A Novel Exophiala Species Associated With Disseminated Granulomatous Inflammation in a Captive Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis)

Abstract

The genus Exophiala is composed of ubiquitous, pigmented, saprotrophic fungi and includes both terrestrial and waterborne species. Though Exophiala species are generally considered opportunistic pathogens, exophialosis can be an important cause of morbidity and mortality in aquatic and semi-aquatic species. Over a 6-year period, a captive 32-year-old male eastern hellbender (Cryptobranchus alleganiensis alleganiensis), was treated for recurring, slow growing, ventral midline cutaneous masses. Excisional biopsies were characterized histologically by granulomatous dermatitis with low numbers of intralesional, pigmented fungal conidia and hyphae. Bacterial and fungal cultures of the masses and skin were negative on two separate submissions. Polymerase chain reaction amplification of a short fragment of the fungal 28S large subunit (LSU) ribosomal RNA was positive with 100% nucleotide sequence identity to several species of Exophiala. Following recurrence after successive rounds of antifungal therapy, euthanasia was elected. At necropsy, similar dermal granulomatous inflammation and intralesional pigmented fungal elements as observed in excisional biopsies formed a thick band in the dermis and extended through the coelomic body wall. Visceral dissemination was noted in the lung and kidney. Postmortem DNA sequence analysis of a large portion of the fungal LSU as well as the internal transcribed spacer (ITS) from a portion of frozen affected dermis identified the fungus as a novel species, Exophiala sp. 1 (UTHSCSA R-5437).

Full text

CASE REPORT
published: 31 January 2020
doi: 10.3389/fvets.2020.00025
Frontiers in Veterinary Science | www.frontiersin.org 1January 2020 | Volume 7 | Article 25
Edited by:
Chris Walzer,
University of Veterinary Medicine
Vienna, Austria
Reviewed by:
Randy Junge,
Columbus Zoo and Aquarium,
United States
Kathryn Christine Gamble,
Lincoln Park Zoo, United States
*Correspondence:
Cynthia Hopf
crh245@cornell.edu
Specialty section:
This article was submitted to
Zoological Medicine,
a section of the journal
Frontiers in Veterinary Science
Received: 31 May 2019
Accepted: 13 January 2020
Published: 31 January 2020
Citation:
Hopf C, Graham EA, Gibas CFC,
Sanders C, Mele J, Fan H,
Garner MM, Wiederhold NP,
Ossiboff R and Abou-Madi N (2020) A
Novel Exophiala Species Associated
With Disseminated Granulomatous
Inflammation in a Captive Eastern
Hellbender (Cryptobranchus
alleganiensis alleganiensis).
Front. Vet. Sci. 7:25.
doi: 10.3389/fvets.2020.00025
A Novel Exophiala Species
Associated With Disseminated
Granulomatous Inflammation in a
Captive Eastern Hellbender
(Cryptobranchus alleganiensis
alleganiensis)
Cynthia Hopf 1
*, Erin A. Graham 2, Connie F. C. Gibas 3, Carmita Sanders 3, James Mele 3,
Hongxin Fan 2, Michael M. Garner 4, Nathan P. Wiederhold3, Robert Ossiboff 2,5 and
Noha Abou-Madi 1
1Department of Clinical Sciences, Cornell University College of Veterinary Medicine, Ithaca, NY, United States, 2Department
of Comparative Diagnostic and Population Medicine, University of Florida College of Veterinary Medicine, Gainesville, FL,
United States, 3Fungus Testing Laboratory & Molecular Diagnostics Laboratory, Department of Pathology and Laboratory
Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, United States, 4Northwest ZooPath,
Monroe, WA, United States, 5Department of Population Medicine and Diagnostic Sciences, Cornell University College of
Veterinary Medicine, Ithaca, NY, United States
The genus Exophiala is composed of ubiquitous, pigmented, saprotrophic fungi
and includes both terrestrial and waterborne species. Though Exophiala species
are generally considered opportunistic pathogens, exophialosis can be an important
cause of morbidity and mortality in aquatic and semi-aquatic species. Over a 6-year
period, a captive 32-year-old male eastern hellbender (Cryptobranchus alleganiensis
alleganiensis), was treated for recurring, slow growing, ventral midline cutaneous masses.
Excisional biopsies were characterized histologically by granulomatous dermatitis with
low numbers of intralesional, pigmented fungal conidia and hyphae. Bacterial and fungal
cultures of the masses and skin were negative on two separate submissions. Polymerase
chain reaction amplification of a short fragment of the fungal 28S large subunit (LSU)
ribosomal RNA was positive with 100% nucleotide sequence identity to several species of
Exophiala. Following recurrence after successive rounds of antifungal therapy, euthanasia
was elected. At necropsy, similar dermal granulomatous inflammation and intralesional
pigmented fungal elements as observed in excisional biopsies formed a thick band in the
dermis and extended through the coelomic body wall. Visceral dissemination was noted
in the lung and kidney. Postmortem DNA sequence analysis of a large portion of the
fungal LSU as well as the internal transcribed spacer (ITS) from a portion of frozen affected
dermis identified the fungus as a novel species, Exophiala sp. 1 (UTHSCSA R-5437).
Keywords: amphibian, chromoblastomycosis, chromomycosis, cryptobranchid, exophialosis, fungus,
phaeohyphomycosis, salamander

Hopf et al. Exophialosis in an Eastern Hellbender
INTRODUCTION
Native to the United States, hellbenders (Cryptobranchus
alleganiensis) are large aquatic salamanders represented by two
subspecies: the eastern hellbender (C.a.alleganiensis) and the
Ozark hellbender (C.a.bishopi). Cryptobranchus a. alleganiensis
ranges from New York to Georgia, through Tennessee and the
Ohio River Valley to the Ozarks and inhabit clear, swift-flowing
streams with rocky bottoms (1). Wild populations have been
declining due to historical overharvesting for the pet trade,
and are now threatened by habitat deterioration and infectious
disease outbreaks (Batrachochytrium dendrobatidis [Bd]) (1–4).
The eastern hellbender was designated as a species of special
concern in New York State in 1983 and is listed as near-
threatened by the IUCN Red List. Conservation efforts have
focused on head-start and reintroduction programs, habitat
restoration, and disease surveillance (1,5). Some reported health
problems in the eastern hellbender include traumatic injuries,
neoplasia (epidermal papilloma, squamous cell carcinoma,
Sertoli cell tumor, and poorly differentiated sarcoma), and
infections with Saprolegnia or Bd (1).
Phaeohyphomycosis is a disease caused by a heterogenous
group of septate dark-walled phaeoid fungi that can cause
both subcutaneous and systemic infections. Over 100 species of
melanized fungi are in this category, including Exophiala and
Cladophialophora, that both belong to the order Chaetothyriales
(6). Exophiala species can be opportunists or pathogens of
immunocompetent humans and have also caused disease in fish,
amphibians, and invertebrates (7). Study of the taxonomy and
clinical disease characteristics of Exophiala species is ongoing
with updates in their classification. This report characterizes a
unique morphologic presentation of fatal phaeohyphomycosis
in an eastern hellbender associated with an undescribed species
of Exophiala.
CASE PRESENTATION
A 32-year-old male eastern hellbender (C.a.alleganiensis), wild
caught as a juvenile, was examined following the discovery of a
chain of small, nodular masses noted along the ventral midline.
The masses were slow-growing and initially involved only the
skin. Two excisional surgeries were performed 31 months apart
due to the recurrence of the condition.
For surgical procedures the hellbender was anesthetized
in a bath solution of 0.1% MS222 (Ethyl 3-aminobenzoate
methanesulfonate, Millipore Sigma, Darmstadt, Germany)
buffered with sodium bicarbonate. Induction was achieved
after 15 min and anesthesia was maintained by covering the
body of the hellbender with gauzes soaked with a buffered
0.05% solution of MS222. Heart rate was monitored using
a Doppler ultrasonic flow detector and depth of anesthesia
was gauged by the lack of righting reflex and response to
surgical stimulation. No increase in the concentration of
MS222 was required throughout the procedure. For both
anesthetic procedures, the hellbender recovered completely
after 2 fresh water bath changes and 20 min after the anesthesia
was discontinued.
During the first surgery, the ventral midline masses involved
only the skin. The larger masses were excised for diagnostic
purposes. Post-operative treatment included enrofloxacin
(Baytril 22.7 mg/ml, Bayer, Shawnee Mission, Kansas 66201
USA) 5 mg/kg given intra-coelomically. Butorphanol (0.5 mg/L)
bath solution (Dolorex 10 mg/ml, Merck, Madison, New Jersey,
07940 USA) was used for analgesia for the first 24 h. Eleven days
post-operatively, the skin wound dehisced over 2–3 mm but the
coelomic wall closure remained intact and the skin healed by
second intention without further complication. Microscopically
the lesions consisted of dermal granulomatous inflammation.
A Fite’s acid fast stain revealed no acid fast positive bacteria
within the lesions. A Gomori methenamine silver (GMS) stain
identified scattered and infrequent spherical yeast-like structures
that were difficult to find in foci of inflammation. Culture and
PCR were not attempted at this time and treatment was declined
as euthanasia was considered. The hellbender healed and no
recrudescence of the masses was noted for several years.
Three and a half years after the initial surgery, new masses
were discovered, and a second surgery was performed. The
masses had invaded into the coelomic wall. They were excised
using radiosurgery and blunt dissection in the anesthetized
animal. The chain of masses removed measured 9 ×4.5 ×2 cm.
Sections of the mass were submitted for aerobic bacterial culture,
fungal culture, and histologic analysis.
Cultures for bacteria and fungi were negative. Histologic
findings were similar to the previous biopsy and were
characterized by granulomatous dermatitis with epidermal
ulceration, edema, and hyperplasia (Figures 1A,B). Low
numbers of pigmented, round to oval-shaped conidia (6.5
to 8.5 µm diameter) were present in small clusters within
granulomatous foci (Figure 1B). Rare non-branching hyphae
with thin, non-parallel, pigmented walls ranging from 5 to 10 µm
in diameter and sporadic septation were also noted. A GMS stain
revealed that the conidia and hyphae were intensely argyrophilic
(Figure 1C) and demonstrated positive cytochemical reactivity
with a Fontana-Masson stain for melanin, consistent with
a pigmented fungus (phaeohyphomycosis) (Figure 1D). No
acid-fast bacteria were highlighted with Ziehl-Neelsen or
Fite-Faraco staining.
Nucleic acids were extracted from scrolls of paraffin-
embedded skin using a commercially available kit (DNeasy Blood
and Tissue Kit, QIAGEN, Germantown, Maryland, USA), and
fungal DNA was amplified by polymerase chain reaction (PCR)
using universal primers that target a variable region within the
large 28S subunit (8). An amplicon of approximately 260 bp
was sequenced bidirectionally (Genomics Facility, Biotechnology
Resource Center, Cornell University), assembled and edited of
primer sequence (Geneious R10; Auckland, New Zealand) for
a final product size of 224 bp, and compared for homology
with sequences in GenBank (https://www.ncbi.nlm.nih.gov/
genbank/). The resultant product exhibited 100% nucleotide
identity to a number of Exophiala species, including Exophiala
opportunistica (KP347962.1), E.bonariae (KR781083.1), and
E.cancerae (KF928502.1).
Post-operative treatment consisted of 5 mg/kg of enrofloxacin
injected subcutaneously (SC) in a pocket of electrolyte solution
Frontiers in Veterinary Science | www.frontiersin.org 2January 2020 | Volume 7 | Article 25

Hopf et al. Exophialosis in an Eastern Hellbender
FIGURE 1 | Granulomatous mycotic dermatitis in an eastern hellbender
(Cryptobranchus alleganiensis alleganiensis). (A) The dermis is expanded by a
band of nodular to diffuse granulomatous inflammation with focal epidermal
attenuation and extension through underlying striated muscle fibers.
Hematoxylin and eosin (HE). (B) Small, sporadic, clusters of extracellular
pigmented conidia are surrounded by numerous epithelioid macrophages and
multinucleated giant cells amid scattered pyknotic and karyorrhectic cellular
debris (HE). (C) Fungal elements are argyrophilic; conidia are accompanied by
rare hyphae with thin, non-parallel walls and sporadic septation. Gomori
methenamine silver (GMS). (D) Conidia and hyphae exhibit positive
cytochemical reactivity within the fungal wall, consistent with a pigmented
(melanin) fungus. Fontana-Masson.
(Normosol-R, Hospira Inc, Lake Forest, IL 60045 USA), and
meloxicam (Metacam 5 mg/ml, Boehringer Ingelheim, St. Joseph,
MO 64506 USA) 0.2 mg/kg also given SC every other day
for 8 and 6 days respectively. In light of the histology
and PCR results, itraconazole (Sporanox, 10 mg/ml, Janssen
Pharmaceutica N.V. Beerse, Belgium) was added at a dose of
5 mg/kg once daily hidden in the prey food item. A total
of only 18 treatments of itraconazole over the course of 30
days were successfully administered due to hypophagia and
poor compliance. Itraconazole was discontinued after the 18th
successful administration. The surgical wound healed without
complication. The hellbender’s appetite improved. Two years
after the second surgery, the masses recurred at the same location
with ulceration of the skin. Due to the recurrence of the lesions
and the extent of the masses, euthanasia was elected.
POST-MORTEM DIAGNOSTICS
Gross necropsy revealed a chain of multinodular, coalescing,
and ulcerated dermal masses of the ventral coelomic body
wall (Figure 2A). The masses had a gross appearance similar
to those of previous surgical excisions, and were concentrated
in the region of the two previous surgical incisions. The
masses extended through the body wall and were multifocally
visible through the coelomic mesothelium. Multiple, white-tan
FIGURE 2 | Multicentric granulomatous mycotic inflammation in an eastern
hellbender (Cryptobranchus alleganiensis alleganiensis). (A) The ventral dermis
is expanded by multinodular coalescing granulomas with ulceration of the
overlying epidermis. (B) Similar nodular granulomas are present within
the lungs.
nodular masses were also present in the lungs (Figure 2B)
and the kidneys. No gross abnormalities were noted in other
visceral organs. Several tissues were sampled and fixed in 10%
neutral buffered formalin for microscopic examination and
frozen for further testing, including the body wall sections of
granulomatous lesion, sections of skin and mass, left distal
thoracic limb, eye, brain, heart, urinary bladder, lungs, liver,
spleen, kidneys, pancreas, intestines, segments of small and large
intestine, and testicular tissue.
Histologic examination confirmed the presence of generalized
granulomatous dermatitis associated with pigmented
fungal elements as noted in historical excisional biopsies
(Figures 1A–D). Granulomatous inflammation extended into
the muscle of the coelomic body wall, but did not penetrate into
the coelomic cavity. Evidence of visceral mycotic dissemination
was noted in the lung and kidney and was characterized by
moderate to severe granulomatous pneumonia and nephritis
with low numbers of intralesional pigmented fungi as described
in the skin lesions.
A 1 cm3sample of frozen, affected dermis collected at the
time of necropsy was lysed in a Precellys R
Evolution tissue
homogenizer (Bertin Technologies, Montigny-le-Bretonneux
FRANCE) and genomic DNA was extracted using EZ1 DNA
Frontiers in Veterinary Science | www.frontiersin.org 3January 2020 | Volume 7 | Article 25

Hopf et al. Exophialosis in an Eastern Hellbender
tissue kit with a BioRobot EZ1 instrument (QIAGEN) following
manufacturer’s instructions. PCR amplification of the internal
spacer region (ITS) and partial large subunit gene of the nuclear
rDNA (LSU) were done using primers specifically designed for
Exophiala: EXO1 and EXO2 (9), and U1 and U2 (8), respectively.
Sequencing was done with the same primers using BigDye
Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems,
Foster City, CA, USA). The sequences generated were deposited
in GenBank, https://www.ncbi.nlm.nih.gov/ with the accession
nos. MK253014 (ITS) and MK253015 (LSU).
Using the obtained ITS sequence, identification was done by
an in-house BLASTn search within a sequence dataset compiled
in Bioedit v7.0.5 program comprising short barcode identifiers
located in the ITS2 region of reference and authentic strains
of Exophiala and related species (10,11). A 100% match was
not found with any of the strains in the dataset. Subsequently,
BLASTn searches in GenBank were performed using partial
ITS and partial LSU sequences. The top ITS results showed
that the isolate (UTHSCSA R-5437) matched 100% with an
unidentified Exophiala sp. S4.3 (KY322615), 96% with E.cancerae
CBS 120420T(NR_1377664), 95.69% with E.opportunistica
CBS 109811T(KF928435) and 95.32% with E.bonariae CBS
139957T(NR_144964). In the partial LSU BLASTn search, a
100% match was observed with E.cancerae (KF928502), and
type strains of E.opportunistica (KF928501) and E.bonariae
(KR781083), 98-99% matches with E.psychrophila CBS 191.87T
(MH873750), E.salmonis CBS 157.67T(MH870616), E.radicis
P2854T(KT723448) and E.pisciphila CBS 537.73T(MH870790).
Phylogenetic analysis was then conducted using only the ITS
sequence because the LSU sequence obtained was short and was
unreliable, and repeat sequencing of LSU failed to obtain a longer
sequence for use.
The ITS sequences of representative Exophiala species and
relatives were obtained from GenBank based on BLASTn
searches and on the datasets of Yong et al. (12) and Borman
et al. (13). The sequences were assembled and aligned using
MUSCLE (14) as implemented in Sequencher version 5.4.6-
Build 46289 (Gene Codes Corp., MI USA) and refined visually
using SE-AL (15). The best fit evolutionary model for this
dataset was determined using the Find Best DNA Models for
maximum likelihood (ML) as implemented in MEGA version 7
(16). General Time Reversible model with Gamma distributed
rate variation and an estimated proportion of invariable sites
(GTR+G+I) (17) was found to be the best fit evolutionary model
for this dataset having the lowest Bayesian Information Criterion
score (BIC) (18). Maximum likelihood analysis was conducted
with GTR+G+I substitution model and 1000 bootstrap (19)
iterations. Bayesian inference was done using MrBayes v3.2.5 (20)
applying the same substitution model. The Markov Chain Monte
Carlo (21) started from a random tree topology and lasted 6
million generations, where every 100th tree was retained and the
first 25% of the trees were discarded as burn in. A 50% majority
rule consensus tree and posterior probabilities were calculated.
Bayesian posterior probabilities (BPP) >0.90 and bootstrap (BT)
values >70% were considered significant.
The phylogenetic tree inferred from maximum likelihood
(Figure 3) showed our isolate within a monophyletic clade
(BPP 0.99/ BT 93%) comprising mostly of waterborne species,
i.e., E.opportunistica,E.lacus,E.cancerae,E.psychrophila,E.
salmonis,E.aquamarina,E.pisciphila (7) and other species
originally isolated from non-aquatic sources, i.e., E.bonariae (22)
and E.equina (7). Within the clade, it is closest to E.cancerae with
high support (BPP 1.00 / BT 86%). ITS pairwise comparison of
UTHSCSA R-5437 with E.cancerae CBS 120420T(NR_1377664)
however, showed 15 nucleotide base pair differences.
DISCUSSION
Phaeohyphomycosis is caused by pigmented filamentous
fungi including Exophiala species (23). Waterborne Exophiala
species are important pathogens in fish including E. salmonis
in cut-throat trout (Salmo clarkii) and the Atlantic salmon
(Salmo salar). Exophiala pisciphila caused an epizootic
in the channel catfish (Ictalurus punctatus), and brain
and skin lesions in smooth dogfish (Mustelus canis) (7).
In amphibians, infection with Exophiala are reported in
marine toads (Bufo marinus) and European blind cave
salamanders (Proteus anguinus) (24). Case reports of
dermal or disseminated disease in reptiles are described in
a Galapagos tortoise (Geochelone nigra) (25), Eastern box
turtle (Terrapene carolina carolina) (26), and Aldabra tortoise
(Geochelone gigantean) (27). Exophiala dermatitidis and E.
oligosperma have both been reported to cause infections in
humans (7).
Melanized fungi including Exophiala spp. can cause disease
more frequently in aquatic animals than in terrestrial animals.
Further, dry, thicker skin, the presence of fur or feathers, and
the absence of sweat glands are thought to provide a degree
of protection against infection in reptiles, birds, and mammals
(7). Infections may occur secondary to traumatic percutaneous
inoculation (23,27,28). Immunosuppression may be a factor in
producing infection, but phaeohyphomycosis has been reported
in immunocompetent animals (7,27–29).
Pathogenicity associated with Exophiala is not described
in C.a.alleganiesis. The source of infection in this case is
unknown but may be percutaneous invasion of the fungus
associated with ventral abrasions. Concurrent diseases that
may have contributed were not identified in this animal. It
is considered possible that the fungus of this report was a
primary pathogen.
The initially observed nodular dermatitis spread to deeper
structures of the body wall and eventually spread systemically.
Over time lesions were identified in the dermis, skeletal muscle
of the body wall, lungs, and kidneys. The lesions in the viscera
suggested hematogenous spread from the skin. The lesions in this
animal were first noted 6 years prior to the first surgery and had
not appeared to change, until a progressive growth was noted.
No observed changes in the hellbender’s environment or health
justified the change in the masses. Detailed reports of lesions from
Exophiala infections are rare and include systemic mycosis with
E. angulospora in an Atlantic halibut (Hippoglossus hippoglossus),
and disseminated mycosis in a colony of Ornate-horned
frogs (Ceratophrys ornata) with cutaneous and visceral lesions
Frontiers in Veterinary Science | www.frontiersin.org 4January 2020 | Volume 7 | Article 25

Hopf et al. Exophialosis in an Eastern Hellbender
FIGURE 3 | Phylogenetic tree inferred from ITS sequences showing the relationship of Exophiala sp. UTHSCSA R-5437 to known Exophiala species. Branch lengths
are proportional to phylogenetic distance. ML bootstrap support values (1000 resampling, Right) >70% and posterior probability values from Bayesian inference (Left)
>0.90 are shown above the branches. Teratosphaeria karinae CBS 128774Twas designated as outgroup. T, type strain; CBS, CBS-KNAW Fungal Biodiversity
Center culture collection, The Netherlands; NBRC, National Biological Resource Center, Japan; NCPF, UK National Collection of Pathogenic Fungi, PHE UK National
Mycology Reference Laboratory and School of Biological Sciences, University of Bristol, Bristol, United Kingdom; UTHSCSA, Fungus Testing Lab, University of Texas
Health Science Center at San Antonio, Texas USA. Sequence accession numbers are indicated in front of species names.
(28,29); however, Exophiala species infections are commonly
encountered in fish and amphibians during routine diagnostic
investigations of captive collections (personal observations, MG).
Diagnostic techniques used in cases of phaeohyphomycoses
include impression smear cytologic exam, histologic exam,
culture, molecular identification, and phylogenetic analysis
Frontiers in Veterinary Science | www.frontiersin.org 5January 2020 | Volume 7 | Article 25

Hopf et al. Exophialosis in an Eastern Hellbender
(23,28,29). In the present case, the fungus was identified
histologically followed by molecular screening. Initial
antemortem PCR results on a short segment of the 28S
ribosomal large subunit identified the species as an Exophiala.
Differentiation from other closely related species was not
possible based on the short size and conserved nature
of the targeted region. Attempts to culture the organism
were unsuccessful. Different Exophiala species have similar
morphologic and physiologic characteristics and can be difficult
to differentiate based on clinical signs, gross necropsy, and
histologic examination. The organism of this report had unique
features in histologic section that differed considerably from
the more commonly observed infections. In hematoxylin and
eosin stained sections of infected tissue, Exophiala typically
forms long, streaming, yellow, slender hyphae and occasional
branching elements with parallel cell walls. In this salamander,
the lesions were chronic, with minimal necrosis, and the fungal
agent was present in low numbers primarily as small yeast-like
or septate conidial forms, with only rare hyphae observed. In
this regard, infection with this organism in hellbenders or other
species could possibly misconstrued as a different fungus, or be
overlooked due to the low density of organism in tissue sections.
Identification of Exophiala species is challenging because they
are highly pleomorphic and produce synanamorphs in their
life cycles (10,30,31). Although these species are generally
recognizable as Exophiala, identification is difficult using
morphological and other phenotypic criteria because almost
identical structures are observed even with phylogenetically
distantly related species (10). DNA sequencing and the increasing
availability of sequences in public databases (e.g., GenBank)
improved fungal identification and led to the discovery of
novel species as well as reclassification of species in their
natural groupings. ITS sequences in Exophiala and related
species have been found to have variabilities caused by stutter
formation in PCR products making this target unreliable for
identification (10). However, short fragments in the ITS2 region
have differences in nucleotide composition that are stable
allowing them to be used as barcodes by clinical diagnostic
laboratories for the identification of medically important black
yeasts and relatives (10). In this case, the barcode did not
give a species identity because the Exophiala strain in this
study did not match with any of the species in the database
and could represent a new species. ITS phylogenetic analysis
showed that this sequence is closely related with E.cancerae
(BPP1.00/ BT 86%); however pairwise ITS sequence comparison
with that of E. cancerae showed 15 base pair differences.
Based on the in-house Bioedit barcode identifier sequence
BLASTn result, BLASTn results in GenBank and phylogenetic
analysis, this organism is as a putatively novel Exophiala species
closely related to E.cancerae. Confirmation and description
of this organism as a new species is not possible at this
time because of the lack of viable culture of the organism
and failure to sequence other gene loci for a multi-gene
phylogenetic analysis.
Treatment options for Exophiala species are limited.
In humans, this includes radiation therapy, amputation,
resection followed by skin grafts, and systemic administration
of amphotericin B and 5-fluorocytosine (32). Ketoconazole
and acridine dyes combined with excisional surgery were
unsuccessful in a marine toad (Bufo marinus) (33). Treatment
was successful in an Aldabra tortoise (Geochelone gigantean)
infected with Exophiala oligosperma (27). In the present case, two
surgeries with aggressive debridement of the masses, followed
by oral itraconazole failed to resolve the infection. Without
a culture, the appropriateness of itraconazole therapy could
not be confirmed. Poor compliance may have been caused
by post-operative stress, the increased feeding frequency to
match the treatment schedule, or a side effect of the drug.
Decreased frequency of the treatment was attempted, but
the hellbender remained hypophagic and the treatment was
ultimately discontinued. Treatment in bath water might
have been successful and was considered but declined due to
husbandry considerations.
DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the
GenBank, https://www.ncbi.nlm.nih.gov/.
ETHICS STATEMENT
The care and treatment of this animal, as well as the testing
performed post-mortem were approved by the Rosamond
Gifford Zoo (Syracuse, NY, USA) Animal Care Committee
and the Rosamond Gifford Zoo (Syracuse, NY, USA)
IACUC committee.
AUTHOR CONTRIBUTIONS
CH, and NA-M performed the medical and surgical management
of this case, the submission of the samples to necropsy, writing
of the manuscript. CG, CS, JM, HF, and NW performed the
molecular and genetic analysis on the samples provided. MG, RO,
and EG processed the tissues for histopathologic examination,
and performed the analysis of the results. All authors discussed
the results and commented on the manuscript.
REFERENCES
1. Junge RE. Hellbender medicine. In: Miller RE, Fowler M, editor. Fowler’s Zoo
and Wild Animal Medicine Current Therapy. (2012). p. 260–4.
2. Souza MJ, Gray MJ, Colclough P, Miller DL.Prevalence of infection
by Batrachochytrium dendrobatidis and ranavirus in Eastern hellbenders
(Cryptobranchus alleganiensis alleganiensis) in eastern Tennessee. J Wildl Dis.
(2012) 48:560–6. doi: 10.7589/0090-3558-48.3.560
3. Bales EK, Hyman OJ, Loudon AH, Harris RN, Lipps G, Chapman E,
et al. Pathogenic chytrid fungus Batrachochytrium dendrobatidis, but not,
B.salamandrivorans, detected on Eastern Hellbendersz. PLoS ONE. (2015)
10:e0116405. doi: 10.1371/journal.pone.0116405
Frontiers in Veterinary Science | www.frontiersin.org 6January 2020 | Volume 7 | Article 25

Hopf et al. Exophialosis in an Eastern Hellbender
4. Wheeler BA, Prosen E, Mathis A, Wilkinson RF. Population declines
of a long-lived salamander: a 20+-year study of hellbenders,
Cryptobranchus alleganiensis.Biol Conserv. (2003) 109:151–6.
doi: 10.1016/S0006-3207(02)00136-2
5. New York State Department of Conservation. Eastern Hellbender Fact Sheet.
Available online at: https://www.dec.ny.gov/animals/7160.html (accessed
January 29, 2019).
6. Nyaoke A, Weber ES, Innis C, Stremme D, Dowd C, Hinckley L. Disseminated
phaeohyphomycosis in weedy, Phyllopteryx taeniolatus, and leafy, Phycodurus
eques, seadragons caused by species of Exophiala, including a novel species. J
Vet Diagnos Investig. (2009) 21:69–79. doi: 10.1177/104063870902100111
7. de Hoog GS, Vicente VA, Najafzadeh MJ, Harrak MJ, Badali H, Seyedmousavi
S. Waterborne Exophiala species causing disease in cold-blooded animals.
Persoonia. (2011) 27:46–72. doi: 10.3767/003158511X614258
8. Sandhu GS, Kline BC, Stockman L, Roberts GD. Molecular probes
for diagnosis of fungal infections. J Clin Microbiol. (1995) 33:2913–9.
doi: 10.1128/JCM.33.11.2913-2919.1995
9. Porteous NB, Grooters AM, Redding SW, Thompson EH, Rinaldi MG,
De Hoog GS, et al. Identification of Exophiala mesophila isolated from
treated dental unit waterlines. J Clin Microbiol. (2003) 41:3885–89.
doi: 10.1128/JCM.41.8.3885-3889.2003
10. Heinrichs G, de Hoog GS, Haase G. Barcode identifiers as a practical tool for
reliable species assignment of medically important black yeast species. J Clin
Microbiol. (2012) 50:3023–30. doi: 10.1128/JCM.00574-12
11. Hall TA. BioEdit: a user-friendly biological sequence alignment editor
and analysis program for windows 95/98/NT. Nucleic Acids Symp Series.
(1999) 41:95–8.
12. Yong LK, Wiederhold NP, Sutton DA, Sandoval-Denis M, Lindner JR,
Fan H, et al. Morphological and molecular characterization of Exophiala
polymorpha sp. nov. isolated from Sporotrichoid lymphocutaneous lesions
in a patient with Myasthenia gravis.J Clin Microbiol. (2015) 53:2816–22.
doi: 10.1128/JCM.00622-15
13. Borman AM, Fraser M, Szekely A, Larcombe DE, Johnson EM. Rapid
identification of clinically relevant members of the genus exophiala by
matrix-assisted laser desorption ionization–time of flight mass spectrometry
and description of two novel species, Exophiala campbellii and Exophiala
lavatrina.J Clin Microbiol. (2017) 55:1162–76. doi: 10.1128/JCM.02459-16
14. Edgar RC. Muscle: a multiple sequence alignment method with
reduced time and space complexity. BMC Bioinformatics. (2004) 5:113.
doi: 10.1186/1471-2105-5-113
15. Rambaut A. Se-Al: Sequence Alignment Editor, version 2.0 alpha 11 Carbon
[Computer program]. Oxford Univercitry (2002). Available online at: http://
tree.bio.ed.ac.uk/software/seal/ (accessed May 21, 2019).
16. Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics
analysis version 7.0 for bigger datasets. Mol Biol Evol. (2016) 33:1870–74.
doi: 10.1093/molbev/msw054
17. Nei M, Kumar S. Molecular Evolution and Phylogenetics. New York, NY:
Oxford University Press (2000).
18. Posada D, Crandall KA. Selecting the best-fit model of nucleotide substitution.
Syst Biol. (2001) 50:580–601. doi: 10.1080/10635150118469
19. Felsenstein J. Confidence limits on phylogenies: an approach
using the bootstrap. Evolution. (1985) 39:783–91. doi: 10.1111/
j.1558-5646.1985.tb00420.x
20. Ronquist F, Huelsenbeck JP. MRBAYES 3: Bayesian phylogenetic inference
under mixed models. Bioinformatics. (2003) 19:1572–4. doi: 10.1093/
bioinformatics/btg180
21. Metropolis NA, Rosenbluth W, Rosenbluth MN, Teller AH, Teller E.
Equations of state calculations by fast computing machines. J Chem Phys.
(1953) 21:1087–91. doi: 10.1063/1.1699114
22. Crous PW, Schumacher RK, Akulov A, Thangavel R, Hernández-Restrepo M,
Carnegie AJ. New and interesting fungi. 2. Fungal Syst Evol. (2019) 3:57–34.
doi: 10.3114/fuse.2019.03.06
23. Pare JA. Fungal diseases of amphibians: an over view. Vet
Clin Exot Anim. (2003) 6:315–26. doi: 10.1016/S1094-9194(03)
00006-9
24. Bizjak-Mali L, Zalar P, Turk M, Babiˇ
c MN, Kostanjšek R, Gunde-Cimerman
N. Opportunistic fungal pathogens isolated from a captive individual of the
European blind cave salamander Proteus anguinus.Dis Aquat Organ. (2018)
129:15–30. doi: 10.3354/dao03229
25. Manharth A, Lemberger K, Mylniczenko N, Pinkerton M, Pessier AP,
Kammeyer P, et al. Disseminated phaeohyphomycosis due to Exophiala
species in a Galapagos tortoise,Geochelone nigra.J Herp Med Surg. (2005)
15:20–6. doi: 10.5818/1529-9651.15.2.20
26. Joyner PH, Shreve AA, Spahr J, Fountain AL, Sleeman JM.
Phaeohyphomycosis in a free-living eastern box turtle (Terrapene Carolina
Carolina). J Wildl Dis. (2006) 42:883–8. doi: 10.7589/0090-3558-42.4.883
27. Stringer EM, Garner MM, Proudfoot JS, Ramer JC, Bowman MR, Heng
HG, et al. Phaeohyphomycosis of the Carapace in an Aldabra Tortoise
(Geochelone gigantean). J Zoo Wildl Med. (2009) 40:160–7. doi: 10.1638/2008-
0035.1
28. Miller EA, Montali RJ, Ramsay EC, Rideout RA. Disseminated
chromoblastomycosis in a colony of ornate-horned frogs (Ceratophrys
ornata). J Zoo Wildl Med. (1992) 23:433–8.
29. Overy DP, Groman D, Giles J, Duffy S, Rommens M, Johnson G. Exophiala
angulospora causes systemic mycosis in atlantic halibut: a case report. J Aquat
Anim Health. (2015) 27:12–9. doi: 10.1080/08997659.2014.953266
30. Cañete-Gibas CF, Wiederhold NP. The black yeasts: an update on species
identification and diagnosis. Curr Fungal Infect Rep. (2018) 12:59–65.
doi: 10.1007/s12281-018-0314-0.
31. de Hoog GS, Takeo K, Yoshida S, Gottlich E, Nishimura K, Miyaji M.
Pleoanamorphic life cycle of Exophiala (Wangiella) dermatitidis. Antonie Van
Leeuwenhoek. (1994) 65:143–53. doi: 10.1007/BF00871755
32. Sudduth EJ, Crumbley AJ. Phaeohyphomycosis due to exophiala species:
clinical spectrum of disease in humans. Clin Infect Dis. (1992) 15:639–44.
doi: 10.1093/clind/15.4.639
33. Bube A, Burkhardt E, Weiss R. Spontaneous chromomycosis in
the marine toad (Bufo marinus). J Comp Pathol. (1992) 106:73–7.
doi: 10.1016/0021-9975(92)90069-7
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
The handling Editor declared a past co-authorship with one of the authors RO.
Copyright © 2020 Hopf, Graham, Gibas, Sanders, Mele, Fan, Garner, Wiederhold,
Ossiboff and Abou-Madi. This is an open-access article distributed under the terms
of the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) and the
copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or
reproduction is permitted which does not comply with these terms.
Frontiers in Veterinary Science | www.frontiersin.org 7January 2020 | Volume 7 | Article 25