Approaches to vaccination against Theileria parva and Theileria annulata.

Despite having different cell tropism, the pathogenesis and immunobiology of the diseases caused by Theileria parva and Theileria annulata are remarkably similar. Live vaccines have been available for both parasites for over 40 years, but although they provide strong protection, practical disadvantages have limited their widespread application. Efforts to develop alternative vaccines using defined parasite antigens have focused on the sporozoite and intracellular schizont stages of the parasites. Experimental vaccination studies using viral vectors expressing T. parva schizont antigens and T. parva and T. annulata sporozoite antigens incorporated in adjuvant have, in each case, demonstrated protection against parasite challenge in a proportion of vaccinated animals. Current work is investigating alternative antigen delivery systems in an attempt to improve the levels of protection. The genome architecture and protein-coding capacity of T. parva and T. annulata are remarkably similar. The major sporozoite surface antigen in both species and most of the schizont antigens are encoded by orthologous genes. The former have been shown to induce species cross-reactive neutralizing antibodies, and comparison of the schizont antigen orthologues has demonstrated that some of them display high levels of sequence conservation. Hence, advances in development of subunit vaccines against one parasite species are likely to be readily applicable to the other.

BoLA-6*01301 and BoLA-6*01302, two allelic variants of the A18 haplotype, present the same epitope from the Tp1 antigen of Theileria parva.

We have recently shown that the BoLA-A18 variant haplotype (BoLA-6*01302) is more prevalent than the BoLA-A18 haplotype (BoLA-6*01301) in a sample of Holstein/Friesian cattle in Kenya. These MHC class I allelic variants differ by a single amino acid polymorphism (Glu97 to Leu97) in the peptide-binding groove. We have previously mapped an 11-mer peptide epitope from the Theileria parva antigen Tp1 (Tp1214-224) that is presented by BoLA-6*01301. Crystal structure data indicates that Glu97 in the MHC molecule plays a role in epitope binding through electro-static interaction with a lysine residue in position 5 of the epitope, which also functions as an additional anchor residue. In contrast to expectations, we demonstrate that the amino acid substitution in BoLA-6*01302 does not divert the CTL response away from Tp1214-224. The two MHC molecules exhibit similar affinity for the Tp1 epitope and can present the epitope to parasite-specific CTLs derived from either BoLA allelic variants. These data confirm that this BoLA polymorphism does not alter Tp1 epitope specificity and that both allelic variants can be used for Tp1 vaccine studies.

A method to discriminate between closely related bovine major histocompatibility complex class I alleles by combining established PCR-SSP assays with RFLPs.

We have developed a polymerase chain reaction-sequence-specific primers-restriction fragment length polymorphism (PCR-SSP-RFLP) method to rapidly differentiate between the A18 and A18 variant (v) BoLA haplotypes and between A14 and A15/A15v BoLA haplotypes in Holstein/Friesian cattle. We used published SSP to PCR amplify BoLA alleles expressed in animals of known haplotype and exposed the amplicons to the restriction enzyme PvuII that was predicted to cut at a unique site in the middle of BoLA-6*01302 (A18v) and BoLA-1*00901 (A15) but not in BoLA-6*01301 (A18) or BoLA-1*02301 (A14) alleles. Whereas the method does not discriminate between the A15 and A15v haplotypes, as the BoLA-1*00902 allele associated with A15v also contains a PvuII site, we are interested in cattle of A18 and A14 haplotype for vaccine related studies. Our results also indicated that the BoLA-6*01302 (A18v) allele is much more abundant than BoLA-6*01301 (A18) in the cattle that we sampled.

EST-based gene indices for the cattle fever tick, the horn fly, and the screwworm fly.

The southern cattle tick, Rhip,cephalus (Boophilus) microplus, the horn fly, Haematobia irritans, and the New World screwworm, Cochl,omyia hominivorax, are economically important parasites of cattle throughout the world. Understanding the biology and genomics of these pests is critical to developing novel control methods. We developed EST sequence databases for these parasites and used bioinformatic analysis to identify conceptual open reading frames and assigned Gene Ontology (GO) terms to those database transcripts that had informative BlasIX hits. Bloinformatic analysis showed that a large fraction of the tick database entries have no clear matches in other sequenced genomes. Several putative tick G protein-coupled receptors (GPCRs) were identified that mightserve as starting points to develop novel control technologies.

Expressed cDNAS from embryonic and larval stages of the horn fly (Diptera: Muscidae).

We used an expressed sequence tag approach to initiate a study of the genome of the horn fly, Hematobia irritans (L.) (Diptera: Muscidae). Two normalized cDNA libraries were synthesized from RNA isolated from embryos and first instars from a field population of horn flies. Approximately 10,000 clones were sequenced from both the 5' and 3' directions. Sequence data from each library was assembled into a database of tentative consensus sequences (TCs) and singletons and used to search public protein databases and annotate the sequences. Additionally, the sequences from both the egg and larval libraries were combined into a single database consisting of 16,702 expressed sequence tags (ESTs) assembling into 2886 TCs and 1,522 singleton entries. Several sequences were identified that may have roles in the horn fly's resistance to insecticides. The availability of this database will facilitate the design of microarray and other experiments to study horn fly gene expression on a larger scale than previously possible. This would include studies designed to investigate metabolic-based insecticide resistance, identify novel antigens for vaccine-based control approaches, and discover new proteins to serve as targets for new pesticide development.

Tick genomics: the Ixodes genome project and beyond.

Ticks and mites (subphylum Chelicerata; subclass Acari) include important pests of animals and plants worldwide. The Ixodes scapularis (black-legged tick) genome sequencing project marks the beginning of the genomics era for the field of acarology. This project is the first to sequence the genome of a blood-feeding tick vector of human disease and a member of the subphylum Chelicerata. Genome projects for other species of Acari are forthcoming and their genome sequences will likely feature significantly in the future of tick research. Parasitologists interested in advancing the field of tick genomics research will be faced with specific challenges. The development of genetic tools and resources, and the size and repetitive nature of tick genomes are important considerations. Innovative approaches may be required to sequence, assemble, annotate and analyse tick genomes. Overcoming these challenges will enable scientists to investigate the genes and genome organisation of this important group of arthropods and may ultimately lead to new solutions for control of ticks and tick-borne diseases.

Serial analysis of gene expression in the southern cattle tick following acaricide treatment of larvae from organophosphate resistant and susceptible strains.

Organophosphate resistant and susceptible tick larvae from laboratory strains of the southern cattle tick, Rhipicephalus (Boophilus) microplus were exposed to low doses of the organophosphate (OP) acaricide, coumaphos. Serial analysis of gene expression (SAGE) was used to analyse differential gene expression in response to OP treatment and to compare the responses of OP-treated and -untreated resistant and susceptible tick larvae. An R. microplus Gene Index was used as an EST database to identify genes which corresponded to SAGE tags whose abundance changed in response to acaricide exposure. Relative quantitative RT-PCR was used to confirm the differential expression results from the SAGE experiments. Of particular interest is a SAGE tag which corresponds to a cytochrome P450-like EST in the Gene Index which was more abundant in untreated OP resistant larvae compared to untreated OP susceptible larvae. This SAGE tag was also more abundant in OP resistant larvae treated with OP compared to OP susceptible larvae treated with OP.

Identification of candidate sialome components expressed in ixodid tick salivary glands using secretion signal complementation in mammalian cells.

Ixodid ticks manipulate mammalian host pathways by secreting molecules from salivary glands. Novel cDNAs containing functional secretion signals were isolated from ixodid tick salivary glands using a signal sequence trap. Only 15/61 Rhipicephalus appendiculatus and 1/7 Amblyomma variegatum cDNAs had significant identity (< 1e-15) to previously identified sequences. Polypeptides that may interact with host pathways included a kinase inhibitor. Two proteins encoded homologues of the yolk protein vitellogenin and seventeen contained glycine-rich motifs. Four proteins without sequence matches had conserved structural folds, identified using a Threading algorithm. Predicted secretion signals were between fifteen and fifty-seven amino acids long. Four homologous polymorphic proteins contained conserved (26/27 residues) signal peptides. Ten functional tick secretion signals could not be unambiguously identified using predictive algorithms.

BmiGI: a database of cDNAs expressed in Boophilus microplus, the tropical/southern cattle tick.

We used an expressed sequence tag approach to initiate a study of the genome of the southern cattle tick, Boophilus microplus. A normalized cDNA library was synthesized from pooled RNA purified from tick larvae which had been subjected to different treatments, including acaricide exposure, heat shock, cold shock, host odor, and infection with Babesia bovis. For the acaricide exposure experiments, we used several strains of ticks, which varied in their levels of susceptibility to pyrethroid, organophosphate and amitraz. We also included RNA purified from samples of eggs, nymphs and adult ticks and dissected tick organs. Plasmid DNA was prepared from 11,520 cDNA clones and both 5' and 3' sequencing performed on each clone. The sequence data was used to search public protein databases and a B. microplus gene index was constructed, consisting of 8270 unique sequences whose associated putative functional assignments, when available, can be viewed at the TIGR website (http://www.tigr.org/tdb/tgi). A number of novel sequences were identified which possessed significant sequence similarity to genes, which might be involved in resistance to acaricides.

Theileria: intracellular protozoan parasites of wild and domestic ruminants transmitted by ixodid ticks.

Theileria are economically important, intra-cellular protozoa, transmitted by ixodid ticks, which infect wild and domestic ruminants. In the mammalian host, parasites infect leukocytes and erythrocytes. In the arthropod vector they develop in gut epithelial cells and salivary glands. All four intra-cellular stages of Theileria survive free in the cytoplasm. The schizont stages of certain Theileria species induce a unique, cancer-like, phenotype in infected host leukocytes. Theileria undergoes an obligate sexual cycle, involving fusion of gametes in the tick gut, to produce a transiently diploid zygote. The existence of sexual recombination in T. parva has been confirmed in the laboratory, and is presumed to contribute to the extensive polymorphism observed in field isolates. Key parameters in T. parva population dynamics are the relative importance of asymptomatic carrier cattle and animals undergoing severe disease, in transmission of the parasite to ticks, and the extent of transmission by nymphs as compared to adult ticks. Tick populations differ in vector competence for specific T. parva stocks. Recombinant forms of T. parva and T. annulata sporozoite surface antigens induce protection against parasite challenge in cattle. In future, vaccines might be improved by inclusion of tick peptides in multivalent vaccines.

Fusion to green fluorescent protein improves expression levels of Theileria parva sporozoite surface antigen p67 in insect cells.

East Coast fever (ECF) is a fatal disease of cattle caused by the protozoan parasite Theileria parva. The development of a subunit vaccine, based on the sporozoite-specific surface antigen p67, has been hampered by difficulties in achieving high-level expression of recombinant p67 in a near-authentic form. Therefore two sets of recombinant baculovirus vectors were constructed. The first set, encoding various regions of p67, produced low levels of the corresponding p67 domains in High Five cells, despite the presence of large amounts of p67 RNA. The second, consisting of p67 domains fused to the carboxy-terminus of GFP expressed significantly higher levels of p67 protein. The GFP:p67 fusion proteins were recognized by a sporozoite-neutralizing monoclonal antibody (TpM12) raised against native p67 whereas non-fused full length p67 expressed in insect cells was not recognized. GFP-tagging therefore, appeared to enhance the stability of p67 and to conserve its folding. The high-level expression of p67 domains in a more authentic form is an important step towards the development of an effective subunit vaccine against ECF.

Trapping parasite secretory proteins in baker's yeast.

Because the function of signal sequences has been conserved during evolution it has been possible to develop both bioinformatics resources to identify them and techniques to clone genes that encode secretory proteins. The latter entail insertion of heterologous signals upstream of signal peptide deleted reporter genes. We discuss the advantages of using Saccharomyces cerevisiae for signal sequence trap technology. The yeast protein-translocation system appears to be less discriminating than that of higher eukaryotes - for example, a Theileria parva cysteine protease gene containing a recessed, nonclassical signal allows access to the secretory pathway--and yeast technology could have general application in studying elements of parasite protein trafficking.

Molecular and immunological characterisation of Theileria parva stocks which are components of the 'Muguga cocktail' used for vaccination against East Coast fever in cattle.

The 'Muguga cocktail' which is composed of three Theileria parva stocks Muguga, Kiambu 5 and Serengeti-transformed has been used extensively for live vaccination against East Coast fever in cattle in eastern, central and southern Africa. Herein we describe the molecular characterisation of the T. parva vaccine stocks using three techniques, an indirect fluorescent antibody test with a panel of anti-schizont monoclonal antibodies (MAb), Southern blotting with four T. parva repetitive DNA probes and polymerase chain reaction (PCR)-based assays detecting polymorphism within four single copy loci encoding antigen genes. The Muguga and Serengeti-transformed stocks exhibited no obvious differences in their reactivity with the panel of MAbs, whereas Kiambu 5 differed with several MAbs. Kiambu 5 DNA was very distinct from the Muguga and Serengeti-transformed isolates in the hybridisation pattern with all four nucleic acid probes, whereas Muguga and Serengeti-transformed isolates exhibited minor differences and could not be discriminated with one of the probes. PCR amplification in combination with restriction fragment length polymorphism analysis indicated that Kiambu 5 was also markedly divergent from the Muguga and Serengeti-transformed stocks within two of the four antigen coding genes. The T. parva Serengeti-transformed stock did not contain a 130 base pair insert within the p67 sporozoite antigen gene, which has been observed previously in most T. parva parasites isolated from buffalo, and could not be discriminated from T. parva Muguga at any of the four single copy loci. Collectively the data indicate that two of the cocktail components T. parva Serengeti-transformed and Muguga are genetically closely related, while the third component Kiambu 5 is quite distinct. Based on the findings, there may be a need to include only one of the T. parva Muguga and Serengeti-transformed components in the immunising cocktail. The study demonstrates the value of molecular characterisation data for monitoring of live vaccines.

Polymorphic open reading frames encoding secretory proteins are located less than 3 kilobases from Theileria parva telomeres.

Polymorphic, multicopy gene families are frequently located in subtelomeric regions of the genomes of parasitic protozoa. Theileria parva telomere-associated (TA) DNA from two chromosomes contained long open reading frames (ORFs) 54% identical at the N-termini, whose 3' ends were 2670 and 2680 bp from the telomeric repeats. Probes derived from these ORFs revealed related sequences close to additional telomeres. The 3' end of an unrelated ORF was approximately 2720 bp from a third telomere. These are among the closest ORFs to telomeres in any organism. Reverse transcription PCR detected transcripts originating within the telomeric multicopy gene family. Additional ORFs, with complex sequence similarities, were located centromeric to the telomere-adjacent ORFs. Transcripts from the schizont stage of T. parva, containing domains with significant amino acid similarity to a 3529 codon ORF located 6900 bp upstream of the telomeric repeats, were mapped to a subtelomeric locus at a fourth telomere. Five telomeric ORFs contained predicted N-terminal signal peptides and one of these signal peptides was functional in a heterologous system. Hybridisation data suggested extensive strain polymorphism between ORFs. Two of the telomere-adjacent ORFs were absent from the genome of a cloned T. parva parasite which can, nonetheless, be passaged through ticks and cattle. T. parva is unusual, among organisms so far studied, in the high density of potential coding sequences located directly adjacent to telomeres and the apparent absence of extensive tracts of repeated sequences within the TA DNA.

Cloning, characterization, and expression of a 200-kilodalton diagnostic antigen of Babesia bigemina.

Current serological tests for Babesia bigemina use semipurified merozoite antigens derived from infected erythrocytes. One of the major drawbacks of these tests is that antigen quality can vary from batch to batch. Since the quality of the antigen contributes to the sensitivity and specificity of serological tests, the use of standardized recombinant antigens should ensure consistency in assay quality. Previously, a 200-kDa merozoite antigen (p200) was identified as a candidate diagnostic antigen for use in a serological assay for the detection of B. bigemina antibodies in infected cattle. In this study, we have cloned, characterized, and expressed p200. A 3.5-kbp cDNA clone encoding p200 was isolated and shown to be almost full length, lacking approximately 300 bp at the 5' end. The predicted amino acid sequence shows that p200 consists of a long, highly charged central repeat region of an uninterrupted alpha helix, indicative of a fibrous protein. Immunoelectron microscopy localized p200 to the merozoite cytoplasm, suggesting that the antigen may be a structural protein involved in forming filament structures within the cytoskeleton. The 3.5-kbp cDNA was expressed in bacteria as a fusion protein with glutathione S-transferase (GST), but the yield was poor. To improve the yield, cDNA fragments encoding antigenic domains of p200 were expressed as fusions with GST. One of these fusion proteins, C1A-GST, is composed of a 7-kDa fragment of the p200 repeat region and contains epitopes that react strongly with sera from cattle experimentally infected with B. bigemina. Recombinant C1A-GST should permit the development of an improved enzyme-linked immunosorbent assay for the detection of antibodies against B. bigemina.

A 32 kDa surface antigen of Theileria parva: characterization and immunization studies.

Previous studies using monoclonal antibody (mAb) 4C9 specific for a 32 kDa antigen (p32) of Theileria parva demonstrated expression of the antigen on the surface of the sporozoite, making it a potential antigen for sporozoite neutralization. A full-length cDNA encoding the major merozoite/piroplasm surface antigen (mMPSA) of T. parva was cloned and expressed in bacteria. The expressed product reacted strongly with mAb 4C9, demonstrating identity between the p32 and mMPSA of T. parva. Using immunoblot analysis and immunoelectron microscopy with mAb 4C9 it was shown that the mMPSA is a major antigen of the merozoite and piroplasm at the cell surface, while lower levels of antigen are expressed in the sporozoite and schizont stages. Upregulation of the mMPSA occurs at merogony and can be induced by culturing schizont-infected lymphocytes at 42 degrees C. Recombinant mMPSA of T. parva induced high titres of specific antibodies in cattle but failed to confer protection against a T. parva sporozoite stabilate challenge. The pre-challenge sera also failed to neutralize infectivity of sporozoites in an in vitro assay. Possible reasons for the lack of parasite neutralization in vivo and in vitro are discussed.

Reciprocal cross-protection induced by sporozoite antigens SPAG-1 from Theileria annulata and p67 from Theileria parva.

Theileria annulata and Theileria parva both possess a major surface antigen on the sporozoite stage of the life-cycle, called SPAG-1 and p67, respectively. In each case, these antigens are vaccine candidates and have been shown to induce a degree of homologous protection in earlier work. These antigens share sequence homology and are serologically cross-reactive. Here, we confirm that these antigens confer protection against homologous species challenge. More importantly, they mutually confer a degree of cross-species protection raising the prospect of a common vaccine in the future.

Cloning, sequence and mRNA expression of bovine interleukin-16.

A lambda gt11 cDNA library was constructed using mRNA isolated from Theileria parva-infected bovine lymphocytes. Sequencing of random clones of this library resulted in the identification of a cDNA encoding bovine interleukin-16 (IL-16). The cDNA has an open reading frame of 1134 bp, and a 3' untranslated region of 275 nucleotides with a polyadenylation signal 16 bases upstream from the poly (A) tail. The protein predicted by the cDNA sequence contains 378 amino acids and the level of amino acid homology with the corresponding part of human precursor IL-16 is 79 %. No information is available about the tissue distribution of IL-16 in cattle, therefore we investigated the expression of IL-16 mRNA in bovine lymphoid tissues by reverse-transcription polymerase chain reaction assays. To investigate the potential of IL-16 as an immunoregulatory molecule we also analysed IL-16 mRNA expression in CD4+ and CD8+T-cell clones derived from T. parva-immunised cattle.