Immunogenic mapping of rDyn-1 and rKDDR-plus proteins and selection of oligopeptides by immunoblotting for the diagnosis of Leishmania infantum-infected dogs

Endemic in Brazil, visceral leishmaniasis (VL) is a zoonotic infection that is among the most important parasitic diseases transmitted by vectors. Dogs are the main reservoirs of canine leishmaniasis (CanL) and their identification is used in some countries as part of disease prevention and control measures in the canine and human population. In this context, serological tests are necessary, composed of antigens capable of correctly identifying infected dogs, minimizing the number of false-negative cases. This study aimed to identify more immunoreactive peptides derived from two previously described whole proteins (rDyn-1 and rKDDR-plus) and compare their performance to the control antigens rK39 and the crude extract for the detection of dogs infected with L. infantum, especially the asymptomatic ones. The three selected peptides and a mixture of them, along with the rDyn-1, rKDDR-plus, rK39, and crude extract antigens were evaluated using indirect ELISA with sera samples from 186 dogs with CanL, being asymptomatic (n = 50), symptomatic (n = 50), co-infected (n = 19), infected with Babesia sp. (n = 7), Ehrlichia sp. (n = 6), T. cruzi (n = 20) and uninfected (n = 34). The results showed that the rDyn-1 protein and the peptide mixture had the highest sensitivity (100% and 98.32%, respectively) and specificity (97.01 and 98.51, respectively). A high degree of kappa agreement was found for rDyn-1 protein (0.977), mixed peptides (0.965), rKDDR-plus protein (0.953), K-plus peptide 1 (0.930) and Dyn-1 peptide (0.893). The mixture of peptides showed the highest likelihood (65.87). The ELISA using the mixture of peptides and the rDyn-1 protein showed high performance for CanL serodiagnosis. More mix combinations of the peptides and additional extended field tests with a larger sample size are recommended. Author summaryCanine leishmaniasis (CanL) is a parasitic zoonotic infection of dogs caused by Leishmania spp. parasites. In Brazil, CanL is among the most important vector-borne parasitic diseases. Dogs are the main reservoirs of the disease and in some countries have been regularly culled as part of government policy to control human leishmaniasis. The identification of infected dogs, regardless of the clinical condition, is necessary as part of the prevention and control measures of the disease in the canine population and consequently in the human population. In this context, it is necessary serological tests, composed of antigens capable of correctly identifying infected, asymptomatic, as well as symptomatic dogs, minimizing the number of false negative cases. Therefore, this study aimed to compare the efficacy between two previously described whole proteins (rDyn-1 and rKDDR-plus) with their more immunoreactive synthetic peptides to sera from symptomatic and asymptomatic dogs selected in immunoblotting assays and to evaluate in ELISA assays. ELISA using the rDyn-1 protein and mixed peptides showed high performance in the diagnosis of CanL.