A three-dimensional air-liquid interface airway epithelial cell model to study pathogen interactions within the bovine respiratory tract

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Background. Bovine respiratory disease (BRD) is a multifactorial condition of cattle that involves complex interactions between different viral and bacterial pathogens and causes significant economic losses to the cattle industry worldwide. Bovine respiratory syncytial virus (BRSV) and bovine herpes virus-1 (BHV-1) are two of the most important viruses and Mannheimia haemolytica is one of the most important bacterial species involved in BRD. There is an urgent need to develop more effective vaccines and antimicrobials against the pathogens responsible for BRD. To achieve this we require a greater understanding of the molecular interactions that take place between pathogens and host. In particular, we need to more fully understand the early events that take place when viruses and bacteria colonise the host respiratory tract. Unfortunately, the methods that are currently available to do this are not well developed and, consequently, a large number of research experiments have to be carried out in cattle. Consequently, there is an urgent need to develop laboratory-based methods that can be used to study the interactions of viruses and bacteria with the bovine respiratory tract. One such method involves the three-dimensional (3-D) culture of differentiated primary airway epithelial cells grown at an air-liquid interface (ALI). Crucially, these cells can be extracted from the lungs of freshly killed cattle at abattoirs; the approach does not involve killing cattle specifically for this purpose.Aims and objectives. The overall aim of the project is to develop a 3-D bovine airway epithelial cell (BAEC) model at an ALI that will allow us to investigate the complex interactions of bacteria and viruses within the bovine respiratory tract. The project comprises four phases. In phase 1, differentiated BAECs grown at an ALI will be characterized and optimized by examining the cells over a period of 42 days using a range of microscopic and other techniques. The optimum "window" during which the cells are at their healthiest for use in subsequent experiments will be identified. In phase 2, the interactions of BRSV and BHV-1, as well as M. haemolytica, with BAECs maintained at an ALI will be studied using a range of microscopy and other techniques. We will also carry out co-infection studies in which we will investigate the effect of viral infection on subsequent infection with M. haemolytica. In phase 3, the immune response of the epithelial cells to "infection" with these pathogens will be studied. Respiratory epithelial cells secrete special immune chemicals (cytokines) when they are infected with pathogens that are important in protecting the epithelial surface from infection. We will study this innate immune response by measuring cytokine secretion. In phase 4, the findings of the laboratory studies will be validated by infecting cattle with a virus alone, with M. haemolytica alone, and with the virus followed by M. haemolytica. The effects of these infections will be assessed by evaluating colonisation of bacteria and the cytokine response within the respiratory tract.Applications and benefits. The development and characterization of this model, and the demonstration that it can be used to study bacterial and viral infections of the bovine respiratory tract, will have a number of important applications and benefits to the study of respiratory disease in cattle. It will provide an invaluable tool for studying the interactions of bacteria and viruses within the bovine respiratory tract in the laboratory without the need to use cattle. Importantly, this approach could also be extended to other animals. In this way, the development of this model will save countless animals from being used in such experiments. In addition, this and future work will further our understanding of bacterial and viral infections within the bovine respiratory tract and this will contribute to the development of improved vaccines and antimicrobials.