In Vitro Infectioion of Bovine Hoof Cells and Skin Explants with Treponema Brennaborense and Treponema Denticola

The aim of this study was to establish an in vitro model that permits in vitro infection of bovine skin with Treponema spp . and enables to study the role of treponemes in the pathogenesis of digital dermatitis (DD). In all experiments, incubation with T. denticola or T. brennaborense was carried out simultaneously. Keratinocytes obtained from the claw were cultivated on cover glasses without antibiotics until they reached sub-confluence. Then they were incubated with OMIZ Pat medium containing treponemes for up to 96 h. Every 24 h two cover glasses were fixed and stained with the DAPI method. Skin explants were obtained from typical sites of DD lesions. First the explants were maintained in medium with antibiotics to eliminate bacterial contamination. Subsequently, they were rinsed thoroughly with medium without antibiotics and incubated with Treponema suspensions for 48 h. The treponemes stayed vital under culture conditions for even up to 96 h. They were still showing their typical spiral shaped morphology and adhered to the cultured keratinocytes at all time points. With prolonged incubation time cultured cells began to show morphologic damage and some cells detached from the cover glasses. Light and electron microscopical investigations of the explants revealed that treponemes were adhering to the surface of the epidermis. They were visible in often-enlarged intercellular spaces. In addition treponemes could be detected in deep epidermal layers and in very high concentrations in the dermis. In periodontal disease, spirochetes were observed in enlarged intercellular spaces. Our results support these findings suggesting that treponemes invade the deeper claw tissue via the intercellular spaces of the epidermis. We suggest that the enlargement of the intercellular spaces can improve an increased infection of deeper tissue layers and facilitates the way for infection with other anaerobic bacteria.