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.
The TaqMan real-time PCR assay was developed from the Blastomyces dermatitidis BAD1 gene promoter. The assay identified all haplotypes of B. dermatitidis and five of six positive paraffin-embedded tissues. The assay sensitivity threshold was 1 pg genomic DNA of the mold form and 2 CFU of the yeast form of B. dermatitidis. No cross-reactivity was observed against other fungal DNA. The assay allowed rapid (5-h) identification of B. dermatitidis from culture and from clinical specimens. Blastomyces dermatitidis is a dimorphic fungal pathogen that causes blastomycosis. The infection generally starts by inhalation of spores of the mold form of the fungus, found in the environment. Upon entry into the hosts, the spores convert to the yeast form. The infection can be self-limiting to the lungs, or it can disseminate to other body parts, mainly to bones and skin. Apart from humans, dogs are highly susceptible to B. dermatitidis infection. Blastomycosis is endemic in the midwestern and southeastern United States and around the Great Lakes (4). Interestingly, blastomycosis has also been reported in humans and dogs in New York State (6,8), but the ecological niche of this fungus has yet to be established in this region. Similarly, blastomycosis has been reported from Colorado and Nebraska, which are all outside known zones where the infection is endemic (7, 13).The laboratory methods most frequently used to diagnose blastomycosis include serology, direct smear, and histopathology. However, the gold standard for diagnosis remains a positive culture. Traditional confirmation of a suspect culture of B. dermatitidis by conversion to the yeast form in the laboratory can take weeks, but identification can be confirmed on the same day by Gen-Probe (Gen-Probe, Inc., San Diego, CA). The Gen-Probe test can be used only with pure cultures of B. dermatitidis (yeast or mold); hence, it has limited application. To overcome these problems, several conventional PCR assays have been developed for the identification of B. dermatitidis from clinical specimens and soil samples (2, 5). These assays used identical primer pair sequences targeting the putative promoter region of the BAD1 (earlier known as WI-1) gene, which codes for an important adhesin molecule and virulence factor (10). Recently, it has been shown that the BAD1 promoter region has a number of nucleotide polymorphisms, which resulted in the identification of four haplotypes of B. dermatitidis (11,12). In addition to nucleotide polymorphism, a major size disparity due to two large insertions in the BAD1 promoter was also found in many B. dermatitidis strains (12). This can complicate the conventional PCR assay due to either insufficient amplification efficiency or misinterpretation as a nonspecific product.In this study, we describe the development of a TaqMan real-time PCR assay using a specific region of the BAD1 promoter to encompass all known haplotypes of B. dermatitidis. Our results indicate that the BAD1 real-time PCR assay is highly specific and rapid, with a turnaround t...
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