Development and characterisation of in vitro models for use in bovine tuberculosis research

Bovine tuberculosis is a pressing agricultural issue that is currently poorly understood and under-researched. The need for further studies on host-pathogen interactions between cattle and its etiological agent Mycobacterium bovis is necessary to improve agricultural economic health and animal welfare. This project first investigates the co-culture epithelial and endothelial cell bovine lung organoid model created by Diane Lee and Mark Chambers and focuses on adapting the model to examine solely the epithelial layer. The model is currently unsuccessful, and a different bovine lung model is explored to examine the response of the alveolar epithelium to M. bovis. This model involves the A549 cell line cultured at air-liquid interface, which allows the apical side of the cells to be exposed to air while being supplied with media from the basolateral side. This model will serve as a surrogate model for bovine alveolar type II (ATII) cells in the alveolar epithelium. While the relationship between M. bovis and alveolar macrophages has been studied extensively, much remains unknown about the relationship between M. bovis and ATII cells. The aim of this project is to explore how the bacterium crosses the alveolar barrier and how it affects tight junctions, mucin production, and cellular viability in the model. Proving a functional model of the response of the alveolar epithelium to M. bovis without the need for animal tissue will present an inexpensive, ethical option for alveolar epithelial host-pathogen studies of an under-researched pathogen. This study draws primarily from the works of Lee and Chambers as well as Wu et al. to create a model of the alveolar epithelium by culturing the popular ATII model cell line A549 at air-liquid interface as well as at a monolayer. Characterisation of the cells cultured as ALI shows tight junction and mucin production, as well as the presence of ATII marker Cytokeratin 18. M. bovis Bacille Calmette-Guerin infection of both the three-dimensional model and the monolayer shows that the three-dimensional model displays higher bacterial uptake than the monolayer. The study also shows that BCG does not cause significant cell death in A549 cells within the first 48 hours of infection.