Development of an air-interface in vitro organ culture model (IVOC) of bovine respiratory epithelium

Project background

Infectious respiratory diseases of cattle are common and major causes of economic and welfare concern globally. Bovine respiratory disease is complex arising from the interaction of a number of contributing factors including physical stresses and bacterial and viral infections. Usually, all three factors are needed for the disease to develop. The most important bacterial pathogen is Mannheimia haemolytica as this causes pleuropneumonia and subsequent septicaemia that is often fatal. M. haemolytica can be isolated from the nasopharynx of clinically healthy animals but the mechanism through which it breeches mucosal defences, gaining access to the lungs is not well understood. Live animals are required to research both mechanisms by which M. haemolytica causes disease and how to counter the infection by either vaccination or by antimicrobials.

Why we funded it

This Project Grant aims to develop an in vitro culture system to replace live animals used in the study of bovine respiratory infections.

The tissue used to develop the culture system in this project is collected at commercial slaughterhouses and a single bovine head/trachea can be used to supply 200 individual tissue pieces. These tissue samples are used for pathogen challenge experiments which would require 54 animals for an experiment covering three timepoints. The model could also be used in screening experiments to assess the impact of mutations on bacterial colonisation. A library of 10,000 mutants is typically applied to live animals, requiring approximately 100 animals in total to be exposed to between 96 and 480 bacterial mutants. The in vitro culture system can replace all of these animals.

Research methods

Fresh bovine respiratory tissues obtained from healthy cattle will be used to establish nasal and tracheal organ culture infection models. The first phase of this project looks to determine the exact culture conditions required to maintain epithelial function and integrity. Baseline characterisation of the cellular, ultrastructural and biochemical features of uninfected tissue will then be analysed, in particular, cytokine production will be measured. This will be compared to tissue infected with respiratory bacteria or viruses to determine any changes caused by the infection.

The aim of this project is to significantly reduce the use of animals in research investigating respiratory tract infections. Specifically we will establish highly physiologically relevant in vitro air-interface respiratory tract organ culture systems that can be used to study the early stages of the pathogenesis of bovine respiratory bacterial and viral infections thereby replacing experimental animals. This will build on and extend similar work that has successfully replaced dogs, pigs and horses for studying respiratory infections in these hosts.

The objectives are:

1. to establish air interface organ culture systems for bovine respiratory tract tissues.
2. to characterise the cellular, ultrastructural, and biochemical features of uninfected cultures
3. to establish the cellular, ultrastructural, and biochemical changes induced in these cultures when infected with wild-type or mutant strains of Mannheimia haemolytica
4. to establish the cellular, ultrastructural, and biochemical changes induced in these cultures when infected with BHV-1
5. to use the system to investigate the pathology of co-infection with BHV-1 and Mannheimia haemolytica

This joint project between the Department of Veterinary Medicine at Cambridge University (CU) and the Royal Veterinary College, London (RVC) will provide a novel and sensitive in vitro method of investigating the molecular basis of pathogenesis of bovine respiratory bacterial and viral infections. The culture systems, techniques and reagents will be made freely available to other laboratories.