Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo

Abstract

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

Figure 1. Acute parasitemia after IV sporozoite inoculation and tick-transmission.

A. Course of parasitemia (parasites/ml whole blood) for horses infected with T. equi sporozoite (∼1×10⁵–10⁶) by intravenous inoculation or by tick-transmission was assessed by quantitative real time PCR. Levels of peak parasitemia (B) and days to peak parasitemia (C) between groups were compared using a t test with α = 0.05. DPI, days post inoculation.

Figure 2. In vitro PBMC infection with T. equi sporozoites.

T. equi (Florida strain) infected PBMC day 9-11 in culture; Diff-Quick stained, cytospin preparation (A), and IFA (B, C, and D). (A) Schizont-infected cells have few to abundant, ∼0.5–1.0 µm diameter, purple, intracytoplasmic parasite nuclei (arrow). Adjacent to schizont-infected cells there are several extracellular merozoites (asterix), schizonts (arrowhead), and cellular debris. (B, C and D) IFA images of cells containing different schizont forms are labeled with anti-EMA 1/2 (green). (B) Infected cell is surface labeled with anti-IgM mAb (B lymphocyte). (C, D) Infected cells did not express detectable leukocyte specific surface markers. Nuclei are stained blue with DAPI.

Figure 3. Immunophenotype of schizont-infected PBMC in vitro.

Flow cytometric (A) and IFA (B) analysis of schizont-infected cells in vitro. (A) Representative flow cytometric data for infect horse H2. Left column: percent of total PBMC dual labeled with leukocyte specific mAbs and T. equi specific mAb (anti-EMA 1/2). Right column: percent infected cells determined on IgM⁺ (B lymphocyte), CD3⁺ (T lymphocyte), or CD172a⁺ (macrophage) gated leukocytes. (B) IFA images of schizont-infected cells dual-labeled with one of the three leukocyte specific mAb and anti-EMA 1/2. The nuclei of all cells were stained blue with DAPI. The anti-EMA 1/2 mAb either formed diffuse signal throughout the cytoplasm of infected cells or discrete signal along the surface of intracytoplasmic macroschizonts. Rare cells had punctate foci of EMA 1/2⁺ immunoreactivity along their outer margin (top left panel; arrow). The space adjacent to cells multifocally contained scant, irregular to round, immunoreactive debris (left panels of second and third row; arrowhead). The anti-IgM, anti-CD3, and anti-CD172a mAb formed punctate to diffuse signal along the outer surface of infected cells. Labeling with an isotype control for the anti-EMA 1/2 mAb did not form any detectable signal (representative data shown in the bottom panels of B).

Figure 4. SCID foal infection with cryopreserved sporozoites.

Course of parasitemia for SCID foals infected with sporozoites by intravenous inoculation. SCID1 = 4×10⁶; SCID2 = 6×10⁶ sporozoites.

Figure 5. Prepatent period: sporozoite vs. merozoite inoculation.

Days to first detectable parasitemia for immunocompetent and SCID foals inoculated intravenously with sporozoites was compared with that for historical SCID controls inoculated with merozoite-parasitized erythrocyte stabilates , , using a t test (α = 0.05).

Figure 6. Immunophenotype of immunocompetent and SCID foal PBMC containing T. equi antigen ex vivo.

PBMC from Foal1 (48 DPI) and SCID1 (15 DPI) were analyzed post-infection by flow cytometry to determine the percentage of CD2⁺, CD8⁺, CD4⁺, and CD172a⁺ cells that contained T. equi antigen (EMA 1/2).