Dermatomycosis Caused by Paranannizziopsis australasiensis in Nonnative Panther Chameleons (Furcifer pardalis) Captured in Central Florida, USA

Emergent fungal pathogens are of growing concern for many wild herpetofauna (Davy et al. 2021; Olson et al. 2021). The fungal family Nannizziopsiaceae within the order Onygenales contains several species that are considered significant reptile pathogens (Sigler et al. 2013; Stchigel et al. 2013; Paré et al. 2020). Fungal pathogens from this group include members of the genera Nannizziopsis and Paranannizziopsis, which have been documented as causes of skin disease (dermatomycosis) in a variety of reptile taxa worldwide (Masters et al. 2016; Paré and Sigler 2016; Webster 2018; Christman et al. 2020; Díaz-Delgado et al. 2020; Peterson et al. 2020; Davy et al. 2021). These pathogens have also been reported in captive species kept outside their native ranges (Paré et al. 1997; Bertelsen et al. 2005; Abarca et al. 2010; Díaz-Delgado et al. 2020). Nannizziopsis barbatae, first described in captive bearded dragons (Pogona barbata), has recently been identified as the cause of fatal dermatomycosis in multiple free-living lizard species across Australia, and may represent a threat to Australia's wild lizard populations (Peterson et al. 2020). Paranannizziopsis australasiensis has been identified as the cause of dermatitis in wild geckos, skinks, and tuatara in New Zealand (Webster 2018). Some outbreaks have been successfully contained in controlled environments (e.g., Díaz-Delgado et al. 2020), but there is precedent for fungal pathogen spillover from nonnative to native species, such as the introduction of Batrachochytrium dendrobatidis to naïve hosts by invasive bullfrogs (Yap et al. 2018). Thus, it is important to monitor disease outbreaks in captive and introduced herpetofauna to be aware of spillover risk to native species.

Ten adult panther chameleons (Furcifer pardalis) were captured from an established nonnative free-living population in central Florida, USA, from October 2019 to February 2020 (n=9) and October 2020 (n=1). Before accession into the study in September 2020, the chameleons were privately housed by the authors and were isolated from other animals. Chameleons were housed indoors in small groups of 2–3 individuals in screen enclosures with Epipremnum spp. and artificial plants, with an incandescent 60-W bulb to provide a basking spot, and an ultraviolet light (Repti-Sun 5.0 UVB, Zoo Med, San Luis Obispo, California, USA) on a 12-h day–night cycle until September 2020 (approximately 9–12 mo after capture). Chameleons were misted 2–3 times daily to provide water and were offered crickets (Acheta domesticus) and superworms (Zophobas morio) every other day, dusted with calcium carbonate without vitamin D3 (Rep-Cal, Rep-Cal Research Labs, Los Gatos, California, USA) at every feeding, except when replaced with a multivitamin (Reptivite, Zoo Med, San Luis Obispo, California, USA) dusting once every other week. While indoors, ambient temperatures were 24 C during the day and 21–22 C at night. In September 2020 (approximately 1 mo before placement in individual experimental enclosures; see the following discussion), chameleon enclosures were moved outdoors, halfway underneath a canopy to provide partial shade, exposed to fluctuating daily temperatures, and watered with an automated sprinkler twice daily. Of the enclosures used, two enclosures had previously housed veiled chameleons (Chameleo calyptratus) and one housed knight anoles (Anolis equestris); all enclosures were thoroughly cleaned with 10% bleach solution prior to housing any F. pardalis. The authors had not housed Australian reptiles, nor any species or families previously reported to carry P. australasiensis (e.g., Sigler et al. 2013; Masters et al. 2016; Webster 2018; Díaz-Delgado et al. 2020) before or during the time chameleons were housed. Of the species kept, no cases of dermatitis had been diagnosed nor any skin lesions observed. From 30 September 2020 to 28 July 2021, nine of the 10 chameleons were placed into an experimental enclosure constructed with stainless steel aviary mesh at the US Department of Agriculture (USDA) Wildlife Services National Wildlife Research Center in Gainesville, Florida, USA. The 10th individual (No. 491) was directly placed from the wild into the enclosure in October 2020. Within the main enclosure, animals were housed individually in screen enclosures with native Florida scrub vegetation and a potted Hibiscus plant. Animals were also exposed to natural sunlight, temperature fluctuation, and rain events. An automated misting system provided mist and dripping water four times daily. Chameleons were fed every other day as detailed above. As part of a thermal acclimation experiment to assess seasonal influence on thermal limits, performance, and preference, chameleons were exposed to natural thermal regimes, including nighttime temperatures as low as 12 C in winter; on nights forecast below 12 C heat emitters were turned on over the cages and tarpaulins used to insulate the sides and roof. On nights forecast below 7 C, chameleons were placed in individual cloth bags and housed indoors. We chose to buffer these cold temperatures because the thermal tolerance of the species was unknown prior to the study. All research was conducted under University of Florida IACUC 201910938 and USDA QA3214.

On 19 December 2020, an adult male chameleon (No. 409) presented with yellow skin crusts with gray–black margins and reddened underlying dermal tissue on the ventral aspect of the right elbow and on the ventrum just cranial to the vent, plus yellow crusts around one eye turret (Fig. 1A; Table 1). The animal was otherwise eating and behaving normally. The crust was gently removed from the eye turret with warm wet cotton applicators. The animal was isolated from the group to an indoor screen enclosure. Biopsies were taken from the lesions on the elbow and cranial to the ventrum on 23 December 2020 and submitted in 10% neutral buffered formalin for histopathology, and fresh skin tissue was submitted for DNA extraction. Microscopic findings included heterophilic and histiocytic inflammation of the dermis and epidermis with epidermal necrosis and hyperkeratosis. Admixed within the inflammation were high numbers of fungal hyphae (2–3 µm diameter) with parallel walls and rare septation (Fig. 2). At the lesion–air interface, there were dense aggregates of 2 × 3 µm cylindrical arthroconidia. Fungal cultures were not performed. We extracted DNA from the fresh skin tissue biopsy with a commercial extraction kit (DNeasy^® Blood and Tissue Kit, Qiagen, Valencia, California, USA) following manufacturer's instructions. We carried out PCR amplification of the internal transcribed spacer 2 (ITS2) domain of the rRNA gene with primers ITS4 and ITS86 as described (Turenne et al. 1999). Products were electrophoresed on a 1% agarose gel and PCR products were purified using a commercial extraction kit (QIAquick gel extraction kit, Qiagen Inc.) followed by direct sequencing using a commercial kit (Big-Dye terminator kit, Applied Biosystems, Foster City, California, USA) on an automated DNA sequencer (ABI 3130, Applied Biosystems). Primer sequences were trimmed off prior to further analysis, and the ITS2 sequence was 245 nucleotides. The sequence was submitted to GenBank under accession number OM570321. The sequence showed 100% homology with P. australasiensis sequences available in GenBank (n=6), 99.5% with available Paranannizziopsis tardicrescens (n=2), 98.64–98.65% with Paranannizziopsis californiensis (n=2), and 95.51% with Paranannizziopsis crustacea (NR111881). Given the diagnosis of P. australasiensis infection, individual 409 remained isolated indoors; daily oral antifungal therapy was commenced, including terbinafine 25 mg/kg (InvaGen Pharmaceuticals, Inc., Hauppauge, New York, USA) and voriconazole 5 mg/kg (Ajanta Pharma USA, Inc., Bridgewater, New Jersey 08807, USA; Van Waeyenberghe et al. 2010; Paré et al. 2020; Table 1).

On 11 February 2021, an adult female from the experimental enclosure (No. 498) was found moribund with poor motor control and balance, and died the next day (Table 1). This individual had yellow crusting lesions on the ventrum, as well as a subdermal mass on her right side. Histopathology confirmed the mass as a fungal granuloma. The lesion on the ventrum was confirmed as P. australasiensis by histopathology and PCR. Fungal culture was not performed.

All chameleons were examined the following week for signs of dermatitis. Of the eight remaining chameleons in the enclosure, three had missing toenails (Fig. 1B), four had swellings on the feet or toes (Fig. 1B, C), two had dermatitis with yellow crusts (Fig. 1D, E), one had a footpad lesion and mass on its side (Fig. 1F), one had dermatitis of the scales of the rostrum and crest with black or black-bordered discoloration (Fig. 1G), and one had no lesions. There did not appear to be a clear pattern of observed lesions (Table 1). Attempts to culture P. australasiensis from crusts on potato dextrose agar and inhibitory mold agar incubated for 4 wk at room temperature were unsuccessful. All chameleons began a course of terbinafine 25 mg/kg and voriconazole 5 mg/kg orally once daily (Fig. 1). The previously isolated individual (No. 409) was returned to the enclosure and continued oral antifungal therapy. About 6 wk later, all chameleons were re-examined (Table 1). Two individuals had developed dermatitis behind the nape that did not develop yellow crusts or black scales and were probably caused by abrasion from restraint during medication administration. The restraint technique was changed and the lesions healed; no new evidence of dermatitis at the nape emerged in other individuals. Five additional individuals presented with cutaneous black or black-bordered dermatitis on the crest. Foot swellings were still present in the individuals, but visibly reduced in two cases. After 11–12 wk of antifungal therapy, foot swellings were reduced to almost normal in two cases, but remained visible in another two. All individuals with former cutaneous lesions on the crest presented with scars in those areas. After 14 wk of treatment, one individual (No. 496), which had not presented with dermatitis, was found moribund and died the following day (Table 1). Necropsy revealed renal and visceral gout, gram-negative septicemia, necrotizing inflammation of the large intestine, and severe inflammation associated with bacteria in the nasal cavity.

During the period chameleons were housed at USDA, they were briefly exposed to temperatures of 45 C and 6 C as part of the thermal limit trials (N.M.C. pers. comm.). These trials were conducted 5 wk before observing signs in No. 409 (autumn trials) and 1–2 wk before observing signs in other chameleons (winter trials). Chameleon No. 409 was not reassessed during the winter trials.

Because of concerns over the possible toxicity and adverse effects of long-term oral antifungal treatment (e.g., Van Waeyenberghe et al. 2010; Alexander 2017) and their potential contribution to the death of individual No. 496, oral medications were discontinued at 15 wk for all chameleons except the two individuals with visible toe swellings (Table 1). After an additional 2 wk, medication was discontinued for the remaining two individuals following visible reduction in toe swellings. On 1 August 2021, the chameleons were removed from the USDA facility following the completion of experimental trials, into private housing.

This is the first report of P. australasiensis in a member of the Chamaeleonidae; there is only one previous report in the suborder Iguania, in a coastal bearded dragon (P. barbata; Masters et al. 2016). Of note, another fungal pathogen from the Nannizziopsiaceae, Nannizziopsis dermatitidis, has been reported in four chameleon species from the genus Chamaeleo: captive Chamaeleo parsonii, Chamaeleo lateralis, and Chamaeleo jacksonii (Paré et al. 1997) and experimentally infected C. calyptratus (Paré et al. 2006). Infection with P. australasiensis has been reported in a handful of disparately related captive terrestrial, arboreal, and semiaquatic reptiles (tuatara, Sphenodon punctatus and coastal bearded dragon, P. barbata, Masters et al. 2016; African bush vipers, Atheris squamigera, Díaz-Delgado et al. 2020; file snakes, Acrochordus unknown sp., Sigler et al. 2013), as well as wild tuatara, skink, and gecko species (Webster 2018). Little is known about the pathogenic mechanisms of P. australasiensis infection (reviewed in Díaz-Delgado et al. 2020). The cutaneous lesions and yellow crusts that we saw were similar to those previously described in tuatara and bearded dragons (Masters et al. 2016). Our observations of swollen toes and subdermal masses may be specific to presentation in chameleons, in which similar lesions have been described in association with other fungal infections (Paré et al. 2006; Sigler et al. 2010; Schmidt et al. 2012). Cultured P. australasiensis shows optimal growth between 20 and 30 C, but can also grow at 12–15 C (Alexander 2017) and survive in tuatara, which regularly experience temperatures below 10 C in winter (Cree et al. 1990). We observed fungal lesions in the chameleons in the winter months following exposure to temperatures below 10 C (even with our buffering protocols) in the experimental enclosure, but not when housed at 21–24 C before the study. The influence of thermal environment on pathogenicity of P. australasiensis in different hosts deserves further study.

The source of initial P. australasiensis infection in these two chameleons is difficult to determine. This pathogen can cause latent infections in other reptiles (Webster 2018). It is unlikely the chameleons contracted the pathogen while privately housed, as the authors had not housed Australian reptiles or any species or families previously reported to carry P. australasiensis (e.g., Sigler et al. 2013; Masters et al. 2016; Webster 2018; Díaz-Delgado et al. 2020) and no cases of dermatitis have been diagnosed in the other captive animals. As P. australasiensis can reside in soil (Webster 2018), there is a possibility that the fungal pathogen was introduced to the experimental enclosure via soil in potted Hibiscus plants, which were obtained from a nursery in Florida. This exposure route could be attributed to nonnative reptile species, which are frequently observed in nurseries in south Florida (N.M.C. pers. obs.); however, no other species reported to carry P. australasiensis are established in Florida (Krysko et al. 2011). Although Acrochordus spp. from the Melbourne Zoo in Australia have been documented with P. australasiensis (Sigler et al. 2013), and A. javanicus is established in a localized area in Florida, the snake is not reported from nurseries (Krysko et al. 2011); Beyond this possibility, it is unlikely that chameleons contracted the pathogen in the experimental enclosure at USDA National Wildlife Research Center Field Station, as P. australasiensis has not yet been reported in any native species in North America and is unlikely to reside in soil there. The enclosures had only housed native passerine birds previously (Carolina chickadee, Poecile carolinensis), and had been vacant for several years and exposed to natural ultraviolet light. It is suspected that chameleons were already infected with the fungus prior to capture. The authors collected an individual that was directly accessioned into the enclosure (No. 491) in October 2020. This individual developed dermatitis similar to the others, but exposure postaccessioning cannot be ruled out. An additional chameleon from the same population was captured 19 March 2021, and was kept isolated from all other reptiles. This chameleon developed dermatitis with black scale discoloration on its rostrum and crest consistent with those exhibited by chameleons in the enclosure, but did not develop any swellings or yellow crusts. Attempts to collect additional individuals were halted after residents became hostile towards collectors, even when demonstrating best practices (C.M.G. pers. comm.). The history of this introduced population of chameleons is not well known, but some residents indicated that chameleons had been in the area for about 11 yr prior to our collecting (C.M.G. pers. comm.).

A possible preaccession infection route is that prior to release or escape of the original founders, the chameleons were maintained in close contact with species infected with P. australasiensis. For example, the bearded dragon (Pogona vitticeps) is popular in trade in the USA, and is a sister taxa to P. barbata, which has been documented with P. australasiensis (Masters et al. 2016). It is also possible that chameleons were infected during a multispecies shipment, or from handling multiple species at a reptile exposition. Infected chameleons may then have escaped or were intentionally released; chameleons are notoriously difficult to care for in captivity, which can be a factor in choices to release animals (Stringham and Lockwood 2018).

Because of their high value combined with their poor acclimation to captivity (Stahl 1996; Carpenter et al. 2004), chameleons are increasingly released into natural areas where they may breed, for future collection (Gillette et al. 2010; Edwards et al. 2014; Episcopio-Sturgeon and Pienaar 2019). It is thus unclear whether the population arose from captive individuals that escaped or were released, or was seeded from existing established populations elsewhere (e.g., Fieldsend et al. 2021). Although this population was in a residential area, many seeded populations are placed in less conspicuous natural areas (Gillette et al. 2010; Daly 2017). This is alarming, because the site we collected these animals from became popular with chameleon hunters (C.M.G. pers. comm.), leading to the increased likelihood that chameleons potentially infected with P. australasiensis have been collected and seeded to establish new populations elsewhere in the state. As P. australasiensis may infect a wide variety of reptile taxa (squamates and tuatara; Sigler et al. 2013; Masters et al. 2016; Webster 2018; Díaz-Delgado et al. 2020), this novel fungal pathogen has the potential to threaten native reptile populations if released and spread into natural areas. Our findings highlight the need for improved pathogen screening and quarantine practices in the reptile trade, especially during import of wild-caught animals.

This work was supported in part by the Department of Wildlife Ecology and Conservation, University of Florida (C.M.R.). Salary for D. Sherman and B. Kluever was provided by the USDA. We would like to thank P. Miller, M. Sandfoss, K. Hengstebeck, D. Juárez-Sánchez, S. Nielson, S. Tillis, and D. Catizone for their assistance in the field. We thank P. Miller for assistance building chameleon enclosures. We thank D. Sherman, P. Harrell, C. Hildenbrand, K. Beloyan, and C. Lackey for assistance with chameleon husbandry. We thank J. Humphrey for retrieving weather station data. Thanks to L. Baeten and M. McBride for assistance coordinating veterinary care, and A. Childress for assistance with laboratory assays. The findings and conclusions in this publication have not been formally disseminated by the USDA and should not be construed to represent any agency determination or policy.

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