Assessing a semi-validated quantitative PCR for high-throughput surveillance of <i>Ichthyophonus</i> sp. in Yukon River Chinook Salmon

OBJECTIVE: We sought to determine the suitability of a semi-validated quantitative PCR (qPCR) assay for assessing the prevalence and density of Ichthyophonus sp. infections in Yukon River Chinook Salmon Oncorhynchus tshawytscha, representing a new species and location for the assay's application. Ichthyophonus sp. causes severe disease that has been implicated in the en route prespawning mortality of this Chinook Salmon stock. The reduced survival of these fish has caused many hardships for the people who rely on them. METHODS: We assessed a modified qPCR assay with stage 1 and partial stage 2 validation per World Organisation for Animal Health criteria for Ichthyophonus sp. surveillance in Yukon River Chinook Salmon. Cohen's kappa tests were used to compare qPCR results of infection status (infected/uninfected) with those of explant culture-the current reference standard. Linear regressions were used to interrogate qPCR results of parasite gene copies per milligram for infection density with parasite counts per cubic millimeter from the reference standard of histopathology. RESULTS: We found that the prevalence of Ichthyophonus sp. DNA by qPCR did not significantly differ from that of the reference standard for explant culture of the parasite. The qPCR demonstrated high analytical sensitivity and estimated diagnostic performance. Gene copies per milligram by qPCR were correlated to the number of schizonts per cubic millimeter with an R2 value of 0.40-0.41, but the R2 increased to 0.92 after we accounted for parasite size in the 2023-2024 data set. CONCLUSIONS: The results from this qPCR with our modifications were equivalent to the prevalences and densities determined by reference standards and fit our purpose for a rapid Ichthyophonus sp. surveillance assay. This could be used in-season to inform fishery managers about disease and mortality associated with Ichthyophonus sp. infections in high-risk regions like the Yukon River, where en route prespawning mortality associated with this parasite occurs periodically.