The impact of tick-borne pathogen infection in Indian bovines is determined by host type but not the genotype of Theileria annulata.

Tick-borne pathogens (TBP) are a major source of production loss and a welfare concern in livestock across the globe. Consequently, there is a trade-off between keeping animals that are tolerant to TBP infection, but are less productive than more susceptible breeds. Theileria annulata is a major TBP of bovines, with different host types (i.e. exotic and native cattle breeds, and buffalo) displaying demonstrable differences in clinical susceptibility to infection. However, the extent to which these differences are driven by genetic/physiological differences between hosts, or by different parasite populations/genotypes preferentially establishing infection in different host breeds and species is unclear. In this study, three different bovine host types in India were blood sampled to test for the presence of various TBP, including Theileria annulata, to determine whether native cattle (Bos indicus breeds), crossbreed cattle (Bos taurus x Bos indicus breeds) or water buffalo (Bubalus bubalis) differ in the physiological consequences of infection. Population genetic analyses of T. annulata isolated from the three different host types was also performed, using a panel of mini- and micro-satellite markers, to test for sub-structuring of the parasite population among host types. We discovered that compared to other host types, "carrier" crossbreed cattle showed a higher level of haematological pathology when infected with T. annulata. Despite this finding, we found no evidence for differences in the genotypes of T. annulata infecting different host types, although buffalo appeared to harbour fewer mixed parasite genotype infections, indicating they are not the major reservoir of parasite diversity. The apparent tolerance/resistance of native breed cattle and buffalo to the impacts of T. annulata infection is thus most likely to be driven by host genotype, rather than differences in the parasite population. Our results suggest that an improved understanding of the genetic factors that underpin disease resistance could help to ameliorate future economic loss due to TBP or tropical theileriosis.

EtCRK2, a cyclin-dependent kinase gene expressed during the sexual and asexual phases of the Eimeria tenella life cycle.

EtCRK2, a cyclin-dependent kinase from the coccidian parasite, Eimeria tenella is closely related to eukaryotic cyclin-dependent kinases that regulate progression of the cell cycle and to several cyclin-dependent kinases identified in the Apicomplexa. Northern blot analyses revealed that EtCRK2 is transcribed during both asexual (first-generation schizogony) and sexual (oocyst sporulation) replicative phases of the parasite life cycle. In addition, it appears to be transcriptionally regulated during meiosis. Recombinant EtCRK2 produced in Escherichia coli has kinase activity which is significantly stimulated by the addition of vertebrate cyclin A. This cyclin-dependent kinase may play a significant role in regulating critical cell cycle events during both asexual proliferation and sexual development of the parasite.

Genetic and phenotypic analysis of Tunisian Theileria annulata clones.

Many parasite species are known to show high levels of genetic diversity, yet the consequences of this diversity for host-parasite interactions are not well understood. Variation in phenotypic traits such as growth rates and the ability to form transmission stages are raw material for natural and artificial selection to act upon with consequences for the evolution of the parasite species and disease control. In order to study genetic and phenotypic diversity amongst Theileria annulata parasites, a collection of 52 parasite clones was generated from cattle isolates and tick material recently collected in Tunisia. Genetic diversity was assessed using PCR-RFLP and monoclonal antibody markers, and genetically distinct clones selected for further study. Clones varied significantly in their growth rates in culture at 37 degrees C, their viability after a period of culture at 41 degrees C and their differentiation rates into transmission stages after culturing at 41 degrees C. The viability of a clone after culturing at 41 degrees C could not be predicted from its growth rate at 37 degrees C, but across clones, differentiation rates were positively correlated with growth rates at 37 degrees C. All 3 in vitro measures are likely to have relevance to parasite-host interactions in animals with clinical theileriosis, and should be acted on by within-host and between-host selection.

Identification of a 40S Ribosomal protein (S17) that is differentially expressed between the macroschizont and piroplasm stages of Theileria annulata.

The nucleotide and protein sequence of the 40S ribosomal protein S17 (RibS17) of the protozoan parasite Theileria annulata has been determined. Southern blot analysis showed the gene was single copy and comparative sequence analysis revealed that the predicted polypeptide had high sequence homology with the RibS17 from other organisms. Northern blot analysis showed that there was a 3-fold increase in the level of RibS17 RNA between the macroschizont and the piroplasm stage of the lifecycle, whereas, there was no difference in expression between the sporozoite and the macroschizont stages. Antisera to the purified fusion protein, corresponding to the terminal 50 amino acids of the protein sequence, were raised in rabbits. Western analysis detected a polypeptide of the predicted size that was more abundant in the piroplasm stage compared with the macroschizont stage. Immunofluorescence analysis with the same antisera revealed a strong signal in the macroschizont and piroplasm stages, but the antiserum did not cross-react with the bovine host cells. The antisera did, however, cross-react with Toxoplasma gondii tachyzoites and Plasmodium falciparum merozoites. The possible functional significance of the stage related increase in abundance of a ribosomal protein is discussed.

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.

Theileria annulata: identification, by differential mRNA display, of modulated host and parasite gene expression in cell lines that are competent or attenuated for differentiation to the merozoite.

To identify both host and parasite genes that show altered expression during differentiation of Theileria annulata from the macroschizont to the merozoite stage of the life cycle, the RNA profiles of two T. annulata-infected clonal cell lines (D7 and D7B12) with the same genetic background have been compared by RNA display. In the cloned cell line D7, T. annulata differentiates from the macroschizont to the merozoite at 41 degrees C, whereas in the cell line D7B12, which was derived by recloning D7, the parasite does not differentiate. Therefore, genes that show altered expression levels in either clone could be modulated by the differentiation event and are possible candidates for regulators of this process. Differential display was carried out initially on RNA extracted from D7 and D7B12 macroschizont-infected cells cultured at 37 degrees C and secondly on RNA extracted from the two cell lines incubated at 41 degrees C to induce differentiation to the merozoite. The first procedure identified 29 cDNA fragments that displayed altered levels between D7 and D7B12, 9 of which were confirmed to be differentially expressed by Northern blot analysis. Of these 9 gene fragments, 8 were found to be of host origin, while 1 was parasite derived. The second RNA display analysis identified 14 transcripts that showed altered levels during a differentiation time course, of which 6 were confirmed to be differentially expressed between D7B12 cells and differentiating D7 cells by Northern blot analysis. Of these 6 gene fragments, 1 was of host and 5 were of parasite origin. The parasite genes either showed levels of RNA consistent with constitutive gene expression or, in one case, a genuine upregulation of mRNA associated with the differentiation process.

Temporal co-ordination of macroschizont and merozoite gene expression during stage differentiation of Theileria annulata.

The bovine parasite, Theileria annulata has a complex life-cycle involving the expression and repression of genes during development of its morphologically distinct life-cycle stages. In order to detail the molecular events that occur during differentiation of the intracellular multinucleate macroschizont to the extra-cellular uninucleate merozoite, we have isolated two genes, Tash1 and Tash2 which are differentially expressed during differentiation. Nuclear run on data show that Tash1 gene expression is controlled, at least in part, at the level of transcription. Immunofluorescence data identify the macroschizont as the location for both Tash1 and Tash2 gene products. Northern blot analysis of these genes indicated that their mRNA levels decrease during differentiation in vitro, at a time point coincident with major elevation in the mRNA levels of the merozoite antigen, Tams1, shown previously to be associated with commitment to merozoite production. Furthermore, experiments where cultures were incubated at 41 degrees C for 4 days and replaced at 37 degrees C for 2 days demonstrated that re-expression of Tash1 occurred and is probably linked to reversion to the macroschizont and decreased expression of Tams1. These results imply that the control of macroschizont and merozoite gene expression during differentiation is closely co-ordinated temporally. In addition, a comparison of Tash2 and Tams1 expression has indicated that translational or post-translational control of gene expression may operate in the undifferentiated macroschizont.

Study of Theileria annulata population structure during bovine infection and following transmission to ticks.

Tams1 is the polymorphic immunodominant merozoite-piroplasm surface protein of Theileria annulata. Evidence for selection of divergent forms of Tams1 has been obtained recently. This study was performed to address whether selection takes place during persistent infection of the bovine host or during passage through the Hyalomma tick vector. Four calves were infected with a T. annulata isolate representing multiple parasite genotypes. The development of the parasite population was analysed by denaturing gradient gel electrophoresis (DGGE) using the Tams1 gene as a marker. In addition, the parasitaemia was measured by a semi-quantitative reverse line blot hybridization assay in order to correlate Tams1 variation to changes in parasitaemia. It was found that both parasitaemia and parasite population displayed limited variation during persistent infection. Ticks were allowed to acquire T. annulata during 2 periods of the bovine infection. Tams1 alleles detected in ticks fed during acute infection were identical to the population in the bovine host. However, ticks fed during the carrier status acquired parasites showing a single Tams1 isotype that represented, in several cases, a minor population in the bovine host at the time of infestation. Although only a limited number of ticks were studied, these preliminary data suggest that specific parasite genotypes may be selected during tick transmission from a carrier animal.

Generation of a mosaic pattern of diversity in the major merozoite-piroplasm surface antigen of Theileria annulata.

The polypeptide Tams1 is an immunodominant major merozoite piroplasm surface antigen of the protozoan parasite Theileria annulata. Generation and selection of divergent antigenic types has implications for the inclusion of the Tams1 antigen in a subunit recombinant vaccine or use in the development of a diagnostic ELISA. In this study a total of 129 Tams1 sequences from parasites isolated in Bahrain, India, Italy, Mauritania, Portugal, Spain, Sudan, Tunisia and Turkey were obtained to estimate the extent of Tams1 diversity throughout a wide geographical range. Significant sequence diversity was found both within and between isolates and many of the sequences were unique. No geographical specificity of sequence types was observed and almost identical sequences occurred in different geographical areas and a panmictic population structure is suggested by our results. A sliding window analysis identified sub-regions of the molecule where selection for amino acid changes may operate. Evidence is also presented for the generation of diversity through intragenic recombination with switching of corresponding variable domains between alleles. Recombination to exchange variable domains appears to occur throughout the length of the gene sequence, and has the potential to generate a mosaic pattern of diversity.

Evidence for localisation of a Theileria parasite AT hook DNA-binding protein to the nucleus of immortalised bovine host cells.

Immortalisation of bovine leukocytes by the macroschizont stage of the tick transmitted protozoan parasite, Theileria annulata, results in the clonal expansion of infected cells and dissemination throughout the bovine host. The parasite-encoded factors which induce this unique transformation event have not been defined to date. In this study, a gene family (TashAT) has been characterised that encodes polypeptides with homology to known DNA-binding proteins. Expression of TashAT genes occurs at the intracellular macroschizont stage of the parasite life cycle and during differentiation to the merozoite, negative regulation of TashAT genes is detected early relative to other macroschizont genes. Interestingly, the early reduction in TashAT expression coincides with the initial decrease in host cell proliferation. One member of the family, TashAT2, was characterised in detail and the predicted polypeptide sequence was found to harbor three AT hook DNA-binding domains. Antisera generated against two distinct regions of TashAT2 both located the antigen to the host cell nucleus and, combined with protein translation inhibition and immunoprecipitation studies, provide evidence that this polypeptide could be transported from the parasite to this location. Further evidence for this postulation was provided by transfection studies which demonstrated that TashAT2 does encode the structural information required for localisation to the nucleus of a mammalian cell. Thus, TashAT2 is a potential candidate for a parasite-encoded factor that modulates host cell gene expression and may be involved in the control of host cell proliferation.

Innate and adaptive immune responses co-operate to protect cattle against Theileria annulata.

For many years it was assumed that Theileria annulata resembled T. parva, parasitizing lymphocytes and causing lymphoproliferative disease, with the two species being controlled by similar protective immune responses. Patricia Preston et al. here review the evidence that has led to a different view of T. annulata. It is now thought that the schizonts of T. annulata inhabit macrophages and B cells, and that tropical theileriosis is not a lymphoproliferative disease. Both innate and adaptive responses contribute to recovery from infection and resistance to challenge and cytokines produced by infected and uninfected cells influence the outcome of infection. Partial protection has been stimulated recently by defined recombinant antigens; efficacy depended upon the delivery system.

Should I stay or should I go now? A stochastic model of stage differentiation in Theileria annulata.

The events that initiate and determine stage differentiation of protozoan parasites are not fully understood. In this article, Brian Shiels suggests that for differentiation to the merozoite in Theileria annulata the process is predetermined by the parasite, but can be initiated and modulated by changes to the extracellular environment. Shiels proposes a mechanism operating on the basis of factors that regulate gene expression reaching a commitment threshold. Similarities across protozoan and higher eukaryotic differentiation systems lead Shiels to speculate that the T. annulata model may be of relevance to other parasites.

Induction of protective immunity to Theileria annulata using two major merozoite surface antigens presented by different delivery systems.

Allelic forms (Tams1-1 and Tams1-2) of the major merozoite surface antigen gene of Theileria annulata have recently been expressed in Escherichia coli and in Salmonella typhimurium aroA vaccine strain SL3261. To test the potential of subunit vaccines against T. annulata infection, we immunized four groups of three calves with either recombinant (re-) (Tams1-1 and Tams1-2) proteins or naked DNA encoding these antigens. Group I was immunized intramuscularly with both re-proteins incorporated into immunostimulating complexes (ISCOMs). Group II was inoculated intramuscularly with naked plasmid DNA encoding Tams1-1 and Tams1-2. Groups III and IV received S. typhimurium SL3261 [pSTams1-1][pIP5] and SL3261 [pSTams1-2] [pIP5] subcutaneously and orally, respectively. A final group of three animals (Group V) served as an unimmunized control group. Four weeks after the last immunization all calves were challenged with a T. annulata stabilate generated from blood of an infected animal with 30% piroplasm parasitaemia. All calves vaccinated with ISCOMs proved to be protected from T. annulata infection and had generated antibodies against both re-(Tams1-1 and Tams1-2) at the time of challenge. In two of these animals the antibody had a surface binding profile by IFAT. Two of three calves immunized with naked DNA also proved to be protected, but none of the animals had generated any detectable antibodies against the recombinants. Salmonella-based delivery of the recombinants did not induce any protection; two of six animals died of theileriosis and there was no difference between subcutaneous or oral administration. These preliminary results show that re-(Tams1-1 and/or Tams1-2) may elicit protective immune responses in cattle, depending on the antigen delivery system.

Expression of genes encoding two major Theileria annulata merozoite surface antigens in Escherichia coli and a Salmonella typhimurium aroA vaccine strain.

The genes, Tams1-1 and Tams1-2, encoding the 30-and 32-kDa major merozoite surface antigens of Theileria annulata (Ta), have recently been cloned and characterized. Both genes encode a protein of 281 amino acids (aa) containing a putative hydrophobic N-terminal signal peptide. Another hydrophobic stretch is predicted at the C terminus which probably functions to anchor the protein in the membrane of the merozoite and piroplasm. Here, we report the successful expression of both Tams1-1 and Tams1-2 in Escherichia coli (Ec) using gene fragments lacking both hydrophobic domains. Attempts to produce high amounts of the entire recombinant (re-) protein, or a fragment containing the N terminus only, were unsuccessful. This is presumably due to the toxicity of these re-proteins. The internal part of both genes was also expressed in Salmonella typhimurium (St) aroA vaccine strain SL3261. We employed a dual-plasmid expression system based on an invertible promoter and selected the most stable St construct in vitro using liquid cultures and a macrophage-like cell line. The re-Tams1-1 protein produced in Ec, as well as in St, was recognized by monoclonal antibody (mAb) 5E1 specific to the 30-kDa protein. Both re-Tams1-1 and re-Tams1-2 were recognized by Ta immune calf serum.

Theileria annulata: altered gene expression and clonal selection during continuous in vitro culture.

Kept in continuous in vitro culture, the protozoan parasite Theileria annulata gradually loses virulence when inoculated into cattle. These attenuated cell lines form the basis of the in vitro live vaccines which have been used successfully to control tropical theileriosis in several endemic regions. In the study reported here, events occurring during in vitro culture of an Indian (Hisar) cell line, which may be associated with the reduction in virulence, have been investigated. Hybridization with two polymorphic DNA probes following Southern blotting showed that selection of particular parasite genotypes occurs very rapidly with culture; a novel hybridization pattern is observed with both probes after 50-100 passages in vitro. In addition to this selection process, immunofluorescence studies using a monoclonal antibody which specifically recognizes virulent T. annulata revealed alterations in antibody reactivity following in vitro culture. This loss of reactivity was observed in three cloned cell lines derived from the early, virulent Hisar line and implies that phenotypic changes resulting from alterations to parasite gene expression are taking place during the attenuation process. When considered with the results from in vivo infections with serial passages of this cell line, it can be proposed that both altered gene expression and selection may be involved in the loss of pathogenicity of T. annulata during continuous in vitro culture.

Selection of diversity at putative glycosylation sites in the immunodominant merozoite/piroplasm surface antigen of Theileria parasites.

The immunodominant merozoite/piroplasm surface antigen of Theileria parasites has potential as a diagnostic reagent and as a component of a sub-unit vaccine. This molecule is known to be antigenically diverse, and it is important to determine the nature and extent of this heterogeneity. In the present study nucleotide sequences, representing alleles of the gene (Tams1) encoding this molecule in Theileria annulata were compared to each other and to sequences of homologous genes in Theileria sergenti, Theileria buffeli and Theileria parva. This analysis revealed that a region of the polypeptide which contains putative N-linked glycosylation sites is particularly diverse and, in analogy to retroviral systems, may indicate selection of variable glycosylation sites or amino acid epitopes to evade the bovine immune response. This conclusion was also made from the results of a phylogenetic analysis which compared the variable region of the genes with a second region, which appeared to show no bias for diversity or functional constraint. The results indicated that the variable sequence encoding putative glycosylation sites has diverged, both within and between Theileria species, at a much faster rate than the rest of the molecule. Southern blot analysis of T. annulata populations from within a single geographical region detected six possible variant Tams1 alleles. However, a correlation between restriction-fragment-length polymorphism (RFLP) patterns detected by the Tams1-1 gene probe and geographical location could not be made. In addition, although a high prevalence of one particular RFLP was found, this is unlikely to be the result of a clonal population structure, as we present evidence for significant parasite genotypic variability within a single endemic region.

A novel CDC2-related protein kinase from Leishmania mexicana, LmmCRK1, is post-translationally regulated during the life cycle.

The p34CDC2 protein kinase is a key component in the regulation of the eukaryotic cell cycle. We have isolated from the protozoan parasite Leishmania mexicana mexicana a CDC2-related kinase gene (Lmmcrk1) encoding a 34-kDa protein kinase (lmmCRK1) which has 56% amino acid identity with the human CDC2 and contains a PCTAIR motif in place of the highly conserved PSTAIR box. lmmCRK1 was detected in all life cycle stages at comparable levels, yet its histone H1 kinase activity was detected in only the promastigote form, indicating that its activity is stage-regulated at a post-translational level. lmmCRK1 did not bind p13suc1 beads and Lmmcrk1 was unable to complement a fission yeast temperature-sensitive cdc2 mutant. These data suggest that Lmmcrk1 is unlikely to be the functional L. mexicana cdc2 homologue. A distinct histone H1 kinase activity that binds p13suc1 beads (SBCRK) was also detected, with activity that correlated with the division status of the developmental forms of the parasite, being present in the dividing stages of the parasite and absent in nondividing metacyclic forms. SBCRK is a candidate for the functional CDC2 homologue, but it does not react with an anti-PSTAIR monoclonal antibody on Western blots when eluted from p13suc1 beads, indicating a divergent PSTAIR box. These data suggest that a family of CDC2-related protein kinases are present in Leishmania. Some share sequence and biochemical properties with CDC2, but significant differences also exist, possibly reflecting the evolutionary distance between Leishmania and higher eukaryotes.

Antigenic diversity of a major merozoite surface molecule in Theileria annulata.

The 30-kDa merozoite surface antigen of Theileria annulata (Ankara) has been characterised by using merozoites derived from differentiating macroschizont-infected cloned cell lines. This molecule was found to be 1 of the major polypeptides in extracts of both merozoites and piroplasms and was strongly detected by serum from an immune cow. Analysis of merozoite extracts derived from cloned cell lines infected with parasites of different genotypes detected 2 forms of this molecule, with respective molecular masses of 30 kDa and 32 kDa. These 2 molecules were shown to be related to each other by peptide mapping. In addition to the observed size differences, the molecules were found to be antigenically divergent. Periodate treatment of western blots demonstrated that at least 1 of the variable antigenic determinants was generated by secondary modification of the polypeptide and that a large amount of the antibody reactivity was directed against similar moieties. Parasite stocks isolated from different geographical regions were distinguished by analysis of the molecules within the 30-32-kDa molecular region. The relationship between T. annulata molecules and similar antigens described for other Theileria species is discussed.