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Diseases of Aquatic Organisms

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DAO 76:141-149 (2007)  -  doi:10.3354/dao076141

Characterization of Neoparamoeba pemaquidensis strains: PCR-RFLP of the internal transcribed spacer region from the amoeba and endosymbiont

Charles G. B. Caraguel1,2, Nathanaëlle Donay1,2, Salvatore Frasca Jr.3, Charles J. O’Kelly4, Richard J. Cawthorn1,2 Spencer J. Greenwood1,2,*

1AVC Lobster Science Centre and 2Department of Pathology & Microbiology, Atlantic Veterinary College, University of Prince Edward Island, 550 University Avenue, Charlottetown, Prince Edward Island C1A 4P3, Canada
3Department of Pathobiology & Veterinary Science, University of Connecticut, 61 North Eagleville Road, Storrs, Connecticut 06269-3089, USA
4Bigelow Laboratory for Ocean Sciences, PO Box 475, 180 McKown Point Road, West Boothbay Harbor, Maine 04575, USA
*Corresponding author. Email:

ABSTRACT: Neoparamoeba pemaquidensis continues to be an ongoing problem for commercial finfish aquaculture and has also sporadically been associated with mass mortalities of commercially relevant marine invertebrates. Despite the ubiquity and importance of this amphizoic amoeba, our understanding of the biology as it applies to host range, pathogenicity, tissue tropism, and geographic distribution is severely lacking. This may stem from the inability of current diagnostic tests based on morphology, immunology, and molecular biology to differentiate strains at the subspecies level. In the present study, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method based on the internal transcribed spacer (ITS) region that can accurately differentiate amoeba strains of N. pemaquidensis. The investigation focused on the complications of the amoeba ITS microheterogeneity in the development of a subspecies marker and the use of the endosymbiont, Ichthyobodo necator related organism (IRO), ITS region as an alternative marker. The combination of host amoeba and endosymbiont ITS PCR-RFLP analyses was successfully used to correctly identify and characterize an N. pemaquidensis isolate from an outbreak of amoebic gill disease in Atlantic salmon Salmo salar from the west coast of North America (Washington State, USA).

KEY WORDS: Internal transcribed spacer · Strain marker · PCR-RFLP · Microheterogeneity · Ichthyobodo necator related organism · Parasome

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