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.

RESUMO A leishmaniose visceral americana é zoonose em expansão no Brasil com transmissão vetorial, onde o cão é o principal reservatório urbano. Partindo de um caso canino da doença no Jacaré, Niterói/RJ, realizouse inquérito epidemiológico canino e levantamento entomológico para avaliar a extensão da enfermidade na localidade. As amostras foram coletadas ao redor do caso e os cães identificados por testes sorológicos (teste rápido de imunocromatografia em dupla plataforma, ensaio imunoenzimático/ELISA, reação de imunofluorescência indireta/RIFI). O diagnóstico parasitológico foi realizado nos animais reagentes em ao menos um desses exames. O levantamento entomológico foi feito com armadilhas luminosas e coleta manual. As associações entre variáveis caninas coletadas e o desfecho (ELISA e RIFI reagentes) foram avaliadas pelo teste qui-quadrado e ajustadas pela regressão logística multivariada para variáveis com p< 0,1 na análise bivariada. Foram detectados 17 casos de LVA entre os 110 cães avaliados (prevalência de 15,5%). Presença de ectoparasitas (OR 6,5; 95% IC 1,1-37,4), animais com sinais clínicos (OR 9,5; 95% IC 1,2-76,6) e casos prévios de LVA canina na residência (OR 17,9; 95% IC 2,2-147,1) foram associados ao desfecho. Não foi detectada Lutzomyia longipalpis. Nossos resultados indicam situação de transmissão contínua na área.

SUMMARY American visceral leishmaniasis is a vector-borne zoonosis in expansion in Brazil. Dogs are the main urban reservoir. Departing from a case of canine visceral leishmaniasis (CVL) in Jacaré, Niterói, Rio de Janeiro State, an epidemiological canine and entomological study was performed to assess the extension of the disease at the location. Sample was collected around the case and the dogs identified by serological tests (rapid double platform immunochromatographic exams, immunoenzymatic assay/ELISA, indirect immunofluorescence/IFAT). The parasitological diagnosis was performed in animals positive in at least one of these tests. The entomological study was carried out by using light traps and manual collection. The associations between canine variables and outcome (ELISA and IFAT reagents) were assessed by the chi-square test and adjusted by multivariate logistic regression for those associations with p < 0.1 in the bivariate analysis. Seventeen cases of CVL were detected among 110 evaluated dogs (prevalence of 15.5%). Presence of ectoparasites (OR 6.5; 95% CI 1.1-37.4), animals with clinical signs (OR 9.5; 95% CI 1.2-76.6), and previous cases of CVL in the same house (OR 17.9; 95% CI 2.2-147.1) were associated with the outcome. Lutzomyia longipalpiswas not detected. Our results are indicative of an ongoing transmission in the area.

This study describes the production of a new avian polyclonal antibody (IgY) against canine IgG, as another tool for the immunodiagnostic using IgY technology. The immunization protocol caused neither deaths nor pathologies, and no decline in egg laying capacity was detected. The total concentration of isolated IgY was constant, without significant difference (p> 0.05), with average of 97.55 mg of IgY/yolk. The IgY revealed a strong sensitivity and specificity in recognition against canine IgG by ELISA. After the immunization, there was a significant increase in the production of IgY specific from the first to the second month (p <0.05), reaching a stable peak without decrease in the production by the end of the analysis period (p > 0.05). The IgY demonstrated a suitable specificity in Western blot against the purified and serum canine IgG, not enabling recognition of canine IgM or IgG of other animal species. The specific IgY in the egg yolks of immunized hens proved to be a molecule with an appropriate purity and desired specificity against the immunizing antigen. Moreover, its constant production in large quantities during the four months analyzed indicated that IgY antibody production technology could be considered as an excellent alternative to the standard methods.

Este estudo avaliou os fatores associados à frequência de anticorpos específicos para Leishmania spp. em cães domiciliados na microrregião de Itaguaí, Rio de Janeiro. Foram colhidas amostras de sangue de 524 cães. As amostras de soro foram submetidas a reação de imunofluorescência indireta (RIFI) e ensaio imunoenzimático indireto (ELISA-teste) para Leishmania spp. A frequência de cães soropositivos foi de 28,24% (n = 148) na microrregião e, entre os três municípios avaliados, a maior frequência (p < 0,05) foi observada em Seropédica (59,46%), seguida de Itaguaí (29,05%) e Mangaratiba (11,49%). Em relação aos fatores associados ao hospedeiro, observou-se que cães sem raça definida e aqueles com idade acima de dois anos apresentaram maior frequência de anticorpos para Leishmania spp. (p < 0,05). Em relação aos fatores relacionados ao ambiente e ao hábito do animal, os cães residentes em áreas rurais (RF = 1,67, p = 0,0002), animais que vivem fora da residência (RF = 1,42, p = 0,0197), com acesso à mata, córregos e pastagens (FR = 2,81, p = 0,0007), que permanecem soltos (RF = 1,66, p = 0,0073), e aqueles que não possuem abrigo (RF = 2,16, p < 0,0001) apresentaram maior chance de serem soropositivos. A leishmaniose canina é uma enfermidade com elevada ocorrência na microrregião de Itaguai, e aspectos como definição racial, idade, hábitos e cuidados estabelecidos pelo proprietário mostraram associação significativa nessa microrregião.

This study evaluated factors associated with the frequency ofLeishmania spp. antibodies in dogs residing in the Itaguai micro-region, State of Rio de Janeiro, Brazil. Blood samples were collected from 524 dogs. The serum samples were submitted to indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) forLeishmania spp. The frequency of seropositive dogs was 28.24% (n = 148) in the micro-region, and among the three municipalities within that region, the highest frequency (p < 0.05) was observed in Seropedica (59.46%), followed by Itaguai (29.05%) and Mangaratiba (11.49%). Regarding factors associated with the host, mongrel dogs and those over the age of two presented higher frequency of antibodies to Leishmaniaspp. (p < 0.05). Concerning factors related to the environment and habits of the animal, dogs residing in rural areas (FR = 1.67, p = 0.0002), living outside the residence (FR = 1.42, p = 0.0197), with access to forest, streams and pastures (FR = 2.81, p = 0.0007), remaining loose (FR = 1.66, p = 0.0073), and those that had no shelter (FR = 2.16, p < 0.0001) were more likely to be seropositive. Canine leishmaniasis is a disease with high occurrence in the Itaguai micro-region, and aspects such as the definition of breed, age, habits and care by owners showed significant association in this micro-region.