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.

Hepatic transcription of the acute-phase alpha 1-inhibitor III gene is controlled by a novel combination of cis-acting regulatory elements.

mRNA coding for the abundant broad-range plasma proteinase inhibitor alpha 1-inhibitor III (alpha 1I3) was detected only in rat liver, while mRNA for the related proteins alpha 1-macroglobulin and alpha 2-macroglobulin was also found in a variety of nonhepatic tissues. cis-Acting control elements necessary for the hepatic transcription of alpha 1I3 were mapped by transfection and expression studies of control-region constructs in cultured hepatic and nonhepatic cells. The promoter-proximal 5'-flanking region contained four control elements, I to IV, located between -109 and -196 base pairs upstream of the transcriptional start site relevant for the hepatic transcription of this gene. Elements II and III were essential, and I and IV exerted strong modulatory effects. Elements I to III acted as positive regulators, and IV acted as a negative element. Element II contained the sequence TGGCA and is probably a binding site for a nuclear factor related to the known transcription factor NF1. The other three elements did not resemble consensus binding sites for known transcription factors that are involved in the hepatocyte-specific transcription of other well-characterized plasma protein genes, such as the prototype factor HNF-1. Thus, the alpha 1I3 gene achieves its highly hepatocyte-specific transcription through a novel combination of cis-acting control elements and trans-acting factors.

A retrospective serological survey on human babesiosis in Belgium.

In Europe, most clinical babesiosis cases in humans have been attributed to Babesia divergens and Babesia sp. EU1. Babesia microti infection of humans occurs mainly in the United States; although a case of autochthonous B. microti infection and serological evidence of infection have been reported in Europe. The Indirect Fluorescent Antibody Test was used to screen sera from 199 anonymous Belgian patients with history of tick bite and clinical symptoms compatible with a tick-borne disease. The serological screen detected positive reactivity in 9% (n = 18), 33.2% (n = 66), and 39.7% (n = 79) of the samples against B. microti, B. divergens, and Babesia sp. EU1, respectively. Thus, evidence of contact among three potentially zoonotic species of Babesia and humans has been confirmed in Belgium. Preventive action and development of better diagnostic tools should help in prevention of clinical cases and to clarify the true burden of such infection for individuals and public health.

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.

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.

Phylogenetic analysis of Theileria and Babesia equi in relation to the establishment of parasite populations within novel host species and the development of diagnostic tests.

The divergence of parasites is important for maintenance within an established host and spread to novel host species. In this paper we have carried out phylogenetic analyses of Theileria parasites isolated from different host species. This was performed with small subunit ribosomal RNA sequences available in the data bases and a novel sequence amplified from Theileria lestoquardi DNA. Similar phylogenetic studies were carried out with sequences representing the major merozoite/piroplasm surface antigen (mMPSA) from the data base, and novel sequences representing 2 mMPSA alleles from T. lestoquardi, a full length sequence of a Theileria taurotragi mMPSA gene and partial sequences of two new allelic variants of the Babesia equi mMPSA gene homologue. The analysis indicated that the pathogenic sheep parasite T. lestoquardi has most probably evolved from a common ancestor of T. annulata. Interestingly, the level of mMPSA sequence diversity found for T. lestoquardi was surprisingly low, while diversity between the B. equi sequences was higher than that found within any of the classical Theileria species. The possible implications of these results for the establishment of Theileria parasites within novel species are discussed. Extensive cross-reactivity of a range of antisera was found when tested against recombinant mMPSA polypeptides from different Theileria (including B. equi) species. The cross-reactivity between mMPSA polypeptides and sequence diversity are relevant for the development of species specific diagnostic tests.

Developmental expression of a Theileria annulata merozoite surface antigen.

Culture of a lymphoblastoid cell line infected with the macroschizont stage of the protozoan parasite Theileria annulata at 41 degrees C induces differentiation to the next stage, the merozoite. We have demonstrated that this development results in the loss of monoclonal antibody epitopes associated with the macroschizont stage, and the appearance of epitopes associated with the piroplasm (the intra-erythrocytic stages). One of the monoclonals (5E1) was shown by immunoelectron microscopy to bind to the surface of heat-induced culture forms which had size and structural characteristics of the merozoite. The monoclonal was found to detect two polypeptides of 30 kDa and 25 kDa in extracts of piroplasms. The 30-kDa polypeptide was also detected in a merozoite extract, but was not detected in an extract derived from macroschizont-infected lymphoblastoid cells. We conclude that the heat-induced differentiation of T. annulata in vitro results in the expression of a 30-kDa molecule which is located at the surface of the merozoite, and discuss the potential of this molecule as a component in a subunit vaccine.

Characterisation of an extrachromosomal DNA element from Theileria annulata.

Extrachromosomal nucleic acid elements are found in all organisms, commonly as organelle, viral or plasmid genomes. In this paper we describe the initial characterisation of a novel 6.5-kb linear, double-stranded extrachromosomal element from Theileria annulata, and a 2.6-kb RNA species. The DNA element is present in different stages of the life cycle and in different stocks of the parasite. Northern blots of total RNA isolated from different stages of the parasite, probed with the purified element, detect three major transcripts, of 1.45, 1.05 and 0.24 kb, present in all life-cycle stages examined. The possible origin and function of this element is discussed, together with its possible use as a transfection vector for the introduction of genes into protozoan cells.

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.

Sequence and expression of a gene from Theileria annulata coding for a 70-kilodalton heat-shock protein.

A library consisting of randomly sheared Theileria annulata genomic DNA fragments in the vector lambda gt11 was screened with a Drosophila DNA segment containing the coding region of the 70-kDa heat-shock protein (hsp) gene. A positive recombinant was isolated and subjected to nucleotide sequence analysis. The DNA segment contains an open reading frame coding for a 71-kDa protein strongly homologous to hsp 70 from other organisms. Using this DNA as a probe, a homologous 2.5-kb RNA species was detected in sporozoites, piroplasms and a macroschizont-infected cell line, showing that this gene is constitutively expressed. The amount of this RNA increased following heat shock in the macroschizont-infected cell line. The T. annulata genome contains other sequences that hybridize weakly with this heat-shock gene.

Characterization of surface polypeptides on different life-cycle stages of Theileria annulata.

We describe the characterisation of polypeptides located on the surface of Theileria annulata sporozoites, macroschizonts, piroplasms and infected lymphoblastoid cells using surface iodination techniques. The sporozoite stage exhibited a complex profile of surface polypeptides. However, using data from experiments with defined monoclonal antibodies, the sporozoite surface appeared to be composed of several distinct groups of related polypeptides. Analysis of the macroschizont detected seven surface polypeptides, while eight polypeptides were identified for the piroplasm stage. On the basis of molecular weight comparisons, one of the surface polypeptides appeared to be common to the sporozoite, macroschizont and piroplasm. Stage cross-reactive monoclonals failed to immunoprecipitate a surface-radiolabelled polypeptide, and this prohibited the characterisation of a stage common surface antigen. From the surface labelling studies of Theileria-infected and uninfected lymphoblastoid cell lines, we concluded that infection results in major changes at the surface of the host cell, including both the appearance and loss of specific polypeptides. By employing monoclonal antibodies which detect infection-associated determinants, and a polyclonal antiserum raised against a glycoprotein fraction of an infected cell lysate, surface-labelled polypeptides were specifically immunoprecipitated from extracts of infected cells. The polypeptide detected by monoclonal antibody 4H5 was characterised as an infection-associated glycoprotein which varies in molecular mass when immunoprecipitated from different infected cell lines. The identification of infection-associated glycoproteins on the surface of the lymphoblastoid cell suggests that these molecules may be recognised by the cytotoxic T cells of immune animals.

Transient transfection of Theileria annulata.

We have developed a method to transiently transfect infective, uninucleate, Theileria annulata sporozoites. Transfection vectors have been constructed using a number of T. annulata 5' gene flanking sequences linked to the enhanced green fluorescence protein (eGFP) reporter gene. Sporozoites were transfected with these constructs using the lipid transfection agent SuperFect, then allowed to infect purified bovine mononuclear cells (PBMs). Green fluorescence was observed in developing trophozoites, 36-40 h post infection, using constructs containing the upstream regions of the T. annulata Hsp70, T. annulata merozite surface antigen 1 (TamS1) and T. annulata macroschizont-specific AT hook-containing protein2 (TashAT2) genes. A construct with the 5' TamS1 upstream sequence in reverse orientation gave no detectable fluorescence indicating fluorescence was derived by expression from the T. annulata promoter. A cytomegalovirus (CMV) promoter construct showed no activity in this stage of the parasite. However, when this construct was introduced directly into schizont-infected cells by electroporation, fluorescence was observed in the bovine cells but not the schizont. We describe the significance of these results in relation to novel control strategies and the fundamental biology of Theileria parasites.

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.

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.

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.

Simplified northern blot hybridization using 5% sodium dodecyl sulfate.

We describe a simplified Northern blot hybridization procedure. This procedure eliminates the need for many reagents commonly used in RNA hybridization and replaces them with two buffers containing 5% sodium dodecyl sulfate for pre-hybridization, hybridization and all post-hybridization washes.

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.

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.

Directing differentiation in Theileria annulata: old methods and new possibilities for control of apicomplexan parasites.

Apicomplexan parasites are major pathogens of humans and domesticated animals. The ability of these organisms to evade the host immune response and the emergence of drug-resistant parasites indicates a need for the identification of novel control strategies. Ideally, selected targets should be shared by a range of apicomplexans and fundamental to parasite biology. One process of apicomplexan biology which may provide this type of target is the molecular regulation of stage differentiation. This paper has reviewed studies carried out on differentiation of Theileria annulata and has highlighted general similarities with other apicomplexan differentiation steps. Similarities include asynchrony of differentiation, the loss (attenuation) of differentiation potential and an association between reduced proliferation and differentiation. In addition, novel data are presented assessing a possible role for a signal transduction mechanism or a direct involvement of classical heat-shock polypeptides in regulating differentiation of T. annulata in vitro. These studies, and previously published data, have led to the postulation that progression to the next stage of the life-cycle can be predetermined and involves the attainment of a quantitative threshold by regulators of gene expression. A modification of this model takes into account that for certain in-vitro systems, or differentiation steps in vivo, the process has to be initiated by alteration of the extracellular environment. Work which has shown that the time taken to achieve differentiation can be increased or decreased is also outlined. The ability to change the timing of differentiation suggests that the associated regulatory mechanism could be manipulated directly to significantly influence the outcome of an apicomplexan infection. The observation that a number of existing drugs and control strategies may exert their protective effect by altering differentiation potential supports this possibility.

Modulation of growth relative to division regulates expression of a Theileria annulata infection associated antigen in HL-60 cells differentiating towards granulocytes.

The protozoan parasite Theileria annulata has the ability to immortalise the bovine leukocyte in which it resides. Immortalisation is known to be associated with a number of molecular and antigenic alterations to the host cell. In this study cells of related lineage were compared with T. annulata infected cells, using a monoclonal antibody which detects an infection associated glycoprotein on the host cell surface. The results show that this antibody recognises a 160 kDa antigen in HL-60 cells, and that expression of this antigen is up-regulated as cells are induced to differentiate towards granulocytes, but not macrophages. Up-regulation was observed to proceed in a quantitative manner, with progression through an intermediate phase, before full antigen expression and granulocyte formation was observed. Limited incubation with inducer (DMSO) for 18 h indicated that intermediate cells could revert to negative cells, while longer exposure resulted in conversion to high level antigen expression and a commitment to differentiate. Alteration of cell culture conditions and modulation of division (DNA synthesis) relative to growth (protein synthesis) by incubation in aphidicolin resulted in an increase in the number of cells expressing antigen at both the intermediate level and the level associated with commitment. Based on these results and related studies we present a model which proposes that differentiation is initiated, and then progresses to a quantitative commitment threshold, by altering the level of key regulators of gene expression relative to their DNA templates.