DAO 102:107-118 (2012)  -  DOI: https://doi.org/10.3354/dao02540

Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica

Ami E. Wilbur1,*, Susan E. Ford2, Julie D. Gauthier3, Marta Gomez-Chiarri

1Department of Biology and Marine Biology, Center for Marine Science, University of North Carolina Wilmington, 5600 Marvin K. Moss Lane, Wilmington, North Carolina 28409, USA
2Rutgers University, Haskin Shellfish Research Laboratory, 6959 Miller Avenue, Port Norris, New Jersey 08349, USA
3Department of Biology, Loyola University, New Orleans, 6363 St. Charles Ave., New Orleans, Louisiana 70118, USA
4Department of Fisheries, Animal and Veterinary Science, University of Rhode Island, CBLS Room 169, Kingston, Rhode Island 02881, USA

ABSTRACT: The continuing challenges to the management of both wild and cultured eastern oyster Crassostrea virginica populations resulting from protozoan parasites has stimulated interest in the development of molecular assays for their detection and quantification. For Haplosporidium nelsoni, the causative agent of multinucleated sphere unknown (MSX) disease, diagnostic evaluations depend extensively on traditional but laborious histological approaches and more recently on rapid and sensitive (but not quantitative) end-point polymerase chain reaction (PCR) assays. Here, we describe the development and application of a quantitative PCR (qPCR) assay for H. nelsoni using an Applied Biosystems TaqMan® assay designed with minor groove binder (MGB) probes. The assay was highly sensitive, detecting as few as 20 copies of cloned target DNA. Histologically evaluated parasite density was significantly correlated with the quantification cycle (Cq), regardless of whether quantification was categorical (r2 = 0.696, p < 0.0001) or quantitative (r2 = 0.797, p < 0.0001). Application in field studies conducted in North Carolina, USA (7 locations), revealed widespread occurrence of the parasite with moderate to high intensities noted in some locations. In Rhode Island, USA, application of the assay on oysters from 2 locations resulted in no positives.


KEY WORDS: MSX · Oysters · qPCR · Diagnostic assay · Histology · Parasite density


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Cite this article as: Wilbur AE, Ford SE, Gauthier JD, Gomez-Chiarri M (2012) Quantitative PCR assay to determine prevalence and intensity of MSX (Haplosporidium nelsoni) in North Carolina and Rhode Island oysters Crassostrea virginica. Dis Aquat Org 102:107-118. https://doi.org/10.3354/dao02540

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