Introduction

Nannizziopsis guarroi is an onygenalean keratinophillic fungus and the causative agent of “yellow fungus disease” in bearded dragons (Pogona vitticeps).^1–4 Additional host species reportedly infected with this fungus include the green iguana (Iguana iguana) and the common agama (Agama agama) although a wider host range is suspected.^2–5 Previously reported under the nomenclature of Chrysosporium anamorph of Nannizziopsis vriessi and Chrysosporium guarroi, this fungus is a significant cause of morbidity and mortality in companion lizard species.^2–4 Clinical signs of infection range from mild scale discoloration to severe, disfiguring, and ulcerative lesions, which can ultimately lead to invasive mycosis and death.^1–8 In bearded dragons, these crusty skin lesions often, but not always, have a yellow discoloration, leading to the now outdated colloquial term “yellow fungus disease.”^1,2,7

The host-pathogen-environment triad is a well-accepted framework to understand the multifactorial reasons for disease manifestation.⁹ Despite this framework, many diagnostic and treatment considerations do not include consideration of the role of the environment on disease outcomes.⁹ Despite the description of N guarroi infecting lizards for over 2 decades,^2–4 the literature is limited to the clinical presentation of the host^5–8 and mycological and molecular description of the microbe.^3,4 Environmental drivers for disease manifestation have not currently been explored although environmental stressors including inappropriate husbandry and fungal environmental persistence have been suspected.²

The literature^2,5–8 has multiple examples of antifungal treatment failure in lizards infected with N guarroi; it is unknown if treatment failure is secondary to antifungal toxicity, lack of response to therapy, or reinfection from the environment after successful therapy. Understanding environmental drivers for disease manifestation, including how long N guarroi persists in the environment, may facilitate improved management of clinical cases and prevention of infection.

The objective of this study was to evaluate the environmental persistence of N guarroi by the detection of viable fungus from 9 clinically relevant substrates. The authors hypothesized that N guarroi would exhibit prolonged persistence on multiple substrates.

Materials and Methods

Nine commonly utilized solid and aqueous substrates were selected. Fabric aquarium liner (10-gallon green terrarium liner; Zilla), pure fir bark mulch (Repti bark; ZooMed), and sand (Calci-sand; ZooMed Reptifresh) were evaluated as these represent common products used as substrates in the husbandry of lizards. Polystyrene hard plastic (Petri dish; VWR) and smooth glass (plain microscope slides; Fisherbrand) were assessed as these products represent common materials used in enclosures that house lizards. Cotton (cheesecloth 100% unbleached cotton; Sceng) and stainless steel (No. 10 sterile surgical blade; Bard-Parker) were both assessed as potentially representing materials that could act as fomites in a veterinary clinical setting. Sterile distilled water and saline solution (0.9% NaCl; Hospira) were aqueous products assessed, the former being one that a lizard would be in contact with both husbandry and clinical settings. Contact with saline solution may occur in clinically affected lizards receiving wound lavage.

Two isolates of N guarroi, molecularly confirmed and previously described in the literature¹⁰ were used in this study. The isolates were derived from clinical cutaneous samples obtained from bearded dragons exhibiting skin lesions consistent with N guarroi infection (crusting, ulceration). Isolates were incubated after inoculation on agar for 10 days at room temperature and normal atmospheric pressure, and then swabs were collected using a sterile technique and mixed into 10 mL of distilled water in a 15-mL conical tube. Large particle debris was removed using vacuum filtration over glass wool in a sterile environment. Both fungal filtrates had their concentration standardized by measuring and diluting each suspension to 1 McFarland densitometry. Three fungal concentrations were made from this stock suspension: the highest fungal concentration measured to 1 McFarland (high concentration), 1:10 McFarland (1:10 concentration, 1 mL of high concentration with 9 mL sterile water), and 1:100 McFarland (low concentration, 1 mL of 1:10 with 9 mL sterile water).

On day 0, contamination of substrates with filtrates occurred in a sterile petri dish (sterilized solid substrates) or a conical tube (sterilized aqueous substrates). The sizes of fabric aquarium liner, cotton, and hard plastic were standardized to 4 X 1-cm pieces, and stainless steel (scalpel blades) and glass (microscope slides) were used as is. One teaspoon of loose substrate was used for sand and mulch. To these substrates, 100 μL of vortexed filtrate was mixed with the solid materials. For absorbent materials (fabric aquarium liner, cotton, sand, wood mulch), after filtrate application, spreading or mixing of the material was performed. Nonabsorbent substrates (stainless steel, hard plastic, glass) had 100 μL of filtrate spread over their superficial surface. Moisture from the filtrates was allowed to evaporate prior to closing the petri dish. Aqueous substrates (water and saline solution) had 0.5 mL of filtrate added to 4.5 mL of the aqueous substrates. Each substrate was exposed to each filtrate concentration (high, 1:10, and low) with biological replication performed for all solid substrates; no biological replication was performed for aqueous substrates. Substrates contaminated with filtrate were held under standard laboratory conditions (room temperature and normal atmospheric pressure) for the entirety of the experiment.

Each biological replication of each filtrate-substrate mixture was sampled for fungal culture on days 1, 3, and 14 to detect the presence of viable N guarroi. Using a sterile technique, the absorbent substrates were sampled by mixing the substrate with the culture swab. The culture swab was passed over the superficial surface of nonabsorbent substrates 4 to 5 times. Samples from filtrate-substrate mixtures were immediately plated onto agar (potato dextrose agar; Thermo Scientific) using a modified quadrant method¹¹ with a duplicate technical repeat. Agar plates were incubated in a standard laboratory setting (room temperature and normal atmospheric pressure) for 10 days when images were collected from each agar plate on a custom stand at a consistent distance from the agar plate. Images were assessed by 2 investigators and categorically characterized as having or lacking growth. Plates with obvious contamination based on visualization of colonies of different morphology from the classically described white fluffy mycelia noted with N guarroi³ were excluded. Samples from all plates with colony growth characteristics for N guarroi (Figure 1) were confirmed with cytologic evaluation. Slides were stained with a commercial Romanowksy stain (Diff-Quik,) following kit instructions before being examined microscopically for the presence of fungal elements (hyphae and conidia; Figure 1).³

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Isolate 2 of Nannizziopsis guarroi colony appearance after 10 days of incubation on potato dextrose agar (A) and microscopic cytologic appearance under 40X objective and commercial Romanowsky stain (B) cultured after 14 days contact with sterile distilled water.

Citation: Journal of the American Veterinary Medical Association 261, S1; 10.2460/javma.22.12.0575

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The incidence of agreement between technical and biological repeats was tabulated. A biological repeat was considered positive for growth if 1 or both technical replicates exhibited growth. An overall positive status for growth was considered if 1 or both biological replicates exhibited growth.

Results

Out of 576 plates representing 3 sampling days, 38 (6.6%) plates were excluded due to gross contamination, 26/38 of the excluded plates were from wood mulch samples. Given the concern that the wood mulch may have been incompletely sterilized with 26 of 72 (36.1%) plates having gross contamination, the data from wood mulch were not evaluated further. After the exclusion of wood mulch from further analysis (72 plates), 504 plates from 8 substrates had 12 (2.4%) plates that were excluded due to gross contamination. In all but 1 instance, the excluded plate was not replicated on a consecutive sampling day, indicating that contamination was unlikely to be present in the source filtrate substrate mixture. In all but 1 instance, excluded plates were incidental and another plate was available because of the duplicate technical repeat design. When considering technical repeats as repeats of a single experiment, there were 251 experimental conditions spanning the 8 substrates, 3 fungal concentrations, and biological repeats (solid substrates only) that could be reported. In 200/251 (79.7%) of the experimental conditions, the results of technical repeats agreed when the excluded data point was assumed to not agree. If the data point was assumed to agree, the incidence of agreement rose to 210/251 (83.7%). When technical repeat data were combined, there were 87/107 (81.3%) biological repeats when findings agreed.

Viable N guarroi, represented by the presence of fungal growth after contact with different substrates, are presented in Table 1. In general, fungal growth after contact with solid substrates was isolate, concentration, and time dependent. At least 1 concentration of at least 1 isolate of N guarroi could be grown from every solid substrate on day 1, every solid substrate except fabric aquarium liner on day 3, and from sand, hard plastic, stainless steel, and cotton on day 14. Both isolates of N guarroi, regardless of concentrations, were able to be grown from both aqueous substrates at each time point.

Discussion

Viable N guarroi persists on many solid and aqueous substrates for at least 14 days, which may represent persistent environmental sources of infection for naïve lizards or reinfection for previously treated lizards. Given that the substrates assessed in this study mirror those that a lizard may encounter while under human care or in a veterinary practice, the environmental persistence of this microbe should be considered in the treatment and prevention of infection; clinicians treating infected lizards should counsel clients about effective environmental disinfection during treatment. Further, veterinarians encountering suspect or confirmed cases of N guarroi should disinfect all potential fomites between patients. Effective disinfection against N guarroi has recently been described and is limited to sodium hypochlorite (bleach).¹²

The environmental persistence of N guarroi on solid substrates was, in general, fungal concentration, isolate, and time dependent. Exposure to higher concentrations of the fungus was associated with a longer environmental persistence while lower concentrations were associated with a shorter environmental persistence. The isolates of N guarroi evaluated in this study exhibited differences in environmental persistence; isolate 1 was more likely to be cultured from substrates and for longer periods of time. The mechanism behind this difference is unknown but may be an avenue for future research. Prior studies have used the same isolates used in this project to assess antifungal minimum inhibitory concentrations and appropriate disinfection; differences between isolates were demonstrated in each of those studies.^10,12 The differences noted suggest a continuum of N guarroi phenotype; a larger number of isolates evaluated would build upon this knowledge. Finally, the ability to culture N guarroi from contaminated solid substrates was time dependent, and although N guarroi could be isolated from most solid substrates at day 14 after contamination, this finding was often limited to a single isolate or only the highest fungal concentration.

Fabric aquarium liner was the substrate that N guarroi was least likely to be cultured from; viable fungus was only detected 1 day after substrate contamination and only after contact with the highest fungal concentrations. This commercially available product is composed of recycled polyester fibers that are infused with a proprietary odor control enzyme. It is unknown if the composition of the product or the presence of the enzyme makes the environment less conducive for the survival of N guarroi. Alternatively, the product is absorbent, and this structure may have drawn the fungal suspension deep within the fibers making them less available for sampling with our superficial sampling technique. Despite not understanding the mechanism behind the low recovery of viable N guarroi during our study, clinicians may consider recommending the use of this substrate for lizards under treatment to reduce the environmental burden of this fungus.

N guarroi was able to be cultured at each time point and each fungal concentration, regardless of the fungal isolate. The persistent ability to culture viable fungus from aqueous substrates (water and saline solution) was surprising to the authors. This fungal microbe has not been associated with aqueous environments as documented infections have been limited to terrestrial lizards.^1–8 Further, the classically infected host, the bearded dragon, is an arid grassland species that is kept in low-humidity environments while under human care.¹³ This finding warrants further investigation into the potential role of aqueous environments in the epidemiology of infection.

Several other onygenalean mycotic agents of veterinary importance have been shown to exhibit environmental persistence. Ophidiomyces ophidiicola, the causative agent of “snake fungal disease,” is a significant cause of morbidity and mortality in individuals and populations of snakes. The fungus persists in the soil, particularly in snake hibernacula, and is considered a potential reservoir for infection.¹⁴ Geomyces destructans, the cause of “white-nose syndrome” in bats, is associated with significant population declines in North American bats. Viable fungus has been detected in caves during times of the year when bats are not present, suggesting that prolonged environmental persistence plays a role in infection dynamics.¹⁵ Coccidioides immitis, Cryptococcus neoformans, and Cryptococcus gatti exhibit prolonged environmental persistence, and contact with environmental reservoirs is an important part of the epidemiology of disease in multiple taxa.^16–18 The role of environment is often poorly understood as it relates to disease manifestation, particularly in the study of veterinary mycoses. Although the environmental presence of fungi suggests an environmental source of infection, this finding must be interpreted within the larger framework of the host-pathogen-environment triad; further studies are necessary to assess transmission in the face of differing host immune function, differences in environmental parameters (such as temperatures, humidity) and differing fungal phenotypes.

Our study design employed methodology to detect viable fungus instead of molecular techniques that would detect DNA, which can potentially be from viable fungus and able to cause infection or non-viable fungus. Despite this, the findings of the study should be interpreted considering its limitations. Although the fungal isolates used were obtained from clinical cases proven to cause disease in lizards, the low number of isolates represents limited diversity of the phenotype of N guarroi, and larger studies utilizing larger numbers of isolates should be performed. Further, N guarroi is 1 of several fungal species within the genus that cause dermatomycosis in lizards and further studies should be performed to assess the environmental persistence of these microbes. Given the controlled nature of the study, confirmation of fungal identification after exposure to substrates was limited to the morphologic appearance of white to cream-colored fluffy colonies and the cytologic confirmation of the presence of hyphae and/or conidia that have been described in the literature.³ Several other fungi may have a similar colony and cytologic morphology that may have led to occult plate contamination not identified with our methodology. Finally, the fungal concentrations used in this study mirror other in vitro fungal studies¹²; however, it is unknown how the concentrations evaluated compare to real-life implications of substrate exposure to infected lizards. Further work is required to understand the minimum infective dose for naïve lizards.

In conclusion, clinicians treating lizards with confirmed or suspected dermatomycosis caused by N guarroi should be aware that this microbe may have environmental persistence after exposure to aqueous and solid substrates. This information should be paired with currently available data on effective disinfection strategies to reduce the environmental burden of the fungus and potentially reduce fomite and environmental transmission.