induced by L. (V.) braziliensis. Our results confirm the potential use of the F1F3 chimera in a multi-species cross-protective vaccine against L. (V.) braziliensis.

Further evidence associating IgG1, but not IgG2, with susceptibility to canine visceral leishmaniasis caused by Leishmania (L.) infantum chagasi-infection.

We present here a cross-sectional study analyzing the IgG1 and IgG2 immune responses to natural canine Leishmania (L.) infantum chagasi-infection and their relationships with delayed-type hypersensitivity (DTH) in 50 mongrel dogs with previous positive serodiagnoses (IFAT-IgG) (56% with subclinical status [= apparently healthy] and 44% clinically sick), living in endemic areas for visceral leishmaniasis in the Brazilian Amazon. IgG1 and IgG2 responses were measured using commercial polyclonal antibodies in ELISA, while DTH was elicited by intradermal skin test using cultured promastigotes L. (L.) i. chagasi-antigen. Data analyses used Chi-square and Pearson's r coefficient (95% confidence interval). Regarding DTH and the clinical statuses of dogs, it was noted that 100% of the animals showing positive DTH (n = 8) were from the subclinical group, while 100% showing negative DTH were from the clinically sick group; higher IgG2 than IgG1 responses were observed in both clinical groups. However, when this comparison was made between the subclinical and sick groups, higher IgG1 responses were noted in the dogs from the sick rather than the subclinical group, while no differences were noted between the IgG2 responses in the dogs from both clinical groups. Additionally, we found lower IgG1 responses in dogs from the subclinical group showing positive DTH than in the dogs from the subclinical or sick groups with negative DTH; no differences were found between the IgG2 responses of these two clinical groups. These findings suggest that the IgG1, but not the IgG2, response is associated with susceptibility to canine visceral leishmaniasis (CVL).

Reduced Leishmania (L.) infantum chagasi parasitic loads in humans exposed to Lutzomyia longipalpis bites in the Amazon region of Brazil

SUMMARY We performed a cross-sectional survey in humans to evaluate Lutzomyia longipalpis, i.e. sand fly vector, bite exposure association with Leishmania (Leishmania) infantum chagasi infection in Bujarú municipality, Northern Brazil, an endemic area for visceral leishmaniasis. In recruited individuals, which were stratified by sex and age, we measured L. (L.) i. chagasi parasitic loads with quantitative polymerase chain reaction (qPCR), exposure to sand fly bites with an anti-saliva immunoglobulin G enzyme-linked immunosorbent assay and performed immunological diagnostic tests, in order to evaluate the association between exposure to sand fly bites, and infection. The prevalence increased from 11% when using immunological diagnostic tests to 28% when using qPCR, being around that value for all age classes, but children below 5 years (40%) and people over 60 years (15%). The association between PCR-based L. (L.) i. chagasi prevalence and saliva exposure was convex, reflecting the fact that at both high and low saliva exposure the PCR-based L. (L.) i. chagasi prevalence decreases. This scenario indicates that low sand fly exposure is likely associated with low parasite transmission, while high anti-saliva prevalence, i.e. a large sand fly bite exposure could be associated with anti-Leishmania protective immune mechanisms driven by vector saliva and/or increased parasite exposure.

Serum Cytokine Responses over the Entire Clinical-Immunological Spectrum of Human Leishmania (L.) infantum chagasi Infection.

The clinical-immunological spectrum of human Leishmania (L.) infantum chagasi infection in Amazonian Brazil was recently reviewed based on clinical, DTH, and IFAT (IgG) evaluations that identified five profiles: three asymptomatic (asymptomatic infection, AI; subclinical resistant infection, SRI; and indeterminate initial infection, III) and two symptomatic (symptomatic infection, SI; American visceral leishmaniasis, AVL; and subclinical oligosymptomatic infection, SOI). TNF-α, IL-4, IL-6, and IL-10 serum cytokines were analyzed using multiplexed Cytometric Bead Array in 161 samples from endemic areas in the Brazilian Amazon: SI [AVL] (21 cases), III (49), SRI (19), SOI (12), AI (36), and a control group [CG] (24). The highest IL-6 serum levels were observed in the SI profile (AVL); higher IL-10 serum levels were observed in SI than in SOI or CG and in AI and III than in SOI; higher TNF-α serum levels were seen in SI than in CG. Positive correlations were found between IL-6 and IL-10 serum levels in the SI and III profiles and between IL-6 and TNF-α and between IL-4 and TNF-α in the III profile. These results provide strong evidence for associating IL-6 and IL-10 with the immunopathogenesis of AVL and help clarify the role of these cytokines in the infection spectrum.

Immucillins ImmA and ImmH Are Effective and Non-toxic in the Treatment of Experimental Visceral Leishmaniasis.

BackgroundImmucillins ImmA (IA), ImmH (IH) and SerMe-ImmH (SMIH) are synthetic deazapurine nucleoside analogues that inhibit Leishmania (L.) infantum chagasi and Leishmania (L.) amazonensis multiplication in vitro without macrophage toxicity. Immucillins are compared to the Glucantime standard drug in the chemotherapy of Leishmania (L.) infantum chagasi infection in mice and hamsters. These agents are tested for toxicity and immune system response.Methodology/Principal FindingsBALB/c mice were infected with 107 amastigotes, treated with IA, IH, SMIH or Glucantime (2.5mg/kg/day) and monitored for clinical variables, parasite load, antibody levels and splenocyte IFN-γ, TNF-α, and IL-10 expression. Cytokines and CD4+, CD8+ and CD19+ lymphocyte frequencies were assessed in uninfected controls and in response to immucillins. Urea, creatinine, GOT and GPT levels were monitored in sera. Anti-Leishmania-specific IgG1 antibodies (anti-NH36) increased in untreated animals. IgG2a response, high levels of IFN-γ, TNF-α and lower levels of IL-10 were detected in mice treated with the immucillins and Glucantime. Immucillins permitted normal weight gain, prevented hepato-splenomegaly and cleared the parasite infection (85–89%) without renal and hepatic toxicity. Immucillins promoted 35% lower secretion of IFN-γ in uninfected controls than in infected mice. IA and IH increased the CD4+ T and CD19+ B cell frequencies. SMIH increased only the proportion of CD-19 B cells. IA and IH also cured infected hamsters with lower toxicity than Glucantime.Conclusions/SignificanceImmucillins IA, IH and SMIH were effective in treating leishmaniasis in mice. In hamsters, IA and IH were also effective. The highest therapeutic efficacy was obtained with IA, possibly due to its induction of a TH1 immune response. Low immucillin doses were required and showed no toxicity. Our results disclose the potential use of IA and IH in the therapy of visceral leishmaniasis.

Topical resiquimod protects against visceral infection with Leishmania infantum chagasi in mice.

New prevention and treatment strategies are needed for visceral leishmaniasis, particularly ones that can be deployed simply and inexpensively in areas where leishmaniasis is endemic. Synthetic molecules that activate Toll-like receptor 7 and 8 (TLR7/8) pathways have previously been demonstrated to enhance protection against cutaneous leishmaniasis. We initially sought to determine whether the TLR7/8-activating molecule resiquimod might serve as an effective vaccine adjuvant targeting visceral leishmaniasis caused by infection with Leishmania infantum chagasi. Resiquimod was topically applied to the skin of mice either prior to or after systemic infection with L. infantum chagasi, and parasite burdens were assessed. Surprisingly, topical resiquimod application alone, in the absence of vaccination, conferred robust resistance to mice against future intravenous challenge with virulent L. infantum chagasi. This protection against L. infantum chagasi infection persisted as long as 8 weeks after the final topical resiquimod treatment. In addition, in mice with existing infections, therapeutic treatment with topical resiquimod led to significantly lower visceral parasite loads. Resiquimod increased trafficking of leukocytes, including B cells, CD4(+) and CD8(+) T cells, dendritic cells, macrophages, and granulocytes, in livers and spleens, which are the key target organs of visceralizing infection. We conclude that topical resiquimod leads to systemic immune modulation and confers durable protection against visceralizing L. infantum chagasi infection, in both prophylactic and therapeutic settings. These studies support continued studies of TLR-modulating agents to determine mechanisms of protection and also provide a rationale for translational development of a critically needed, novel class of topical, preventative, and therapeutic agents for these lethal infections.

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.

Polymerase chain reaction and real-time PCR for diagnosing of Leishmania infantum chagasi in dogs

The importance of dogs as a reservoir for Leishmania infantumchagasi in urban environments has stimulated numerous studies assessing diagnostic techniques. When performed properly, such procedures are an important step in preventing leishmaniasis in humans. Molecular methods have become prominent for this purpose. The aim of the present study was to determine the performance of the polymerase chain reaction (PCR) and real-time PCR (qPCR) for diagnosing of canine visceral leishmaniasis (CVL) using different biological samples. For this, 35 dogs from an area endemic for CVL were used. Bone marrow aspirate and lymph node and spleen fragments from these dogs were used for the molecular diagnosis. In the present study, qPCR was able to detect a greater number of positive animals than seen with PCR. Among the different biological samples used, there was no significant difference in L. infantumchagasi DNA detection between PCR and qPCR. However, considering that lymph nodes are easy to acquire, these can be considered to be the best samples for making molecular diagnoses of L. infantum chagasi infection.

A cross-sectional study on canine Leishmania (L.) infantum chagasi infection in Amazonian Brazil ratifies a higher prevalence of specific IgG-antibody response than delayed-type hypersensitivity in symptomatic and asymptomatic dogs

This was a cross-sectional study which analyzed the prevalence and the clinical and immunological spectrum of canine Leishmania (L.) infantum chagasi infection in a cohort of 320 mongrel dogs living in an endemic area of American visceral leishmaniasis in the Amazonian Brazil by using, mainly, the indirect fluorescence antibody test (IFAT-IgG) and the delayed-type hypersensitivity (DTH), and the parasite research by the popliteal lymph node aspiration. The IFAT and DTH reactivity recognized three different immune response profiles: (1) IFAT((+))/DTH((-)) (107 dogs), (2) IFAT((-))/DTH((+)) (18 dogs), and (3) IFAT((+))/DTH((+)) (13 dogs), providing an overall prevalence of infection of 43% (138/320). Thus, the specific prevalence of IFAT( (+) )/DTH( (-) ) 33.4% (107/320) was higher than those of IFAT( (-) )/DTH( (+) ) 5.6% (18/320) and IFAT( (+) )/DTH( (+) ) 4.0% (13/320). Moreover, the frequency of these profiles among 138 infected dogs showed that the IFAT( (+) )/DTH( (-) ) rate of 77.5% (107/138) was also higher than those of 13.0% (18/138) of IFAT( (-) )/DTH( (+) ) and 9.5% (13/138) of IFAT( (+) )/DTH( (+) ) rates. The frequency of asymptomatic dogs (76%-105) was higher than those of symptomatic (16.6%-23) and oligosymptomatic ones (7.4%-10). A total of 16 (11.6%) L. (L.) i. chagasi isolates were obtained from infected dogs, all from the IFAT( (+) ) /DTH( (-) ) profile: 41% (9/22) from symptomatic, 33.3% (3/9) from oligosymptomatic, and 5.2% (4/76) from asymptomatic dogs. These findings strongly suggested that despite the higher frequency of asymptomatic dogs (76%-105), the majority (72.4%-76) was characterized by the IFAT( (+) ) /DTH( (-) ) profile with a doubtful immunogenetic resistance against infection.

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

This study examined the susceptibility of peritoneal macrophage (PM) from the Neotropical primates: Callithrix jacchus, Callithrix penicillata, Saimiri sciureus, Aotus azarae infulatus and Callimico goeldii to ex vivo Leishmania (L.) infantum chagasi-infection, the etiological agent of American visceral leishmaniasis (AVL), as a screening assay for evaluating the potential of these non-human primates as experimental models for studying AVL. The PM-susceptibility to infection was accessed by the PM-infection index (PMI) at 24, 72 h and by the mean of these rates (FPMI), as well as by the TNF-α, IL-12 (Capture ELISA) and Nitric oxide (NO) responses (Griess method). At 24h, the PMI of A. azarae infulatus (128) was higher than those of C. penicillata (83), C. goeldii (78), S. sciureus (77) and C. jacchus (55). At 72h, there was a significant PMI decrease in four monkeys: A. azarae infulatus (128/37), C. penicillata (83/38), S. sciureus (77/38) and C. jacchus (55/12), with exception of C. goeldii (78/54). The FPMI of A. azarae infulatus (82.5) and C. goeldii (66) were higher than C. jacchus (33.5), but not higher than those of C. penicillata (60.5) and S. sciureus (57.5). The TNF-a response was more regular in those four primates which decreased their PMI at 24/72 h: C. jacchus (145/122 pg/mL), C. penicillata (154/130 pg/mL), S. sciureus (164/104 pg/mL) and A. azarae infulatus (154/104 pg/mL), with exception of C. goeldii (38/83 pg/mL). The IL-12 response was mainly prominent in A. infulatus and C. goeldii which presented the highest FPMI and, the NO response was higher in C. goeldii, mainly at 72 h. These findings strongly suggest that these New World primates have developed a resistant innate immune response mechanism capable of controlling the macrophage intracellular growth of L. (L.) i. chagasi-infection, which do not encourage their use as animal model for studying AVL.

Killed but Metabolically Active Leishmania infantum as a Novel Whole-Cell Vaccine for Visceral Leishmaniasis

There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.

The transmission of Leishmania infantum chagasi by the bite of the Lutzomyia longipalpis to two different vertebrates

BackgroundSandflies are vectors of Leishmania, the causative agent of leishmaniasis in mammalian hosts, including humans. The protozoan parasite is transmitted by the sandfly bite during salivation that occurs at the moment of blood feeding. The components of vector saliva include anticlotting and vasodilatory factors that facilitate blood flow and immunomodulatory factors that inhibit wound healing and quell the immune response. Not surprisingly, these factors also play important roles in the establishment of Leishmania infection. To date, the majority of knowledge that has been generated regarding the process of Leishmania infection, including L. infantum chagasi transmission has been gathered by using intradermal or subcutaneous inoculation of purified parasites.FindingsThis study presents the establishment of a transmission model of Leishmania infantum chagasi by the bite of Lutzomyia longipalpis, the vector of American visceral leishmaniasis. The parasites were successfully transmitted by infected sandfly bites to mice and hamsters, indicating that both animals are good experimental models. The L. infantum chagasi dose that was transmitted in each single bite ranged from 10 to 10, 000 parasites, but 75% of the sandflies transmitted less than 300 parasites.ConclusionsThe strategy for initiating infection by sandfly bite of experimental animals facilitates future investigations into the complex and dynamic mechanisms of visceral leishmaniasis. It is important to elucidate the transmission mechanism of vector bites. This model represents a useful tool to study L. infantum chagasi infection transmitted by the vector.

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

In Amazonian Brazil, the Cebus apella monkey (Primates: Cebidae) has been associated with the enzootic cycle of Leishmania (V.) shawi, a dermotropic parasite causing American cutaneous leishmaniasis (ACL). It has also been successfully used as animal model for studying cutaneous leishmaniasis. In this work, there has been investigated its susceptibility to experimental Leishmania (L.) infantum chagasi-infection, the etiologic agent of American visceral leishmaniasis (AVL). There were used ten C. apella specimens, eight adult and two young, four males and six females, all born and raised in captivity. Two experimental infection protocols were performed: i) six monkeys were inoculated, intra-dermal via (ID), into the base of the tail with 2 x 10(6) promastigotes forms from the stationary phase culture medium; ii) other four monkeys were inoculated with 3 x 10(7) amastigotes forms from the visceral infection of infected hamsters by two different via: a) two by intravenous via (IV) and, b) other two by intra-peritoneal via (IP). The parameters of infection evaluation included: a) clinical: physical exam of abdomen, weigh and body temperature; b) parasitological: needle aspiration of the bone-marrow for searching of amastigotes (Giemsa-stained smears) and promastigotes forms (culture medium); c) immunological: Indirect fluorescence antibody test (IFAT) and, Delayed-type hypersensitivity (DTH). In the six monkeys ID inoculated (promastigotes forms) all parameters of infection evaluation were negative during the 12 months period of follow-up. Among the four monkeys inoculated with amastigotes forms, two IV inoculated showed the parasite in the bone-marrow from the first toward to the sixth month p.i. and following that they cleared the infection, whereas the other two IP inoculated were totally negative. These four monkeys showed specific IgG-antibody response since the third month p.i. (IP: 1/80 and IV: 1/320 IgG) toward to the 12th month (IP: 1/160 and IV: 1/5120). The DTH-conversion occurred in only one IV inoculated monkey with a strong (30 mm) skin reaction. Considering these results, we do not encourage the use of C. apella monkey as animal model for studying the AVL.

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.

Further evidences on a new diagnostic approach for monitoring human Leishmania (L.) infantum chagasi infection in Amazonian Brazil

This was a prospective study carried out during a period over 2 years (May/2006-September/2008) with a cohort of 1,099 individuals of both genders, aged 1 year old and older, from an endemic area of American visceral leishmaniasis (AVL) in Pará state, Brazil. The object was to analyze the prevalence and incidence of human Leishmania (L.) infantum chagasi infection as well as the dynamics evolution of its clinical-immunological profiles prior identified: (1) asymptomatic infection (AI); (2) symptomatic infection (SI = AVL); (3) sub-clinical oligosymptomatic infection (SOI); (4) sub-clinical resistant infection (SRI) and; (5) indeterminate initial infection (III). The infection diagnosis was performed by using both the indirect fluorescent antibody test and leishmanin skin test with amastigotes and promastigotes antigens of L. (L.) i. chagasi, respectively. A total of 187 cases of infection were recorded in the prevalence (17%), 117 in the final incidence (6.9%), and 304 in the accumulated prevalence (26.7%), which provided the following distribution into the clinical-immunological profiles: AI, 51.6%; III, 22.4%; SRI, 20.1%; SOI, 4.3%; and SI (=AVL), 1.6%. The major finding regarding the dynamics evolution of infection was concerned to III profile, from which the cases of infection evolved to either the resistant profiles, SRI (21 cases, 30.8%) and AI (30 cases, 44.1%), or the susceptible SI (=AVL; 1 case, 1.5%); the latter 16 cases remained as III till the end of the study. These results provided the conclusion that this diagnostic approach may be useful for monitoring human L. (L.) i. chagasi infection in endemic area and preventing the high morbidity of severe AVL cases.