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PLoS One. 2016 Mar 1;11(3):e0150401. doi: 10.1371/journal.pone.0150401. eCollection 2016.

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

Author information

1
Institute for Genome Sciences, University of Maryland School of Medicine, 801 West Baltimore Street, Baltimore Maryland, United States of America.
2
Animal Disease Research Unit, Agricultural Research Service, US Department of Agriculture, Pullman, Washington, United States of America.
3
Department of Veterinary Microbiology & Pathology, Washington State University, Pullman, Maryland, United States of America.
4
Swiss Tropical and Public Health Institute, Socinstrasse 57, 4002 Basel, Switzerland.
5
University of Basel, Petersplatz 1, 4003 Basel, Switzerland.
6
International Livestock Research Institute, P.O. Box 30709, Nairobi 00100, Kenya.
7
Department of Microbiology and Immunology, University of Maryland School of Medicine, 685 West Baltimore Street, Baltimore Maryland, United States of America.

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.

PMID:
26930209
PMCID:
PMC4773007
DOI:
10.1371/journal.pone.0150401
[Indexed for MEDLINE]
Free PMC Article

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