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Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand

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Abstract

Although wildlife rehabilitation and translocations are important tools in wildlife conservation in New Zealand, disease screening of birds has not been standardized. Additionally, the results of the screening programmes are often difficult to interpret due to missing disease data in resident or translocating avian populations. Molecular methods have become the most widespread method for diagnosing avian malaria (Plasmodium spp.) infections. However, these methods can be time-consuming, expensive and are less specific in diagnosing mixed infections. Thus, this study developed a new real-time PCR (qPCR) method that was able to detect and specifically identify infections of the three most common lineages of avian malaria in New Zealand (Plasmodium (Novyella) sp. SYAT05, Plasmodium elongatum GRW6 and Plasmodium spp. LINN1) as well as a less common, pathogenic Plasmodium relictum GRW4 lineage. The assay was also able to discern combinations of these parasites in the same sample and had a detection limit of five parasites per microlitre. Due to concerns relating to the presence of the potentially highly pathogenic P. relictum GRW4 lineage in avian populations, an additional confirmatory high resolution (HRM) qPCR was developed to distinguish between commonly identified P. elongatum GRW6 from P. relictum GRW4. The new qPCR assays were tested using tissue samples containing Plasmodium schizonts from three naturally infected dead birds resulting in the identified infection of P. elongatum GRW6. Thus, these rapid qPCR assays have shown to be cost-effective and rapid screening tools for the detection of Plasmodium infection in New Zealand native birds.

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Notes

  1. Kate McInnes, Department of Conservation, Wellington, New Zealand. kmcinnes@doc.govt.nz

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Acknowledgements

We thank Dr. Isabel Castro and Dr. Dan Tompkins for their valuable suggestions to this manuscript. This project received funding from the Morris Animal Foundation study grant ID number D13ZO-811: Do Translocations for Species Restoration Cause Pathogen Pollution? Further funding was received from Julie Alley Bursary, Marion Cunningham Memorial Fund, Forest and Bird J S Watson Trust, Massey University (IAE) and the New Zealand Veterinary Association Wildlife Society.

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Authors and Affiliations

  1. Institute of Agriculture and Environment (IAE), Ecology, Massey University, Palmerston North, New Zealand

    E. R. Schoener

  2. Institute of Veterinary, Animal and Biomedical Sciences (IVABS), Massey University, Palmerston North, New Zealand

    S. Hunter & L. Howe

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  1. E. R. Schoener
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  2. S. Hunter
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  3. L. Howe
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Correspondence to L. Howe.

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All birds were caught at Bushy Park/Wanganui in February 2013 during a study examining avian malaria under the Department of Conservation permit TW-32756-FAU and the approval by the Animal Ethics Committee at Massey University, MUAEC Protocol 11/59. Birds were banded under institutional banding permit No. 2012/009.

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Schoener, E.R., Hunter, S. & Howe, L. Development of a rapid HRM qPCR for the diagnosis of the four most prevalent Plasmodium lineages in New Zealand. Parasitol Res 116, 1831–1841 (2017). https://doi.org/10.1007/s00436-017-5452-8

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