FLUORESCENCE IN-SITU HYBRIDIZATION SHOWS SPATIAL-DISTRIBUTION OF AS YET UNCULTURED TREPONEMES IN BIOPSIES FROM DIGITAL DERMATITIS LESIONS

Fluorescence in situ hybridization (FISH) was performed on sections of plastic-embedded tissue using 16S rRNA-directed oligonucleotide probe s to visualize uncultured treponemes in skin biopsies of cows with dig ital dermatitis. Plastic as embedding material allowed sectioning of h ard and soft tissue with a defined thickness, avoiding the risk of dra gging bacteria into the tissue while sectioning. Furthermore, it provi ded a good signal-to-noise ratio. Using this method the spatial distri bution of three different bacterial phylotypes was visualized simultan eously within the tissue. Whereas debris covering the ulcers contained a mixture of different micro-organisms, a layering of certain trepone mal phylotypes was observed deeper in the epidermis, Confocal laser sc anning microscopy and subsequent three-dimensional reconstruction of s eries of optical sections confirmed that the treponemes migrated inter cellularly around the cells, most of them directed towards the dermis. In situ hybridization on tissue embedded in plastic proved to be a us eful method to study mixed bacterial infections since it combines exce llent histological conservation of tissue with identification of bacte rial species by simultaneous use of probes labelled with different flu orescent dyes. This technique may have implications for in situ detect ion, identification and localization of microorganisms in veterinary a s well as in human medicine.