Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

Abstract

bOriental 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.T ick-borne diseases (TBDs) pose a major threat to livestock production worldwide and can have a significant impact on farming communities due to economic losses (1). Theileriosis is one of the important TBDs of cattle, sheep, and/or other ruminants, mainly in tropical and subtropical regions of the world (2). In cattle, East Coast fever (ECF) and Mediterranean/tropical theileriosis are due to Theileria parva and Theileria annulata, respectively, whereas oriental theileriosis is caused by Theileria orientalis. The prevalence of various forms of theileriosis in different parts of the world is dependent on the occurrence of suitable tick vectors for their transmission (3).Oriental theileriosis is caused by one or more genotypes of the T. orientalis complex and is transmitted by ixodid ticks, primarily Haemaphysalis spp. (4-6). Presently, 11 genotypes of T. orientalis complex (designated Chitose or type 1, Ikeda or type 2, Buffeli or type 3, types 4 to 8, and N-1 to N-3) have been identified using a number of molecular markers, including major piroplasm surface protein (MPSP) (7, 8), 23-kDa piroplasm membrane protein (p23) (9-11, 60), small-subunit (SSU) rRNA gene (8, 12, 13), and/or the first and second internal transcribed spacers of nuclear ribosomal DNA (ITS-1 and ITS-2, respectively) (12, 14). Of these genotypes, Ikeda and Chitose are recognized to be associated with clinical outbreaks of oriental theileriosis, mainly in the Asia-Pacific region (15-21). The major clin...

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

“…The UIC multiplex qPCR assay developed here was both sensitive and specific for T. orientalis detection compared to cPCR and reliably identified the clinically relevant Ikeda and Chitose genotypes. Furthermore, the UIC assay was more specific and had near-identical sensitivity to that reported for a recently published multiplexed tandem PCR assay (28). The U component of the UIC multiplex targets highly conserved regions of the MPSP gene in order to account for the genotypic diversity within T. orientalis (11 types observed currently) (13,15,16).…”

“…While real-time assays that detect T. orientalis have been developed, most have focused on species-level detection (not discrimination of genotypes) (34), discrimination of different Theileria spp. (43,44), or are only semiquantitative (28). The UIC multiplex qPCR assay developed here was both sensitive and specific for T. orientalis detection compared to cPCR and reliably identified the clinically relevant Ikeda and Chitose genotypes.…”

“…As a genuine multiplex assay, the UIC qPCR can be applied to high-throughput clinical testing, enabling hundreds of UIC assays to be performed in a single day. The UIC qPCR also offers a considerable advantage over other available assays which require dilution of DNA extracts to overcome PCR inhibition (6,9,27,28). Indeed, the number of assays required per sample to achieve a result is reduced from six, via cPCR detection, to just a single assay with the UIC qPCR.…”

Inadequate Differentiation of Theileria orientalis Genotypes buffeli and ikeda in a Multiplexed Tandem PCR (MT-PCR) Assay Using the p23 Gene as a Marker

“…Ltd., being insufficient to achieve selectivity from ikeda. Previously, when we tested DNA samples using the MPSP gene, we did not observe any significant variation in peak melting temperatures or SSCP profiles among MT-PCR amplicons from genotype buffeli (6). Additionally, we did not find any discrepancy in the assignments of three genotypes (buffeli, chitose, and ikeda) of T. orientalis using the MPSP gene or the p23 gene (6,16).…”

“…Previously, when we tested DNA samples using the MPSP gene, we did not observe any significant variation in peak melting temperatures or SSCP profiles among MT-PCR amplicons from genotype buffeli (6). Additionally, we did not find any discrepancy in the assignments of three genotypes (buffeli, chitose, and ikeda) of T. orientalis using the MPSP gene or the p23 gene (6,16). However, sequence variation in the p23 gene region identified in this study is likely due to population variation within T. orientalis (buffeli) in blood samples from cattle and buffaloes from locations outside Victoria, Australia (New South Wales and South Australia within Australia, and Ethiopia, New Zealand, and Pakistan) (7)(8)(9)(10)(11)(12); at the time, the p23 gene had not been assessed for variation within T. orientalis outside Victoria, Australia (16).…”