Linear peptide specificity of bovine antibody responses to p67 of Theileria parva and sequence diversity of sporozoite-neutralizing epitopes: implications for a vaccine.
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A stage-specific surface antigen of Theileria parva, p67, is the basis for the development of an anti-sporozoite vaccine for the control of East Coast fever (ECF) in cattle. By Pepscan analysis with a series of overlapping synthetic p67 peptides, the antigen was shown to contain five distinct linear peptide sequences recognized by sporozoite-neutralizing murine monoclonal antibodies. Three epitopes were located between amino acid positions 105 to 229 and two were located between positions 617 to 639 on p67. Bovine antibodies to a synthetic peptide containing one of these epitopes neutralized sporozoites, validating this approach for defining immune responses that are likely to contribute to immunity. Comparison of the peptide specificity of antibodies from cattle inoculated with recombinant p67 that were immune or susceptible to ECF did not reveal statistically significant differences between the two groups. In general, antipeptide antibody levels in the susceptible animals were lower than in the immune group and neither group developed high responses to all sporozoite-neutralizing epitopes. The bovine antibody response to recombinant p67 was restricted to the N- and C-terminal regions of p67, and there was no activity against the central portion between positions 313 and 583. So far, p67 sequence polymorphisms have been identified only in buffalo-derived T. parva parasites, but the consequence of these for vaccine development remains to be defined. The data indicate that optimizations of the current vaccination protocol against ECF should include boosting of relevant antibody responses to neutralizing epitopes on p67. (+info)
Bovine gammadelta T-cell responses to the intracellular protozoan parasite Theileria parva.
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T cells bearing the gammadelta antigen receptor (gammadelta T cells) can constitute up to 50% of T cells in the peripheral blood and lymphoid organs of young cattle. We present data showing that gammadelta T cells are involved in immune responses against Theileria parva. gammadelta T cells isolated from peripheral blood mononuclear cells (PBMC) of T. parva-naive and -immune cattle proliferated in the presence of fixed or unfixed autologous T. parva-infected lymphoblasts (TpL) and heat-stressed concanavalin A (ConA)-induced blasts (ConA blasts) but not untreated ConA blasts. The specificity of response was further evaluated with a panel of gammadelta T-cell lines and clones. T-cell reactivity was blocked by GB21A, a monoclonal antibody (MAb) specific for the gammadelta T-cell receptor, but not by MAbs specific for class I and class II major histocompatibility complex (MHC) molecules. In addition, TpL but not ConA blasts from a variety of MHC-mismatched animals induced proliferation of the gammadelta T-cell lines and clones. These gammadelta T cells were found to respond to TpL infected with several different parasite stocks and failed to recognize TpL after elimination of the parasite by the theilericidal drug BW 720C. Assays for cytotoxic activity of gammadelta T cells sorted from bulk cultures of immune PBMC restimulated several times with autologous TpL demonstrated that effector cells whose specificity is similar to that of proliferating cells are generated. These results suggest that bovine gammadelta T cells are activated by and lyse T. parva-infected cells by recognizing conserved parasite-induced or parasite-derived antigens in an MHC-unrestricted fashion. (+info)
The intracellular parasite Theileria parva protects infected T cells from apoptosis.
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Parasites have evolved a plethora of strategies to ensure their survival. The intracellular parasite Theileria parva secures its propagation and spreads through the infected animal by infecting and transforming T cells, inducing their continuous proliferation and rendering them metastatic. In previous work, we have shown that the parasite induces constitutive activation of the transcription factor NF-kappaB, by inducing the constitutive degradation of its cytoplasmic inhibitors. The biological significance of NF-kappaB activation in T. parva-infected cells, however, has not yet been defined. Cells that have been transformed by viruses or oncogenes can persist only if they manage to avoid destruction by the apoptotic mechanisms that are activated on transformation and that contribute to maintain cellular homeostasis. We now demonstrate that parasite-induced NF-kappaB activation plays a crucial role in the survival of T. parva-transformed T cells by conveying protection against an apoptotic signal that accompanies parasite-mediated transformation. Consequently, inhibition of NF-kappaB nuclear translocation and the expression of dominant negative mutant forms of components of the NF-kappaB activation pathway, such as IkappaBalpha or p65, prompt rapid apoptosis of T. parva-transformed T cells. Our findings offer important insights into parasite survival strategies and demonstrate that parasite-induced constitutive NF-kappaB activation is an essential step in maintaining the transformed phenotype of the infected cells. (+info)
N-acetylcysteine blocks apoptosis induced by N-alpha-tosyl-L-phenylalanine chloromethyl ketone in transformed T-cells.
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The serine protease inhibitor N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) can interfere with cell-cycle progression and has also been shown either to protect cells from apoptosis or to induce apoptosis. We tested the effect of TPCK on two transformed T-cell lines. Both Jurkat T-cells and Theileria parva-transformed T-cells were shown to be highly sensitive to TPCK-induced growth arrest and apoptosis. Surprisingly, we found that the thiol antioxidant, N-acetylcysteine (NAC), as well as L- or D-cysteine blocked TPCK-induced growth arrest and apoptosis. TPCK inhibited constitutive NF-kappaB activation in T. parva-transformed T-cells, with phosphorylation of IkappaBalpha and IkappaBbeta being inhibited with different kinetics. TPCK-mediated inhibition of IkappaB phosphorylation, NF-kappaB DNA binding and transcriptional activity were also prevented by NAC or cysteine. Our observations indicate that apoptosis and NF-kappaB inhibition induced by TPCK result from modifications of sulphydryl groups on proteins involved in regulating cell survival and the NF-kappaB activation pathway(s). (+info)
Immunization against diseases caused by Theileria parva: a review.
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Theileria parva is the causative agent of three epidemiologically different diseases, East Coast fever (ECF), Corridor disease and January disease, caused by 3 types of T. parva, T. p. parva, T. p. lawrencei and T. p. bovis, respectively. The history of immunization against these diseases has been marked by salient discoveries such as the immune status in recovered animals, the activity of tetracyclines during the incubation period, the possibility for cryopreserving supernatant of prefed ticks and the development of useful serological tests. The possibility of simultaneous administration of stabilate and long-acting tetracycline have greatly contributed to making the infection and treatment method operational. The importance of antigenic diversity in T. parva has been reflected in the difficulties related to the selection of the immunizing stock or combinations of stocks: a 'cocktail' of East African isolates may give broad protection against field challenge by ECF (T. parva parva), but Corridor disease is more problematic. On the other hand, certain single isolates may give equally good protection against ECF field challenge. Studies on the immunology of T. parva infection and the application of molecular tools have led to the discovery that sera of recovered animals neutralize sporozoites of various isolates, and to the p67 molecular vaccine; yet so far the only available method of immunizing against T. parva infections is the infection and treatment method or, in the case of T. parva bovis, the use of sublethal stabilate doses. Infection and treatment is applied on a fairly large scale in Zambia, and on a more limited scale in a few other countries. Immunity by this rather crude method is long-lasting and solid, but cross-immunity problems against some field strains remain. Furthermore, as immunized animals remain carriers, immunization may contribute to attaining and improving endemic stability in endemic areas in indigenous breeds with an adequate level of genetic tolerance to ECF. On the other hand, carrier animals may constitute a risk for spreading the disease into ECF-free areas where the vector is present. Other disadvantages of the method are that immunization of cattle during the incubation of naturally contracted East Coast fever will not prevent the disease and jeopardize its reputation. Furthermore, stabilates have to be cryopreserved, often a technical drawback, and contamination with undesirable pathogens may occur in tick-derived material. Therefore the need remains for the development of effective molecular vaccines and it must be remembered that immunization must be cost-effective and sustainable and it is only one aspect of integrated control of theileriosis and other tick-borne diseases. There is no universally valid strategy. Several factors have to be considered: value and susceptibility of cattle to theileriosis and to other tick-borne and tick-associated diseases, infestation by various ticks present in the area, the type of theileriosis (ECF, Corridor disease or January disease) and the epidemiological situation where immunization is taking place. The optimal age for immunization of the calves in endemic areas needs to be determined: when calf mortality by naturally occurring theileriosis is a problem, the sooner calves are immunized the better, but a proportion will have contracted natural infection before they can be reached, and immunization of very young calves might not be fully effective. (+info)
Molecular epidemiology of Theileria parva in the field.
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Molecular tools based on seminested RFLP-PCR techniques to characterize field parasites in bloodspots dried on filter paper permitted investigation of the extent and the dynamics of diversity of Theileria parva populations in the field. Parallel molecular studies explored the long-term genome stability of various isolates by probing Southern blots of EcoRI digested total genomic DNA with four different reference nucleic acid probes. Three polymorphic single copy loci encoding for antigen genes were developed for seminested PCR detection in order to apply them for a multilocus approach in population genetic studies. Seven alleles were identified for the polymorphic immunodominant molecule (PIM) locus by using restriction enzymes, and 4 alleles each for the p150 and p104 loci. A simple DNA extraction method gave good results in amplifying these loci from carrier animals using samples of blood dried on filter papers. Results from probing Southern blots of cultures taken at sequential timepoints indicate relative genome stability in T. parva in comparison to other parasitic protozoa such as Plasmodium. Comparatively homogeneous profiles in sympatric isolates from Zambia were identified using all four probes and PCR amplified products which contrasted with the variety found amongst Kenyan stocks. Preliminary characterization of T. parva field samples from the Southern Province of Zambia strongly suggest clonal expansion of one of the components of a non-Zambian trivalent vaccine used on a limited scale in the Province from 1985 until 1992. (+info)
Differences in the epidemiology of theileriosis on smallholder dairy farms in contrasting agro-ecological and grazing strata of highland Kenya.
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A prospective cohort study was conducted in five purposively-sampled agro-ecological zone (AEZ)-grazing system strata in Murang'a District, Kenya, between March 1995 and June 1996. The study strata were selected based on a preliminary characterization study to represent the widest range of risks to East Coast fever (ECF) in the District and included zero-grazing and open-grazing farms. In total, 225 calves from 188 smallholder farms were examined from birth to 6 months of age and visited within the first 2 weeks of life and thereafter at bi-weekly intervals for up to 14 visits. The purpose of the study was to characterize the differences in epidemiology (risks of infection, morbidity and mortality) and potential control of ECF between the selected strata. Evidence of Theileria parva infection was assessed by increased antibody levels as measured in an indirect ELISA assay by the percent positivity (PP) of serum samples relative to a strong positive reference serum. Sero-conversion risks of T. parva were highest in the open-grazing strata. Antibody prevalence in adult cattle and ECF morbidity and mortality risks were also highest in open-grazing strata. While different, all five AEZ-grazing strata were considered to be endemically unstable for ECF. East Coast fever challenge was low in all zero-grazing strata and this challenge is likely to remain low due to continuing intensification of smallholder farming in the central highlands. In the open-grazing strata, there was higher challenge and a greater impact of ECF. (+info)
Constitutive exclusion of Csk from Hck-positive membrane microdomains permits Src kinase-dependent proliferation of Theileria-transformed B lymphocytes.
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Infection of bovine T cells and B cells with the intracellular protozoan parasite Theileria parva induces a transformed phenotype with characteristics comparable to leukemic cells. The transformed phenotype reverts on drug-induced parasite death, and the cured lymphocytes acquire a resting phenotype and eventually die by apoptosis if not further stimulated. Here, we show that both lymphocyte proliferation and activation of the transcription factor AP-1 are mediated by Src-family protein tyrosine kinases (PTKs) in a parasite-dependent fashion. Src-family PTKs are known to be present in glycolipid-enriched microdomains (GEMs), also called lipid rafts, and to be negatively regulated by PTK Csk complexed to tyrosine-phosphorylated transmembrane adapter protein PAG (phosphoprotein associated with GEMs) also called Cbp (Csk-binding protein). We, therefore, purified GEMs from proliferating infected B cells and from growth-arrested cells that had been drug-cured of parasites. Proliferation arrest led to a striking increase of PAG/Cbp expression; correspondingly, the amount of Csk associated with PAG/Cbp in GEMs increased markedly, whereas PTK Hck accumulation in GEM fractions did not alter on growth arrest. We propose that Theileria-induced lymphocyte proliferation and permanent activation of Hck stems from down-regulation of PAG/Cbp and the concomitant constitutive loss of the negative regulator Csk from the GEMs of transformed B cells. (+info)