Supplemental Figure S1. Architecture of the Theileria parva Muguga genome and associated new structural annotation and RNAseq expression data.Data are shown for each of the 4 T. parva nuclear and one mitochondrial chromosomes. The five concentric rings, from outer-to innermost, represent (i) the boundaries of each contig (blue), with the last and most AT-rich contig representing the mitochondrion, (ii) the deviation from the average GC percentage (red), (iii) the genes on the forward strand (green = SVSP family genes, red = Tpr genes, purple = TpHN genes), (iv) the genes on the reverse strand (same colors as forward), and (v) RNAseq coverage depth (blue, on light purple background). The figure was generated using the Circleator software. The apicoplast genome was not re-annotated since only three of its 70 currently annotated genes had representative RNAseq coverage, and their existing annotation was consistent with the RNAseq data. Supplemental Figure S2. Updated gene annotation efforts in theTheileria parva genome reveal the existence of many genes that overlap adjacent genes at either UTR or CDS sequences. (A) Boxplots of the distribution of the total overlap length by genes that overlap by only the untranslated regions (UTR), or where the overlap includes a protein coding sequence (CDS) on either the same or opposite strands. (B) Shown are boxplots for the distribution of CDS length overlap in adjacent genes. Distributions were compared with a one-way ANOVA using alpha = 0.05. Significance is shown as follows: * 0.05>p≥0.01; ** 0.01>p≥0.001; *** p<0.001. Bar plot lines represent the mean and 5-95% percentiles from the mean. Supplemental Figure S3. RNA-seq reads that map to introns in the Theileria parva Muguga genome support the existence of genes where a subset of introns are not spliced. Read coverage per intron and protein coding sequence (CDSs) was calculated using HT-seq and their ratio plotted as a frequency histogram. While most introns had a coverage of zero (hence an intron_coverage/CDS_coverage ratio of zero), 1,610 introns had some read coverage, 701 of which had an intron_coverage/CDS_coverage ratio above or equal to 1.0. These 1,610 introns correspond to 744 genes. Supplemental Figure S4. Histogram of mRNA length across all annotated transcripts in the current T. parva Muguga annotation.All mRNAs were used to make this graph, including those for which no untranslated region was annotated. SupplementaryFigure S5. Types of transcription initiated in Theileria parva. Transcription in T. parva Muguga results from potential bidirectional (A) and cryptic promoters (B). Antisense transcription can be spliced (C), and may result in run-through transcription overlapping another gene (D). The model of transcription that emerges from these data is one of ubiquitous sense transcription of most genes in the schizont stage, but with a wide range of expression levels. Transcription can arise from potential bidirectional and cryptic promoters with highly prevalent antisense transcription. Blue boxes indicate exo...

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Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle- and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle- and buffalo-derived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is ~20 kb larger than the genome from the reference, cattle-derived, Muguga strain, and contains 25 new potential genes. The average non-synonymous nucleotide diversity (πN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average Wright’s fixation index (FST), genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle- and buffalo-derived T. parva parasites more commonly seen between, rather than within, species. These findings present clear implications for vaccine development, further demonstrated by the ability to assemble nearly all known antigens in the buffalo-derived strain, which will be critical in design of next generation vaccines. The DNA capture approach used provides a clear advantage in specificity over alternative T. parva DNA enrichment methods used previously, such as those that utilize schizont purification, is less labor intensive, and enables in-depth comparative genomics in this apicomplexan parasite.

AbstractBackgroundThe apicomplexan parasite Theileria parva causes a livestock disease called East coast fever (ECF), with millions of animals are at risk in sub-Saharan East and Southern Africa, the geographic distribution of T. parva. Over a million bovines die each year of ECF, with a tremendous economic burden to pastoralists in endemic countries. Comprehensive, accurate parasite genome annotation can facilitate the discovery of novel chemotherapeutic targets for disease treatment, as well as elucidate the biology of the parasite. However, genome annotation remains a significant challenge because of limitations in the quality and quantity of the data being used to inform the location and function of protein-coding genes and, when RNA data are used, the underlying biological complexity of the processes involved in gene expression. Here, we apply our recently published RNAseq dataset derived from the schizont life-cycle stage of T. parva to update structural and functional gene annotations across the entire nuclear genome.ResultsThe re-annotation effort lead to evidence-supported updates in over half of all protein-coding sequence (CDS) predictions, including exon changes, gene merges and gene splitting, an increase in average CDS length of approximately 50 base pairs, and the identification of 128 new genes. Among the new genes identified were those involved in N-glycosylation, a process previously thought not to exist in this organism and a potentially new chemotherapeutic target pathway for treating ECF. Alternatively-spliced genes were identified, and antisense and multi-gene family transcription were extensively characterized.ConclusionsThe process of re-annotation led to novel insights into the organization and expression profiles of protein-coding sequences in this parasite, and uncovered a minimal N-glycosylation pathway that changes our current understanding of the evolution of this post-translation modification in apicomplexan parasites.

Parasite-specific CD8 T cell responses play a key role in mediating immunity againstTheileria parva in cattle (Bos taurus) and there is evidence that efficient induction of these responses requires CD4 T cell responses. However, information on the antigenic specificity of the CD4 T cell response is lacking. The current study used a high-throughput system for antigen identification using CD4 T cells from immune animals to screen a library of ~40,000 synthetic peptides representing 499 T. parva gene products. Use of CD4 T cells from 12 immune cattle, representing 12 class II MHC types, identified 26 antigens. Unlike CD8 T cell responses, which are focused on a few dominant antigens, multiple antigens were recognised by CD4 T cell responses of individual animals. The antigens had diverse properties, but included proteins encoded by two multi-member gene families -five haloacid dehalogenases and five subtelomere-encoded variable secreted proteins (SVSPs). Most antigens had predicted signal peptides and/or were encoded by abundantly transcribed genes, but neither parameter on their own was reliable for predicting antigenicity. Mapping of the epitopes confirmed presentation by DR or DQ class II alleles and comparison of available T. parva genome sequences demonstrated that they included both conserved and polymorphic epitopes. Immunisation of animals with vaccine vectors expressing two of the antigens demonstrated induction of CD4 T cell responses capable of recognising parasitised cells. The results of this study provide detailed insight into the CD4 T cell responses induced by T. parva, and identify antigens suitable for use in vaccine development. Key points  Multiple CD4 T cell antigens identified by screening a T. parva peptide library  Antigens have diverse properties and include polymorphic and conserved proteins  Parasite infection and viral-vector delivered antigen induce similar CD4 responses

Theileria parva is an economically important, intracellular, tick-transmitted parasite of cattle. A live vaccine against the parasite is effective against challenge from cattle-transmissible T. parva but not against genotypes originating from the African Cape buffalo, a major wildlife reservoir, prompting the need to characterize genome-wide variation within and between cattle-and buffalo-associated T. parva populations. Here, we describe a capture-based target enrichment approach that enables, for the first time, de novo assembly of nearly complete T. parva genomes derived from infected host cell lines. This approach has exceptionally high specificity and sensitivity and is successful for both cattle-and buffaloderived T. parva parasites. De novo genome assemblies generated for cattle genotypes differ from the reference by ~54K single nucleotide polymorphisms (SNPs) throughout the 8.31 Mb genome, an average of 6.5 SNPs/kb. We report the first buffalo-derived T. parva genome, which is larger than the genome from the reference, cattle-derived, Muguga strain. The average non-synonymous nucleotide diversity (pN) per gene, between buffalo-derived T. parva and the Muguga strain, was 1.3%. This remarkably high level of genetic divergence is supported by an average FST, genome-wide, of 0.44, reflecting a degree of genetic differentiation between cattle-and buffalo-derived T. parva parasites more commonly seen between, rather than within, species, with clear implications for vaccine development. The DNA capture approach used provides clear advantages over alternative T. parva DNA enrichment methods used previously and enables in-depth comparative genomics in this apicomplexan parasite.