RESUMO

Leishmania infantum causes canine leishmaniasis. Using parasitological and molecular analyses, we identified L. infantum in the reproductive organs of male and female dogs. Using histochemistry, immunohistochemistry, and PCR, we examined tissue samples from the reproductive organs of 8 male dogs and 16 female dogs diagnosed with leishmaniasis. Despite the absence of macroscopic or microscopic lesions in these organs, we observed L. infantum amastigotes in tissue samples from the testis and the uterus. PCR and sequencing of these tissues revealed sequences that matched 100% with L. infantum DNA available at GenBank. The presence of L. infantum amastigotes and DNA in testicular and uterine tissue samples suggested that these organs can harbor the parasite without associated macroscopic or microscopic lesions, and this can be especially important in the vertical and venereal transmission of leishmaniasis in dogs.(AU)

Leishmania infantum é agente etiológico da leishmaniose canina. Por meio de análises parasitológicas e moleculares, a presença do parasita foi investigada em órgãos reprodutivos de cães machos e fêmeas. Amostras de tecidos dos órgãos reprodutivos de 8 cães machos e 16 fêmeas diagnosticados com leishmaniose foram avaliadas por histoquímica, imunohistoquímica e PCR. Apesar de não terem sido observadas lesões macroscópicas ou microscópicas nos órgãos reprodutivos desses cães, formas amastigotas de L. infantum foram observadas em amostras teciduais do testículo e útero. A PCR e o sequenciamento do DNA extraído desses tecidos revelaram sequências 100% idênticas a L. infantum depositadas no GenBank. Nossos resultados sugerem que os testículos e o útero podem abrigar o parasita, sem associação com lesões macroscópicas ou microscópicas, o que pode ter uma grande importância na transmissão venérea e vertical da leishmaniose entre cães.(AU)

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RESUMO

Leishmania infantum causes canine leishmaniasis. Using parasitological and molecular analyses, we identified L. infantum in the reproductive organs of male and female dogs. Using histochemistry, immunohistochemistry, and PCR, we examined tissue samples from the reproductive organs of 8 male dogs and 16 female dogs diagnosed with leishmaniasis. Despite the absence of macroscopic or microscopic lesions in these organs, we observed L. infantum amastigotes in tissue samples from the testis and the uterus. PCR and sequencing of these tissues revealed sequences that matched 100% with L. infantum DNA available at GenBank. The presence of L. infantum amastigotes and DNA in testicular and uterine tissue samples suggested that these organs can harbor the parasite without associated macroscopic or microscopic lesions, and this can be especially important in the vertical and venereal transmission of leishmaniasis in dogs.

Leishmania infantum é agente etiológico da leishmaniose canina. Por meio de análises parasitológicas e moleculares, a presença do parasita foi investigada em órgãos reprodutivos de cães machos e fêmeas. Amostras de tecidos dos órgãos reprodutivos de 8 cães machos e 16 fêmeas diagnosticados com leishmaniose foram avaliadas por histoquímica, imunohistoquímica e PCR. Apesar de não terem sido observadas lesões macroscópicas ou microscópicas nos órgãos reprodutivos desses cães, formas amastigotas de L. infantum foram observadas em amostras teciduais do testículo e útero. A PCR e o sequenciamento do DNA extraído desses tecidos revelaram sequências 100% idênticas a L. infantum depositadas no GenBank. Nossos resultados sugerem que os testículos e o útero podem abrigar o parasita, sem associação com lesões macroscópicas ou microscópicas, o que pode ter uma grande importância na transmissão venérea e vertical da leishmaniose entre cães.

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Serum levels of interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 6 (IL-6), interleukin 10 (IL-10), interleukin 17 (IL-17), interferon gamma (IFN-γ), tumor necrosis factor α (TNF-α), and interleukin 1ß (IL-1ß), cytokines involved in the immune response, were investigated in 75 Leishmania-positive blood donors living in endemic areas. Based on their status in 2011 and 2015, the subjects were clustered into three groups: positive for at least one diagnostic method in both years, but lacking clinical progression to disease (G1); positive on at least one method in 2011 but negative in 2015 (G2); negative on all methods in both years (G3). Donors were interviewed for sociodemographic data collection and underwent clinical evaluation and laboratory tests. Serum cytokines were quantified using a CBA Flex set (BD Biosciences). Significant differences were found for all the cytokines evaluated, with lower concentrations in consistently Leishmania-negative individuals. The exception was IFN-γ, with similar levels among all donors. No changes consistent with active disease were observed in the laboratory results for Leishmania-positive donors who underwent clinical evaluation, none of whom progressed to disease. This suggests that infection control is associated with serum IL-17 levels. Resolution of Leishmania infection in positive donors may be related to high levels of IL-17 and low levels of IL-10, highlighting the role played by IL-17 in asymptomatic Leishmania-infected individuals.

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ABSTRACT Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14 kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500 ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n = 114), including suspect (n = 30) and positive (n = 50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n = 34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density = 0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.

Assuntos

RESUMO

Cysteine proteinases are well-known virulence factors of Leishmania spp. with demonstrated actions in both experimental mouse infection and human infection. However, studies on these enzymes in canine leishmaniasis are scarce. Here, we show, for the first time, the reactivity of sera from dogs living in an endemic area to a recombinant protein from the COOH-terminal region of cysteine B protease. In this work, enzyme-linked immunosorbent assays were performed using a 14kDa rcyspep protein obtained through a pET28-a expression system in Escherichia coli. First, 96-well plates were coated with rcyspep (500ng/well) and incubated with sera from dogs (1:100). Subsequently, IgG antibody detection was performed using rabbit anti-dog IgG antibodies conjugated with peroxidase. Sera from dogs (n=114), including suspect (n=30) and positive (n=50) dogs from a leishmaniasis-endemic area and dogs from a nonendemic area, (n=34), negative for leishmaniasis, were assessed. The results showed that sera from the suspect (42%) and positive (68%) groups responded differently to the antigen titers tested above the cut-off (Optical Density=0.166). This finding suggests that the immune response detected against cyspep may be related to clinical disorders present in these animals. Collectively, the data gathered here suggest that cyspep can sensitize the immune systems of dogs from a leishmaniasis-endemic area to elicit a humoral response, an immunological parameter indicating the contribution of this protein in host-parasite interaction.

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In this study, we evaluated the performance of a new enzyme-linked immunosorbent assay (ELISA) variant known as indirect "plasmonic ELISA" (pELISA) for the detection of Leishmania spp. infection. Serum samples from 170 dogs from an area where canine leishmaniosis (CanL) is endemic and from 26 healthy dogs from a nonendemic area were tested by indirect pELISA, and the results were compared to those of an indirect ELISA (both with recombinant antigen rK28) and those of an immunochromatographic test (dual-path platform, TR-DPP®) using real-time PCR on blood samples or conjunctival swabs as the gold standard. The pELISA, indirect rK28 ELISA and the TR-DPP® immunochromatographic test presented sensitivities of 94.7%, 89.5% and 79.0% and specificities of 100%, 92.7% and 91.5%, respectively. The analysis of the results revealed that the specificity of the indirect pELISA was greater than that of the method recommended by the Ministry of Health in Brazil and may increase the feasibility of diagnosis in resource-constrained countries because it does not require sophisticated instruments to read. Thus, this method can be used as an additional tool for the detection of Leishmania spp. infection in these areas.

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Antibodies to pathogens of public health importance were investigated in 33 free-ranging six-banded armadillos (Euphractus sexcinctus) from Brazil. The frequency of seropositive animals for Toxoplasma gondii, Leishmania spp., and Leptospira spp. were two, three, and two, respectively.

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The role of cats in the epidemiological cycle of leishmaniasis remains unclear. To better understand the occurrence of leishmaniasis in cats, we studied the frequency of Leishmania in serum samples of 100 cats living in an endemic region for canine and human leishmaniasis by serological, parasitological, and molecular methods. Of the 100 cats, 54 were seropositive for Leishmania antibodies by immunofluorescence antibody test. None of the bone marrow aspirates collected from these cats tested positive for the parasite in culture or upon polymerase chain reaction (PCR) analysis. Biopsy samples of the ears also tested negative for Leishmania upon PCR analysis. These findings may indicate that the region is endemic for canine leishmaniasis and cats are infected by Leishmania; or that cross-reaction with antibodies against other parasites increases the frequency of seropositivity; or that cats respond to Leishmania infection by producing antibodies when few or no parasites are present in bone marrow and tissue samples. Overall, our results suggest that cats can be infected by Leishmania ; however, we failed to demonstrate feline parasitosis. These findings highlight the need to study leishmaniasis in cats, since sandflies feed on cats, these animals may act as a reservoir for the parasite.(AU)

O papel dos gatos no ciclo epidemiológico da leishmaniose ainda não está claro. Para entender melhor a ocorrência de leishmaniose em gatos, estudou-se a frequência de Leishmania em amostras de soro de 100 gatos, os quais vivem em uma região endêmica para leishmaniose canina e humana, por métodos sorológicos, parasitológicos e moleculares. Dos 100 gatos, 54 foram soropositivos para anticorpos de Leishmania por teste de anticorpos de imunofluorescência. Nenhum dos aspirados de medula óssea coletados desses gatos mostrou-se positivo para o parasita em cultura, ou após a realização da reação em cadeia da polimerase (PCR). Amostras de biópsia das orelhas também foram negativas para Leishmania submetidas a PCR. Esses achados indicam que na região estudada endêmica para leishmaniose canina, os gatos podem se infectar por Leishmania; ou que a reação cruzada com anticorpos contra outros parasitas aumenta a frequência de soropositividade; ou que os gatos respondem à infecção por Leishmania produzindo anticorpos quando poucos ou nenhum parasita estão presentes na medula óssea e em amostras de tecido. Em geral, os resultados sugerem que os gatos podem ser estar infectados por Leishmania spp. No entanto, não foi possível demonstrar parasitismo felino. Essas descobertas evidenciam a necessidade de estudar a leishmaniose em gatos, uma vez que, como os flebotomíneos se alimentam em gatos, e esses animais podem atuar como um reservatório para o parasita.(AU)

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Transfusion-transmitted leishmaniasis has been a concern in regions endemic for the disease. Whether immediate or delayed, the risks posed by this mode of transmission call for careful assessment. The purpose of this study was to detect Leishmania infection in blood donors living in an endemic area and to investigate progression to the disease in these individuals. Immunofluorescent antibody test, enzyme-linked immunosorbent assay, leishmaniasis rapid test, and the polymerase chain reaction were applied to 430 donors in an initial evaluation. Of those donors with at least one positive test, 50 were reevaluated four years later by the same methods, as were 25 controls who had been negative on the same tests. In the first evaluation, Leishmania infection was detected in 41.4% (95% CI: 36.7-46.1) of donors (n = 430). None of the 75 reevaluated individuals had developed the disease, but retesting revealed positivity in at least one test in 36.0% (95% CI: 25.1-46.9) of donors. Of the 50 initially testing positive, 50% remained so on retesting. Of the 25 initially negative controls, two tested positive in the subsequent evaluation. The severity of the parasitosis and the risk of transfusion transmission warrant investigation of the potential inclusion of methods for Leishmania detection into blood banks for effective screening of infected donors.

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Abstract The aim of this study were to detect antibodies anti-Leishmania spp. and anti-Trypanosoma cruzi in two different populations of domestic cats (Felis catus domesticus) from North Paraná referred for surgical castration and to determine which characteristics of the animals studied may be associated with seropositivity. Serum samples from 679 cats were analyzed using enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence antibody test (IFAT) in series. Associations between age, sex, race, year of care and animal group were verified using the simple logistic regression. Percentage of 8.5% (58/679) of cats were positive for Leishmania spp. and 7.6% (51/673) for T. cruzi by the tests ELISA and IFAT. Animals collected by non-governmental animal protection organizations presented more seropositivity for Leishmania spp. (p<0.0001). Results shown that Leishmania spp. and T. cruzi are present in domestic cats in the northern part of the state of Paraná, as well as, owners of non-governmental animal protection organizations may be more exposed to leishmaniasis when compared to other animal owners evaluated in the present study.

Resumo O objetivo desse estudo foi detectar a presença de anticorpos IgG anti-Leishmania spp. e anti-Trypanosoma cruzi em duas populações de gatos domésticos (Felis catus domesticus) do Norte do Paraná encaminhados para castração cirúrgica e determinar quais as características dos animais estudados que podem estar associadas à soropositividade. Amostras de soro de 679 gatos foram analisadas utilizando-se os testes imunoenzimático (ELISA) e a reação de imunofluorescência indireta (RIFI), em série. Associações entre idade, sexo, raça, ano de atendimento e grupo animal ​​foram verificadas usando regressão logística simples. Um percentual de 8,5% (58/679) dos gatos apresentou positividade para Leishmania spp. e 7,6% (51/673) para T. cruzi pelos testes ELISA e RIFI. Gatos mantidos em organizações não governamentais de proteção animal apresentaram maior sororeatividade para Leishmania spp. (p<0.0001). As sorologias reativas para Leishmania spp. e Trypanosoma cruzi mostram que esses agentes estão presentes em gatos domésticos na parte norte do estado do Paraná, bem como, os proprietários de organizações não governamentais de proteção animal podem estar mais expostos à leishmaniose quando comparados com outros proprietários de animais avaliados no presente estudo.

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