Seropositive dogs have significantly higher frequencies of T. cruzi -specific PBMCs and reactive T cell compartment. A, Interferon (IFN)-γ enzyme-linked immmunosorbent spot (ELISpot) well images of 4 × 105 PBMCs cultured with media, PMA/ionomycin (PMA/ion), or T. cruzi lysate (TcLysate/Tc) and 1 × 104 PBMCs stimulated with PMA/ionomycin (PMA/ion (10 000)). Individual dog identification (ID) and results of multiplex serology are denoted above wells. B-C, Numbers of IFNγ-producing PBMCs in the ELISpot assay responding to T. cruzi parasite lysate (B) or PMA (C) adjusted to 1 × 106 or per 1 × 104 PBMCs, respectively. Each square and dot represents an individual dog, while each diamond represents one of three US dog controls to ensure plate reproducibility. Dogs are grouped by multiplex serology negative (Sero -) or serology positive (Sero +) from Argentina or seronegative dogs from United States. Statistical significance was determined by one-way ANOVA using Prism v4.0c (GraphPad). Horizontal line, threshold for positive/negative response.