Absolute quantification of the host-to-parasite DNA ratio in Theileria parva-infected lymphocyte cell lines

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

Re-annotation of the Theileria parva genome refines 53% of the proteome and uncovers essential components of N-glycosylation, a conserved pathway in many organisms

The apicomplexan parasite Theileria parva causes a livestock disease called East coast fever (ECF), with millions of animals 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.; The 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.; The 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-translational modification in apicomplexan parasites

Capture-based enrichment of Theileria parva DNA enables full genome assembly of first buffalo-derived strain and reveals exceptional intra-specific genetic diversity

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

Genome-wide diversity and gene expression profiling of Babesia microti isolates identify polymorphic genes that mediate host-pathogen interactions

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

Specificity of PCR reaction in biological sample.

<p>Amplification specificity of <i>ama1</i> (<b>A</b>) and <i>hprt1</i> (<b>B</b>) was confirmed by comparing the average melting peaks for the respective primer sets using as template the plasmid DNA (colored line) or the DNA extracted from a biological sample consisting of a lymphocyte cell line, from bovine BV115, infected with the <i>T</i>. <i>parva</i> Muguga isolate (black line). Gel electrophoresis of the products was performed on a 2% agarose gel. The average peak melting temperature for <i>hprt1</i> was 74.9°C and 75.9°C, respectively, for the plasmid and the biological sample used as the source of DNA; for <i>ama1</i>, they were 79.8°C and 79.5°C, respectively for plasmid and biological sample.</p

Primer sequences for quantitative and conventional PCR.

<p>Primer sequences for quantitative and conventional PCR.</p

Estimation of <i>T</i>. <i>parva</i> and bovine in four biological samples.

<p>Plasmid copy numbers were inferred from C_q values based on the respective standard curve. Genomic copy number and the relative amount of parasite and host DNA were determined as described in Methods (A). The mean percentages are 1.94%, 3.05%, 0.92%, and 1.72% for Muguga, Marikebuni, Uganda, and buffalo 7014 (<i>T</i>. <i>parva</i> lawrencei) (B), respectively. Mean values were obtained by averaging over 3 replicates. In each case, the third qPCR replicate was done in a different day.</p

Quantification of <i>T</i>. <i>parva</i> and bovine DNA using standard reference curves for <i>ama1</i> and <i>hprt1</i>.

<p>The standard curves were constructed with seven serial 10-fold dilutions of TOPO-ama (<b>A</b>) and TOPO-hprt (<b>B</b>). Each of the plasmid dilution (n = 3 replicates per dilution; squares) and the four biological samples (<i>n</i> = 3 replicates per sample; circles) was amplified by qPCR. For each gene, the C_q was plotted against the logarithm of the concentration. The standard curve was generated by logarithmic regression of the average C_q value on the concentration of the dilutions. The original bovine and <i>T</i>. <i>parva</i> genome copy number of each biological sample was estimated by converting their respective C_q values into plasmid equivalents (ng/μL) using the respective regression equations and using Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150401#pone.0150401.e001" target="_blank">1</a>) in methods, to obtain the corresponding gene copy number.</p

C_q measurements to establish standard curves.

<p>C_q measurements to establish standard curves.</p