History

A group of 8 squarespot anthias (Pseudanthias pluerotaenia), an Indo-Pacific marine tropical fish, were received for necropsy by the Department of Pathology, College of Veterinary Medicine at the University of Georgia. The fish were part of a mixed-sex group of approximately 700 wild-caught animals that were placed in a 20,000-L closed recirculating quarantine tank; while in the tank, an outbreak of ulcerative dermatitis and an increasing number of deaths occurred. During the epizootic, the mean ± SD temperature of the seawater was 24.2 ± 0.4°C, salinity was 32.97 ± 4.98 ppt, and pH was 8.29 ± 0.14. The tank system had been disinfected, drained, and dried prior to stocking with these fish. A low number of deaths occurred 1 week after an uneventful, approximately 24-hour shipment, but the mortality rate accelerated dramatically over the next 5 days despite the application of a continuous 12-day waterborne prophylactic regimen of trimethoprim-sulfathiazole (30 mg/L) beginning on the day of arrival and placement in the tank.

Clinical and Gross Findings

Within the large population, individual fish were difficult to observe and sick fish were generally found dead or moribund. The fish submitted for necropsy were in good physical condition and had a mean length to the tail fork of 7.2 ± 0.6 cm. Gross necropsy findings were limited to relatively inconspicuous pale epithelial erosions and ulcers of variable size and shape that were randomly distributed on dorsolateral aspect of the trunk and peduncle regions (sparing the fins; Figure 1). There were no notable internal abnormalities, and bacterial cultures of liver and spleen samples on blood agar plates yielded no growth.

Photograph of a female squarespot anthias (1 of a mixed-sex group of approx 700 wild-caught animals) that was found dead 12 days after placement in a closed recirculating quarantine tank. A week after the arrival of the group of fish, a low number of deaths had been noted; the mortality rate increased rapidly during the next 5 days. In this representative fish, notice the large, irregular, shallow ulcer on the left side of the trunk region. The ulcer has ragged loosely adhered margins and is partially bordered by a narrow reddened zone. Bar = 1 cm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Photograph of a female squarespot anthias (1 of a mixed-sex group of approx 700 wild-caught animals) that was found dead 12 days after placement in a closed recirculating quarantine tank. A week after the arrival of the group of fish, a low number of deaths had been noted; the mortality rate increased rapidly during the next 5 days. In this representative fish, notice the large, irregular, shallow ulcer on the left side of the trunk region. The ulcer has ragged loosely adhered margins and is partially bordered by a narrow reddened zone. Bar = 1 cm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Photograph of a female squarespot anthias (1 of a mixed-sex group of approx 700 wild-caught animals) that was found dead 12 days after placement in a closed recirculating quarantine tank. A week after the arrival of the group of fish, a low number of deaths had been noted; the mortality rate increased rapidly during the next 5 days. In this representative fish, notice the large, irregular, shallow ulcer on the left side of the trunk region. The ulcer has ragged loosely adhered margins and is partially bordered by a narrow reddened zone. Bar = 1 cm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Formulate differential diagnoses from the history, clinical findings, and Figure 1—then turn the page →

Cytologic and Histopathologic Findings

Skin scrapings of the margins of ulcers in the 8 fish were obtained, and wet-mount preparations of the scrapings were examined microscopically. Large numbers of highly motile, holociliated, oval to pyriform (approx 20 × 40-μm) protozoans, each with a prominent long, nonmotile caudal cilium, were detected (Figure 2). The appearance of the protozoans was consistent with that of scuticociliates.

Photomicrograph of a wet-mount preparation of a skin scraping obtained from the fish in Figure 1. Notice the scuticociliate with abundant cytoplasmic vacuoles and prominent caudal cilium (arrow). Bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Photomicrograph of a wet-mount preparation of a skin scraping obtained from the fish in Figure 1. Notice the scuticociliate with abundant cytoplasmic vacuoles and prominent caudal cilium (arrow). Bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Photomicrograph of a wet-mount preparation of a skin scraping obtained from the fish in Figure 1. Notice the scuticociliate with abundant cytoplasmic vacuoles and prominent caudal cilium (arrow). Bar = 20 μm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Histologically, lesions in the 8 fish varied from epithelial erosion and lepidorthosis (edema of the scale pockets) to deep ulceration. The epithelium bordering the ulcers was typically spongiotic, lifted from the dermis, and infiltrated by small numbers of lymphocytes and granulocytes. Ciliated protozoans were limited to the necrotic dermis and scale pockets in some fish, whereas parasites infiltrated perimysial connective tissues, were present intercellularly, and often penetrated to the retroperitoneal adventitia in more severely affected fish. Isolated myofibers had loss of tinctorial properties and striation, sarcoplasmic coagulation, and coarse vacuolation. Endomysial areas contained multiple small foci of hemorrhage and necrotic cellular debris. Rarely, ciliates were detected within vascular lumens and sinusoids of parenchymal organs, including the liver and kidney. With the exception of scattered macrophages and lymphocytes, which were confined primarily to the dermis, inflammatory changes were largely absent.

In tissue sections, the scuticociliates appeared round to ovoid or pyriform, and measured approximately 12 × 20 μm to 20 × 45 μm (Figure 3). The abundant foamy pale basophilic cytoplasm contained numerous large hyaline inclusions, interpreted as remnants of ingested erythrocytes, and small intensely basophilic granules. The large round basophilic nuclei were centrally located to slightly eccentric. In sections of skeletal muscle treated with a polyclonal rabbit antibody against Uronema nigricans, positive immunostaining of the parasites was evident.

Photomicrographs of skeletal muscle subadjacent to the skin ulcer of the fish in Figure 1. Notice the group of scuticociliates among necrotic intercellular debris. The parasites have a prominent round nucleus and contain phagocytized erythrocytes. H&E stain; bar = 100 μm. The inset illustrates parasites from the same muscle lesion following immunostaining with a polyclonal rabbit antibody against Uronema nigricans, which accentuates the fine cilia. Uronema nigricans-specific immunohisto-chemical stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Photomicrographs of skeletal muscle subadjacent to the skin ulcer of the fish in Figure 1. Notice the group of scuticociliates among necrotic intercellular debris. The parasites have a prominent round nucleus and contain phagocytized erythrocytes. H&E stain; bar = 100 μm. The inset illustrates parasites from the same muscle lesion following immunostaining with a polyclonal rabbit antibody against Uronema nigricans, which accentuates the fine cilia. Uronema nigricans-specific immunohisto-chemical stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Photomicrographs of skeletal muscle subadjacent to the skin ulcer of the fish in Figure 1. Notice the group of scuticociliates among necrotic intercellular debris. The parasites have a prominent round nucleus and contain phagocytized erythrocytes. H&E stain; bar = 100 μm. The inset illustrates parasites from the same muscle lesion following immunostaining with a polyclonal rabbit antibody against Uronema nigricans, which accentuates the fine cilia. Uronema nigricans-specific immunohisto-chemical stain; bar = 50 μm.

Citation: Journal of the American Veterinary Medical Association 238, 3; 10.2460/javma.238.3.301

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Morphologic Diagnosis

Moderate to severe, acute, multifocal, ulcerative dermatitis and subadjacent necrotizing myositis, with intralesional ciliated protozoans.

Comments

Ulcerative skin lesions are a common pathological finding in sick fish and have a wide range of noninfectious and infectious causes, including trauma, neoplasia, bacterial and fungal infections, and ectoparasite infestations. A minimal diagnostic evaluation should include microscopic examination of impressions or scrapings of lesion margins; bacterial culture of those samples or possibly biopsy of lesion margins should be considered when pathogens are not detected microscopically. In the fish of this report, necropsy and microscopic findings were consistent with scuticociliate-induced dermatitis and subsequent systemic invasion.

Pathogenic scuticociliates that affect fish, most notably Uronema marinum and Miamiensis avidis, are opportunistic histophagous or tissue-consuming parasites that have the potential for systemic invasion of stressed hosts.^1–3 The scuticociliates are increasingly considered to be important pathogens of ornamental and farmed marine fish. First described as facultative parasites of captive seahorses⁴ and other marine fish in public aquaria,¹ representatives of the group are now known to cause mass mortality events in various cultured species and affect marine aquaculture worldwide.⁵

In scuticociliate-infected fish, gross pathological changes are limited predominantly to epidermal sloughing and ulceration, with exposure and hemorrhage of underlying muscle.^1,3 Microscopic lesions are characterized by tissue necrosis and hemorrhage, with minimal inflammatory response. Typically, large numbers of ciliates infiltrate affected tissues and numerous ingested erythrocytes and cellular debris are evident within their cytoplasm. Lesions are variable, ranging from skin ulceration and muscle necrosis to systemic spread with invasion of scale pockets, fins, cornea, gills, blood vessels, heart, kidney, urinary bladder, spinal canal, brain, and gastrointestinal lamina.^1,3,5 Well-vascularized red muscle may be preferentially invaded.¹ The extent to which specific host-parasite interactions determine the degree of spread of the infection and the scuticociliates' tissue predilection site is unclear. Seahorses appear highly susceptible to widespread systemic involvement,¹ whereas tuna develop severe CNS disease.⁶

The scuticociliates have little host specificity, and in experimental challenges of olive flounder (Paralichthys olivaceus), mortality rates have approached 100%.⁵ Epizootics have occurred in fish maintained in a broad range of conditions (eg, salinity ranging from 15 to 35 ppt and temperature ranging from 8° to 28°C).^1,2 Traditionally considered free-living bacteriophagic opportunists, the roles of certain species as primary and secondary pathogens have recently been investigated.⁵ However, the occurrence and severity of outbreaks have a strong association with adverse environmental factors, including temperature stress, crowding, transport, and high bacterial counts.^1,2,6 Once infection is established, proliferation and spread of the parasite occurs rapidly.³ The short time course of disease is evidenced by the fact that affected fish are typically in good physical condition and may have ingesta in their digestive tracts.¹ The pathogenesis of infection remains poorly understood, but there is evidence to suggest that the invasion of host tissues and avoidance of defensive barriers may be mediated through the liberation of substances such as phospholipases, superoxide dismutase, and catalase, which destroy phagocytes and protect against their oxidative killing mechanisms.^7–9 Extensive skin ulceration is believed to result in osmoregulatory disturbances that ultimately lead to death.¹⁰

In the outbreak of this report, the feral fish were placed into a tank system that had been thoroughly disinfected prior to stocking, which suggests that low numbers of the parasite were present in the shipment. Furthermore, the rapid onset of clinical signs indicates that the epizootic may have been precipitated by transport-associated stress. Before, during, and after transport, fish can be exposed to several stressors, including netting, rough handling, crowding, fluctuations in water temperature, and perturbations in water quality (eg, low concentration of dissolved oxygen, buildup of organic wastes, and high concentration of ammonia). In the outbreak of this report, 85% of the 700 fish died, illustrating the resistance of this parasite to chemical treatment. Following the diagnosis of scuticociliatosis, a continuous treatment with copper sulfate (0.2 mg of free copper ion/L of water) was applied for 5 days, followed immediately by treatment with chloroquine (10 mg/L) for 7 days. Deaths again occurred in low numbers 2 weeks later, and metronidazole (25 mg/L) was then administered continuously in the water for 14 days. Thereafter, there were no other deaths among the remaining fish.

Early stages of scuticociliatosis in fish can reportedly be controlled by various combinations of freshwater dips and immersions in methylene blue, nitrofurazone, and formalin.^11,12 The commonly used chemotherapeutants malachite green, formalin, and hydrogen peroxide have been effective in in vitro trials, but have not been critically evaluated in affected fish in vivo.² Some success has also been reported for treatment with metronidazole.¹³ However, fish with systemic and deep muscle infections have a poor prognosis, regardless of the treatment protocol.¹¹ Despite this, use of chemotherapeutants may reduce parasite numbers in tank water, thereby limiting spread of the organisms to unaffected animals. Furthermore, the removal of decaying organic matter may limit the proliferation of heterotrophic bacteria on which the ciliates feed. Prompt removal of dead and moribund fish from the environment may also minimize proliferation and dissemination of the parasite.