Can domestic dogs be considered a good reservoir of Leishmania (L.) infantum chagasi in an endemic area of nonulcerated cutaneous leishmaniasis in Southern Honduras?

ABSTRACT Dogs are considered to be the main domestic reservoir associated with the transmission of Leishmania (L.) infantum chagasi to humans in endemic areas of visceral leishmaniasis in America. However, little is known about the role of canines as a source of infection in endemic areas of nonulcerated cutaneous leishmaniasis (NUCL). Therefore, the objective of the present study was to investigate the role of dogs as a possible reservoir of the parasite in Southern Honduras. Dogs (n = 107) living with individuals affected by NUCL were clinically examined and biological material was collected for parasitological and immunological diagnosis. Most animals showed a healthy appearance and a few presented slight weight loss (64%), alopecia (7%), onychogryphosis (5%) and skin lesions (1%). The overall seroprevalence of Leishmania infection based on the DDP ® quick test and/or in-house ELISA serological test was 41%. The presence of the parasite's DNA was confirmed in 94% of the dogs; however, the average parasite load in the buffy coat was low at 6.09 parasites/µL, ranging between 0.221 and 50.2. The skin of seropositive dogs examined by histopathology using paraffin sections stained by hematoxylin and immunohistochemistry did not show cutaneous lesions or parasite amastigotes. Based on the absence of parasites in the skin and the low parasite load detected in the buffy coat, it seems that the dog does not represent a good source of infection for the vector in the endemic area of NUCL transmission in Southern Honduras. Other domestic and/or wild animals should be investigated.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil

BACKGROUND Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS To the best of our knowledge, this is the first detection of LRV2 in the New World.

Leishmania enriettii visceralises in the trachea, lungs, and spleen of Cavia porcellus

BACKGROUND Leishmania (Mundinia) enriettii is a species commonly found in the guinea pig, Cavia porcellus. Although it is a dermotropic species, there is still an uncertainty regarding its ability to visceralise during Leishmania life cycle. OBJECTIVE Here, we investigated the ability of L. enriettii (strain L88) to visceralise in lungs, trachea, spleen, and liver of C. porcellus, its natural vertebrate host. METHODS Animals were infected sub-cutaneously in the nose and followed for 12 weeks using histological (hematoxilin-eosin) and molecular tools (polymerase chain reaction-restriction fragment length polymorphism - PCR-RFLP). To isolate parasite from C. porcellus, animals were experimentally infected for viscera removal and PCR typing targeting hsp70 gene. FINDINGS Histological analysis revealed intense and diffuse inflammation with the presence of amastigotes in the trachea, lung, and spleen up to 12 weeks post-infection (PI). Molecular analysis of paraffin-embedded tissues detected parasite DNA in the trachea and spleen between the 4th and 8th weeks PI. At the 12th PI, no parasite DNA was detected in any of the organs. To confirm that the spleen could serve as a temporary site for L. enriettii, we performed additional in vivo experiments. During 6th week PI, the parasite was isolated from the spleen confirming previous histopathological and PCR observations. MAIN CONCLUSION Leishmania enriettii (strain L88) was able to visceralise in the trachea, lung, and spleen of C. porcellus.

In situ cellular immune response in non-ulcerated skin lesions due to Leishmania (L) infantum chagasi infection

Skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi are characterized by lymphohistiocytic inflammatory infiltrate associated with epithelioid granuloma and scarce parasitism. However, the in situ cellular immune response of these patients is unclear. Therefore, the aim of the present study was to characterize the cellular immune response in the skin lesions of patients affected by NUCL. Methods Twenty biopsies were processed by immunohistochemistry using primary antibodies to T lymphocytes (CD4, CD8), NK cells, B lymphocytes, macrophages, nitric oxide synthase and interferon-gamma. Results Immunohistochemistry revealed higher expression of all cellular types and molecules (IFN-γ, iNOS) in the dermis of diseased skin compared to the skin of healthy individuals (p < 0.05). Morphometric analysis performed in the skin lesions sections showed the predominance of CD8+ T lymphocytes in the mononuclear infiltrate, followed by macrophages, mostly iNOS+, a response that could be mediated by IFN-γ. Conclusion Our study improves knowledge of the cellular immune response in non-ulcerated or atypical cutaneous leishmaniasis caused by L. (L.) infantum chagasi in Central America and pointed to the pivotal participation of CD8+ T lymphocytes in the host defense mechanisms against the parasite in patients with NUCL.(AU)

Exposure to Leishmania spp. infection and Lutzomyia spp. in individuals living in an area endemic for visceral leishmaniasis in Brazil

Abstract INTRODUCTION: This study aimed to investigate human exposure to Leishmania spp. infection and sandflies in an area endemic for the disease. METHODS: The presence of antibodies specific for Leishmania spp. and saliva of Lutzomyia spp. and that of L. infantum DNA in blood were evaluated. RESULTS: Antibodies against Leishmania spp. and sandfly saliva were observed in 20.8% and 37.7% of individuals, respectively. DNA of Leishmania spp. was amplified from the blood of one patient. CONCLUSIONS: The results suggest that Leishmania spp. infection may be underdiagnosed in this area.

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.(AU)

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

Background: Lipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells. Methods: In the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice. Results: The aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice. Conclusions: Results presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.

Serodiagnosis of visceral and cutaneous leishmaniasis in human and canine populations living in Indigenous Reserves in the Brazilian Amazon Region

ABSTRACT INTRODUCTION: Leishmaniasis is endemic to the Northern, Northeastern, Central-Western, and Southeastern regions of Brazil. We aimed to assess the epidemiological situation of leishmaniasis in humans and dogs in indigenous villages located in the States of Mato Grosso and Tocantins using a serological survey conducted in May 2011. METHODS: Serum samples were collected from 470 humans and 327 dogs living in villages of the Urubu Branco and Tapirapé Karajá indigenous reserves. The samples were analyzed for the presence of Leishmania spp. antibodies using the indirect fluorescent antibody test (IFAT), enzyme-linked immunosorbent assay (ELISA) with a crude antigen (CA) and soluble antigen (SA), and Dual Path Platform (DPP®) immunoassay for canine visceral leishmaniasis. RESULTS: Of 470 human samples tested, two (0.4%) were positive using IFAT. Among 327 dog samples tested, 28 (8.6%) were positive using ELISA CA, five (1.5%) using ELISA SA, two (0.6%) using IFAT, and none using DPP® immunoassay with Leishmania infantum chagasi antigen. When Leishmania amazonensis antigen was used, 20 (6.1%) samples were positive using ELISA CA and four (1.2%) using IFAT. CONCLUSIONS: There was a low prevalence of infection in the region, and significant differences among the main serological methods used for the diagnosis of leishmaniasis. These findings indicated that the detection of Leishmania spp. requires further study and improvement.

Serological, parasitological and molecular tests for canine visceral leishmaniosis diagnosis in a longitudinal study / Testes sorológicos, parasitológicos e moleculares para o diagnóstico da leishmaniose visceral canina em estudo longitudinal

Abstract One of the measures to control visceral leishmaniosis (VL) in Brazil is the identification and culling of the canine reservoir. There is much controversy concerning this strategy, including the proper identification of positive dogs and the fact that the host-parasite relationship changes over time make it more challenging. A dynamic cohort of 62 dogs was followed every three months using serological and parasitological examinations and PCR. Positivity by PCR was higher than by serology and by parasitological examinations and showed a tendency to decrease over time, while serology tended to increase after six months. Concomitant positivity in all tests was observed in 10.4% of the samples, and negativity in 29.1%. Overall sensitivity ranged from 43.6 to 64.1%, and was not uniform over time. The proportion of dogs with or without clinical signs was not different by cytology or PCR but PCR was able to identify a larger number of asymptomatic dogs compared to ELISA and immunochromatography. PCR can be useful for surveillance of areas where cases of canine VL have not yet been detected and in which control strategies can be implemented to limit the spread of the disease. Despite the advance in diagnostic tools CVL diagnosis remains a challenge.

Resumo Uma das medidas de controle da leishmaniose visceral (LV) no Brasil se baseia na identificação e eliminação do reservatório canino. Existe considerável controvérsia relativa a esta estratégia incluindo a correta identificação dos cães positivos e a variação temporal da relação hospedeiro-parasita, o que torna esta medida ainda mais desafiadora. Uma coorte dinâmica de 62 cães foi acompanhada trimestralmente utilizando-se métodos sorológicos, parasitológicos e a PCR. A taxa de positividade por PCR foi maior em comparação à dos métodos sorológicos e parasitológicos, e mostrou tendência à diminuição com o passar do tempo, enquanto que a positividade sorológica apresentou tendência a aumento, após seis meses. Observou-se positividade concomitante em todos os testes em 10,4% das amostras e, negatividade concomitante, em 29,1%. A sensibilidade geral variou de 43,6% a 64,1%, não sendo uniforme ao longo do estudo. A proporção de cães com e sem sinais clínicos que foram positivos ao exame parasitológico ou à PCR não foi estatisticamente diferente. Contudo, foi possível identificar como positivos um maior número de animais assintomáticos por meio da técnica de PCR, em comparação aos testes ELISA e imunocromatográfico. A PCR pode ser bastante útil para a vigilância epidemiológica de áreas onde casos de LV canina ainda não tenham sido descritos e onde estratégias de controle podem ser implantadas para limitar a disseminação da doença. Não obstante o avanço nas ferramentas diagnósticas, diagnosticar a LVC continua um desafio.

Serological and infection statuses of dogs from a visceral leishmaniasis-endemic area / Status sorológico e de infecção canina em área endêmica de leishmaniose visceral

OBJECTIVE This study investigated the serological status of dogs living in a visceral leishmaniasis-endemic area and its correlation with the parasitological condition of the animals. METHODS Canine humoral response was evaluated using the sera of 134 dogs by enzyme-linked immunosorbent assay and immunohistochemistry to detect parasites in the skin, lymph node, and spleen of the animals. The specific antibodies investigated were IgG, IgG1, IgG2, and IgE. RESULTS According to the parasitological, laboratory, and clinical findings, the dogs were placed into one of four groups: asymptomatic with (AP+, n = 21) or without (AP-, n = 36) Leishmania tissue parasitism and symptomatic with (SP+, n = 52) or without (SP-, n = 25) parasitism. Higher IgG and IgE levels were positively correlated with the infection condition and parasite load, but not with the clinical status. In all groups, total IgG was the predominant antibody, which occurred at the expense of IgG2 instead of IgG1. Most of the infected dogs tested positive for IgG (SP+, 98.1%; AP+, 95.2%), whereas this was not observed with IgE (SP+, 80.8%; AP+, 71.2%). The most relevant finding was the high positivity of the uninfected dogs for Leishmania-specific IgG (SP-, 60.0%; AP-, 44.4%), IgE (SP-, 44.0%; AP-, 27.8%), IgG1 (SP-, 28.0%; AP-, 22.2%), and IgG2 antibodies (SP-, 56.0%; AP-, 41.7%). CONCLUSIONS The serological status of dogs, as determined by any class or subclass of antibodies, did not accurately distinguish dogs infected with L. (L.) infantum chagasi from uninfected animals. The inaccuracy of the serological result may impair not only the diagnosis, but also epidemiological investigations and strategies for visceral leishmaniasis control. This complex serological scenario occurring in a visceral leishmaniasis-endemic area highlights the challenges associated with canine diagnosis and points out the difficulties experienced by veterinary clinicians and coordinators of ...

OBJETIVO Foi investigado o status sorológico de cães, em área endêmica de leishmaniose visceral, e sua correlação com a infecção parasitológica dos animais. MÉTODOS A resposta humoral canina foi avaliada no soro de 134 cães pelo método ELISA e pela imuno-histoquímica, para detectar parasitos na pele, linfonodo e baço desses animais. Os anticorpos específicos investigados foram IgG, IgG1, IgG2 e IgE. RESULTADOS De acordo com os achados parasitológicos, laboratoriais e clínicos, os cães foram alocados em um dos quatro grupos: assintomáticos com (AP+, n = 21) e sem (AP-, n = 36) parasitismo tecidual por Leishmania e sintomáticos com (SP+, n = 52) ou sem (SP-, n = 25) parasitismo. Níveis mais elevados de IgG e IgE se correlacionaram positivamente com o status de infecção e a carga parasitária, mas não com a condição clínica. Em todos os grupos, IgG total foi o anticorpo predominante, com maior concentração de IgG2 que IgG1. O anticorpo IgG foi positivo em proporção elevada nos animais infectados (SP+ 98,1%; AP+ 95,2%), mas não o IgE (SP+ 80,8%; AP+ 71,2%). O achado mais relevante refere-se aos cães não infectados que apresentaram elevada positividade para anticorpos IgG anti-Leishmania (SP- 60,0%; AP- 44,4%), IgE (SP- 44,0%; AP- 27,8%), IgG1 (SP- 28,0%; AP- 22,2%) e IgG2 (SP- 56,0%; AP- 41,7%). CONCLUSÕES O status sorológico dos cães, determinado por qualquer classe ou subclasse de anticorpos, não distinguiu com acurácia cães infectados por L. (L.) infantum chagasi daqueles não infectados. A imprecisão do resultado sorológico pode prejudicar não só o diagnóstico, mas também as investigações epidemiológicas e as estratégias para o controle da leishmaniose visceral. Esse complexo ...

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. .

Severe visceral leishmaniasis in children: the relationship between cytokine patterns and clinical features

Introduction The relationship between severe clinical manifestations of visceral leishmaniasis (VL) and immune response profiles has not yet been clarified, despite numerous studies on the subject. This study aimed to investigate the relationship between cytokine profiles and the presence of immunological markers associated with clinical manifestations and, particularly, signs of severity, as defined in a protocol drafted by the Ministry of Health (Brazil). Methods We conducted a prospective, descriptive study between May 2008 and December 2009. This study was based on an assessment of all pediatric patients with VL who were observed in a reference hospital in Maranhão. Results Among 27 children, 55.5% presented with more than one sign of severity or warning sign. Patients without signs of severity or warning signs and patients with only one warning sign had the highest interferon-gamma (IFN-γ) levels, although their interleukin 10 (IL-10) levels were also elevated. In contrast, patients with the features of severe disease had the lowest IFN-γ levels. Three patients who presented with more than two signs of severe disease died; these patients had undetectable interleukin 2 (IL-2) and IFN-γ levels and low IL-10 levels, which varied between 0 and 36.8pg/mL. Conclusions Our results showed that disease severity was associated with low IFN-γ levels and elevated IL-10 levels. However, further studies with larger samples are needed to better characterize the relationship between disease severity and cytokine levels, with the aim of identifying immunological markers of active-disease severity. .

Leishmania sp. infection in dogs from Florianópolis, Santa Catarina, SC, Brazil / Infecção por Leishmania sp. em cães de Florianópolis, Santa Catarina, SC, Brasil

The aim of the present study was to investigate the occurrence of Leishmania sp. infection in dogs (N = 491) living in the municipality of Florianópolis, Santa Catarina (SC), Brazil, which was considered a disease-free region for visceral leishmaniasis until 2011, when autochthonous cases of canine disease were notified. Seroprevalence in this population was assessed by ELISA (0.4%; 2/491) and IFAT (4.09%; 24/491). Only one dog exhibited seroreactivity in both serological methods, comprising a total of 25 (5.3%) seroreagent animals. Leishmania sp. DNA, obtained from a sample of whole blood of this animal, was amplified by both conventional and Real-Time PCR. Sequencing of the amplified DNA and, thereby, determination of the Leishmania species involved, was not possible. Our results suggest the necessity of a thorough epidemiological investigation in Florianópolis.

O objetivo do presente estudo foi pesquisar a ocorrência de infecção por Leishmania sp. em cães (N = 491) domiciliados no município de Florianópolis, Santa Catarina, considerada uma região indene para leishmaniose visceral até o ano de 2011, quando foram notificados casos autóctones da doença canina. A soroprevalência na população foi avaliada por ELISA (0,4%; 2/491) e RIFI (4,09%; 24/491). Somente um cão apresentou sororeatividade em ambos os métodos soro-lógicos, totalizando 25 (5,3%) animais sororeagentes. O DNA de Leishmania sp., obtido de uma amostra do sangue total desse animal, foi amplificado por PCR convencional e PCR em Tempo Real. Não foi possível realizar o sequenciamento do DNA amplificado e, deste modo, determinar a espécie de Leishmania envolvida. Os nossos resultados sugerem a necessidade de uma investigação epidemiológica minuciosa em Florianópolis.

Caracterização clínica e epidemiológica das leishmanioses em cães no Estado de São Paulo / Clinical and epidemiological characterization of leishmaniasis in dogs in São Paulo State

Caracterizou-se retrospectivamente, a partir do ano de 1997 até março de 2008, a ocorrência de casos de leishmaniose, visceral ou tegumentar, dentre os cães dermatopatas atendidos em serviço especializado de hospital-escola veterinário de Faculdade de Medicina Veterinária e Zootecnia da Universidade de São Paulo. Para o diagnóstico em 427 animais, foram utilizados testes sorológicos (Ensaio Imunoenzimático Indireto, Imunofluorescência Indireta e/ou Fixação de Complemento), parasitológicos diretos e moleculares (Reação em Cadeia da Polimerase), que resultaram na confirmação de 117 (27,4%) casos positivos. Da amostragem, 96 (82,1%) eram cães de raça definida, os demais 21 (17,9%) não dispunham de plena definição racial, sendo 57 (48,7%) fêmeas e 60 (51,3%) machos. Quanto à faixa etária, 26 (22,2%) apresentavam idade inferior a 36 meses, 48 (41%) entre 36 e 84 meses e 43 (36,8%) ultrapassavam o sétimo ano de vida. Considerou-se 64 (54,7%) cães como oligossintomáticos, 44 (37,6%) sintomáticos e os nove (7,7%) remanescentes como assintomáticos. As principais alterações clínicas observadas foram, em ordem decrescente, lesões tegumentares, linfonodomegalia, anemia, disorexia, prostração e esplenomegalia. Quanto às alterações hematológicas, a mais importante foi a linfopenia relativa e absoluta. Dos animais positivos para a enfermidade, 96 (82,1%) correspondeu a casuística alóctone ao Município de São Paulo.

The occurrence of visceral or cutaneous canine leishmaniasis cases among dermatophatic dogs admitted to the veterinary teaching hospital School of Veterinary Medicine and Animal Husbandry - University of São Paulo from 1997 to 2008 was retrospectively characterized. For the diagnosis of 427 animals, serological (Enzyme-Linked Immunosorbent Assay, Indirect Immunofluorescence and/or Complement Fixation), parasitological and molecular (Polymerase Chain Reaction) tests were performed, which resulted in 117 (27.4%) confirmed positive cases. Of these, 96 (82.1%) were pure breed dogs and the others 21 (17.9%) were cross breed, being 57 (48.7%) females and 60 (51.3%) males. In terms of age, 26 (22.2%) were younger than 36 months, 48 (41%) were between 36 and 84 months, and 43 (36.8%) were older than seven years old. Sixty-four (54.7%) dogs were considered oligosymptomatic, 44 (37.6%) symptomatic, and the remaining nine (7.7%) asymptomatic. The main clinical findings were, in descending order, dermal lesions, lymphadenopathy, anemia, dysorexia, prostration and splenomegaly. As for hematological disorders, relative and absolute lymphopenia was the most important findings. Ninety-six (82.1%) of the positive animals correspond to allochthonus cases from São Paulo municipality.

Susceptibility of peritoneal macrophage from different species of neotropical primates to Ex vivo Leishmania (L.) infantum chagasi-infection / Susceptibilidade do macrófago peritoneal de diferentes espécies de primatas neotropicais para a infecção ex vivo por Leishmania (L.) infantum chagasi

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.

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.

Alterações eletromiográficas e histopatológicas da musculatura de cães naturalmente infectados por Leishmania infantum / Muscular electromyographic and histopathologic changes in dogs naturally infected by Leishmania infantum

O objetivo do presente estudo foi avaliar as alterações eletromiográficas e histopatológicas de músculos estriados esqueléticos de cães naturalmente infectados por Leishmania infantum. Foram selecionados 25 cães adultos, sem raça definida, com diagnósticos parasitológico, molecular e sorológico estabelecidos para a infecção. Os músculos avaliados foram: tríceps braquial, extensor carpo radial, bíceps femoral e gastrocnêmio. Um cão possuía problemas locomotores, com paresia de membros posteriores associada à intensa atrofia muscular. Vinte e três (92%) apresentavam algum tipo de alteração muscular, sendo que em 22 (88%) tais alterações foram identificadas diretamente pela eletromiografia. Mesmo sem sinais clínicos, em dez cães (40%) foram evidenciadas alterações eletromiográficas e histopatológicas. Antígenos de Leishmania foram identificados na musculatura de quatro (16%) cães. Os resultados eletromiográficos indicaram a ocorrência de polimiosite crônica em 13 (52%) cães, presença de músculos com inflamação tanto aguda quanto crônica em quatro (16%), miopatia aguda em dois (8%), e ausência de alterações eletromiográficas em três (12%). As alterações histopatológicas mais frequentemente observadas foram degeneração e necrose de miofibras e presença de infiltrado inflamatório verificadas em 12 (48%) cães. Outras alterações, quando comparado com as amostras de cães normais, foram do tamanho de grupos de fibras musculares em 15 (60%) e fibrose peri ou endomisial em 14 (56%) animais. As alterações observadas no presente estudo permitiram concluir que mesmo na ausência de sinais clínicos de comprometimento muscular, a maior parte dos cães infectados por L. infantum apresenta polimiosite crônica.

The aim of this study was to evaluate the electromyographic and histopathological changes in skeletal muscles of dogs naturally infected by L. infantum. Twenty five mixed breed adult dogs with parasitological, molecular and serological diagnosis were selected. The evaluated muscles were: triceps brachial, extensor carpi radialis, biceps femoris and gastrocnemius. One dog had locomotor clinical signs with hind limbs paresis associated with severe muscle atrophy. Twenty-three (92%) had some type of muscular change, and in 22 (88%) such changes were directly identified by electromyography. Even without any clinical signs of the disease, 10 (40%) dogs had electromyographic and histopathological changes. Leishmania antigens were detected in muscles of four (16%) dogs. The electromyographic evaluation indicated the occurrence of chronic polymyositis in 13 (52%) dogs, the presence of both acute and chronic muscle inflammation four (16%), acute myopathy in two (8%) and absence of electromyographic abnormalities in three (12%) dogs. The most frequently observed histopathological changes were degeneration and necrosis of myofibers and inflammatory infiltration observed in 12 (48%) dogs. Other changes were decreased diameter of muscle fibers in 15 (60%) and peri or endomysial fibrosis in 14 (56%) animals. The changes observed in the present study showed that even in the absence of clinical signs, most dogs infected by Leishmania infantum have chronic polymyositis.

Estudo da imunopatogenia das lesões no sistema nervoso central de cães naturalmente acometidos por leishmaniose visceral / Study of the immunopathogenesis of central nervous system lesions in dogs naturally affected by visceral leishmaniasis

A Leishmaniose visceral em cães é descrita como uma doença de caráter crônico na qual os principais sintomas são perda progressiva de peso, caquexia e lesões dermatológicas. Recentemente, a doença tem sido relacionada com alterações neurológicas. Um total de 40 cães portadores de leishmaniose visceral foi dividido em dois grupos. O primeiro composto por cães sem sintomas neurológicos (n=30) e o segundo grupo composto por cães com sintomas neurológicos (n=10). Amostras de encéfalo foram coletadas e armazenadas em formalina tamponada, para realização de imunoistoquímica para a pesquisa de formas amastigotas de Leishmania (Leishmania) infantum chagasi, linfócitos T CD3+, CD4+ e CD8+ e macrófagos. A reação de imunoistoquímica não revelou formas amastigotas do parasita. Linfócitos T estavam presentes em 24/30 (80%) dos cães sem sintomas neurológicos e em todos os cães do segundo grupo (p=0,0011). Linfócitos CD4+ e CD8+ raramente foram observados, apresentando imunomarcação para CD4+ em 10/40 (25%) dos cães e em metade dos animais do grupo neurológico (p=0,0090). A presença de CD8+ foi detectada em 4/10 (40%) cães com doenças neurológicas (p=0,0021). Macrófagos foram observados em 38/40 (95%) cães, sem diferença estatística significante entre os dois grupos (p= 0,7664).

Visceral leishmaniasis in dogs is described as a chronic disease whose main symptoms are progressive weigth loss, cachexy and dermatologic lesions. Recently, the disease has been associated to neurologic disorders. A total of 40 dogs with visceral leishmaniasis were divided into two groups. The first composed of dogs without neurological signs (n=30) and the second by dogs with neurological disorders (n=10). Brain samples were collected, stored in 10% buffered formalin and subjected to immunohistochemical examination for amastigotes forms of Leishmania (Leishmania) infantum chagasi, CD3+, CD4+ and CD8+ T lymphocytes and macrophages. Imunnohistochemistry evaluation revealed no amastigote forms of the parasite. CD3+ T lymphocytes were present in 24/30 (80%) dogs without neurological signs and in all dogs from the second group (p=0.0011). CD4+ and CD8+ were rarely observed, with CD4+ immunostaining in 10/40 (25%) dogs, from which half of them had neurological disease (p=0.0090). The presence of CD8+ was detected only in 4/10 (40%) dogs from neurological group (p=0.0021). Macrophages were detected in 38/40 (95%) dogs, without significant differences between groups (p=0.7664).

Susceptibility of Cebus apella monkey (Primates: Cebidae) to experimental Leishmania (L.) infantum chagasi-infection / Susceptibilidade do macaco Cebus apella (Primata: Cebidae) à infecção experimental por Leishmania (L.) infantum chagasi

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.

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 2x10(6) formas promastigotas em fase estacionária de crescimento; ii) outros quatro macacos foram inoculados com 3x10(7) 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.

Detecção de anticorpos anti-Leishmania infantum syn chagasi em cães de São José do Rio Preto, São Paulo / Anti-Leishmania infantum syn chagasi antibodies in dogs from São José do Rio Preto, São paulo

A leishmaniose visceral (LV) é uma zoonose que vem se expandido por todo o território paulista desde 1998, quando foi identificado o primeiro caso canino autóctone, no município de Araçatuba. O objetivo do presente estudo foi determinar a ocorrência de anticorpos anti-Leishmania infantum syn chagasi em amostras de soro de 584 cães de São José do Rio Preto, São Paulo, área não endêmica para a doença. Cinco cães (0,86%) foram soro reagentes pela técnica de ELISA e um (0,17%) por imunocromatografia. A reação de imunofluorescência indireta, realizada em 138 animais que possuíam densidades ópticas acima ou próximas ao ponto de corte do ELISA evidenciou dois cães (1,45%) com títulos acima de 1:40. Apenas um animal foi sororeagente nas três técnicas sorológicas. Apesar deste cão não apresentar histórico de deslocamento para áreas endêmicas, havia sido adquirido em região com casos caninos e humanos de LV. Os resultados obtidos no presente estudo fazem supor que não existiam casos autóctones de LV canina na população estudada.

Visceral leishmaniasis (VL) has been a widespread zoonosis in São Paulo since 1998, when the first autochthonous canine case was identified in Araçatuba. The aim of this study was to determine the occurrence of anti–Leishmania infantum syn chagasi antibodies in serum samples of 584 dogs from São José do Rio Preto, São Paulo, a non endemic area for the disease. Five dogs (0.86%) seroconverted by ELISA and one (0.17%) by immunochromatography. The indirect immunofluorescent reaction, carried out in 138 animals whose optical densities were above or close to ELISA’s cutt-off point, evidenced two dogs (1.45%) with titers above 1:40. Only one dog was serum-reactive on the three techniques. Although there was not a history of displacing this animal to endemic areas, the dog had been acquired in a region with canine and human cases of VL. These results suggests that there were no autochthonous cases of canine VL in this population.

Canine visceral leishmaniasis due to Leishmania (L.) infantum chagasi in Amazonian Brazil: comparison of the parasite density from the skin, lymph node and visceral tissues between symptomatic and asymptomatic, seropositive dogs / Leishmaniose visceral canina causada por Leishmania (L.) infantum chagasi na Amazônia brasileira: comparação da densidade parasitária da pele, linfonodo e vísceras entre cães soropositivos, sintomáticos e assintomáticos

Canine visceral leishmaniasis (CVL) is recognizable by characteristic signs of disease and is highly lethal. The infection, however, may be quite inapparent in some seropositive dogs, and this has raised the polemic question as to whether or not such animals can be a source of infection for Lutzomyia longipalpis, the vector of American visceral leishmaniasis (AVL). In this study we have examined 51 dogs with acute CVL from an AVL area in Pará State, northern Brazil, and compared the parasite density, amastigotes of Leishmania (L.) infantum chagasi, in the skin, lymph node and viscera of symptomatic with that of nine asymptomatic but seropositive dogs (IFAT-IgG). Post-mortem biopsy fragments of these tissues were processed by immunohistochemistry, using a polyclonal antibody against Leishmania sp. The X² and Mann Whitney tests were used to evaluate the means of infected macrophage density (p < 0.05). There was no difference (p > 0.05) in the skin (10.7/mm² x 15.5/mm²) and lymph node (6.3/mm² x 8.3/mm²), between asymptomatic and symptomatic dogs, respectively. It was higher (p < 0.05), however, in the viscera of symptomatic (5.3/mm²) than it was in asymptomatic (1.4/mm²) dogs. These results strongly suggest that asymptomatic or symptomatic L. (L.) i. chagasi-infected dogs can serve as a source of infection, principally considering the highest (p < 0.05) parasite density from skin (10.7/mm² x 15.5/mm²), the place where the vetor L. longipalpis takes its blood meal, compared with those from lymph node (6.3/mm² x 8.3/mm²) and viscera (1.4/mm²x 5.3/mm²).

A leishmaniose visceral canina (LVC) é reconhecida pelas características clínicas da doença e é altamente letal. A infecção, entretanto, pode ser totalmente assintomática em alguns cães soropositivos, o que tem levantado questão polêmica sobre a possibilidade desses animais, serem ou não uma fonte importante da infecção para o flebotomíneo, Lutzomyia longipalpis, o principal vetor da leishmaniose visceral americana (LVA). Neste estudo foram examinados 51 cães com LVC aguda, provenientes de área endêmica de LVA no Estado do Pará, Brasil, e a carga parasitária, formas amastigotas de Leishmania (L.) infantum chagasi, na pele, linfonodo poplíteo e vísceras (fígado e baço) foi comparada com a de nove cães assintomáticos soropositivos (IFAT-IgG). Fragmentos de biópsia desses tecidos obtidos post-mortem foram processados para análise através de imunohistoquímica, usando um anticorpo policlonal contra Leishmania sp. Os testes do Qui-quadrado (X²) e Mann Whitney foram usados para avaliar as médias da densidade de macrófagos infectados (p < 0,05). Os resultados mostraram que não houve diferença (p > 0,05) na densidade de macrófagos infectados da pele (10,7/mm² x 15,5/mm²) e do linfonodo (6,3/mm² x 8,3/mm²) entre cães assintomáticos e sintomáticos. Entretanto, a densidade de macrófagos infectados da víscera de cães sintomáticos (5,3/mm²) foi maior (p < 0,05) que a de cães assintomáticos (1,4/mm²). Estes resultados sugerem, fortemente, que cães naturalmente infectados por L. (L.) i. chagasi, assintomáticos ou sintomáticos, podem servir como fonte de infecção, principalmente, considerando-se que a densidade de macrófagos infectados da pele (10,7/mm² x 15,5/mm²), local onde o flebotomíneo vetor Lu. longipalpis realiza a hematofagia, foi maior (p < 0,05) que as do linfonodo (6,3/mm² x 8.3/mm²) e vísceras (1,4/mm²x 5,3/mm²).