Histopathologic criteria to confirm white-nose syndrome in bats
Carol Uphoff Meteyer,1 Elizabeth L. Buckles, David S. Blehert, Alan C. Hicks, D. Earl Green,
Valerie Shearn-Bochsler, Nancy J. Thomas, Andrea Gargas, Melissa J. Behr
Abstract. White-nose syndrome (WNS) is a cutaneous fungal disease of hibernating bats associated with a
novel Geomyces sp. fungus. Currently, confirmation of WNS requires histopathologic examination. Invasion
of living tissue distinguishes this fungal infection from those caused by conventional transmissible
dermatophytes. Although fungal hyphae penetrate the connective tissue of glabrous skin and muzzle, there
is typically no cellular inflammatory response in hibernating bats. Preferred tissue samples to diagnose this
fungal infection are rostral muzzle with nose and wing membrane fixed in 10% neutral buffered formalin. To
optimize detection, the muzzle is trimmed longitudinally, the wing membrane is rolled, and multiple cross-
sections are embedded to increase the surface area examined. Periodic acid–Schiff stain is essential to
discriminate the nonpigmented fungal hyphae and conidia. Fungal hyphae form cup-like epidermal erosions
and ulcers in the wing membrane and pinna with involvement of underlying connective tissue. In addition,
fungal hyphae are present in hair follicles and in sebaceous and apocrine glands of the muzzle with invasion of
tissue surrounding adnexa. Fungal hyphae in tissues are branching and septate, but the diameter and shape of
the hyphae may vary from parallel walls measuring 2 mm in diameter to irregular walls measuring 3–5 mm in
diameter. When present on short aerial hyphae, curved conidia are approximately 2.5 mm wide and 7.5 mm in
curved length. Conidia have a more deeply basophilic center, and one or both ends are usually blunt.
Although WNS is a disease of hibernating bats, severe wing damage due to fungal hyphae may be seen in bats
that have recently emerged from hibernation. These recently emerged bats also have a robust suppurative
inflammatory response.
Key words: Bats; emerging disease; fungus; Geomyces sp.; hibernation; Myotis; skin erosion.
White-nose syndrome (WNS) has caused mortality in
hundreds of thousands of little brown bats (Myotis
lucifugus) since first reported by biologists in 2007, yet
the disease is still poorly understood.1 Initially detected
near Albany, New York, this fungal disease of cave- and
mine-hibernating bats has spread rapidly to hibernacula
in Vermont, Massachusetts, Connecticut, Pennsylvania,
New Jersey, Virginia, West Virginia, and New Hamp-
shire. In addition to little brown bats, other species in
which WNS has since been diagnosed include tricolored
bats (Pipistrellus subflavus), northern long-eared bats
(Myotis septentrionalis), and endangered Indiana (Myo-
tis sodalis) and big brown (Eptesicus fuscus) bats.
The classic presentation of WNS in affected bats
living in caves and mines includes the delicate,
exuberant, white filaments that obscure the muzzle.1
Fungus on the wings of these bats can appear as an
opaque white, tacky film of varying density (Fig. 1A).
Grossly visible fungus is not always seen on bats with
WNS, and the white facial plume and white fungus on
the surface of wings is usually lost when bats are
removed from hibernacula and prepared for shipping.
Once received in the laboratory, gross signs of fungal
infection in bats are subtle and difficult to detect. The
small size of little brown bats (5–7 g) make lighted
magnifying loops an asset for examining skin. Changes
seen in the skin of affected bats are inconsistent and
nonspecific, including patches of rough skin on the
face, ears, forearms, wing membranes, and feet as well
as pinpoint white foci that resemble comedones on the
muzzle. Less obvious changes are loss of sheen on
glabrous skin and irregular pigmentation with areas of
contraction or small tears in wing membranes. Back-
lighting of extended wings using a light box improves
detection of these changes (Fig. 1B).
Although gross lesions can be suggestive of WNS,
histopathologic examination is necessary to confirm
this disease. To optimize microscopic detection of
fungal hyphae, surface area of examined skin was
maximized. Muzzle and nose, once dissected from
underlying bone and fixed in 10% neutral buffered
formalin, were trimmed into multiple longitudinal
sections perpendicular to the surface of skin. The skin
From the National Wildlife Health Center, U.S. Geological
Survey, Madison, WI (Meteyer, Blehert, Green, Shearn-Bochsler,
Thomas), Cornell University, Ithaca, NY (Buckles), the New
York Department of Environmental Conservation, Albany, NY
(Hicks), the New York State Department of Health, Wadsworth
Center, Axelrod Institute, Albany, NY (Behr), and Symbiology
LLC, Middleton, WI (Gargas).
1 Corresponding Author: Carol Uphoff Meteyer, National
Wildlife Health Center, 6006 Schroeder Road, Madison WI 53711.
cmeteyer@usgs.gov
J Vet Diagn Invest 21:411–414 (2009)
411

was embedded with trimmed surfaces presented for
sectioning to maintain the orientation of the fungus to
dermal structures.
Wing membrane was sampled in multiple rectangu-
lar pieces. Each piece of membrane was dipped in
formalin, carefully unfolded over a gloved finger, and
rolled along the short end onto wooden dowels
approximately 0.2 cm in diameter and 3 cm long. After
fixation, the dowels were removed from the rolls of
wing membrane, and skin rolls were trimmed into
multiple cross-sections with the cut surfaces embedded
for sectioning and staining. Various fungal stains were
initially applied to the tissue sections, but periodic acid–
Schiff stain (PAS) proved superior for the microscopic
detection of nonpigmented fungal hyphae, appreciation
of the pattern of fungal skin invasion (Fig. 2A–2C),
and recognition of conidia (Fig. 2D).
The wing membrane is composed of 2 single-cell
layers of epidermis separated by a thin layer of
connective tissue with elastin fibers.3 Adnexa are only
present in wing membrane near the arms and legs.
The mildest microscopic changes seen in the wing
membranes were cup-like epidermal erosions that
were filled with fungal hyphae. Ulceration and fungal
invasion of underlying connective tissue was common
(Fig. 2B–2D) and could span the full thickness of the
wing membrane. When the muzzle was involved,
fungal hyphae filled hair follicles, invaded sebaceous
and apocrine glands, and extended into the regional
connective tissue obscuring epithelial boundaries of
the adnexa (Fig. 3A, 3B). Typically, there was an
absence of inflammation in the skin of hibernating
bats even with extensive fungal invasion (Figs. 2, 3),
which was random and nonangiotrophic. Samples of
wing membranes from bats euthanized in caves and
immediately fixed in formalin had more abundant
aerial fungal growth and conidia on the surface of the
skin, as well as the characteristic epidermal ulceration
and connective tissue invasion (Fig. 2C, 2D).
Lack of inflammation in response to fungal hyphae
was surprising. However, tissue invasion noted in
samples fixed immediately after euthanasia of bats
within hibernacula provided evidence that invasion of
living tissue was an antemortem event. When inflam-
mation was present in the skin of hibernating bats,
edema and neutrophils were observed microscopically
in the regional connective tissue, occasionally with
intradermal abscesses. The presence of bacteria was
inconsistent but common in bats with inflammation.
Typical transmissible dermatophytes of mammals only
invade the nonliving structures of skin (keratin, hair,
and nails),4 in contrast to the extensive invasion of skin
and underlying connective tissue in bats with WNS.
Winter bats with heavy fungal burdens were emaciated,
but there were no consistent microscopic lesions in
tissues other than skin.
Unlike bats in hibernation, bats with visibly
damaged wings that were collected outside hibernac-
ula in May had severe inflammation associated with
fungal infection. Histologic changes included suppu-
rative dermatitis with folliculitis, edema, and scat-
tered infiltrates of macrophages. Frequent serocellu-
lar inflammatory crusts containing fungal hyphae
were present over the intact epidermis (Fig. 3C). Bats
collected shortly after hibernation also had small
quiescent packets of fungal hyphae within the dermis
that were surrounded by a thin layer of acellular
material (Fig. 3D).
Fungal hyphae in tissue sections were branching
and septate (Fig. 3B) with variable morphology
ranging from parallel walls measuring 2 mm in
diameter to irregular, bulging, or globose walls
measuring 3–5 mm in diameter (Fig. 3B). When
present on short aerial hyphae, curved conidia were
Figure 1. Myotis lucifugus with white-nose syndrome, New York. A, little brown bat with white foci of fungus on the face
(arrowhead) and an irregular pattern of white fungus on the wing (long arrows) and ear (short arrow). B, little brown bat submitted to
the National Wildlife Health Center, Madison, Wisconsin. The image illustrates the taping method used to extended wings, which are
backlit on a light box. Damage to wing with contraction of wing membrane (white arrow) and loss of pigmentation (black arrows). Bar
5 2.5 cm.
412 Meteyer et al.