Development and application of a qPCR assay targeting Ichthyophthirius multifiliis in environmental water samples

Ichthyophthirius multifiliis (Ich) is a globally distributed, freshwater parasitic ciliate that infects wild and cultured fishes. It has a direct, temperature-dependent life cycle that enables rapid multiplication when hosts are plentiful and environmental conditions are favorable. Accurate detection is central to the control of Ich infections and prevention of host mortality, particularly in wild systems where chemical treatments are not feasible. In the Klamath River, California, USA, the parasite threatens pre-spawning adult salmon Oncorhynchus spp. Currently, Ich is monitored by lethal sampling of fish hosts and visual quantification of parasite load. This method is insensitive to light infections, contributes to pre-spawn mortality of wild salmon, and does not allow for population-level disease risk assessments. We developed and applied an alternate sampling method based on molecular analysis of water samples for parasite DNA. We sequenced the small subunit ribosomal DNA (ssrDNA) of Ich isolates collected from the Klamath River, and then developed and validated a novel qPCR assay (SYTO9) that targets Ich ssrDNA. Our assay has better specificity than previously published assays, with strong linearity, efficiency and repeatability. The limit of detection was 50 copies of ssrDNA, equivalent to ~2 theronts in a sample. We found that Ich abundance in environmental water samples collected from the lower Klamath River from July to October, 2014 through 2016, related to observed parasite load on salmon sampled concurrently, indicating that the qPCR assay could be a useful monitoring tool for Ich in the Klamath River, with applications beyond the region.