Macrophages and lymphocytes support the development of Theileria equi pre-erythrocytic stages, but neither B nor T lymphocytes are required to establish infection In vivo

Abstract

Theileria equi is a tick-transmitted apicomplexan hemoprotozoan parasite that causes acute hemolytic disease (equine piroplasmosis) and persistent infection of wild and domestic equids worldwide. The life cycle of Theileria equi is biphasic in the horse, with a period of intraleukocyte development (pre-erythrocytic phase) followed by patent erythrocytic parasitemia. The specific phenotype of schizont-infected leukocytes is not defined for T. equi and represents a significant gap in our understanding of its pathogenesis. In closely related apicomplexan parasites of cattle (Theileria annulata and T. parva) the phenotype of infected leukocytes is well described and known to impact pathogenesis, adaptive immunity, and strain virulence. To resolve this gap in T. equi pathogenesis, PBMC were isolated from three immunocompetent horses and co-cultured with T. equi sporozoites. On day 9-14 post-inoculation (DPI) the immunophenotype of infected leukocytes was assayed by flow cytometry and IFA using a panel of three mAb for cell surface labeling [anti-IgM, anti-CD3, or anti-CD172a] and a second mAb to detect infection [anti-equine merozoite antigen (EMA ) 1/2 ]. Previous work has shown anti-EMA 1/2 mAb specifically labels the pre-erythrocytic stages of T. equi. To determine if B and T-lymphocytes are required to establish infection in vivo, two SCID foals were infected with T. equi sporozoites by intravenous inoculation and monitored for parasitemia by real time PCR. Flow cytometric analysis of in vitro infected PBMC showed a subset of cells in each culture were dual labeled with a leukocyte-specific mAb and anti-EMA 1/2 mAb. Further analysis demonstrated that a relatively high proportion of the B-lymphocytes and macrophages were infected (37% and 30%, respectively), as compared to the low proportion of T-lymphocytes (9%), suggesting that T. equi preferentially infects B-lymphocytes and macrophages. IFA performed on the same cultures verified that the dual labeled cells were infected with schizonts. IFA also demonstrated that signal generated by leukocyte-specific mAb was unevenly distributed and less abundant on infected cells. Infection of SCID foals with T. equi sporozoites resulted in patent merozoite parasitemia 11 and 13 DPI, and demonstrated that B and Tlymphocytes are not required to establish infection in vivo. The results of in vitro experiments demonstrate that the leukocyte tropism of T. equi is not limited to lymphocytes, as previously reported, but extends to included monocytes/macrophages. The establishment of infection in foals lacking B and T-lymphocytes (SCID foals) with sporozoites provides strong in vivo support of this finding. T. annulata has a similar host cell range, suggesting that the immune responses that control pre-erythrocytic T. annulata (i.e. cytostatic macrophages and cytotoxic T-cells) may also be important for controlling T. equi. This information will therefore help focus the search for host immune responses that can stop T. equi infection from progressing to the persistent erythrocytic stage.