Diminished CD4+/CD25+ T cell and increased IFN-gamma levels occur in dogs vaccinated with Leishmune in an endemic area for visceral leishmaniasis.

The Leishmune vaccine has been used in endemic areas to prevent canine visceral leishmaniasis in Brazil, but cytokine production induced by vaccination has rarely been investigated in dogs. This study aimed to evaluate the immune response of dogs vaccinated with Leishmune FML vaccine (Fort Dodge) against total antigen of Leishmania (Leishmania) chagasi (TAg) and FML. Twenty healthy dogs from Araçatuba, São Paulo, Brazil, an endemic leishmaniasis area, received three consecutive subcutaneous injection of Leishmune vaccine at 21-day intervals. PBMC were isolated before and 10 days after completing vaccination and lymphoproliferative response and antibody production against FML or total promastigote antigen were tested. Cytokines IFN-gamma, IL-4 and TNF-alpha were measured in culture supernatant and CD4+/CD25+ and CD8+/CD25+ T cell presence was determined. Analysis of the data indicated that the vaccine conferred humoral responses (100%) against both antigens and cellular immunity to FML (85%) and total antigen (80%), the supernatant of cultured cells stimulated with TAg and FML showed an increase in IFN-gamma (P<0.05), and the vaccine reduced CD4+/CD25+ T cell presence compared to that observed before vaccination. These responses may constitute part of the immune mechanism induced by Leishmune.

Clinical and parasitological evaluation of dogs naturally infected by Leishmania (Leishmania) chagasi submitted to treatment with meglumine antimoniate.

Aiming to evaluate the efficacy of the treatment of canine visceral leishmaniasis, to verify the occurrence of a possible disease relapse, and to search for the presence of the parasites after the end of the treatment, seven dogs naturally infected by Leishmania (Leishmania) chagasi were used. The dogs were subjected to a treatment with 75 mg/kg meglumine antimoniate subcutaneously every 12 h for 21 days, and followed-up for a period of 6 months. During the whole experimental period the animals wore deltamethrin collars and were kept in a screened kennel to avoid reinfection. Lymph node and bone marrow aspiration biopsy was carried out to search for the parasite at seven moments: before the treatment, 30, 60, 90, 120, 150 and 180 days after the start of the treatment. After the end of the experiment all dogs were humanely euthanized. Then, spleen and liver "imprints" and in vitro cultures were carried out to search for amastigote forms of the parasite. During the treatment all animals presented remission of symptoms. However, two dogs were observed to present new symptoms in the course of the experiment. At the end of the experiment, the presence of amastigote forms of the parasite was evidenced in five of the seven dogs. This enabled us to conclude that the treatment promoted clinical cure but did not eliminate the parasites completely.

Estudo histológico e imunoistoquímico do estudo nervoso central de cães naturalmente infectados por Leishmania (Leishmania) chagasi / Histological and immunohistochemical study of the central nervous system of dogs naturally infected by Leishmania (Leishmania) chagasi

O objetivo do presente estudo foi caracterizar as alterações histopatológicas e detectar por imunoistoquímica, a presença de formas amastigotas de Leishmania no sistema nervoso central de cães com e sem sintomatologia neurológica da doença. Foram utilizados dois grupos de animais; o primeiro foi composto de 18 cães com leishmaniose visceral sem evidência clínica de envolvimento neurológico, e o segundo, composto de 21 cães com leishmaniose visceral e sintomas neurológicos. As mais freqüentes alterações histopatológicas encontradas no sistema nervoso central de cães de ambos os grupos foram degeneração neuronal com neuronofagia, gliose, leptomeningite, congestão vascular, presença de infiltrado linfoplasmocitário perivascular e áreas de microhemorragia. Não foram observadas marcações antigênicas para formas amastigotas de Leishmania em nenhum fragmento do sistema nervoso central de cães de ambos os grupos; entretanto, a maioria deles apresentava marcação em parede de vasos sangüíneos, o que sugere a presença de antígenos parasitários circulantes.

The present study aimed to characterize the histopathological alterations and to detect, by immunohistochemistry, the presence of amastigote forms of Leishmania in CNS tissue of dogs with and without neurological clinical signs of the disease. Two groups of animals were used: the first was composed of 18 dogs with visceral leishmaniasis without clinical evidence of neurological involvement, and the second, composed of 21 dogs with visceral leishmaniasis and neurological symptoms. The most frequent histopathological alterations found in the CNS of dogs of both groups were neuronal degeneration with neuronophagia, gliosis, leptomeningitis, vascular congestion, presence of perivascular lymphoplasmacytic infiltrate and areas of focal microhemorrhage. Antigen labeling for whole forms of Leishmania amastigotes was not observed in any fragment of the CNS of the dogs of either groups; however, most of them presented labeling of blood vessels walls, which suggests the presence of circulating parasite antigens.

Avaliação do proteinato sérico de cães com e sem sintomas neurológicos, naturalmente infectados por Leishmania chagasi / Evaluation of serum proteinogram of dogs naturally infected with Leishmania chagasi with and without neurological symptoms

A leishmaniose visceral é uma antropozoonose causada por um protozoário do gênero Leishmania. A infecção no cão usualmente causa uma doença sistêmica crônica, que, clinicamente, é similar à humana. No Brasil, o Ministério da Saúde exige a eutanásia de animais portadores de leismaniose visceral e não recomenda o tratamento dos mesmos. Entretanto, em muitos países esses animais são tratados, e a avaliação da resposta à terapia baseia-se nos títulos de anticorpos anti-leishmania e no proteinograma sérico. O presente trabalho teve como objetivos determinar a concentração de proteína total e suas frações eletroforéticas correlacionando-as com a presença de imunoglobinas no soro de cães com leishmaniose visceral, com e sem acometimento neurológico. Foram utilizados grupos de animais, sendo o primeiro constituído de 18 cães portadores de leishmaniose visceral, sem evidências clínicas de envolvimento neurológico , e o segundo formado por 21 cães portadores de leishmaniose visceral com quadro neurológico. A proteína sérica total foi determinada através do método de biureto e em seguida realizou-se a migração eletroforética para separação das frações protéicas do soro sanguíneo. Ambos os grupos apresentaram uma intensa gamaglobulinemia, uma hipoalbuminemia e uma diminuição da fração alfaglobulina. A elevação dos níveis de gamaglobulina, no grupo controle apresentou uma correlação com uma maior positividade no teste de ELISA no soro destes animais; 88,9 dos cães do grupo controle, contra apenas 28

Presença de anticorpos anti-Neospora caninum e Toxoplasma gondii em cães com leishmaniose visceral da região de Araçatuba, São Paulo, Brasil / Presence of anti-Neospora caninum and Toxoplasma gondii antibodies in dogs with visceral leishmaniosis from the region of Araçatuba, São Paulo, Brazil

Dogs that had positive and negative sera to Leishmania chagasi from the region of Araçatuba, São Paulo, Brazil, were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies as potential co-infecting agents. Blood samples were collected from 204 dogs and out of them 98 were carriers of leishmaniosis. Sera were tested for the presence of anti-L. chagasi antibodies by ELISA, and anti-T. gondii and anti-N. caninum by an indirect fluorescent antibody test (IFAT). Age, gender, and association between the presences of anti-L. chagasi antibodies and seroprevalence to N. caninum and T. gondii were analyzed by chi-square test. Out of the 204 sera investigated, 36 (17.6%) were positive for N. caninum (IFAT=50) and 75 (36.8%) to T. gondii (IFAT=16) with titers that varied from 50 to 6400 for N. caninum, and from 16 to 16384 for T. gondii. The co-presence of anti-L. chagasi, N. caninum and T. gondii antibodies was observed in 17 (8.3%) dogs. Antibodies to N. caninum were observed in four (3.8%) out of 106 dogs that were negative for L. chagasi, and in 32 (32.6%) out of the 98 dogs that were positive for L. chagasi. Anti-T. gondii antibodies were found in 40 (41.0%) and in 35 (33.0%) of the 98 positive dogs and in 106 negative dogs for L. chagasi, respectively. An association between the presence of antibodies against L. chagasi and a positive response to N. caninum (p<0.001) was observed. The gender and age of the dogs did not show an association between the presence of antibodies and any of the agents studied (p>0.05), with the exception of age and presence of anti-L. chagasi antibodies, in which only a slight association was observed (p=0.038). Within this interaction, a higher number of dogs, older than four years, were positive for this agent when compared to other age groups.

Cães soropositivos e soronegativos à Leishmania chagasi da região de Araçatuba, São Paulo, Brasil, foram avaliados quanto à presença de possível co-infecção por Neospora caninum e Toxoplasma gondii. Amostras de sangue de 204 cães, sendo 98 portadores de leishmaniose, foram coletadas e os soros testados quanto à presença de anticorpos anti-L. chagasi pelo método de ELISA e anti-T. gondii e N. caninum pela reação de imunofluorescência indireta (RIFI). Idade, sexo e possíveis associações entre a presença de anticorpos anti-L. chagasi e a soroprevalência de N. caninum e T. gondii foram analisados pelo teste do Qui-quadrado. Dos 204 soros examinados, 36 (17,6%) foram positivos para N. caninum (RIFI ³50) e 75 (36,8%) para T. gondii (RIFI ³16) com títulos que variaram de 50 a 6.400 para N. caninum, e de 16 a 16.384 para T. gondii. A co-presença de anticorpos anti-L. chagasi, N. caninum e T. gondii foi observada em 17 (8,3%) cães. Anticorpos anti- N. caninum foram observados em quatro (3,8%) dos 106 cães negativos e em 32 (32,6%) dos 98 cães positivos à L. chagasi. Anticorpos anti-T. gondii foram encontrados em 40 (41%) e em 35 (33%) dos 98 cães positivos e dos 106 cães negativos à L. chagasi, respectivamente. Foi observada associação entre a presença de anticorpos anti-L.chagasi e a presença de anticorpos anti-N. caninum (p<0,001). O sexo e a idade dos cães não apresentaram associação com a presença de anticorpos para nenhum dos agentes estudados (p>0,05), com exceção da idade e presença de anticorpos anti-L. chagasi , que apresentou uma associação fraca (p=0,038), com maior número de cães com mais de quatro anos de idade positivos a esse agente quando comparado às outras idades.