Annulata-infected Cells Research Articles

Cimicifugin, a broad-spectrum inhibitor of Theileria annulata and Plasmodium falciparum CDK7.

Cyclin-dependent kinase 7 is an attractive therapeutic target for the treatment of cancers, and a previous report suggested that Plasmodium falciparum CDK7 is a potential drug target for developing new anti-malarial drugs. In this study, we aimed to characterize and evaluate the drug target potential of Theileria annulata CDK7. Theileria annulata is responsible for tropical theileriosis, which induces a phenotype similar to cancerous cells like immortalization, hyperproliferation, and dissemination. Virtual screening of the MyriaScreen II library predicted 14 compounds with high binding energies to the ATP-binding pocket of TaCDK7. Three compounds (cimicifugin, ST092793, and ST026925) of these 14 compounds were non-cytotoxic to the uninfected bovine cells (BoMac cells). Cimicifugin treatment led to the activation of the extrinsic apoptosis pathway and induced autophagy in T. annulata-infected cells. Furthermore, cimicifugin also inhibited the growth of P. falciparum, indicating that it has both anti-theilerial and anti-malarial activities and that TaCDK7 and PfCDK7 are promising drug targets.

Comparative transcriptional analysis identifies genes associated with the attenuation of Theileria parva infected cells after long-term in vitro culture

Autologous administration of attenuated Theileria parva-infected cells induces immunity to T. parva in cattle. The mechanism of attenuation, however, is largely unknown. Here, we used RNA sequencing of pathogenic and attenuated T. parva-infected T-cells to elucidate the transcriptional changes underpinning attenuation. We observed differential expression of several host genes, including TRAIL, PD-1, TGF-β and granzymes that are known to regulate inflammation and proliferation of infected cells. Importantly, many genes linked with the attenuation of the related T. annulata-infected cells were not dysregulated in this study. Furthermore, known T. parva antigens were not dysregulated in attenuated relative to pathogenic cells, indicating that attenuation is not due to enhanced immunogenicity. Overall this study suggests that attenuation is driven by a decrease in proliferation and restoration of the inflammatory profile of T. parva-infected cells. Additionally, it provides a foundation for future mechanistic studies of the attenuation phenotype in Theileria-infected cells.

Epidrugs: alternative chemotherapy targeting Theileria annulata schizont stage parasites.

The growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata-infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC50) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata-infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCETheileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata, contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper's significance in advancing veterinary science and improving livestock health globally.

Activation of telomerase activity and telomere elongation of host cells by Theileria annulata infection.

Theileria annulata-transformed cells share many phenotypes with cancer cells, including uncontrolled proliferation, immortalization, and dissemination. Telomeres are DNA-protein complex at the end of eukaryotic chromosomes that function to maintain genome stability and cell replicative capacity. Telomere length maintenance is primarily dependent on telomerase activity. In up to 90% of human cancer cells, telomerase is reactivated through expression of its catalytic subunit TERT. However, the effect of T. annulata infection on telomere and telomerase activity in bovine cells has not yet been described. In the present study, we confirmed that telomere length and telomerase activity are upregulated after T. annulata infection in three types of cell lines. This change depends on the presence of parasites. After eliminating Theileria from cells with antitheilerial drug buparvaquone, telomerase activity and the expression level of bTERT were decreased. In addition, inhibition of bHSP90 by novobiocin led to decreased AKT phosphorylation levels and telomerase activity, indicating that the bHSP90-AKT complex is a potent factor modulates telomerase activity in T. annulata-infected cells.

Metabolomic profiling of bovine leucocytes transformed by Theileria annulata under BW720c treatment

BackgroundWhen Theileria annulata infects host cells, it undertakes unlimited proliferation as tumor cells. Although the transformed cells will recover their limited reproductive characteristics and enter the apoptosis process after treatment with buparvaquone (BW720c), the metabolites and metabolic pathways involved are not clear.MethodsThe transformed cells of T. annulata were used as experimental materials, and the buparvaquone treatment group and DMSO control group were used. Qualitative and quantitative analysis was undertaken of 36 cell samples based on the LC–QTOF platform in positive and negative ion modes. The metabolites of the cell samples after 72 h of drug treatment were analyzed, as were the different metabolites and metabolic pathways involved in the BW720c treatment. Finally, the differential metabolites and metabolic pathways in the transformed cells were found.ResultsA total of 1425 metabolites were detected in the negative ion mode and 1298 metabolites were detected in the positive ion mode. After drug treatment for 24 h, 48 h, and 72 h, there were 56, 162, and 243 differential metabolites in negative ion mode, and 35, 121, and 177 differential metabolites in positive ion mode, respectively. These differential metabolites are mainly concentrated on various essential amino acids.ConclusionBW720c treatment induces metabolic disturbances in T. annulata-infected cells by regulating the metabolism of leucine, arginine, and l-carnitine, and induces host cell apoptosis.Graphical abstract

Theileria annulata surface protein (TaSP) is a target of cyclin-dependent kinase 1 phosphorylation in Theileria annulata-infected cells.

Leucoproliferative Theileria parasites possess the unique capability to transform their bovine host cell, resulting in tumour-like characteristics like uncontrolled proliferation. The molecular mechanisms underlying this parasite-dependent process are only poorly understood. In the current study, bioinformatic analysis of the Theileria annulata surface protein (TaSP) from different T.annulata isolates identified a conserved CDK1 phosphorylation motif T131 PTK within the extracellular, polymorphic domain of TaSP. Phosphorylation assays with radioactively labelled ATP as well as ELISA-based experiments using a phospho-threonine-proline (pThr-Pro) antibody revealed, that CDK1-cyclin B specifically phosphorylates T131 , identifying TaSP as a substrate in vitro. Confocal microscopy and proximity ligation assays suggest an interaction between CDK1 and TaSP in T.annulata-infected cells. Further studies demonstrated a nearly complete co-localization of the pThr-Pro signal and TaSP only in cells in interphase, pointing towards a cell cycle-dependent event. Immunostainings of isolated, non-permeabilized schizonts confirmed the presence of the pThr-Pro epitope on the schizont's surface. Lambda phosphatase treatment abolished the pThr-Pro signal of the schizont, which was reconstituted by the addition of CDK1-cyclin B. Treatment of T.annulata-infected cells with the CDK1 inhibitor purvalanol A resulted in morphological changes characterized by tubulin-rich cell protrusions and an extension of the schizont, and a dose-dependent reduction of BrdU incorporation and Ki67 staining of T.annulata-infected cells, demonstrating a clear impact on the Theileria-dependent proliferation of the bovine host cell. Our data reveal the parasite surface protein TaSP as a target for the host cell kinase CDK1, a major player during cell division. Targeting the uncontrolled proliferation of Theileria-infected cells is a novel and reasonable approach to limit parasite load in order to facilitate a successful cellular immune response against the parasite.

Characterization of HSP90 isoforms in transformed bovine leukocytes infected with Theileria annulata.

SummaryHSP90 chaperones are essential regulators of cellular function, as they ensure the appropriate conformation of multiple key client proteins. Four HSP90 isoforms were identified in the protozoan parasite Theileria annulata. Partial characterization was undertaken for three and localization confirmed for cytoplasmic (TA12105), endoplasmic reticulum (TA06470), and apicoplast (TA10720) forms. ATPase activity and binding to the HSP90 inhibitor geldanamycin were demonstrated for recombinant TA12105, and all three native forms could be isolated to varying extents by binding to geldanamycin beads. Because it is essential, HSP90 is considered a potential therapeutic drug target. Resistance to the only specific Theileriacidal drug is increasing, and one challenge for design of drugs that target the parasite is to limit the effect on the host. An in vitro cell culture system that allows comparison between uninfected bovine cells and the T. annulata‐infected counterpart was utilized to test the effects of geldanamycin and the derivative 17‐AAG. T. annulata‐infected cells had greater tolerance to geldanamycin than uninfected cells yet exhibited significantly more sensitivity to 17‐AAG. These findings suggest that parasite HSP90 isoform(s) can alter the drug sensitivity of infected host cells and that members of the Theileria HSP90 family are potential targets worthy of further investigation.

Intracellular Theileria annulata Promote Invasive Cell Motility through Kinase Regulation of the Host Actin Cytoskeleton

The intracellular, protozoan Theileria species parasites are the only eukaryotes known to transform another eukaryotic cell. One consequence of this parasite-dependent transformation is the acquisition of motile and invasive properties of parasitized cells in vitro and their metastatic dissemination in the animal, which causes East Coast Fever (T. parva) or Tropical Theileriosis (T. annulata). These motile and invasive properties of infected host cells are enabled by parasite-dependent, poorly understood F-actin dynamics that control host cell membrane protrusions. Herein, we dissected functional and structural alterations that cause acquired motility and invasiveness of T. annulata-infected cells, to understand the molecular basis driving cell dissemination in Tropical Theileriosis. We found that chronic induction of TNFα by the parasite contributes to motility and invasiveness of parasitized host cells. We show that TNFα does so by specifically targeting expression and function of the host proto-oncogenic ser/thr kinase MAP4K4. Blocking either TNFα secretion or MAP4K4 expression dampens the formation of polar, F-actin-rich invasion structures and impairs cell motility in 3D. We identified the F-actin binding ERM family proteins as MAP4K4 downstream effectors in this process because TNFα-induced ERM activation and cell invasiveness are sensitive to MAP4K4 depletion. MAP4K4 expression in infected cells is induced by TNFα-JNK signalling and maintained by the inhibition of translational repression, whereby both effects are parasite dependent. Thus, parasite-induced TNFα promotes invasive motility of infected cells through the activation of MAP4K4, an evolutionary conserved kinase that controls cytoskeleton dynamics and cell motility. Hence, MAP4K4 couples inflammatory signaling to morphodynamic processes and cell motility, a process exploited by the intracellular Theileria parasite to increase its host cell's dissemination capabilities.

Identification and Characterization ofTheileria annulataHeat-Shock Protein 90 (HSP90) Isoforms

Heat-shock proteins (HSPs) refer to a group of proteins whose synthesis is enhanced upon sudden increase in temperature or exposure to a variety of other stressors. In this study, Theileria annulata (T. annulata) HSP90 was identified and characterized as a first step to understand the function of this molecule in T. annulata-infected cells. Our results indicated the existence in the genome of T. annulata of two HSP90 genes: one located in chromosome one (TaHSP90-Chr1) and the other in chromosome four (TaHSP90-Chr4). The amino acid alignment between the two isoforms has shown identity and similarity values of 23.52% and 30.26%, respectively. Theileria annulata recombinant HSP90 proteins were expressed using a bacterial expression system and could be recognized in Western blots by rabbit anti-serum raised against an antigenic peptide derived from a unique sequence of TaHSP90-Chr1. On the other hand, bovine HSP90 was detected in T. annulata-infected cells using Western blot and immunocytostaining. To demonstrate the effect of the inhibition of HSP90 on the survival of T. annulata-infected cells, Geldanamycin (GA), a specific inhibitor for HSP90, was used. Upon GA treatment, p53 was observed to translocate into the host cell nucleus, a phenomenon that occurs in cells undergoing apoptosis. Using flowcytometry, a significant increase (P = 0.028) in cell death (%) was observed in T. annulata-infected cells treated with two different GA concentrations, 0.5 and 1 μm, and incubated for 24, 48 and 72 h.

Study on Cryo-preservation of Theileria annulata Schizont Infected Cell Line Vaccine Strain S15

The p urpose of this study was to examine the potential application of Sorbitol, Sucrose and Gelatin in combination with glycerol in order to Theileria annulata infected cell line cryo-preservation. The efficiency of the freezing methods was compared by assessing the viability and plating efficiency of cells after resuscitation, dilution and equilibration in cell culture medium. As a result, the conventional cryopreservative medium containing 5% PEG treated Bovine Serum and 10% glycerol successfully cryopreserved cell as efficiently as the standard medium containing 80% FBS and 10% glycerol and was superior to all thirteen different experimental cryo-preservative media. A crucial result was obtained among the thirteen different serum-free media which showed that the 0.2M sorbitol+10% glycerol method yielded cells with viability of 54% and good plating efficiency results. It was concluded that the conventional method would be the best and ideal for the cryo-preservation and storage of Theileria annulata-infected cells for vaccine purposes to date.

The protozoan parasite Theileria annulata alters the differentiation state of the infected macrophage and suppresses musculoaponeurotic fibrosarcoma oncogene (MAF) transcription factors

The tick-borne protozoan parasite Theileria annulata causes a debilitating disease of cattle called Tropical Theileriosis. The parasite predominantly invades bovine macrophages (mϕ) and induces host cell transformation by a mechanism that has not been fully elucidated. Infection is associated with loss of characteristic mϕ functions and phenotypic markers, indicative of host cell de-differentiation. We have investigated the effect of T. annulata infection on the expression of the mϕ differentiation marker c-maf. The up-regulation of c-maf mRNA levels observed during bovine monocyte differentiation to mϕ was suppressed by T. annulata infection. Furthermore, mRNA levels for c-maf and the closely related transcription factor mafB were significantly lower in established T. annulata-infected cell-lines than in bovine monocyte-derived mϕ. Treatment of T. annulata-infected cells with the theileriacidal drug buparvaquone induced up-regulation of c-maf and mafB, which correlated with altered expression of down-stream target genes, e.g. up-regulation of integrin B7 and down-regulation of IL12A. Furthermore, T. annulata infection is associated with the suppression of the transcription factors, Pu.1 and RUNX1, and colony stimulating factor 1 receptor (CSF1R) which are also involved in the regulation of monocyte/mϕ differentiation. We believe these results provide the first direct evidence that T. annulata modulates the host mϕ differentiation state, which may diminish the defence capabilities of the infected cell and/or promote cell proliferation. Musculoaponeurotic fibrosarcoma oncogene (MAF) transcription factors play an important role in cell proliferation, differentiation and survival; therefore, regulation of these genes may be a major mechanism employed by T. annulata to survive within the infected mϕ.

Infection of bovine cells by the protozoan parasite Theileria annulata modulates expression of the ISGylation system

The apicomplexan parasite, Theileria annulata, dedifferentiates and induces continuous division of infected bovine myeloid cells. Re-expression of differentiation markers and a loss of proliferation occur upon treatment with buparvaquone, implying that parasite factors actively maintain the altered status of the infected cell. The factors that induce this unique transformation event have not been identified. However, parasite polypeptides (TashAT family) that are located in the infected leucocyte nucleus have been postulated to function as modulators of host cell phenotype. In this study differential RNA display and proteomic analysis were used to identify altered mRNA and polypeptide expression profiles in a bovine macrophage cell line (BoMac) transfected with TashAT2. One of the genes identified by differential display was found to encode an ubiquitin-like protease (bUBP43) belonging to the UBP43 family. The bUBP43 gene and the gene encoding its ubiquitin-like substrate, bISG15, were expressed at a low level in T. annulata-infected cells. However, infected cells were refractory to induction of elevated bISG15 expression by lipopolysaccharide or type 1 interferons while TashAT2-transfected cells showed no induction when treated with camptothecin. Modulation of the ISGylation system may be of relevance to the establishment of the transformed infected host cell, as ISGylation is associated with resistance to intracellular infection by pathogens, stimulation of the immune response and terminal differentiation of leukaemic cells.

Characterisation of a cluster of genes encoding Theileria annulata AT hook DNA-binding proteins and evidence for localisation to the host cell nucleus.

Infection of bovine leukocytes by the apicomplexan parasite Theileria annulata results in alteration of host cell gene expression and stimulation of host cell proliferation. At present, the parasite-derived factors involved in these processes are unknown. Recently, we described the characterisation of a parasite gene (TashAT2), whose polypeptide product bears AT hook DNA-binding motifs and may be transported from the parasite to the host nucleus. We now describe the isolation of a further two genes (TashAT1 and TashAT3) that are very closely related to TashAT2. All three TashAT genes are located together in a tight cluster, interspersed by two further small open reading frames, all facing head to tail. TashAT2 was shown to be expressed in all T. annulata cell lines examined, whereas TashAT1 and TashAT3 were expressed in the sporozoite stage of the parasite, and also in infected cell lines, where their expression was found to vary between different cell lines. Evidence for transport was provided by antisera raised against TashAT1 and TashAT3 that reacted with the host nucleus of T. annulata-infected cells. Reactivity was particularly strong against the host nuclei of the T. annulata-infected cloned cell line D7B12, which is attenuated for differentiation. A polypeptide in the size range predicted for TashAT3 was preferentially detected in host enriched D7B12 nuclear extracts. DNA-binding analysis demonstrated that fusion proteins containing the AT hook region of either TashAT1 or TashAT2 bound preferentially to AT rich DNA.

The proliferation-associated nuclear protein Ki-67 in the bovine system: partial characterisation and its application for determination of the proliferation of Theileria-infected bovine cells.

Theileria annulata-infected bovine cells as well as mitogen-stimulated bovine peripheral blood mononuclear cells (PBMC) express a proliferation-associated nuclear protein equivalent to the human Ki-67 protein. In analogy to the human system, the expression of the bovine Ki-67 protein is restricted to proliferating cells only, since (a) Ki-67 expression paralleled [3H]-thymidine incorporation in concanavalin A (Con A)-stimulated bovine PBMC, (b) Ki-67 was not detectable in quiescent bovine cells, and (c) Ki-67 expression in Theileria-infected cells is related to the presence of the parasites within the cytoplasm of the host cells; upon treatment with the theilericidal drug buparvaquone the parasites are destroyed and the cells cease to proliferate and to express the Ki-67 protein. Western-blot analysis of lysates of proliferating bovine cells revealed that the prototype monoclonal antibody Ki-67 and the new equivalent antibody MIB-1 detected one prominent protein band with an apparent molecular weight of 430 kDa. Two cDNA clones (pUC18.B1.Ki-67 and pUC18.B2.Ki-67) were isolated from a lambdagt11 cDNA library of T. annulata-infected bovine cells by immunoscreening with the monoclonal antibody MIB-1. Comparison of these cDNA sequences with those of the human Ki-67 protein revealed 60-70% identity. Within the "Ki-67 motif", identity proved to be 80% at the amino acid level. The remarkable identity between bovine and human Ki-67 proteins suggests that MIB-1 can be used as a marker for cell proliferation in animal research. In this context we could identify proliferating cells in lymph nodes of Theileria-infected animals and, furthermore, we could distinguish between infected and uninfected proliferating cells using MIB-1 and an antiserum against a recombinant parasite protein designated SA288.

Is interleukin 2 necessary for the autocrine proliferation of Theileria-infected bovine cells?

Theileria-infected bovine lymphoblastoid cells are induced to proliferate permanently such that the division of the parasites and that of their host cells occur synchronously. The mechanism by which the parasites induce the transformation of their host cells is unknown. We investigated the growth-factor dependency of a number of Theileria-infected cell lines. Supernatants of the majority of the cell lines tested in our study showed no growth-enhancing activity. However, supernatants of a limited number of cell lines occasionally enhanced the growth of parasitized cells that were used as responder cells. Thus, they contained a growth factor whose biological effect was not eliminated by an anti-interleukin-2 (anti-IL-2) antibody. Moreover, neither the proliferation of T. parva-infected cells nor that of T. annulata-infected cells was impaired by this antibody. In contrast, the anti-IL-2 antibody substantially prevented bovine peripheral blood mononuclear cells from undergoing a proliferative response upon stimulation with concanavalin A (Con A). In line with these results we observed that unlike Con A-stimulated lymphocytes, the infected cell lines did not express IL-2 mRNA. Taken together, our results suggest that Theileria-infected cells do not secrete IL-2 and that IL-2 does not play an important role in the autocrine proliferation of the parasitized host cells.

Cytokine production/T-cell-stimulatory ability of Theileria annulata-infected cells and post-vaccinal reactions.

Cytokine production/T-cell-stimulatory ability of Theileria annulata-infected cells and post-vaccinal reactions.

Macroschizonts of Theileria annulata as vaccine and diagnostic tools.

T. annulata-infected cells present infection-associated peptides. These peptides represent target molecules of the cytotoxic acting cells. Their preparation and characterization may help to develop a sub-unit vaccine. Our studies show that macroschizont-infected bovine cells can be used as parasite antigen in serology for the detection of parasite-specific antibodies in serum of infected animals. Primers derived from the macroschizont of T. annulata can be used as molecular tools for the detection of parasite DNA in blood samples of carrier cattle.

Bovine gamma/delta TcR+ T lymphocytes are stimulated to proliferate by autologous Theileria annulata-infected cells in the presence of interleukin-2.

An in vitro model system has been developed in which freshly isolated resting WC1+ gamma/delta TcR+ T cells proliferate in response to cells transformed by the protozoan parasite Theileria annulata, providing a strategy in which the basis of activation of naive gamma/delta T cells can be investigated. Irradiated parasite-transformed cells stimulate the proliferation, but not cytolytic activity, of autologous peripheral blood mononuclear cells (PBMC) from non-immune animals. The proliferating cells are mainly WC1+ gamma/delta T cells. The majority of WC1+ gamma/delta T cells in freshly isolated PBMC express CD25 at a low level that increases when stimulated with T. annulata-infected cells. Purified WC1+ gamma/delta T cells fail to proliferate when cultured with irradiated T. annulata-infected cells and produce a small proliferative response to IL-2, but proliferate strongly to irradiated or lightly fixed Theileria-infected cells in combination with IL-2. The Theileria-infected cells express cytokine transcripts encoding IL-1 alpha, IL-1 beta, IL-6 and IL-10, but not IFN gamma, IL-2, IL-4 and IL-7. Purified WC1+ gamma/delta T cells stimulated with T. annulata-infected cells with or without IL-2 fail to produce IL-2 transcripts, but do produce those for TNF alpha. These experiments show that WC1+ gamma/delta T cells recognize a surface determinant on T. annulata-infected cells, that together with a second signal, which can be provided by exogenous IL-2, stimulates their proliferation.

Proliferation of theileria annulata and Theileria parva macroschizont-infected bovine cells in scid mice

Theileria annulata and Theileria parva macroschizont-infected bovine cells formed tumours at the inoculation site when injected subcutaneously into C.B.-17 scid mice. T. annulata tumours showed more vigorous growth than T. parva tumours. The tumours did not regress and infected cells spread to other tissues. Intraperitoneal injection of high doses of T. annulata-infected cells resulted in the development of ascites; the infected cells colonized abdominal organs, in particular mesenteric tissue. Low doses of cells did not establish when administered by this route. Evidence for a role for macrophages in controlling proliferation of Theileria-infected cells was provided by finding (i) that uninfected bovine cells did not survive for as long in the peritoneal cavities of scid mice as in Balb/c mice; (ii) peritoneal macrophages both proliferated in vivo in the presence of infected cells and were activated as assessed by production of interleukin-1. Evidence against a role for NK cells was provided by (i) the failure of an in vivo assay for allogeneic lymphocyte cytotoxicity to reveal any activity against bovine cells in the lungs or liver, i.e. the sites usually associated with NK cell cytotoxicity, and (ii) the lack of correlation between tumour regression and NK cell activity in the spleens of mice with chronic T. annulata tumours.

Production of interferon by Theileria annulata- and T. parva-infected bovine lymphoid cell lines.

Theileria annulata and T. parva-infected lymphoblastoid cells were examined for their capacity to produce interferon (IFN). Supernatants of such cells were tested in biological assay for their antiviral activity. Only T. parva-infected cells of T-cell origin were capable of producing IFN-gamma. Supernatants of some but not all T. annulata-infected cells showed also antiviral activity, which was greatly reduced after exposure to a pH of 2. Northern-blot analysis of the cells using an IFN-gamma cDNA probe confirmed the results obtained for T. parva-infected cells in a biological assay. No IFN-gamma mRNA was detected in T. annulata-infected cells. The importance of IFN for the pathogenesis of theileriosis is discussed.