Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called "Infection and Treatment Method" (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Identification of Theileria lestoquardi Antigens Recognized by CD8+ T Cells.

As part of an international effort to develop vaccines for Theileria lestoquardi, we undertook a limited screen to test T. lestoquardi orthologues of antigens recognised by CD8+ T lymphocyte responses against T. annulata and T. parva in cattle. Five MHC defined sheep were immunized by live T. lestoquardi infection and their CD8+ T lymphocyte responses determined. Thirteen T. lestoquardi orthologues of T. parva and T. annulata genes, previously shown to be targets of CD8+ T lymphocyte responses of immune cattle, were expressed in autologous fibroblasts and screened for T cell recognition using an IFNγ assay. Genes encoding T. lestoquardi antigens Tl8 (putative cysteine proteinase, 349 aa) or Tl9 (hypothetical secreted protein, 293 aa) were recognise by T cells from one animal that displayed a unique MHC class I genotype. Antigenic 9-mer peptide epitopes of Tl8 and Tl9 were identified through peptide scans using CD8+ T cells from the responding animal. These experiments identify the first T. lestoquardi antigens recognised by CD8+ T cell responses linked to specific MHC class I alleles.

Evidence for a role of the host-specific flea (Paraceras melis) in the transmission of Trypanosoma (Megatrypanum) pestanai to the European badger.

We investigated the epidemiology of Trypanosoma pestanai infection in European badgers (Meles meles) from Wytham Woods (Oxfordshire, UK) to determine prevalence rates and to identify the arthropod vector responsible for transmission. A total of 245 badger blood samples was collected during September and November 2009 and examined by PCR using primers derived from the 18S rRNA of T. pestanai. The parasite was detected in blood from 31% of individuals tested. T. pestanai was isolated from primary cultures of Wytham badger peripheral blood mononuclear cells and propagated continually in vitro. This population was compared with cultures of two geographically distinct isolates of the parasite by amplified fragment length polymorphism (AFLP) and PCR analysis of 18S rDNA and ITS1 sequences. High levels of genotypic polymorphism were observed between the isolates. PCR analysis of badger fleas (Paraceras melis) collected from infected individuals at Wytham indicated the presence of T. pestanai and this was confirmed by examination of dissected specimens. Wet smears and Giemsa-stained preparations from dissected fleas revealed large numbers of trypanosome-like forms in the hindgut, some of which were undergoing binary fission. We conclude that P. melis is the primary vector of T. pestanai in European badgers.

Construction of a genetic map for Theileria parva: identification of hotspots of recombination.

The tick-borne protozoan parasite Theileria parva is the causal agent of East Coast Fever (ECF), a severe lymphoproliferative disease of cattle in eastern, central and southern Africa. The life cycle of T. parva is predominantly haploid, with a brief diploid stage occurring in the tick vector that involves meiotic recombination. Resolved genetic studies of T. parva are currently constrained by the lack of a genome-wide high-definition genetic map of the parasite. We undertook a genetic cross of two cloned isolates of T. parva to construct such a map from 35 recombinant progeny, using a genome-wide panel of 79 variable number of tandem repeat markers. Progeny were established by in vitro cloning of cattle lymphocytes after infection with sporozoites prepared from Rhipicephalus appendiculatus ticks fed on a calf undergoing a dual infection with the two clonal parental stocks. The genetic map was determined by assigning individual markers to the four chromosome genome, whose physical length is approximately 8309 kilobasepairs (Kb). Segregation analysis of the markers among the progeny revealed a total genetic size of 1683.8 centiMorgans (cM), covering a physical distance of 7737.62 Kb (∼93% of the genome). The average genome-wide recombination rate observed for T. parva was relatively high, at 0.22 cM Kb(-1) per meiotic generation. Recombination hot-spots and cold-spots were identified for each of the chromosomes. A panel of 27 loci encoding determinants previously identified as immunorelevant or likely to be under selection were positioned on the linkage map. We believe this to be the first genetic linkage map for T. parva. This resource, with the availability of the genome sequence of T. parva, will promote improved understanding of the pathogen by facilitating the use of genetic analysis for identification of loci responsible for variable phenotypic traits exhibited by individual parasite stocks.

Influence of host immunity on parasite diversity in Theileria parva.

We examined the influence of host immunity on the genotypic diversity of the intracellular transforming cattle parasite Theileria parva. By tracking the emergence of discrete parasite genotypes in an animal challenged with a bulk stabilate following immunization with its major component clone, we observed a profound modulation of genotypic frequencies in the breakthrough schizont population. In particular, no incidences of the immunizing clone were observed and a progressive decline was apparent in the relatedness of breakthrough genotypes to it. These observations were reflected in the genotypic profile of transmissible parasite stages that emerged in the erythrocyte fraction of the animal and in parasite progeny generated by tick pickup. In a separate experiment, genotypic profiles of breakthrough parasite populations were observed to vary between unrelated immune animals selected on the basis of the major histocompatibility complex (MHC) class I phenotype, a known determinant of the specificity of the immune response. Furthermore, immunization and challenge of calves with molecularly distinct but cross-protective parasite populations revealed that infection results in transmissible erythrocyte forms in spite of a protective immune response. These observations suggest that immunity does not prevent transmission of challenge parasites and that its impact on the parasite at a population level is influenced by herd MHC diversity.

Immunostimulatory CpG oligodeoxynucleotides enhance the induction of bovine CD4+ cytotoxic T-lymphocyte responses against the polymorphic immunodominant molecule of the protozoan parasite Theileria parva.

Enhancement of the induction of cytotoxic T-cell responses by immunostimulatory CpG oligodeoxynucleotides has been described in humans and mouse models. The present study attempted to address whether CpG has a similar effect in cattle. Immunisation of cattle with a recombinant form of the polymorphic immunodominant molecule from Theileria parva emulsified with immunostimulatory CpG oligodeoxynucleotides in adjuvant had no effect on the induction of antibody responses including the isotype profile, but significantly enhanced the induction of cytolytic responses that were mediated by CD4+CD3+ T cells utilizing the perforin-granzyme pathway.

Extensive genotypic diversity in a recombining population of the apicomplexan parasite Theileria parva.

We evaluated sexual recombination in the apicomplexan parasite Theileria parva using genome-wide marker analysis of haploid sporozoite populations obtained from infected Rhipicephalus appendiculatus ticks. Analysis of 231 parasite clones derived by in vitro infection of bovine lymphocytes revealed 48 distinct combinations of 64 polymorphic marker loci. One genotype accounted for more than 75% of the clones, and the population was highly inbred with respect to this. The occurrence of frequent recombination was evident from reassortment of contiguous markers in blocks, with some recombination occurring within blocks. Analysis of four polymorphic loci encoding antigens targeted by protective cytotoxic-T-lymphocyte responses confirmed that these loci reassort, both within and between chromosomes, suggesting that recombination may influence immune recognition. Marker analysis of a panel of 142 clones derived from the population after an additional passage through a calf and the same tick colony revealed 18 genotypes, with the original dominant genotype accounting for 75% of the population and a higher level of inbreeding with respect to it in the remaining clones. Selected marker analysis of genomic DNA from these stabilates and the two preceding generations of the isolate, each derived from distinct tick colonies, revealed shifts in population structure with each generation, suggesting that the tick vector may impose nonrandom selective pressure on the parasite.