Morphological and molecular characterizations of psychrophilic fungus Geomyces destructans from New York bats with White Nose Syndrome (WNS)

Abstract

Background: 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. 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 findings: We 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/significance: Further 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.

Figure 1. Microscopic and histopathological evidence of G. destructans in bats with WNS.

(A) Direct lactophenol cotton blue mount prepared from skin scrape taken from the muzzle of a little brown bat from Graphite Mine on April 6, 2008 revealed fungal hyphae and curved conidia, bar 10 µm. (B) Control, [Bi] and infected muzzle tissue section [Bii] stained with PAS revealed epidermal colonization by fungal hyphae and spores; the sample was from a little brown bat from Williams Hotel Mine on March 27, 2008. Notably, a few neutrophils are present in the underlying dermis (arrows), bar 10 µm. Bacteria are also seen in this sample (C). SEM photomicrograph of muzzle sample from bat from Williams Hotel Mine showing characteristic curved conidia and septate hyphae spread over bat skin tissues. Note heavy fungal growth with profuse curved conidia covering the skin and hair shaft (Ci, muzzle, bar 100 µm; Cii, higher magnification of a portion of muzzle, bar 10 µm; Ciii & Cvi, higher magnifications, bar 10 µm).

Figure 2. G. destructans in culture from bat tissues.

(A). Original culture tubes of Sabouraud agar supplemented with nine antibiotics and incubated at 4°C for six- or eight-weeks; notice the profuse growth of G. destructans strains. (B) Some fungal contamination on individual isolates was visible as depicted in the close-up of a culture tube. (C) Enrichment and recovery of pure fungal colonies by treating a culture contaminated with bacteria with hydrochloric acid.

Figure 3. G. destructans in bat tissues and culture are similar.

(A) SEM of photomicrograph prepared from bat tissues samples, examined from Fig. 1C at high magnification, showed fungal hyphae and spores on the surface. (B) SEM photomicrograph prepared from G. destructans culture isolated from bat tissue samples collected from Williams Hotel Mine; note curved conidia borne in whorls on septate hyphae; this pattern is similar to SEM image in Fig. 3A, bar is 2 µm. All images are pseudo-colored in Adobe Photoshop 9.0.

Figure 4. Molecular analysis of bat tissues and fungal cultures.

(A) ITS PCR analysis of bat tissues and fungal cultures from DNA extracted from bat tissues and from pure G. destructans isolates. PCR amplification was carried out with primer set V47/V50. PCR amplicons were electrophoresed on 2% agarose gel, stained with ethidium bromide and photographed with a imaging software. Four bat tissues and respective fungal isolates showed perfect matches (blue connectors); one tissue DNA amplicon did not match with G. destructans amplicon obtained from pure culture (green connector). Also shown are amplicons from two additional G. destructans isolates (MYC80280, MYC80282) where corresponding tissues samples were not processed. (B) ITS PCR analysis of bat tissue samples positive for G. destructans. Ten bat tissues including five untreated samples and five paraffin-fixed samples were positive for G. destructans DNA (details in Table 1). (C-D) Molecular typing of G. destructans was performed with RAPD primers. (C) Results shown were obtained by PCR of fungal genomic DNA with M-13 and (GACA)₄ primers, amplicons were run on 2% agarose gels and band patterns were used to construct dendrograms with Applied Math software. Geomyces pannorum (UAMH 1062 and UAMH 2586) were used as outgroup. (D) Results shown were obtained by PCR of genomic DNA with Operon Technology 10-mer primers OPA1, OPA2 and OPA3; outgroup strains are similar to panel in C. Genotyping with five different primers showed that all six G. destructans culture isolates obtained from two sites, approximately 200-km apart, had indistinguishable band patterns. These preliminary results raised the possibility of involvement of a single strain of G. destructans in the outbreak of WNS in bats in upstate NY.

Figure 5. Phylogenetic analysis of nucleotide sequences from G. destructans.

(A) Phylogenetic tree was constructed by parsimony analysis of ITS sequences. The evolutionary history of representative isolates of G. destructans from this study and the sequences in the databases were inferred using the Maximum Parsimony method and bootstrap consensus tree from 1000 replicates conducted in MEGA 4.1 . After elimination of gaps and missing data, the dataset contained 448 positions of which 109 were parsimony informative. Percentage of replicate trees shown indicate clustering of associated tax in 1000 bootstrap replicates. Asterisks denote sequences deposited by other investigators from bats with WNS in US and France . (B) Phylogenetic tree constructed by parsimony analysis of 28S ribosomal sequences. The evolutionary history of representative isolates of G. destructans from this study and additional related fungi sequenced in our laboratory, were inferred using the Maximum Parsimony method and bootstrap consensus tree from 1000 replicates conducted in MEGA 4.1. After elimination of gaps and missing data, the dataset contained 537 positions of which 88 were parsimony informative. The consensus phylogenetic tree shown was inferred from 94 most parsimonious trees.

Figure 6. Growth characteristics of G. destructans isolates.

Colony morphology and growth rates were compared on Sabouraud dextrose agar and potato dextrose agar at -10°C, 4°C, 15°C, and 25°C. (A). Close up of fungal colonies of the isolate MYC80254 incubated at 4°C (Fig. 6A) and 15°C (Fig. 6B) for 28 days on potato dextrose agar, marker 10 mm. The initial colony appearance was white, velvety, glabrous turning grayish green, powdery in texture. Reverse with no pigmentation initially (Fig. 6Ai) later on revealing diffusible dark brown pigment (Fig. 6Bii). Older colony also exhibited exudates on surface, marker 10 mm. (Fig. 6Bi). Colony diameters of five G. destructans strains isolated from bat tissues and incubated for 28 days on Sabouraud dextrose agar at 4°C and 15°C. Exponential growth was seen at both temperatures with larger colony diameters at 15°C (Fig. 6Biii) than at 4°C (Fig. 6Aiii). The results represent average of two separate experiments. There was no growth in cultures incubated concurrently at −10°C or at 25°C (data not shown).

Figure 7. G. destructans proteolytic activities.

Results from a representative strain, G. destructans MYC80-0251, showed secretory proteases after 28-days growth on albumin agar (Ai-ii, 4°C front & reverse; Aiii-iv, 15°C front & reverse), Casein agar(Bi-ii, 4°C front & reverse; Biii-iv, 15°C front & reverse), Geleatin agar(Ci-ii, 4°C front & reverse; Ciii-iv, 15°C front & reverse) or Urea agar at 4°C and 15° (Di-ii, 4°C front & reverse; Diii-iv, 15°C front & reverse), marker is 10 mm. Similar patterns of secretory proteases were seen with remaining four G. destructans strains.