Controlling enteric pathogens of poultry: Host/microbiota interactions, risk assessment and effective management interventions

Lead Research Organisation: Royal Veterinary College
Department Name: Pathology and Pathogen Biology

Abstract

The global human population is predicted to increase by a third over the next 25 years, with countries of the developing world hosting 97% of this expansion. The ability to 'feed 9Bn people sustainably by 2050' is an urgent priority Indian and UK governments with the 12th Indian Five Year Plan requiring growth of the agricultural sector at 4% per annum to achieve food security. Poultry farming is a highly efficient and cost-effective system for producing animal protein for human consumption, but circulating infectious diseases compromise gut health and impact dramatically on farm economics, animal welfare and occasionally human health through transmission of zoonoses. Poultry gastrointestinal infections of most concern in UK and India are caused by avian pathogenic Escherichia coli (APEC), Campylobacter jejuni, Clostridium perfringens, Eimeria and Salmonella.

Susceptibility to gut colonisation and the outcomes of infection are directly influenced by many factors including host genotype, immune status, age at infection, strain of infecting microbe, composition of commensal enteric microbiota and presence of other acute or chronic infections. There are significant interactions between host and microbe biology, genetics, epigenetics, the environment and farm practices. Changes to diet, use of vaccines or antimicrobials, and flock-level interventions such as 'thinning', can have profound effects on intestinal health and the evolution and spread of disease-causing microbes and may be amplified by genetic variation in host and microbe populations. Whilst major advances in genomics and genotyping of commercial poultry lines is facilitating the identification of loci linked to susceptibility or resistance, the impact of host and pathogen diversity on disease and production outcomes remains largely unexplored.

There is rich genetic diversity in India's native poultry breeds, and the hybrid exotic lines often used in Indian commercial production are distinct from the majority of poultry reared in the UK. The prevalence and dynamics of gastrointestinal infection at farm-level has a direct bearing on economic risk to individual farmers and contributes to overall global concerns of food security and food safety.
Gaps in current knowledge prompt four fundamental questions around which this proposal is framed:

1. What is the epidemiology of specified gastrointestinal infections, and co-infections, across UK and Indian poultry production systems?

2. Does host genotype exert an influence on (a) the prevalence, evolution and transmission of specified microbes and (b) the composition of flock-level enteric microbiota?

3. What is the level of genetic variation within specific microbial populations in Indian and UK poultry production?

4. What on-farm factors affect the risk of enteric colonisation and carriage of specified microbes and how can changes in poultry husbandry and management practices mitigate this risk?

The proposal brings together UK and India experts in poultry genetics, animal health, epidemiology, pathology and pathogen biology. A multidisciplinary approach combining metagenomic sequencing, high density SNP-based QTL mapping, bacteriology, parasitology, molecular epidemiology and mathematical modelling will be used to quantify and predict disease risks at farm and national levels and to inform the development of intervention and management strategies, including future breeding and husbandry planning.

Technical Summary

Our overarching hypothesis is that QTL controlling resistance to micro-organisms of veterinary and zoonotic importance are segregating in native Indian and modern commercial chickens. Further, we hypothesise that the composition of the commensal enteric microbiota, and thus its influence on colonisation by a panel of pathogenic micro-organisms, is also underpinned by host QTL. Finally, we hypothesise that the impact of management strategy on this dynamic system is also shaped by QTL.

We will perform whole genome association (WGA) for traits including resistance to enteric colonisation by a panel of pathogens with veterinary and zoonotic relevance, as well as variation in microbiome composition, as defined by metagenomic next-generation sequencing and qPCR of the caecal microbiota. These data will also be used to investigate genetic diversity among the targeted micro-organisms, supplemented by whole genome shotgun sequencing of up to 40 cultured field isolates, informing on the occurrence of antimicrobial resistance and virulence factors. These data will support molecular epidemiological characterisation of microbial flow and development of existing mathematical models of pathogen interactions and spread within farmed chickens. Ultimately these interlinked data will be integrated into a probabilistic risk assessment of poultry management to identify optimal parameters for production using local and global commercial chicken lines.

The overall aim is to identify markers (SNPs), candidate genes and eventually causative mutations for resistance/susceptibility to colonisation by specific micro-organisms. The resistance-associated genotypes will inform commercial breeding programmes to reduce risk to the secure supply of safe animal protein and improve animal welfare. In a broader context these studies will develop a series of recommendations for effective and safe poultry production utilising native Indian breeds and regional production strategies.

Planned Impact

Details of the research and recommendations developed will be disseminated through traditional means including peer-reviewed publication, prioritising open access journals, and public presentation to audiences including students, professional forums, academics and politicians as well as the two planned BBSRC/DBT events. Priority audiences in India and the UK will be informed through workshop-based training to farmers, public servants and industry, supplemented by scientific and lay-article publications and web-based releases. More specifically we have identified the following stakeholders:

Industry stakeholder meetings. The applicants have contacts with all of the major international animal production and health companies (i.e. Aviagen, Cobb-Vantress, Merck and Pfizer) and their national representatives (e.g. Suguna Foods and the VH Group). Company representatives will be invited to each annual programme meeting. Additionally, each company's views will be sought during the first six months of the project and our results will be shared in UK and Indian workshops early in the final year of the project.
Impact on policy. The research undertaken is likely to have implications for policy makers and regulatory bodies in the public sector concerned with the poultry/livestock industry and food safety. We will continue to engage with representatives of the key UK and Indian bodies to ensure the application of our research for the benefit of all potential users including the farming industry, pharmaceutical industry and the general public. This will be achieved via forums such as the British and Indian Veterinary Poultry Associations, the British Poultry Council and the Indian Poultry Science Association, with whom the applicants have regular dialogue (two or more times per annum). Exploitation of results has been described in section iv.

Poultry producers. The applicants regularly interact with poultry producers through presentations, workshops and practical training. Research we have generated in India under BBSRC/DFID project BB/H009337/2 has already been made publically available online (Indian Coccidia Alert Network) and used to prepare a series of six pamphlets in seven different languages/dialects in India. From the start, we will aim to collaborate closely with animal health services to create awareness, build capacity and elaborate feasible strategies that take into account local practices and culture.

Academic impact. Novel data will be disseminated via publications in peer-reviewed journals of high standing, and via presentations at national and international symposia. Raw data will be made publically available as appropriate after reasonable consideration of IP.
Training and Capability. Project staff will be expected to contribute substantially to the dissemination activities, developing skills in diverse areas including project management, knowledge transfer and presentation skills, supported by training courses.

Deliverables
- Publication in academic journals (more than three over the course of the grant).
- Presentations at relevant conferences (more than once in each of years 2 and 3).
- Industry events including representation at annual project meetings.
- Press releases on significant findings aimed at the public when relevant.
- Deposition of SNP and sequencing data in the public domain.
- At least one article to be authored for the popular scientific press.
- Staff training in public engagement activities.

Publications


10 25 50
 
Title Journal cover image 
Description Front cover image for the International Journal for Parasitology 
Type Of Art Artwork 
Year Produced 2016 
Impact Improved awareness of the associated research article and linked blog. 
URL http://www.sciencedirect.com/science/journal/00207519/46/9
 
Title Parasitology journal cover image 
Description Journal cover image 
Type Of Art Artwork 
Year Produced 2017 
Impact Improved awareness of the associated research article. 
URL https://www.cambridge.org/core/journals/parasitology/issue/F2BB6612AC2CA7F1FA269B440448EEDA
 
Description BBSRC DTP (LIDo)
Amount £76,594 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 07/2014 
End 09/2017
 
Description BBSRC-GCRF Impact Accelerator Award
Amount £60,000 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Department BBSRC Impact Award
Sector Academic/University
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 12/2016 
End 03/2017
 
Description Commonwealth scholarship and fellowship plan
Amount £30,000 (GBP)
Organisation Government of the UK 
Department Commonwealth Scholarship Commission
Sector Public
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2016 
End 09/2017
 
Description KTN CASE Studentships
Amount £110,000 (GBP)
Organisation Knowledge Transfer Network 
Sector Charity/Non Profit
Country United Kingdom of Great Britain & Northern Ireland (UK)
Start 10/2016 
End 09/2020
 
Title Quantitative PCR for Eimeria parasite genome counting during in vivo infection 
Description A pre-existing quantitative PCR (qPCR) assay used to count Eimeria parasite genomes has been complemented by addition of a second qPCR to quantify host genomes. The assay has been validated and compared with traditional parasitological measures such as total oocyst output per bird. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact This research tool will be essential to map the genetic basis of chicken susceptibility/resistance to infection and disease caused by Eimeria species parasites where experimental group sizes will exceed capacity for traditional measures of parasite replication. The technique can also be applied to field studies of coccidiosis and offers considerable opportunitites to improve bird welfare since: 1. In some experimental models there is now no need to cage birds individually to assess parasite replication. Housing birds in groups in floor pens allows them to express more natural behaviours (REFINEMENT). 2. The precision achieved using qPCR improves statistical power, facilitating the use of smaller group sizes (REDUCTION). A manuscript describing this work is currently in preparation. 
 
Title Chicken caecal 16S microbiome 
Description Illumina 16S amplicon sequencing + analysis derived from >700 chicken caecal microbiome samples 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact The provision of such a large NGS microbiome sequence database provides a valuable resource for genetic, microbiome and husbandry analyses. To be complemented by host genotypes in due course. 
 
Title Chicken caecal 18S NGS 
Description Illumina 18S amplicon sequencing + analysis derived from >700 chicken caecal microbiome samples 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact The provision of such a large NGS microbiome sequence database provides a valuable resource for genetic, microbiome and husbandry analyses. To be complemented by host genotypes in due course. 
 
Title Chicken caecal microbioms shotgun 
Description Illumina whole microbiome shotgun sequencing + analysis derived from >700 chicken caecal microbiome samples 
Type Of Material Database/Collection of data 
Year Produced 2017 
Provided To Others? Yes  
Impact The provision of such a large NGS microbiome sequence database provides a valuable resource for genetic, microbiome and husbandry analyses. To be complemented by host genotypes in due course. 
 
Title Indian chicken husbandry survey 
Description Access database including surveys from >150 chicken producers in India describing health, husbandry and social parameters. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact This work is ongoing. 
 
Description Accelerating impact of RVC poultry health projects in South Asia 
Organisation Chittagong Veterinary and Animal Sciences University
Country Bangladesh, People's Republic of 
Sector Academic/University 
PI Contribution Workshop exploring future applications for research and pathways to impact
Collaborator Contribution Workshop exploring future applications for research and pathways to impact
Impact Still active, outputs are pending.
Start Year 2016
 
Description Accelerating impact of RVC poultry health projects in South Asia 
Organisation Poultry Research Institute
Country Pakistan, Islamic Republic of 
Sector Academic/University 
PI Contribution Workshop exploring future applications for research and pathways to impact
Collaborator Contribution Workshop exploring future applications for research and pathways to impact
Impact Still active, outputs are pending.
Start Year 2016
 
Description Accelerating impact of RVC poultry health projects in South Asia 
Organisation United Nations (UN)
Department Food and Agricultural Organisation (FAO)
Country Italy, Italian Republic 
Sector Charity/Non Profit 
PI Contribution Workshop exploring future applications for research and pathways to impact
Collaborator Contribution Workshop exploring future applications for research and pathways to impact
Impact Still active, outputs are pending.
Start Year 2016
 
Description Accelerating impact of RVC poultry health projects in South Asia 
Organisation University of Peradeniya
Country Sri Lanka, Democratic Socialist Republic of 
Sector Academic/University 
PI Contribution Workshop exploring future applications for research and pathways to impact
Collaborator Contribution Workshop exploring future applications for research and pathways to impact
Impact Still active, outputs are pending.
Start Year 2016
 
Description British Society for Immunology vaccine affinity group meeting 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation keynote/invited speaker
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact ~60 people attended the presentation, which prompted questions and discussion afterwards.

I have become a reviewer for a new journal as a result of increased awareness of RVC science.
Year(s) Of Engagement Activity 2014
 
Description Countryfile appearance 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Countryfile appearance focused on the occurrence of apicomplexan parasites and the risks to farming and the general public.

As a direct result of this appearance D. Blake was acted as external examiner for a PhD thesis at the University of Liverpool.
Year(s) Of Engagement Activity 2014
 
Description Further education college visit (Bedfordshire) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Undergraduate students
Results and Impact 15 pupils attending a short lecture on DNA fingerprinting and its application, followed by two practical sessions. In the first session we extracted genomic DNA. In the second we set up a PCR and resolved the product by agarose gel electrophoresis. The sessions sparked questions and discussion, including some email correspondence over the following week.

Several of the students reported that they had enjoyed the sessions, prompting them to consider registering for a third year degree upgrade programme.
Year(s) Of Engagement Activity 2014,2015
 
Description Parasites and Vectors blog 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact We provided a blog to the BugBitten site describing Eimeria genome sequencing and its value to global chicken production. Special mention was included of the possible impact on poultry production in the developing world.

The blog was Tweeted and recommended on Facebook.
Year(s) Of Engagement Activity 2014
URL http://blogs.biomedcentral.com/bugbitten/2014/09/
 
Description Poultry Diseases group meeting 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact ~20 people, including veterinary practitioners, animal production and health company representatives, government officials and academics, attended the meeting. During this meeting we briefly presented and discussed our work to map the genetic basis of resistance or suscpetibility to Eimeria in chickens.

Discussion about provision of samples and future data dissemination.
Year(s) Of Engagement Activity 2014,2015
 
Description RVC Bioinformatics day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact ~50 people including undergraduate and postgraduate students, as well as early career researchers, attended a workshop focused on improving awareness and understanding of next-generation sequencing technologies. Prompted questions and discussion, as well as one embryonic collaboration.

Support offered to a postgraduate towards application for research fellowship funding.
Year(s) Of Engagement Activity 2014