Analysis of schizont-infected PBMC in vitro. The capacity of T. equi (Florida) to infect PBMC in vitro was assessed by light microscopy (A, B) and IFA (C, D). Fresh PBMC from adult Arabian horses were cocultured with tick salivary gland homogenates containing T. equi sporozoites. Infected and uninfected control PBMC were sampled daily for cytospin preparation and Diff-Quick staining [representative photomicrographs of uninfected control cultures (A) and T. equi infected cultures (B) on day 9]. Panel B includes three schizont-infected leukocytes, with multiple, oval to round, 1–2 μm diameter, purple nuclei (developing merozoites). To confirm the intracytoplasmic organisms were T. equi, uninfected control and infected cultures were labeled with antibody specific for equine merozoite antigens 1 and 2 [mAb 36/133.97 (anti-EMA 1/2)]. In the infected culture wells (D), intracytoplasmic schizonts and developing merozoites were specifically labeled with anti-EMA 1/2 (secondary goat anti-mouse IgG1 conjugated with FITC-green; Nuclear stain = DAPI). Cells from the uninfected control cultures were not labeled with anti-EMA 1/2 (representative data in panel C). Scale bar = 10 μm.