Understanding inflammatory processes in ovine footrot to inform rational vaccine design

Lead Research Organisation: University of Nottingham
Department Name: School of Veterinary Medicine and Sci

Abstract

Footrot is one of the most important diseases affecting the health and welfare of sheep in the UK and throughout the world, and impacts on productivity and profitability within the sheep industry. It is in the top five most important diseases of sheep and globally affects the income of the poorest farmers. Approximately 14 million sheep get footrot in the UK each year.
Footrot is caused by bacteria that invade the skin of the foot, and cause pain and swelling. In severe cases, the hoof horn capsule, the hard outer surface of the foot, separates from the underlying and sensitive living tissue, rather like a fingernail coming away from the finger. Affected sheep can be treated with an antibiotic injection, however to do this sheep have to be caught. Sheep are fearful animals and are very stressed by being caught and handled, so although it is good for their health and welfare to be treated, the very act of catching them to administer the treatment also reduces their welfare; 14 million treatments means a lot of very stressed sheep. If sheep are left untreated they lose weight, remain lame for some time and are less likely to become pregnant. If they do have lambs they produce less milk and so cannot feed their lambs as well as those ewes that are not lame, as well as impacting on their profitability.
We know that treatment by antibiotic injection early in disease leads to dramatic recovery within a few days. In the long term however, we want to stop using antibiotics to treat farm animals so that we reduce the risk of antibiotic resistance and avoid antibiotics being present in livestock farmed for human consumption. In addition, we want to reduce stress in sheep and avoid the need to catch and treat lame sheep. Ideally, rather that treating disease, we would prefer to prevent disease by vaccination. There is a vaccine available to assist in control of footrot, but less than 10% of UK farmers use this vaccine: it is neither long lasting (5 months maximum) nor very effective (60% effective at best). This lack of effectiveness is thought to be due to a failure to stimulate the best possible host immune response. Our research will help us to understand how sheep respond to the bacteria that cause footrot during the very early stages of infection, and will ultimately help in the development of alternative approaches to preventing disease, including a new vaccine in the longer term.
We will do this by studying the local immune defences in the sheep foot, how the bacteria causing footrot are recognised by the immune system and the immune response to these bacteria. In addition, we will investigate the relationships between individual strains of the bacterium that causes disease and the potential for certain strains to be associated with disease. We think that footrot might occur when the sheep immune system responds too much to the bacteria, rather like human intolerance to milk, when our gut reacts to milk proteins when it should not. Alternatively, this could be due to lack of regulation of the inflammatory response leading to excessive collateral damage to the surrounding tissues. If we are right, then this will explain why antibiotics are so good at aiding recovery from footrot - because they kill the bacteria and so reduce the sheep immune response. We will be able to see this by studying tissue samples taken from the feet of sheep affected and unaffected with footrot. We will also use tissue samples taken from healthy sheep that we will culture in the laboratory and expose to bacteria that cause footrot.
By the end of this project we will be able to explain better how the clinical signs of footrot are caused and how the immune system contributes to this. This will help to inform on new approaches to disease management and ultimately disease prevention.

Technical Summary

The aetiology of footrot is complex and multifactorial. Physical damage to the interdigital skin is required to initiate disease following exposure to damp conditions. The incidence of footrot could therefore increase in line with the climate change predictions of warmer and wetter summers in the UK. Bacterial replication in the damaged skin leads to interdigital dermatitis where the superficial epidermal layers are inflamed, damaged and slough off. This can progress to footrot with separation of the hoof horn from the underlying tissue. The essential bacterium causing footrot is Dichelobacter nodosus. However, the role of a second bacterium, Fusobacterium necrophorum, in the pathogenesis of footrot is unclear. The host response to the invasion of bacteria is characterised by inflammation of the epidermis and pressure in the hoof horn capsule. Consequently the hoof horn separates from the underlying tissue. The host immune response is a double-edged sword, while it is required for successful defence, if unbalanced it can lead to pathology. Given that separation of the hoof horn is characterised by immune-mediated inflammation, investigating how this is initiated and controlled will dramatically improve our understanding of footrot pathogenesis. Analysing the global host immune response to footrot-associated bacteria is a first step in understanding local inflammation. Together with an insight into the molecular epidemiology of D. nodosus and analysis of the correlation of sequence type with disease presentation, this will greatly enhance our understanding of the aetiology of footrot.
This proposal will help to inform the design and delivery of novel therapeutic and vaccine approaches, with the ultimate aim of facilitating strategic effective vaccine development. This proposal aligns with solutions to food security, reducing antibiotic usage in meat production and the potential for resistance, and providing a sustainable, secure supply of good quality food.

Planned Impact

Footrot is a bacterial infection of the interdigital skin of the sheep foot resulting in lameness, and is the greatest welfare and economic concern for sheep farmers and veterinarians worldwide. In England, more than 95% of sheep flocks have footrot, with a mean daily prevalence of ~10% and an estimated cost to the UK sheep industry of £24-84 million per annum. The results from this project will help to improve our understanding and management of footrot, a disease that impacts this industry and those concerned with animal welfare. It is therefore of relevance to the sheep industry (both pedigree and commercial sheep farmers), their consultants including veterinarians and policy makers, Farm Animal Welfare Committee and the RSPCA. We will ensure that our work has high visibility through peer-reviewed publications, trade publications and through presentations at scientific, veterinary and farmer meetings. We will continue work with EBLEX, the levy body for beef and sheep farmers, and transfer knowledge as appropriate throughout the project allowing interaction and promotion of results to key stakeholders. The results are also of potentially high media interest and could lead to a radical change in the approach to managing the foot health of sheep. The farming press (Farming Today, Farmer's Weekly) are routes to communicating the outputs of this project to the farming industry. In addition, outcomes will inform teaching practice and have the potential to change prevention and treatment of footrot.
This is a collaborative, multidisciplinary project and we envisage that the proposed research will benefit a wide range of academics working in diverse fields. It will benefit immunologists studying innate immunity to bacterial infections by elucidating host recognition and signalling responses to bacteria in a farm animal species of veterinary importance. It will also provide valuable tools (ovine TLR expression constructs, ovine skin fibroblast cell lines) to study othe host - microbe interactions in sheep. This research will be of interest to scientists studying mechanisms of inflammation and immunopathology in response to infections. The generated and freely available mammalian transcriptome and pathogen genome and community profiling data will benefit the research community for diverse future research. In addition, the MLST analysis is a web based portable system that will allow others to 'plug and play' with their own data, thus developing an ever growing database of such information on D. nodosus globally, enabling bespoke global adaptation of project findings.
The public have a strong interest in the health and welfare of animals that are farmed for human consumption. This includes concerns over the use of antibiotics to control disease in food animals and the impact of disease on productivity that ultimately influences the quality and purchase cost of meat. Footrot in sheep impacts on all of these areas and is therefore pertinent to the global Food Security agenda. The applicants have expertise in communicating their science to broad audiences beyond the academic arena. Drs Tötemeyer and Coffey are involved in outreach activities including Science Week, Sutton Trust Summer Schools and as STEM ambassadors aiming to communicate veterinary science to pupils, including pupils from widening participation backgrounds. Dr Tötemeyer also reviewed for the Wikivet project. Prof Entrican and Dr Wattegedera have communicated their science to farmers at The Royal Highland Show and open days at Moredun Research Institute. They have presented to the public at The Edinburgh International Science Festival and have delivered immunology workshops to school teachers addressing the learning objectives relating to immunology and infectious disease within the new Curriculum for Excellence.

Publications

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Maboni G (2017) A Novel 3D Skin Explant Model to Study Anaerobic Bacterial Infection. in Frontiers in cellular and infection microbiology

 
Description 1. Development of a bacterial typing method. Bacterial species exist in many different subtypes, which can be seen by variations in their genes. These differences are thought to help the bacteria survive and spread which presents an additional challenge for vaccine development. Considering this, we have developed a new method for identifying subtypes of the bacterium that principally causes footrot in sheep, Dichelobacter nodosus. This new typing scheme identifies variations in several selected genes, allowing for subtle differences to be detected. The application of this method on bacteria isolated from within the U.K. has shown that each farm seems to have a specific collection of bacterial subtypes which differs from others, even those in close geographic proximity. Also, whilst in other countries where populations comprise of one or two subtypes the U.K. is highly variable making design of an efficacious difficult. Blanchard et al 2018, DOI 10.3389/fmicb.2018. 00551 .

2. Understanding the bacterial population during the progression of ovine footrot. Although D. nodosus is the bacterium that principally causes footrot, there are other factors and bacteria that are associated with disease. Progression from the initial publication: A distinct bacterial dysbiosis associated skin inflammation in ovine footrot (Maboni et al 2017, DOI 10.1038/srep45220): We have identified bacterial populations in the tissue and on the skin surface and how those change in footrot. The use of Dual RNASeq has allowed us to identify the transcriptional profile for the host showing that genes relating to inflammation and chronic wounds are significantly higher expresses in footrot affected feet, while genes relating to the barrier function of the skin are significantly lower expressed comparted to healthy feet from the same sheep.

3. In vitro infection models to investigate host response to footrot.
We have developed a novel 3D skin explant model to study anaerobic bacterial infection such as footrot (Maboni et al 2017, DOI: 10.3389/fcimb.2017.00404). We have isolated primary ovine fibroblasts and keratinocytes from tissue samples to determine cellular host response. The host response of those primary cells to D. nodsus includes the expression of proinflammatory cytokines and metalloproteases. Transfection studies suggest a role for TLRs in the recognition of D nodosus.
Exploitation Route Our new typing scheme and the associated database will be of use to researchers interested in understanding genetic exchange between footrot bacteria and how the bacteria evolve. The information will also be valuable to researchers interested in finding new vaccines to prevent footrot.

The influence of microbiome communities on disease development and progression is very topical. The new information we are generating on the microbiome in footrot will be of interest to researchers who are aiming to draw associations between 'good' and 'bad' bacteria in the feet of healthy and diseased sheep. This could ultimately be linked to studies on the use of antibiotics in treatment of disease and the effect on the microbiome.
Sectors Agriculture, Food and Drink

 
Description AHDB Studentship
Amount £69,000 (GBP)
Organisation Agricultural and Horticulture Development Board 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description BBSRC UK Veterinary Summer Studentship Program
Amount £1,900 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 07/2018 
End 09/2018
 
Description Bridging the Gap
Amount £9,735 (GBP)
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 11/2016 
End 02/2017
 
Description Students into Work Grant
Amount £2,000 (GBP)
Organisation Society for Applied Microbiology 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2017 
End 09/2017
 
Title A tiered Multi-Locus Sequence Typing (MLST) scheme for the Footrot causing pathogen Dichelobacter nodosus 
Description MLST uses DNA sequence variation in a set of genes required for basic cellular maintenance, to define allelic differences, and has become the gold standard for population analyses of bacterial pathogens. The main benefits of MLST are that it is portable and highly reproducible. The analysis is also automated through server based databases removing the bias potentially incorporated through different users and the complexity of data analysis. Sequence types are created by the generation of an allelic profile. This is a series of numbers based on novel sequence variation present in the seven alleles, for example the first loci combination for isolate one will be designated the starting profile (1-1-1-1-1-1-1). Any subsequent variation within an allele will generate allele two (e.g. 1-1-2-1-1-1-1) and so on for all unique loci for each isolate. With numerous Dichelobacter nodosus (D. nodosus) isolates publically available in the NCBI sequence read archive (SRA ID: ERP005873) the development of a tiered MLST scheme was undertaken with the aim of developing a robust typing method to define STs, which would be accessible to all, regardless of budget or experience. 
Type Of Material Improvements to research infrastructure 
Year Produced 2017 
Provided To Others? Yes  
Impact This study has addressed the lack of knowledge on the global relatedness of D. nodosus and interestingly highlights the lack of recombination at many levels of analysis. The will allow future researchers to add their isolates to the database to further inform the population structure of D. nodosus potentially leading to advances in determining vaccine targets. 
URL https://pubmlst.org/dnodosus/
 
Description C Bryant - Cambridge 
Organisation University of Cambridge
Department Department of Veterinary Medicine
Country United Kingdom 
Sector Academic/University 
PI Contribution Intellectual input
Collaborator Contribution expertise, intellectual input, training of staff
Impact see AB 072017 internship
Start Year 2015
 
Description C Muelling - Leipzig 
Organisation University of Leipzig
Department Institute of Veterinary Anatomy
Country Germany 
Sector Academic/University 
PI Contribution Expertise, intellectual input
Collaborator Contribution intellectual input, training of staff
Impact see AB 022016 internship
Start Year 2015
 
Description Ian Carr - Metagenomics 
Organisation St James's University Hospital (Jimmy's)
Country United Kingdom 
Sector Hospitals 
PI Contribution Participating in novel application of technology
Collaborator Contribution Discount on novel application of technology
Impact no outcomes yet
Start Year 2017
 
Description M Christodoulides - Southampton 
Organisation University of Southampton
Country United Kingdom 
Sector Academic/University 
PI Contribution Development few MLST scheme for D nodosus
Collaborator Contribution Provided whole genome sequencing data for MLST scheme
Impact Additional data to MLST data base
Start Year 2016
 
Description Stuart Carter 
Organisation University of Liverpool
Department School of Engineering
Country United Kingdom 
Sector Academic/University 
PI Contribution Provided D nodosus isolates
Collaborator Contribution Discussion
Impact no outcomes yet
Start Year 2016
 
Description ARC Dissemination Event 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Dissemination event to update partners of the ARC consortium on progress and to discuss data and research with professional colleagues also funded by BBSRC ARC, allevents were hosted in Edinburgh
09/10 Mar 2015
03/04 Feb 2016
01/02 Nov 2016
30/31 Oct 2017
11/12 Dec 2018
Year(s) Of Engagement Activity 2015,2016,2017,2018
 
Description ARC Dissemination Event 2016 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Attendance at ARC dissemination event attended by scientists and industry where the project was presented and discussed.
Year(s) Of Engagement Activity 2016
 
Description Antimicrobial Resistance Awareness Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact In the World Antibiotic Awareness week on 18/11/2015 an Information stall and 'hunt for new antibiotics' in collaboration with 'Swab & Send@ initiative based at UCL; whereby 200 swabs were handed out to members of public, staff (particularly aiming at admin staff who not normally engage with science) and students ac; a group of students also visited a local primary school. 114 swabs were returned were sent for culture, 22 isolates producing compounds that inhibit bacterial growth have been identified from 18 different swabs. The most interesting isolate was one that inhibited a multi resistant E. coli, Micrococcus and MRSA. Identification of some of the isolates is still ongoing and the outcomes will be presented to admin staff, students and school children involved. The event was publicised via Veterinary Schools Council Press release and on Twitter
Event was repeated in 2016 with additional funding obtained from Bridging the Gap and UoN Hermes Fellowship to set up a 'Hunt for New antimicrobial producers' and with an additional competition to 'Make a Giant Microbe'. Event was publicized via UoN press release & reported in Vet Record
http://www.nottingham.ac.uk/news/pressreleases/2016/november/vet-school-students-raise-awareness-of-antibiotic-resistance.aspx.
The event was repeated in 2017 with 'bacteria games' and feedback from a student project on using 'Hunt for new antimicorbial producers' to also investigate attitudes to antimicrobial use in members of the public participating in such activities. Event was publicized via UoN press release & reported in Vet Times: http://www.nottingham.ac.uk/news/pressreleases/2017/november/students-reveal-superbug-fighting-microbes-at-amr-awareness-day.aspx
https://www.vettimes.co.uk/news/superbug-fighting-microbes-revealed-at-amr-awareness-day/
Year(s) Of Engagement Activity 2015,2016,2017
URL http://dx.doi.org/10.1136/vr.i6445
 
Description CPD event for Sheep Farmers 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Discussion with Farmers that participate din our study, feedback form our research as well as general discussion about footrot and lameness in sheep
Year(s) Of Engagement Activity 2018
 
Description Final ARC Project Meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Study participants or study members
Results and Impact A final meeting of the 'Understanding Inflammatory Processes in Ovine Footrot to Inform on Rational Vaccine Design' project team (BB/M012085/1 & BB/M011941/1) was held at the University of Nottingham on 22 February 2019 in association with the related ARC Project 'Is Multistrain Infection by Dichelobacter nodosus Important in the Severity of Footrot and Management of Disease?' (BB/M012980/1). The aims of the meeting were to review the outcomes of the research and identify potential future projects. The meeting was attended by a total of 20 investigators, researchers and students who had contributed to the projects. There were 12 presentations, followed by a round-table discussion which focussed on 'knowns' and 'unknowns' of ovine footrot. This generated a number of ideas which were captured and will be used to formulate future funding applications.
Year(s) Of Engagement Activity 2019
 
Description Final BBSRC ARC Dissemination Event 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Attended the final BBSRC ARC Dissemination Event 11-12th December in Manchester, UK. The project was presented to ARC partners and there was discussion about the findings. Plans were put in place for a final project meeting in Nottingham in early 2019 that would be attended by members of the other ARC Footrot Project (University of Warwick).
Year(s) Of Engagement Activity 2019
 
Description Pint of Science - 'Unhappy Feet - Why sheep with Footrot don't tap dance' 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation as part of Pint of Science - 'Unhappy Feet - Why sheep with Footrot don't tap dance' with the aim of making university research more accessible to the general public. Event also included hands on DNA isolation.
The audience got engaged and asked questions
Year(s) Of Engagement Activity 2018
URL https://pintofscience.co.uk/event/food-for-thought-the-science-behind-agriculture
 
Description SB Fest (University of Nottingham) 
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 Public/other audiences
Results and Impact A public event showcasing different activities based on the Nottingham University Sutton Bonington Campus. This included interactive tasks specifically aimed at families about microbes and infection. This was part of a science hub showcasing veterinary medicine related public engagement activities.
Year(s) Of Engagement Activity 2016,2017,2018,2019
 
Description School visits (Wreak Valley Academy/Charnwood College Loughborough, Limehurst Academy Loughborough, Woodbrook Vale Loughborough, John Ferneley College Melton Mowbray ) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Careers talk and hands on science workshop. Used footrot and development of a tissue culture model as an example. This sparked questions and discussion.
Year(s) Of Engagement Activity 2016,2017
 
Description Sheep Breeders Round Table Meeting 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Posters were presented at Sheep Breeders Round Table meetings in 2015 and 2017 that sparked questions and discussion. These are AHDB organized stakeholder events.
Year(s) Of Engagement Activity 2015,2017
URL http://www.nationalsheep.org.uk/SBRT/