While white-nose syndrome (WNS) has decimated hibernating bat populations in the Nearctic, species from the Palearctic appear to cope better with the fungal skin infection causing WNS. This has encouraged multiple hypotheses on the mechanisms leading to differential survival of species exposed to the same pathogen. To facilitate intercontinental comparisons, we proposed a novel pathogenesis-based grading scheme consistent with WNS diagnosis histopathology criteria. UV light-guided collection was used to obtain single biopsies from Nearctic and Palearctic bat wing membranes non-lethally. The proposed scheme scores eleven grades associated with WNS on histopathology. Given weights reflective of grade severity, the sum of findings from an individual results in weighted cumulative WNS pathology score. The probability of finding fungal skin colonisation and single, multiple or confluent cupping erosions increased with increase in Pseudogymnoascus destructans load. Increasing fungal load mimicked progression of skin infection from epidermal surface colonisation to deep dermal invasion. Similarly, the number of UV-fluorescent lesions increased with increasing weighted cumulative WNS pathology score, demonstrating congruence between WNS-associated tissue damage and extent of UV fluorescence. In a case report, we demonstrated that UV-fluorescence disappears within two weeks of euthermy. Change in fluorescence was coupled with a reduction in weighted cumulative WNS pathology score, whereby both methods lost diagnostic utility. While weighted cumulative WNS pathology scores were greater in the Nearctic than Palearctic, values for Nearctic bats were within the range of those for Palearctic species. Accumulation of wing damage probably influences mortality in affected bats, as demonstrated by a fatal case of Myotis daubentonii with natural WNS infection and healing in Myotis myotis. The proposed semi-quantitative pathology score provided good agreement between experienced raters, showing it to be a powerful and widely applicable tool for defining WNS severity.

Objectives The aim of this report is to describe the identification of a novel vitamin D metabolite, a C-3, alpha-epimer of 25-hydroxycholecalciferol (3-epi-25(OH)D), in serum and plasma extracts of cat blood and compare its abundance in cat, dog and rat serum to 25-hydroxycholecalciferol (25(OH)D), a conventional marker of vitamin D status. Methods Serum 25(OH)D and 3-epi-25(OH)D concentrations were measured in healthy cohorts of cats (n = 8), dogs (n = 8) and rats (n = 17) using validated reverse and normal-phase high-performance liquid chromatography methods. The methods were verified using liquid chromatography tandem mass spectrophotometry. Dietary intake and dietary concentrations of vitamin D were also measured for evaluation of species differences and effect of dietary change on vitamin D metabolite concentrations. Differences between cat serum and plasma metabolite concentrations were determined. Results Detectable concentrations of 3-epi-25(OH)D were observed in all cats and rats. No 3-epi-25(OH)D was detected in dogs, where our limit of detection was 5 ng/ml. There were significant differences ( P <0.05) in serum concentrations of 25(OH)D and 3-epi-25(OH)D among species, with cats having the greatest concentrations of both metabolites. Serum and plasma results were not significantly different. A diet change, which resulted in an increase in vitamin D intake among the cats, affected serum concentration with an increase ( P = 0.004) in 3-epi-25(OH)D but no significant change in 25(OH)D. Conclusions and relevance Serum and plasma of cats contain 3-epi-25(OH)D in varied and extraordinary concentrations, much greater than in rats and certainly than that of dogs, a species for which the metabolite was not detected. Importantly, this finding indicates a C-3 epimerization pathway is quantitatively significant for vitamin D metabolism in domestic cats, making 3-epi-25(OH)D assays essential for the evaluation of vitamin D status in cats and positioning the cat as a novel model for study of this pathway.

Pseudogymnoascus destructans colonizes the wing membrane of hibernating bats with the potential to form dense fungal hyphae aggregates within cupping erosions. These fungal cupping erosions emit a characteristic fluorescent orange-yellow color when the wing membrane is transilluminated with 385 nm UV light. The purpose of this study was to create and validate the R package, countcolors, for quantifying the distinct orange–yellow ultraviolet (UV) fluorescence in bat-wing membrane lesions caused by P. destructans. Validation of countcolors was completed by first quantifying the percent area of 20, 2.5 cm2 images. These generated images were of two known pixel colors ranging from 0% to 100% of the pixels. The countcolors package accurately measured the known proportion of a given color in each image. Next, 40, 2.5 cm2 sections of UV transilluminated photographs of little brown bat ( Myotis lucifugus) wings were given to a single evaluator. The area of fluorescence was both manually measured and calculated using image analysis software and quantified with countcolors. There was good agreement between the two methods (Pearson's correlation=0.915); however, the manual use of imaging software showed a consistent negative bias. Reproducibility of the analysis methods was tested by providing the same images to naive evaluators who previously never used the software; no significant difference ( P=0.099) was found among evaluators. Using the R package countcolors takes less time than does manually measuring the fluorescence in image analysis software, and our results showed that countcolors can improve the accuracy when quantifying the area of P. destructans infection in bat wing-membranes.