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Applied and Environmental Microbiology, February 2006, p. 1551-1557, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1551-1557.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Molecular Assays for Detecting Aphanomyces invadans in Ulcerative Mycotic Fish Lesions

Mark W. Vandersea,1* R. Wayne Litaker,1 Bryan Yonnish,2 Emilio Sosa,3 Jan H. Landsberg,3 Chris Pullinger,4 Paula Moon-Butzin,4 Jason Green,4 James A. Morris,1 Howard Kator,5 Edward J. Noga,6 and Patricia A. Tester1

Center for Coastal Fisheries and Habitat Research, National Ocean Service, National Oceanic Atmospheric Administration, Beaufort, North Carolina,1 Duke University, Duke University Medical Center, Purple Zone, Durham, North Carolina,2 Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, St. Petersburg, Florida,3 North Carolina Division of Water Quality, Raleigh, North Carolina,4 Virginia Institute of Marine Science, The College of William and Mary, Gloucester Point, Virginia,5 College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina6

Received 15 March 2005/ Accepted 15 November 2005

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.

* Corresponding author. Mailing address: Center for Coastal Fisheries and Habitat Research, National Ocean Service, National Oceanic Atmospheric Administration, 101 Pivers Island Rd., Beaufort, NC 28516-9722. Phone: (252) 728-8774. Fax: (252) 728-8784. E-mail: Mark.W.Vandersea{at}noaa.gov.

Applied and Environmental Microbiology, February 2006, p. 1551-1557, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1551-1557.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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