Doreen Beyer

Characterization of a polymorphic Theileria annulata surface protein (TaSP) closely related to PIM of Theileria parva: implications for use in diagnostic tests and subunit vaccines.

Theileria annulata is a tick-transmitted protozoan that causes tropical theileriosis, an often fatal leukoproliferative disorder of cattle. To characterize and identify parasite proteins suitable as diagnostic antigens and/or vaccine candidates, a cDNA clone encoding a macroschizont stage protein was isolated and characterized (here designated TaSP). The gene, present as a single copy within the parasite genome, is transcribed in the sporozoite and schizont stage and codes for a protein of about 315 amino acids, having a predicted molecular weight of 36 kDa. Allelic variants were found within single parasite isolates and between isolates originating from different geographical regions. The N-terminal part contains a predicted signal peptide and the C-terminal section encodes membrane-spanning regions. Comparison of a number of cDNA clones showed that both these sequence regions are conserved while the central region shows both size and amino acid sequence polymorphism. High identity of the N- and C-terminal regions with the polymorphic immunodominant molecule (PIM) of Theileria parva (identity of 93%), the existence of a central polymorphic region and two short introns within genomic clones suggest that the presented gene/protein may be the T. annulata homologue of PIM. However, the central region of TaSP has no significant identity with PIM, contains no repetitive peptide motifs and is shorter, resulting in a lower molecular weight. The existence of the predicted secretion signal peptide and membrane spanning regions suggest that TaSP is located at the parasite membrane.

Application of the recombinant Theileria annulata surface protein in an indirect ELISA for the diagnosis of tropical theileriosis

The recombinant surface protein of Theileria annulata (TaSP) was used in the standardization and validation of an enzyme linked immunosorbent assay (ELISA) for the detection of circulating antibodies against tropical theileriosis. ELISA data were expressed as the percentage positivity (PP) of the reactivity of an internal positive control. A total of 50 sera samples from a disease-free area were used for the calculation of the cut-off value which served as a threshold between the positive and the negative sera samples. This was determined as the mean PP plus two standard deviations or the twice the mean PP of the results obtained with these negative samples. The obtained thresholds were 17.8% and 18.3%, respectively. Accordingly, the reactivity of 140 field sera samples collected at random from an area known to be endemic for tropical theileriosis in Sudan was determined as PP values which were then compared to the results obtained using the indirect fluorescence antibody test (IFAT) from the same samples. Both tests showed a high degree of correlation. The TaSP-ELISA had a sensitivity of 99.1% and specificity of 90.47% when taking the IFAT as a reference test. Our test has proved its suitability for the diagnosis of tropical theileriosis and could be used in serological surveys to map out the prevalence of the disease or to monitor vaccination efficiencies in disease-free populations.

Development of a recombinant indirect ELISA for the diagnosis of Theileria sp. (China) infection in small ruminants

Theileria sp. (China) causes severe limitations on the development of the livestock industry in the north-west of China. An enzyme-linked immunosorbent assay (ELISA) based on merozoite homogenate of the parasite for diagnosis of infection has been established; however, cross-reactivity with other small ruminant-infecting piroplasms could not be excluded. Thus, a prerequisite for epidemiological surveys and diagnosis was the establishment of a recombinant protein-based ELISA. To this end, serum from Theileria sp. (China)-infected sheep was used to screen a Theileria lestoquardi expression library, resulting in the identification of a specifically reacting clone with a high identity to the heat shock protein 70 (HSP70) of Theileria parva and Theileria annulata and thus named TlHSP70. An HSP70 homologue was also confirmed to be expressed by Theileria sp. (China) merozoites (TcHSP70). A part of the TlHSP70 protein, found to be conserved in TcHSP70, was recombinantly expressed and used to establish an ELISA. A total of 260 field serum samples tested resulted in a sensitivity and specificity of 94.3 and 89.5%, respectively, in comparison with the merozoite homogenate ELISA. The potentials of the application of the test in epidemiological surveys to map out the prevalence of the disease and for routine diagnostics are described.

Schizonts of Theileria annulata interact with the microtubuli network of their host cell via the membrane protein TaSP

Intracellular leukoproliferative Theileria are unique as eukaryotic organisms that transform the immune cells of their ruminant host. Theileria utilize the uncontrolled proliferation for rapid multiplication and distribution into host daughter cells. The parasite distribution into the daughter cells is accompanied by a tight association with the host cell mitotic apparatus. Since the molecular basis for this interaction is largely unknown, we investigated the possible involvement of the immunodominant Theileria annulata surface protein, TaSP, in the attachment of the parasite to host cell microtubule network. Confocal microscopic analyses showed co-localization of the TaSP protein with alpha-tubulin and reciprocal immuno-co-precipitation experiments demonstrated an association of TaSP with alpha-tubulin in vivo. In addition, the partially expressed predicted extracellular domain of TaSP co-localized with the mitotic spindle of dividing cells and was co-immunoprecipitated with alpha-tubulin in transiently transfected Cos-7 cells devoid of other T. annulata expressed proteins. Pull-down studies showed that there is a direct interaction between TaSP and polymerized microtubules. Analysis of the interaction of TaSP and host microtubulin during host cell mitosis indicated that TaSP co-localizes and interacts with the spindle poles, the mitotic spindle apparatus and the mid-body. Moreover, TaSP was demonstrated to be localized to the microtubule organizing center and to physically interact with gamma-tubulin. These data support the notion that the TaSP—microtubule interaction may be playing a potential role in parasite distribution into daughter host cells and give rise to the speculation that TaSP may be involved in regulation of microtubule assembly in the host cell.

Identification of antigenic proteins of a Theileria species pathogenic for small ruminants in China recognized by antisera of infected animals.

Abstract: The antigenic proteins of the piroplasm stage of Theileria species (China), the causative agent of theilerosis of small ruminants in China, were analyzed by Western blot, revealing several specific immunoreactive proteins of different predicted molecular weights. Furthermore, sera from Theileria species (China)‐infected animals were probed for reactivity with the TaSP protein of T. annulata, for which a homologue has been described in Theileria species (China). Affinity chromatography demonstrated the presence of TaSP‐ reactive antibodies, and the majority of the sera showed reactivity with this protein both in Western blots and in ELISA. The identified parasite antigens and TaSP will be assessed for their suitability for developing diagnostic methods as well as evaluated for their capacity to stimulated host immune competent cells.

Influence of subculturing on gene expression in a Theileria lestoquardi-infected cell line

In this study potential molecular markers for identification of attenuation in a Theileria lestoquardi-infected cell line to be used in vaccination trials were identified. Two markers associated with attenuation in Theileria annulata vaccine strains were analyzed (metalloproteinase activity and TNF? mRNA expression). The result showed a decreased activity of MMP 9 and decreased mRNA expression of TNF? with increasing passage number. Suppression subtractive hybridization was used to identify potential new markers of attenuation. Random screening revealed nine differentially expressed genes, one from the parasite and eight from the host. Quantitative real time-PCR confirmed mRNA expression of the parasite vacuolar H+ATPase to be downregulated at higher passages.

Initiation of translation and cellular localization of Theileria annulata casein kinase IIα: Implication for its role in host cell transformation

Theileria annulata and T. parva are protozoa that infect bovine leukocytes which leads to subsequent transformation and uncontrolled proliferation of these cells. It has been proposed that the CKIIα subunit of T. parva induces mitogenic pathways of host leukocytes by being exported into the host cell. The evidence for this is the existence of a predicted N‐terminal secretion signal‐like peptide. We tested this hypothesis by analyzing gene structure, translation, and protein localization of the T. annulata CKIIα (TaCKIIα). The determined TaCKIIα‐ORF potentially codes for a 50 kDa protein with an N‐terminal extension including a possible signal sequence not present in CKIIα proteins of non‐Theileria species. However, antisera raised against TaCKIIα recognized a protein of a molecular weight of about 40 kDa and, therefore, inconsistent with this predicted molecular weight. We demonstrate by in vitro transcription/translation that this discrepancy is due to translation from a downstream initiation site omitting the putative N‐terminal signal sequence and thus excluding the notion that the protein product is secreted via the classical secretory pathway. In corroboration immunofluorescence investigations suggest that the TaCKIIα subunit is confined to the parasite schizonts within the host cell. On the basis of the above findings it seems highly unlikely that export via the classical pathway of the parasite CKIIα is the way in which this protein possibly contributes to host cell transformation. J. Cell. Physiol. 196: 444–453, 2003.

Immune Response of Theileria sp.‐infected Sheep to Recombinant Theileria Proteins

Sheep and goats in northwest China suffer from theileriosis from infection with Theileria sp. (China), resulting in large economic losses. To investigate the immune response to infection with Theileria sp. (China), parameters of cellular and humoral immunity of experimentally infected sheep against two recombinantly expressed Theileria proteins were investigated. The in vitro proliferative response of blood mononuclear cells to a recombinant T. annulata membrane protein and a recombinant Theileria sp. (China) homologue to T. annulata surface protein, both putative membrane proteins, was significantly elevated and significant amounts of specific immunoglobulins were produced against both.