Serological diagnosis of visceral leishmaniasis by an enzyme immunoassay using protein A in naturally infected dogs

摘要

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Leishmania chagasi using total antigen from lysed promastigotes. Fifty symptomatic mixed breed dogs from a region of high incidence of visceral leishmaniasis in Brazil were examined. The results showed that in the positive animals, diagnosed by cytological examination, the ELISA using protein A assay system (mean optical density ?± SD / 2.078 ?± 0.631) detected more antibodies than the anti-IgG assay (mean optical density ?± SD / 1.008 ?± 0.437), while in the negative animals, the results by both systems were similar. These results suggest that the ELISA assay using protein A peroxidase conjugated could be useful to detect early infected animals in endemic areas, and thus help to control the spread of the infection. Index terms: Leishmania chagasi, visceral leishmaniasis, protein A, dogs. RESUMO Um ensaio de imunoadsorção enzimática para detecção de anticorpos contra Leishmania chagasi, utilizando antígeno total de formas promastigotas lisados foi desenvolvido. Cinqüenta cães com sintomas clínicos de leishmaniose visceral foram examinados. Esta técnica utilizou anti-IgG de cão conjugado a peroxidase ou proteína A conjugado a peroxidase. Foi verificado que nos animais positivos diagnosticados por exame parasitológico direto o ensaio ELISA utilizando proteína A conjugada a peroxidase (média da densidade óptica ?± desvio padrão 2,078 ?± 0,631) detecta mais anticorpos do que o sistema utilizando anti-IgG de cão conjugado a peroxidase (média da densidade óptica ?± desvio padrão 1,008 ?± 0,437), enquanto para os animais negativos o resultado obtido nos dois sistemas de detecção são similares. Esse resultado sugere que o sistema de ELISA utilizando proteína A conjugado a peroxidase pode ser útil na detecção de animais na fase aguda da infecção e desta forma auxiliar na identificação dos animais positivos e no controle desta importante zoonose. Termos de indexação: Leishmania chagasi, ELISA, leishmaniose visceral, cães.     INTRODUCTION Zoonotic visceral leishmaniasis (VL), caused by both Leishmania infantum and Leishmania chagasi, represents 20% of human visceral leishmaniasis in the world (100,000 cases annually) and its incidence is growing in urban and peri-urban areas of the tropics (Dye 1996). Dogs constitute the main domestic reservoir of this parasite and play a central role in the transmission cycle of the parasite to humans by phlebotomine sand flies (Moreno & Avar 2002). The diagnosis of VL is based on the demonstration of the causative parasites in bone marrow aspirates, spleen, lymph nodes or liver biopsies by microscopy or culture (Alvar et al., 2004). The demonstration of specific serum antibodies also permits clinical confirmation, albeit indirect, diagnosis of VL (Bray 1976). Recently, molecular methods also have been used (Cortes et al. 2004). In dogs the disease symptoms include fever, hypergamma-globulinemia, hepatosplenomegaly and anemia. From the analysis of the clinical and pathological symptoms accompanying canine VL disease, two main groups of responses to infection have been evidenced in both naturally and experimentally infected dogs. Most of the infected animals are susceptible and develop active disease characterized by high anti-Leishmania antibody titers and depressed lymphoproliferative abilities, whereas a small percentage is resistant to the infection, with delayed type hypersensitivity, few, if any, specific circulating antibodies, and enhanced lymphoproliferative response. The latter group does not develop the disease or, when it does, it heals spontaneously (Abranches et al. 1991, Pinelli et al. 1994). The high levels of specific antibodies found in symptomatic animals with visceral leishmaniasis improve serological analysis and diagnosis. Several methods are used to detect specific antibodies including immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and Dot-ELISA. These methods generally show similar specificity and sensitivity (Senaldi et al. 1996, Fisa et al. 1997). Previous studies showed the binding of protein A (Staphilococcus aureus) and immunoglobulin in mammalian sera (Lindmark et al. 1983). The present work compares groups of sera using anti-IgG peroxidase conjugate and protein A from S. aureus peroxidase conjugate in an ELISA assay to detected antibodies to Leishmania chagasi in naturally infected dogs.   MATERIALS AND METHODS The antigen source was lysed promastigotes. The antigen was prepared from Leishmania chagasi promastigotes. The parasites were grown at 28?oC in RPMI 1640 (Gibco, Pisley, UK) supplemented with 100IU/ml penicillin, 100?μg/ml streptomycin, 2mM L-glutamine and