During intensive monitoring of bat hibernation in France, 1 bat (Myotis myotis) found on March 12, 2009, near Périgueux (45°8′N, 0°44′E), showed a powdery, white fungal growth on its nose (, panel A), which is characteristic of WNS. Sterile dry cotton swabs were used to collect fungus material from the nose of the bat. The bat was then weighed, measured, and released. Swabs were moistened with 50 μL of sterile water and streaked onto plates containing potato dextrose agar supplemented with 0.1% mycologic peptone. Plates (9 cm in diameter) were sealed with parafilm and incubated inverted at 10°C. A dense fungus growth developed within 14 days (, panel B).
Figure 1 A) Myotis myotis bat found in a cave on March 12, 2009, in France, showing white fungal growth on its nose (arrow). B) Fungus colony on malt extract medium after incubation for 3 weeks at 10°C. Scale bar = 1 cm. C) Clusters of unstained spores (more ...)
Cultures were established by transferring inoculum to other mycologic media, including malt extract agar and Sabouraud agar. Colonies on malt extract agar were initially white but after spore production and aging they quickly darkened from the center to a dull gray, often showing a faint green hue. Spores were hyaline, irregularly curved, broadly crescent-shaped (typically 6–8 μm long and 3–4 μm wide), and narrowed at each end, one of which was broadly truncate, often showing an annular frill (, panel C). Fungal cultures have been deposited in the culture collection of the Industrial Microbiology Department of University College Dublin (Reference IMD Z2053).
Microscopic examination of the original swab samples showed numerous spores with the above-mentioned features. The psychrophilic nature of the fungus and its species-specific morphologic features (Technical Appendix
) led to the conclusion that this fungus was G
, which was recently isolated from WNS-positive bats in the northeastern United States (6
Two molecular markers were sequenced from 6 randomly chosen fungus cultures to confirm species identity. DNA was extracted by using a Blood and Tissue Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions with slight modifications (after step 3, we added an incubation time of 10 min at 70°C). The internal transcribed spacer (ITS) regions (ITS1, 5.8S, and ITS2) and the small subunit (SSU) of the rRNA gene were amplified separately.
PCRs were performed in 25-μL volumes containing 1 μL of DNA (10–75 ng/μL), 1.5 mmol/L MgCl2
, 0.4 μmol/L of each primer, 0.2 mmol/L dNTP, 1× PCR buffer, and 1 U of Platinum Taq DNA Polymerase High Fidelity (Invitrogen, Carslbad, CA, USA). Identical PCR cycling conditions were used for both fragments: an initial step at 95°C for 15 s; 10 cycles at 95°C for 30 s, 60°C (reduction of 2°C every 2 cycles) for 1 min and 45 s, and 72°C for 1 min; 30 cycles at 95°C for 30 s, 50°C for 1 min and 45 s, and 72°C for 1 min; and a final step at 72°C for 10 min. PCR products were purified and sequenced in both directions by using primers listed in the . Complementary sequences were assembled and edited for accuracy by using CodonCode Aligner version 3.0.3 (www.codoncode.com/aligner/download.htm
Primers used for PCR amplification and sequencing of fungus in bats, France*
The ITS and SSU sequences from the 6 WNS fungus cultures were identical. They were deposited in GenBank as single sequences: ITS (GQ489024) and SSU (GQ489025). Sequences obtained for the 2 genetic markers showed a 100% sequence identity with the described G. destructans fungus (, panels A, B). Thus, morphologic and genetic data support the presence of G. destructans infection in a bat in France.
Figure 2 Genetic distance between fungus A) internal transcribed spacer (ITS) (474 nt) and B) small subunit (SSU) rRNA (1,865 nt) gene sequences and other closely related fungus species present in GenBank. Results are based on pairwise sequence comparisons with (more ...)