Footrot is a highly contagious disease affecting the foot of sheep, causing heavy economic losses due to lameness and reducing animal welfare. The causative bacterium is the gram negative anaerobe Dichelobacter nodosus. The aim of this study was to develop a reconstructed sheep epidermis (RSE) by 1) selecting the most appropriate skin digestion protocol; 2) comparing two conditions of cell culture; 3) performing functional and histological characterization of RSE. Among 6 digestion protocols tested on 5 skin samples, the protocol based on 1 hour of incubation in 0.17% of trypsin at 37°C provided the most efficient result in terms of keratinocyte count (851.217±405.797 cells/mL) and viability 24h after digestion (85,5±3,7%). Keratinocyte cultures were performed with and without Rho kinase (ROCK) inhibitor Y-27632. In presence of Y-27632, isolated keratinocytes showed an increase in proliferation and delayed differentiation while cultures without inhibitor stopped growing. RSE were prepared with third passage keratinocytes seeded onto polycarbonate fiber and exposed to an air-liquid interface with a medium without Y-27632 allowing differentiation. Differentiated RSE were obtained 11 days after seeding. Histological staining using hematoxylin-erythrosine and immunohistological staining allowed morphological analysis of RSE confirming differentiation of basal, spinous, granular and cornified layer. Transepithelial electrical resistance was measured and confirmed functional integrity of RSE (12.589±310 Ω cm2, n=6). This study describes the development of a RSE and provides a promising tool for the characterization of mechanisms involved in Dichelobacter nodosus infection, such as adhesion, invasion and proliferation of bacterium in epidermis. Moreover, this work revealed that ROCK inhibitor Y-27632 seems to improve the reconstruction of epidermis from isolated keratinocytes cultures.

Elaboration of reconstructed sheep epidermis