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Babesia microti, a tick-transmitted, intraerythrocytic protozoan parasite circulating mainly among small mammals, is the primary cause of human babesiosis. While most cases are transmitted by Ixodes ticks, the disease may also be transmitted through blood transfusion and perinatally. A comprehensive analysis of genome composition, genetic diversity, and gene expression profiling of seven B. microti isolates revealed that genetic variation in isolates from the Northeast United States is almost exclusively associated with genes encoding the surface proteome and secretome of the parasite. Furthermore, we found that polymorphism is restricted to a small number of genes, which are highly expressed during infection. In order to identify pathogen-encoded factors involved in host-parasite interactions, we screened a proteome array comprised of 174 B. microti proteins, including several predicted members of the parasite secretome. Using this immuno-proteomic approach we identified several novel antigens that trigger strong host immune responses during the onset of infection. The genomic and immunological data presented herein provide the first insights into the determinants of B. microti interaction with its mammalian hosts and their relevance for understanding the selective pressures acting on parasite evolution.

Theileria parva is a tick-transmitted intracellular apicomplexan pathogen of cattle in sub-Saharan Africa that causes East Coast fever (ECF). ECF is an acute fatal disease that kills over one million cattle annually, imposing a tremendous burden on African small-holder cattle farmers. The pathology and level of T. parva infections in its wildlife host, African buffalo (Syncerus caffer), and in cattle are distinct. We have developed an absolute quantification method based on quantitative PCR (qPCR) in which recombinant plasmids containing single copy genes specific to the parasite (apical membrane antigen 1 gene, ama1) or the host (hypoxanthine phosphoribosyltransferase 1, hprt1) are used as the quantification reference standards. Our study shows that T. parva and bovine cells are present in similar numbers in T. parva-infected lymphocyte cell lines and that consequently, due to its much smaller genome size, T. parva DNA comprises between 0.9% and 3% of the total DNA samples extracted from these lines. This absolute quantification assay of parasite and host genome copy number in a sample provides a simple and reliable method of assessing T. parva load in infected bovine lymphocytes, and is accurate over a wide range of host-to-parasite DNA ratios. Knowledge of the proportion of target DNA in a sample, as enabled by this method, is essential for efficient high-throughput genome sequencing applications for a variety of intracellular pathogens. This assay will also be very useful in future studies of interactions of distinct host-T. parva stocks and to fully characterize the dynamics of ECF infection in the field.

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...

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.