Use of ELISA employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of canine visceral leishmaniasis.

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

Immunoperoxidase technique using an anti-Leishmania (L.) chagasi hyperimmune serum in the diagnosis of culture-confirmed American tegumentary leishmaniasis.

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.

Use of elisa employing homologous and heterologous antigens for the detection of IgG and subclasses (IgG1 and IgG2) in the diagnosis of Canine visceral leishmaniasis / A utilização do ELISA empregando antígenos homólogos e heterólogos para a detecção de IgG e subclasses (IgG1 e IgG2) no diagnóstico de Leishmaniose visceral canina

Indirect immunofluorescence is the method recommended for the diagnosis of visceral leishmanisis in dogs, however, the accuracy of this technique is low and its use on a large scale is limited. Since ELISA does not present these limitations, this technique might be an option for the detection of IgG or specific IgG1 and IgG2 subclasses. Canine ehrlichiosis is an important differential diagnosis of American Visceral Leishmaniasis (AVL). The present study compared ELISA using Leishmania chagasi and Leishmania braziliensis antigen for the detection of anti-Leishmania IgG and subclasses in serum samples from 37 dogs naturally infected with L. chagasi (AVL) and in samples from four dogs co-infected with L. braziliensis and L. chagasi (CI). The occurrence of cross-reactivity was investigated in control serum samples of 17 healthy dogs (HC) and 35 infected with Ehrlichia canis (EC). The mean optical density obtained for the detection of IgG was significantly higher when L. chagasi antigen was used, and was also higher in subgroup VLs (symptomatic) compared to subgroup Vla (asymptomatic). The correlation between IgG and IgG1 was low. The present results suggest that IgG ELISA using homologous antigen yields the best results, permitting the diagnosis of asymptomatic L. chagasi infection and the discrimination between cases of AVL and ehrlichiosis in dogs.

A imunofluorescência indireta é o método recomendado para o diagnóstico de leishmaniose visceral em cães, entretanto, a acurácia dessa técnica é baixa e seu uso em grande escala é limitado. Uma vez que o ELISA não apresenta essas limitações, essa técnica poderia ser uma opção para a detecção de IgG ou subclasses IgG1 e IgG2 específicas. A ehrlichiose canina é um importante diagnóstico diferencial de Leishmaniose Visceral Americana (LVA). O presente estudo comparou o ELISA usando antígenos de Leishmania chagasi e Leishmania braziliensis para a detecção de IgG e subclasses anti-Leishmania em amostras de soro de 37 cães naturalmente infectados com L. chagasi (LVA) e em amostras de quatro cães co-infectados (CI). A ocorrência de reatividade cruzada foi investigada em amostras de soro controle de 17 animais saudáveis (HC) e 35 de infectados por Ehrlichia canis (EC). A média de densidade óptica obtida para a detecção de IgG foi significantemente maior quando o antígeno de L. chagasi foi usado e também mais elevada no subgrupo LVs (sintomático) quando comparado ao subgrupo LVa (assintomático). A correlação entre IgG e IgG1 foi baixa. O presente resultado sugere que ELISA IgG empregando antígeno homólogo, produz os melhores resultados, permitindo o diagnóstico de infecção assintomática por L. chagasi e a discriminação entre casos de LVA e ehrlichiose em cães.

Is Leishmania (Viannia) braziliensis preferentially restricted to the cutaneous lesions of naturally infected dogs?

Nineteen dogs naturally infected with Leishmania (Viannia) braziliensis were studied in order to determine the presence of the parasite outside cutaneous lesions. Eleven (57.9%) animals showed single cutaneous or mucosal lesions and eight (42.1%) presented two or three lesions. Twenty-eight active lesions were biopsied. Isolation in culture and characterization by enzyme electrophoresis were possible in 100% of cases and amastigote forms were visualized upon histopathological examination in three samples (n=25, 12%). Isolation of the parasite in culture from peripheral blood and intact skin fragments obtained from the scapular region was negative in all animals, as was the histopathological analysis of skin from this region. Serological reactivity determined by an immunofluorescent antibody test and/or enzyme-linked immunosorbent assay was demonstrated in 15 animals. The results obtained suggest that L. braziliensis preferentially remains at the site of lesion, in contrast to the systemic distribution of parasites observed in dogs infected with L. (Leishmania) chagasi. A better understanding of this aspect may help direct diagnostic and control strategies applicable to areas characterized by the simultaneous occurrence of the cutaneous and visceral forms of leishmaniasis, as is the case for the Municipality of Rio de Janeiro, Brazil.

Immunoperoxidase technique using an anti-Leishmania (L. ) chagasi hyperimmune serum in the diagnosis of culture-confirmed American tegumentary leishmaniasis / Técnica da imunoperoxidase utilizando um soro hiperimune anti-Leishmania (L. ) chagasi no diagnóstico da leishmaniose tegumentar americana confirmada por cultura

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.

O presente estudo relata a produção do soro policlonal de coelho anti-Leishmania (L.) chagasi, a padronização da técnica de imunohistoquímica (IHQ) e sua aplicação em lesões de leishmaniose cutânea (LC) diagnosticadas por isolamento de Leishmania sp. em cultura. Foram examinados 30 fragmentos de lesões ativas de LC e 10 fragmentos de lesões de etiologia fúngica, utilizados como grupo controle. A IHQ mostrou-se mais sensível na detecção de amastigotas que a coloração em hematoxilina-eosina (HE), sendo positiva em 24 fragmentos de LC (80%) e ao passo que a HE foi positiva em 16 (53%) (p = 0,028). A IHQ também marcou diferentes espécies de fungos causadoras de micoses cutâneas. Adicionalmente, verificou-se positividade no citoplasma de células mononucleares e células endoteliais. Entretanto, esse achado esteve presente no grupo controle. Conclui-se que o método de IHQ apresentou boa sensibilidade na detecção de formas amastigotas.