PCR-based detection of DNA from the human pathogen Blastomyces dermatitidis from natural soil samples.

Blastomyces dermatitidis is the dimorphic fungal agent of blastomycosis, a disease that primarily affects humans and dogs. The clinical appearance of this mycosis is well characterized, but there is still little known about its environmental niche, having been isolated from nature only 21 times. We have developed a PCR-based assay to detect B. dermatitidis from soil samples using primers specific to a portion of the promoter region of the BAD1 virulence gene. An internal standard control, pTJV2, was constructed to validate the results from soil samples. Amplification of this control indicated adequate removal of ambient soil inhibitors. The PCR detection limits for the control plasmid and B. dermatitidis genomic DNA were 0.1 and 500 femtograms, respectively. No PCR cross-reactivity was observed against bacteria, actinomycetes, and 13 other fungi that were genetically related or found in the same geographic areas. In spiked soil samples, this method was sensitive to 304 copies of pTJV2 DNA and 8,450 live B. dermatitidis yeast cells. Three of eight natural soil samples from a dog kennel near Lexington, KY in which dogs suffered from blastomycosis were positive using the described method, demonstrating its utility in detecting B. dermatitidis in its natural surroundings.

[1]  T. Herrmann,et al.  Nested PCR Assays for Detection of Blastomyces dermatitidis DNA in Paraffin-Embedded Canine Tissue , 2003, Journal of Clinical Microbiology.

[2]  M. Sogin,et al.  Microbial Diversity of Hydrothermal Sediments in the Guaymas Basin: Evidence for Anaerobic Methanotrophic Communities , 2002, Applied and Environmental Microbiology.

[3]  T. Mikawa,et al.  Phylogenetic analysis of the non-pathogenic genusSpiromastix (Onygenaceae) and related onygenalean taxa based on large subunit ribosomal DNA sequences , 2001 .

[4]  B. Klein,et al.  Selective expression of the virulence factor BAD1 upon morphogenesis to the pathogenic yeast form of Blastomyces dermatitidis: evidence for transcriptional regulation by a conserved mechanism , 2001, Molecular microbiology.

[5]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[6]  D. Baumgardner,et al.  The in vitro isolation of Blastomyces dermatitidis from a woodpile in north central Wisconsin, USA. , 1999, Medical mycology.

[7]  T. C. White,et al.  Comparison of six extraction techniques for isolation of DNA from filamentous fungi. , 1998, Medical mycology.

[8]  D. M. Sander,et al.  Genetic diversity in clinical isolates of the dimorphic fungus Blastomyces dermatitidis detected by a PCR-based random amplified polymorphic DNA assay , 1995, Journal of clinical microbiology.

[9]  D. Baumgardner,et al.  The epidemiology of blastomycosis in dogs: north central Wisconsin, USA. , 1995, Journal of medical and veterinary mycology : bi-monthly publication of the International Society for Human and Animal Mycology.

[10]  J. W. Taylor,et al.  Phylogeny of five fungus-like protoctistan Phytophthora species, inferred from the internal transcribed spacers of ribosomal DNA. , 1992, Molecular biology and evolution.

[11]  B. Varkey,et al.  Isolation of Blastomyces dermatitidis in soil associated with a large outbreak of blastomycosis in Wisconsin. , 1986, The New England journal of medicine.

[12]  Denton Jf,et al.  Additional isolations of Blastomyces dermatitidis from natural sites. , 1979 .

[13]  E. S. McDonough,et al.  Isolation of Blastomyces dermatitidis from Soil , 1961, Science.

[14]  D. Baumgardner,et al.  Studies on the molecular ecology of Blastomyces dermatitidis , 2004, Mycopathologia.

[15]  W. Untereiner,et al.  Phylogeny of Ajellomyces, Polytolypa and Spiromastix (Onygenaceae) inferred from rDNA sequence and non-molecular data. , 2002 .

[16]  Blastomycosis--Wisconsin, 1986-1995. , 1996, MMWR. Morbidity and mortality weekly report.

[17]  B. Elewski,et al.  Cutaneous blastomycosis. , 1992, Cutis.

[18]  A. Disalvo,et al.  Additional isolations of Blastomyces dermatitidis from natural sites. , 1979, The American journal of tropical medicine and hygiene.