DISEASE IN WILDLIFE OR EXOTIC SPECIES

Dermatophytosis caused by Trichophyton spp.

in a Tenerife Lizard (Gallotia galloti): an
Immunohistochemical Study
J. Or os
*
, J. D. Hern andez

, J. Gallardo
*
, P. Lupiola
*
and H. E. Jensen

*Veterinary Faculty, University of Las Palmas de Gran Canaria (ULPGC), Trasmontana s/n, 35416 Arucas,

Serviexotic,
Vega de San Mateo, Las Palmas, Spain and

Department of Pharmacology and Pathobiology, The Royal Veterinary and
Agricultural University, 13 Bulowsvej, DK-1870 Frederiksberg C, Copenhagen, Denmark
Summary
Reports of dermatophytosis in reptiles are rare. This report describes the microscopical and immunohistochem-
ical ndings in a case of dermatophytosis caused by Trichophyton spp. in a 2-year-old Tenerife lizard (Gallotia
galloti) with ulcerative and pustular skin lesions. Microscopically, the lesions were characterized by supercial
epidermal pustules containing heterophils with numerous fungal hyphae that stained by periodic acideSchiff
and Grocotts stain. Fungal culture was not performed, but a panel of polyclonal antibodies specic for different
fungal genera was applied to tissue sections. These immunohistochemical studies demonstrated reactivity of the
hyphae only with antiserum specic for Trichophyton spp.
2012 Elsevier Ltd. All rights reserved.
Keywords: dermatophytosis; immunohistochemistry; Tenerife lizard; Trichophyton spp.
The term dermatophytosis describes infection by kera-
tinophilic fungi of the genera Microsporum, Trichophyton
and Epidermophyton (Pare and Jacobson, 2007). Derma-
tophytes are rarely isolated from reptiles (Hazell et al.,
1985; Miller et al., 2004) and it has been suggested
that there is insufcient evidence to support the occur-
rence of dermatophytosis in these animals (Pare and
Jacobson, 2007).
Fungal infections in reptiles are often diagnosed by
culture or histopathology, but inthe absence of culture,
immunohistochemistry (IHC) provides an alternative
diagnostic approach. Reports of immunohistochemical
diagnosis of mycotic infections in reptiles are scarce
(Oros et al., 2004a, b; 2011). The present report de-
scribes a case of dermatophytosis causedby Trichophyton
spp. in a Tenerife lizard (Gallotia galloti).
A captive 2-year-old, 205 g, female Tenerife lizard,
developed several irregular pustular and ulcerative
cutaneous lesions (0.1 cm diameter) in the interscap-
ular region. The lizard had been taken from the wild
fromVi~ na de las Arenas (Tenerife) when it was young
because it had lost its right hindlimb after a cat attack.
The lizard had been kept alone in a 60 50 50 cm
terrarium with articial grass as substrate and cork
bark sheets as hiding places. The diet consisted of
crickets coated with a vitamin/mineral powder and
some vegetables and grapes. Using a commercial
heat mat and sunlight for a basking site the ambient
temperature in the terrarium was maintained at
24e28

C. Due to nancial constraints, haematologi-

cal examination and microbiological cultures were
not performed. The lizard was treated with enroox-
acin (Baytril, Bayer, Leverkusen, Germany) at
15 mg/kg orally q24h for 21 days and 1% chlorhexi-
dine solution (Menalmina, Orravan S. L., Sant
Joan Desp, Spain) was applied topically. The health
of the lizard declined, with the animal developing
lethargy and inappetence before dying.
At necropsy examination, the lizard was cachectic,
with muscle atrophy. Several irregular pustular and
ulcerative skin lesions (0.3 cm diameter) were ob-
served in the interscapular region (Fig. 1). Both
kidneys contained numerous round white foci
Correspondence to: J. Oros (e-mail: joros@dmor.ulpgc.es).
0021-9975/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jcpa.2012.11.245
J. Comp. Path. 2013, Vol. 149, 372e375
Available online at www.sciencedirect.com
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(1e2 mm diameter). No gross lesions were present in
other organs. Tissue samples from all major organs
were xed in 10% neutral buffered formalin, pro-
cessed routinely and embedded in parafn wax.
Sections (4 mm) were stained with haematoxylin
and eosin (HE). Selected samples were also stained
with periodic acideSchiff (PAS) and Grocotts
methenamine silver nitrate (GMS) stain.
Microscopically, there was severe supercial epi-
dermal pustule formation and these pustules con-
tained mainly heterophils. Secondary erosion and
ulceration was observed in some areas. Numerous
PAS- and GMS-positive fungal hyphae were associ-
ated with the lesions. These hyphae were branched,
septate and approximately 1.4e3.6 mm in diameter.
Some hyphae were present in the dermis where
they were associated with an inammatory inltra-
tion of macrophages and lymphocytes. A discrete
number of small granulomata were also present in
the dermis and these contained some hyphae in
the central necrotic areas. No bacterial colonies
were observed. Both kidneys showed numerous ur-
ate tophi associated with a mixed inammatory re-
action. No histological lesions were detected in
other major organs.
Because fungal cultures were not taken at the time
of necropsy examination, an immunohistochemical
study was carried out in order to determine the iden-
tity of the organism. Sections of the cutaneous lesions
were mounted on adhesive slides (Superfrost Plus,
Menzel-Glaser, Braunschweig, Germany) and kept
at 4

C until processed. Primary reagents for immuno-

labelling included monoclonal antibodies specic for
antigens of Aspergillus spp. (WF-AF-1; Dako,
Glostrup, Denmark) and the Mucorales group
(WSSA-RA-1; Dako) and rabbit polyclonal anti-
bodies specic for Candida spp. and Trichophyton spp.
(Trichophyton verrucosum and Trichophyton mentagro-
phytes) (Jensen et al., 1996). Genus-specic rabbit
polyclonal antibodies raised against Aspergillus fumiga-
tus, Aspergillus avus, Aspergillus niger, Geotrichum candi-
dum, Fusarium solani and Scedosporium apiospermum
were also applied (Jensen et al., 1996). All polyclonal
antibodies were absorbed against other fungal organ-
isms (Okuda et al., 1987; Jensen et al., 1996). The spec-
icity and dilutions of primary antibodies are
summarized in Table 1. A commercial detection sys-
tem (Power-vision+ Poly-HRP Histostaining Kit;
Immunovision Technologies Co., Burlingame,
California, USA) was used to visualize immunolab-
elling. Application of this detection system was
followed by incubation for 6 min with 3-amino-9-
ethyl-carbazole (AEC) solution. The sections were
counterstained with Harriss haematoxylin for
10 sec. Tissues from mice and rabbits infected exper-
imentally with reference fungi were used as positive
controls (Jensen et al., 1996). In addition, cutaneous
lesions induced experimentally in two green iguanas
(Iguana iguana) by Chrysosporium anamorph of Nanniz-
ziopsis vriesii (University of Alberta Microfungus Col-
lection and Herbarium UAMH number 7583) were
used as negative tissue controls.
The fungal elements within the cutaneous lesions
were labelled only by the antiserum specic for
Trichophyton spp. (Fig. 2). No immunolabelling was
Fig. 1. Interscapular region of a Tenerife lizard (Gallotia galloti)
showing pustular and ulcerative skin lesions (arrow).
Table 1
Specicity and dilution of primary antibodies used in IHC
Antibody Fungal species Fungal specicity Dilution
Monoclonal Aspergillus spp. Aspergillus spp. 1 in 2
Monoclonal Mucorales (Zygomycetes) Mucorales (Zygomycetes) 1 in 4
Polyclonal Trichophyton mentagrophytes Trichophyton spp. 1 in 600
Polyclonal Trichophyton verrucosum Trichophyton spp. 1 in 600
Polyclonal Aspergillus fumigatus Aspergillus spp. 1 in 64
Polyclonal Candida albicans Candida spp. 1 in 256
Polyclonal Geotrichum candidum Geotrichum candidum 1 in 32
Polyclonal Fusarium solani Fusarium spp. 1 in 16
Polyclonal Scedosporium apiospermum Scedosporium spp. 1 in 16
Dermatophytosis in a Tenerife Lizard 373
observed in the tissues from iguanas infected experi-
mentally with Chrysosporium anamorph of N. vriesii.
There are no reports of Trichophyton spp. causing
skin disease or systemic mycotic infection in reptiles
of the family Lacertidae and reports of dermatophyto-
sis in reptiles are rare. Trichophyton terrestre was isolated
fromlesions in a group of blue-tongued skinks (Tiliqua
scincoides) with progressive digital necrosis (Hazell
et al., 1985), but causality was unclear (Pare and
Jacobson, 2007). In addition, the recognition over
the last decade of the Chrysosporium anamorph of
N. vriesii as a major cause of hyalohyphomycosis in
reptiles (Pare et al., 1997, 2006; Abarca et al., 2009;
Hellebuyck et al., 2010), casts some doubt on the au-
thenticity of previously reported cases of infection
with Trichophyton spp., Geotrichum spp., Chrysosporium
spp., Malbranchea spp. and Trichosporon spp., as these
fungi may be readily confused with each other (Pare
and Jacobson, 2007).
However, Trichophyton spp. was isolated from two
green anacondas (Eunectes murinus) with dermal
lesions (Miller et al., 2004) and recently, IHC was
used to identify Trichophyton spp. associated with sys-
temic lesions in an olive ridley sea turtle (Lepidochelys
olivacea) (Oros et al., 2011). The positive immunolab-
elling of the fungal elements in this lizard with anti-
bodies specic for Trichophyton spp. and the absence
of immunoreaction when using these antibodies on
tissues containing the Chrysosporium anamorph of
N. vriesii, conrms the specicity of the antibodies
and the aetiology of the mycotic infection.
The origin of the fungal infection in this lizard
remains unknown. The genus Trichophyton includes
anthropophilic, zoophilic and geophilic species, but
cross infections are frequent (Oros et al., 2011). Super-
cial dermatomycoses occur when the skin comes in
contact with spores or conidia of pathogenic fungi
that colonize the outer epidermal layers. Invasion of
the fungal hyphae into the stratum spinosum and
stratum germinativum triggers a heterophilic re-
sponse, typically, but not always, leading to coagula-
tive and liquefactive necrosis of the epidermis
surrounding the fungal elements (Pare and
Jacobson, 2007).
The diagnosis of cutaneous or systemic infections in
reptiles is usually achieved by culture or histopathol-
ogy. More recently, molecular techniques for fungal
identication frompure cultures have been developed
(Borman et al., 2008). However, a rm diagnosis of
mycosis can never rely solely on isolation of a fungus
from clinical material and can only be established if
fungal elements are demonstrated histologically
within the lesions and inammation is present around
the fungal elements. IHC has proven to be a powerful
tool for the accurate diagnosis of a number of impor-
tant mycoses in man and domestic animals (Jensen
et al., 1996) and in sea turtles (Oros et al., 2004a, b;
2011), especially when cultures are not available.
This case suggests that Trichophyton spp. should be
included as a possible aetiology in the differential di-
agnosis of cutaneous mycoses in lizards. In addition,
exotic animal veterinarians should be aware of the
availability of immunohistochemical techniques for
identifying fungi in reptiles.
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Received, September 24th, 2012

Accepted, November 29th, 2012

Dermatophytosis in a Tenerife Lizard 375