Semiquantitative multiplexed tandem PCR for detection and differentiation of four Theileria orientalis genotypes in cattle

Abstract

Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region.

FIG 1

Detection of various genotypes of the Theileria orientalis complex using the MT-PCR assay. Cycling (A) and melting (B) curves for the genotypes Buffeli, Chitose, Ikeda, and type 5 of T. orientalis are shown. (C) Cycling curves of a blood DNA sample showing partial inhibition/delayed amplification of the spike control. (D and E) Cycling curves using undiluted template and showing no inhibition when the template was diluted at 1:10 (D) and 1:100 (E).

FIG 2

Prevalence of genotypes of the Theileria orientalis complex detected by the MT-PCR assay. Letters C, B, I, and T denote single infections by genotypes Chitose, Buffeli, Ikeda, and type 5, respectively. Various combinations of letters with the plus sign denote mixed infections with two or more genotypes.

FIG 3

Box plot diagrams showing the number of DNA copies of genotypes in mixed infections as indicated on each panel. The DNA copy number recorded for genotype Ikeda was divided by 2 to determine the DNA copy numbers shown in the figure.

FIG 4

Diagnostic sensitivity and specificity of the MT-PCR at different cutoff points.