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Objective: We sought to evaluate accurate and reproducible detection of Myxobolus cerebralis (Mc), the causative agent of whirling disease, by using nested polymerase chain reaction (nPCR) and three previously established real-time quantitative PCR (qPCR) assays: K18S (Kelley 18S), C18S (Cavender 18S), and Hsp70 (heat shock protein 70). We used a "fit for purpose" approach combined with intra- and interlaboratory testing to identify a molecular testing method that would be equivalent to the currently accepted nPCR procedure for Mc., Methods: Assay performance was compared using a combination of intra- and interlaboratory testing that used synthetic gBlocks along with naturally and experimentally infected fish tissue. North American isolates representing geographically distinct locations were also tested using all three assays., Result: The K18S and C18S assays exhibited high assay sensitivity, intra- and interlaboratory repeatability of sample replicates, and reproducible identification of all test samples across multiple laboratories. In contrast, the Hsp70 assay failed to detect several positive samples at low DNA concentrations during intra- and interlaboratory testing. The K18S assay was the only procedure that demonstrated perfect detection accuracy when testing geographically distinct Mc isolates. Results demonstrated the K18S assay is robust under variable test conditions, is more accurate than the C18S and Hsp70 assays, and provides detection capabilities equivalent to those of the currently accepted nPCR confirmation assay "gold standard" that is described in the American Fisheries Society-Fish Health Section (AFS-FHS) Blue Book., Conclusion: The "fit for purpose" approach and preliminary completion of the World Organization for Animal Health validation pathway demonstrate that the K18S assay provides an alternate method for Mc testing. This work provides the foundation for acceptance of the K18S assay into the AFS-FHS Blue Book as a standardized test procedure for Mc., (© 2024 American Fisheries Society.)

Over the past century, populations of Lake Trout Salvelinus namaycush have declined throughout the Great Lakes basin due to overfishing, habitat destruction, introduction of invasive species, and associated recruitment issues from high thiaminase, as well as emerging infectious diseases. To combat these declines, state and federal fishery management agencies undertook substantial stock enhancement efforts, including more stringent regulation of sport and commercial catch limits and increasing hatchery propagation of Lake Trout stocked into Great Lakes basin waterways. One state fish hatchery involved in these rehabilitation efforts experienced mass mortality events in 2012 and 2017. In 2012, following a period of abnormally heavy rain, hatchery staff observed abnormal behavior followed by increased mortalities in two strains of Lake Trout fingerlings, reaching upwards of 20% mortality and totaling a loss of approximately 100,000 fish. In 2017, following another heavy-rain season, 6-8% of 2-year-old Lake Trout experienced morbidity and mortality similar to that observed in 2012. During the 2012 event, Brook Trout Salvelinus fontinalis and splake (Lake Trout × Brook Trout hybrid) reared in flow-through systems receiving water from diseased Lake Trout remained clinically unaffected. Molecular analyses revealed all lots of affected Lake Trout were infected with the salmonid herpesvirus-3 (epizootic epitheliotropic disease virus [EEDV]), a disease that caused complete depopulation of this hatchery in the late 1980s and until 2012 was never again detected in this hatchery or in Michigan. Further sampling detected EEDV in apparently healthy 5-year-old Lake Trout and in wild Mottled Sculpin Cottus bairdii collected in the hatchery source water. The ability of the virus to replicate in tissues of infected fish was verified by exposing naïve Lake Trout to the filtered tissue homogenates of infected fish resulting in similar disease signs. Despite the virus going undetected for many years, these two EEDV episodes clearly demonstrate the continued presence of this deadly herpesvirus in the Great Lakes basin., (© 2019 American Fisheries Society.)