Oral Presentation The Prato Conference on the Pathogenesis of Bacterial Infections of Animals 2016

3D culture model for ovine footrot: generating an alternative to in vivo research (#34)

Grazieli Maboni 1 , Rebecca Davenport 1 , Kate Sessford 1 , Adam Blanchard 1 , Gary Entrican 2 , Sean Wattegedera 2 , Catrin Rutland 1 , Sabine Totemeyer 1
  1. University of Nottingham, Loughborough, LE, United Kingdom
  2. Moredun Research Institute, Edinburgh , United Kingdom

Ovine footrot is characterized by the separation of the hoof from the underlying skin. Dichelobacter nodosus is a bacterium essential to initiate footrot (1, 2). In vivo research on footrot involves ethical issues regarding sheep welfare, hence in vitro models represent an alternative to accomplish the 3Rs (Replacement, Reduction and Refinement) of animal experimentation. Therefore, we aimed to develop an ex vivo organ culture (EVOC) that allows us to mimic the ovine interdigital environment in a 3D culture, hence generating an alternative to in vivo research.

Biopsies were taken from the interdigital skin of post slaughtered sheep. The viability of the biopsy was assessed by tissue integrity (H&E stain) and cell death (H&E and TUNNEL staining) over 72 hours. The development of the infection protocol was carried out with virulent and benign strains of D. nodosus, which were quantified in the biopsies by qPCR. Localisation of the bacteria within the biopsy was achieved by in tissue Gram stain. Expression of pro-inflammatory cytokines in repose to D. nodosus infection was measured.

 Preliminary data suggests that tissues are viable for up to 48 hours of culture. D. nodosus was quantified in all samples infected with both virulent and benign strains. Gram negative rods were found in the epidermis which could be putatively D. nodosus. Expression of pro-inflammatory cytokines was detected in all biopsies.

 This novel model is the first of its kind for investigating footrot in alignment with the 3Rs. EVOC allows the investigation on how bacteria infect these tissues and the early stages of host response. This is a fundamental step that will in a long term underpin the design of an efficacious vaccine.

(1) EGERTON, J. R. et al (1969). Journal of Comparative Pathology.

(2) KENNAN, R. M. et al (2010). PLoS Pathogens.