Canine leishmaniosis (CanL) is a zoonotic disease caused by Leishmania infantum, where increased interferon-gamma (IFN-γ) levels are associated with controlling the infection and mild to moderate disease. Therefore, monitoring IFN-γ concentrations is essential for monitoring the immune responses in CanL. This study compared a faster, cost-effective IFN-γ release whole blood assay in tubes (WBA-T) with a standardized version (WBA-S) in 41 dogs at different states of L. infantum infection. WBA-T was performed at 24, 48, and 72 h of incubation with three conditions: blood, blood with L. infantum-soluble antigen (LSA), and blood with mitogen ConA. WBA-S was performed in plates, with blood diluted and incubated for five days using the same conditions. Supernatants (WBA-S) or plasma (WBA-T) were harvested for IFN-γ measurement by ELISA. No significant differences were observed in terms of IFN-γ concentration between WBA-T and WBA-S under LSA conditions. However, the 48 h incubation period during WBA-T showed the highest median of IFN-γ concentration compared to other incubation periods and WBA-S. The IFN-γ concentrations under ConA stimulation in WBA-S were significantly higher than in WBA-T at all incubation times studied. In conclusion, WBA-T stimulated with LSA at 48 h incubation time was shown to be the most appropriate for assessing IFN-γ production.

Most Leishmania infantum infected dogs remain healthy, with low parasitic loads and negative to low antibody levels, making detection challenging. rKDDR-plus has shown outstanding diagnostic performance in ELISA but has never been tested in dogs thoroughly characterized by physical examination and routine laboratory tests. This study aimed to evaluate the diagnostic performance of rKDDR-plus ELISA in dogs with different L. infantum infection states (n = 94), classified as healthy seronegative (n = 25), healthy seropositive (n = 54), and sick (n = 15). In total, 37 seropositive dogs were classified as low and 32 as high. Diagnostic performance was evaluated in five groups: (G1) all dogs, (G2) healthy seronegative + sick (n = 40), (G3) healthy seronegative + healthy seropositive (n = 79), (G4) healthy seronegative + high seropositive (n = 57), and (G5) healthy seronegative + low seropositive (n = 62). Negative percent agreement was 100% in all groups, while positive percent agreement was perfect in G2 (100%), followed by G4 (96.88%), G1 (82.61%), G3 (77.78%), and G5 (70.27%). Accuracy ranged from 85.14% to 100%, and the area under the ROC curve (AUC-ROC) was as follows: G2 (1.000), G4 (0.978), G1 (0.951), G3 (0.937), and G5 (0.928). Agreement (kappa) was almost perfect in G2 and G4, and substantial in the remaining groups. In conclusion, rKDDR-plus ELISA shows overall good diagnostic performance.