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

Bogema, DR; Deutscher, AT; Fell, S; Collins, D; Eamens, GJ; Jenkinsa, C

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

Bogema, DR

Deutscher, AT Fell, S Collins, D Eamens, GJ Jenkinsa, C

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Publication Type:: Journal Article
Citation:: Journal of Clinical Microbiology, 2015, 53 (3), pp. 941 - 950
Issue Date:: 2015-03-01

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Field	Value	Language
dc.contributor.author	Bogema, DR https://orcid.org/0000-0001-7013-7326	en_US
dc.contributor.author	Deutscher, AT	en_US
dc.contributor.author	Fell, S	en_US
dc.contributor.author	Collins, D	en_US
dc.contributor.author	Eamens, GJ	en_US
dc.contributor.author	Jenkinsa, C	en_US
dc.date.issued	2015-03-01	en_US
dc.identifier.citation	Journal of Clinical Microbiology, 2015, 53 (3), pp. 941 - 950	en_US
dc.identifier.issn	0095-1137	en_US
dc.identifier.uri	http://hdl.handle.net/10453/34324
dc.description.abstract	Copyright © 2015, American Society for Microbiology. All Rights Reserved. Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease.	en_US
dc.relation.ispartof	Journal of Clinical Microbiology	en_US
dc.relation.isbasedon	10.1128/JCM.03387-14	en_US
dc.subject.classification	Microbiology	en_US
dc.subject.mesh	Blood	en_US
dc.subject.mesh	Animals	en_US
dc.subject.mesh	Cattle	en_US
dc.subject.mesh	Theileria	en_US
dc.subject.mesh	Theileriasis	en_US
dc.subject.mesh	Cattle Diseases	en_US
dc.subject.mesh	Oligonucleotide Probes	en_US
dc.subject.mesh	Sensitivity and Specificity	en_US
dc.subject.mesh	Sequence Analysis, DNA	en_US
dc.subject.mesh	Molecular Sequence Data	en_US
dc.subject.mesh	Asia	en_US
dc.subject.mesh	Australia	en_US
dc.subject.mesh	New Zealand	en_US
dc.subject.mesh	Real-Time Polymerase Chain Reaction	en_US
dc.subject.mesh	Parasite Load	en_US
dc.title	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	en_US
dc.type	Journal Article
utslib.citation.volume	3	en_US
utslib.citation.volume	53	en_US
utslib.for	0707 Veterinary Sciences	en_US
utslib.for	0605 Microbiology	en_US
utslib.for	06 Biological Sciences	en_US
utslib.for	07 Agricultural and Veterinary Sciences	en_US
utslib.for	11 Medical and Health Sciences	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
utslib.copyright.status	closed_access
pubs.issue	3	en_US
pubs.publication-status	Published	en_US
pubs.volume	53	en_US

Abstract:

Copyright © 2015, American Society for Microbiology. All Rights Reserved. Theileria orientalis is an emerging pathogen of cattle in Asia, Australia, and New Zealand. This organism is a vector-borne hemoprotozoan that causes clinical disease characterized by anemia, abortion, and death, as well as persistent subclinical infections. Molecular methods of diagnosis are preferred due to their sensitivity and utility in differentiating between pathogenic and apathogenic genotypes. Conventional PCR (cPCR) assays for T. orientalis detection and typing are laborious and do not provide an estimate of parasite load. Current real-time PCR assays cannot differentiate between clinically relevant and benign genotypes or are only semiquantitative without a defined clinical threshold. Here, we developed and validated a hydrolysis probe quantitative PCR (qPCR) assay which universally detects and quantifies T. orientalis and identifies the clinically associated Ikeda and Chitose genotypes (UIC assay). Comparison of the UIC assay results with previously validated universal and genotype-specific cPCR results demonstrated that qPCR detects and differentiates T. orientalis with high sensitivity and specificiy. Comparison of quantitative results based on percent parasitemia, determined via blood film analysis and packed cell volume (PCV) revealed significant positive and negative correlations, respectively. One-way analysis of variance (ANOVA) indicated that blood samples from animals with clinical signs of disease contained statistically higher concentrations of T. orientalis DNA than animals with subclinical infections. We propose clinical thresholds to assist in classifying high-, moderate-, and low-level infections and describe how parasite load and the presence of the Ikeda and Chitose genotypes relate to disease.

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http://hdl.handle.net/10453/34324