Fig 1.
Histopathology grades induced by natural Pseudogymnoascus destructans skin infection in Holarctic bats.
(A) Myotis myotis: fungal skin-surface colonisation with aerial hyphae developing conidia (g1, black arrow), not classified as WNS in absence of other findings; (B) M. myotis: a single cupping erosion (g4, black arrow) eroding to the epidermal/dermal interface; (C) M. myotis: three confluent cupping erosions (g5, black arrows); (D) M. daubentonii: necrotic wing membrane (witnessed as loss of dermal tissue stainability; g9, white asterisk) next to multiple cupping erosions packed with P. destructans hyphae (g5, black arrows) that also breached the basement membrane (g6, white arrowheads); (E) M. daubentonii: infection of hair follicle (g2, black arrow) and associated glands (g3, black arrowhead). Surface skin colonisation (g1), multiple cupping erosions (g5), inflammatory cells (g8) and necrotic tissue (g9, white asterisk) are also present in the section; (F) M. dasycneme: an outline of a cupping erosion (g4, black arrow) clearly visible together with full thickness fungal invasion (g7) replacing the necrotic wing membrane (g9) and sporadic neutrophils (g8, white asterisk); (G) M. lucifugus: marked inflammatory response (g8, black asterisks) to fungal invasion of several cupping erosions (g5, black arrows) on both sides of the wing membrane; (H) M. dasycneme: fungal sequestration (g11, black arrow) with neutrophils (g8, black asterisk) from the wing membrane. Periodic acid-Schiff stain. Scale bar– 50 μm.
Fig 2.
Skin infarction and necrosis associated with progressive white-nose syndrome lesions in Myotis daubentonii.
Samples for histopathology were collected on Day 7 of the time series documented using UV and daylight photography. Extensive Pseudogymnoascus destructans infection of the wing membrane produced confluent cupping erosions (g5, black arrows) resulting in skin necrosis (g9, white asterisk), characterised as loss of identifiable skin structures (A). Intraluminal neutrophilic infiltration (g8, black asterisk) in distended blood vessels was associated with the compromised wing membrane (B). Other skin lesions in this bat included haemorrhagic infarcts (g10, black arrowhead) with stagnant blood and skin necrosis (g9, white asterisk) (C). Periodic acid-Schiff stain.
Table 1.
Prevalence of histopathology severity grades associated with natural skin infection by Pseudogymnoascus destructans.
Prevalence was calculated as the percentage of positive biopsies at the given grade in the dataset of all bats positive for WNS based on histopathology. All infection grades apply to the wing membrane and, aside from fungal skin surface colonisation in the absence of any other findings, are classified as white-nose syndrome (WNS). Tested = number of individuals examined for histopathology. WNS histo+ = number of individuals confirmed positive for WNS. Std. error (%) = , where n is the overall number of scored grades for the given species and p is the proportion of positively scored grades. The respective severity grades are shown in Figs 1 and 2. Numbers in brackets represent WNS pathology grade weighting—see text for details.
Fig 3.
Species-specific weighted cumulative white-nose syndrome pathology score (histoSum).
Average sum of weighted qualitative scoring for white-nose syndrome severity grades displayed in Figs 1 and 2 (± std. error). Animals with histoSum = 1 not classified as positive for WNS on histopathology are included in the figure. Species sampled on multiple continents are presented separately. See Table 1 for sample sizes.
Table 2.
Inter-rater agreement in scoring white-nose syndrome (WNS) pathology according to Fleiss’ κ.
Experienced pathologists (n = 5) scored 30 photographs of randomly drawn histopathology slides, each group of naïve raters (n ∈{27, 20, 25}) scored a subset of 10 photographs. Standard errors for the given sample sizes were 0.047, 0.017, 0.023 and 0.018, respectively. Negative κ values indicate no inter-rater agreement.
Fig 4.
Time series of a Myotis daubentonii wing showing progression of white-nose syndrome lesions to fatality.
Extensive white-nose syndrome infection was recognised on a M. daubentonii bat at a hibernaculum in the Podyjí National Park (Czech Republic). The bat was kept under euthermic conditions at a rescue centre, fed ad libitum and supplied with drinking water. The wing was extended over a Wood’s lamp at 366 nm wavelength and photographed in a darkroom. (A) = Day 0, (B) = Day 1, (C) = Day 2, (D) = Day 3, (E) = Day 5, (F) = Day 6, (G) = Day 7, white arrows indicate biopsy punch sites (results presented in Fig 5). Day 7 was also documented using daylight photography (H). Scale bar = 1 cm. This time series spanned seven days from capture at the hibernaculum to death in the rescue centre. Wing membrane areas with extensive Pseudogymnoascus destructans infection became dry and necrotic within two days of euthermy, whereupon they contracted and tore around the white-nose syndrome lesions in a proximal-to-distal pattern. The animal displayed loss of skin tone, elasticity and surface sheen, and ceased eating one day prior to death.
Fig 5.
UV transillumination time series of a Myotis myotis wing with decrease in fluorescence corresponding to white-nose syndrome lesions over time.
The bat was captured at the end of the hibernation period, kept in captivity at euthermy, fed ad libitum with cockroaches and mealworms and supplied with drinking water, and released after the white-nose syndrome lesions had healed. The wing was extended over a Wood’s lamp at 366 nm wavelength and photographed in a darkroom. A = Day 0, B = Day 3, C = Day 5, D = Day 7, E = Day 11, F = Day 15. The top-left corner matches in each image, wing deformation is due to variable handling of the live animal during sampling. Scale bar = 1 cm.
Fig 6.
Healing of a white-nose syndrome lesion in Myotis myotis.
Samples for histopathology were collected on Day 15 of the time series described in Fig 5. A cupping erosion-like structure (black arrow) packed with fungal hyphae within a scab covering the healing wing membrane of M. myotis. Periodic acid-Schiff stain.
Fig 7.
Relationship between data from non-destructive diagnostic methods and selected histopathology severity grades.
Logistic regressions of skin surface colonisation by the fungus (black circle, dotted line; coefficients: (A) β0 = 3.38, β1 = 0.74, (B) β0 = 2.45, β1 = 2.32), single cupping erosion (orange cross, dashed line; coefficients: (A) β0 = 2.60, β1 = 0.68, (B) β0 = 1.49, β1 = 1.88) and multiple cupping erosions (red star, solid line; coefficients: (A) β0 = -0.09, β1 = 0.24, (B) β0 = -0.06, β1 = 1.44) dependent on fungal load detected on qPCR in Palearctic bats (A) or number of UV fluorescent spots in Holarctic bats (B). Shaded area represents 95% confidence interval on predicted probability.
Fig 8.
Increase in weighted cumulative white-nose syndrome pathology score with progressing UV fluorescence.
Phylogenetic generalised least-squares accounts for phylogeny and intraspecific variability in evaluating the relationship between weighted cumulative white-nose syndrome pathology score dependent on unit number of UV fluorescent lesions (β0 = 62.1, β1 = 16.41; solid line). Linear regression without phylogenetic correction (α = 42.38, β = 11.59, F1,131 = 9.12, p = 0.003, r2 = 0.07; dotted line).