An Overview of Reptile Fungal Pathogens in the Genera Nannizziopsis, Paranannizziopsis, and Ophidiomyces
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An Overview of Reptile Fungal Pathogens
in the Genera Nannizziopsis, Paranannizziopsis,
and Ophidiomyces
Jean A. Paré1, DMV, DVSc, DACZM, Lynne Sigler2, MSc
1. Wildlife Conservation Society, Zoological Health Program, 2300 Southern Boulevard, Bronx, NY
10460, USA
2. University of Alberta, Microfungus Collection and Herbarium, Edmonton, AB T6G 2E1, Canada
Abstract: Two recent molecular analyses of morphologically similar fungal isolates formerly grouped
under the appellation “Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex” led to major
taxonomic revisions and revealed new insights into the biology of these reptile pathogens. All CANV-
complex isolates differed from N. vriesii and were assigned to 16 species, either within Nannizziopsis or
within the new genera Paranannizziopsis and Ophidiomyces, and 14 of these species were newly described.
From these revisions, a trend in host specificity clearly emerged that was not previously apparent.
Nannizziopsis now includes nine species associated with chamaeleonid, gekkonid, cordylid, teiid, agamid,
and iguanid lizards; crocodiles; and human hosts. Paranannizziopsis includes four species that infect
squamates and tuataras. Ophidiomyces, with the single species Ophidiomyces ophiodiicola (formerly
Chrysosporium ophiodiicola), is only associated with terrestrial or semiaquatic snakes. Nannizziopsi guarroi
(formerly Chrysosporium guarroi) is the main causal agent of yellow fungus disease in captive bearded
dragons (Pogona vitticeps), and O. ophiodiicola is the cause of mycoses in captive snakes and of snake fungal
disease, an emergent global threat to populations of endangered wild snakes. Histopathology, polymerase
chain reaction assays, and culture are crucial for confirming a diagnosis of fungal infection in reptiles;
however, because fungal identification based on morphologic and physiologic features alone is difficult,
sequencing should be sought to speciate reptile fungal isolates. Information gathered from PCR assays and
molecular speciation will help in outlining pathogenic potential and contagion risks associated with each of
these newly recognized fungal species and allow for a more comprehensive understanding of the ecology,
distribution, and host range of these pathogens.
Key Words: Chrysosporium anamorph of Nannizziopsis vriesii, Paranannizziopsis, Ophidiomyces
ophiodiicola, mycosis, reptiles.
Introduction and culture. Affected reptiles included crocodilians (Thomas
et al., 2002), lizards (Paré et al., 1997; Martel et al., 2006;
Fungi once referred to as Chrysosporium anamorph of Nan- Bowman et al., 2007; Abarca et al., 2008, 2009; Han et al.,
nizziopsis vriesii (CANV) complex or as Chrysosporium spe- 2010; Hellebuyck et al., 2010; Johnson et al., 2011; Toplon et
cies have clearly emerged as major reptile fungal pathogens. al., 2012), snakes (Nichols et al., 1999; Bertelsen et al., 2005;
Nannizziopsis vriesii, originally named Rollandina vriesii, is a Paré and Jacobson, 2007; Bicknese, 2009; Eatwell, 2010;
sexually reproducing ascomycetous fungus that was first iso- McLelland et al., 2010), and tuataras (Sphenodon punctatus)
lated from the skin and lungs of a captive teiid lizard (Ameiva (Masters et al., 2016), leaving the host range of CANV-com-
sp.) in Europe (Paré et al., 1997). This fungus, only known plex fungi ill-defined. Additionally, Chrysosporium guarroi
from the original Ameiva isolate and from a second isolate was described for CANV-complex isolates cultured from a
recovered from soil in California in 1985, produces solitary bearded dragon (Pogona vitticeps) and green iguanas (Iguana
asexual conidia (aleurioconidia) typical of the genus iguana) in Spain (Abarca et al., 2008, 2009, 2010),whereas a
Chrysosporium and this state predominates when it is grown fungal isolate causing facial granulomas in a rat snake was
in culture (Paré et al., 1997). Over the last three decades, described as Chrysosporium ophiodiicola (Rajeev et al.,
many isolates morphologically resembling N. vriesii but 2009). Infections caused by these fungi were typically severe
lacking a sexual stage (teleomorph) have been cultured from and often fatal and were seemingly contagious among ani-
reptile lesions, and in the vast majority of these cases, a mals housed together or in close proximity to each other.
causal relationship was strongly supported. Since none of Two complementary studies of CANV-complex isolates
these isolates yielded the sexual stage in culture, they became based on DNA sequence data and morphology were pub-
known as CANV-complex isolates in contrast to true N. lished in 2013 that provided strong support for the assign-
vriesii isolates. ment of 96 clinical isolates from reptile and human sources
CANV-complex isolates have been recovered from sick to 16 species within Nannizziopsis or within two new genera,
reptiles belonging to a broad variety of taxa, and a diagnosis Paranannizziopsis and Ophidiomyces, all in the order Ony-
in all these cases was confirmed by histopathology of lesions genales (Sigler et al., 2013; Stchigel et al., 2013). Results con-
46 Journal of Herpetological Medicine and Surgery Volume 26, No. 1–2, 2016
firmed that all CANV-complex isolates differed from N.
vriesii, that no reptile or human isolates belong to the same
species within Nannizziopsis, and that the genera Paranan-
nizziopsis and Ophidiomyces are associated only with reptiles
(Sigler et al., 2013). Chrysosporium guarroi and C. ophiodii-
cola were reclassified as Nannizziopsis guarroi and Ophidio-
myces ophiodiicola, respectively. Data from these and the
studies cited above confirm that these two species are the
most prevalent and important fungal pathogens of reptiles.
Newly described species that are only associated with rep-
tiles include Nannizziopsis arthrosporioides, Nannizziopsis
barbata, Nannizziopsis chlamydospora, Nannizziopsis croco-
dili, Nannizziopsis dermatitidis, Nannizziopsis draconii, Nan-
nizziopsis pluriseptata, Paranannizziopsis australasiensis,
Paranannizziopsis californiensis, Paranannizziopsis crusta-
cea, and Paranannizziopsis longispora (formerly Chrysospo-
rium longisporum), whereas species associated only with
humans include Nannizziopsis hominis, Nannizziopsis infre-
quens, and Nannizziopsis obscura (Sigler et al., 2013; Stchigel Figure 1. Extensive epidermal necrosis over the neck, dor-
et al., 2013). sum, and right forelimb of an inland bearded dragon with yel-
Morphological and physiological descriptions are avail- low fungus disease, a mycosis usually caused by Nannizziopsis
able for all new species and complement the molecular data guarroi. The crust is sloughing, exposing the dermis. Photo
(Sigler et al., 2013; Stchigel et al., 2013). Isolates all produce courtesy of Bruce Henderson, DVM, and VIN.
whitish dense colonies. Young colonies may appear moist
and demonstrate yeast-like budding cells. All isolates are HF547869; Steininger et al., 2005; Stchigel et al., 2013), they
cycloheximide-resistant and, therefore, will grow on derma- were later considered closer to N. hominis and N. obscura,
tophyte media. These fungi are also keratinolytic and have respectively, based on high internal transcribed spacer (ITS)
the ability to perforate hair. All species produce single-celled region similarity and thermotolerance (Sigler et al., 2013).
aleurioconidia borne on the sides of the hyphae or on short The status of N. infrequens as an infectious agent is not yet
stalks. Most species additionally produce arthroconidia determined. The only isolate was obtained from a bronchial
formed in adjacent chains as well as undulate hyphal washing of a HIV+ man who resided in an area endemic for
branches, and both of these features are highly characteris- Histoplasma. It was not considered to be the cause of pneu-
tic for this group of fungi (Sigler et al., 2013). Although monia in this patient but was sent for further identification
conidia vary in size and length with each species, their after it tested positive in the AccuProbe Histoplasma culture
dimensions are not sufficiently distinct to aid identification identification test (Gen-Probe, San Diego, CA) (Brandt et
based on morphology. Perhaps of significance is the fact al., 2005). Similar cross-reactivity was also shown by an iso-
that these fungi exhibit varying thermotolerance, with late of N. hominis that tested positive with the AccuProbe
human-associated species growing well at 35°C (95°F) and Blastomyces test (Sigler et al., 2013). Molecular data were
most reptile-associated species being unable to grow or used to confirm that none of the Nannizziopsis isolates from
strongly inhibited at 35°C (Sigler et al., 2013; Schtigel et al., the human source group included Nannizziopsis species
2013). Exceptions are N. chlamydospora, N. guarroi, N. pluri- comprising reptile isolates, thereby mitigating concerns
septata, and N. vriesii, all from lizards, that are only moder- about the zoonotic potential of these fungi for reptile care-
ately inhibited at 35°C. givers and handlers.
The information generated by these recent studies impacts
the medical care of individual reptiles with mycosis and the Reptile-associated Nannizziopsis species and Paranannizziop-
management of other reptiles potentially at risk within a sis: Nannizziopsis guarroi (formerly C. guarroi) is the pri-
population or collection. Here, we briefly review our current mary etiologic agent of a deep granulomatous dermatomy-
understanding of these fungal taxa. cosis in inland bearded dragons that is commonly referred to
as “yellow fungus disease” (YFD). Lesions of YFD initially
Human-associated species N. infrequens, N. hominis, and N. appear as patchy yellowing of the skin that turns dark and
obscura: Nannizziopsis hominis and N. obscura are confirmed becomes necrotic and thickened. Crusts may slough off,
agents of fungal infection in humans, having been recovered exposing the dermis (Fig. 1). Infection often progresses to
from lesions in which histopathology revealed morphologi- granulomatous inflammation of the deeper dermis. Infec-
cally compatible fungal elements (Stillwell et al., 1984; Sigler tion in bearded dragons is often fatal and may disseminate
et al., 2013). Affected patients had similar backgrounds, and involve organs such as the liver (Bowman et al., 2007;
with most cases involving individuals with underlying immu- Schmidt-Ukaj et al., 2014). The first N. guarroi isolate was
nosuppression. Two patients resided in the United States but recovered in 1999 from a pet bearded dragon in Wisconsin
had a history of association with Nigeria. Similarly, Stchigel with facial lesions typical of YFD, and an additional five
et al. (2013) examined two isolates obtained from human isolates were obtained in subsequent years, all from different
patients with disseminated disease who also had Nigerian bearded dragons infected with YFD (Bowman et al., 2007;
associations. Although these isolates were identified origi- Sigler et al., 2013). Nannizziopsis guarroi dermatomycosis
nally as N. guarroi (UTHSC R-4317; GenBank No. has been recorded in pet bearded dragons in Europe, as well
HF547876) and N. vriesii (RKI 04-0104; GenBank No. as in several green iguanas in Spain and South Korea
Volume 26, No. 1–2, 2016 Journal of Herpetological Medicine and Surgery 47
meleons (Chamaeleo calyptratus) challenged with N. derma-
titidis conidia developed skin lesions, and the fungus acted
as a primary pathogen in this lizard species (Paré et al.,
2006).
The pathogenic potential of N. pluriseptata and N.
arthrosporioides, each represented by a single isolate, remains
unclear because neither details of a pathogenic process nor
procedures for fungal isolation were described (Stchigel et
al., 2013). The former was recovered from a southeastern
five-lined skink (Eumeces inexpectatus), whereas the latter
was obtained from a captive water dragon (Physignathus
sp.). The taxonomic affinity of a CANV-complex fungus
causing an outbreak of severe dermatitis and cellulitis in a
commercial breeding colony of leopard geckos (Euplepharis
macularius) in Florida was not precisely determined. Phylo-
genetic analysis of the ITS sequence placed it intermediately
between N. dermatitidis and N. vriesii (Sigler et al., 2013;
Toplon et al., 2012), but a relationship between this leopard
gecko isolate and N. arthrosporioides is currently surmised
due to high ITS sequence similarity (577/579 base pairs) and
Figure 2. Cytology of a touch preparation from a skin lesion production of arthroconidia in terminal chains. The fungus
of an inland bearded dragon with yellow fungus disease. Rect- from leopard geckos caused a rapidly progressing and severe
angular arthroconidia, typical of Nannizziopsis spp., are readily epizootic involving 80 animals (Toplon et al., 2012).
identifiable. Diff Quick, 100×. Photo courtesy of Kenny Craw- All isolates of N. crocodili were recovered during two out-
ford, DVM, and VIN. breaks of mycosis in farmed saltwater crocodiles (Crocody-
lus porosus) in northern Australia in 1994 and 1997 (Thomas
(Abarca et al., 2008, 2009; Han et al., 2010; Van Waeyenber- et al., 2002). Affected animals exhibited skin disease charac-
ghe et al., 2010; Sigler et al., 2013; Schmidt-Ukaj et al., terized by plaque-like lesions, and over 48 hatchlings died of
2014). Cytology of scotch tape impression preparations or the infection. Environmental Fusarium solani and Purpureo-
lesion scrapings may reveal arthroconidiating hyphae and be cillium lilacinum (formerly Paecilomyces lilacinus) isolates,
useful in diagnosing YFD (Fig. 2) or similar dermatomyco- both occasionally reported as opportunistic reptile patho-
gens, were also frequently recovered; however, histopathol-
ses in other reptiles species caused by related fungi. Although
ogy revealed the presence of numerous arthroconidia, typi-
all published cases involve lizards, a single N. guarroi was
cal of infection by Nannizziopsis species. There are currently
collected from an unnamed snake under circumstances that
no other records of this fungus.
were not clarified (Stchigel et al., 2013). Nannizziopsis
The genus Paranannizziopsis accommodates four species,
chlamydospora and N. draconii are two other species recov-
three of which were recovered exclusively from captive ten-
ered from P. vitticeps with lesions typical of YFD in the tacled snakes (Erpeton tentaculatum) in North America.
United States and Europe (Stchigel et al, 2013). Based on Paranannizziopsis crustacea, P. californiensis, and P. longis-
later analysis of ITS sequences, these two species were found pora (formerly C. longisporum) cause extensive and severe
to group closer to the human-associated N. infrequens than necrotic dermatitis in tentacled snakes in zoological institu-
to N. guarroi (Sigler et al., 2013). Inland bearded dragons tions (Bertelsen et al., 2005; Nichols, 2009; Sigler et al., 2013;
appear particularly sensitive to disease caused by these three Stchigel et al., 2013). Paranannizziopsis crustacea was iso-
fungi, and YFD is an important infectious disease that lated from four sick snakes in Ontario, Canada, whereas P.
impacts the commercial bearded dragon pet trade. A severe californiensis was recovered from three snakes in California,
granulomatous skin disease similar to YFD occurred in a all of which died despite therapy (Nichols, 2009; Sigler et al.,
group of captive coastal bearded dragons (Pogona barbata) 2013). Skin specimens from sick tentacled snakes in a New
in Australia and was attributed to N. barbata (Johnson et al., York zoo also yielded P. crustacea (JAP, unpublished data)
2011), a species that has yet to be isolated from inland (Figs. 3, 4). Histopathology revealed branching, septate
bearded dragons. An isolate recovered from a skin infection hyphae deep within the lesions. All species demonstrated no
in a free-ranging eastern water dragon (Physignathus lesueu- or restricted growth at 35°C. Dermatomycosis caused by
rii) in Australia showed high ITS similarity (98.5%) with the Paranannizziopsis species has emerged as the most devastat-
N. barbata type culture (LS, unpublished data). Only ing infectious disease of captive tentacled snakes in North
through recovery and sequencing of additional isolates from America.
infected animals can we learn more about these dragon– The fourth Paranannizziopsis species, P. australasiensis,
associated species and the diseases they cause. has been recovered from a less restricted group of hosts, one
Nannizziopsis dermatitidis is another potent fungal patho- that includes lizards, snakes, and tuataras, but only in Aus-
gen of lizards with reported cases involving three species of tralia and New Zealand. This fungus was isolated from two
chameleons (Paré et al., 1997) and newly imported day geck- captive file snakes (Acrochordus sp.) housed in a zoological
oes (Phelsuma sp.) in Germany (Schildger et al., 1991). Ani- institution in Victoria (Sigler et al., 2013). Snakes displayed
mals in all cases had extensive and severe skin lesions, with disseminated, circular or punctate whitish skin lesions (Paré
dissemination to the lungs and kidney recorded in one cha- and Jacobson, 2007). Coincidentally, a condition dubbed
meleon (Schildger et al., 1991; Paré et al., 1997). Veiled cha- “white spot fungus” is often described in file snakes in the
48 Journal of Herpetological Medicine and Surgery Volume 26, No. 1–2, 2016
Figure 3. Tentacled snake. Multifocal discoloration of the Figure 4. Photomicrograph of the necrotic epidermis in the
skin with swelling and thickening of the epidermis caused by tentacled snake with Paranannizziopsis crustacea infection from
infection with Paranannizziopsis crustacea. Photo courtesy of Figure 3. Massive and dense arthroconidiation, or arthroconid-
Wildlife Conservation Society. ial tuft, at the epidermal surface. H&E, 100×. Microphoto-
graph courtesy of Wildlife Conservation Society.
pet trade and may well be caused by this or a similar fungus.
Paranannizziopsis australasiensis was confirmed by histopa- colony obtained from the wild in Guam and transferred to
thology, culture, and ITS sequencing as the cause of nodular an American facility (Nichols et al., 1999). The first isolate
to ulcerative dermatitis in four captive adult and one juve- of O. ophiodiicola was obtained in 1985 from a captive ball
nile Northern tuataras in a zoological institution in New python (Python regius) in England that presented with sub-
Zealand (Sigler et al., 2013; Masters et al., 2016). The same cutaneous granulomas (Sigler et al., 2013). Further isolates
fungus was isolated from raised yellowish lesions on a single were confirmed as O. ophiodiicola from within the United
captive coastal bearded dragon housed in the same facility. States and were from a corn snake (Pantherophis guttata)
Infection in the dragon progressed rapidly, and postmortem with subcutaneous granulomas in New York in 1986, a milk
examination revealed granulomas with hyphae in the liver. snake (Lampropeltis sp.) in Wisconsin, recently caught salt
In contrast, infection in the tuataras progressed more slowly, marsh snakes (Nerodia clarkii) in Florida, an eastern dia-
and no animal died of infection. Lesions in the tuataras mondback rattlesnake (Crotalus adamanteus) in Tennessee,
resolved following therapy with itraconazole and topical ter- and four green anacondas (Eunectes murinus) in a zoo in
binafine but often recurred over several years of monitoring California (Bicknese, 2009; Sigler et al., 2013).
(Masters et al., 2016). Additional isolates from overseas were recovered from a
Paranannizziopsis species differ in their production of garter snake (Thamnophis sp.) in Germany (originally iden-
arthroconidia in culture and in the types of conidia pro- tified as C. queenslandicum by Vissiennon et al., 1999) and
duced in lesions. Typical cylindrical arthroconidia are pro- two from Australia, including a file snake (Acrochordus sp.)
duced in culture by P. crustacea and often occur near the on display in a crocodile farm in Queensland (Sigler et al.,
surface of dermal lesions of infected animals (Bertelsen et 2013) and an adult male broad-headed snake (Hoplocepha-
al., 2005) (Fig. 4). Arthroconidia are absent in P. californien- lus bungaroides) in a breeding program at a zoological insti-
sis and P. australasiensis. In P. californiensis lesions, the pres- tution in South Australia (McLelland et al., 2010). Fungal
ence of aleurioconidia were noted, which is an unusual find- dermatitis in three captive carpet snakes (Morelia spilotes
ing thus far not reported in any other case (Sigler et al, 2013) variegata) in Queensland was attributed to Geotrichum can-
and sometimes incorrectly interpreted as arthroconidia didum but may well represent the earliest presentation of O.
(Nichols, 2009). ophiodiicola infection in Australian snakes (McKenzie et al.,
1976). Infections in all cases were confirmed by histopathol-
Ophidiomyces ophiodiicola from snakes: Ophidiomyces ophio- ogy demonstrating hyphae, usually with presence of arthro-
diicola is an important cause of morbidity and mortality in conidia that sometimes occurred in aggregates or tufts at the
captive and free-ranging snakes and is the etiological agent surface of the lesion or within granulomas, and isolation of
of a cutaneous fungal disease syndrome known as snake the fungus in culture, or by isolation of the same fungus
fungal disease (SFD). The first report of O. ophiodiicola from multiple animals (McKenzie et al., 1976; Nichols et al.,
infection is often considered to be associated with a captive 1999; Vissiennon et al., 1999; Rajeev et al., 2009; Allender et
black rat snake (Elaphe obsolete obsoleta, now Pantherophis al., 2011; Sigler et al., 2013).
alleghaniensis) with facial fungal granulomas (Rajeev et al., Over the past five years, SFD has been recognized as a
2009), however, an earlier published case of a rapidly pro- threat to populations of endangered wild snakes, mainly in
gressing fatal cutaneous dermatomycosis in four captive the north central and northeastern parts of the United
brown tree snakes (Boiga irregularis) was confirmed as O. States but clearly represents a global threat as evidenced by
ophiodiicola by re-examination of case isolates (Nichols et repeated isolation of O. ophiodiicola in sick snakes from the
al., 1999; Sigler et al., 2013). The animals were part of a overseas countries listed above. Free-ranging eastern massa-
Volume 26, No. 1–2, 2016 Journal of Herpetological Medicine and Surgery 49
Figure 5. Timber rattlesnake with multifocal labial and tem- Figure 6. Distortion of rostral facial structures from soft tis- poral scale necrosis and underlying soft tissue swelling from sue swelling and necrosis of overlying scales in a timber rattle- Ophidiomyces ophiodiicola infection. Necrotic scales occlude snake with Ophidiomyces ophiodiicola infection. Photo courtesy the loreal pit. Photo courtesy of Wildlife Conservation Society. of Wildlife Conservation Society. saugas (Sistrurus catenatus catenatus) in Illinois were ini- a free-ranging eastern fox snake (Pantherophis gloydi) with tially described with extensive, disfiguring facial lesions, the skin lesions for which the etiology was confirmed by culture result of deep granulomatous fungal dermatitis in which O. and ITS sequencing (unpublished data). Cottonmouths ophiodiicola was demonstrated using PCR (Allender et al., (Agkistrodon piscivorus) have been diagnosed with infection 2011). Around that time, timber rattlesnake population in the wild (Latney and Wellehan, 2013) and have been declines in New England were attributed to a fungal derma- shown to be susceptible to experimental infection (Allender titis analogous to that in massasaugas (Clark et al., 2011) et al., 2014). Although SFD is increasingly recognized in and later found also to be caused by O. ophiodiicola (McBride wild snakes, the isolation of O. ophiodiicola from multiple et al., 2015). Lesions of ophidiomycosis in timber rattle- captive snakes in North America, Europe, and Australia snakes were mild to moderate, often located on the head, precedes the emergence of SFD in wild North American and reminiscent of what field biologists over the years have snakes by more than 20 years and sheds a new historical called “hibernation sores” or “hibernation blisters” (Figs. perspective on this disease (Sigler et al., 2013). 5–6). Colubrid snakes sympatric with affected crotalid pop- ulations are also occasionally found with lesions of SFD, perhaps a spillover of infection in rattlesnakes. Although lesions in timber rattlesnakes were not as exuberant as those in massasaugas, the emergence of SFD seemed to coincide with precipitated declines in affected populations (Clark et al., 2011; McBride et al., 2015). Ophidiomyces ophiodiicola was isolated or was confirmed in tissue using conventional PCR or more sensitive real time PCR (qPCR) in practically all wild snakes with SFD (Allender et al., 2015; McBride et al., 2015). Other than massasaugas and timber rattlesnakes, O. ophiodiicola infection has also been confirmed in pygmy rattlesnakes (Sistrurus miliarus), eastern racers (Coluber constrictor), ring-necked snakes (Diadophis punctatus), northern water snakes (Nerodia sipedon), and milk snakes (Lampropeltis triangulum), as well as in Plains garter snakes (Thamnophis radix), northern copperheads (Agkistrodon contortrix), mud snakes (Farancia abacura), northern pine snakes (Pituophis melanoleucus), and black rat snakes in over 10 states (Murray, 2013; Sleeman, 2013; Dolinski et al., Figure 7. Ophidomyces ophiodiicola arthroconidia and hyphae 2014; Fenton et al., 2015; JAP, unpublished data). The first in the necrotic epidermis of the rattlesnake from Figure 6. case of O. ophiodiicola in Canada was diagnosed in 2015 in Gomori methenamine silver stain. 50 Journal of Herpetological Medicine and Surgery Volume 26, No. 1–2, 2016
The epidemiology of O. ophiodiicola in ecosystems remains plated onto a culture medium containing cycloheximide
poorly understood. The fungus was not cultured from the (e.g., Mycosel agar, Becton, Dickinson and Company,
skin of 38 healthy wild massasaugas (Allender et al., 2013) Franklin Lakes, NJ) and an antibiotic such as chloramphen-
but was detected by PCR assays in skin samples of nearly all icol. Cycloheximide allows for selective recovery of derma-
sympatric massasaugas and timber rattlesnakes with lesions tophytes and other onygenalean fungi. Plating a section of
of SFD (Allender et al., 2011, 2015; McBride et al., 2015), the specimen onto a second fungal culture medium contain-
suggesting it is not part of the healthy cutaneous mycobiota ing an antibiotic but lacking cycloheximide is recommended
of these wild snakes and that exposure to the fungus often in case another type of fungus is involved in the infection.
results in disease. This is supported by a survey of fungi on Cultures are incubated at 30°C (86°F) and held for up to
the skins of healthy captive squamates in which O. ophiodii- three weeks and monitored at regular intervals for growth of
cola was cultured only once out of 91 snake exuvia sampled characteristic white powdery colonies emanating from the
(Paré et al., 2003). This isolate, from a captive African rock sample. Because both bacterial and fungal contamination
python (Python sebae) in a southwestern zoological institu- can occur, regular monitoring and early subculture to fresh
tion, is the only one that has not been recovered from an culture medium will improve the chance of isolation. Iso-
actual lesion. In most snakes, O. ophiodiicola arthroconidi- lated colonies should be held for further work-up, including
ates massively at the surface of infected skin. It seems logical molecular speciation. Fungal isolates should be forwarded
to surmise that propagules on the skin of sick animals would to depositories so that they remain accessible for subsequent
result in contamination of the soil or objects that snakes studies or research.
crawl over or against. This might be particularly relevant in Increasingly sophisticated molecular techniques aiming at
hibernacula where snakes congregate annually for prolonged detection of fungal DNA in clinical specimens have become
periods of time. Furthermore, shed exuvia with arthroco- available. Real-time PCR assays have recently been shown to
nidia likely serve as focal sources of infection in the environ- reliably detect O. ophiodiicola from skin swabs and to be
ment. Ophidiomyces ophiodiicola shows restricted growth at more sensitive and specific than conventional PCR (Allen-
15°C (59°F) (Rajeev et al., 2009) but does survive freezing. der et al., 2015; Bohuski et al., 2015). Some commercial labs
Declining temperatures in dens following ingress and rising offer PCR assays to detect N. guarroi from the skin of liz-
temperatures before emergence might well allow for fungal ards with lesions suggestive of YFD. Culture or PCR assays
growth at a time where the immune system of snakes is slug- do not infer causality and histopathology is crucial in deter-
gish, creating seemingly optimal conditions for disease to mining the role of any fungal isolate in the disease process.
occur. Snakes from infected hibernacula have been seen In tissue, hyphae of O. ophiodiicola, Nannizziopsis spp., and
basking outside in winter months, possibly in an attempt to Paranannizziopsis spp. are hyaline, 3 to 6 µm wide, septate,
raise their body temperature and improve immune response. and parallel-walled, with occasional branching. Aleurioco-
Mortality occurring in dens or hibernacula is logistically dif- nidia are rarely present with hyphae, but arthroconidia are
ficult to determine but could be substantial. more often seen (Figs. 2,7). The presence of arthroconidial
tufts, consisting of massive arthroconidiation at the skin
Diagnosis and treatment of reptiles infected with Ophidiomy- surface, is practically pathognomonic for infection with one
ces ophiodiicola, Nannizziopsis species, and Paranannizziop- of these fungi (Fig. 4). Yeast-like elements may also be seen,
sis species: Dermatomycosis should be among the list of especially deeper in the dermis and within granulomas. Ide-
differential diseases for reptiles presenting with skin lesions. ally, in situ DNA hybridization should be used to confirm
A firm diagnosis of dermatomycosis, however, can only be the identity of fungi in tissues, but this diagnostic modality
established through histopathology of biopsies collected is not currently available.
from the live patient or at necropsy. Fungi are ubiquitous on Disease caused by fungi in these three genera is typically
the integument of reptiles, so culture and/or detection limited to skin lesions but sometimes will disseminate to
through molecular techniques of any isolate from a lesion internal viscera. Dermatomycosis, however, is usually exten-
needs to be supported by the presence of fungal elements in sive and progressive with substantial necrotic and prolifera-
tissue sections that are morphologically consistent with the tive lesions that often result in death. Provision of adequate
isolate. Skin biopsies are typically easily collected in anes- thermal, fluid, and nutritional support is imperative as is a
thetized reptiles. Multiple biopsy sites allow for some to be review of captive husbandry to identify and correct inade-
submitted for histopathology and some to be submitted for quacies. Because infections with these fungi appear conta-
culture or PCR modalities. Isolation of Nannizziopsis spp., gious, isolation of sick reptiles and implementation of
Paranannizziopsis spp., and O. ophiodiicola from infected tis- proper biosecurity measures are important to prevent spread
sues or other reptile samples is often complicated by the to other animals.
quality of the specimen, presence of bacterial contamina- Lesions should be surgically debulked or debrided, as
tion, use of inappropriate culture media, and length of time dead skin likely contains large amounts of infective conidia
the culture is held. Recommended specimens are skin sam- out of reach of systemically administered antifungals. Biop-
ples pulled from the lesion or biopsies, because swabs are sies of lesions are always indicated, because a diagnosis of
less effective for culturing the fungus. Samples are better mycosis is impossible without histological confirmation.
fresh but may be frozen, if needed, because culture has been Fungal culture, with subsequent speciation through sequenc-
successful from such material. Bacterial contamination of ing or PCR assays, should be sought whenever fungal ele-
samples is often heavy, and fungal retrieval may be facili- ments are present in tissue sections if we are to learn more
tated by immersing the specimen in enrofloxacin (Baytril about these reptile fungal pathogens. Topical preparations
Injectable Solution 22.7%, Bayer Health Care LLC, Shaw- such as antifungals or antiseptics may be helpful, and cover-
nee Mission, KS) for a few minutes after aseptically dividing age of secondary bacterial infections with systemic antibiot-
the specimen for culture (unpublished data). Specimens are ics is probably indicated in most cases. Systemic antifungal
Volume 26, No. 1–2, 2016 Journal of Herpetological Medicine and Surgery 51
drugs are always indicated because infection is rarely limited tum) caused by the Chrysosporium anamorph of Nannizziop-
to the epidermis, and lesions are often too extensive and sis vriesii. J Zoo Wildl Med, 36(1):82–87.
deep for topical treatment alone to succeed. Bicknese E. 2009. Itraconazole treated CANV (Chrysosporium
In vitro susceptibility of some isolates to amphotericin B, anamorph of Nannizziopsis vriesii) in green anacondas
fluconazole, terbinafine, itraconazole, and voriconazole has (Eunectes murinus murinus). Proc ARAV, 157–158.
been performed (Paré et al., 2005). No isolate showed sus- Bohuski E, Lorch JM, Griffin KM, Blehert DS. 2015. TaqMan
ceptibility to fluconazole. Seven O. ophiodiicola isolates were real-time polymerase chain reaction for detection of Ophidi-
susceptible to terbinafine, itraconazole, and voriconazole, as omyces ophiodiicola, the fungus associated with snake fungal
were all tested isolates of N. dermatitidis and N. crocodili. A disease. BMC Vet Res, 11:95.
single isolate of P. crustacea was similarly susceptible to all Bowman MR, Paré JA, Sigler L, Naeser JP, Sladky KK, Han-
three drugs, suggesting these could be used in tentacled ley CS, Helmer P, Phillips LA, Brower A, Porter R. 2007.
snakes with Paranannizziopsis infections. Isolates of N. guar- Deep fungal dermatitis in three inland bearded dragons
roi were also sensitive to terbinafine and voriconazole but a (Pogona vitticeps) caused by the Chrysosporium anamorph of
little less to itraconazole (Paré et al., 2005). Higher MICs of Nannizziopsis vriesii. Med Mycol, 45(4):291–296.
N. guarroi to itraconazole were also reported from Europe, Brandt ME, Gaunt D, Iqbal N, McClinton S, Hambleton S,
and voriconazole yielded better clinical results in bearded Sigler L. 2005. False-positive Histoplasma capsulatum Gen-
dragons with YFD (Van Waeyenberghe et al., 2010). Data Probe chemiluminescent test result caused by a Chrysospo-
are lacking for the remaining reptile-associated Nannizziop- rium species. J Clin Microbiol, 43(3):1456–1458.
sis species Clark RW, Marchand MN, Clifford BJ, Stechert R, Stephens S.
2011. Decline of an isolated timber rattlesnake (Crotalus hor-
Conclusions ridus) population: interactions between climate change, dis-
ease, and loss of genetic diversity. Biol Conserv, 144(2):886–
Reptile fungi once referred to as CANV-complex isolates 891.
were shown through two molecular studies to differ from the Dolinski AC, Allender MC, Hsiao V, Maddox CW. 2014. Sys-
true N. vriesii and, therefore, were reassigned to novel taxa. temic Ophidiomyces ophiodiicola infection in a free-ranging
From this reclassification, morphological and physiological plains garter snake (Thamnophis radix). J Herp Med Surg,
properties, as well as host trends and sensitivity patterns, 24(1–2):7–10.
have emerged for each novel taxon that were not evident Eatwell K. 2010. Suspected fatal Chrysosporium anamorph of
before. Because identification of these common reptile Nannizziopsis vriesii (CANV) dermatitis in an albino boa
pathogens is largely based on molecular data, sequencing of constrictor (Constrictor constrictor). J Small Anim Pract,
reptile isolates should be sought by reptile clinicians when- 51(5):290.
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