Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines.
Absolute Quantification of the Host-To-Parasite DNA Ratio in Theileria parva-Infected Lymphocyte Cell Lines.
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DOI：
10.1371/journal.pone.0150401
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Silva JC
中科院分区：
文献类型：
--
作者：
Gotia HT;Munro JB;Knowles DP;Daubenberger CA;Bishop RP;Silva JC
微小泰勒氏菌是一种在撒哈拉以南非洲地区引起东海岸热(ECF)的牛的扁虱传播的细胞内尖端复合体病原体。ECF是一种急性致命疾病，每年导致100多万头牛死亡，给非洲小农养牛户带来巨大负担。野生动物宿主非洲水牛(Syncerus Caffer)和牛的微小毛滴虫感染的病理和水平是不同的。我们建立了一种基于定量聚合酶链式反应(QPCR)的绝对定量方法，以含有针对寄生虫(顶膜抗原1基因，ama1)或宿主(次黄嘌呤磷酸核糖基转移酶1，hprt1)的单拷贝基因的重组质粒作为定量参考标准。我们的研究表明，在感染了微小毛滴虫的淋巴细胞系中，微小毛滴虫和牛细胞的数量相似，因此，由于其基因组大小要小得多，所以从这些品系中提取的DNA样本中，微小毛滴虫DNA占0.9%至3%。这种对样本中寄生虫和宿主基因组拷贝数的绝对定量分析提供了一种简单可靠的方法来评估感染的牛淋巴细胞中的微小旋毛虫病毒载量，并且在广泛的宿主与寄生虫DNA比率范围内都是准确的。通过这种方法，了解样本中靶DNA的比例，对于各种细胞内病原体的高效高通量基因组测序应用是必不可少的。该方法在未来研究不同宿主-T的相互作用方面也将非常有用。PARVA种群，并充分描述现场ECF感染的动态。
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.