Use of multiplexed tandem PCR to estimate the prevalence and intensity of Theileria orientalis infections in cattle

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Highlights

  • MT-PCR was used to estimate the prevalence and intensity of T. orientalis genotypes.

  • Genotype buffeli had the highest prevalence.

  • Genotype ikeda had the highest average intensity of infection.

  • Intensity of ikeda was higher (P < 0.001) than buffeli in symptomatic cows.

  • Intensity of ikeda was higher in symptomatic cows (P = 0.004) than asymptomatic cows.

Abstract

This study employed a semi-quantitative, multiplexed tandem PCR (MT-PCR) to assess the prevalence and infection intensity of four genotypes (buffeli, chitose, ikeda and type 5) of Theileria orientalis in cattle in Australia. Genomic DNA samples from blood samples (n = 448) collected from 27 to 32 dairy cows from each of 15 dairy herds with a history of recent theileriosis outbreaks (Group 1), and from blood samples available from 24 cows with or without oriental theileriosis (Group 2) were tested using MT-PCR. Results revealed that all four genotypes were present in Group 1 cattle; genotype buffeli had the highest prevalence (80.5%), followed by genotypes ikeda (71.4%), chitose (38.6%) and type 5 (20.3%). Genotype ikeda had the highest average infection intensity in the cattle (relating to 55,277 DNA copies), followed by buffeli, chitose and type 5 (6354–51,648 copies). For Group 2, results indicated that genotype ikeda had a significantly higher average intensity of infection than buffeli in symptomatic cattle (P < 0.001), and symptomatic cattle had a higher intensity of ikeda than asymptomatic cattle (P = 0.004). Future studies should assess the utility of the present MT-PCR assay as a diagnostic and epidemiological tool in other parts of Australasia and the world.

Introduction

Oriental theileriosis of cattle is caused by Theileria orientalis, a group of haemoprotistan parasites transmitted by ixodid ticks. This disease is manifested by pyrexia, haemolytic anaemia, productivity losses, abortion and/or mortality (Izzo et al., 2010, Aparna et al., 2011, Islam et al., 2011, McFadden et al., 2011, Perera et al., 2014). To date, 11 distinct genotypes of T. orientalis have been characterised using the major piroplasm surface protein (MPSP) gene (reviewed by Sivakumar et al., 2014); five of these genotypes (i.e., buffeli, chitose, ikeda, and types 4 and 5) have been reported in Australia (Islam et al., 2011, Kamau et al., 2011, Cufos et al., 2012, Perera et al., 2013). Recently, some T. orientalis genotypes (including chitose and ikeda) have been inferred to be involved in significant outbreaks of oriental theileriosis in cattle in the Asia–Pacific region (Izzo et al., 2010, Aparna et al., 2011, Islam et al., 2011, McFadden et al., 2011, Perera et al., 2014).

Traditionally, the diagnosis of oriental theileriosis was based on the detection of piroplasms of T. orientalis in blood smears, and/or the use of serological or conventional molecular techniques (Becerra et al., 1983, Kawazu et al., 1992, Tanaka et al., 1993, Kakuda et al., 1998, Jeong et al., 2005, Altay et al., 2008). However, each of these methods has limitations, such as low diagnostic sensitivity and/or specificity. Recently, we established and validated a multiplexed-tandem PCR (MT-PCR) assay (Perera et al., 2015) to overcome these limitations. This method allows the simultaneous detection and differentiation of the four commonest genotypes (i.e., buffeli, chitose, ikeda and type 5) of the T. orientalis complex in Australasia as well as their semi-quantitation in bovine blood samples. This assay is a cost-effective, practical and specific diagnostic tool whose analytical sensitivity is ∼1000 times greater than conventional PCR (Perera et al., 2015), suggesting that it will provide a useful epidemiological tool for rapidly tracking disease outbreaks and for the surveillance of infections. Here, we assessed the prevalence and intensity of infections of four genotypes of T. orientalis in dairy cattle in an endemic region in south-eastern Australia, following recent outbreaks of theileriosis. In addition, the infection intensity of pathogenic and apathogenic genotypes of T. orientalis was assessed using blood samples available from a previous study from symptomatic and asymptomatic cattle suffering from oriental theileriosis.

Section snippets

Farms, collection of blood samples and haematological examination

For this study, 448 blood samples (Group 1) were collected by registered practicing veterinarians from 27 to 32 cows from each of 15 dairy herds randomly selected in three geographical regions (Bairnsdale, Leongatha and Maffra) of the state of Victoria, Australia, following oriental theileriosis outbreaks between September 2012 and June 2013 (Table 1). Each cow was clinically examined under the supervision or by a registered veterinarian (bovine specialist: J.M.). The clinical signs of

Prevalence of T. orientalis genotypes

In Group 1, 53.8% (241/448) of cattle were test-positive for T. orientalis by MT-PCR (Table 1), and the highest prevalence (63.8%; 95/149) was recorded in the Bairnsdale region, followed by Leongatha (51.7%; 76/147) and Maffra (46.1%; 70/152). Epidemiological data also revealed that the highest number (n = 36) of cow deaths had occurred in the Bairnsdale region (Table 1). All four genotypes (i.e., buffeli, chitose, ikeda and type 5) were detected in all three geographical regions, and buffeli had

Discussion

This is the first application of semi-quantitative MT-PCR to estimate the prevalences and intensity of four common genotypes (i.e., buffeli, chitose, ikeda and type 5) of T. orientalis complex in dairy cattle populations in Victoria, Australia. The prevalence of T. orientalis determined herein is more accurate than conventional PCR (detection of 1 pg of genomic DNA) (Perera et al., 2013) due to the higher (1000 times) sensitivity of the MT-PCR compared with conventional PCR (Perera et al., 2015

Concluding remarks

In conclusion, this study demonstrates the utility and usefulness of the MT-PCR assay for assessing the prevalences and intensities of four common genotypes of T. orientalis (buffeli, chitose, ikeda and type 5) in cattle in Victoria, Australia. MT-PCR might also be useful to predict the risk of clinical disease developing in cattle, based on threshold levels of infection intensity for individual genotypes. This aspect requires detailed evaluation using a well-defined sampling strategy and large

Acknowledgements

This project was partially supported by the Department of Agriculture Fisheries and Forestry (DAFF), the Victorian Cattle Compensation Fund, the Department of Environment and Primary Industries of Victoria, a Collaborative Research Grant (the University of Melbourne) (A.Ja.) and the Australian Research Council (ARC) (R.B.G. et al.). Funding from the CASS Foundation and The Ian Potter Foundation is also gratefully acknowledged (A.Ja.). P.P. is a grateful recipient of the International

References (18)

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    To circumvent these limitations, we recently developed a multiplexed tandem PCR (MT-PCR) that allows the simultaneous detection, differentiation and semi-quantitation of four common genotypes (buffeli, chitose, ikeda and type 5) of T. orientalis (see Perera et al., 2015b). We showed the utility of this MT-PCR assay to rapidly estimate both prevalence and intensity of T. orientalis genotypic infections following outbreaks in Australia (Perera et al., 2015c) and New Zealand (Perera et al., 2015d). This assay has a high diagnostic specificity (97.0%) and sensitivity (98.8%) and has proved to be cost effective and practical as a laboratory-based assay (Perera et al., 2015b); it achieves semi-quantitative genotypic diagnosis, has an analytical sensitivity that is ∼1000 times greater than conventional PCR and can detect 0.25 parasites per microliter of blood (Perera et al., 2015b).

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