RESUMO
Background: Domestic cats have been implicated as accidental hosts of Leishmania sp. However, in recent years, the recurrent description of new cases in endemic and nonendemic areas draw attention to the potential epidemiological role of cats as reservoir hosts. Although dogs are considered urban reservoirs, cats could act as a secondary natural reservoirs in these areas. Thus, feline leishmaniasis has become an emerging disease in several countries worldwide. Case presentation: This study aimed to describe the first case of feline leishmaniasis in a stray animal that presented lesions compatible with the disease in Belém, Pará, Brazil, an important urban area in eastern Amazon. Serological tests for Leishmania infantum (ELISA and IFA) were nonreactive, whereas histopathological examination indicated infectious dermatitis caused by Leishmania spp. or Toxoplasma gondii. Cytopathological study of lesion aspirate confirmed the presence of Leishmania sp. amastigotes within macrophages. Finally, molecular analyses revealed that the feline infection was caused by Leishmania (Leishmania) infantum chagasi. Conclusion: To the best of the authors' knowledge, this study reports the first case of natural infection by Leishmania (Leishmania) infantum chagasi in a feline from eastern Amazon. These findings suggest domestic cats as potential secondary reservoir hosts of Leishmania spp. in Belém, which reinforces the importance of further epidemiological investigation of feline leishmaniasis, especially in urban areas with human cases.
RESUMO
In Central America, infection by Leishmania (Leishmania) infantum chagasi causes visceral leishmaniasis and non-ulcerated cutaneous leishmaniasis (NUCL). This work aimed to evaluate the participation of subpopulations of antigen-presenting cells in skin lesions of patients affected by NUCL through double-staining immunohistochemistry using cellular and intracellular markers. Twenty-three skin biopsies from patients affected by NUCL were used. Histological sections stained by HE were used for histopathological study. Immunohistochemical studies were performed using primary antibodies against Langerhans cells, dermal dendritic cells, T lymphocytes, and the cytokines IL-12, IFN-γ, TNF-α, iNOS, and IL-10. The histopathological lesions were characterized by an inflammatory infiltrate, predominantly lymphohistiocytic, of variable intensity, with a diffuse arrangement associated with epithelioid granulomas and discreet parasitism. Double-staining immunohistochemistry showed higher participation of dendritic cells producing the proinflammatory cytokine IL-12 in relation to the other evaluated cytokines. Activation of the cellular immune response was marked by a higher density of CD8 Tc1-lymphocytes followed by CD4 Th1-lymphocytes producing mainly IFN-γ. The data obtained in the present study suggest that antigen-presenting cells play an important role in the in situ immune response through the production of proinflammatory cytokines, directing the cellular immune response preferentially to the Th1 and Tc1 types in NUCL caused by L. (L.) infantum chagasi.
Assuntos
Leishmania infantum , Leishmaniose Cutânea , Leishmaniose Visceral , Humanos , Citocinas , Células Apresentadoras de Antígenos , Interleucina-12RESUMO
Background: Domestic cats have been implicated as accidental hosts of Leishmania sp. However, in recent years, the recurrent description of new cases in endemic and nonendemic areas draw attention to the potential epidemiological role of cats as reservoir hosts. Although dogs are considered urban reservoirs, cats could act as a secondary natural reservoirs in these areas. Thus, feline leishmaniasis has become an emerging disease in several countries worldwide. Case presentation: This study aimed to describe the first case of feline leishmaniasis in a stray animal that presented lesions compatible with the disease in Belém, Pará, Brazil, an important urban area in eastern Amazon. Serological tests for Leishmania infantum (ELISA and IFA) were nonreactive, whereas histopathological examination indicated infectious dermatitis caused by Leishmania spp. or Toxoplasma gondii. Cytopathological study of lesion aspirate confirmed the presence of Leishmania sp. amastigotes within macrophages. Finally, molecular analyses revealed that the feline infection was caused by Leishmania (Leishmania) infantum chagasi. Conclusion: To the best of the authors' knowledge, this study reports the first case of natural infection by Leishmania (Leishmania) infantum chagasi in a feline from eastern Amazon. These findings suggest domestic cats as potential secondary reservoir hosts of Leishmania spp. in Belém, which reinforces the importance of further epidemiological investigation of feline leishmaniasis, especially in urban areas with human cases.(AU)
Assuntos
Animais , Gatos/microbiologia , Leishmania infantum/genética , Leishmania/genética , Brasil , Área Urbana , Técnicas de Diagnóstico Molecular/veterináriaRESUMO
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/Th17 kit 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.
Assuntos
Leishmania infantum , Leishmania , Leishmaniose Cutânea , Leishmaniose Visceral , Humanos , Imunidade Celular , Imunoglobulina G , Leishmaniose Visceral/diagnósticoRESUMO
Skin lesions in nonulcerated cutaneous leishmaniasis (NUCL) caused by Leishmania (L.) infantum chagasi are characterized by a mononuclear inflammatory infiltrate in the dermis, which is composed mainly of lymphocytes, followed by macrophages, few plasma cells and epithelioid granulomas with mild tissue parasitism. Previous studies have shown that the main population of lymphocytes present in the dermal infiltrate is CD8+ T cells, followed by CD4+ T cells, which are correlated with IFN-γ+ cells. To improve the knowledge of cellular immune responses in NUCL, skin biopsies were submitted to immunohistochemistry using anti-ROR-γt, anti-IL-17, anti-IL-6, anti-TGF-ß, and anti-IL-23 antibodies to characterize the involvement of Th17 cells in the skin lesions of patients affected by NUCL. ROR-γt+ , IL-17+ , IL-6+ , TGF-ß+ and IL-23+ cells were observed in the dermal inflammatory infiltrate of NUCL skin lesions. A positive correlation between CD4+ T-lymphocytes and ROR-γt+ and IL-17+ cells suggests that some of the CD4+ T-lymphocytes in NUCL could be Th17 lymphocytes. Moreover, a positive correlation between ROR-γt+ cells and TGF-ß+ , IL-6+ , IL-17+ and IL-23+ cells could indicate the role of these cytokines in the differentiation and maintenance of Th17 lymphocytes. Our findings improve knowledge of the pathogenesis of this rare and atypical clinical form of leishmaniasis.
Assuntos
Imunidade Celular , Leishmania infantum/imunologia , Leishmaniose Cutânea/imunologia , Células Th17/imunologia , Adolescente , Adulto , Idoso , Animais , América Central , Criança , Citocinas/imunologia , Feminino , Humanos , Imuno-Histoquímica , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Macrófagos/imunologia , Masculino , Pessoa de Meia-Idade , Pele/imunologia , Pele/parasitologia , Pele/patologia , Adulto JovemRESUMO
Leishmania infantum chagasi is the primary etiological agent of visceral leishmaniasis in Latin America, a lethal disease that afflicts hundreds of thousands of people worldwide every year. Previous studies have shown that the parasite releases microvesicles known as exosomes, which prolong and exacerbate infection in the vertebrate vector. However, little is known of their role in the insect vector, the sand fly Lutzomyia longipalpis. Exosomes were isolated from cultured L. i. chagasi in logarithmic (procyclic) (LOG) and stationary phase (metacyclic-like) (STAT) growth stages, which are the parasite stages found in the vector, and submitted to proteomic analysis. Our studies showed that exosomes from LOG and STAT L. i. chagasi display discrete protein profiles. The presence of approximately 50 known virulence factors was detected, including molecules for immunomodulation and evasion (GP63, EF1α, Oligopeptidase), increased pathogenicity (Casein kinase, KMP-11, Cysteine Peptidase and BiP) and parasite protection (Peroxidoxin). Additionally, the majority of ontological terms were associated with both exosome phases, and no substantial ontological enrichment was observed associated with any of the two exosomal stages. We demonstrated that LOG exosomes show a marked increase in protein number and abundance, including many virulence factors, compared to STAT L. i. chagasi exosomes. SIGNIFICANCE: The knowledge of the role of Leishmania exosomes on leishmaniasis opened up a new world of potential and complexity regarding our understanding of the disease. In Brazil the majority of visceral leishmaniasis cases are caused by the parasite Leishmania infantum chagasi and transmitted by the vector Lutzomyia longipalpis. While Leishmania exosomes were found to play an active role in the mammalian host, little is understood about their effects on the sand fly, or how they might impact on the insect infection by the parasite. For this reason, we isolated exosomes from two developmental stages of L. i. chagasi that occur within the insect with a view to identifying and describing the alterations they undergo. We have identified many hundreds of proteins within both exosome phases and have developed a structure by which to examine potential candidates. Our findings regarding the composition of the exosome proteome raise many questions regarding their function and provide compelling evidence that exosomes play an active role in the parasite's development within the sand fly.
Assuntos
Exossomos , Leishmania infantum , Leishmaniose Visceral , Psychodidae , Animais , Brasil , ProteômicaRESUMO
Visceral leishmaniasis (VL) is epidemic in Brazil with an increasing incidence of human cases and canine reservoirs, with host hypergammaglobulinemia. Conventional enzyme-linked immunosorbent assay (cELISA) based on several parasitic antigens is the main method for diagnosis and indication of treatment. Dissociative ELISA (dELISA) uses acidic treatment to free immunoglobulin G (IgG) from immune complexes, and its use revealed a significant positive fraction of suspected cases with negative serology. Looking for small molecules or haptens that block IgG antibodies, we purified by molecular exclusion chromatography, 1000-3000 MW molecules from promastigote soluble extract, mostly oligosaccharides comprising 6-13 sugar residues using MALDI-TOF analysis. Glycan-BSA complex (GBC) was constructed by conjugating promastigote glycans to BSA molecules, allowing their use in the solid support in cELISA or dELISA. Sera from experimentally infected hamsters showed higher levels of blocked monomeric IgG during infection, mostly against GBC, which was also present in lower concentrations in the promastigote soluble extract dELISA. Those data show that most of the specific monomeric IgG in serum are blocked by haptens composed by glycans produced by the parasite, better detected in the high dilution of sera in the dELISA assays. dELISA is a useful technique for detecting blocked monomeric antibodies that could have difficult clearance from blood, which could result in hypergammaglobulinemia.
Assuntos
Complexo Antígeno-Anticorpo/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Imunoglobulina G/sangue , Leishmaniose Visceral/imunologia , Polissacarídeos/sangue , Animais , Brasil/epidemiologia , Cricetinae , Modelos Animais de Doenças , Epidemias , Humanos , Concentração de Íons de Hidrogênio , Hipergamaglobulinemia , Leishmaniose Visceral/epidemiologia , Polissacarídeos/metabolismo , Soroalbumina Bovina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Physical contact between dendritic cells (DCs) and T cell lymphocytes is necessary to trigger the immune cell response. CCL19 and CCL21 chemokines bind to the CCR7 receptor of mature DCs, and of T cells and regulate DCs migration to the white pulp (wp) of the spleen, where they encounter lymphocytes. In visceral leishmaniasis (VL), cellular immunosuppression is mediated by impaired DC migration due to the decreased chemokine secretion by endothelium and to the reduced DCs CCR7 expression. The Leishmania (L.) donovani nucleoside hydrolase NH36 and its C-terminal domain, the F3 peptide are prominent antigens in the generation of preventive immunity to VL. We assessed whether these vaccines could prevent the migrating defect of DCs by restoring the expression of CCR7 receptors. C57Bl6 mice were vaccinated with NH36 and F3 and challenged with L. (L.) infantum chagasi. The F3 vaccine induced a 100% of survival and a long-lasting immune protection with an earlier CD4+Th1 response, with secretion of higher IFN-γ and TNF-α/IL-10 ratios, and higher frequencies of CD4+ T cells secreting IL-2+, TNF-α+, or IFN-γ+, or a combination of two or the three cytokines (IL-2+TNF-α+IFN-γ+). The CD8+ T cell response was promoted earlier by the NH36-vaccine, and later by the F3-vaccine. Maximal number of F3-primed DCs migrated in vitro in response to CCL19 and showed a high expression of CCR7 receptors (26.06%). Anti-CCR7 antibody treatment inhibited DCs migration in vitro (90%) and increased parasite load in vivo. When transferred into 28-day-infected mice, only 8% of DCs from infected, 59% of DCs from NH36-vaccinated, and 84% of DCs from F3-vaccinated mice migrated to the wp. Consequently, immunotherapy of infected mice with F3-primed DCs only, promoted increases in corporal weight and reductions of spleen and liver parasite loads and relative weights. Our findings indicate that vaccination with F3-vaccine preserves the maturation, migration properties and CCR7 expression of DCs, which are essential processes for the generation of cell-mediated immunity. The F3 vaccine is more potent in reversing the migration defect that occurs in VL and, therefore, more efficient in immunotherapy of VL.
Assuntos
Antígenos de Protozoários/imunologia , Células Dendríticas/imunologia , Imunoterapia , Vacinas contra Leishmaniose/imunologia , Leishmaniose Visceral/terapia , N-Glicosil Hidrolases/imunologia , Receptores CCR7/genética , Animais , Movimento Celular , Citocinas/imunologia , Epitopos de Linfócito T/imunologia , Feminino , Imunidade Celular , Leishmania donovani , Leishmaniose Visceral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Receptores CCR7/imunologiaRESUMO
Although serological assays have been widely used for the diagnosis of canine visceral leishmaniasis (CVL), they present different performances depending on the clinical profile of the dogs. This study evaluated the accuracy of serological tests, immunochromatographic (Dual Path Platform: DPP®) and enzyme-linked immunosorbent (ELISA EIE®), for CVL in relation to the detection of Leishmania DNA through real-time polymerase chain reaction (real-time PCR) in samples from symptomatic and asymptomatic dogs from a non-endemic area in the state of Rio Grande do Sul, Southern Brazil. Serum from 140 dogs (39 symptomatic and 101 asymptomatic) was tested by DPP and ELISA followed by real-time PCR. From a total of 140 samples evaluated, Leishmania DNA was detected by real-time PCR in 41.4% (58/140). Moreover, 67.2% of samples positive in real-time PCR were positive in both DPP and ELISA (39/58), showing moderate agreement between methods. In the symptomatic group, one sample non-reactive in both serological assays was positive in real-time PCR, whereas in the asymptomatic group, 17.8% non-reactive or undetermined samples in serological assays were positive in the molecular method. Leishmania DNA was not detected in 17.9% reactive samples by serological assays from the symptomatic group, and in 3.9% from asymptomatic dogs. Real-time PCR demonstrated greater homogeneity between symptomatic and asymptomatic groups compared with DPP and ELISA. The molecular method can help to establish the correct CVL diagnosis, particularly in asymptomatic dogs, avoiding undesirable euthanasia.
Assuntos
Cromatografia de Afinidade/veterinária , Doenças do Cão/diagnóstico , Ensaio de Imunoadsorção Enzimática/veterinária , Leishmania infantum/isolamento & purificação , Leishmaniose Visceral/diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Animais , Infecções Assintomáticas , Brasil , Cromatografia de Afinidade/métodos , Doenças do Cão/parasitologia , Cães , Ensaio de Imunoadsorção Enzimática/métodos , Leishmaniose Visceral/parasitologia , Reação em Cadeia da Polimerase em Tempo Real/métodosRESUMO
Because of visceral leishmaniasis (VL) urbanization and spreading of the human immunodeficiency virus (HIV) infection to rural areas, coinfection has become more common. Here, we compared the accuracy of Kalazar Detect® (KD), an rK39-based immunochromatographic (IC) test, and OrangeLife® (OL), an rK39 + rK28 IC test, for diagnosing VL in patients coinfected with HIV in an endemic area in Brazil. Seventy-six VL patients and 40 patients with other diseases, of which 31 and 21 patients, respectively, were infected with HIV, were examined. The sensitivity of OL and KD tests was 88.89 and 95.45%, respectively, in patients without HIV. The sensitivity dropped to 67.74 and 61.29%, respectively, in coinfected patients. The decrease in sensitivity was not related to a decrease in the production of Leishmania-specific IgG. Because of the low sensitivity of rk39 test in HIV-infected patients, we suggest that patients with negative rK39 results should undergo further investigation with additional serological tests that are not based only on the rK39 antigen and examination of bone marrow aspirates.