Defining early entry mechanisms of Mycobacterium avium paratuberculosis into the host
Lead Research Organisation:
Moredun Research Institute
Department Name: Vaccines and Diagnostics
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
Technical Summary
Johne's disease of ruminants is a disease of major economic and animal health concern for which current control measures are inadequate. The disease is complex with divergent disease states, differences between host species and genetically diverse strains of the causative organism MAP. The ability of MAP to evade immune detection whilst persisting within gut and lymphatic tissues during the early subclinical phase of infection is likely to determine the nature and severity of disease. The effector mechanisms utilised by MAP are not yet fully understood.
The project will comprise comprehensive analyses of host responses, taking advantage of murine models where cell types can be depleted, or over-expressed, unique access to tissue culture systems (gut organoids) and surgical models enabling access to the gut. We will utilise a combination of cellular and molecular techniques including flow cytometry, confocal microscopy and bioimaging alongside transcriptomic analysis to define host cellular responses and gene expression profiles. We will use immunohistochemistry and culture to identify MAP and to assess bacterial survival and growth.
In parallel, we will identify the bacterial factors that determine intracellular net replication and pathogenesis using transposon-directed insertion-site sequencing and generation of specific mutants.
This project will produce an integrated analysis of host-pathogen interactions occurring in vivo following exposure to MAP and will facilitate identification of targets for disease intervention through improved diagnosis and/or vaccination.
The project will comprise comprehensive analyses of host responses, taking advantage of murine models where cell types can be depleted, or over-expressed, unique access to tissue culture systems (gut organoids) and surgical models enabling access to the gut. We will utilise a combination of cellular and molecular techniques including flow cytometry, confocal microscopy and bioimaging alongside transcriptomic analysis to define host cellular responses and gene expression profiles. We will use immunohistochemistry and culture to identify MAP and to assess bacterial survival and growth.
In parallel, we will identify the bacterial factors that determine intracellular net replication and pathogenesis using transposon-directed insertion-site sequencing and generation of specific mutants.
This project will produce an integrated analysis of host-pathogen interactions occurring in vivo following exposure to MAP and will facilitate identification of targets for disease intervention through improved diagnosis and/or vaccination.
Planned Impact
Johne's disease (JD) is a fatal infectious enteritis principally of ruminants caused by Mycobacterium avium paratuberculosis (MAP). The disease impacts on animal health and welfare, food security and is responsible for significant economic losses to the livestock industries. JD is very difficult to diagnose and control. There is no single test that can reliably detect all stages of the disease and subclinically infected animals can spread infection via intermittent shedding of MAP in faeces and milk. Diagnosis and control could be significantly improved if it were possible to detect animals early in infection and determine which animals would progress to clinical disease. This project aims to study the early infection events and identify key cellular responses and concurrent changes in bacterial function and gene expression. As such, the research and academic communities are the primary beneficiaries of our project outputs. In the long term, the new knowledge could lead to the identification of specific targets for diagnostics or vaccine development or novel strategies for intervening and reducing early infection such as dietary supplements. This would ultimately improve animal health and welfare, food security and economic competitiveness of the livestock industries.
The project will enhance interaction and communication between academics and research workers and build research capacity by encouraging the next generation of research workers to embrace and expand the new technologies developed during this project. There will be training opportunities within the project for students, post-doctoral scientists and technicians in molecular microbiology, cellular immunology, pathology, surgery and anaesthetics. Knowledge exchange to researchers out with the project team and other stakeholders such as policy makers will be achieved through conferences, peer reviewed publications, agricultural shows and the workshop planned for year three.
Information from the project will help farmers and veterinarians to understand the disease process and the rationale behind current management and control recommendations. We will engage with these beneficiaries via the Moredun Foundation and Moredun Scientific Limited, which are well connected to the farming, veterinary, animal health and livestock industries and host events and focus groups across the UK to raise awareness of JD. This will encourage more farmers to carry out JD testing and control and reduce the impacts of JD.
The project results will have both societal and economic benefits. The information will be available to dairy and beef producers, associated food industries, supermarkets and policy makers to design and implement effective JD management and control measures. This will improve food quality and safety for society and increase the economic competitiveness of these industries. Through organisation of public events at shows and festivals we will improve public awareness of the potential impacts of JD on the economies of farming and livestock associated industries, the quality and safety of our food supply and the possible effect of MAP on human health.
The project will enhance interaction and communication between academics and research workers and build research capacity by encouraging the next generation of research workers to embrace and expand the new technologies developed during this project. There will be training opportunities within the project for students, post-doctoral scientists and technicians in molecular microbiology, cellular immunology, pathology, surgery and anaesthetics. Knowledge exchange to researchers out with the project team and other stakeholders such as policy makers will be achieved through conferences, peer reviewed publications, agricultural shows and the workshop planned for year three.
Information from the project will help farmers and veterinarians to understand the disease process and the rationale behind current management and control recommendations. We will engage with these beneficiaries via the Moredun Foundation and Moredun Scientific Limited, which are well connected to the farming, veterinary, animal health and livestock industries and host events and focus groups across the UK to raise awareness of JD. This will encourage more farmers to carry out JD testing and control and reduce the impacts of JD.
The project results will have both societal and economic benefits. The information will be available to dairy and beef producers, associated food industries, supermarkets and policy makers to design and implement effective JD management and control measures. This will improve food quality and safety for society and increase the economic competitiveness of these industries. Through organisation of public events at shows and festivals we will improve public awareness of the potential impacts of JD on the economies of farming and livestock associated industries, the quality and safety of our food supply and the possible effect of MAP on human health.
Publications
Chapuis A
(2024)
Expression and functional characterization of bovine receptor activator of NF-?B ligand (RANKL)
in Veterinary Immunology and Immunopathology
Smith D
(2022)
Advancing animal health and disease research in the lab with three-dimensional cell culture systems.
in The Veterinary record
Smith D
(2021)
The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions.
in Frontiers in cellular and infection microbiology
Description | The overall purpose of the work funded through this award is to define the early entry mechanisms of the bacteria Mycobacterium avium paratuberculosis (MAP) into the host. Infection with MAP leads to Johne's disease which has a significant negative impact on animal health and welfare with knock-on effects to productivity and profit. 1) New research methods - We have developed novel 3D cell culture systems ('enteroids') from the part of the small intestine that is the target of early infection by MAP. In the laboratory we infected these enteroids with MAP bacteria that was tagged with marker (green fluorescent protein, GFP) to allow us to track the location of the bacteria. Using a fluorescence microscope we showed that the bacteria can infect the enteroids. This is an important first step in demonstrating that the enteroids can be used to model MAP infection in the intestine of cattle. By using a robust and valid non-animal model we can reduce the number of animals used in research. 2) New research methods/New knowledge generated - MAP infects the intestine via a specialised type of cell called an M cell. In mice, it has been shown that the number of M cells can be increased by treating with a protein called RANKL. However, the link between RANKL and M cells has not been proven in cattle. We tried using RANKL from mice to stimulate M cells in enteroids from cattle but were not successful. We think this is because the RANKL was from one species - i.e. mice - and the enteroids were from a different species - i.e. cattle. Therefore, we used the protein sequence of RANKL from cattle to synthesise bovine RANKL and used this to treat the cattle enteroids. We found that the bovine RANKL increased the numbers of M cells in the enteroids, similar to what had been reported in mice. This is an important new research method since it will allow us to investigate in detail the interactions between MAP bacteria and M cells in the enteroids. 3) Increased research capability/Improved research methods and skills - The live animal phase of this work involved performing surgery on calves to create discrete "loops" in the intestine, similar to a string of sausages. Each loop was injected with MAP bacteria and, after a defined period of time, each loop was removed from the animal and divided into a range of samples. By testing each sample we investigated the interactions between the MAP bacteria and the cells lining the intestine. This was the first time that this gut loop model had been carried out by members of this research team. During training for the surgery and sample collection we identified and implemented a number of changes to improve animal welfare and sample quality. In addition, optimisation of this research method allowed a number of researchers to expand their skillset in this area. |
Exploitation Route | Ex-vivo enteroid cell culture model - This model has already been utilised for further work involving MAP as well as gastrointestinal nematodes. |
Sectors | Agriculture Food and Drink Pharmaceuticals and Medical Biotechnology |
Description | APHA Cattle Expert Group annual meeting (JM) |
Geographic Reach | National |
Policy Influence Type | Contribution to new or improved professional practice |
Description | EPIC Workpackage IV, Syndromic Surveillance Strategy workshop (JM) |
Geographic Reach | National |
Policy Influence Type | Contribution to a national consultation/review |
Description | Additional UKRI funding to mitigate delays due to coronavirus restrictions |
Amount | £208,798 (GBP) |
Funding ID | EP/V52119X/1 |
Organisation | United Kingdom Research and Innovation |
Sector | Public |
Country | United Kingdom |
Start | 08/2020 |
End | 02/2021 |
Description | Moredun-IGM pathology |
Organisation | Instituto de Ganadería de Montaña |
Country | Spain |
Sector | Public |
PI Contribution | Demonstration of surgical technique for gut loop surgery. Provision of training in sample collection and processing for histopathological and immunohistochemical examination of gut loop samples. |
Collaborator Contribution | Two collaborators from IGM came to the UK to assist with the sample collection phase of the study. This facilitated rapid sample collection and processing. IGM processed a subset of the samples and examined them for histopathological changes in the tissue and the presence/absence of bacteria of interest. |
Impact | Building a strong working relationship between institutes. Having two additional people present during the sample collection phase of this study significantly reduced the time between sample collection and processing, thus maximising the quality of the pathology samples and data generated from these samples. |
Start Year | 2021 |
Description | Moredun-RDSVS anaesthesia |
Organisation | University of Edinburgh |
Department | Royal School of Veterinary Studies |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Provision of the opportunity for Royal (Dick) School of Veterinary Studies anaesthesia residents to gain experience in maintaining calves under general anaesthesia for a prolonged period. |
Collaborator Contribution | Provision of expertise in large animal anaesthesia which allowed the study animals to be induced and maintained under anaesthesia for prolonged periods without any adverse impact on animal welfare. |
Impact | Successful induction and maintenance of anaesthesia of study animals without any adverse impact on animal welfare. |
Start Year | 2022 |
Description | Moredun-Roslin invitro organoid model development |
Organisation | University of Edinburgh |
Department | The Roslin Institute |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | The Moredun team brings expertise in culture and infection of organoids generated from tissues from cattle and sheep. |
Collaborator Contribution | The Roslin team brings expertise in culture and infection of organoids generated from tissues from wild-type and transgenic mice. |
Impact | Publication (in preparation) comparing and contrasting the culture, infection and fluorescence imaging of organoids from cattle, sheep and mouse following infection with Mycoplasma avium ssp paratubercuolsis (MAP). |
Start Year | 2022 |
Description | Moredun-STEM Cell Technologies |
Organisation | Stemcell Technologies |
Department | STEMCELL Technologies UK Ltd. |
Country | United Kingdom |
Sector | Private |
PI Contribution | Expertise in organoid cell culture models to facilitate validation and optimisation of culture media |
Collaborator Contribution | STEMCELL provided samples of culture media that are still in pre-commercial development |
Impact | None to date |
Start Year | 2022 |
Description | Moredun-nanoString |
Organisation | NanoString Technologies |
Country | United States |
Sector | Private |
PI Contribution | Expertise in target probe selection, experimental design, selection and preparation of tissue samples, optimisation of sample preparation protocols. Access to tissue samples. Staff time for above activities. |
Collaborator Contribution | Expertise for preparation of RNA probe libraries. Provision of facilities and training to analyse samples, generate data, QC data, analyse data, generate outputs. Staff time for above activities. |
Impact | Creation of a novel bovine RNA probe set (n=200) Application of a novel bovine RNA probe set to bovine and ovine tissue samples Multidisciplinary - Yes. Disciplines involved: parasitology, virology, bacteriology, histopathology, pathology, spatial transcriptomics |
Start Year | 2021 |
Description | Animal Disease Research Funders, Oral presentation on MAP Gut Loop (JM) |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Policymakers/politicians |
Results and Impact | 8 representatives attended from research funders including the UK Science Partnership for Animal and Plant Health, Bill and Melinda Gates Foundation, Scottish Government, Foreign, Commonwealth and Development Office, DEFRA, Wellcome Trust, and UKRI BBSRC. Moredun researchers presented on projects in the current research portfolio which sparked questions and discussion of current and future research directions and funding strategies. |
Year(s) Of Engagement Activity | 2023 |
Description | Oral presentation, Edinburgh Infectious Diseases Early Career Researcher Symposium, October 2021 (AC) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | Oral presentation at the Edinburgh Infectious Diseases Early Career Researcher Symposium (October 2021). Title - Development of bovine and ovine "apical-out" gut organoid models for the study of pathogen versus host interactions in John's Disease. |
Year(s) Of Engagement Activity | 2021 |
Description | Poster presentation, Digital Pathology and AI Congress, December 2023 (JM) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Poster title - Spatial transcriptomics in veterinary health: applications of Nanostring GeoMx in ruminant gastrointestinal disease. |
Year(s) Of Engagement Activity | 2023 |
Description | Poster presentation, HRH The Princess Royal - Celebrating 100 years of Moredun, November 2021 (AC) |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Supporters |
Results and Impact | Poster presentation to HRH Princess Anne, title "Miniature organs (organoids) for modelling infectious diseases of animals in the lab". |
Year(s) Of Engagement Activity | 2021 |
Description | Poster presentation, International Veterinary Immunology Society Symposium, November 2023 (AC) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Poster title - Characterization of bovine and ovine ileal organoids after polarity exchange |
Year(s) Of Engagement Activity | 2023 |
Description | Poster presentation, International Veterinary Immunology Society Symposium, November 2023 (AC) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Poster title - Fc-tagged fusion proteins as tools to define veterinary cytokines and investigate immunological receptor/ligand interactions. |
Year(s) Of Engagement Activity | 2023 |
Description | Poster presentation, Organ Modelling Congress, April 2022 (AC) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Other audiences |
Results and Impact | Poster title - Development of bovine and ovine "apical-out" gut organoid models for the study of pathogen versus host interactions. Activity description - The Organ Modelling Congress addresses an important and expanding field which provides new understandings in organ functionality and novel approaches to drug development. These engineered human organs using Organ-on-a-Chip, Tissue-on-a-Chip and Organoids are a remarkable tool with the potential to facilitate studies that better predict drug efficacy and thereby reduce cost, time, and failure rates in clinical trials. Further, patient-derived organoids hold promise for personalised medicine, as they have the potential to produce individualised predictions of drug response. |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.rsc.org/events/detail/73825/organ-modelling-uk-in-person |
Description | School Visit, Saint Mary's Music School, Edinburgh, November 2022 (AC) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | 2 researchers from the research organisation visited the school to introduce the students to the possibilities of a career in science. The students were interested and asked questions about the research carried out at the research organisation in particular animal studies. One student appeared interested in pursuing a career in science. |
Year(s) Of Engagement Activity | 2022 |
Description | Scottish Rural & Environment Science & Analytical Services (RESAS) visit to Pentlands Science Park, February 2023 (AC) |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Policymakers/politicians |
Results and Impact | 10 representatives from the Scottish Rural & Environment Science & Analytical Services (RESAS) visited the research organisation to learn more about the research activities carried out at the institute. We presented results on development of a new cell culture system (organoids) to study bacterial infection in vitro. The visitors expressed interest in and asked questions about the technical aspects and wider applications of the cell culture model. |
Year(s) Of Engagement Activity | 2023 |
Description | Secondary school teachers visit Moredun Research Institute, May 2022 (AC) |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Schools |
Results and Impact | This event was coordinated by SSERC and Royal Society of Biology Secondary. School teachers from urban, rural and remote areas of Scotland visited the research organisation to learn more about new scientific projects that have the potential to be incorporated into their teaching curricula. Research areas included parasitology, immunology, microbiology and 3D cell culture models (organoids). We had a very positive response including multiple requests for researchers to come and present their work and tell the students more about careers in science. |
Year(s) Of Engagement Activity | 2022 |
Description | Visit of Norwegian farmers to the Pentlands Science Park, July 2022 (AC) |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Industry/Business |
Results and Impact | Approximately 30 sheep farmers visited the research organisation to learn more about progress in livestock animal health and welfare. Two researchers gave a presentation on the development of a novel 3D cell culture system (organoids) and applications to research on bacteria and parasites. Farmers and veterinary practitioners were interested to learn more about the best method to collect samples that could contribute to further test development. |
Year(s) Of Engagement Activity | 2022 |