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Dermatitis and Cellulitis in Leopard Geckos (Eublepharis macularius) Caused by the Chrysosporium Anamorph of Nannizziopsis vriesii D. E. Toplon, S. P. Terrell, L. Sigler and E. R. Jacobson Vet Pathol published online 16 November 2012 DOI: 10.1177/0300985812465324 The online version of this article can be found at: http://vet.sagepub.com/content/early/2012/11/16/0300985812465324

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Veterinary Pathology OnlineFirst, published on November 16, 2012 as doi:10.1177/0300985812465324

Dermatitis and Cellulitis in Leopard Geckos (Eublepharis macularius) Caused by the Chrysosporium Anamorph of Nannizziopsis vriesii

Veterinary Pathology 00(0) 1-5 ª The Author(s) 2012 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985812465324 http://vet.sagepub.com

D. E. Toplon1, S. P. Terrell1, L. Sigler2, and E. R. Jacobson1

Abstract An epizootic of ulcerative to nodular ventral dermatitis was observed in a large breeding colony of 8-month to 5-year-old leopard geckos (Eublepharis macularius) of both sexes. Two representative mature male geckos were euthanized for diagnostic necropsy. The Chrysosporium anamorph of Nannizziopsis vriesii (CANV) was isolated from the skin lesions, and identification was confirmed by sequencing of the internal transcribed spacer region of the rRNA gene. Histopathology revealed multifocal to coalescing dermal and subcutaneous heterophilic granulomas that contained septate fungal hyphae. There was also multifocal epidermal hyperplasia with hyperkeratosis, and similar hyphae were present within the stratum corneum, occasionally with terminal chains of arthroconidia consistent with the CANV. In one case, there was focal extension of granulomatous inflammation into the underlying masseter muscle. This is the first report of dermatitis and cellulitis due to the CANV in leopard geckos. Keywords Chrysosporium anamorph of Nannizziopsis vriesii, dermatitis, dermatomycosis, Eublepharis macularius, fungus, internal transcribed spacer sequencing, leopard gecko, skin

An epizootic of ulcerative dermatitis occurred in an established commercial breeding colony of approximately 2100 leopard geckos (Eublepharis macularius), with cases in both males and females, ranging in age from 8 months to 5 years old. The animals were maintained in groups of 3 to 5 females and 1 male in 26-liter plastic containers and were provided with newspaper substrate and nest boxes containing peat moss and vermiculite. They were fed mealworms and provided with a calcium block. Color morphs derived through selective breeding were initially affected, and the number of cases increased over several weeks to include approximately 80 animals, including wild-color-type geckos. Due to the number of geckos affected and the rapid spread through the colony, infectious etiology was suspected. A pooled sample of lesional skin from 3 geckos submitted for fungal culture by the referring veterinarian to the Florida Department of Agriculture and Consumer Services Kissimmee Diagnostic Laboratory resulted in isolation of Lichtheimia corymbifera (formerly known as Absidia corymbifera). Two representative mature male geckos with multifocal skin lesions were submitted to the University of Florida Veterinary Medical Hospitals for additional testing. The geckos were humanely euthanized with an intracoelomic injection of a concentrated barbiturate and monitored until they no longer responded to a painful stimulus.

Pathological Findings At necropsy, case No. 1 was in good body condition, weighed 26 g, and had a 9.7-cm snout–vent length. Case No. 2 was obese, weighed 46 g, and had a 10.1-cm snout–vent length. Gross findings were similar for both geckos. There were 10 to 15 punctate to 4-mm-diameter, slightly raised, frequently dark red and ulcerated nodules scattered across the ventral skin from the ventral mandibular region to the tail (Figs. 1, 2). Case No. 1 had retained shed skin over the tail and right hind distal limb, and case No. 2 had retained shed overlying the entire trunk and all 4 limbs. Representative samples of skin and major organs were collected in 10% neutral buffered formalin, processed routinely for histopathology, sectioned at 6 mm, and stained with hematoxylin and eosin. Selected skin specimens were also 1 University of Florida, College of Veterinary Medicine, Veterinary Medical Hospitals, Gainesville, Florida 2 University of Alberta Microfungus Collection and Herbarium, Devonian Botanic Garden, Edmonton, Alberta, Canada

Corresponding Author: Scott Terrell, Disney’s Animal Kingdom, Department of Animal Health, PO Box 10000, Lake Buena Vista, Florida, 32830 Email: [email protected]


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Figure 1. Leopard gecko, case No. 1. Note the skin ulcer overlying the ramus of the mandible. Figure 2. Leopard gecko, case No. 2. Note several raised ulcerated nodules overlying the ventral mandible bilaterally. Figure 3. Scaled skin; leopard gecko, case No. 1. Note the subcutaneous heterophilic granuloma (G), moderate epidermal hyperplasia (E), subcorneal pustule (P), and hyperkeratosis (H). HE. Figure 4. Scaled skin; leopard gecko, case No. 1. Detail depicting the hyperkeratotic stratum corneum. Note several parallel-walled septate branching fungal hyphae within the stratum corneum, occasional arthroconidia (arrow), and occasional erumpent hyphae (arrowhead). Periodic acid–Schiff.

stained with Gomori methenamine silver, periodic acid–Schiff, Brown and Brenn Gram, Giemsa, and Warthin-Starry stains. Histologic findings were similar in both cases. Within the subcutis and occasionally the dermis, there were multifocal to coalescing, up to 4-mm-diameter heterophilic granulomas (Fig. 3). The granulomas contained dense aggregates of degenerate and necrotic heterophils surrounding numerous 3- to 5-mm-wide, parallel-walled fungal hyphae. The hyphae were frequently septate, with infrequent acute-angle nondichotomous branching, and stained well with periodic acid–Schiff (Fig. 4) but lightly with Gomori methenamine silver and very lightly with hematoxylin and eosin. There was occasional moderate to marked parakeratotic hyperkeratosis, spongiosis, and hyperplasia of the overlying epidermis. Within

the hyperkeratotic stratum corneum, there were moderate densities of similar hyphae, along with scattered heterophils and occasional small intracorneal pustules. Near the surface of the stratum corneum, occasional hyphae fragmented to form chains of 2- to 3-mm-diameter arthroconidia (Fig. 4). Occasional erumpent hyphae penetrated the surface of the stratum corneum. There was multifocal mild mononuclear perivascular cuffing within the dermis and subcutis near the granulomas. In case No. 1, granulomatous inflammation extended through the right masseter muscle to the lateral surface of the ramus of the mandible, with a moderate chronic periosteal reaction. The remaining special stains were negative for secondary bacterial infection. Both geckos were mildly infected with strongylid nematodes in the lumen of the large


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Figure 5. Colony of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) on potato dextrose agar (PDA) after 21 days at 30 C. Figure 6. Microscopic appearance of CANV on PDA showing pyriform aleurioconidia and terminal arthroconidia (arrows). Figure 7. Colony streaked on PDA showing mycelial and yeastlike colonies (arrows) after 6 days at 30 C. Figure 8. Yeastlike cells (arrows), germinating cell (ellipse), and cylindrical arthroconidia (arrowhead) of CANV on PDA.

intestine, without associated pathology. There were no other significant lesions affecting internal organs.

Culture and Molecular Diagnostics Samples of skin and sterile oral swabs collected aseptically from both geckos postmortem were inoculated onto inhibitory mold agar plates (Hardy Diagnostics, Santa Maria, California) and incubated at 25 C to 28 C for 8 days. Cultures of oral swabs yielded no growth. Cultures of skin lesions from each gecko yielded few colonies of a presumptive Chrysosporium sp. Subcultures of each isolate were submitted for further identification to the University of Alberta Microfungus Collection and Herbarium (UAMH), Edmonton, Canada, where they were accessioned as UAMH 11231 and UAMH 11232. The isolates were identified as the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) by morphology and confirmed by sequencing. Colonies on potato dextrose agar

(PDA; Difco, Becton Dickinson and Company, Sparks, Maryland) were yellowish-white, powdery, and slightly zonate and reached 5 cm in diameter after 21 days at 30 C (Fig. 5). Growth was strongly inhibited on PDA at 35 C. Microscopic examination revealed pyriform to ovate aleurioconidia borne on the sides of the hyphae or on stalks and occasional arthroconidia (Fig. 6). Subcultures streaked onto PDA demonstrated a mixture of mycelial and moist, yeastlike colonies in 3 to 6 days at 30 C (Fig. 7). The yeastlike colonies consisted of ovoid to subcylindrical yeastlike cells, some of which germinated to form filaments, and numerous cylindrical arthroconidia (Fig. 8). The yeastlike growth was transitory, and colonies became mycelial upon further incubation. Sequencing of the internal transcribed spacer (ITS) region of the rRNA gene was done using primers BMBC-R, ITS1, ITS2, and ITS4 and followed methods described previously.9 The ITS sequence from strain UAMH 11231 was 99% similar to sequences derived from the ex-type culture of N. vriesii


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(in-house sequence from strain UAMH 3527 and GenBank No. AJ131687). The sequence was deposited in GenBank under accession no. JX457149.

Discussion The CANV was confirmed as the etiologic agent of multifocal heterophilic granulomatous dermatitis, cellulitis, and focal masseter myositis in the leopard geckos by the presence of intralesional hyaline septate branching hyphae with typical arthroconidia, by the isolation of the fungus from skin samples of both geckos cultured, and also by sequencing of the ITS region. These findings ruled out a potential etiological role for the zygomycete L. corymbifera, which has broad, nonseptate hyphae. As this fungus was cultured initially from a single pooled skin swab, it was likely a superficial contaminant or a colonizer of damaged skin. This is the first report of infection with the CANV in leopard geckos. The CANV has been associated with dermatitis in multiple terrestrial, semiaquatic, and aquatic reptile species in North America, Europe, and Australia, and infections are frequently highly contagious and often fatal.2–7,10 The CANV produces both aleurioconidia and arthroconidia in culture.5,6 The infectious propagules are presumably the arthroconidia, which, as in this case, have been observed at the skin surface in experimentally infected veiled chameleons (Chamaeleo calyptratus)5 and in naturally infected reptiles.2,10 In culture, arthroconidia may occur together with yeastlike cells in early stages of growth, as described here in the gecko isolates. In CANV dermatomycosis, lesions initially consist of hyperkeratotic plaques, with colonization of the superficial epidermis and mild heterophilic epidermitis, and progress to epidermal necrosis and ulceration. The hyphae then penetrate the dermis and occasionally deeper tissues, causing granulomatous dermatitis and cellulitis.5,6 Cases of dermatomycosis with the CANV have been reported mainly in squamates, including bearded dragons (Pogona vitticeps, P. barbata),3,4 3 chameleon species (Chamaeleo spp),6,7 tentacled snakes (Erpeton tentaculatum),2 brown tree snakes (Boiga irregularis),6 and others.6 Dermatitis associated with the CANV has also been reported in saltwater crocodiles (Crocodylus porosus).10 Ongoing molecular work suggests that the CANV represents a species complex, with possible host specificity of individual isolates (Sigler, unpublished data). Two related species have been described recently, including Chrysosporium guarroi, associated with dermatomycosis in pet green iguanas (Iguana iguana) and bearded dragons in Spain1 and C. ophiodiicola causing mycotic granuloma in a black rat snake (Elaphe obsoleta obsoleta).8 Outbreaks of the CANV have mainly affected young, recently wild-caught, or crowded animals, leading to the suggestion that stress and suboptimal temperature predispose to infection.2,10 Crowding may have played a role in the present outbreak, as the breeder observed that the larger groups of geckos appeared to be more frequently affected. The breeder’s observation that color

variants were initially affected suggests that immune incompetence may be an important predisposing factor, as these were likely the most inbred animals and thus likely had reduced diversity of specific immune cell receptor phenotypes. Attempts to gain more information from the breeder about further evolution of the outbreak were unsuccessful. The CANV can be difficult to isolate from skin lesions of affected animals due to the presence of contaminant fungi, as occurred here with the original isolation of L. corymbifera from pooled skin samples, or due to heavy bacterial contamination, which strongly inhibits growth of the fungus. Therefore, use of selective fungal culture media, including the antifungal agent cycloheximide and antibacterial antibiotics, is recommended for optimum recovery. The fungus may also be difficult to identify, because the aleurioconidia are similar to those of other Chrysosporium or Trichophyton species, and the arthroconidia resemble those of Geotrichum or Trichosporon species.6 However, isolation of a white, powdery fungus exhibiting both aleurioconidia and cylindrical arthroconidia is strongly indicative of CANV, especially if typical hyphae and arthroconidia are observed on histology. Similarly, these findings will rule out fungi such as Fusarium spp, Aspergillus spp, or Paecilomyces lilacinus that may produce similar hyphae in lesions, as well as other fungi that exhibit distinct histologic morphology such as the zygomycete L. corymbifera. In conclusion, the CANV is a common contagious cause of ulcerative heterophilic granulomatous dermatitis in squamates, including leopard geckos, and should be suspected with any ulcerative or nodular skin lesion. Diagnosis should be based on histopathology and microbiology, including fungal culture by a laboratory familiar with reptile mycoses. Acknowledgments We wish to thank Randall Eisel, DVM, for providing fungal culture results from previous case submissions and Crystal Schuman for initial culture of the current cases.

Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding The author(s) disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported in part by the Natural Sciences and Engineering Research Council of Canada (grant Nos. G121160004, G121210359 to L Sigler).

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3. Hedley J, Eatwell K, Hume L. Necrotising fungal dermatitis in a group of bearded dragons (Pogona vitticeps). Vet Rec. 2010;166: 464–465. 4. Johnson RSP, Sangster CR, Sigler L, et al. Deep fungal dermatitis caused by the Chrysosporium anamorph of Nannizziopsis vriesii in captive coastal bearded dragons (Pogona barbata). Austr Vet J. 2011;89:515–519. 5. Pare´ JA, Coyle KA, Sigler L, et al. Pathogenicity of the Chrysosporium anamorph of Nannizziopsis vriesii for veiled chameleons (Chamaeleo calyptratus). Med Mycol. 2006;44:25–31. 6. Pare´ JA, Jacobson ER. Mycotic diseases of reptiles. In: Jacobson ER, ed. Infectious Diseases and Pathology of Reptiles. New York, NY: Taylor & Francis Group; 2007:527–570. 7. Pare´ JA, Sigler L, Hunter DB, et al. Cutaneous mycoses in chameleons caused by the Chrysosporium anamorph of

Nannizziopsis vriesii (Apinis) Currah. J Zoo Wildl Med. 1997; 28:443–453. 8. Rajeev S, Sutton DA, Wickes BL, et al. Isolation and characterization of a new fungal species, Chrysosporium ophiodiicola, from a mycotic granuloma of a black rat snake (Elaphe obsoleta obsoleta). J Clin Microbiol. 2009;47: 1265–1268. 9. Sigler L, Gibas CF, Kokotovicab B, et al. Disseminated mycoses in veiled chameleons (Chamaeleo calyptratus) caused by Chamaeleomyces granulomatis, a new fungus related to Paecilomyces viridis. J Clin Microbiol. 2010;48:3182–3192. 10. Thomas AD, Sigler L, Peucker S, et al. Chrysosporium anamorph of Nannizziopsis vriesii associated with fatal cutaneous mycoses in the salt-water crocodile (Crocodylus porosus). Med Mycol. 2002;40:143–151.
