Development and use of advanced in vitro culture systems to interrogate host-pathogen interactions in livestock species
Project Lead
Challenges
Most infectious agents affecting livestock enter the body or reside on mucosal surfaces of the gut, lung and nasal tracts, and mammary gland. Mucosal surfaces are lined by epithelial cells, provide a barrier against infectious agents, and play a key role in initiating immune responses. Researching these interactions is critically important for developing novel ways to control infections. However, developing studies can be technically challenging due to: (i) the inaccessibility of most mucosal surfaces for studies within the animal; (ii) available epithelial cell culture systems do not accurately reflect the range of cell types present in vivo, nor the morphology of the epithelial surface. There is a need to generate more relevant epithelial culture models which represent the in vivo epithelium.
A key development in this area in recent years has been the ability to generate epithelial organoids from epithelial stem cells, which form three-dimensional tissue constructs that mimic the corresponding in vivo epithelium in terms of morphology and cell types. These organoid cultures consist of a central lumen lined by epithelial cells, although they can be altered to allow the apical (luminal facing) surface of the epithelium to be on the outside of the organoid (‘apical-out’ organoids). These organoids can be passaged in vitro and cryopreserved, meaning that multiple experiments can be performed from organoids generated from a single individual. This greatly reduces the number of animals required for host-pathogen studies, in line with the 3Rs principles (Replacement, Reduction and Refinement).
Epithelium additionally plays a key role in alerting the immune system to infection, either by producing signalling molecules to activate and recruit immune cells, or uptake of antigens via microfold or M-cells. These cells are highly specialised in the uptake of particulate antigens from the luminal surface which are then sampled by sub-epithelial antigen-presenting cells (APCs). M-cells are located within the follicle-associated epithelium (FAE) overlying organised lymphoid tissue within the mucosa, known as Mucosa Associated Lymphoid Tissue (MALT). It is both here and in draining lymph nodes that APC present antigen to mucosal T cells to initiate the adaptive (antigen-specific) immune response. In cattle and sheep, MALT is concentrated in specific mucosal sites, including nasal, bronchial, intestinal, and rectal mucosa. These sites represent key targets for vaccines aimed at inducing strong mucosal immune responses, which may be key for several pathogens.
Questions
- For key diseases for livestock in Scotland, which pathogen component and host immune responses will prove useful to exploit as targets for potential vaccines or accurate detection and diagnoses of infection?
Solutions
In this project, we are extending the range of epithelial organoid culture systems in ruminants to include respiratory (nasal and lower lung), mammary, small intestinal and rectal organoids. Nasal and rectal organoids are inductive sites readily accessible for mucosally delivered vaccines, we are ensuring organoids contain enough M-cells to allow testing of particle-based vaccine delivery systems.
We are using respiratory, gastro-intestinal and mammary organoids to investigate the following pathogens, all of which cause significant economic losses and welfare issues in Scottish ruminant livestock: ovine and bovine gastro-intestinal nematodes (GIN), parasitic gastro-enteritis; Mycobacterium avium subsp. paratuberculosis (MAP), Johne’s disease in sheep and cattle; Jaagsiekte sheep retrovirus (JSRV), Ovine Pulmonary Adenocarcinoma (OPA) in sheep; and mastitis in cattle and sheep.
Overall, we are underpinning novel vaccines and diagnostic tests with the potential to have a significant impact on animal health, welfare, and productivity. Improvements in production efficiency will enhance Scotland’s food security and rural and national economies and reduce the environmental impact of livestock farming. We are validating alternatives to animal experimentation which complement ‘gold-standard’ animal infection models and reduce use of animals in research, addressing a key societal issue.
Project Partners
Progress
We have developed a range of different culture systems derived from sheep and cattle epithelial stem cells ('organoid'), generated pathogens/pathogen products and optimised organoid cultures for subsequent infection studies and developed materials (gels and particles) to facilitate delivery of vaccines to mucosal surfaces. Three dimensional (3D) organoids have been generated from intestinal, mammary and respiratory epithelial cells of sheep and cattle, and different orientations of the organoids for infection studies have been developed. Importantly, developed organoids have been characterised by gene expression and cell type analysis. For future studies of sheep and cattle gastro-intestinal (GI) parasite infections, we have isolated molecules excreted or secreted by the parasites (ES products) which are likely to affect the GI epithelium. Different orientations of intestinal organoid cultures (e.g., 2D, 3D) have been tested to identify the best culture system to study epithelial infection by MAP, the causative agent of Johne's disease in cattle and sheep, from Year 2 onwards. Organoids have been generated from sheep lungs to provide a culture system for future isolation and growth of Jaagsiekte sheep retrovirus, the causative agent of OPA. Bacteria have been isolated from sheep with mastitis of varying severities to allow organoid infection studies of sheep mammary organoids with bacteria of different disease-causing potential. Finally, a range of gels and nanoparticles with adhesive properties have been generated to allow efficient delivery of vaccines onto the epithelial surface.
Project Impact
The expertise in organoid cultures within this group and their utility in studying host-pathogen interactions has been presented to visiting groups of the Scottish Government Animal Health and Welfare Division and Rural and Environmental Science and Analytical Services, with an emphasis on the use of these systems replacing studies involving live animals. Direct knowledge exchange with industry stakeholders have occurred through industry events, such as the Royal Highland Show, and through visits and meetings with five commercial partners that shares an interest in the technology. This has resulted in a new collaboration with a leading company in organoid technologies to beta-test novel organoid media formulations for use in livestock species. The research generated during this project has been presented at national and international meetings, including invited talks at the Microbiology Society, ICOPA 2023 (during which we also organised a session on organoids), as well as the American Association of Veterinary Parasitologists Annual Meeting 2023. This project was also presented at the Hannah Dairy Research Foundation Next Generation Dairying Workshop 2022, the Biggar and Dundee Science Festivals as well as the Moredun Annual Press Day. We have also created a YouTube video on the use of organoids to model host-parasite interactions.
Related Projects
Disease mechanisms: The aim of this work is to provide increased understanding of how pathogens (viruses, bacteria and parasites) interact with their host to cause disease. This new knowledge will underpin the development of next-generation control strategies for a range of infectious diseases of importance to livestock production both nationally and internationally. A broad...
- Animal Disease
- 2016-2022
The aim of this RD is to develop safe, highly effective, optimised, novel vaccines for the control of the most production- and welfare-limiting endemic diseases of Scottish livestock. The key drivers for this research are:
- Prevention is better than cure. Anthelmintics (chemotherapeutics for the control of intestinal worms) and antibiotics are used to treat diseases but can leave...
- Animal Disease
- 2016-2022