Research Article: Lymphocytes and Macrophages Are Infected by Theileria equi, but T Cells and B Cells Are Not Required to Establish Infection In Vivo

Date Published: October 7, 2013

Publisher: Public Library of Science

Author(s): Joshua D. Ramsay, Massaro W. Ueti, Wendell C. Johnson, Glen A. Scoles, Donald P. Knowles, Robert H. Mealey, Ulrike Gertrud Munderloh.


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.

Partial Text

Theileria equi is a tick-transmitted apicomplexan hemoprotozoan parasite that causes acute hemolytic disease (equine piroplasmosis) and persistent infection of wild and domestic equids throughout the world [1], [2]. The life cycle of Theileria equi is biphasic in the mammalian host, with a period of intraleukocyte development (pre-erythrocytic schizogony) followed by patent erythrocytic parasitemia [3], [4]. The pre-erythrocytic stage of T. equi has not been associated with clinical disease in equids and relatively little work has been done to characterize host-parasite interaction during this phase of infection. In vitro and in vivo, tick-transmitted T. equi sporozoites infect mononuclear leukocytes and differentiate into multinucleated schizonts (schizogony), which further divide to form erythroinvasive merozoites [4]. Based on morphology, schizont-infected cells have been characterized as lymphocytes, but this finding has not been confirmed [3], [4]. Conversely, the leukocyte tropism is very well described for two close relatives of T. equi, Theileria annulata and Theileria parva[5]. The aforementioned Theileria spp. are bovine pathogens that infect both leukocytes and erythrocytes; however, unlike T. equi, the diseases caused by T. annulata (Tropical Theileriosis) and T. parva (East Coast Fever) are largely due to the transformation and dissemination schizont-infected leukocytes and lymphoproliferation [6]–[9].

In previous studies, T. equi schizont-infected cells have been characterized in vitro and ex vivo as lymphocytes based on their microscopic and ultrastructural morphology [4], [41], [42]. The subtle differences between lymphocyte and monocyte/macrophage morphology (e.g. amount of cytoplasm, nuclear shape) are, however, distorted by intracytoplasmic schizont development and precludes determining the specific identity of infected cells. The present study was designed to resolve the phenotype of schizont-infected cells in vitro and determine if lymphocytes were required to establish infection in vivo. Flow cytometric analysis of PBMC infected in vitro demonstrated that T. equi infected B lymphocytes, T lymphocytes, and monocyte/macrophages. Subsequent immunefluorescent microscopic analysis confirmed that the vast majority of cells labeled with T. equi antigen specific mAb contained schizonts within their cytoplasm; however, there were rare cells that had small, punctate foci of immunoreactivity with the EMA 1/2 mAb along the outer margin of the cell. Since all cultures examined contained merozoites released from infected leukocytes, it could not be determined if these small foci of immunoreactivity were sporozoites arrested during initial invasion, merozoites attached to the host cell membrane, or simply immunoreactive debris within the culture. Regardless, these findings demonstrate for the first time that the host cell range of T. equi extends to include both lymphocytes and monocyte/macrophages, making it the most promiscuous among related Theileria spp. (T. annulata and T. parva).