Abstract
Ophidiomycosis (snake fungal disease) is the most common cause of skin lesions in free-ranging snakes in North America. Naturally infected snakes with ophidiomycosis (9 carcasses, 12 biopsies) were examined grossly and histologically. These cases comprised 32% of the 66 snake cases submitted to the Canadian Wildlife Health Cooperative-Ontario/Nunavut Node in 2012 through 2018. Affected species included the eastern foxsnake (Pantherophis vulpinus; n = 15), gray ratsnake (Pantherophis spiloides; n = 3), eastern massasauga (Sistrurus catenatus; n = 2), and queensnake (Regina septemvittata; n = 1). Severity of disease varied widely from mild microscopic skin lesions to fatal, necrotizing, and ulcerative facial lesions.
Key clinical message:
Ophidiomycosis should be the primary differential diagnosis for skin lesions in wild snakes, particularly in southern Ontario.
Résumé — Pathologie associée avec l’ophidiomycose chez des serpents sauvages en Ontario, Canada
L’ophidiomycose (maladie fongique du serpent) est la cause la plus fréquente de lésions cutanées chez les serpents en liberté en Amérique du Nord. Les serpents infectés naturellement avec l’ophidiomycose (9 carcasses, 12 biopsies) furent examinés macroscopiquement et histologiquement. Ces cas comprenaient 32 % des 66 cas de serpents soumis au Réseau canadien pour la santé de la faune – Centre régional de l’Ontario et du Nunavut entre 2012 et 2018. Les espèces affectées incluaient la couleuvre fauve de l’est (Pantherophis vulpinus; n = 15), la couleuvre obscure (Pantherophis spiloides; n = 3), la massasauga (Sistrurus catenatus; n = 2) et la couleuvre royale (Regina septemvittata; n = 1). La sévérité de la maladie variait grandement allant de lésions cutanées microscopiques à une forme fatale, nécrosante et lésions faciales ulcératives.
Message clinique clé :
L’ophidiomycose devrait être le diagnostic différentiel primaire pour les lésions cutanées chez les serpents sauvages, particulièrement dans le sud de l’Ontario.
(Traduit par Dr Serge Messier)
Introduction
Ophidiomycosis, also known as snake fungal disease, is caused by the fungus Ophidiomyces ophiodiicola and can cause facial swelling, granulomatous dermatitis, and death in both captive and free-ranging snakes (1–3). The disease was first described in a free-ranging population of timber rattlesnakes (Crotalus horridus) in 2011 in New Hampshire, and has since been documented in over 30 species, encompassing 6 families of wild snakes across North America and in some areas of Europe (1,3–5). Furthermore, O. ophiodiicola is thought to be the most common cause of skin lesions in free-ranging snakes in the eastern United States (3,6). The observed prevalence and severity of ophidiomycosis lesions vary seasonally in some species. Clinical disease in snakes often corresponds to their emergence from brumation, a time of severe energy restriction and communal behavior in snakes (1,7,8). In free-ranging snakes, the development of ophidiomycosis is likely multifactorial, and the mechanisms by which it causes mortality are currently unknown (1,8). Thirteen of Ontario’s native, free-ranging snake species are considered species at risk (9), 4 of which have been found with ophidiomycosis as discussed in this study. They are subject to numerous threats, including habitat loss and degradation, vehicular trauma, and deliberate persecution by humans, in addition to the potential threat posed by infectious diseases, such as ophidiomycosis (9). We sought to describe the pathology of ophidiomycosis in naturally infected free-ranging snakes in Ontario, Canada, at the northern limit of the currently recognized range of O. ophiodiicola.
Materials and methods
All snake cases diagnosed with ophidiomycosis at the Canadian Wildlife Health Cooperative (CWHC) Ontario/Nunavut node from 2012–2018 (including whole carcasses and snake skin biopsies) that met the following criteria were reviewed: available formalin-fixed, paraffin-embedded tissues, a positive quantitative polymerase chain reaction (qPCR) result for O. ophiodiicola, and microscopically visible fungal elements. For each snake, the species, sample submission type, diagnosed cause of death, and gross lesions were recorded. Before 2012, no snakes submitted to the CWHC in Ontario were reported to have lesions consistent with ophidiomycosis. Before 2015, qPCR for O. ophiodiicola was not available at the provincial diagnostic laboratory (Animal Health Laboratory, University of Guelph, Guelph, Ontario) and thus, frozen skin samples collected from 2012 and 2014 were retrospectively tested by qPCR. Whole carcasses were typically frozen before submission. After thawing, the skin over the dorsal, ventral, and lateral body walls was swabbed, as well as any grossly visible skin lesions, with a polyester-tipped applicator and a standard necropsy was performed. A common alternative to carcass submission was field collection of a skin biopsy sample from live snakes along with an additional section of fresh skin or a skin swab for the qPCR test. Biopsy samples submitted in lysis buffer were placed in 10% neutral-buffered formalin for at least 24 h before processing for histopathology. Formalin-fixed samples were routinely processed, embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin (H&E), as well as Gomori’s methenamine silver (GMS) stain. When a fresh skin or swab sample was not available, the lysis buffer fluid that contained the skin biopsy was tested by qPCR. In 1 case, formalin-fixed paraffin-embedded tissue was deparaffinized and used for qPCR analysis. When multiple tissues were submitted for qPCR from the same snake, the sample with the greatest amount of fungal DNA, as indicated by the lowest cycle threshold value, was used for analysis.
Histopathology scoring was performed on the most affected skin section from each snake, based on estimated percentage of the section with a lesion. Inflammation and necrosis were subjectively scored based on depth and amount of tissue affected as 0, 1, 2, or 3. The presence or absence of serocellular crusting and ulceration was also scored. A histologic grade (Table 1) was then assigned to each snake skin as 0 (i.e., absent), I (i.e., mild; Figure 1a), II (i.e., moderate; Figure 1b), and III (i.e., severe; Figure 1c). In many biopsy samples, deep muscle involvement could not be assessed due to superficial sampling; thus, the maximum number of points for skin inflammation in these cases was 2.
Table 1.
Grading scheme for Ophidiomyces ophiodiicola microscopic skin lesions in snakes.
| Skin histologic feature score | ||
|---|---|---|
|
| ||
| Feature | Description | Points |
| Inflammation | • None | 0 |
| • Heterophils in epidermis | 1 | |
| • Mixed inflammation, dermal involvement | 2 | |
| • Mixed inflammation, muscle involvement | 3 | |
| Necrosis | • None | 0 |
| • Minimal | 1 | |
| • Coalescing areas, < 1 scale width | 2 | |
| • Coalescing areas, > 1 scale width | 3 | |
| Other | • Serocellular crust | 1 |
| • Ulceration | 1 | |
|
| ||
| Skin grade determination | ||
|
| ||
| Histologic grade | Total points | |
|
| ||
| 0 | 0 | |
| I | 1–3 | |
| II | 4–5 | |
| III | 6–8 | |
Figure 1.
Natural Ophidiomyces ophiodiicola infections in snakes from Ontario, Canada. a — Skin, Pantherophis vulpinus, grade I lesion with heterophilic inflammation limited to the epidermis and serocellular crusting with intralesional fungal hyphae, antemortem biopsy sample, snake W.B.01. Hematoxylin and eosin (H&E) stain. Scale bar = 50 μm. b — Skin, P. vulpinus, grade II lesion with heterophilic and granulomatous inflammation extending into the dermis, with a small amount of necrosis, serocellular crusting and ulceration, postmortem specimen, snake W.C.02. H&E stain. Scale bar = 100 μm. c — Skin, P. vulpinus, grade III lesion with heterophilic and granulomatous inflammation extending into the skeletal muscle, with extensive necrosis, serocellular crusting and ulceration, antemortem biopsy sample, snake W.B.05. H&E stain. Scale bar = 500 μm. d — Head, Sistrurus catenatus, severe, locally extensive swelling, crusting and ulceration with oral and ocular involvement, postmortem specimen, snake W.C.08. e — Head, S. catenatus, severe, heterophilic and granulomatous, necroulcerative dermatitis, cellulitis, and myositis (black arrowhead), stomatitis (black chevron), with serocellular crusts over the head and eyes (asterisk), postmortem specimen, snake W.C.09. There is also osteomyelitis and encephalitis in this specimen, not visible at this low magnification. H&E stain. Scale bar = 2 mm. f — Scale, S. catenatus, a large number of O. ophiodiicola hyphae (black arrowhead) and arthroconidia (black chevron) in the keratin of a scale overlying necrotic tissue, postmortem specimen, snake W.C.09. Gomori’s methenamine silver stain. Scale bar = 20 μm. g — Lung, S. catenatus, heterophilic and granulomatous fungal pneumonia, postmortem specimen, snake W.C.08. H&E stain. Scale bar = 200 μm. h — Skin, Pantherophis spiloides, thickened, irregular and dull scales on the lateral body wall (white asterisk), postmortem specimen, snake W.C.07.
Nucleic acid (DNA) was extracted from fresh-frozen skin swabs, fresh-frozen skin, and formalin-fixed paraffin-embedded samples using a commercially available kit (DNeasy Plant mini Kit; Qiagen, Valencia, California, USA) following the manufacturer’s instructions. A TaqMan qPCR assay was performed using primers and probe from IDT (Coralville, Iowa, USA) and master mix from Roche (Laval, Quebec) to amplify the internal transcribed spacer between the 18S and 5.8S ribosomal RNA gene specific for O. ophiodiicola as described in Allender et al (10). All histology processing and staining and qPCR were performed at the Animal Health Laboratory (Guelph, Ontario).
Results
Of the 66 snake cases submitted to the CWHC from 2012 to 2018, 21 (32%) met the criteria of having both histological lesions consistent with ophidiomycosis and a positive qPCR result for O. ophiodiicola (9 carcasses, 12 skin biopsies; Table 2). Species included the eastern foxsnake (Pantherophis vulpinus; n = 15), gray ratsnake (P. spiloides; n = 3), eastern massasauga (Sistrurus catenatus; n = 2), and queensnake (Regina septemvittata; n = 1).
Table 2.
Summary of gross and microscopic lesions and qPCR results from carcasses and biopsy samples of wild snakes naturally infected with Ophidiomyces ophiodiicola in Ontario, Canada, 2012–2018.
| Submission | Case | Species | Year | Gross lesions | Skin | qPCR | |||||
|---|---|---|---|---|---|---|---|---|---|---|---|
|
|
|
||||||||||
| Inflammation | Necrosis | Crust | Ulceration | Histologic grade | Sample | Cycle threshold | |||||
| Carcass | W.C.01 | Pantherophis vulpinus | 2012 | − | 1 | 0 | 1 | 0 | Ia | fresh skin | 38.69 |
| Carcass | W.C.02 | P. vulpinus | 2014 | + | 2 | 1 | 1 | 1 | IIa | fresh skin | 23.35 |
| Carcass | W.C.03 | P. vulpinus | 2016 | − | 1 | 1 | 1 | 0 | IIa | skin swab | 34.42 |
| Carcass | W.C.04 | P. vulpinus | 2016 | − | 1 | 1 | 1 | 0 | Ia | skin swab | 26.85 |
| Carcass | W.C.05 | P. vulpinus | 2017 | + | 3 | 2 | 1 | 0 | IIIa | skin swab | 34.62 |
| Carcass | W.C.06 | P. vulpinus | 2018 | + | 3 | 3 | 1 | 1 | IIIa | skin swab | 36.83 |
| Carcass | W.C.07 | P. spiloides | 2018 | + | 2 | 2 | 1 | 0 | IIa | skin swab | 30.46 |
| Carcass | W.C.08 | Sistrurus catenatus | 2018 | + | 3 | 3 | 1 | 1 | IIIa | skin swab | 30.79 |
| Carcass | W.C.09 | S. catenatus | 2018 | + | 3 | 3 | 1 | 1 | IIIa | skin swab | 29.71 |
| Biopsy | W.B.01 | P. vulpinus | 2015 | + | 1 | 0 | 1 | 0 | Ia | shed skin | 21.48 |
| Biopsy | W.B.02 | P. vulpinus | 2016 | + | 2 | 3 | 1 | 1 | IIIa | fresh skin | 33.61 |
| Biopsy | W.B.03 | P. vulpinus | 2016 | + | 2 | 1 | 1 | 1 | IIa | FFPE | 23.10 |
| Biopsy | W.B.04 | Regina septemvittata | 2016 | + | 2 | 1 | 1 | 0 | IIa | skin swab | 33.95 |
| Biopsy | W.B.05 | P. vulpinus | 2017 | + | 3 | 3 | 1 | 1 | IIIa | skin swab | 27.69 |
| Biopsy | W.B.06 | P. spiloides | 2018 | + | NA | NA | 1 | NA | NAa | skin swab | 22.65 |
| Biopsy | W.B.07 | P. spiloides | 2018 | + | NA | NA | 1 | NA | NAa | skin swab | 33.66 |
| Biopsy | W.B.08 | P. vulpinus | 2018 | + | 2 | 3 | 1 | 1 | IIIa | lysis buffer | 24.27 |
| Biopsy | W.B.09 | P. vulpinus | 2018 | + | 2 | 3 | 1 | 1 | IIIa | lysis buffer | 20.49 |
| Biopsy | W.B.10 | P. vulpinus | 2018 | + | 2 | 3 | 1 | 1 | IIIa | lysis buffer | 19.49 |
| Biopsy | W.B.11 | P. vulpinus | 2018 | + | 2 | 3 | 1 | 1 | IIIa | lysis buffer | 24.68 |
| Biopsy | W.B.12 | P. vulpinus | 2018 | + | 3 | 3 | 1 | 1 | IIIa | lysis buffer | 24.73 |
NA — not applicable; + — present; − — absent; FFPE — formalin-fixed paraffin-embedded tissue.
Fungal hyphae observed in affected tissue.
The cause of death was attributed to ophidiomycosis in 3 of the 21 (14%) snakes (W.C.05, W.C.08, and W.C.09), 2 of which were eastern massasaugas. The 2 rattlesnakes had severe gross lesions with brown crusting over the head, affecting the spectacles and nasolabial pits, and swelling of the rostrum (Figure 1d). Caudal to the crusted areas, there was loosely adhered, unshed skin, indicating dysecdysis. The ventral and dorsal subcutis of both snakes had approximately twelve, 1- to 3-mm diameter, raised, firm, nodules randomly scattered along the length of the body. Snake W.C.09 had 2, firm, 5-mm diameter nodules within the caudal lung. Both snakes were in good nutritional condition with abundant intracoelomic and pericardial adipose tissue, as these 2 snakes were held in a rehabilitation facility and force fed for 5 wk before submission. Microscopically, these lesions corresponded to severe granulomatous and heterophilic, necroulcerative dermatitis, cellulitis, myositis, keratitis, stomatitis, and osteomyelitis with intralesional fungal hyphae and conidia consistent with O. ophiodiicola (Figure 1e). Fungal hyphae were amphophilic, 4 to 6 μm wide, septate, and had undulating walls and acute angle branching consistent with O. ophiodiicola. Conidia were rectangular and approximately 3 to 8 × 3 μm. The fungus stained positive with GMS (Figure 1f ). Both snakes had extensive, ≤ 150-μm deep foci of serocellular crusting and necrosis of the epidermis and dermis, affecting approximately 20% to 80% of the skin over the head. In the snout of snake W.C.09, there was extensive necrosis with aggregates of fungal hyphae that effaced the epidermis, dermis, skeletal muscle, bone, and nasal turbinates. In the more caudal portions of the head, fungal hyphae and associated inflammation extended from the skin into underlying bone and, to a lesser extent, the brain (Figure 1e). In the epidermis, fungal hyphae and conidia most often formed dense aggregates within the keratin and serocellular crust, underlain by a layer of heterophils and epithelioid macrophages. In the deeper tissues, fungal hyphae were most often found in a central area of necrosis, surrounded by a ring of epithelioid macrophages with interspersed heterophils that increased in number towards the periphery. The pulmonary nodules in snake W.C.09 were fungal granulomas, which were also seen in the lung of snake W.C.08, although they were not recognized grossly. Inflammation, hyphae and debris extended from the faveolar septa into airspaces and the central lumen (Figure 1g).
The third snake in which mortality was directly attributed to ophidiomycosis was an eastern foxsnake (W.C.05). The cause of death was determined to be emaciation (i.e., empty gastrointestinal tract, no fat bodies, and reduced muscle mass) secondary to facial ophidiomycosis lesions. There was an opaque, right, ocular spectacle and a 5-mm diameter, firm swelling at the caudal aspect of the right mandible. Microscopically, a heterophilic, serocellular crust containing fungal hyphae and conidia covered the corneal surface, with a granuloma in the adjacent eyelid and extraocular connective tissue. Over the mandibular skin, there was serocellular crusting and a 2-mm diameter granuloma in the underlying dermis, with heterophils and macrophages in the subjacent skeletal muscle.
In 2 additional snakes (W.C.06 and W.C.07), ophidiomycosis was considered to be an incidental finding or potentially contributing to death. In snake W.C.06, an eastern foxsnake, anesthetic complications during transmitter implantation surgery were considered the proximate cause of death. Gross lesions attributed to ophidiomycosis included three, 1- to 2-mm diameter crusts over the caudodorsal head, which corresponded to grade III skin lesions with dermal and skeletal muscle granulomas, ulceration and crusting with intralesional fungi. The proximate cause of death in snake W.C.07, a gray ratsnake, was determined to be septicemia secondary to traumatic tail amputation. There were two, 1.5 × 1.0 cm foci of crusting and irregular scales in the skin over the lateral body wall (Figure 1h), which corresponded to skin lesions with a histologic grade of II with inflammation in the dermis, and a moderate amount of necrosis and serocellular crusting.
Two snakes (eastern foxsnakes, W.C.01 and W.C.03) had microscopic lesions compatible with ophidiomycosis and tested qPCR positive for O. ophiodiicola, but the fungal infection was considered incidental due to lack of gross lesions and mild (grade I) histologic lesions. Snake W.C.01 was egg bound with resulting sepsis. Microscopically, W.C.01 had serocellular crusting affecting half of a single scale on the head, with rare fungal hyphae, and a few heterophils in the underlying dermis with no necrosis. Snake W.C.03 had extensive damage to the cranial portion of the body, attributed to vehicular-induced trauma. Microscopically, W.C.03 had a very similar lesion on the head, with a small amount of necrosis.
The cause of death was not determined in 2 snakes (W.C.02 and W.C.04, both eastern foxsnakes) with microscopic ophidiomycosis lesions that were considered subclinical (i.e., mild and of limited distribution) and positive qPCR results. In snake W.C.02, there were a few thickened scales grossly over the dorsal aspect of the head, which microscopically correlated to grade II skin lesions with primarily heterophilic inflammation. In snake W.C.04 there were no gross lesions of ophidiomycosis, but the snake was in poor nutritional condition and had numerous nematodes in the lungs, and a transmitter had been previously implanted in the coelomic cavity. Microscopically, there was a grade I skin lesion with heterophilic inflammation.
Of the 12 snakes for which skin biopsy samples were submitted, all were reported by the submitter to have had gross lesions. All samples had serocellular crusting with heterophils and visible fungal hyphae. Samples from 2 snakes (W.B.06 and W.B.07) were limited to crusts removed from the skin (presumed supraepidermal), with no epidermis or dermis for evaluation; these samples could not be graded. Among the remaining 10 samples, 1 had grade I skin lesions, 2 had grade II skin lesions, and 7 had grade III skin lesions. Only 2 skin samples (snakes W.B.05 and W.B.12) were sufficiently deep to assess the underlying skeletal muscle for involvement, which included inflammation in both cases (Figure 1c).
Discussion
All snakes in this study displayed varying degrees of heterophilic and granulomatous dermatitis, cellulitis, and myositis, as well as additional lesions potentially attributable to invasive or disseminated O. ophiodiicola infection, consistent with previous reports of ophidiomycosis in the United States and Europe (1,5).
Lesion severity associated with O. ophiodiicola infection varied greatly among individual snakes. Previous studies have suggested that species within the family Viperidae may be more susceptible to the effects of ophidiomycosis (4,11,12). Indeed, 2 of the 3 snakes we observed with fatal ophidiomycosis were viperids. The potential population-level impacts of ophidiomycosis on free-ranging snakes were first recognized in a population of eastern massasaugas in Illinois (11). The eastern massasauga is Ontario’s only extant, native species in the family Viperidae (13). However, many other snake species, including colubrids, also can develop severe infections (1). Both eastern massasaugas in this study had extensive facial lesions, including fungal stomatitis, keratitis, osteomyelitis, and encephalitis. This most likely led to fungal pneumonia in both snakes through either inhalation or direct extension, rather than hematogenous embolization, given the presence of fungal hyphae and debris in the central airspaces. All of the other snakes diagnosed with naturally acquired ophidiomycosis in this study were species within the family Colubridae, which encompasses the majority of native, free-ranging snake species in Ontario. Lesions in these snakes were primarily confined to the superficial skin. Eastern foxsnakes are overrepresented among the naturally infected species in this study, as many samples came from a single, free-ranging population that is intensively monitored and sampled as part of a research initiative.
In the colubrids in the present study, ophidiomycosis was usually considered an incidental finding, or potentially contributing to the cause of death. The only colubrid in which ophidiomycosis was considered the cause of death was an eastern foxsnake, which was emaciated, presumably secondary to facial ophidiomycosis. Lesions involving the head are thought to decrease the snake’s ability to obtain food and lead to a negative caloric balance and subsequent immunosuppression (1,14). The function of the innate immune system could also be compromised by direct fungal damage to the physical barrier of the skin and dysecdysis. Ophidiomycosis can also lead to behavioral changes (e.g., increased time spent basking), which may predispose to other mortality causes (e.g., vehicular trauma, predation). This may have been the case with an eastern massasauga, which had multiple ophidiomycosis skin lesions, but died from sepsis due to a traumatic tail amputation. Affected snakes of some species are often observed basking in the open more often than uninfected snakes in both experimental and natural settings (4,15). This behavior may in part be an adaptation toward combatting pathogens, as basking increases body temperature and metabolic rate (15–17).
Skin from biopsied snakes in the present study displayed a wide range of lesion severity. Although the samples that consisted only of supraepidermal crusts were sufficient for demonstrating fungus in the lesion, the deeper sections allowed for assessment of skeletal muscle involvement, which may have been prognostically significant. When collecting biopsy samples from snakes for ophidiomycosis assessment, the benefits of a deeper, more complete biopsy sample must be weighed against the negative effects of a more invasive procedure that may be detrimental to the snake.
The ophidiomycosis microscopic skin lesion grading scheme described in the present study proved a useful tool to create semi-quantitative data based on lesion characteristics and severity. The scheme allowed us to compare skin lesions in both carcasses and biopsy samples. This grading scheme provides a template for future assessments of ophidiomycosis, allowing for more consistent lesion comparisons across studies. It may also prove useful as a prognostic indicator, but this would require further studies (e.g., a prospective study).
Although the population-level impacts of ophidiomycosis are unclear, snake species in Ontario are subject to various significant threats, primarily due to human activity, and it is essential that all risk factors, including disease, be considered in conservation initiatives (9). Ophidiomycosis is a complex disease, especially in natural settings in which season, stress, food availability and habitat degradation play a role in pathogenesis (1,7,15,18–20). Further targeted studies as well as ongoing surveillance, including population monitoring, are required to determine the potential impacts of ophidiomycosis on snakes in Ontario, as well as in the rest of Canada.
Acknowledgments
We are indebted to Doug Campbell, recently retired from the Canadian Wildlife Health Cooperative (CWHC), for conducting the initial examination of all naturally infected snakes prior to August 2018. We are grateful to the CWHC Ontario/Nunavut staff and to those who submitted case material. We thank the Animal Health Laboratory, especially Hugh Cai, as well as the histotechnology team. CVJ
Footnotes
Use of this article is limited to a single copy for personal study. Anyone interested in obtaining reprints should contact the CVMA office (hbroughton@cvma-acmv.org) for additional copies or permission to use this material elsewhere.
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