James W. Wynne , Anca G. Rusu , Ben T. Maynard , Megan L. Rigby , Richard S. Taylor
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引用次数: 0
Abstract
The ability to distinguish between viable and non-viable protozoan parasites is central to improved human and animal health management. While conceptually simple, methods to differentiate cell viability in situ remain challenging. Amoebic gill disease, caused by Neoparamoeba perurans is a parasitic disease impacting Atlantic salmon aquaculture globally. Although commercial freshwater treatments alleviate AGD, viable amoebae remain on gills or in used treatment water. Existing PCR-based assays are able to quantify N. perurans abundance but cannot discriminate amoeba viability. We investigated the use of propidium monoazide (PMA) application, prior to real-time PCR, to distinguish between alive and dead cells. We demonstrate that 200 μM PMA can significantly reduce amplification from non-viable (isopropanol treated) cultured amoebae across at least three logs of cell concentrations. Using a serial dilution of viable and non-viable cells, we show that non-PMA PCR amplifies both viable and non-viable amoebae, while PMA exposure suppresses (but does not completely inhibit) amplification from non-viable amoebae. The effect of freshwater treatment on N. perurans viability was assessed using the PMA-PCR. Following PMA exposure, amplification from freshwater treated amoebae was reduced by approximately 94–97 %. Taken together this study demonstrates that PMA combined with traditional real-time PCR can estimate amoeba viability.
期刊介绍:
Protist is the international forum for reporting substantial and novel findings in any area of research on protists. The criteria for acceptance of manuscripts are scientific excellence, significance, and interest for a broad readership. Suitable subject areas include: molecular, cell and developmental biology, biochemistry, systematics and phylogeny, and ecology of protists. Both autotrophic and heterotrophic protists as well as parasites are covered. The journal publishes original papers, short historical perspectives and includes a news and views section.