Detection of Nanophyetus salmincola in Water, Snails, and Fish Tissues by Quantitative Polymerase Chain Reaction

Abstract

We report the development and validation of two quantitative PCR (qPCR) assays to detect Nanophyetus salmincola DNA in water samples and in fish and snail tissues. Analytical and diagnostic validation demonstrated good sensitivity, specificity, and repeatability of both qPCR assays. The N. salmincola DNA copy number in kidney tissue was significantly correlated with metacercaria counts based on microscopy. Extraction methods were optimized for the sensitive qPCR detection of N. salmincola DNA in settled water samples. Artificially spiked samples suggested that the 1-cercaria/L threshold corresponded to an estimated log₁₀ copies per liter ≥ 6.0. Significant correlation of DNA copy number per liter and microscopic counts indicated that the estimated qPCR copy number was a good predictor of the number of waterborne cercariae. However, the detection of real-world samples below the estimated 1-cercaria/L threshold suggests that the assays may also detect other N. salmincola life stages, nonintact cercariae, or free DNA that settles with the debris. In summary, the qPCR assays reported here are suitable for identifying and quantifying all life stages of N. salmincola that occur in fish tissues, snail tissues, and water.

Received April 13, 2017; accepted August 6, 2017 Published online September 22, 2017

Acknowledgments

This is Publication Number 11 from the Salish Sea Marine Survival Project (marinesurvivalproject.com), an international, collaborative research effort designed to determine the primary factors affecting the survival of juvenile salmonid fish in the combined marine waters of Puget Sound and the Strait of Georgia. Funding was provided by the USGS Fisheries Program, Ecosystems Mission Area. All raw data and machine-readable metadata are accessible through sciencebase.gov (https://doi.org/10.5066/F7FN14QN). Assistance with snail collections and experiments was provided by A. MacKenzie, M. Wilmot, and J. Gregg (USGS, MMFS). Water samples from the Nisqually River were provided by Megan Moore (National Oceanic and Atmospheric Administration Fisheries) and Jed Moore (Nisqually Indian Tribe). Nanophyetus salmincola metacercaria counts in the posterior kidneys of infected fish were provided by M. Chen. Animal experiments were approved by the Institutional Animal Care and Use Committee at the USGS Western Fisheries Research Center (Protocol Number 2008-37). Use of any trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government.