Introduction

The eastern tiger salamander (Ambystoma tigrinum) is a freshwater, terrestrial, urodelian species kept as pets, displayed in zoological facilities, and occasionally used as research models for studying infectious disease transmission in amphibians (Collins et al., 2004; IUCN, 2022). Tiger salamanders are considered a species of least concern for conservation by the IUCN with stable populations throughout much of the middle and eastern United States (IUCN, 2022); however, the species is currently considered state endangered in Delaware, Maryland, and Virginia. Populations face threats from habitat loss, degradation, and modification in the form of dams, high climate variability, and the introduction of nonnative salmonids (IUCN, 2022). Infectious disease, often in association with poor environmental conditions and/or husbandry, also presents challenges (Pessier, 2002). Ranavirosis and chytridiomycosis cause significant mortality events in wild and captive amphibians with extensive study in tiger salamanders (Pessier, 2002; Schilliger et al., 2023). Other causes of cutaneous infections in amphibians include water molds such as Saprolegnia spp., bacteria including Aeromonas hydrophila and Mycobacterium spp., herpesviruses, and various protozoal and metazoan parasites (Pessier, 2002). Although rarely reported in tiger salamanders, case series and individual reports of neoplasia have described cutaneous mastocytoma, soft tissue sarcoma, suspect eosinophilic leukemia, amelanotic melanoma, and odontoma (Harshbarger et al., 1999; Sleeman et al., 1999; Boylan et al., 2021).

This report details a case of chromomycosis associated with the melanized fungus Veronaea botryosa in a laboratory-reared population of tiger salamanders.

Case Report

Gross and histopathology

At necropsy, the adult female salamander weighed 36 g and was considered in adequate body condition with minimal autolysis. Grossly, the skin along the dorsal midline had a focally extensive, well-defined and irregularly marginated, 6.5 cm × 1.2 cm region of dark discoloration and roughened surface extending from the neck caudally to the base of the tail (Fig. 1). There were multifocal to coalescing, raised, gray to pink, dermal to subcutaneous nodules ranging in size from 0.5 cm × 0.5 cm × 0.4 cm to 0.8 cm × 0.6 cm × 0.5 cm. On cut surfaces, the nodules were soft, pale pink, and solid with central regions of pallor and a variably distinct capsule. No other significant external or internal lesions were identified. Representative samples of lesions and major organs were placed into 10% neutral buffered formalin and processed routinely for histopathologic examination. Skin swabs and lesioned tissue were also collected for bacteriology and mycology.

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Microscopically, the dermis and underlying skeletal muscle were disrupted by well-demarcated, densely cellular masses (Fig. 2A). The masses were composed of sheets of epithelioid macrophages, multinucleated giant cells, fewer eosinophils, and multifocal regions of amorphous, eosinophilic cellular debris and karyorrhectic nuclei, consistent with necrosis. Individual and clusters of melanized sclerotic bodies (muriform cells) and rare hyphae were scattered intracellularly and extracellularly within the inflammation and necrosis (Fig. 2B). The muriform cells were round, approximately 9-12 μm in diameter, and had a thin, darkly pigmented capsule with a central, foamy, clear to lightly basophilic cytoplasm and occasionally a midline division. The muriform cells stained bright magenta with periodic acid–Schiff (PAS) stain (Fig. 2C). The hyphae were rare, septate, lacked parallel walls, had bulbous projections, and exhibited similar PAS staining (Fig. 2D). The inflammation dissected superficially into the adjacent underlying skeletal muscle. The overlying epidermis was occasionally ulcerated. No bacteria were identified superficially or within the lesions.

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Discussion

Emerging and unusual fungal infections are receiving increased attention in reptile and amphibian conservation and research (Schilliger et al., 2023). Major fungal pathogens contributing to amphibian population declines include Batrachochytrium dendrobatidis and B. salamandrivorans, the causative agents of chytridiomycosis. These fungal agents contribute to superficial cutaneous infections on the ventral body and limbs, resulting in epidermal hyperplasia, hyperkeratosis, erythema, and discoloration (Schilliger et al., 2023). Melanized fungi are less commonly reported from amphibians and include Exophiala spp., Cladophialophora spp., Phialophora spp., Cladosporium spp., Ochroconis spp., Fonsecaea spp., and V. botryosa (Elkan and Philpot, 1973; Bube et al., 1992; Pessier, 2002; de Hoog et al., 2011; Hosoya et al., 2015; Hopf et al., 2020). Melanized fungi typically contribute to cutaneous or systemic granulomatous lesions with associated melanized hyphae (phaeohyphomycosis) or sclerotic bodies (chromomycosis) (Bube et al., 1992; Hopf et al., 2020). The melanin within the fungal cell wall is considered a virulence factor, acting to neutralize free radicals produced by the immune system and confer other advantageous characteristics (Cordero and Casadevall, 2017). The host inflammatory response can form tumor-like masses that must be clinically and diagnostically differentiated from neoplasms.

In amphibians, V. botryosa has been isolated from several anuran species (Hosoya et al., 2015; Mayer et al., 2022). In 15 captive, cohoused White’s tree frogs, the fungus was associated with ulcerative dermatitis of the ventrum and distal extremities and progression to vasculitis and osteomyelitis in one animal (Mayer et al., 2022). Although husbandry conditions were considered appropriate, the frogs had been previously housed with freshwater fish, which were considered a potential source for the agent. Similarly, another White’s tree frog from a separate report had been previously cohoused with goldfish (Hosoya et al., 2015). No previous cohabitation with fish was documented with the tiger salamanders from this report. Disease onset and progression ranged considerably from a few days to over 8 months, similar to the chronic onset in the salamander from this report (Mayer et al., 2022).

In other aquatic vertebrates, V. botryosa causes systemic infections in Siberian and white sturgeon (Acipenseridae family) presenting with ulcerative skin lesions, coelomic hemorrhage and effusion, and multisystemic organomegaly due to granulomatous inflammation (Steckler et al., 2014; Soto et al., 2017). Under experimental challenges, lesions and mortality manifested in sturgeon held at various temperatures; however, heat-stressed groups exhibited significantly greater mortality and systemic dissemination (Coleman et al., 2018). This suggests increased pathogenicity in individuals held at upper thermal tolerances (Coleman et al., 2018). Vaccine development using inactivated molds and yeasts yielded a potential candidate that was safe, immunostimulatory, and decreased fungal burden and histopathologic lesion severity, but did not exhibit significantly increased survivability (McDonald et al., 2024). In addition to sturgeon, V. botryosa was isolated with several other melanized fungi from granulomatous masses that partially obstructed the tracheal lumen in three immature female green sea turtles (Chelonia mydas) (Donnelly et al., 2015).

Although V. botryosa is readily cultured on standard fungal media, cases may require molecular confirmation, as the gross appearance and hyphal morphology can resemble related species. Also, cultures from external tissues are often complicated by the presence of normal or opportunistic flora. In the reports of V. botryosa infection in White’s tree frogs, heavy bacterial overgrowth obscured the recovery of fungi from superficial skin samples or swabs on culture (Hosoya et al., 2015; Mayer et al., 2022). Instead, confirmatory diagnosis in the frog cases relied on histopathology and deeper cultures from lesioned tissue, followed by PCR and sequencing. Recovery of mixed bacterial flora has also been reported in V. botryosa infections in sturgeon (Steckler et al., 2014). With the tiger salamander cases, mixed gram-positive and gram-negative bacteria, but no fungal hyphae, were observed on antemortem superficial skin scrapes. Veronaea botryosa and low numbers of mixed bacteria were later recovered from tissue lesions sampled postmortem. In these cases, the lack of a predominant bacterial species and absence of bacterial disease on histopathology suggests V. botryosa was the primary cause of cutaneous disease and bacteria were relatively nonpathogenic. Nonetheless, the authors recommend performing both bacterial and fungal cultures in cases of nonspecific skin disease in herpetofauna, and if possible, combining culture results with histopathology to determine the significance of the observed pathogens with lesions.

Effective long-term treatments targeting melanized fungal infections in amphibians have not been established (Bube et al., 1992; Hosoya et al., 2015; Hopf et al., 2020; Mayer et al., 2022). Itraconazole had no effect on disease progression of V. botryosa in multiple anuran species (Hosoya et al., 2015). A marine toad (Bufo marinus) treated with ketoconazole combined with surgical excision showed brief improvement before being euthanized because of disease recurrence (Bube et al., 1992). Several White’s tree frogs received varying treatments during an outbreak of V. botryosa, including topical silver sulfadiazine, nonprescription triple antibiotic ointment and saline baths, enrofloxacin, amikacin, and itraconazole, without clinical success (Mayer et al., 2022). An eastern hellbender (Cryptobanchus alleganiensis) required multiple excisional surgeries over a 31-month period but ultimately succumbed to disseminated infection with an Exophiala sp. (Hopf et al., 2020).

This report details a case of chromomycosis caused by V. botryosa in the tiger salamander, which was associated with persistent cutaneous lesions, diagnosed by histopathology, and confirmed via fungal culture and PCR with sequencing. This represents the first description of disease caused by V. botryosa in a urodelian species. Based upon work in other aquatic vertebrates, temperature modification combined with surgical and/or medical intervention may improve treatment outcomes, though this requires experimental validation (Coleman et al., 2018; Boylan et al., 2021). Veronaea botryosa is considered a zoonotic pathogen, as it has been isolated from cutaneous and systemic infections in both immunocompromised and immunocompetent humans. As such, timely diagnosis and intervention are necessary to develop optimal treatments and protect people handling affected individuals.