title: A highly specific PCR assay for detecting the fish ectoparasite Amyloodinium ocellatum.
abstract: Amyloodiniosis, caused by the dinoflagellate ectoparasite Amyloodinium ocellatum, is one of the most serious diseases affecting marine fish in warm and temperate waters. Current diagnostic methods rely entirely on the microscopic identification of parasites on the skin or gills of infested fish. However, subclinical infestations usually go undetected, while no method of detecting the free-swimming, infective (dinospore) stage has been devised. Targeting the parasite's ribosomal DNA region, we have developed a sensitive and specific PCR assay that can detect as little as a single cell from any of the 3 stages of the parasite's life cycle (trophont, tomont, dinospore). This assay performs equally well in a simple artificial seawater medium and in natural seawater containing a plankton community assemblage. The assay is also not inhibited by gill tissue. Sequence analysis of the internal transcribed spacer region of 5 A. ocellatum isolates, obtained from fish in the Red Sea (Israel), eastern Mediterranean Sea (Israel), Adriatic Sea (Italy), Gulf of Mexico (Florida), and from an unknown origin, revealed insignificant variation, indicating that all isolates were the same species. However, 3 of these isolates propagated in cell culture varied in behavior and morphology, and these differences were consistent during at least 2 yr in culture. Thus, our findings do not eliminate the possibility that different strains are in fact 'subspecies' or lower taxa, which may also differ in pathogenic and immunogenic characteristics, environmental tolerance, and other features.
authors.affiliation: College of Veterinary Medicine, North Carolina State University, 4700 Hillsborough Street, Raleigh, North Carolina 27606-1499, USA. mike_levy@ncsu.edu
authors.forename: Michael G
authors.initials: MG
authors.lastname: Levy
authors.forename: Matthew F
authors.initials: MF
authors.lastname: Poore
authors.forename: Angelo
authors.initials: A
authors.lastname: Colorni
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Noga
authors.forename: Mark W
authors.initials: MW
authors.lastname: Vandersea
authors.forename: R Wayne
authors.initials: RW
authors.lastname: Litaker
mesh_terms: D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004141:Dinoflagellida / Q000235:genetics* / Q000302:isolation & purification; D004478:Ectoparasitic Infestations / Q000175:diagnosis / Q000662:veterinary*; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D011528:Protozoan Infections / Q000175:diagnosis; D011529:Protozoan Infections, Animal; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000662:veterinary; D033761:Spores, Protozoan / Q000166:cytology
chemical_list: D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal