Introduction - The Theileria parva Genome Database

Theileria parva is the causative agent of East Coast fever (ECF), an acute, tick-borne disease causing high rates of morbidity and mortality in cattle in 12 countries in sub-Saharan Africa (1). One million cattle die each year from ECF with annual economic costs estimated to be $168 million (2). As the livelihood of smallholder farms, often managed by women, depend on one or two cattle, the financial burden due to loss of income and livestock products impacts on the quality of all aspects of family life. Research at the International Livestock Research Institute (ILRI) in Nairobi, Kenya, one of 16 agricultural research institutes operated by the Consultative Group on International Agricultural Research (CGIAR), is aimed at the development of vaccines to control ECF. ILRI and TIGR sequenced the entire T. parva genome (3), primarily to assist in vaccine development, but also to learn more about the mechanisms used by the parasite to transform host lymphocytes, and to compare its genome to those of related organisms such as the malaria causing parasite Plasmodium. The genome of Theileria annulata was sequenced by the Wellcome Trust Sanger Institute (4).

T. parva is transmitted by the brown-ear tick Rhipicephalus appendiculatus (Figure 1). Sporozoites introduced into the host animal by the bite of an infected tick invade host lymphocytes (Figure 2), where they further develop into intracytoplasmic multinucleated schizonts. By mechanisms not completely understood, the presence of the parasite within the lymphocyte induces the malignant transformation of the host cell. The host cell and the schizont divide synchronously, resulting in the clonal expansion of the infected lymphocytes. Infected animals develop a lymphoma-like disorder that is rapidly fatal, with most animals dying within 3-4 weeks of infection. Some parasites form merozoites and are released into the bloodstream by rupture of the host cell, where they invade erythrocytes and develop into intra-erythrocytic forms called piroplasms. Ticks ingest the piroplasms during a blood meal. Following a sexual cycle in the gut, kinetes migrate to the salivary glands of the tick. Sporogony is initiated when the tick attaches to a host animal, resulting in the release of sporozoites into the salivary glands, ready for transmission to the host.

Cattle that naturally recover from infection or those immunized by infection and treatment, a process that involves simultaneous inoculation of cryopreserved sporozoites and a long acting tetracycline, exhibit long lasting immunity to a homologous parasite re-challenge (5). Several lines of evidence indicate that cellular immune responses, including CD8+ T cells (Figure 3), directed against the schizont infected lymphocytes are responsible for protection, but the protective antigens have not been identified. In addition to cellular immunity acquired by infection there is also an antibody response to sporozoite antigens (6), and an experimental subunit vaccine comprised of the major sporozoite surface protein p67 has been shown to reduce cases of severe ECF by 50%. These results suggest that it should be possible to construct practical subunit vaccines to control ECF.

Development of subunit vaccines, potentially the most effective method of ECF control, is a major goal of research conducted by ILRI. A major impediment facing researchers has been the difficulty of identifying parasite antigens that could serve as the target(s) of a protective immune response. Because T. parva is an intracellular parasite, conventional approaches to identification of parasite antigens have not been very successful. Prior to the genome project, only a handful of T. parva genes had been sequenced and no parasite antigens expressed in schizont-infected cells that were targets of cytotoxic T cell mediated protective immunity had been found. Using new in vitro assays developed at ILRI, and the T. parva genome sequence, several schizont stage antigens that are recognized by cytotoxic T cells from immune cattle have been identified. These antigens are being further characterized and tested for protective efficacy (E. Taracha, S. Graham, et al., submitted for publication).

A characteristic feature of T. parva infection in cattle is a lymphoma-like disorder caused by the induction of host cell proliferation by the intracellular schizont. The host cell and schizont divide in synchrony resulting in the clonal expansion of infected cells. Schizont infected cell lines behave like immortalized cells in vitro, exhibiting several phenotypes characteristic of cancerous cells (7). The capacity to cause host cell transformation is unique among eukaryotic pathogens, and is present in only some members of the genus. Thus, comparative studies with T. annulata, which exhibits this phenomenon mainly in bovine macrophages/monocytes and perhaps B cells (8), and the non-transforming species T. sergenti and T. mutans (1) should provide insights into induction and maintenance of cell-specific transforming events.

Association of the schizont with the host cell nuclear spindle ensures that daughter host cells remain infected during cytokinesis (9). Although schizont and host cell divide synchronously, schizont DNA synthesis occurs as the host cell enters mitosis and is immediately followed by division when the host cell is in metaphase (10). What drives host cells to proliferate and to maintain this phenotype is unknown but it is dependent on the parasite and thus it is reasonable to propose that the parasite usurps pathways controlling the host cell cycle. However, because the transformed phenotype can be reversed by treatment with antiparasitic agents (11), the alterations to the host cell apparently do not involve such permanent changes to the host cell genome such as mutations or chromosomal translocations. Considerable progress has been made towards the analysis of the pathways that regulate cell proliferation in schizont infected cells. These studies are discussed in a recent review (12). The complete T. parva genome sequence and associated microarray studies will assist in the identification of parasite gene products involved in the transformation of host lymphocytes.

Theileria parva is closely related to other apicomplexans that are major human pathogens including Plasmodium (malaria), Toxoplasma (toxoplasmosis), and Crytosporidium (cryptosporidiosis). There are approximately 250 million new cases of malaria and 2-4 million deaths due to malaria per year (13). Toxoplasma and Crytosporidium infections are especially dangerous to immunocompromised individuals. Comparison of the T. parva genome with the genomes of these related parasites will provide insights into many aspects of apicomplexan biology, including metabolic pathways, antigenic variation, mechanisms of host cell invasion, regulation of gene expression, and the roles of the plastid and mitochondria, etc. Information gleaned from these studies will contribute to the development of new drugs and vaccines for infectious diseases such as malaria.

Sequencing of the T. parva genome was supported by the TIGR Board of Trustees, ILRI, and J. Craig Venter. Additional support was provided by the U.K. Department for International Development, the U.S. Agency for International Development, and the Rockefeller Foundatation.

Figure 1. Life cycle of Theileria parva (1).

Figure 2. Immuno-electron micrograph of a sporozoite entering a bovine cell, stained with an antibody to p67, the sporozoite surface coat protein (Clive Wells, ILRI).

Figure 3. Live/dead assay of a schizont-infected cell mixed with parasite specific CTLs. The bovine CTLs are alive and stain green while the parasite infected cell is dead and stains red (Clive Wells, ILRI).