Developing high throughput quantitative PCR assays for diagnosing Ikeda and other Theileria orientalis types common to New Zealand in bovine blood samples

Abstract

<div><p></p><p>AIMS: To develop rapid, quantitative PCR (qPCR) assays using high resolution melt (HRM) analysis and type-specific TaqMan assays for identifying the prevalent types of <i>Theileria orientalis</i> found in New Zealand cattle; and to evaluate their analytical and diagnostic characteristics compared with other assays for <i>T. orientalis</i>.</p><p>METHODS: Nucleotide sequences aligned with <i>T. orientalis</i> Buffeli, Chitose and Ikeda types, obtained from DNA extracted from blood samples from infected cattle, were used to design HRM and type-specific probe-based qPCR assays. The three type-specific assays were also incorporated into a single-tube multiplex qPCR assay. These assays were validated using DNA extracted from blood samples from cattle in herds with or without clinical signs of <i>T. orientalis</i> infection, other veterinary laboratory samples, as well as plasmids containing <i>T. orientalis</i> type-specific sequences. Diagnostic specificity (DSp) and sensitivity (DSe) estimates for the qPCR assays were compared to blood smear piroplasm results, and other PCR assays for <i>T. orientalis</i>. Copy number estimates of Ikeda DNA in blood were determined from cattle exhibiting anaemia using the Ikeda-specific qPCR assay.</p><p>RESULTS: The <i>T. orientalis</i> type-specific and the HRM qPCR assays displayed 100% analytical specificity. The Ikeda-specific qPCR assay exhibited linearity (R²=0.997) with an efficiency of 94.3%. Intra-assay CV were ≤0.08 and inter-assay CV were ≤0.095. For blood samples from cows with signs of infection with <i>T. orientalis</i>, the DSp and DSe of the multiplex probe qPCR assay were 93 and 96%, respectively compared with blood smears, and 97 and 100%, respectively, compared with conventional PCR assays. For the Ikeda-specific qPCR assay, the number of positive samples (n=66) was slightly higher than a conventional PCR assay (n=64). The concentration of Ikeda genomes in blood samples from 41 dairy cows with signs of infection with <i>T. orientalis</i> ranged between 5.6×10⁴ and 3.3×10⁶ genomes per µL of blood.</p><p>CONCLUSIONS: All qPCR assays had improved specificity and sensitivity over existing conventional PCR assays for diagnosis of <i>T. orientalis</i> Ikeda. The burden of Ikeda DNA in blood was demonstrated using an Ikeda-specific qPCR assay with titrated synthetic gene target.</p><p>CLINICAL RELEVANCE: Adoption of high-throughput DNA extraction and qPCR reduced <i>T. orientalis</i> and Ikeda diagnosis times. The Ikeda-specific qPCR assay provides a specific diagnosis for Ikeda in animals with signs of infection with <i>T. orientalis</i> and can be used to monitor the parasite load of Ikeda in blood.</p></div