The farm environment: an overlooked source of Mycobacterium bovis?

Lead Research Organisation: University of Warwick
Department Name: School of Life Sciences

Abstract

Bovine tuberculosis (bTB) is a cattle disease which can be transmitted to people (although human infections are rare in the UK). Despite decades of efforts to eradicate it, the disease has spread across much of western Britain. The disease seems to persist because the tests used to detect and remove infected cattle miss some infected animals, and because wildlife (especially badgers) can also catch the disease and pass it on to cattle.
Most cattle catch bTB by breathing in specks of mucus or saliva from other cattle. However, new research shows that badgers avoid cattle, and very seldom come close enough to transmit (or catch) the disease in this way. Because there is very strong evidence that badgers give bTB to cattle, and that cattle give bTB to badgers, this finding strongly suggests that cattle can both transmit and catch bTB without coming into close contact with another animal.
Both cattle and badgers excrete bTB bacteria, which can survive in the environment for weeks or months. We can quantify these bacteria in the environment by using genetic methods which count the number of copies of bacterial DNA in a sample of soil, faeces, or water. Our preliminary studies have detected such bacteria in cattle slurry, and in the soil of fields where cattle graze, as well as in badger faeces collected on cattle farms. It is likely that cattle can catch bTB from these sources; however, current bTB management does little to address the risk posed by bTB in the environment.
Our project aims to address six research questions:
(1) Where in the farm environment are bTB bacteria concentrated?
(2) Are the bTB bacteria found in the environment still alive and hence likely to infect cattle?
(3) To what extent do cattle come into contact with bTB bacteria in the environment?
(4) Does spreading slurry onto fields contribute to bTB bacteria in the environment?
(5) Do the bTB bacteria found in the environment come mostly from cattle or from badgers?
(6) How might farms be managed to avoid cattle catching bTB from the environment?
Our project will seek to answer these six questions by a careful study of 20 farms located in Cornwall, where rates of cattle bTB are very high. Our team will collect thousands of samples of cattle dung, slurry, soil and badger faeces, as well as swabs from inside barns and cattle troughs. These samples will be brought back to our laboratory where we shall extract DNA from them and count the numbers of copies of bTB genetic code from each one. These analyses will allow us to map where on the farms bTB bacteria are most abundant, and to explore whether the numbers and distribution of bacteria vary between seasons. At the same time, we shall track the movements of both cattle and muckspreaders (used to spread slurry) using technology similar to that present in vehicle satnav systems. Tracking muckspreaders will help us to assess whether bTB is more abundant in fields where slurry has been spread recently, and tracking cattle will help us to assess where they are at greatest risk of encountering bTB bacteria in the environment.
We shall test whether bTB found in the environment is still alive and also explore how long bTB bacteria survive in cattle dung and badger faeces held inside small open-air enclosures inaccessible to animals.
To assess where environmental bacteria come from, we plan to compare the DNA of bTB bacteria found in soil, barns and troughs with that of bacteria known to have come from cattle (found in fresh cattle dung) and from badgers (found in fresh badger faeces). If, for example, the DNA strains found in soil were similar to those found in cattle dung, but different from those found in badger faeces, it would suggest that most environmental contamination comes from cattle.
To ensure that our research findings can be translated into practical guidance for farm management, our project steering committee includes representatives from the farming industry and the veterinary profession.

Technical Summary

The focus of the research is to better understand how the environment can act as a source of Mycobacteria bovis and provide mitigation strategies to significantly reduce the potential for transmission, as we hypothesise that environmental contamination is a vital component in the transmission of this disease between animals on farms. We aim to provide a definitive study of the prevalence and detection frequency of M. bovis on 20 farms in Cornwall, a high bTB incidence area. This will be the first study to combine detailed molecular detection of a wide range of environmental samples including cattle slurry, totaling 18,000 samples with detailed cattle movement information. This will be carried out contemporaneously with intensive farm monitoring including tracking cattle movements and approaches to slurry handling. The farms have already been the subject of a three year study of cattle and badger movements so there is an extensive database available to augment with environmental prevalence. A heatmap of the location, presence and quantity of M. bovis DNA shed into the environment will be produced and combined with information on with these sources to determine the relative risk of cattle exposure to M. bovis in various locations within the farm environment. Furthermore, the viability of environmental M. bovis detected on these farms will be assessed to establish whether these environmental pools of M. bovis may be potentially infectious. The longevity of environmental M. bovis will be determined using exclosure experiments where the M. bovis load and viability of faeces will be assessed over time. Metagenomic techniques will be developed during this study to retrieve whole genomes directly from environmental samples without the need to culture. This will enable the genome of strains present in the environment on these farms to be determined and potentially attributed to a source as well as information on the longevity of strains.

Planned Impact

This proposal has the potential for very significant impacts on both mitigation of risk for exposure of cattle to sources of M. bovis in the farm environment, establish appropriate biosecurity measures and inform policy for suitable eradication measures at the farm scale to reduce the risks of bTB herd breakdowns. The research work will provide data for development of statistical models which can be used to evaluate biosecurity methods and indeed evaluate a wide range of strategies to control risk such as vaccination, culling and isolation.
We aim to identify processes influencing the distribution of sources of M. bovis which provide risk for onward transmission to cattle and other animals and wildlife. Thus we will evaluate appropriate intervention strategies including BCG vaccination, and provide added value to the implementation of molecular detection tools by establishing appropriate sampling regimes and high risk areas on farms. This will provide farmers and vets with vital information for often simple and effective control measures in addition to informing policy makers of the most appropriate measures to develop biosecurity strategies for high risk areas. A primary beneficiary of these results is Defra who have a continuing interested in badger management, and predicted conditions for badger-cattle and cattle-badger transmission, particularly in the edge zone where bTB herd breakdowns are advancin. Vets, farmers, Defra and wildlife enthusiasts are interested in developments of non-invasive molecular tools for environmental monitoring of M. bovis to increase farm biosecurity against bTB in cattle, in surveillance of badger populations to inform badger management, and to measure efficacy of badger BCG vaccination and culling against M. bovis shedding into the environment.

The heat maps, models and strain diversity data coupled with strain dissemination and persistence data will be a valuable resource and influence the decision making process that impacts on livestock farmers' economic activities. Farmers can take responsibility for biosecurity and we aim to assist directly in this process. Stakeholders will benefit from direct interaction with the research and this can be implemented via the steering committee consisting of pathogenicity experts, vets, farmers and commercial kit detection company LGC Group. Methods for environmental monitoring of disease exposure risks are widely sought as we become more aware of the import

Data generated from along the geographical edge of endemic bTB in the UK will provide insights into the processes regulating seeding of M. bovis in advance of bTB becoming established in cattle and/or badgers. We envisage a future bolt-on proposal to conduct whole genome analysis of M. bovis isolates from badger and cattle along this frontier to determine the role played by the environment in disease transmission. In fact our work in Tanzania monitoring shedding into environmental sites of both M. bovis and M. tuberculosis has provided both vets and clinicians with opportunities for disease surveillance and this will apply for bTB in the UK without the need for invasive and expensive animal tests. This will aid in the evaluation of herd breakdown data and also information on wildlife infection status.
The work will contribute to enhancing quality of life and health of the farming community suffering from chronic bTB and economic difficulties. It provides materials for education at all levels, in providing direct access to the scientists and results disseminated through lectures, seminars and talks, and providing the epidemiological data and biological samples for UG and PG projects. The wider public will be informed by unbiased delivery at public meeting, museum talks, encouraging more engagement and public interest in science.
 
Description Farms were sampled to detect the infectious agent for bovine TB, Mycobacterium bovis, in a range of substrates including soil, slurry, cow pats, water troughs and soils around badger setts. Many thousands of assays were done and when the data was analysed and unblinded we have been able to show hot spots for the presence of M. bovis DNA indicating potential sources of infection and transmission. The large amount of data has provided a basis for continued analysis and current statistical methods provided evidence for a correlation with bTB breakdowns on farms and disease status of other animals on farmland including badgers.
It was evident that M. bovis can survive in the environment and be detected in badger faeces, cow manure and slurry in addition to water troughs. We also developed long range PCR primers to amplify variable regions of the genome of M. bovis and then use the portable Oxford nanopore MinION to undertake long read sequencing. Furthermore, we developed and tested a custom bioinformatics pipeline to assemble variable repeat regions of the M. bovis genome from long range amplicon sequencing. We were ultimately able to assign recognised strain type resolution to our data set and develop a custom Perl scrip specifically designed to handle and process long read sequence data for this task. In a paper in submission (bioRxiv) we present data on the specificity and sensitivity of this assay on pure culture isolates of BCG, wild type M. bovis isolates, M. tuberculosis isolates as well as spiked environmental samples. Furthermore, we demonstrated the use of this technique on wild type M. bovis samples collected from the environment that consist of raw badger faeces and water. This has allowed us to identify the limit of detection of this assay through qPCR data as well as to enable us to assign geographically relevant strain types to both known and unknown environmental samples.
Exploitation Route Assist farmers in improving on-farm biosecurity and help Defra to formulate policies regarding the control of bTB. We will provide a risk map to illustrate how shedding of M. bovis occurred across a stylised farm to illustrate how the environment can become contaminated and may pose a risk to cattle.
Sectors Agriculture, Food and Drink,Environment

 
Description The findings confirm that there are high risk areas on farms where the causal agent of bTB has been shed and was readily detected by qPCR assay. This information was mapped onto images indicating farming activities and will be used to inform farmers of shedding patterns and using camera trap data indicate to them where possible sources of contamination might be coming from. The combined data sets produced by this project are hugely important for dairy and beef farming communitities badly hit by bTB outbreaks Also the study produced data relevant for sheep and deer farmers wanting to protect their livestock from infection.
First Year Of Impact 2020
Sector Agriculture, Food and Drink,Environment
Impact Types Cultural,Societal,Economic,Policy & public services

 
Description Meetings with farmers and farm consortia in England and Wales to establish use of environmental screening to aid in improved farm biosecurity against bTB infection and dissemination.
Geographic Reach National 
Policy Influence Type Influenced training of practitioners or researchers
Impact If farmers can avoid contamination of cattle from infected material this will help in the fight against bTB and onward transmission of the disease. This will result in a substantial economic benefit and improve agricultural sustainability.
URL http://www.metagenomics.uk
 
Description Participated in policy round table on TB control with Defra Secretary of State, Minister of State, Chief Veterinarian and Chief Scientist
Geographic Reach National 
Policy Influence Type Membership of a guideline committee
 
Description Conference invited speaker travel funding
Amount £1,000 (GBP)
Organisation National University of Sciences and Technology 
Sector Academic/University
Country Pakistan
Start 02/2019 
End 02/2019
 
Description LC 2018 conference travel and accommodation funding
Amount £500 (GBP)
Organisation Oxford Nanopore Technologies 
Sector Private
Country United Kingdom
Start 05/2018 
End 05/2018
 
Description LC 2019 conference travel and accommodation funding
Amount £550 (GBP)
Organisation Oxford Nanopore Technologies 
Sector Private
Country United Kingdom
Start 05/2019 
End 05/2019
 
Description Poster presentation at international conference.
Amount $500 (USD)
Organisation Oxford Nanopore Technologies 
Sector Private
Country United Kingdom
Start 09/2018 
End 09/2018
 
Title Laboratory magnetic separator 
Description Magnetic separator design for 3D printing. Released open source and open access under creative common protection Attribution-NonCommercial-ShareAlike. 
Type Of Material Biological samples 
Year Produced 2018 
Provided To Others? Yes  
Impact Researchers and members of the public are able to create a standard laboratory magnetic rack for DNA separation at low cost. Users are able to re design for purpose. 
URL https://www.thingiverse.com/thing:2990155/files
 
Title M. bovis visual detection assay 
Description A PCR free, strain specific, visual colour changing assay for the rapid identification of Mycobacterium bovis. 
Type Of Material Technology assay or reagent 
Year Produced 2018 
Provided To Others? No  
Impact Cultures enritched or isolated from complex environmental microbial communities are now able to be screened for the presence of Mycobacterium bovis without the time and cost associated with PCR based approaches. 
 
Title Novel use of minION for diagnostics 
Description Novel approach to diagnostics for typing pathogens in faecal samples directly 
Type Of Material Technology assay or reagent 
Year Produced 2017 
Provided To Others? No  
Impact New approach for direct typing of a pathovar or spoligotype directly from cells in a faecal or other samples for example soil, blood, tissue. This will allow rapid identification of pathogens in the environment and help track dissemination pathways. 
 
Title Long-read metagenomic assembly pipeline 
Description Detecting and strain typing Mycobacterium bovis in environmental samples is difficult due to the sample impurity and community complexity. Long read sequencing provides a potential means to achieve this aim using both PCR enriched and whole community mete-genomic approaches. However, order to assemble long read environmental meta-genomic data, novel approaches to read mapping and polishing are required. Pipelines and workflows for PCR amplified and whole community meta-genomics have been developed in collaboration with third parties (Dr Andrew Millard, Dr Christopher Quince). 
Type Of Material Data analysis technique 
Year Produced 2019 
Provided To Others? No  
Impact Strain type Mycobacterium bovis from environmental samples to the resolution of spoligotype or better. 
 
Title Mycobacterium strain collection 
Description Curated strain collection of Mycobacterium species. 
Type Of Material Database/Collection of data 
Year Produced 2019 
Provided To Others? No  
Impact Available strain collection for laboratory use. Containment level 3. 
 
Description Collaboration with veterinary practices 
Organisation West Ridge Veterinary Practice ltd
Country United Kingdom 
Sector Private 
PI Contribution Exchange of expertise and working closely with vets to better understand on farm biosecurity and gain samples of cow faeces and other materials.
Collaborator Contribution Expertise given on animal husbandry and data on test performance
Impact Improved diagnostic methods for detection of contamination in the farm environment
Start Year 2017
 
Description Development of point of care diagnostic test for pathogens in the Mycobacterium tuberculosis complex 
Organisation Bee Robotics Ltd
Country United Kingdom 
Sector Private 
PI Contribution Establish the molecular targets, design of the LAMP primers and establish the approaches to extraction procedures. Defined new loci for targeting detection of pathogen DNA in samples and beta tested specificity and sensitivity of the assay. Tried a range of extraction procedures, confirmed amplicon by minION long read sequencing.
Collaborator Contribution Provision of chemistry expertise, design of housing for the test and approaches to producing field based test.
Impact Multi-disciplinary collaboration still in progress
Start Year 2019
 
Description Environmental reservoirs of microbial pathogens 
Organisation National University of Sciences and Technology
Department Institute of Environmental Sciences and Engineering (IESE)
Country Pakistan 
Sector Academic/University 
PI Contribution Working on developing bids for GCRF and other funding agencies to halt the spread of microbial pathogens and AMR in the environment
Collaborator Contribution Hosted a workshop and paid our team's expenses to visit and work in a collaborative pilot study in addition to talks and visits to field sites
Impact The collaboration resulted in the analysis of field sites to consider the environmental reservoirs of microbial pathogens and understanding transmission pathways through water and food chain routes.
Start Year 2019
 
Description Environmental reservoirs of microbial pathogens 
Organisation University College of Islamabad
Country Pakistan 
Sector Academic/University 
PI Contribution Working on developing bids for GCRF and other funding agencies to halt the spread of microbial pathogens and AMR in the environment
Collaborator Contribution Hosted a workshop and paid our team's expenses to visit and work in a collaborative pilot study in addition to talks and visits to field sites
Impact The collaboration resulted in the analysis of field sites to consider the environmental reservoirs of microbial pathogens and understanding transmission pathways through water and food chain routes.
Start Year 2019
 
Title Nanosync 
Description This python script synchronises fast5 files from an internal SSD to an external SSD in near real time as they are being generated by the sequencing platform. This permits large sequencing runs to take place without data loss during standard transfer mechanisms. 
Type Of Technology Software 
Year Produced 2018 
Impact A 25 Gbp metagenomic sequencing run has now been performed on a single flow cell originating from a soil sample. This data set was approximately 862 Gb in storage size and required real time syncing to retain all data produced. 
 
Description Presentation to MRC sponsored scientists and panel members 
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 Gave a presentation entitled "AMR in Soil" MRC workshop Antimicrobial Resistance Cross Council Initiative, London July 4-5th.
Year(s) Of Engagement Activity 2017
 
Description BBSRC, Defra, NC3Rs Bovine tuberculosis Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact The workshop was comprised of a collective of international scientists, veterinary practitioners and government organisation representatives currently undertaking research into the spread and control of bovine tuberculosis. Representatives from each research group presented information on their current research projects then joined breakout sessions detailing future pathways to the eradication and control of Mycobacterium bovis on both a national and global scale.
Year(s) Of Engagement Activity 2017
 
Description Bioinformatics Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Bioinformatics workshop for students from the Nanyang Technological University focusing on handling long read sequence data and pathogen identification. Part of the WESIC overseas research program.
Year(s) Of Engagement Activity 2018
 
Description Gatcombe Farm meeting with Chief Scientific Adviser at the Department for Environment, Food and Rural Affairs 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Event attended by DEFRA representatives, Politicians, Farmers, Veterinarian representatives and landowners and members of the scientific community to discuss the future of bovine tuberculosis control in the UK. Hosted at Gatcombe farm by Dick Sibley and Robert Reed.
Year(s) Of Engagement Activity 2018
 
Description Presentation at Workshop 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Workshop for CartNet European PhD fellows focusing on next generation sequencing technology, pathogen detection in the environment (M. bovis) and a two day practical session. This workshop was comprised of lecture sets and bioinformatics sessions which accompanied a "bring your own DNA sample" sequencing practical. This workshop was funded by the Mari Curie CartNet program and was attended by all European students from the most recent cohort of awards.
Year(s) Of Engagement Activity 2019
 
Description Presentation at workshop 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Presentation entitled "PCR-guided badger culling: could it work?" Bovine TB Symposium 2017 Imperial College London March 28th
Year(s) Of Engagement Activity 2017
 
Description Twitter feed @Robert_S_James 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Personal twitter feed for work undertaken as part of the Wellington group.
Year(s) Of Engagement Activity 2019
URL https://twitter.com/Robert_S_James
 
Description Workshop 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Participated in an international workshop with Indian scientists across a range of disciplines to understand teh issues of AMR in India
Year(s) Of Engagement Activity 2017