Ophidiomycosis, commonly called snake fungal disease, has been linked to significant morbidity of free-ranging snakes in North America and Europe. Diagnosis of ophidiomycosis currently requires detection of skin lesions via physical exam or characteristic histopathology as well as detection of the causative agent, Ophidiomyces ophidiicola, through quantitative (q)PCR or fungal culture of a skin swab or tissue sample. While reliable, these methods require specialized training, invasive procedures (e.g., biopsy), and several days or weeks to receive results. Additionally, screening entire populations can quickly become costly. A fast, easy-to-use, cost-efficient, and sensitive screening tool is needed to optimize conservation strategies and treatment intervention. Our objective was to investigate the association between skin fluorescence under long-wave ultraviolet (UV) light (365 nm) and the detection of Ophidiomyces ophidiicola DNA using qPCR. Fifty-eight Lake Erie watersnakes (Nerodia sipedon insularum) collected in June of 2018 and 2019 from islands in western Lake Erie, Ottawa County, Ohio, US were visually inspected for skin lesions, photographed under natural light and UV light, and swabbed for qPCR analysis. Fluorescence was highly associated with the presence of skin lesions, and the presence of at least one fluorescent skin lesion was 86% sensitive and 100% specific for identifying animals with apparent ophidiomycosis, with a positive predictive value of 100%. While we recommend performing standard diagnostics along with fluorescence, our study supports the use of visual UV fluorescence identification as a preliminary, affordable, noninvasive, and field-applicable method to screen populations for ophidiomycosis.

Emerging fungal diseases have become increasingly common in wildlife and have been associated with widespread mortality and even extinction (Berger 1998; Turner 2011; Fisher 2016). Ophidiomycosis, commonly referred to as snake fungal disease, is caused by the keratinophilic fungus Ophidiomyces ophidiicola (Lorch et al. 2015). Ophidiomyces infection typically causes skin swelling, crusts, and ulcerations, but more-severe infections invade deeper into muscle and bone and, in some cases, cause fatal systemic disease (Lorch et al. 2016; Baker et al. 2019).

Diagnosing ophidiomycosis requires detection of skin lesions, either through physical exam or histopathology and a positive quantitative (q)PCR result or culture for O. ophidiicola from a skin swab or tissue sample (Baker et al. 2019; Table 1). While reliable, established diagnostic methods have the risk of subtle lesions being overlooked, a long lag time, high cost, and can be invasive in the case of skin biopsies. A fast, reliable, affordable, patient-side screening method for ophidiomycosis would be advantageous in field settings to improve disease prevalence assessments in new species and locations.

Table 1

Number of Lake Erie watersnakes (Nerodia sipedon insularum) collected in June of 2018 and 2019 from islands in western Lake Erie, Ottawa County, Ohio, USA. The criteria of lesion presence and Ophidiomyces ophidiicola quantitative (q)PCR results are listed for each of the four previously defined ophidiomycosis categories (Baker et al. 2019) as well as the number of snakes tested in this study and the number of snakes with skin lesions that fluoresced under ultraviolet (UV) light (365 nm) in each category.

Number of Lake Erie watersnakes (Nerodia sipedon insularum) collected in June of 2018 and 2019 from islands in western Lake Erie, Ottawa County, Ohio, USA. The criteria of lesion presence and Ophidiomyces ophidiicola quantitative (q)PCR results are listed for each of the four previously defined ophidiomycosis categories (Baker et al. 2019) as well as the number of snakes tested in this study and the number of snakes with skin lesions that fluoresced under ultraviolet (UV) light (365 nm) in each category.
Number of Lake Erie watersnakes (Nerodia sipedon insularum) collected in June of 2018 and 2019 from islands in western Lake Erie, Ottawa County, Ohio, USA. The criteria of lesion presence and Ophidiomyces ophidiicola quantitative (q)PCR results are listed for each of the four previously defined ophidiomycosis categories (Baker et al. 2019) as well as the number of snakes tested in this study and the number of snakes with skin lesions that fluoresced under ultraviolet (UV) light (365 nm) in each category.

Long-wave ultraviolet (UV) light has been used to diagnose fungal dermatophyte infections in domestic species (Koenig and Schneckenburger 1994) and white nose syndrome (WNS) in bats (Turner 2014). While the diversity of fungi that produce fluorescence is not well characterized, particularly in snakes, applying this technique to snakes could prove useful for immediate and affordable preliminary ophidiomycosis screening and to help biologists target individual snakes and skin areas for further diagnostic testing. The objectives of this study were to develop a protocol to detect skin UV fluorescence in Lake Erie watersnakes (LEWS; Nerodia sipedon insularum) and to determine if skin UV fluorescence is associated with lesion presence and detection of Ophidiomyces ophidiicola DNA by qPCR.

In June of 2018 and 2019, wild LEWS were captured from five islands in western Lake Erie, Ottawa County, Ohio, US (Table 2) via visual encounter surveys. Snakes were visually examined for skin lesions and demographics and morphometric measurements were recorded. When present, the locations of skin lesions were noted and each lesion was classified as a crust, displaced scale, necrotic scale, or ulcer (Baker et al. 2019). If no lesions were present, 2×2-cm areas of apparently normal skin were selected for evaluation. Snakes were then placed in a 20×16.5, 10.5-cm black plastic box with 4.5-cm holes on both sides of the box to allow the handler to manually restrain the snake from outside and appropriately orient lesions toward the UV light and camera (Fig. 1).

Table 2

Demographic data for Lake Erie watersnakes (Nerodia sipedon insularum) evaluated for Ophidiomyces ophidiicola infection and lesion fluorescence under ultraviolet light (365 nm) in 2018 and 2019 from islands in western Lake Erie, Ottawa County, Ohio, USA.

Demographic data for Lake Erie watersnakes (Nerodia sipedon insularum) evaluated for Ophidiomyces ophidiicola infection and lesion fluorescence under ultraviolet light (365 nm) in 2018 and 2019 from islands in western Lake Erie, Ottawa County, Ohio, USA.
Demographic data for Lake Erie watersnakes (Nerodia sipedon insularum) evaluated for Ophidiomyces ophidiicola infection and lesion fluorescence under ultraviolet light (365 nm) in 2018 and 2019 from islands in western Lake Erie, Ottawa County, Ohio, USA.
Figure 1

Field setup for photographing snakes under ultraviolet (UV) light to assess skin fluorescence in 2018 and 2019 on islands in western Lake Erie, Ottawa County, Ohio, USA. Each Lake Erie watersnake (Nerodia sipedon insularum) was manually restrained with a portion of the body inside the plastic box. Lesions or preselected areas of unaffected skin were oriented toward the UV light (365 nm) placed over a hole in the box lid. Skin swabs were taken of each photographed area for detection of Ophidiomyces ophidiicola by quantitative PCR to determine if fluorescent areas were associated with ophidiomycosis.

Figure 1

Field setup for photographing snakes under ultraviolet (UV) light to assess skin fluorescence in 2018 and 2019 on islands in western Lake Erie, Ottawa County, Ohio, USA. Each Lake Erie watersnake (Nerodia sipedon insularum) was manually restrained with a portion of the body inside the plastic box. Lesions or preselected areas of unaffected skin were oriented toward the UV light (365 nm) placed over a hole in the box lid. Skin swabs were taken of each photographed area for detection of Ophidiomyces ophidiicola by quantitative PCR to determine if fluorescent areas were associated with ophidiomycosis.

Close modal

The UV light (Model UV502, 120V, 365 nm, Phillips Burton, Addison, Illinois, USA) was placed over a 12.5×16 cm opening in the lid of the box. This setup provided a consistent distance from the light source to the snake's skin of approximately 9.5 cm, dependent on the diameter of the snake's body. Photos were taken through the magnifying lens on the UV light and presence or absence of fluorescence was determined by a single observer (K.V.). After photography, all lesions or preselected areas of apparently normal skin were swabbed with a cotton-tipped applicator. Swabs were placed in separate 2-mL Eppendorf tubes and stored at –20 C until processing. We extracted DNA from swabs, and qPCR specific for O. ophidiicola was performed as previously reported (Allender et al. 2015). Each snake was classified into one of four ophidiomycosis categories, as previously defined by Baker et al. (2019; Table 1).

Skin fluorescence (present or absent) and O. ophidiicola qPCR status (positive or negative) were modeled using robust generalized linear models (package brglm2; Kosmidis 2018) in RStudio version 3.5.1 (R Core Team 2018) at an alpha level of 0.05. Fixed effects included island, sex, lesion presence, lesion location (head, body, ventrum, or tail), and lesion type. Variance inflation factors (VIF) and generalized variance inflation factors (GVIF) were used to identify and exclude problematic collinear predictors (VIF or GVIF1/(2×DF)>5) in multivariable models (function vif, package car; Fox and Monette 1992; Fox and Weisberg 2011). Post hoc between-group differences were evaluated using the contrast function in the lsmeans package with a Bonferroni correction to control for multiple comparisons (Lenth 2016). The relationship between skin fluorescence and Ophidiomyces qPCR cycle threshold (Ct) value was modeled using robust generalized linear models, and optimal Ct cutpoints were calculated using the cutpointr package (Thiele 2020).

Swabs and photographs were collected from 175 lesions from 58 individual LEWS (Table 2). Fifteen individuals had no skin lesions and 43 had at least one lesion consistent with ophidiomycosis (median=1 lesion, range=1–8 lesions). Fluorescence was observed in 76% of all lesions and not in any individuals without skin lesions (Figs. 2AD, 3AC). Forty-three snakes had apparent ophidiomycosis; 11 were categorized as Ophidiomyces present and four were categorized as ophidiomycosis negative (Table 1).

Figure 2

Photographs of Lake Erie watersnakes (Nerodia sipedon insularum) taken during exposure to ultraviolet light (365 nm) in 2018 and 2019 on islands in western Lake Erie, Ottawa Couty, Ohio, USA. Ophidiomyces ophidiicola quantitative (q)PCR results in fungal copy number per nanogram DNA from a skin swab of the photographed area are included on each image. (A) qPCR and fluorescence positive, (B) qPCR positive and fluorescence negative, (C) pPCR negative and fluorescence positive, and (D) qPCR and fluorescence negative.

Figure 2

Photographs of Lake Erie watersnakes (Nerodia sipedon insularum) taken during exposure to ultraviolet light (365 nm) in 2018 and 2019 on islands in western Lake Erie, Ottawa Couty, Ohio, USA. Ophidiomyces ophidiicola quantitative (q)PCR results in fungal copy number per nanogram DNA from a skin swab of the photographed area are included on each image. (A) qPCR and fluorescence positive, (B) qPCR positive and fluorescence negative, (C) pPCR negative and fluorescence positive, and (D) qPCR and fluorescence negative.

Close modal
Figure 3

Photographs of Lake Erie watersnakes (Nerodia sipedon insularum) when exposed to ultraviolet light (365 nm) in 2018 and 2019 on islands in western Lake Erie, Ottawa County, Ohio, USA. Snakes with diffuse lesions present show a diffuse and distinct yellow-green fluorescence. (A) Diffuse fluorescence along the entire dorsum, (B) diffuse fluorescence on the proximal dorsum and head, and (C) diffuse fluorescence on the middorsum.

Figure 3

Photographs of Lake Erie watersnakes (Nerodia sipedon insularum) when exposed to ultraviolet light (365 nm) in 2018 and 2019 on islands in western Lake Erie, Ottawa County, Ohio, USA. Snakes with diffuse lesions present show a diffuse and distinct yellow-green fluorescence. (A) Diffuse fluorescence along the entire dorsum, (B) diffuse fluorescence on the proximal dorsum and head, and (C) diffuse fluorescence on the middorsum.

Close modal

Positive skin fluorescence appeared as a yellow-green glow and was seen in discrete foci (Fig. 2A, C) or diffuse patterns on the body (Fig. 3AC). Fluorescence was highly associated with the presence of skin lesions (odds ratio=114, 95% confidence interval [CI]=6–2,135, P=0.001). The odds of a swab testing qPCR positive for Ophidiomyces were 15 times higher (95% CI=4–6, P<0.0001) if a skin lesion was present and threefold higher (95% CI=6–1,955, P=0.001) if the skin fluoresced. Island, sex, lesion type, and lesion location were not significantly associated with qPCR status or fluorescence (P>0.05).

When considering individual lesions, skin fluorescence was 69% sensitive (95% CI=59–78%) and 62% specific (95% CI=32–86%) for identifying lesions that were qPCR positive for Ophidiomyces DNA. An Ophidiomyces qPCR Ct cutoff value of 34.8 was 76% accurate, 96% sensitive, and 33% specific for differentiating between fluorescent and non-fluorescent areas of skin (area under the curve=0.64), indicating that lesions with low fungal burdens are less likely to fluoresce under UV light. When considering snakes as a whole, the presence of any fluorescent lesion was 86% sensitive (95% CI=73–95%) and 100% specific (95% CI=78–100%) for identifying animals with apparent ophidiomycosis, with a positive predictive value of 100% (95% CI=85–100%) and a negative predictive value of 71% (95% CI=54–84%).

Long-wave UV light is a promising screening tool for ophidiomycosis in wild and managed snakes. It was highly sensitive for identifying snakes with apparent ophidiomycosis, while snakes in the Ophidiomyces present category were not identified using skin fluorescence (Fig. 2B). In mammalian dermatophyte infections, skin fluorescence is due to the presence of phosphors, which are produced by the interaction between invading fungus and host tissues (Moriello 2017). We suspect that O. ophidiicola infection causes a similar reaction in snake skin, resulting in UV fluorescence. This may explain why only snakes with gross disease, specifically those in the apparent ophidiomycosis category, were identifiable using UV screening. In addition, strain variability can influence the production of UV fluorescence in fungal dermatophytes such as Microsporum spp. (Moriello 2017). The presence of multiple O. ophidiicola strains or other pathogens that cause a similar reaction in host tissues could explain the observed differences in clinical signs and skin fluorescence in LEWS, so investigation of Ophidiomyces genetics and other causes of skin disease in snakes are important avenues of future research.

Our study utilized qPCR for O. ophidiicola detection but, without the histologic observation of a host response to the presence of fungal elements, we cannot confirm ophidiomycosis in these snakes. Additionally, skin swab qPCR was imperfect at detecting ophidiomycosis, so the sensitivity and specificity for UV detection, which were calculated relative to skin swab qPCR, may differ when compared to gold-standard diagnostic approaches. Further studies to compare fluorescence to the most rigorous diagnostic criteria (lesions present, qPCR positive, and characteristic histopathologic changes present) would give a more accurate assessment of this technique. Histopathology was beyond the scope of this study due to the invasiveness of obtaining skin biopsies, but is an important next step in validating UV fluorescence as a diagnostic method for ophidiomycosis.

Comparing sensitivity and specificity values between studies is challenging due to differences in disease definitions and lack of consensus on gold-standard testing (Moriello et al. 2017). Despite this, UV fluorescence is considered to have high positive predictive value for detecting dermatophytosis in domestic species (Moriello et al. 2017) and white nose syndrome in bats (Turner et al. 2014). We found that UV fluorescence also had high sensitivity, specificity, and positive predictive value for identifying apparent ophidiomycosis in LEWS. Positive predictive value depends heavily on disease prevalence, thus future studies should investigate UV fluorescence in populations with lower ophidiomycosis prevalence as well as in populations with animals in the possible ophidiomycosis category.

Ophidiomycosis lesions are commonly subtle, and UV fluorescence can help to train observers who are unfamiliar with identifying lesions and to target diagnostic swabbing toward areas with skin disease. However, observers should also recognize the potential for false positive results because urates, loose unshed skin, and the All-Weather™ Paintstik© (LA-CO©, Elk Grove, Illinois, USA) livestock marker used to distinguish recently captured snakes also fluoresced under UV light. Close examination of fluorescent areas is necessary to rule out such noninfectious causes, and snakes with fluorescence should be swabbed or biopsied for a definitive diagnosis using fungal culture or qPCR. Overall, UV fluorescence is a preliminary, immediate, and affordable population screening tool for ophidiomycosis. Application of this technique in clinical and field settings may facilitate diagnosis, expedite treatment, and support positive research and conservation outcomes.

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