2023 article
What is your diagnosis? Ulcerative shell lesions from a diamond-backed terrapin (Malaclemys terrapin)
Hastain, S. A. A., Buchy, J. M. M., Dombrowski, D. S. S., Womble, M. A. A., Armwood, A. R. R., & Gruber, E. J. J. (2023, July 27). VETERINARY CLINICAL PATHOLOGY.
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Added: August 14, 2023
An adult female intact diamond-backed terrapin (Malaclemys terrapin) at the North Carolina Museum of Natural Sciences presented for an episode of scute sloughing and cystic ulcerative pitting lesions on the carapace and plastron. The patient was wild-caught as a hatchling or juvenile by another institution in 2004 and obtained by the museum in 2007. Since acquisition, this turtle has had a chronic history of pitting shell lesions, keratin scute flaking, and excessive keratin scute buildup. This turtle had two notable episodes with deep shell lesions and scute sloughing, the first of which completely resolved with topical antibiotics. During the second episode, the multifocal deep ulcerative lesions cultured positive for multiple aerobic bacteria that were initially susceptible to topical and injectable antibiotics. Healing was prolonged due to the development of antibiotic resistance, and antibiotic therapy was altered according to sensitivity results. Complete gross resolution occurred after 4 months of treatment. On presentation, multiple ulcerated and hemorrhagic lesions were present on the carapace after acute sloughing of the scutes, most notably between the right first and second costal scutes. The deep ulcers were associated with tan to yellow dry caseous granuloma-like exudative lesions that penetrated into the bone. Swabs from these lesions were submitted for cytology (Figure 1). The patient was otherwise active and eating well. Fungal hyphae with mild heterophilic inflammation The sample is of low cellularity consisting mostly of keratinized squamous epithelial cells. Focal areas of the slides contain low numbers of poorly preserved heterophils (not pictured). Numerous variably staining fungal hyphae are associated with many of the keratin aggregates. The hyphae are found individually and in mats. Hyphae are approximately 2–4 μm in diameter and septate, with thin nonstaining walls that are mostly parallel but occasionally nonparallel. They display internal complexity, often containing variably sized clear, distinct vacuoles. Rare acute and right-angled lateral branching is observed. Gross examination of the dermal fragments and multiple fragments of surrounding tissue reveals multifocal to coalescing pale tan plaques, nodules, and pitting lesions (Figure 2). On histopathologic examination, the superficial dermis is expanded by epithelial inclusion cysts. Cyst structures are lined by well-differentiated stratified squamous epithelium with central keratinization and the accumulation of lamellated layers of keratin debris (Figure 3A). Embedded within the central layers of keratin are numerous fungal hyphae highlighted by Grocott's methenamine silver stain (Figure 3B). Hyphae are approximately 4 μm in diameter with nonparallel cell walls and display acute and right-angle branching and septation. The dermal fragments display marked orthokeratotic to parakeratotic hyperkeratosis, with conglomerates of keratin debris, degenerative leukocytes, and eosinophilic fluid also containing the fungal hyphae (not pictured). A pooled swab prepared from the oral cavity, cloaca, and shell submitted to the University of Illinois College of Veterinary Medicine Zoological Pathology Program Molecular Diagnostic Laboratory tested positive for Emydomyces testavorans by quantitative PCR. Ulcerative shell disease is a common cause of morbidity and mortality in captive and free-ranging populations of turtles,1, 2 and clinical signs include shell erosions, lethargy, and emaciation.3 Reported causes of ulcerative shell disease include trauma, malnutrition (eg, calcium and vitamin D3 deficiencies), and various bacterial and fungal etiologies, though the cause is often unknown.1, 4 In captive individuals, poor husbandry practices such as inappropriate temperature, humidity, lighting, and/or nutrition are considered predisposing factors.4 Infectious causes are most often identified as gram-negative commensals within the Enterobacteriae order, including Escherichia, Klebsiella, and Enterobacter spp.4 Bacterial ulcerative shell disease has the potential to progress to septicemic cutaneous ulcerative disease (SCUD), in which bacterial dermatitis and osteomyelitis may be identified.1 Fungal causes of ulcerative shell disease are rarely reported, and the majority of published reports are from nonaquatic turtle species.2 Emydomyces testavorans is a keratinophilic fungal organism in the Onygenales order.5 Onygenalean fungi have been identified as primary pathogens for ulcerative dermatitis in captive and free-ranging reptilian species, including crocodilians, lizards, and snakes.1 E. testavorans has only been recently described and has been isolated from ulcerative shell lesions in aquatic turtles, causing keratin discoloration, flaking and textural change, erosion, ulceration, and osteonecrosis.2, 5 The most consistent and striking histopathologic lesion with E. testavorans infection is the presence of epithelial inclusion cysts, identified in more than 90% of cases.2 Epithelial inclusion cysts are cystic spaces lined by stratified squamous epithelium with central keratinization. E. testavorans hyphae are typically embedded in keratin debris.5 Although the underlying mechanism of epithelial inclusion cyst formation with E. testavorans infection is unclear, it is presumed that the excessive keratinization occurs secondary to the fungal infection rather than fungal infection with tropism for previously established epithelial inclusion cysts. Since epithelial inclusion cysts in other species are not often associated with fungal infections, it is possible that immunosuppression is contributing to the pathogenesis in aquatic turtles. Definitive diagnosis of E. testavorans typically requires PCR with or without DNA sequencing. Although culture can be performed, it may be complicated by sample contamination with other common environmental bacterial and fungal organisms and by the requirement for reptile keratin-enriched media. Preferred samples are lesion swabs and biopsy material that should be refrigerated for culture and frozen for molecular diagnostics.5 In summary, this case described the cytologic features of the onygenalean fungus E. testavorans, a newly described cause of ulcerative shell lesions in aquatic turtles. The frequent association of fungal hyphae with keratin and concurrent inflammation is consistent with histologic findings of epithelial inclusion cysts associated with E. testavorans.2 The heterophilic inflammation, in this case, could be in response to the fungus, keratin, and/or concurrent bacterial infection. Although bacteria were not identified cytologically, bacterial culture was not performed to exclude this possibility. A thorough examination of keratin aggregates for fungal hyphae elements is warranted in samples from ulcerative shell lesions in aquatic turtles. The authors have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial compensation with, the subject matter or materials discussed in this article.