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.

In the spring of 1998, the Florida Fish and Wildlife Research Institute received numerous reports of lesioned or ulcerated fish primarily from the St. Lucie Estuary on the southeast coast of Florida, an area known since the late 1970s for lesions of the ulcerative mycosis (UM) type. From these and archived reports, as well as others received from different areas of Florida, we documented that diseased specimens had randomly distributed skin ulcers (usually reddened or hemorrhagic) with raised irregular margins and, in some cases, deeply penetrating hyphae in the surrounding muscle tissue. Since 1998, 256 fish (comprising 18 species) with ulcerative lesions (from 15 different locations) were confirmed with hyphae in fresh squash preparation or by histological evaluation. Squash preparations revealed nonseptate, sparsely branching, thick-walled hyphae; histological sections revealed mycotic granulomas in the dermis that occasionally penetrated into the skeletal muscle. These pathological characteristics were consistent with UM caused by the oomycete Aphanomyces invadans in Southeast Asia, Japan, Australia, and the United States. For specific identification, six isolates from ulcerated fish were cultured and prepared for molecular characterization using established diagnostic methods. Ribosomal RNA gene sequence analysis identified three isolates as Aphanomyces invadans, one as the oomycete Achlya bisexualis, and two as the ascomycete Phialemonium dimorphosporum. A more extensive survey of 67 ulcerated skin samples from fish collected between 1998 and 2003 was performed using a polymerase chain reaction assay specific for Aphanomyces invadans. Of these, 26 (38.8%) samples from seven fish species and nine collection locations were positive. Confirmation of UM associated with Aphanomyces invadans represents new host records in Florida for the sheepshead Archosargus probatocephalus, striped mullet Mugil cephalus, white mullet Mugil curema, silver perch Bairdiella chrysoura, black drum Pogonias cromis, largemouth bass Micropterus salmoides, and American shad Alosa sapidissima.

Based on isolations from naturally infected fish in Florida, we investigated the role of the fungi Aphanomyces invadans, Achlya bisexualis, and Phialemonium dimorphosporum in the etiology of ulcerative mycosis (UM) in striped mullet Mugil cephalus. We injected healthy striped mullet subcutaneously with secondary zoospores of four oomycete isolates: two concentrations (50 and 115 zoospores/mL) of SJR (an endemic isolate of Aphanomyces invadans in American shad Alosa sapidissima from the St. Johns River); two concentrations each of CAL (25 and 65 zoospores/mL) and ACH (1,400 and 2,000 zoospores/mL; endemic isolates of Aphanomyces invadans and Achlyva bisexualis, respectively, in striped mullet from the Caloosahatchee River); and two concentrations of the ascomycete culture MTZ (2,500 and 3,500 zoospores/mL; endemic isolate of P. dimorphosporum from whirligig mullet M. gyrans in the Matanzas Inlet). All fish injected with either concentration of SJR developed granulomatous ulcers after 8 d and died within 21 d. Eighty percent (8/10) of fish injected with the high dose of CAL developed ulcers after 13 d and died within 28 d, but only 30% (3/10) of fish injected with the low dose of CAL developed ulcers. Four of the ulcerated fish died within 28 d, and the remaining fish were terminated after 32 d. Fish injected with zoospores of Aphanomyces invadans developed ulcers that were grossly and histologically similar to those observed in naturally infected striped mullet with UM from several estuaries or rivers in Florida. These hemorrhagic skin ulcers were characterized by myonecrosis and the presence of mycotic granulomas. None of the fish injected with ACH, MTZ, or sterile water developed ulcers. This study fulfilled Koch's postulates and demonstrated that ulcers could be experimentally induced in striped mullet after exposure via injection to secondary zoospores of an endemic Florida strain of Aphanomyces invadans.