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.