Morphological and Molecular Characterizations of Psychrophilic Fungus Geomyces destructans from New York Bats with White Nose Syndrome (WNS)

Vishnu Chaturvedi,Melissa J Behr,Deborah J Springer,Xiaojiang Li,Alan C Hicks,Calvin M Butchkoski,Rama Ramani,Britta Wood,Robert J Rudd,Ping Ren,William A Samsonoff,Ward B Stone,Dianna J Bopp,Marcia K Peck,Sudha Chaturvedi,Eleftherios Mylonakis

doi:10.1371/journal.pone.0010783

Abstract

BackgroundMassive die-offs of little brown bats (Myotis lucifugus) have been occurring since 2006 in hibernation sites around Albany, New York, and this problem has spread to other States in the Northeastern United States. White cottony fungal growth is seen on the snouts of affected animals, a prominent sign of White Nose Syndrome (WNS). A previous report described the involvement of the fungus Geomyces destructans in WNS, but an identical fungus was recently isolated in France from a bat that was evidently healthy. The fungus has been recovered sparsely despite plentiful availability of afflicted animals.Methodology/Principal FindingsWe have investigated 100 bat and environmental samples from eight affected sites in 2008. Our findings provide strong evidence for an etiologic role of G. destructans in bat WNS. (i) Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans. (ii) G. destructans DNA was directly amplified from infected bat tissues, (iii) Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples. (iv) RAPD patterns for all G. destructans cultures isolated from two sites were indistinguishable. (v) The fungal isolates showed psychrophilic growth. (vi) We identified in vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans.Conclusions/SignificanceFurther studies are needed to understand whether G. destructans WNS is a symptom or a trigger for bat mass mortality. The availability of well-characterized G. destructans strains should promote an understanding of bat–fungus relationships, and should aid in the screening of biological and chemical control agents.

Highlights

Bats, which are ‘keystone species’ in many ecosystems, play notable roles in plant pollination, forest regeneration and control of insect populations [1,2]
Our findings provide strong evidence for an etiologic role of G. destructans in bat White Nose Syndrome (WNS). (i) Direct smears from bat snouts, Periodic Acid Schiff-stained tissue sections from infected tissues, and scanning electron micrographs of bat tissues all showed fungal structures similar to those of G. destructans (ii) G. destructans DNA was directly amplified from infected bat tissues (iii) Isolations of G. destructans in cultures from infected bat tissues showed 100% DNA match with the fungus present in positive tissue samples (iv) random amplification of polymorphic DNA (RAPD) patterns for all G. destructans cultures isolated from two sites were indistinguishable (v) The fungal isolates showed psychrophilic growth (vi) We identified in vitro proteolytic activities suggestive of known fungal pathogenic traits in G. destructans
Sections of skin from muzzles of four humanely euthanized little brown bats collected from the Williams Hotel Mine on March 27, 2008, showed epidermal colonization with focal dermatitis including a few neutrophils in the underlying dermis and fungal hyphae and spores intermixed with bacteria near the surface (Fig. 1Bi, ii)

Summary

Introduction

Bats, which are ‘keystone species’ in many ecosystems, play notable roles in plant pollination, forest regeneration and control of insect populations [1,2]. No mass mortality was reported until recently from bat sites that had been surveyed for almost three decades by the New York State Department of Environmental Conservation. Little brown bats have been found to be dying in large numbers at many hibernation sites in upstate New York [13]. This problem has spread to other States in the Northeastern US (Figure S1). Massive die-offs of little brown bats (Myotis lucifugus) have been occurring since 2006 in hibernation sites around Albany, New York, and this problem has spread to other States in the Northeastern United States. The fungus has been recovered sparsely despite plentiful availability of afflicted animals

Methods

Results

Conclusion