Generation of IFN-gamma-producing cells that recognize the major piroplasm surface protein in Theileria orientalis-infected bovines.

Theileriosis is a tick-borne protozoan disease caused by Theileria species. The Theileria species are classified into two groups depending on the cell type in which they proliferate and the clinical symptoms. The first group consists of lymphoproliferative Theileria species (T. parva and T. annulata), which mainly proliferate in lymphocytes, causing uncontrolled lymphocyte proliferation. The other group consists of a nonlymphoproliferative Theileria species (T. orientalis, also known as T. sergenti) that proliferates in erythrocytes and causes hemolytic anemia. Based on reports of generation of antigen-specific CD4(+) and CD8(+) T cells in lymphoproliferative theileriosis, we investigated whether T cells specific to the T. orientalis antigen are present in the nonlymphoproliferative form of the disease. In this study, we developed a new assay based on an enzyme-linked immunospot (ELISpot) to detect interferon-gamma (IFN-gamma)- and interleukin-10 (IL-10)-secreting cells in a series of cryogenically preserved bovine peripheral blood mononuclear cells (PBMCs). We first determined that IFN-gamma- and IL-10-secreting T cells were present in PBMCs by stimulating them with phytohemagglutinin L (PHA-L=red kidney bean lectin L, known as T cell stimulator), and then determined whether T. orientalis-specific T cells are present in T. orientalis-infected bovines. Peptides derived from T. orientalis major piroplasm surface protein (MPSP) were used as a T. orientalis-specific stimulator in the ELISpot assay, and peptides from glycoprotein B (gB) of the bovine herpes virus-1 (BHV-1) were used as a BHV-1-specific stimulator as a control for monitoring the immune response. Compared with results obtained using the BHV-1 (gB peptides)-specific IFN-gamma ELISpot assay to assess BHV-1-immunized Holsteins, prominent T. orientalis MPSP peptide-specific IFN-gamma and IL-10 positive spots were detected in T. orientalis-infected Holsteins but weak positive responses were exhibited by T. orientalis-infected Angus and Japanese Black cattle. As far as we are aware, this is the first report to show direct evidence of the presence of T. orientalis-specific T cells in T. orientalis-infected bovines using an antigen-specific ELISpot assay system and that T. orientalis-specific, IFN-gamma- and IL-10-producing T cells are produced in T. orientalis-infected Holsteins. Copyright 2010 Elsevier B.V. All rights reserved.