Abstract
Avian malaria, caused by Plasmodium spp., has been linked to the mortality and population-level declines in native birds in some regions. While molecular diagnostic methods have greatly improved our ability to detect infections of both human and bird malaria, failing to identify false negatives remains an important handicap, particularly for avian malaria due to host DNA presence in the bird blood cells. In an attempt to improve the accuracy of diagnostics by PCR, we evaluated the performance of a commercial silica-membrane-based DNA extraction kit by modifying the protocol with four unpooled elution volume alternatives. Our results suggest that the best template is the DNA extract obtained from the second eluate of a first 50 μL elution step. In one case, the only band visible was from this second eluate and, thus, may not have been identified as positive for Plasmodium spp. if a different elution protocol had been followed. Our results are likely explained by the concept of size exclusion chromatography by which particles of different sizes will elute at different rates. Overall, first elution templates may consist of a lower ratio of parasite to host DNA, while second eluates may contain a higher parasite to host DNA ratio. A low ratio of parasite to host DNA is a concern in detecting chronic infections, in which birds typically carry low levels of parasitemia, making accurate diagnostics imperative when identifying reservoirs of disease that could lead to spillback events.
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Acknowledgments
Thanks to Robert Poulin and Daniel Tompkins for providing feedback on earlier drafts of the manuscript as well as Tania King for laboratory support. Additional thanks to the New Zealand Department of Conservation and the staff of Nelson Lakes National Park for their continued support of wildlife disease research. Funding was provided by the Department of Zoology, University of Otago and Landcare Research. Birds were sampled under Otago University Animal Ethics Approval 2/13 and New Zealand Department of Conservation permit 35873-FAU.
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Niebuhr, C.N., Blasco-Costa, I. Improving detection of avian malaria from host blood: a step towards a standardised protocol for diagnostics. Parasitol Res 115, 3905–3911 (2016). https://doi.org/10.1007/s00436-016-5157-4
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DOI: https://doi.org/10.1007/s00436-016-5157-4