Telomeric features of Theileria parva mitochondrial DNA derived from cycle sequence data of total genomic DNA.
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Animals↗
Biomedical subjects
V Nene
Publications and source records attributed to V Nene.
Organisation and informational content of the Theileria parva genome.
When compared with other Apicomplexan organisms, Theileria parva has an exceptionally small, 10-12 Mbp, genome. There are only 4 chromosomes, each in the Mbp range, and a complete physical map, based on SfiI linking data, is available for each one. A number of genes and cDNAs have been mapped to specific SfiI fragments. Telomeres consist of the simple repeat sequence typical of chromosomal ends but sub-telomeric homologies do not extend beyond 5 kbp. The only dispersed repetitive sequences identified to date are minisatellites, but these are found on a subset of SfiI fragments. There are clusters of distinct multicopy sequences which contain ORFs. However, the majority of parasite protein coding genes are present in a single copy. They have a compact structure, exhibit a bias in codon usage and non-translated regions are small. Introns, if present, are unusually short. Overall, the genome contains remarkably little repetitive, non-coding DNA. The parasite mitochondrial DNA is linear in structure, has a limited protein coding capacity and fragmented rRNA genes and its telomeres contain inverted repeat sequences.
Animals↗
Different vaccine strategies used to protect against Theileria annulata.
SPAG-1, a sporozoite surface antigen of T. annulata, has previously been shown to elicit partial protection when used, as an hepatitis B core antigen fusion, to immunize cattle. The objective of this study was to try and improve the protective capacity of this antigen by enlisting different vaccine strategies. Cattle were immunized with SPAG-1, as a fusion protein with a His6 tag, either incorporated into ISCOMs, with or without the merozoite antigens TAMS 1-1 and 1-2, or with RWL as adjuvant three times at monthly intervals. Another group of cattle were immunized with p67, the T. parva sporozoite antigen, in RWL to assess whether any cross-protection could be induced. The animals were then challenged with an estimated LD50 of T. annulata sporozoites, and their ability to resist the infection was investigated. Serum responses and T-cell proliferative responses were analyzed throughout the trial. Post-challenge analyses included lymph node biopsies and blood smears to check for the presence of parasites, routine hematological parameters, and observation for clinical manifestations of the disease. The results of this trial will be discussed.
Animals↗
An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule.
Field and experimental bovine infection sera were used in immunoblots of sporozoite and schizont lysates of Theileria parva to identify candidate diagnostic antigens. Four parasite antigens of Mr 67,000 (p67), 85,000 (the polymorphic immunodominant molecule, PIM), 104,000 (p104), and 150,000 (p150) were selected for a more detailed analysis. The p67 and p104 antigens were present only in the sporozoite lysates, whereas PIM and p150 were found in both sporozoite and schizont lysates. The four antigens were expressed as recombinant fusion proteins and were compared with each other in an enzyme-linked immunosorbent assay (ELISA) and in the whole-schizont-based indirect fluorescent antibody test (IFAT) in terms of their ability to detect antibodies in sera of experimentally infected cattle. The PIM-based ELISA provided a higher degree of sensitivity and specificity than did the ELISA using the other three recombinant antigens or the IFAT. Further evaluation of the PIM-ELISA using experimental sera derived from cattle infected with different hemoparasites and field sera from endemic and nonendemic T. parva areas showed that the assay had a sensitivity of > 99% and a specificity of between 94% and 98%.
Animals↗
Subunit vaccines for the control of tick-borne diseases: implications for the future.
Tick-borne parasites are a major constraint to the improvement of livestock productivity in the developing world. These parasites include Theileria parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale and Cowdria ruminantium. The impact of these diseases is currently limited by the use of acaricides to decrease transmission by the tick vectors and immunization of the host animals using live vaccines. The use of acaricide is hampered by the development of acaricide resistance and live vaccines are dependent on cold chain facilities, which are generally unreliable in developing countries. There is therefore a requirement for improved vaccines that circumvent these problems. Candidate vaccine antigens have been identified for most of these parasites and are currently being evaluated for their capacity to induce solid protection.
Anaplasma↗
Characterization of a small variable surface glycoprotein from Trypanosoma vivax.
Several biochemical properties of a variant surface glycoprotein (VSG) from the parasite Trypanosoma (Duttonella) vivax have been determined. ILDat 2.1 VSG is approximately 40 kDa in size making this the smallest trypanosome VSG described to date. The glycolipid anchor of ILDat 2.1 VSG is resistant to treatment with T. brucei-derived phospholipase C and data based on lectin affinity chromatography, incorporation of radiolabelled sugar and treatment with endoglycosidase H suggest that the T. vivax VSG bears little carbohydrate. cDNA to ILDat 2.1 VSG mRNA has been cloned and the encoded protein sequence includes the N-terminal amino acid peptide sequence derived from native VSG. The molecular weight of the VSG predicted from the translated cDNA sequence is similar to that of the native molecule and in support of the biochemical data it is devoid of sites for N-linked glycosylation. Examination of the deduced ILDat 2.1 VSG protein sequence reveals that it is most similar to T. congolense VSGs in the distribution of Cys residues and like the former it does not contain any of the defined VSG C-terminal domain types. However, unlike T. congolense VSGs it does not readily fit into the currently described VSG N-terminal domain types. Our studies suggest that ILDat 2.1 VSG is distinct from any of the previously characterized VSGs.
Amino Acid Sequence↗
Conservation of neutralizing determinants between the sporozoite surface antigens of Theileria annulata and Theileria parva.
The sporozoite surface antigens SPAG-1 of Theileria annulata and p67 of Theileria parva are postulated to contain determinants necessary for host cell invasion and/or recognition and are both being considered as candidates for inclusion in subunit vaccines. Preliminary data suggest that these are related molecules. In this paper we describe the investigation of the relationship between these sporozoite antigens further by analysis of the immunological cross-reactivity using Mabs and sera raised against each antigen. The cross-reactions were examined by carrying out Western blots, IFA tests, and in vitro sporozoite neutralization assays. In addition, sequence comparison data which clearly establish that these surface antigens are encoded by related genes are presented. The regions of SPAG-1 identified as containing cross-reactive epitopes recognized by p67 antiserum correlated to regions of high predicted homology between p67 and SPAG-1, which are located at their respective N- and C-termini. Furthermore, p67 and SPAG-1 were found to contain cross-reactive determinants responsible for neutralization of sporozoite infectivity in vitro, and at least some of these were located in the C-termini of both molecules. The relevance of these findings to the possible roles for these molecules in host cell invasion is discussed.
Amino Acid Sequence↗
Prospects for subunit vaccines against tick-borne diseases.
Tick-borne parasites are a serious impediment to the improvement of live-stock production in the developing world. The major parasites affecting cattle include Theileria parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale and Cowdria ruminantium. The control of these infections is dependent on the use of acaricides to decrease transmission by the tick vectors, and immunization of susceptible animals with live vaccines. The use of acaricide is hampered by the development of resistance, and live vaccines require cold chain facilities, which are generally unreliable in developing countries. There is therefore a need for improved vaccines that can circumvent these problems. There is a subunit vaccine being developed for T. parva based on the major surface antigen of the sporozoite (p67). A similar antigen, SPAG 1, has been identified as a candidate for T. annulata. Although several candidate antigens have been identified for Babesia spp., progress towards development of a subunit vaccine based on these antigens has been hampered by polymorphism among isolates and between species, and lack of knowledge of the immune effector mechanisms responsible for protection. The search for protective antigens of A. marginale has focused on outer membrane proteins; immunization with a variety of these antigens alone or in combination, has yielded promising results. As with Babesia, further definition of immune effector mechanisms is needed to optimize immunization strategies. The work on identifying the protective antigens of C. ruminantium is in its embryonic stages; however, two antigens have been identified and are currently being evaluated. There is high expectancy for subunit vaccines for all these diseases; however there is need for further work to elucidate the immune mechanisms in order to select appropriate antigen delivery systems.
Anaplasma↗
Conservation of the sporozoite p67 vaccine antigen in cattle-derived Theileria parva stocks with different cross-immunity profiles.
Immunity to Theileria parva infection in cattle is often parasite stock specific. The antigenic diversity which is expressed at the schizont stage of the parasite together with a wild reservoir of the organism in buffalo has complicated the development of effective disease control by immunization. We have previously shown that about 70% of cattle inoculated with recombinant forms of p67, a sporozoite stage-specific surface antigen from the cattle-derived Muguga stock of the parasite, are immune to a homologous challenge. Thus, immune responses to p67 can play a role in immunity. The genes encoding this protein in five other parasite stocks have been sequenced. Here, we report that the p67 molecule encoded by four cattle-derived parasite stocks (Boleni, Uganda, Mariakani, and Marikebuni) that fall into different cross-immunity groups is identical in sequence to Muguga p67. The protein encoded by a buffalo-derived parasite exhibits 95% sequence identity with Muguga p67, the major difference being the presence of a 43-residue peptide insert. As predicted by these data, cattle inoculated with recombinant p67 can resist a heterologous cattle-derived parasite challenge. Seven of 12 cattle receiving a homologous Muguga challenge and 6 of 11 cattle receiving a heterologous Marikebuni challenge were immune to East Coast fever. These results extend earlier data suggesting that p67 is a conserved molecule and confirm its potential as a broad-spectrum vaccine antigen for the control of T. parva infection.
Amino Acid Sequence↗
Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva.
Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5' and 3' regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3' region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.
Amino Acid Sequence↗
Structure and sequence variation of the genes encoding the polymorphic, immunodominant molecule (PIM), an antigen of Theileria parva recognized by inhibitory monoclonal antibodies.
The polymorphic, immunodominant molecule (PIM) of Theileria parva is the predominant antigen recognized by sera from infected cattle and by monoclonal antibodies (mAb) used to differentiate parasite strains. As such, the antigen is under consideration as a diagnostic antigen, and since the mAbs can neutralize sporozoite infectivity in vitro, in immunization experiments. Initial comparison of two PIM cDNA sequences suggested that the PIM genes consist of conserved 5' and 3' termini flanking a central variable region. We present further evidence, based on sequence analysis, supporting this general structure for the PIM genes. Evidence is also presented for a single copy of the PIM gene per haploid genome, implying that the different versions of PIM are encoded by distinct alleles. The central variable region of the PIM allele from the T. parva (Marikebuni) stock was found to contain 13 copies of the tetrapeptide repeat Gln-Pro-Glu-Pro. We also detected point mutations in the 5' and 3' termini of the PIM alleles, including regions recognized by the neutralizing and typing mAb. This contrasted with the high sequence conservation of the two introns of the genes, suggesting that the protein is undergoing rapid evolution. Sequence comparison of PIM genes from buffalo- and cattle-derived parasites supported earlier results that the parasites infecting buffaloes constitute a more heterogeneous population than those from cattle.
Amino Acid Sequence↗
Characterization of an insect cell-derived Theileria parva sporozoite vaccine antigen and immunogenicity in cattle.
Previous data showed that six out of a group of nine cattle inoculated with NS1-p67, a recombinant form of a 67-kDa Theileria parva sporozoite surface protein, were immune to East Coast fever. This bacterially expressed antigen encoded all 709 amino acid residues of p67 fused to the C-terminal end of 87 residues derived from NS1, a structural protein of influenza virus, and a linker DNA sequence. NS1-p67 lacked reactivity with TpM 12, a monoclonal antibody to native p67, and had an estimated molecular mass of 110 kDa, as opposed to the calculated mass of 85,000 Da. We have used the baculovirus expression system in an attempt to express this parasite protein in a native form and thereby increase the protective capacity of the antigen. However, Spodoptera frugiperda SF21AE cells infected with recombinant virus expressed p67 as a 100-kDa molecule. The host cells exhibited a limited capacity to glycosylate this molecule to a 110-kDa form, and p67 was not exported to the surface membrane. TpM 12 did not bind to these recombinant forms but, at time points late during viral infection, reacted with a molecule of about 70 kDa. Since the bulk of insect cell-derived p67 was not expressed in an appropriate form, we tested the immunogenicity of these partially processed recombinant p67 forms in cattle. Two groups of three cattle were inoculated with antigen formulated either with saponin or Freund's adjuvant. As seen previously with NS1-p67, all animals developed high levels of anti-p67 antibodies that neutralized sporozoite infectivity in vitro, but antigen-specific T-cell proliferative responses were not detected in peripheral blood. Given the caveat of the small number of cattle analyzed, insect cell-derived p67 does not appear to be superior to NS1-p67 as an immunogen, and the latter remains the molecule of choice for the development of vaccines against East Coast fever.
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A 7.1 kb linear DNA molecule of Theileria parva has scrambled rDNA sequences and open reading frames for mitochondrially encoded proteins.
Theileria parva, an intralymphocytic protozoan parasite of cattle, contains a linear 7.1 kb DNA element with terminal inverted repeat sequences. The molecule is transcribed into low molecular weight RNA, and both DNA strands encode short stretches of unique sequences, usually < 100 nucleotides, which are similar to large (LSU) or small (SSU) ribosomal subunit RNA. Phylogenetically conserved conformational rRNA domains were assembled from the discontinuous rDNA sequences using comparative secondary structure modelling. For example, a minimum of four predicted sequences, two derived from each DNA strand, is required to assemble domain V of LSU rRNA which participates in peptidyl transferase activity. The discontinuities in the identified rRNA domains fall within regions of no known functional significance. Hence, it is likely that the element encodes fragmented rDNA genes and the mature rRNA is unconventional, consisting of several fragments of RNA, primarily held together by intermolecular and intramolecular base pairing. The element also has ORFs for components of the last two mitochondrial electron transport enzyme complexes. The structure of the parasite DNA element, its protein coding capacity and scrambled rDNA gene sequences, are reminiscent of the mitochondrial genome of Chlamydomonas reinhardtii. We propose that the 7.1 kb element is equivalent to the mitochondrial DNA of T. parva, although a number of its features are unusual for this family of extrachromosomal DNA molecules.
Amino Acid Sequence↗
Evidence for two single copy units in Theileria parva ribosomal RNA genes.
Bacteriophage clones containing ribosomal RNA genes of Theileria parva were isolated from genomic DNA libraries. Physical mapping studies revealed 2 ribosomal DNA units, which were distinguishable by restriction enzyme site polymorphisms in flanking sequences. The cloned ribosomal DNA units were mapped to 2 separate T. parva chromosomes. Analysis of sequences contained in lambda EMBL3 recombinants, together with Southern blot analysis of genomic DNA and data on the copy number of the rRNA genes, suggested that the rDNA units were not tandemly repeated. This organisation of ribosomal transcription units is similar to that described for other genera of apicomplexan protozoa, but 2 rDNA units, each containing single copies of the rRNA coding genes, would be the lowest copy number described for any eukaryote in which amplification of rRNA genes is not known to occur. EcoRI restriction fragment length polymorphisms, which were revealed using rRNA gene probes, separated T. parva stocks into 2 categories. Nucleotide sequence analysis of polymerase chain reaction-amplified internal transcribed spacer DNA revealed 2 different ITS sequences derived from rDNA transcription units within the genome of a cloned T. parva parasite. Polymorphism was also observed between ITS sequences amplified from the DNA of different T. parva stocks. A synthetic oligonucleotide derived from T. parva Uganda ribosomal ITS DNA sequences hybridised to DNA from the T. parva Uganda stock, but not to the DNA of the T. parva Muguga stock. This oligonucleotide is potentially useful as a marker for the T. parva Uganda stock.
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Bovine T cells specific for Trypanosoma brucei brucei variant surface glycoprotein recognize nonconserved areas of the molecule.
The specificity of bovine CD4+ T-cell responses to Trypanosoma brucei variant surface glycoprotein (VSG) has been examined by using a panel of seven T-cell clones and nested deletions of the ILTat 1.3 VSG gene expressed in Escherichia coli. All clones recognized the polymorphic N-terminal domain of the antigen, and the recognition sites of five of the clones were resolved to three areas with lengths of 14, 18, and 21 amino acids. Comparison of these regions with corresponding areas of other VSG molecules, including those derived from the same trypanosomal serodeme, has shown that the sites are not conserved. In the light of recent observations that VSG-specific T-cell responses are induced in mice infected with T. brucei, these results confirm with the belief that immune pressure from T cells may contribute to the generation of antigenic diversity on the surface of African trypanosomes.
Amino Acid Sequence↗
A Sporozoite-based vaccine for Theileria parva.
East Coast fever, which is caused by Theileria parva infection in cattle, is of major economic importance in eastern and central Africa. Until recently, the only available method of immunization against East Coast fever was the infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. This method has two major disadvantages: (I) it uses live organisms; and (2) the immunity engendered is parasite strain specific. In this article, Antony Musoke, Vishvonath Nene and Subhosh Morzoria review the progress made in developing an alternative method o f immunization based on a defined sporozoite antigen.
Journal Article↗
A recombinant sporozoite surface antigen of Theileria parva induces protection in cattle.
At present immunization against Theileria parva is by infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. This method has major limitations in that live organisms are used and the immunity engendered is parasite stock specific. In an attempt to develop an alternative immunization procedure, the gene encoding p67, a major surface antigen of sporozoites, has been expressed by using the plasmid expression vector pMG1. The gene, which has been characterized previously, encodes 709 amino acid residues, contains a single intron of 29 base pairs, and is only transcribed during sporogony. The recombinant p67 sequences were fused to the first 85 amino acid residues derived from a nonstructural gene (NS1) of influenza virus A. Immunization with a partially purified recombinant antigen emulsified in 3% saponin induced sporozoite neutralizing antibodies in cattle and provided protection in six of nine animals on homologous challenge with T. parva sporozoites. This recombinant antigen is therefore a candidate for development of a vaccine against T. parva.
Animals↗
Characterisation of the gene encoding a candidate vaccine antigen of Theileria parva sporozoites.
We have cloned and characterised the gene encoding the 67-kilodalton stage-specific surface antigen, p67, of Theileria parva (Muguga) sporozoites. The gene which is present in a single copy, is divided into 2 exons by an intron 29 bp long and is transcribed into mRNA of about 2500 nucleotides. The gene is present in all stocks of T. parva and there is a related gene in Theileria annulata. The deduced amino acid sequence of 709 residues predicts that p67 is a membrane protein and that it lacks tandemly repeated sequences. Recombinant p67 has been expressed in Escherichia coli as a fusion protein with Sj-26, a glutathione-S-transferase of Schistosoma japonicum. Antibodies to purified recombinant proteins containing residues 9-316 or 397-709 of p67 bind to p67 in immunoblots and neutralise sporozoite infectivity in vitro. Recombinant p67 is, therefore, a candidate antigen for development of an anti-sporozoite vaccine for East Coast fever in cattle.
Amino Acid Sequence↗