Teste de ELISA indireto para diagnóstico sorológico de leishmaniose visceral em canídeos silvestres

Abstract

In South America, some wild canids are considered natural reservoirs of Leishmania chagasi. The immunological response of wild canids to Leishmania is not well understood, and the development of diagnostic methods is necessary for such purpose. In the present study, the standardization of an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of visceral leishmaniasis (VL) in Brazilian species of wild canids is described. Serum and plasma samples from 12 captive wild canids were studied: seven from maned wolves (Chrysocyon brachyurus), three from hoary foxes (Lycalopex vetulus), and two from crab-eating foxes (Cerdocyon thous). Samples from C. brachyurus and L. vetulus, both captive in an endemic area for VL, presenting clinical disease and positivity in Indirect Immunofluorescence Reaction and Polymerase Chain Reaction tests were used as positive controls. The antibody anti-dog IgG and Protein A, both conjugated with horseradish peroxidase, were compared in indirect ELISA tests which detected four (04/12) and three (03/12) seropositive C. brachyurus for anti-Leishmania antibodies, respectively. The ELISA tests were able to clearly distinguish negative from positive samples, as the mean optical density (OD) of the negative samples was 4.8 and 15.5 times lower than those of the positive ones either using anti-dog IgG and Protein A, respectively. Samples from three ELISA -positive C. brachyurus were analyzed by Western blotting and identified immunodominant bands of 19, 22, 24, 45 and 66 kDa, among 22 protein bands detected. The ELISAs with protein A and anti-dog IgG showed respectively excellent (Kappa = 1.0; p<0.001) and moderate (Kappa = 0.8; p<0.0015) agreement with the Western blotting assay. The ELISA tests showed to be adequate for screening studies to identify antibody responses, thus indicating contact with Leishmania infection by wild canids.INDEX TERMS: Wild canids, Leishmania, visceral leishmaniasis, ELISA, protein A.

New insights on severe clinical manifestations and deaths from visceral leishmaniasis in free-living crab-eating foxes (Cerdocyon thous) in Brazil

“…Molecular diagnosis by PCR identified L. infantum DNA in the animals assessed in our study and therefore confirms the occurrence of VL in foxes (Gontijo and Melo 2004). Likewise, some studies have already demonstrated the existence of a wild VL cycle, and the species C. thous as a potential reservoir for the parasite (Kruse et al 2004;Gomes et al 2007;Luppi et al 2008;Oliveira et al 2012;Ferreira et al 2013;Almeida et al 2018). Several factors can be associated with the occurrence of Leishmania among foxes.…”