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Institut für Mikrobiologie und Hygiene, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, Dorotheenstr. 96, D-10117 Berlin, Germany
Institut für Veterinär-Pathologie der Freien Universität Berlin, Straße 518, Nr. 15, D-14163 Berlin, Germany
Institut für Mikrobiologie und Tierseuchen der Freien Universität Berlin, Luisenstr. 56, D-10117 Berlin, Germany
Department of Oral Biology, Yonsei University, 134 Shinchon-Dong, 120-752 Seoul, Korea
Technische Universität München, Lehrstuhl für Mikrobiologie, Arcisstr. 16, D-80290 München, Germany
ABSTRACT
Fluorescence in situ hybridization (FISH) was performed on sections of plastic-embedded tissue using 16S rRNA-directed oligonucleotide probes to visualize uncultured treponemes in skin biopsies of cows with digital dermatitis. Plastic as embedding material allowed sectioning of hard and soft tissue with a defined thickness, avoiding the risk of dragging bacteria into the tissue while sectioning. Furthermore, it provided a good signal-to-noise ratio. Using this method the spatial distribution of three different bacterial phylotypes was visualized simultaneously within the tissue. Whereas debris covering the ulcers contained a mixture of different micro-organisms, a layering of certain treponemal phylotypes was observed deeper in the epidermis. Confocal laser scanning microscopy and subsequent three-dimensional reconstruction of series of optical sections confirmed that the treponemes migrated intercellularly around the cells, most of them directed towards the dermis. In situ hybridization on tissue embedded in plastic proved to be a useful method to study mixed bacterial infections since it combines excellent histological conservation of tissue with identification of bacterial species by simultaneous use of probes labelled with different fluorescent dyes. This technique may have implications for in situ detection, identification and localization of microorganisms in veterinary as well as in human medicine.
*Author for correspondence: Ulf B. Göbel. Tel: +49 30 2093 4715. Fax: +49 30 2093 4703.
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