Development of a Simple IFN-γ Release Whole Blood Assay for the Assessment of Leishmania infantum Specific Cellular Immunity in Dogs

Canine leishmaniosis (CanL) is a zoonotic disease caused by a parasite that can be transmitted by a mosquito-like insect called a sandfly. In dogs, the disease can range from mild to very severe, depending on the state of the dog's immune system. For this reason, it is important to detect the infection early. Whole blood assays (WBAs) are easy tests that allow for the rapid detection of immunity in response to a pathogen. As a result, the WBA is an important diagnostic tool for immune monitoring in CanL. In dogs, however, few tests are available to monitor their immune response to pathogens. This study aims to fill this gap by adapting a human-derived WBA technique for canine use. We compare a novel, faster, and cost-effective WBA in tubes (WBA-T) with a standardized, longer version (WBA-S) in dogs at various stages of Leishmania infantum infection. The results obtained showed that WBA-T performed similarly to WBA-S. Therefore, by implementing the WBA-T technique, results can be obtained more quickly and simply, which favors the identification of infected animals at an earlier stage, permitting early control of the infection and thus contributing to minimizing this infectious disease. 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.