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

Pesquisa Veterinária Brasileira

Correa, Ana Paula Ferreira Lopes²  Biazzono, Luciane³  Lima, Valéria Marçal Felix de¹  Luvizotto, Maria Cecília Rui³  Silva, Ana Cláudia² 

[1] Universidade Estadual Paulista, Araçatuba, Brazil;FCAV-Unesp, Jaboticabal;UEL, Londrina

关键词: Leishmania chagasi;    visceral leishmaniasis;    protein A;    dogs.;   

DOI  :  10.1590/S0100-736X2005000400005

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 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.     INTRODUCTIONZoonotic 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 28ºC in RPMI 1640 (Gibco, Pisley, UK) supplemented with 100IU/ml penicillin, 100µg/ml streptomycin, 2mM L-glutamine and 10% heat-inactivated fetal calf serum (FCS, Gibco). After reaching the stationary phase, the parasites were harvested, washed in phosphate-buffered saline (PBS), and lysed by repetitive freeze-thaw cycles until they were completely disintegrated as determined by microscopic inspection. The protein concentration in the lysed parasites was determined by bicinchoninic acid protein assay (Pierce, Rockford, IL, USA) (Lima et al. 2003).The antigen was coated to the plate overnight at 4ºC, with a protein concentration of 20 µg/ml (Riera et al. 1999) in carbonate buffer 0.005 M (pH 9.6), the plate was washed three times in PBS containing 0.05 % tween 20 (washing buffer) and saturated for 1 hour at room temperature with 100 µl/well of a mixture of PBS and 10% fetal calf serum. Then they were washed again three times with washing buffer, and blocking buffer/tween was added to each well and incubated at 37ºC for 3 hours at room temperature, followed by three washes with washing buffer. Blocking buffer/tween consisted of 100 µl of serum sample at a dilution of 1/400 in PBS, pH 7.4, containing 0.05% tween 20 and 10% fetal calf serum (Gibco). Subsequently, 100µl/well rabbit anti-dog IgG peroxidase conjugated (Sigma) or protein A peroxidase conjugate (Sigma) in an appropriate dilution in blocking buffer/tween was added, incubated at 37ºC for 1 hour at room temperature and washed.Substrate solution was added at 100µl/well and developed for 5 minutes at room temperature. The substrate solution consisted of 0.4 mg/ml O-phenylendiamine (Sigma) and 0.4 µl/ml H2O2 in phosphate citrate buffer, pH 5.0. The reaction was stopped with 50µl of H2SO4 3M and optical densities were measured at 490 hm using a microplate reader (Titertek Multiskan Plus MK II -Flow Laboratories International SA, Lugano, Switzerland). Negative and positive controls were included on each plate. The cut-off was determined using mean + 3 standard deviation of the readings obtained from serum of healthy dogs (n=20) from leishmaniasis non endemic areas (Voller et al. 1980). Paired comparisons between groups were carried out by Student's test.The canine sera tested were obtained from fifty symptomatic dogs from the leishmaniasis focus of Araçatuba (São Paulo, Brazil) with positive diagnosis by cytological examination of lymph node aspirates and from thirty-eight healthy adult dogs from non endemic areas.In addition, the cross-reaction between total antigen from promastigotes and sera colleted in non edemic area, taken from dogs with other confirmed infectious diseases, i.e., ehrlichiosis (n=15), toxoplasmosis (n=8), babesiosis (n=10) and dirofilariosis (n=20) was available.  RESULTS AND DISCUSSIONThe groups of sera were compared using protein A peroxidase conjugate anti-dog IgG peroxidase conjugate. As seen in figure 1, sera from dogs with visceral leishmaniasis had significantly higher absorbance values when reacted with protein A (P<0.0001). The absorbance values for sera from healthy animals (negative sera) did not differ significantly in the two detection methods (P=0.3202). The same was observed with the sera from other disease groups tested.      The results showed that all sera from positive animals by microscopical observation analyzed were positive for antibodies against Leishmania chagasi antigen, corresponding to a sensitivity of 100% in both methods tested.The higher optical density values observed in the ELISA assay with the use of protein A (Fig.1a) could be related to the detection not only of IgG, but also of IgM and IgA. The reactivity of protein A with immunoglobulins from dog is quite strong. Protein A reacts with all subclasses of IgG and partially reacts with dog IgA and IgM (Goudswaard et al. 1978).The carbohydrates present in parasite induce IgM production; this subclasse of Ig commonly plays a

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