Abstract

Snake fungal disease (SFD) is an emerging disease of wildlife believed to be caused by Ophidiomyces ophiodiicola. Although geographic and host ranges have yet to be determined, this disease is characterized by crusty scales, superficial pustules, and subcutaneous nodules, with subsequent morbidity and mortality in some snake species. To confirm the presence of SFD and O. ophiodiicola in snakes of eastern Virginia, US, we clinically examined 30 free-ranging snakes on public lands from April to October 2014. Skin biopsy samples were collected from nine snakes that had gross lesions suggestive of SFD; seven of these biopsies were suitable for histologic interpretation, and eight were suitable for culture and PCR detection of O. ophiodiicola. Seven snakes had histologic features consistent with SFD and eight were positive for O. ophiodiicola by PCR or fungal culture.

Snake fungal disease (SFD) is an emerging wildlife disease associated with Ophidiomyces (formerly Chrysosporium) ophidiicola; both the disease and the fungus have been increasingly reported and recognized in wild snake populations in the eastern and midwestern US since 2006 (Latney and Wellehan 2013; Dolinski et al. 2014; Sutherland et al. 2014; McBride et al. 2015). Snake fungal disease has been associated with morbidity and mortality in a wide variety of species of North American snakes, including the eastern massasauga rattlesnake (Sistrurus catenatus), eastern rat snake (Pantherophis alleghaniensis), corn snake (Pantherophis guttatus), timber rattlesnake (Crotalus horridus), eastern diamondback rattlesnake (Crotalus adamanteus), salt marsh snake (Nerodia clarkii), milk snake (Lampropeltis sp.), and plains garter snake (Thamnophis radix) (Rajeev et al. 2009; Allender et al. 2011; Latney and Wellehan 2013; Sigler et al. 2013; Dolinski et al. 2014; McBride et al. 2015). Currently, the population impacts of SFD are unknown. To our knowledge, SFD has never been confirmed in Virginia, US.

Common clinical signs of fungal disease associated with O. ophiodiicola include scabs or crusty scales, with superficial pustules and subcutaneous nodules, dysecdysis, and ocular cloudiness (Dolinski et al. 2014; McBride et al. 2015; Tetzlaff et al. 2015); some crusty or pustular scales may shed to reveal pinkish-red ulcers. In extreme cases, facial swelling and sometimes severe facial disfiguration have been reported (Rajeev et al. 2009; Allender et al. 2011; Latney and Wellehan 2013; Tetzlaff et al. 2015). The precise mechanism resulting in mortality among some snakes with SFD remains unclear and is likely multifactorial. Reported pathologic findings of infections associated with O. ophiodiicola include cutaneous hyperkeratosis, granulomatous dermatitis, and ulceration, with necrosis and inflammation that extends into the dermis, skeletal muscle, and bones near areas of hyphal invasion (Allender et al. 2011; Latney and Wellehan 2013; McBride et al. 2015).

Native, nonvenomous snakes were captured opportunistically on a variety of public lands in eastern Virginia from April to October 2014. Five primary sites were chosen based on where snakes were observed regularly, indicating adequate population density for easy collection and where there had been sightings of snakes with ocular and facial lesions. Study sites were sampled repeatedly throughout the study period; snakes were typically captured between 0800 and 1300 hours. Appropriate permits were obtained from state and local governmental authorities (Virginia Department of Game and Inland Fisheries permit 048445, Virginia Department of Conservation and Recreation permit FC-RCP-062413, US Fish and Wildlife Service [USFWS) Back Bay National Wildlife Refuge [NWR) permit BKB-A Guthrie, USFWS Great Dismal Swamp NWR permit R2014-09). This study was approved by the animal care and use committee of the Virginia Zoo (13-006).

Snake species included in this study were the brown water snake (Nerodia taxispilota), rainbow snake (Farancia erytrogramma), northern water snake (Nerodia sipedon), eastern black racer (Coluber constrictor), eastern ribbon snake (Thamnophis sauritus sauritus), eastern rat snake (Pantherophis alleghaniensis), eastern king snake (Lampropeltis getula getula), and eastern garter snake (Thamnophis sirtalis sirtalis).

Snakes were manually restrained by staff who wore clean latex or nitrile exam gloves that were changed after each snake restraint. Each snake was given a thorough physical exam, and standard morphometric data were collected. A transponder (AVID Identification Systems, Inc., Norco, California, USA) was placed subcutaneously in the left cranial one third of the body for permanent identification in all snakes weighing >100 g. Photographs were taken of each snake, and any skin lesions were measured and recorded. Sex was determined for each snake using standard methods (Mader 2006). The capture location of each snake was recorded with a handheld GPS unit and coordinates were mapped using ArcGIS 10.3 (ESRI, Redlands, California, USA; Fig. 1).

Figure 1. 

Locations of free-ranging snakes collected from April to October 2014 in eastern Virginia, USA. Snakes were evaluated for gross skin lesions and the presence of the fungus Ophidiomyces ophiodiicola by histopathology and PCR. Symbols represent positive (nine snakes; skin lesions and confirmed for O. ophiodiicola infection via histopathology or PCR), negative (18 snakes; no skin lesions), or suspect (three snakes; skin lesions but no diagnostics performed).

Figure 1. 

Locations of free-ranging snakes collected from April to October 2014 in eastern Virginia, USA. Snakes were evaluated for gross skin lesions and the presence of the fungus Ophidiomyces ophiodiicola by histopathology and PCR. Symbols represent positive (nine snakes; skin lesions and confirmed for O. ophiodiicola infection via histopathology or PCR), negative (18 snakes; no skin lesions), or suspect (three snakes; skin lesions but no diagnostics performed).

Thirty snakes were examined, and biopsy samples were collected from nine snakes with obvious skin lesions (Table 1). Approximately 5 min before biopsy, local anesthesia was provided with 2 mg/kg lidocaine (20 mg/mL) and 1 mg/kg bupivacaine (5 mg/mL) diluted in a small volume of sterile water (all from Hospira, Inc., Lake Forest, Illinois, USA) (Carpenter 2013). In snakes with large lesions, two full-thickness skin biopsies were taken at the margin of the skin lesion by using a sterile disposable 5-mm biopsy punch tool. For snakes with mild scale lesions or pustules, scales were removed easily with tissue forceps. For one eastern rat snake, the distal end of the tail was desiccated and necrotic and was manually removed. One tissue sample was placed in a small tube containing 10% neutral buffered formalin for subsequent histopathologic examination. When more than one area could be biopsied, a second tissue sample was stored frozen in a cryovial, without preservative, for fungal culture and PCR-based detection of O. ophiodiicola. Each biopsy site was sutured with absorbable 4-0 polyglactin suture (Vicryl, Ethicon, LLC, Guaynabo, Puerto Rico) with a single cruciate suture. Snakes were given a meloxicam injection (5 mg/mL, Norbrook Laboratories Limited, Corby, UK) 0.5 mg/kg subcutaneously for pain management (Carpenter 2013). Snakes were handled for 15 min or less and then released at the capture location. All surgical instruments and equipment were cleaned of debris and disinfected between snakes by using 10% chlorine bleach solution that was allowed to air dry.

Table 1. 

Summary of snakes captured and evaluated for snake fungal disease in eastern Virginia, USA, 2014. Blank cells indicate that snakes had no gross skin lesions and skin biopsy was not performed.

Summary of snakes captured and evaluated for snake fungal disease in eastern Virginia, USA, 2014. Blank cells indicate that snakes had no gross skin lesions and skin biopsy was not performed.
Summary of snakes captured and evaluated for snake fungal disease in eastern Virginia, USA, 2014. Blank cells indicate that snakes had no gross skin lesions and skin biopsy was not performed.
Table 1. 

Continued.

Continued.
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For histopathology, formalin-fixed skin biopsies were processed routinely, embedded in paraffin, and sectioned at approximately 5 μm. Tissue sections were stained with H&E and the periodic acid-Schiff reaction (PAS) and examined by light microscopy.

For O. ophiodiicola detection, frozen skin biopsies were thawed and divided in half. One portion of each biopsy was placed on dermatophyte test medium for culture analysis and incubated at 24 C. Cultured fungi were identified by amplifying and sequencing DNA from a portion of the internal transcribed spacer region according to Lorch et al. (2015). The remaining portion of each biopsy was screened for O. ophiodiicola by real-time PCR according Bohuski et al. (2015). Samples for which the cycle threshold was ≤36 were considered positive for O. ophiodiicola.

Of 30 snakes captured for this study, 12 (40%) had gross lesions consisting of pustules, dry thickened and crusty scales, dysecdysis, or ocular opacity (Fig. 2A, B). Two gravid females and one snake weighing <100 g with skin lesions were exempted from biopsy. Biopsies examined from two of nine affected snakes were considered nondiagnostic because dermis was not present in the histologic sections. Biopsies from the remaining seven affected snakes had histologic features consistent with infections associated with O. ophiodiicola, including epidermal thickening, inflammation and necrosis, ulcerative dermatitis, and granulomatous cellulitis with intralesional PAS-positive fungal hyphae (Allender et al. 2011; Latney et al. 2013; McBride et al. 2015). The majority of the fungal hyphae measured 2–5 µm in diameter, but occasional hyphae were as large as 10 μm in diameter, and the morphology of fungi observed in the sections was similar to those previously reported for infections associated with O. ophiodiicola (Allender et al. 2011; Dolinski et al. 2014; Fig. 2C, D).

Figure 2. 

Gross images and histopathology from free-ranging snakes, Virginia, USA, 2014, infected with the fungus Ophidiomyces ophiodiicola. (A) Skin, rainbow snake (Farancia erytrogramma). Multiple dry, thickened, and slightly raised scales and subcutaneous pustules. (B) Skin, eastern racer (Coluber constrictor). Multiple large areas of dry, thickened, and crusty scales. (C) Skin, eastern racer (Coluber constrictor). The superficial epidermis is thickened by necrotic debris and mixed inflammatory cells with Periodic acid-Schiff (PAS)–positive fungal hyphae (double arrow). Low numbers of granulocytes and macrophages are present in the dermis. PAS. Bar=50 µm. (D) Skin, northern water snake (Nerodia sipedon). The epidermis is replaced by a thick layer of necrotic debris admixed with numerous 2–5 µm in diameter PAS-positive fungal hyphae with parallel to undulating walls and occasional septations (arrow) and branching. Arthroconidia (*) are present on the epidermal surface. PAS. Bar=20 µm.

Figure 2. 

Gross images and histopathology from free-ranging snakes, Virginia, USA, 2014, infected with the fungus Ophidiomyces ophiodiicola. (A) Skin, rainbow snake (Farancia erytrogramma). Multiple dry, thickened, and slightly raised scales and subcutaneous pustules. (B) Skin, eastern racer (Coluber constrictor). Multiple large areas of dry, thickened, and crusty scales. (C) Skin, eastern racer (Coluber constrictor). The superficial epidermis is thickened by necrotic debris and mixed inflammatory cells with Periodic acid-Schiff (PAS)–positive fungal hyphae (double arrow). Low numbers of granulocytes and macrophages are present in the dermis. PAS. Bar=50 µm. (D) Skin, northern water snake (Nerodia sipedon). The epidermis is replaced by a thick layer of necrotic debris admixed with numerous 2–5 µm in diameter PAS-positive fungal hyphae with parallel to undulating walls and occasional septations (arrow) and branching. Arthroconidia (*) are present on the epidermal surface. PAS. Bar=20 µm.

Eight of nine biopsied snakes were screened for O. ophiodiicola by PCR or fungal culture; the remaining animal was evaluated by histopathology only. Of these, five (62%) were culture positive and all eight were PCR positive for O. ophiodiicola. Female snakes were twice as likely to have skin lesions (odds ratio = 2.33), although the fungus was detected in both sexes. A large proportion of snakes captured in April (73%) had skin lesions compared to snakes captured in May (0%) and June (25%). No snakes with skin lesions were captured after mid-July. Although this may be due to the small number of snakes captured after July, it is possible that snakes emerge from brumation with skin lesions and either succumb to the infection shortly thereafter or the condition of their skin improves and they recover by summer. Snake fungal disease–like skin lesions observed in two gravid snakes captured in July suggest the stress of pregnancy may, however, predispose females to an increased risk of exposure or the inability to clear the infection.

In this study, O. ophiodiicola was confirmed in three northern water snakes, one rainbow snake, two eastern racers, and two brown water snakes, in eastern Virginia. The diversity of snakes involved at one study site (Back Bay NWR) supports O. ophiodiicola transmission via an environmental reservoir. Ophidiomyces ophiodiicola is a host-specific, keratinophilic fungus that may act as a primary pathogen (Allender et al. 2013, 2015; Cabanes et al. 2014). Infection most commonly presents as a dermatomycosis, but may become systemic and cause mortality in snakes (Latney et al. 2013; Dolinski et al. 2014; McBride et al. 2015; Tetzlaff et al. 2015). Snakes in this study were in good body condition and seemed healthy overall. Affected snakes exhibited mild skin lesions, but we observed no emaciation or severe facial disfiguration as reported in massasaugua rattlesnakes from Illinois, US (Allender et al. 2011). This suggests that the severity of disease caused by this fungus is likely variable between individuals or snake species. We believe that these results represent the first confirmed cases of fungal dermatitis associated with O. ophiodiicola in Virginia and the first reports of SFD in the rainbow snake and brown water snake. Further research into this potentially significant emerging wildlife disease is warranted.

This project was made possible through the research grant awarded generously by the Virginia Herpetological Society. Special thanks to Kory Steele for GIS mapping work and to Yohn Sutton for enthusiasm and dedication to helping capture snakes in the field. Thanks to the Virginia Zoo for tremendous support throughout this project. We thank Elizabeth Bohuski, Kathryn Griffin, Katie Schmidt, and Stephanie Steinfeldt at the US Geological Survey–National Wildlife Health Center for assistance with laboratory testing. Special thanks to local field biologists for observational information and recommended locations for finding snakes. Thanks to all the volunteers who helped capture snakes. The use of trade, product or firm names is for descriptive purposes only and does not imply endorsement by the US government.

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