Prokaryotic Expression and Antigenic Characterization of Three RecombinantLeishmaniaAntigens for Serological Diagnosis of Canine Leishmaniasis

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Abstract
Three recombinant antigens ofLeishmania chagasi(=L. infantum) were expressed in prokaryotic systems and evaluated (using a panel of dog sera characterized by parasitological and serological immunofluorescent antibody test [IFAT] techniques) as diagnostic markers of infection. The whole open reading frame encoding K9, the gene fragment encoding the repetitive sequence of K26, and the 3′-terminal gene fragment encoding a single 39-amino-acid subunit of the kinesin-related protein K39 (K39sub) were amplified fromL. infantumDNA and cloned into a pGEX-2T expression vector in frame with glutathioneS-transferase (GST). The sensitivity and specificity of enzyme-linked immunosorbent assays (ELISAs) using K26 as an antigen (evaluated with sera from 20 parasitologically positive and 20 parasitologically negative dogs) were both 100% (95% confidence interval [CI] = 83.2 to 100). When K9 and K39sub were used, sensitivity was 95% (95% CI = 75.1 to 99.9) and specificity was 100% (95% CI = 83.2 to 100). Using 182 field sera, a good agreement was found between the recombinant K26 ELISA and IFAT (K= 0.92; 95% CI = 0.86 to 0.98) results and between the K9 and K39sub ELISA (used in parallel) and IFAT (K= 0.87; 95% CI = 0.80 to 0.95) results. The results demonstrate that each antigen carries immunodominant epitopes and that their combination may further increase the sensitivity of currently available serological tests.