Generation of selected gene list
Preliminary contigs of the T. parva genome were searched against a non-redundant database of proteins extracted from GenBank. The search results were used to produce a set of putative T. parva genes that encoded highly conserved eukaryotic genes. This set of genes was complemented with a number of full length genes isolated from a cDNA library made from purified schizonts and were used to train the gene finding programs GlimmerM  and Phat , which were subsequently run against all of the preliminary contigs to produce gene models for the entire preliminary genome sequence. The proteins encoded by the predicted T. parva genes on chromosome 1 were searched against a non-redundant protein database using WU-BLAST , and predictions of signal peptides and signal anchors  and transmembrane domains  analysed. The results were reviewed and a set of 55 genes encoding candidate antigens was selected for cloning and screening .
Cloning of selected genes
Open reading frames (ORFs) less than 3.1 kb were successfully amplified by OneStep RT-PCR kit (QIAGEN Ltd., Crawley, UK) using RNA purified from T. parva (Muguga) schizont-infected lymphoblasts. Thermal cycles were: Genes up to 1 kb: 50°C 30 minutes; 95°C 15 minutes; 94°C 30 seconds; 55°C 30 seconds; 72°C 1 minute; 35 times from 94°C cycle and finally 72°C 10 minutes. Genes between 1 and 2 kb: 50°C 30 minutes; 95°C 15 minutes; 94°C 1 minute; 55°C 1 minute; 72°C 1 minute; 35 times from 94°C cycle and finally 72°C 10 minutes. Genes between 2 and 3 kb: 45°C 30 minutes; 95°C 15 minutes; 94°C 10 seconds; 55°C 1 minute; 68°C 3 minutes; 35 times from 94°C cycle and finally 68°C 10 minutes. Amplified genes were purified from agarose gels by QIAquick Gel Extraction kit (QIAGEN) and cloned into eukaryotic expression T-vector pTargeT (Promega, Madison, WI, USA). Ligated samples were electroporated into JM109, and colonies were screened by PCR using gene specific internal forward primers and vector specific reverse primer (5'GAGCGGATAACATCACACAGG3'). Positive colonies were cultured in 2 ml and plasmids purified by QIAprep Spin Miniprep kit (QIAGEN) and sequenced by ABI PRISM 377 DNA sequencer (Applied Biosystems, Foster City, CA, USA). Although 51 of 55 selected genes were amplified, 36 were cloned successfully in pTargeT . Some of the genes were consistently cloned in wrong orientation suggesting the gene products were toxic to E. coli. Some genes were amplified by the reverse primer only and missed the 5' end.
Analysis of the temporal expression of Tp2
Total RNA from sporozoites, schizonts and piroplasms was reverse-transcribed into ss-cDNA by reverse transcriptase using oligo-(dT) as primer following the recommendation of the supplier (Invitrogen). After removal of RNA complementary to the cDNA by treatment with RNase H and purification with phenol-chloroform, the ss-cDNA was PCR-amplified in the presence of specific primers used to generate the Tp2 ORF (5'-ATGAAATTGGCCGCCAGA-3' and 5'-CTATGAAGTGCCGGAGGCTTCTCC-3'). Northern blots of RNA from the schizont and piroplasm stages of T. parva were probed with the Tp2 ORF DNA fragment. This probe was also used to screen a schizont cDNA library and restriction enzyme analysis was performed by digestion with Eco RI and Bam HI.
Infection and treatment immunization of cattle and generation of CTL
All animal experimentation was reviewed and approved by the ILRI Institutional Animal Care and Use Committee. Four Boran (Bos indicus), 1 Jersey (Bos taurus), 1 Holstein-Friesian (Bos taurus) and 4 crossbreds, selected on the basis of BoLA type, were immunized against T. parva Muguga stock, challenged after 3 months and schizont-specific polyclonal CTL lines and clones established as described .
Transient transfection of immortalized skin fibroblasts
Immortalized skin fibroblasts (iSF) were transfected in 96-well plates with cDNA clones (100 ng/well) using Fugene-6 (Roche Diagnostics GmbH, Mannheim, Germany) and cultured for 24 hours.
IFN-γ ELISpot and 51Chromium release assays
Transfectants were co-cultured with schizont-specific CTL and recognition assessed using an IFN-γ ELISpot assay . Transfectants and schizont-infected cells were labelled with 51Chromium (Amersham Biosciences Europe GmbH, Freiburg, Germany) and lysis by schizont-specific CTL lines assessed .
Identification of CTL epitopes with synthetic peptides
A peptide library of Tp2 was generated that contained every 12 mer, 11 mer, 10 mer and 9 mer offset by 2 amino acids from the protein sequence (Cleaved PepSet; Mimotopes, Clayton, Australia). Peptides were prepared by truncations of 12 mers at the N-terminus and supplied lyophilized with each tube containing a nominal 12 mer and the 9, 10, 11 mer truncations with the same C-terminus. Peptides were dissolved in 400 μl 50% (v/v) DNA synthesis grade acetonitrile/water (Applied Biosystems). Peptides were further diluted to 10 μg/ml in complete RPMI-1640 and 10 μl added to triplicate wells of an ELISpot plate. Autologous iSF were adjusted to a density of 4 × 105/ml and 50 μl added to wells containing peptides. The plates were incubated at 37°C for 1 hour before 50 μl of CTL at a density of 2 × 105/ml were added to each well. Plates were incubated and developed as described above. Based on the results of screening the peptide library, individual 8, 9, 10 or 11 mer peptides were synthesized and tested as described above. Peptide-pulsed iSF were prepared as targets for 51Chromium release assays by overnight incubation with peptide at 1 μg/ml in complete DMEM. Cells were harvested, labelled and assayed as described above.
Ex vivo detection of Tp2 specific CD8+ T cell responses
Immune cattle, BW002, BW013 and BW014, whose schizont-specific CTL had recognized Tp2, were challenged with a lethal dose of T. parva (Muguga) sporozoites and bled daily from day 6 to 13 post-challenge. CD8+ T cells and CD14+ monocytes were purified from PBMC by MACS magnetic cell sorting according to the manufacturer's instructions (Miltenyi Biotec, Gergisch Gladbach, Germany). CD8+ T cells were sorted indirectly using a monoclonal antibody specific for bovine CD8 (IL-A105; ILRI, Nairobi, Kenya) followed by incubation with goat anti-mouse IgG microbeads (Miltenyi Biotec). CD14+ monocytes were sorted directly by incubation with CD14 microbeads (Miltenyi Biotec). CD8+ T cells (2.5 × 105/well) and CD14+ monocytes (2.5 × 104/well) were added to IFN-γ ELISpot plates containing Tp2 peptides (1 μg/ml final concentration) and were incubated and developed as described . To recall Tp2 specific CTL responses, PBMC were stimulated with autologous infected lymphoblasts 14 days post-challenge, viable cells were harvested 7 days post-stimulation and lytic activity against infected lymphoblasts and Tp2 peptide pulsed iSF assessed as described above.
Analysis of variance (ANOVA) was used for the analysis of fixed effects on different traits using SAS Release 8.2 (SAS Institute Inc., Cary, USA).