New tools for investigating enteric host-pathogen interactions in livestock species

Infections of the gut are extremely common in humans and animals. On farms they cause production losses and, because infections of the gut are often passed between people and animals, can also contribute to human illness. Rising antibiotic resistance means that new and better drugs and vaccines against these diseases are needed. In this proposal we wish to develop novel tools for researchers who want to study the gut and in particular, investigate how disease-causing microbes interact with cells in the gut. These tools will be of great importance to those researching how we can maintain healthy and productive animals and also improve gut health in humans.

The earliest interactions between a pathogen and host intestine are crucial for induction of protective immunity, and are therefore attractive targets for drug or vaccine-induced protection. Therefore, to reveal new strategies for drug and vaccine design, we would like to develop new models for studying these early interactions. Until now, if we wanted to study the infection process in detail we have been forced to do so using very simple tissue culture systems, most of which are very distant from the real-life situation. This is a particular problem in farm animals, where we are often forced to use cell lines from other species. In this project we want to make a transformational change to our technology by growing more complex, gut-cell structures in test-tubes - in effect, "mini guts" called "organoids". We will determine the best culture conditions to generate organoids from cattle and pigs, delivering more relevant models to study gut health in livestock. We will also determine the best methods of freezing organoids for long-term storage. Finally, we will also determine the best methods of infecting organoids with microbes, and look to see if they respond in a similar way to the natural situation. Together, this will lead to the availability of organoid cultures as routine tools for researchers interested in studying gut infections. We envisage that this technology will be widely used in the future, assisting with the design of new drugs and vaccines.

Looking beyond the funding period of the grant, we plan to make these tools useful for the wider community and to ensure that the technology does not remain confined to a few specialist laboratories. To do this, we have formed collaborative partnerships with scientists at the the European Collection of Cell Cultures (ECACC). Our collaborators at ECACC will assist us in optimising the cryopreservation of organoids, so that they can be stored for long periods of time and distributed to scientists around the world. ECACC has many years of experience in freezing tissues, authenticating them and shipping them; it is also highly experienced in administering not-for-profit business models, so that scientific resources such as these are sustainable and can be distributed cheaply amongst scientists. This unique combination of expertise will enable both the development and dissemination of an important new technology, which will be important in the design of new drugs and vaccines in the future. It is also represents an important step in reducing the number of experimental animals used in universities and industry for medical and veterinary research.

The earliest interactions between pathogens and the intestinal epithelium are crucial for induction of protective immunity and are major targets for drug or vaccine-induced protection. However, our understanding of these early molecular interactions is limited by a lack of physiologically relevant in vitro infection models. This is particularly true in the veterinary field, where the majority of studies are performed on heterologous cell lines. Stem cell-derived organoids have the potential to be powerful models for investigating the interaction of pathogens with species-matched, fully differentiated, three-dimensional epithelial surfaces that closely resemble those encountered in vivo. However, three key issues have prevented the application of this technique to veterinary infectious disease: (1) the ability to develop stable and fully differentiated organoids from livestock species, (2) the methodology for long-term cryopreservation of organoids, and (3) the development of organoids as tractable and relevant infection models, including restriction of pathogen entry to the luminal epithelial surface. None of these issues can be easily solved by adapting existing organoid technologies. Instead a concerted and methodical approach is required to identify and validate novel techniques for the culture, cryopreservation and infection of livestock organoids. Here, we will develop novel organoid models from the intestines of livestock species and refine these as tractable infection models that mimic in vivo host-microbe interactions. We will evaluate the suitability of organoids as models by investigating how they respond to infection, adopting a systems biology approach using live-cell imaging, immunological analyses and proteomic signatures. We will also develop standardised methods for the cryopreservation of organoids, allowing the technology to be disseminated widely through the research community.

Enteric infections are important contributors to mortality and morbidity in humans and food producing animals, having a significant impact on welfare and productivity. Rising antibiotic resistance and a lack of effective disease control means there is an urgent need to develop new vaccines and drugs against gastrointestinal disease, and to better understand gut health in general. Essential to developing new vaccines is a deeper understanding of the molecular interactions of pathogens with the gastrointestinal tract; this should yield novel targets for intervention. The technology required to generate stable and fully differentiated organoids from livestock species, to use organoids as models for enteric infection, and to cryopreserve organoids for dissemination to the scientific community, has yet to be established. Providing these novel tools for studying enteric host-microbe interactions in livestock is the focus of this proposal and will lead to long-lasting impacts in academic research, industry and wider society.

This proposal addresses a number of BBSRC strategic priorities and themes including: developing next generation vaccines to combat major infectious diseases of livestock, studies that underpin improvements in both human and animal health and wellbeing, systems approaches to the biosciences, and the replacement, refinement and reduction (3Rs) in research using animals.

We aim to ensure high impact and maximum possible exposure of the organoid culture techniques we develop to the international science community. To achieve this, we are partnering with The European Collection of Cell Cultures (ECACC) to optimise cryopreservation of organoids, so that they can be stored for long periods of time and distributed to scientists as fully validated organoid tool-kits. ECACC has many years of experience in freezing tissues, authenticating them and shipping them; it is also highly experienced in administering not-for-profit business models, so that scientific resources such as these are sustainable and can be distributed cheaply amongst scientists. Looking ahead, we will also institute an on-going programme of dissemination and training incorporating the use of gastrointestinal organoids into the ECACC's existing training programmes, thus making use of their excellent existing infrastructure. We anticipate that this new technological resource will eventually have major impact on the use of live animals in research (3Rs). Organoid models can be used specifically as in vitro substitutes for infectivity studies, for infection biology studies aimed at improving our knowledge of basic host-pathogen interactions, and for drug testing and/or vaccine candidate screening.