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.

Tissue samples of skin affected by digital dermatitis were studied histopathologically together with post-mortem findings and anamnestic datas to achieve better understanding of this disease. There are hyperplastic, ulcerative, proliferative and combined forms distinguishable. The epidermal cells show signs of degeneration, hypertrophy and hyperplasia, combined with differing extensions of cell destruction by bacteria and spirochetes. Dermal reactions contain arteriosclerosis-like plaques, activation of endothelial cells, thrombosis and different patterns of perivascular infiltration. It is concluded that epidermal lesions are a sequel of dermal malperfusion. Partial or total ischaemia leads to hyperplasia and ulceration, respectively. Endotoxins of gram negative bacteria taking part in other diseases in the same cattle are strongly presumed as causing agents. This indicates a multifactorial pathogenesis.