Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines

Abstract

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.

Fig 1. Specificity of PCR reaction in biological sample.

Amplification specificity of ama1 (A) and hprt1 (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 T. parva Muguga isolate (black line). Gel electrophoresis of the products was performed on a 2% agarose gel. The average peak melting temperature for hprt1 was 74.9°C and 75.9°C, respectively, for the plasmid and the biological sample used as the source of DNA; for ama1, they were 79.8°C and 79.5°C, respectively for plasmid and biological sample.

Fig 2. Quantification of T. parva and bovine DNA using standard reference curves for ama1 and hprt1.

The standard curves were constructed with seven serial 10-fold dilutions of TOPO-ama (A) and TOPO-hprt (B). Each of the plasmid dilution (n = 3 replicates per dilution; squares) and the four biological samples (n = 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 T. parva 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 (1) in methods, to obtain the corresponding gene copy number.

Fig 3. Estimation of T. parva and bovine in four biological samples.

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 (T. parva 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.