Detection of Ophidiomyces ophiodiicola in wild European snakes

A total of 336 wild snake samples from Great Britain (GB) (n = 33 carcasses and 302 moulted skins) and the Czech Republic (n = 1 moulted skin) were examined for macroscopic skin lesions consistent with O. ophiodiicola infection (Table 1)5, 9. Skin lesions were observed in 80 (23.8%) of the samples and these were screened for the presence of the fungus using real-time PCR. Ophidiomyces ophiodiicola was detected in 25 samples from snakes with macroscopic skin lesions, comprising 24 from British grass snakes (Natrix natrix) (eight carcasses and 16 moulted skins) and a moulted skin from a dice snake (Natrix tessellata) from the Czech Republic (Table 1). Panfungal PCR and DNA sequencing of the D1-D2 region of the large subunit of the ribosomal RNA gene of one of the PCR-positive grass snakes further corroborated the identity of the fungus as O. ophiodiicola (99% sequence identity to available O. ophiodiicola strains in GenBank; GenBank Accession LT607735). Dates of collection of the PCR-positive samples ranged from 2010–2016 with a wide geographical distribution in GB. The Czech Republic detection indicates that the fungus also occurs in mainland Europe (Supplementary Fig. S1). Swab samples from 11 grass snake carcasses and eight adder carcasses (Vipera berus) with no detected macroscopic lesions were similarly tested. A single snake, an adder, without macroscopic skin lesions was PCR-positive.

Table 1 The number of European snake species examined according to sample, if macroscopic lesions were detected and whether the samples were positive for O. ophiodiicola on real-time PCR. Full size table

Fungal culture conducted on skin lesions from 18 of the PCR-positive snakes led to the isolation of O. ophiodiicola from six of these animals (the dice snake and five grass snakes) (Supplementary Worksheet S1). In each case, the fungus was identified by PCR and sequencing of the internal transcribed spacer (ITS) region.

Pathological investigations of Ophidiomyces ophiodiicola infections in European snakes

A systematic post-mortem examination and histopathology were conducted on eight of the European snakes with skin lesions from which suitable formalin-fixed tissues were available. Of these, five grass snakes from GB were positive for O. ophiodiicola on PCR examination (Supplementary Worksheet S2). Macroscopic lesions in these five cases had varied appearance, with multifocal areas of brown discoloration and thickening primarily affecting the ventral body scales (Fig. 1a). Lesions were observed particularly along the scale edges and in the crypts between affected scales, often with irregular caudal scale margins. Suspected dysecdysis (e.g., areas of flaking skin) was evident in two grass snakes. Microscopic examination (Fig. 1b) identified epidermal thickening and necrosis, of variable severity, with intralesional fungal hyphae (2–4 µm -diameter, parallel-walled, septate, branching) and mixed bacterial colonies (Fig. 1b). Arthroconidia with a size and morphology consistent with O. ophiodiicola were detected in three of the grass snakes with SFD (Fig. 1c). There was evidence of dermatitis in four snakes and associated myositis in two of these cases. Snake fungal disease was considered likely to be a direct or indirect cause of mortality in four of the five grass snakes with O. ophiodiicola infection: cachexia and dehydration were noted in three cases, one of which was euthanized for humane reasons, and predation in two cases. Accidental electrocution was considered the likely cause of death in the fifth grass snake with O. ophiodiicola infection. No other significant pathogens or concurrent significant infectious disease processes were detected in any of these snakes (Supplementary Worksheet S2).

Figure 1 (a) Macroscopic lesions of snake fungal disease in a grass snake (Natrix natrix) showing thickened, yellow-brown areas mostly at the edges of the ventral scales with irregular margins (case: XT1041-16); (b) Microscopic lesions of snake fungal disease in a grass snake showing epidermal thickening and necrosis and dermatitis (case: X1041-16), HE stain, 50x magnification; (c) Focused area of microscopic lesion showing presence of arthroconidia in grass snake (case: XT804-15), PAS stain, 1000x magnification. Full size image

Macroscopic lesions in the three grass snakes that were negative for O. ophiodiicola infection on PCR examination had a varied appearance (Supplementary Worksheet S2). Microscopic examination identified epidermal necrosis with intralesional bacteria in all three cases. Dermatitis of variable severity was present in two of these PCR-negative snakes. Fungal hyphae were observed infrequently in the skin of one case and suspected fungal hyphae were present in a second case but no arthroconidia were detected in any of these three snakes.

Lesions observed in all the moulted skin samples positive for O. ophiodiicola were mild, consisting of single to multifocal areas of brown discoloration and thickening affecting the ventral scales that ranged in size from 0.5 × 0.5 mm to 10 × 6 mm. Histopathology was not conducted on the moulted skins due to the nature of the samples.

Characterisation of European isolates of Ophidiomyces ophiodiicola

To understand the relationship of European isolates of O. ophiodiicola with the fungus in North America, a phylogenetic analysis was performed using multiple chromosomal loci (ITS and partial sequences of the actin [ACT] and translation elongation factor 1-α [TEF] genes) from a subset of six European and six North American isolates (Supplementary Table S1). DNA sequences generated from O. ophiodiicola isolates cultured from wild snakes in GB were 100% identical to one another at all three loci; the Czech isolate differed from the GB isolates at each locus by one single nucleotide polymorphism (SNP; i.e., three SNPs over the region examined). The GB and Czech isolates formed a single, well-supported clade using both maximum likelihood and Bayesian analyses. This “European clade” was distinct from isolates of O. ophiodiicola that originated from snakes in eastern North America (Fig. 2). The isolates from both wild European snakes and North American snakes were genetically divergent from a strain of O. ophiodiicola previously isolated from a captive ball python (Python regius) in GB (UAMH Centre for Global Microfungal Biodiversity isolate number 6688;10).

Figure 2 Phylogenetic tree resulting from Bayesian analysis of three concatenated loci (internal transcribed spacer region, and portions of the actin and translation elongation factor 1-α genes) of Ophidiomyces ophiodiicola isolates from eastern North America and Europe. The consensus tree resulting from a maximum likelihood analysis had the same topology. Posterior probabilities (Bayesian)/bootstrap support values (maximum likelihood) are displayed at nodes when the probabilities and bootstrap values were above 0.95 and 65, respectively. The tree is rooted with Pseudoamauroascus australiensis. Isolates of O. ophiodiicola from wild European snakes formed a clade distinct from isolates of the fungus from eastern North America. Isolate UAMH 6688 originated from captive snake at a zoological park in the United Kingdom in 1985 and is divergent from both clades. Full size image

During the process of culturing O. ophiodiicola from samples in this study, it was noted that the European isolates appeared to grow more slowly than those from snakes in eastern North America. To assess this quantitatively, experiments were conducted to compare the growth rates of the various O. ophiodiicola strains at 30 °C. The six isolates from wild European snakes had colony expansion rates that were, on average, 4.8 times slower than those previously isolated from the eastern United States (Supplementary Fig. S2). This phenotypic difference, along with their genetic distinctiveness, suggests that European isolates represent novel strains of O. ophiodiicola that differ from those associated with SFD emergence in North America.