Content uploaded by Priscilla H Joyner
Author content
All content in this area was uploaded by Priscilla H Joyner on Mar 30, 2014
Content may be subject to copyright.
Content uploaded by Priscilla H Joyner
Author content
All content in this area was uploaded by Priscilla H Joyner on Mar 30, 2014
Content may be subject to copyright.
Journal of Wildlife Diseases, 42(4), 2006, pp. 883–888
#
Wildlife Disease Association 2006
Phaeohyphomycosis in a Free-Living Eastern Box Turtle (Terrapene
carolina carolina)
Priscilla H. Joyner,
1,6
Allison A. Shreve,
1,4
John Spahr,
2
Andrea L. Fountain,
3
and Jonathan M. Sleeman
1,5
1
The Wildlife Center of Virginia, PO Box 1557, Waynesboro, Virginia 22980, USA;
2
Blue Ridge Pathologists,
Fishersville, Virginia 22939, USA;
3
Department of Microbiology, Augusta Medical Center, Fishersville, Virginia
22939, USA;
4
Current address: VCA Tampa Bay Animal Clinic, 8501 La Due Lane, Tampa, Florida 33614, USA;
5
Current address: Virginia Department of Game and Inland Fisheries, 4010 West Broad Street, Richmond,
Virginia 23230, USA;
6
Corresponding author (email: phjoyner@seadevil.net)
ABSTRACT:
A free-ranging eastern box turtle
(Terrapene carolina carolina) was referred to
the Wildlife Center of Virginia with a three-
month history of marked swelling of the right
hind limb initially diagnosed as chromomycosis
by histopathology. Hematology revealed severe
anemia (9%), leukocytosis (12.8 cells310
3
/ml),
heterophilia (6.14 cells310
3
/ml), and monocy-
tosis (0.51 cells310
3
/ml). Gross necropsy re-
vealed a firm, encapsulated 331 cm subcuta-
neous mass filled with dark brown-black, friable
necrotic material of the distal right hind limb.
Microscopically, the mass was characterized by
a granulomatous inflammatory process with
numerous multinucleated histiocytic giant cells.
Fungal elements were present within necrotic
centers and associated with multinucleated
cells. Special stains revealed numerous phaeoid
hyphae and yeast; Exophiala jeanselmei was
isolated by routine mycologic culture. Phaeo-
hyphomycosis was diagnosed based on the
histologic appearance of the fungal elements
within the mass and culture results. There was
no histopathological evidence of systemic in-
fection. This is the first report of phaeohypho-
mycosis caused by fungi of the genus Exophiala
in free-living reptiles.
Key words: Eastern box turtle, Exophiala
jeanselmei, mycosis, phaeohyphomycosis, Ter-
rapene carolina carolina.
Limited information is available regard-
ing fungal disease in free-ranging reptiles
with very few reports (McAllister et al.,
1993; Jacobson and Cheatwood, 2000;
Rose et al., 2001; Cheatwood et al.,
2003). Furthermore, there are fewer
reports in the literature describing in-
fection due to phaeohyphomycotic agents,
a group of dematiaceous (pigmented)
fungal organisms, in reptiles (Weitzman
et al., 1985; Jacobson and Cheatwood,
2000). These fungi are ubiquitous in soil
and decaying matter and are opportunistic
pathogens of vertebrates usually intro-
duced by traumatic inoculation. Although
infection is most frequently reported in
immunocompetent humans, in animals it
may be associated with a compromised or
suppressed immune system (Lobetti,
1996). This paper describes a case of
phaeohyphomycosis in an eastern box
turtle (Terrapene carolina carolina) and
to our knowledge is the first report of
clinical infection caused by the genus
Exophiala in a free-living reptile.
An adult male eastern box turtle was
referred to the Wildlife Center of Virginia
(WCV) in December 2003. Initially, the
turtle was presented to a wildlife re-
habilitation facility in Fairfax County
Virginia by a private citizen. Three months
prior to referral, the animal was treated
for marked swelling of the right hind
limb. A mass involving the right proximal
tibia was removed at the referring facility
and diagnosed as chromomycosis by his-
topathology. Treatment included sup-
portive care, a series of antimicrobial
treatments, and a 1-mo course of 1 mg
itraconazole administered orally once dai-
ly. On presentation to WCV, the turtle
weighed 0.45 kg and was well hydrated
and in good body condition. Swelling with
localized edema was visible surrounding
and distal to the right stifle, and a circum-
ferential mass was palpable on the right
proximal tibia. The lateral two digits were
missing from the right hind foot. No
additional abnormalities were noted on
physical examination.
Two-tenths milliliters of blood was
collected from the subcarapacial sinus into
a heparinized 1-ml syringe using a 25-
gauge needle. Hematocrit was determined
883
by microhematocrit centrifugation, and
plasma protein levels were approximated
using a refractometer. Within 10 min of
obtaining the blood sample, a manual
white blood cell count was performed
using the eosinophil UnopetteH 5877
system (Becton Dickinson and Company,
Franklin Lakes, New Jersey, USA) and
a hemocytometer. The total leukocyte
count was determined, and the white
blood cell differential was calculated by
the indirect method (Campbell, 1988).
Results were compared with physiological
reference ranges for the common box
turtle (Terrapene carolina) and presented
as mean 6SD (International Species In-
ventory System, 2002). The hematocrit
was decreased at 9% (mean522.266.6%),
and the hemogram revealed a leukocyto-
sis (12.80 cells310
3
/ml; mean57.056
4.61310
3
/ml) with heterophilia (6.14
cells310
3
/ml; mean52.4762.05310
3
/ml)
and monocytosis (0.51 cells310
3
/ml;
mean5 0.1260.07 cells310
3
/ml). Lateral
and caudo-cranial radiographs revealed
marked soft tissue swelling of the entire
right hind limb, particularly the caudal
and plantar aspects. The third and fourth
digits of the right hind limb were absent.
No other abnormalities were noted on
radiographic examination. The box turtle
was euthanized with intravenous pento-
barbital because of the severity and extent
of the soft tissue involvement and guarded
prognosis for return to function and sub-
sequent survival in the wild.
At necropsy, the right hind limb was
swollen with a palpably firm subcutaneous
mass surrounding the right proximal tibia.
The third and forth digits were absent
from the right hind limb. Subcutaneously,
a3cm31 cm encapsulated mass extend-
ed distally along the caudal aspect of the
right tibia. A subcutaneous adhesion was
associated with the mass on the caudolat-
eral aspect of the right midtibia where
a remnant of suture material was present.
The capsule was filled with dark brown-
black, friable necrotic material (Fig. 1).
The liver was diffusely pale, and a black
branching line was visible on the surface
of the right lobe. The lungs had diffuse
multifocal patches of dark red discolor-
ation on the surface. No other gross
abnormalities were noted.
Histopathologic examination of the sub-
cutaneous soft tissue mass revealed a gran-
ulomatous inflammatory process including
lymphoid cells, eosinophils, and histio-
cytes (Fig. 2). Numerous multinucleated
histiocytic giant cells, often arranged in
ringed groups around necrotic debris,
were present. Numerous phaeoid (brown)
fungal elements consisting of hyphae and
F
IGURE
1. Necropsy specimen of the right hind
limb of an eastern box turtle (Terrapene carolina
carolina). The skin is reflected distally to expose
a subcutaneous mass. Note the small opening and
pigmentation in the caudodistal portion of the
capsule, as well as edema.
884 JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 4, OCTOBER 2006
yeastlike cells were seen within the
necrotic centers and associated with mul-
tinucleated cells. Gomori methenamine
silver stains revealed chains of ovoid
yeastlike bodies (conidia) as well as short
rectangular hyphae with occasional right-
angle branching. The coelomic viscera
were unremarkable, except for the liver,
which had pale and clear hepatocytes.
There was no microscopic evidence of
systemic fungal infection in any of the
other tissues examined including lung,
heart, esophagus, stomach, spleen, pan-
creas, liver, kidney, testes, intestine, cloa-
ca, and right hind limb integument. A
histopathologic diagnosis of chromomyco-
sis of the subcutaneous tissue was made.
A sterile swab of the pigmented lesion
was obtained at necropsy and submitted
for fungal culture at the Augusta Medical
Center, Fishersville, Virginia, USA. The
specimen was cultured on Brain Heart
Infusion Agar with 10% sheep blood,
gentamicin, and chloramphenicol, Inhibi-
tory Mold Agar with gentamicin, Myco-
sel
TM
Agar, and Sabouraud Dextrose Agar
(BBL
TM
Prepared Plated Media, BD Di-
agnostic Systems, Sparks, Maryland, USA)
and incubated at 30 C. After 48 hr, several
small, white colonies were observed on the
Sabouraud plate and were identified as
Candida (Torulopsis) glabrata using the
API20C yeast identification system (bio-
Merieux, 595 Anglum Road, Hazelwood,
Missouri, USA). After 7 days, numerous
colonies (small, black, and shiny with black
reverse [colony color on back of petri
dish]) were seen on all media. Colonies
changed to dark gray, velvety colonies
after an additional 7-day incubation peri-
od. A slide culture was performed using
Cornmeal Agar (BBL
TM
Prepared Plated
F
IGURE
2. Photomicrograph of the subcutaneous lesion on the right hind limb of an eastern box turtle
(Terrapene carolina carolina). Note the granuloma with peripheral multinucleated histiocytic giant cells and
a necrotic center with brown pigmented fungal hyphae and conidia (arrow). H&E. Bar510 mm.
SHORT COMMUNICATIONS 885
Media) incubated at 30 C and was exam-
ined microscopically after sufficient
growth was observed on the coverslip.
Microscopic examination revealed septate
hyphae with slender, tubular annellides,
occasionally branched with tapered tips,
supporting clusters of small, oval conidia
2–3 mm in length. Based on growth rate
and macroscopic and microscopic appear-
ance, the organism was identified as
Exophiala jeanselmei (Larone, 1995).
Chromomycosis is a general term for
a group of clinicopathologic syndromes
caused by fungal agents classified as
chromoblastomycoses and phaeohyphomy-
coses (de Hoog et al., 2000a). The distinc-
tion lies in the fungal form present in the
tissues: dermal muriform cells or sclerotic
bodies in chromoblastomycosis versus hy-
phal fungal forms in phaeohyphomycosis
(de Hoog et al., 2000a). Infection can be
further classified based on location in-
cluding superficial, cutaneous, subcutane-
ous, and deep mycoses. Colonization is
often associated with traumatic inoculation
(Wagner, 2000) and/or immunocompro-
mised hosts (Lobetti, 1996). Deep mycoses
may occur secondary to inhalation, in-
gestion, or dissemination via blood and
lymphatic systems (de Hoog et al., 2000b).
Infection can be diagnosed via cytology or
histology, but the causative agent must be
isolated by culture (de Hoog et al., 2000a).
Chromomycosis has been described in
domestic mammals, birds, reptiles, am-
phibians, fish, and crustaceans (Bube et
al., 1992; Aiello, 1998; Reavill and
Schmidt, 2004). Although mycotic infec-
tions are relatively common in captive
reptiles (Schumacher, 2003), reports of
phaeohyphomycosis are scarce in the
reptile literature. A microscopic descrip-
tion of Scolecobasidium, a phaeohyphomy-
cotic agent of fish, has been reported in an
eastern box turtle (Weitzman et al., 1985).
Chromomycosis has been reported in
a mangrove snake (Boiga dendrophila);
however, cultures were unsuccessful, and
it was not possible to identify the organism
(Jacobson, 1984).
Various antifungal drugs have been
used in the treatment of phaeohyphomy-
coses with questionable efficacy (Wagner,
2000). Excision or debulking is recom-
mended before chemotherapy begins and
is potentially curative (de Hoog et al.,
2000a; Meletiadis et al., 2000; Wagner,
2000). Surgical debulking of the lesion was
not performed in this case because of the
extent of local tissue invasion and the
possibility of disseminated disease due to
chronic infection.
Exophiala jeanselmei is a saprophytic
dematiaceous fungus most commonly
found in decaying wood and soil that is
enriched with organic waste as well as
polluted water and sewage (de Hoog et al.,
2000a; Nucci et al., 2002). It is considered
an opportunistic pathogen, but phaeohy-
phomycosis caused by Exophiala species
has been reported in both immunosup-
pressed and immunocompetent human
patients (Wagner, 2000). Fungal disease
caused by Exophiala has been reported in
humans, domestic animals (Lobetti, 1996;
Helms and McLeod, 2000), wild turkeys
(Meleagris gallopavo) (Davidson et al.,
1989), fruit-eating bats (Eidolon helvum)
(Muotoe-Okafor and Gugnani, 1993), and
Atlantic salmon (Salmo salar L.) (Otis et
al., 1985). Although it was not possible in
this case to confirm the presence of
Exophiala in tissue by immunohistochem-
istry (IHC) or polymerase chain reaction
(PCR), the histologic sections clearly
showed a phaeoid fungus in the appropri-
ate inflammatory reaction, implicating this
as the infectious agent. The cultural
isolation of E. jeanselmei validates the
histopathology, especially the finding of
yeastlike cells, which are often seen in
young colonies of this genus (de Hoog et
al., 2000b). Histopathology with corrobo-
rating microbiology is a time-honored
standard for defining a specific infectious
etiology. As fungal elements in affected
tissues were phaeoid and not consistent
with the morphology of C. glabrata,we
suspect Candida was a contaminant, and
its isolation was considered incidental.
886 JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 4, OCTOBER 2006
This represents the first substantiated
report of Exophiala-associated phaeohy-
phomycosis in a free-living reptile.
The origin of infection is unknown.
Phaeohyphomycotic infections are gener-
ally caused by traumatic implantation into
the skin. When the soft tissue mass was
dissected, there was a small opening in the
caudodistal portion of the subcutaneous
capsule suggestive of a possible site of
inoculation. Although the integument
showed no obvious evidence of trauma,
the absent digits of the right hind limb
indicated possible previous traumatic in-
oculation. In addition, infection has been
associated with a local or systemic immu-
nosuppression. Lymphopenia has been
reported as a common factor in previous
cases of phaeohyphomycosis in dogs (Lo-
betti, 1996); however, lymphopenia was
not present in this case. Other means of
assessing immune function were not
explored; therefore, we were unable to
determine definitively whether this in-
fection was the result of direct inoculation,
immunosuppression, or both.
We thank the staff at the Wildlife
Center of Virginia and Caroline Seitz for
referral of this case.
LITERATURE CITED
A
IELLO
, S. E. (ed.). 1998. The Merck veterinary
manual. 8th Edition. Merck & Co., Whitehouse
Station, New Jersey.
B
UBE
, A., E. B
URKHARDT
,
AND
R. W
EIB
. 1992.
Spontaneous chromomycosis in the marine toad
(Bufo marinus). Journal of Comparative Pathol-
ogy 106: 73–77.
C
AMPBELL
, T. W. 1988. Avian hematology and
cytology. Iowa State University Press, Ames,
Iowa, 101 pp.
C
HEATWOOD
, J. L., E. R. J
ACOBSON
,P.G.M
AY
,T.M.
F
ARREL
,B.L.H
OMER
,D.A.S
AMUELSON
,
AND
J.
W. K
IMBROUGH
. 2003. An outbreak of fungal
dermatitis and stomatitis in a free-ranging
population of pigmy rattlesnakes (Sistrurus
miliarius barbouri) in Florida. Journal of Wild-
life Diseases 39: 329–337.
D
AVIDSON
, W. R., E. B. S
HOTTS
,J.T
ESKA
,
AND
D. W.
M
ORELAND
. 1989. Feather damage due to
mycotic infections in wild turkeys. Journal of
Wildlife Diseases 25: 534–539.
D
E
H
OOG
, G. S., F. Q
UEIROZ
-T
ELLES
,G.H
AASE
,G.
F
ERNANDEZ
-Z
EPPENFELDT
,D.A.A
NGELIS
,A.H.G.
G. V
AN
D
EN ENDE
,T.M
ATOS
,H.P
ELTROCHE
-
L
LACSAHUANGA
,A.A.P
IZZIRANI
-K
LEINER
,J.R
AINER
,
N. R
ICHARD
-Y
EGRES
,V.V
ICENTE
,
AND
F. Y
EGRES
.
2000a. Black fungi: Clinical and pathogenic
approaches. Medical Mycology 38: 243–250.
———, ———, J. G
UARRO
,J.G
ENE
,
AND
M. J.
F
IGUERAS
. 2000b. Atlas of clinical fungi. 2nd
Edition. Centraalbureau voor Schimmelcul-
tures, Utrecht, The Netherlands, 1126 pp.
H
ELMS
, S. R.,
AND
C. G. M
C
L
EOD
. 2000. Systemic
Exophiala jeanselmei infection in a cat. Journal
of the American Veterinary Medical Association
217: 1858–1861.
I
NTERNATIONAL
S
PECIES
I
NVENTORY
S
YSTEM
. 2002.
Reference ranges for physiological values in
captive wildlife. International species inven-
tory system, 2002 Edition, Apple Valley, Minne-
sota.
J
ACOBSON
, E. R. 1984. Chromomycosis and fibrosar-
coma in a mangrove snake. Journal of the
American Veterinary Medical Association 185:
1428–1430.
———,
AND
J. L. C
HEATWOOD
. 2000. Mycotic
diseases of reptiles. Seminars in Avian and
Exotic Pet Medicine 9: 94–101.
L
ARONE
, D. H. 1995. Medically important fungi:
A guide to identification. 3rd Edition. ASM
Press, Washington, D.C. 214 pp.
L
OBETTI
, R. G. 1996. Leukogram and serum globulin
values in two dogs with systemic Xylohypha
bantiana infection. Journal of the South African
Veterinary Association 67: 91–92.
M
C
A
LLISTER
, C. T., S. R. G
OLDBERG
,H.J.H
OLSHUH
,
AND
S. E. T
RAUTH
. 1993. Disseminated mycotic
dermatitis in a wild-caught timber rattlesnake,
Crotalus horridus (Serpentes: Viperidae), from
Arkansas. Texas Journal of Science 45: 279–281.
M
ELETIADIS
, J., J. F. G. M. M
EIS
,G.S.D
E
H
OOG
,
AND
P. E. V
ERWEIJ
. 2000. In vitro susceptibilities of 11
clinical isolates of Exophiala species to six
antifungal drugs. Mycoses 43: 309–312.
M
UOTOE
-O
KAFOR
, F. A.,
AND
H. C. G
UGNANI
. 1993.
Isolation of Lecythophora mutabilis and Wan-
giella dermatitidis from the fruit-eating bat,
Eidolon helvum. Mycopathologia 122: 95–100.
N
UCCI
, M., T. A
KITI
,G.B
ARREIROS
,F.S
ILVEIRA
,S.
R
EVANKAR
,B.L.W
ICKES
,D.A.S
UTTON
,
AND
T. F.
P
ATTERSON
. 2002. Nosocomial outbreak of Exo-
phiala jeanselmei fungemia associated with
contamination of hospital water. Clinical In-
fectious Diseases 34: 1475–1480.
O
ROS
, J., C. D
ELGADO
,L.F
ERNANDEZ
,
AND
H. E.
J
ENSEN
. 2004. Pulmonary hyalohyphomycosis
caused by Fusarium spp in a Kemp’s ridley sea
turtle (Lepidochelys kempi): An immunohisto-
chemical study. New Zealand Veterinary Journal
52: 150–152.
O
TIS
, E. J., R. E. W
OLKE
,
AND
V. S. B
LAZER
. 1985.
Infection of Exophiala salmonis in Atlantic
SHORT COMMUNICATIONS 887
salmon (Salmo salar L.). Journal of Wildlife
Diseases 21: 61–64.
R
EAVILL
, D. R.,
AND
R. E. S
CHMIDT
. 2004. Reptile
mycotic infections from the literature and 55
cases. In Proceedings of the Association of
Reptilitan and Amphibian Veterinarians, Joint
Conference May 6-11, Naples, Florida, pp. 62–71.
R
OSE
, F. L., J. K
OKE
,R.K
OEHN
,
AND
D. S
MITH
. 2001.
Identification of the etiological agent for necro-
tizing scute disease in the Texas tortoise. Journal
of Wildlife Diseases 37: 223–228.
S
CHUMACHER
, J. 2003. Fungal diseases of reptiles.
The Veterinary Clinics of North America, Exotic
Animal Practice 6: 327–335.
W
AGNER
, K. F. 2000. Agents of chromomycosis. In
Mandell, Douglas, and Bennett’s principles and
practice of infectious diseases, G. L. Mandell, J.
E. Bennett and R. Dolin (eds.). Harcourt Health
Sciences, Philadelphia, Pennsylvania, pp. 2699–
2702.
W
EITZMAN
, I., S. A. R
OSENTHAL
,
AND
J. L. S
HUPACK
.
1985. A comparison between Dactylaria gallo-
pava and Scolecobasidium humicola: First report
of an infection in a tortoise caused by S.
humicola. Sabouraudia 23: 287–293.
Received for publication 31 March 2005.
888 JOURNAL OF WILDLIFE DISEASES, VOL. 42, NO. 4, OCTOBER 2006
