Polymerase Chain Reaction Evidence That Hortaea werneckii Causes PIX Skin Disease in the American Alligator (Alligator mississippiensis)
IAAAM Archive
Heather Townsend1; Paul Cardeilhac1; Roger Reep1; Don Samuelson1; James Kimbrough2; Peter McGuire3; AnnMarie Clark4
1College of Veterinary Medicine, University of Florida, Gainesville, FL, USA; 2College of Agricultural and Life Sciences, University of Florida, Gainesville, FL, USA; 3College of Medicine, University of Florida, Gainesville, FL, USA; 4Interdisciplinary Center for Biotechnology Research, University of Florida, Gainesville, FL, USA

In November 1999, small spherical, opaque lesions were identified on alligator skins in Florida. These lesions have since appeared in every southeastern state where alligator farming occurs. Samples were taken from farms in Florida and Louisiana and submitted for microbiology, histology, and polymerase chain reaction (PCR) identification using a 6mm biopsy punch. The microbiology samples yielded several colonies that are considered normal flora for the alligator. The only isolate that was considered a primary pathogenic fungus was Hortaea werneckii. This fungal pathogen can produce a condition known as tinea nigra in humans. Hortaea werneckii is a dematiaceous yeast that inhabits the soil and plants of subtropical climates and is extremely halophilic. The only other animal for which Hortaea werneckii is a known pathogen is a single case of a guinea pig that showed lesions similar to those found in humans. Hortaea werneckii is found along the coastal regions of the United States, consistent with its high affinity for salt. Histology samples were sectioned, stained, and some were found to contain areas of inflammation. These areas of inflammatory response occasionally developed into granulomas. The granulomas were found in the peripheral areas of the dermis and were shown to press on the epidermal layers of the integument. The stratum corneum became displaced, allowing a break in the skin to occur which resulted in an indentation during the tanning process. Upon closer examination using a confocal microscope, fungal hyphae appeared inside the granuloma, along with giant cells, macrophages and other cell types indicative of an inflammatory response. Further investigation was needed to confirm that the fungus was indeed inside the alligator tissue. PCR has shown that the DNA found in the alligator tissue matched the DNA sequence of Hortaea werneckii. Two primers were obtained to carry out the PCR; an internal transcribed spacer (ITS) universal fungal primer was used along with a Hortaea werneckii specific primer. The 6 mm punched tissue was further reduced to 2mm to ensure that alligator DNA was minimized. Isolations were performed along with PCR amplifications of the samples. Positive PCR products were found from farms in both Florida and Louisiana. Wild alligators have also been observed to have these lesions; however, tissue samples from free ranging alligators are not available at this time. These are the first cases of widespread Hortaea werneckii being found in an animal species, specifically the alligator. This project will continue to include an expanded study of where the fungal pathogen is found with confirmation by PCR amplification and electron microscopy.

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Forrest I. Townsend, Jr., DVM
Bayside Hospital for Animals
Ft. Walton Beach, FL, USA