Dead reckoning of protist viability with propidium monoazide (PMA)-quantitative PCR; a case study using Neoparamoeba perurans

• Published in: Protist 2024-12, Vol.175 (6), p.126068, Article 126068
• Main Authors: Wynne, James W., Rusu, Anca G., Maynard, Ben T., Rigby, Megan L., Taylor, Richard S.
• Format: Article
• Language: English
• Subjects: AGD; Amebiasis - drug therapy; Amebiasis - parasitology; Amoebic gill disease; Amoebozoa - drug effects; Amoebozoa - genetics; Animals; Azides - pharmacology; Cell Survival - drug effects; Fish Diseases - parasitology; Live/dead differentiation; Propidium - analogs & derivatives; Propidium - pharmacology; Real-Time Polymerase Chain Reaction; Salmo salar - parasitology
• ISSN: 1434-4610; 1618-0941; 1618-0941
• DOI: 10.1016/j.protis.2024.126068

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.