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Diseases of Aquatic Organisms

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DAO 104:69-81 (2013)  -  DOI:

Development and validation of a quantitative PCR assay for Ichthyophonus spp.

Vanessa C. White1,*, J. Frank Morado1, Lisa M. Crosson2, Brent Vadopalas2, Carolyn S. Friedman

1National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Alaska Fisheries Science Center, Resource Assessment and Conservation Engineering Division, Seattle, Washington 98115, USA
2School of Aquatic and Fishery Sciences, College of the Environment, University of Washington, Seattle, Washington 98105, USA

ABSTRACT: Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus.

KEY WORDS: Ichthyophonus sp. · qPCR · Validation · Detection methods · Theragra chalcogramma

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Cite this article as: White VC, Morado JF, Crosson LM, Vadopalas B, Friedman CS (2013) Development and validation of a quantitative PCR assay for Ichthyophonus spp.. Dis Aquat Org 104:69-81.

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