Clonal genotype of Geomyces destructans among bats with white nose syndrome, New York, USA.
(www.fws.gov/whitenosesyndrome/pdf/NWRS_WNS_ Guidance_Final1.pdf). Thus, an understanding of the dispersal mechanism of G. destructans is urgently needed to formulate effective strategies to control bat geomycosis.
We applied multiple gene genealogic analyses in studying G. destructans isolates; this approach yields robust results that are easily reproduced by other laboratories (5). Sixteen G. destructans isolates recovered from infected bats during 2008-2010 were analyzed. These isolates originated from 7 counties in New York and an adjoining county in Vermont, all within a 500-mile radius (Table 1). The details of isolation and identification of G. destructans from bat samples have been described (2). One isolate of a closely related fungus G. pannorum M1372 (University of Alberta Mold Herbarium, Edmonton, Alberta, Canada) was included as a reference control. To generate molecular markers, 1 isolate, G. destructans (M1379), was grown in yeast extract peptone dextrose broth at 15[degrees]C, and high molecular weight genomic DNA was prepared according to Moller et al. (6). A cosmid DNA library was constructed by using pWEB kit (Epicenter Biotechnologies, Madison, WI, USA) by following protocols described elsewhere (7). One hundred cosmid clones, each with [approximately equal to]40-Kb DNA insert, were partially sequenced in both directions by using primers M13 and T7. The nucleotide sequences were assembled with Sequencher 4.6 (Gene Codes Corp., Ann Arbor, MI, USA) and BLAST (www.ncbi.nlm.nih.gov/ BLAST) homology searches identified 37 putative genes. Sequences of 10 genes, including open reading frames, 3' and/or 5' untranslated regions, and introns, were evaluated as potential markers for analyzing G. pannorum and G. destructans. Our screening approach indicated that 8 gene targets could be amplified from both G. destructans and G. pannorum by PCR (Table 2).
To obtain DNA sequences from 1 G. pannorum and 16 G. destructans isolates, we prepared genomic DNA from mycelia grown in yeast extract peptone dextrose broth through conventional glass bead treatment and phenolchloroform extraction and then ethanol precipitation (7). AccuTaq LA DNA Polymerase (Sigma-Aldrich, St. Louis, MO, USA) was used for PCR: 3 min initial denaturation at 94[degrees]C, 35 amplification cycles with a 15-sec denaturation at 94[degrees]C, 30-sec annealing at 55[degrees]C, and 1-min extension at 68[degrees]C and a 5-min final extension at 68[degrees]C. PCR products were treated with ExoSAP-IT (USB Corp., Cleveland, OH, USA) before sequencing. Both strands of amplicons were sequenced by the same primers used for PCR amplification (Table 2). A database was created by using Microsoft Access (Microsoft, Redmond, WA, USA) to deposit and analyze the sequences. Nucleotide sequences were aligned with ClustalW version 1.4 (www.clustal.org) and edited with MacVector 7.1.1 software (Accelrys, San Diego, CA, USA). Phylogenetic analyses were done by using PAUP 4.0 (8) and MEGA 4 (9).
We cloned and sequenced [approximately equal to] 200 Kb of the G. destructans genome and identified genes involved in a variety of cellular processes and metabolic pathways (Table 2). DNA sequence typing by using 8 gene fragments showed that all 16 G. destructans isolates had identical nucleotide sequences at all 8 sequenced gene fragments but were distinct from G. pannorum sequences. A maximum-parsimony tree generated from the 8 concatenated gene fragments indicated a single, clonal genotype for the 16 G. destructans strains (Figure 1). This consensus tree included 4,470 aligned nucleotides from all targeted gene sequences with 545 variable sites that separate the G. destructans clonal genotype from G. pannorum. Further analyses of the same concatenated gene fragments with exclusion of 50 insertions and deletions between G. destructans and G. pannorum yielded a tree with a shorter length (495 steps instead of 545 steps) but an identical topology (online Technical Appendix Figure 1, www.cdc.gov/EID/ content/17/7/1273-Techapp.pdf). This pattern remained unchanged when different phylogenetics models were used for analysis (online Technical Appendix Figure 2). The lack of polymorphism among the 16 G. destructans isolates was unlikely because of evolutionary constraint at the sequenced gene fragments. We found many synonymous and nonsynonymous substitutions in target genes among a diversity of fungal species, including between G. destructans and G. pannorum (10) (online Technical Appendix Figure 3).
Our finding of a single clonal genotype in G. destructans population fits well with the rapid spread of geomycosis in New York (Figure 2). Our sampling population covered both spatial and temporal dimensions, and the numbers of isolates analyzed were adequate in view of difficulties encountered in obtaining pure isolations of G. destructans (11). Although the affected New York sites are separated by sizable distances and include geographic barriers, a role for the natural dissemination of the fungus through air, soil, and water cannot be ruled out. Indeed, several fungi with geographic distributions similar to that in our study have shown major genetic variation among strains (12,13). It is also possible that humans and/or animals contributed to the rapid clonal dispersal. In such a scenario, the diseased or asymptomatic bats might act as carriers of the fungus by their migration into new hibernation sites where new animals get infected and the dissemination cycle continues (4). Similarly, the likely roles played by humans and/or other animals in the transfer of the fungal propagules from an affected site to a clean one cannot be ruled out from our data.
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Virulent clones of human and plant pathogenic fungi that spread rapidly among affected populations have been recognized with increasing frequency in recent years (12,14). However, other pathogens, such as the frog-killing fungus Batrachochytrium dendrobatidis, have emerged with both clonal and recombining populations (13). Our data do not eliminate the possibility that the G. destructans population undergoes recombination in nature. This process to generate genetic variability would require some form of sexual reproduction, which remains unknown in G. destructans. In addition, the fungus might have both asexual and sexual modes in its saprobic life elsewhere in nature, but it exists only in asexual mode on bats (15).
In conclusion, our data suggest that a single clonal genotype of G. destructans has spread among affected bats in New York. This finding might be helpful for the professionals involved in devising control measures. Many outstanding questions remain about the origin of G. destructans, its migration, and reproduction, all of which will require concerted efforts if we are to save bats from predicted extinction (3).
We acknowledge the Wadsworth Center Applied Genomic Technologies Core for DNA sequencing and Media, Glassware and Tissue Culture Unit for specialized culture media. We thank Jared Mayron for creating a Microsoft Access database.
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Address for correspondence: Vishnu Chaturvedi, Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Ave, Albany, NY 12208, USA; email: email@example.com
Author affiliations: New York State Department of Health, Albany, New York, USA (S.S. Rajkumar, X. Li, R.J. Rudd, S. Chaturvedi, V. Chaturvedi); New York State Department of Environmental Conservation, Albany (J.C. Okoniewski); McMaster University, Hamilton, Ontario, Canada (J. Xu); and State University of New York at Albany, Albany (S. Chaturvedi, V. Chaturvedi)
Dr Rajkumar is a postdoctoral research affiliate in the Mycology Laboratory at the Wadsworth Center, New York State Department of Health, Albany, New York, USA. His research interests are molecular genetics, genomics, and antifungal drugs.
Table 1. Geomyces destructans isolates studied, New York, USA Isolate Date obtained Site, county * M1379 ([dagger]) 2008 Mar 28 Williams Hotel Mine, Ulster M1380 ([dagger]) 2008 Mar 28 Williams Hotel Mine, Ulster M1381 ([dagger]) 2008 Mar 28 Williams Hotel Mine, Ulster M1383 ([dagger]) 2008 Apr 11 Graphite Mine, Warren M2325 2010 Jan 25 Westchester M2327 2010 Feb 2 Dewitt, Onondaga M2330 2009 Mar 5 Lancaster, Erie M2331 2009 Mar 9 White Plains, Westchester M2332 2009 Mar 11 Dannemora, Clinton M2333 2009 Mar 11 Dannemora, Clinton M2334 2009 Mar 12 Newstead, Erie M2335 2009 Mar 16 Ithaca, Tompkins M2336 2009 Oct 6 Bridgewater Mine, Windsor, VT M2337 2010 Feb 9 Akron Mine, Erie M2338 2010 Mar 4 Hailes Cave, Albany M2339 2010 Mar 11 Letchworth Tunnel, Livingston * All locations in New York state except Bridgewater Mine, Windsor, Vermont. ([dagger]) Previously analyzed by randomly amplified polymorphic DNA typing. Table 2. Geomyces destructans and G. pannorum target gene fragments used for multiple gene genealogic analyses, New York, USA Amplicon size/ Homology (GenBank sequence used for Gene * accession no.) comparison, bp ALR Penicillium marneffei 654/534 (XP_002152078.1) Bpntase Glomerella graminicola 921/745 (EFQ33509.1) DHC1 Sordaria macrospora 597/418 (CBI53717.1) GPHN Ajellomyces capsulatus 659/525 (EEH06836.1) PCS A. capsulatus 920/749 (EEH08767.1) POB3 Pyrenophora tritici-repentis 653/417 (XP_001937502.1) SRP72 A. dermatitidis 941/640 (EEQ90678.1) VPS13 Verticillium albo-atrum 665/545 (XP_003001174.1) G. destructans/G. Primer sequence, 5' [right arrow] pannorum GenBank Gene * 3't ([dagger]) accession nos. ALR V1905 (f): CGGAGTGAGATTTATGACGGC HQ834314- V1904 (r): CGTCCATCCCAGACGTTCATC HQ834329/HQ834330 Bpntase V1869 (f): TCAGACGGACTCGGAGGGCAAG HQ834331- V1926 (r): TCGGTTACAGAGCCTCAGTCG HQ834346/HQ834347 DHC1 V1906 (f): GGATGATTCGGTCACCAAACAG HQ834348- V1907 (r): ACAGCAAACACAGCGCTGCAAG HQ834363/HQ834364 GPHN V1918 (f): CACTATTACATCGCCAGGCTC HQ834365- V1919 (r): CTAAACGCAGGCACTGCCTC HQ834380/HQ834381 PCS V1929 (f): AGGCTGCGATTGCTGAGTGC HQ834382- V1873 (r): CCTTATCCAGCTTTCCTTGGTC HQ834397/HQ834398 POB3 V1908 (f): CACAGTGGAGCAAGGCATCC HQ834399- V1909 (r): ACATACCTAGGCGTCAAGTGC HQ834414/HQ834415 SRP72 V1927 (f): AAGGGAAGGTTGGAGAGACTC HQ834416- V1895 (r): CAAGCAGCATTGTACGCCGTC HQ834431/HQ834432 VPS13 V1922 (f): GAGACAACGCTTGTTTGCAAGG HQ834433- V1923 (r): ACATGCGTCGTTCCAAGATCTG HQ834448/HQ834449 * Genes: ALR, [alpha]-L-rhamnosidase; Bpntase, 3'(2'),5'-bisphosphate nucleotidase; DHC1, Dynein heavy chain; GPHN, Gephyrin, molybdenum cofactor biosynthesis protein; PCS, peroxisomal-coenzyme A synthetase; POB3, FACT complex subunit; SRP72, signal recognition particle protein 72; VPS13, vacuolar protein sorting-associated protein. ([dagger]) f, forward; r, reverse.
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|Author:||Rajkumar, Sunanda S.; Li, Xiaojiang; Rudd, Robert J.; Okoniewski, Joseph C.; Xu, Jianping; Chaturved|
|Publication:||Emerging Infectious Diseases|
|Date:||Jul 1, 2011|
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