Molecular Assays for DetectingAphanomyces invadansin 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.

Lack of Specificity of the Molecular Diagnostic Method for Identification of Aphanomyces Astaci

“…Thus, to accurately identify the presence of A. astaci, it is necessary to carry out new approaches based on other sequences of ITS regions or other genes. A PCR-based test for A. invadans was successfully and allowed distinguishing this species from A. astaci and A. frigidophilus (VANDERSEA et al, 2006). In A. astaci, a new PCR-based method has been recently developed to detect this parasite in North American crayfish (OIDTMANN et al, 2006).…”

Development and preliminary evaluation of a real-time PCR assay for Halioticida noduliformans in abalone tissues

“…Mitochondria are also highly abundant in cells and evolve at a quicker rate than nuclear genes, also making them particularly useful as species-specific markers for discriminating between closely related species. Ribosomal DNA and mitochondrial genes have consequently been widely utilized for the development of specific, sensitive and rapid PCR-based diagnostic techniques (Li et al 1996, Zhou et al 2000, Vandersea et al 2006.…”

“…Mitochondria are also highly abundant in cells and evolve at a quicker rate than nuclear genes, also making them particularly useful as species-specific markers for discriminating between closely related species. Ribosomal DNA and mitochondrial genes have consequently been widely utilized for the development of specific, sensitive and rapid PCR-based diagnostic techniques (Li et al 1996, Zhou et al 2000, Vandersea et al 2006.Effective management of abalone tubercle mycosis in South Africa, and elsewhere in the world, requires the development of techniques that will provide accurate and rapid identification of Halioticida noduli formans. Therefore, the primary objective of the present study was to develop a practical, specific, sensitive and rapid diagnostic tool that uses PCR to detect and identify H. noduliformans in infected abalone.…”

Epizootic ulcerative syndrome caused by Aphanomyces invadans in captive bullseye snakehead Channa marulius collected from south Florida, USA

“…The etiology for this disease may involve multiple pathogens; however, most consistently, Aphanomyces invadans (Family: Leptolegniaceae) has been isolated from lesioned fishes in the USA (Blazer et al 1999, 2002, Vandersea et al 2006, Sosa et al 2007b. A. invadans is an oomycete that is widespread in distribution and has been linked with cases of EUS since it was first documented in Japan in 1971 (Chinabut 1998, Lilley et al 1998.…”

“…skin and skeletal muscle lesions) and 2 gDNA samples were obtained for each fish. Approximately 50 ng of purified gDNA was added to the PCR reaction mixtures and the fish tissues were PCR-assayed using the methods described in Vandersea et al (2006). The PCR assay included a positive and negative control.…”