A Perspective: Molecular Detections of New Agents in Finfish—Interpreting Biological Significance for Fish Health Management

Abstract

The increased sensitivity of advanced molecular techniques greatly exceeds the sensitivities of traditional detection methods for infectious agents. This sensitivity causes difficulty in interpreting the biological significance of such detections in fish (and shellfish), especially when the agent(s) cannot be cultured in the laboratory. In the Pacific Northwest, including Canada and Alaska, molecular detections of “new” (unknown or known but discovered in a different geographic location or fish host) potentially infectious agents in fish have received extensive media attention and misinterpretation that call for resource agencies to change current fish health surveillance practices or policies to include these agents. Fish health specialists from several of these agencies and organizations (see Acknowledgments) advise that any policy changes should be made only after further investigations to avoid wasting resources to conduct surveillance for organisms that are not significant to fish health or for noninfectious genetic material that does not represent a viable agent. Molecular detection is not proof of agent viability within or on host tissues and requires further investigation regarding the agent's ability to replicate and evidence that the agent causes substantial risk of disease to exposed fish populations. This document provides examples of molecularly detected agents causing public concern that were accompanied by little or no data to provide context and assessment of biological significance, highlights important questions to be answered regarding these detections, and provides a suggested pathway of investigative criteria to determine viability and pathogenicity of such agents that are necessary for consideration of any changes to aquatic animal health practices and policies.

British Columbia freshwater salmon hatcheries demonstrate minimal contribution to piscine orthoreovirus (PRV) regional occurrence with no evidence for nonendemic strain introductions

“…Although qPCR has demonstrated both high diagnostic specificity and sensitivity--often greater than 98% specificity in stringently controlled laboratories (Polinski et al 2021a;Yang et al 2022;Delphino et al 2023) --false positive detections become increasingly likely as sample size increases. Further, false positive detections are also most likely to manifest at or near the lower limit of detection of the qPCR assay due to a combination of increased probability for low-quantity aerosol contamination to occur or nonspecific fluorescence changes to manifest in late qPCR cycles (Polinski et al 2021a;Meyers and Hickey 2022). In this study, all three inconclusive detections (1 coho, 1 Chinook, and 1 Atlantic) as well as one positive detection from a Chinook salmon were at or below the lower effective limit of detection of the qPCR assay (i.e., less than 1-3 theoretical copies per reaction; Ct > 35) and represent <0.08% of screened samples, implying that theoretical diagnostic specificity was 99.92% or greater in this study even if these detec-tions were false.…”

Detection, sequencing, and tissue distribution of piscine orthoreovirus 2–like virus in diseased coho salmon in Alaska

“…Although detection of this novel PRV2-like virus in coho salmon does not prove causation, localization of viral RNA by ISH within the histologic lesions identifies a strong association worthy of further investigation. 18 We used various testing methods in this investigation, including histopathology, VI, real-time RT-PCR, WGS, and ISH. Of these methods, we found that real-time RT-PCR and ISH were of the greatest utility when attempting to detect the PRV2-like virus.…”