ABSTRACT
Hubbs-SeaWorld Research Institute (HSWRI) maintains a population of broodstock California halibut (Paralichthys californicus) at its facility in San Diego, California. Eggs are hatched in 1600 L incubators and fish are moved to small stacked raceways at the time of settlement until they are 2 g in size. These nursery raceways are maintained with recirculated water using UV sterilization. Once they outgrow the raceways, they are moved into flow-through circular fiberglass pools or concrete raceways without any UV sterilization for growout.
Once in the flow through tanks, the last two cohorts of halibut developed reddened circular skin lesions of various sizes often localized to the head and along the mouth. More chronic lesions were ulcerated and covered by a tan to white pseudomembrane. Histopathology of the ulcerated skin lesions showed that the pseudomembranes were composed of thick mats of densely packed macrophages interspersed with fibrin and far fewer neutrophils and lymphocytes. The above inflammatory infiltrates minimally to extensively radiated into the dermis and underlying connective tissue, skeletal muscle and in the most severely affected areas abut the underlying bone and reach into and fill the bone cavities. Embedded within the mats of inflammatory cells were moderate numbers of delicate fungal hyphae that were light brown on H&E sections and light brown on a serial section stained with a Fontana Masson stain. Grocott's Methenamine Silver (GMS) stain revealed more numerous hyphae. The hyphae had parallel walls that were approximately 10μm in diameter, were separated into variable lengths by transverse septae and irregularly branched at 90° angles. Fungal hyphae were present at all levels of the inflammation. Internal examination of the fish revealed low numbers of distinct tan to white granulomas in the posterior kidney. Histopathology showed that the granulomas were composed of densely packed elongate to spindloid epithelioid macrophages interspersed with delicate strands of collagen and low numbers of loosely sprinkled lymphocytes and eosinophilic granular cells. A serial stain with GMS silver stained revealed very rare fungal hyphae that were morphologically similar to those described in the skin.
Fungal cultures were initiated from both the skin and kidney lesions on Sabouraud's dextrose agar and mycobiotic agar. Only the skin lesions resulted in fungal growth, likely due to a smaller amount of fungus present within the kidneys. After DNA extraction from paraffin embedded tissue blocks, the D1D2 region of the 28S ribosomal RNA gene was amplified by PCR using universal fungal primers. The sequence most closely matched that of Exophiala pisciphila and E. salmonis. In order to discriminate between these two species, the ITS region was also amplified by PCR and sequenced. This sequence was closest to E. pisciphila. Based on morphological characteristics of the fungal hyphae, it appears that Exophiala pisciphila was also responsible for the lesions seen in the kidneys.
Infections with Exophiala species have been previously reported in several marine fish and have been responsible for similar looking skin lesions as well as internal infections in kidneys, spleen, and neurological tissue.1-5 Exophiala species are known as black yeasts and are common environmental pathogens. They have widespread distribution in decaying wood, soil, and water. The infection was probably introduced in the water supply once the fish were moved to a flow-through system. Care should be taken when dealing with affected fish as Exophiala spp. are zoonotic pathogens.6
Treatment of fungal diseases is difficult in aquaculture. Treatment of this pathogen was made more difficult due to its systemic status. Currently, there are no antifungal drugs approved for use in food fish in the United States.7 In this group of fish, treatment was primarily directed towards improved husbandry. Flatfish spend the majority of their time on the bottom of the tank and increased water flow can help prevent build up of detritus which can serve as a source of infection for these fish. By improving the water flow, the percentage of mortalities decreased but the infection remained persistent.
References
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