title: Molecular assays for detecting Aphanomyces invadans in ulcerative mycotic fish lesions.
abstract: The pathogenic oomycete Aphanomyces invadans is the primary etiological agent in ulcerative mycosis, an ulcerative skin disease caused by a fungus-like agent of wild and cultured fish. We developed sensitive PCR and fluorescent peptide nucleic acid in situ hybridization (FISH) assays to detect A. invadans. Laboratory-challenged killifish (Fundulus heteroclitus) were first tested to optimize and validate the assays. Skin ulcers of Atlantic menhaden (Brevoortia tyrannus) from populations found in the Pamlico and Neuse River estuaries in North Carolina were then surveyed. Results from both assays indicated that all of the lesioned menhaden (n = 50) collected in September 2004 were positive for A. invadans. Neither the FISH assay nor the PCR assay cross-reacted with other closely related oomycetes. These results provided strong evidence that A. invadans is the primary oomycete pathogen in ulcerative mycosis and demonstrated the utility of the assays. The FISH assay is the first molecular assay to provide unambiguous visual confirmation that hyphae in the ulcerated lesions were exclusively A. invadans.
authors.affiliation: Center for Coastal Fisheries and Habitat Research, National Ocean Service, National Oceanic Atmospheric Administration, 101 Pivers Island Rd., Beaufort, NC 28516-9722, USA. Mark.W.Vandersea@noaa.gov
authors.forename: Mark W
authors.initials: MW
authors.lastname: Vandersea
authors.forename: R Wayne
authors.initials: RW
authors.lastname: Litaker
authors.forename: Bryan
authors.initials: B
authors.lastname: Yonnish
authors.forename: Emilio
authors.initials: E
authors.lastname: Sosa
authors.forename: Jan H
authors.initials: JH
authors.lastname: Landsberg
authors.forename: Chris
authors.initials: C
authors.lastname: Pullinger
authors.forename: Paula
authors.initials: P
authors.lastname: Moon-Butzin
authors.forename: Jason
authors.initials: J
authors.lastname: Green
authors.forename: James A
authors.initials: JA
authors.lastname: Morris
authors.forename: Howard
authors.initials: H
authors.lastname: Kator
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Noga
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Tester
mesh_terms: D000818:Animals; D044744:Aphanomyces / Q000235:genetics* / Q000302:isolation & purification* / Q000472:pathogenicity; D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D005393:Fish Diseases / Q000382:microbiology*; D005399:Fishes; D017404:In Situ Hybridization, Fluorescence / Q000379:methods / Q000662:veterinary*; D008969:Molecular Sequence Data; D009181:Mycoses / Q000382:microbiology / Q000662:veterinary*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012883:Skin Ulcer / Q000382:microbiology / Q000662:veterinary; D013045:Species Specificity
chemical_list: D004271:DNA, Fungal