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Journal of Clinical Microbiology, January 2003, p. 205-208, Vol. 41, No. 1
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.1.205-208.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Nested PCR Assays for Detection of Blastomyces dermatitidis DNA in Paraffin-Embedded Canine Tissue

Ralf Bialek,^1* Anna Cascante Cirera,^1, Tanja Herrmann,¹ Christian Aepinus,² Valerie I. Shearn-Bochsler,^3, and Alfred M. Legendre⁴

Institute for Tropical Medicine, University Hospital Tübingen, Tübingen,¹ Institute for Hygiene and Microbiology, University of Würzburg, Würzburg, Germany,² Department of Pathology,³ Department of Small Animal Clinical Sciences, University of Tennessee, Knoxville, Tennessee⁴

Received 27 March 2002/ Returned for modification 27 September 2002/ Accepted 25 October 2002

A Blastomyces dermatitidis nested PCR assay targeting the gene encoding the Wisconsin 1 (WI-1) adhesin was developed and compared with a nested PCR targeting the 18S rRNA gene (rDNA) of members of the family Onygenaceae. We examined 73 paraffin-embedded tissue samples obtained from nine dogs which died of blastomycosis and nine dogs which succumbed to lymphosarcoma according to autopsy findings; amplifiable canine DNA was extracted from 25 and 33 specimens from the two groups, respectively. The B. dermatitidis PCR amplified DNA from 8 of 13 tissue samples in which yeast cells were detected by microscopy. Sequencing revealed that all PCR products were homologous to the B. dermatitidis WI-1 adhesin gene. No PCR product was amplified from 12 microscopically negative biopsy specimens from dogs with blastomycosis or from 33 biopsy specimens from dogs with lymphosarcoma. The 18S rDNA PCR amplified DNA from 10 and 9 tissue samples taken from dogs which died of blastomycosis and lymphosarcoma, respectively. Only six products were identified as being identical to B. dermatitidis 18S rDNA; they were exclusively obtained from specimens positive by the B. dermatitidis nested PCR. For specificity testing, 20 human biopsy specimens proven to have histoplasmosis were examined, and a specific H. capsulatum product was amplified by the 18S rDNA PCR from all specimens, whereas no product was obtained from any of the 20 samples by the B. dermatitidis PCR assay. In conclusion, the PCR targeting a gene encoding the unique WI-1 adhesin is as sensitive as but more specific than the PCR targeting the 18S rDNA for detection of B. dermatitidis in canine tissue.

Present address: Molecular and Medical Genetics Center, IRO, Hospital Duran i Reynals, 08907 L'Hospitalet de Llobregat, Spain.

Present address: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, WI 53706-1102.

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