CamChain - Campylobacter in chicken production: survival, virulence and control

Lead Research Organisation: University of Cambridge
Department Name: Veterinary Medicine

Abstract

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Technical Summary

We comprise groups in Europe, Thailand and Viet Nam. The Thai government will fund work there and that in Viet Nam will be funded by Wellcome Trust and the Dutch Government. Our work will address knowledge gaps on the behaviour of Campylobacter in the poultry chain. We need to better understand interactions between Campylobacter, chickens and the environment and how these affect food safety. We will create a holistic picture of Campylobacter behaviour in chicken production and fundamental data on survival, how environmental exposures affect virulence and on-farm population biology and on pre- and/or probiotic intervention on-farm to reduce Campylobacter levels entering the food chain. We will use modelling and risk assessment tools to identify and test the potential efficacy of different interventions that can be utilised by the international poultry industry. We will determine the role of flies as vectors of Campylobacter to broilers and develop improved surveillance tools in order to reduce the number of Campylobacter in broiler meat
We aim to better understand the behaviour of Campylobacter in poultry production to improve control. We will generate understanding of mechanisms for:
In-flock transmission and risk in production including bird to bird spread in-house and downstream in the production chain
Virulence encompassing both broiler colonisation and extra-intestinal spread
Interventions for improving host resistance based on enhancing gut health and, in so doing, finding points for control
Environmental survival and the impact of environmental exposures on virulence. During these processes will be deterine pathogen source and persistence mechanisms
Population evolution to examine how the production chain selects certain sub-types of Campylobacter and whether the selection processes change virulence potential

Planned Impact

Campylobacter is the most important food borne zoonosis in the UK and the wider EU. In the UK it is estimated that there are 700000 cases of infection each year and that chicken-associated Campylobacter infection costs the UK economy ~£1 billion per year. Chicken is overwhelmingly the most important vehicle for human infection and is believed to be responsible for up to 80% of infections. ~80% of chickens on sale in the UK are Campylobacter-positive. Contaminated chicken presents two health threats. Surface contamination levels can be very high and contamination of deep muscle and liver tissues has been reported in up to 27 and 60% of samples tested respectively. The project seeks to better understand the processes that allow Campylobacter, and principally Campylobacter jejuni, to survive in the poultry production chain in the EU and in Thailand and to determine how exposure to potentially hostile environments affects virulence and bacterial population structures. We will also provide a comprehensive understanding of the molecular mechanisms used by C. jejuni to colonise the chicken gut and to leave there and infect edible tissues. We will examine the role of chicken gut health in the processes of Campylobacter infection and explore the use of pro- and prebiotics to better protect the birds from this major zoonotic pathogen.
The project is in partnership with the EU poultry industry and that in Thailand and all major UK retailers. Thus the beneficial impacts of our work can quickly be transferred to stakeholders.
 
Description 1. What were the most significant achievements from the award?
The most significant finding from the award was that C. jejuni flagella/motility is the most important factor for colonisation, survival and virulence of C. jejuni in all models tested.

2. To what extent were the award objectives met? If you can, briefly explain why any key objectives were not met?
The grant was part of an extended network collaboration. The involvement of the University of Cambridge (Partner 3) with particular work packages (WP) and Tasks was as follows:

WP1: THE INTERACTIONS OF C. JEJUNI AND BROILERS WITH THE PRODUCTION ENVIRONMENT
PARTNER 3: Will create C. jejuni mutant libraries and individual allelic replacement mutants; will identify TraDIS mutants showing different behaviours both in and ex vivo and will determine the gene expression profiles of C. jejuni strains in the environment and in flies

Task 1.3: C. jejuni virulence mechanisms in chicken colonisation
Task 1.3.2: Identification of molecular mechanisms: To investigate the molecular mechanisms of C. jejuni chicken colonization, interaction with host epithelial cells, and survival in the environment and the food processing environment, Partner 3 completed the construction of large transposon mutant libraries in three well-characterized C. jejuni isolates; M1, 81-176, and 11168. To facilitate rapid analysis of C. jejuni transposon mutant libraries a new mariner transposon element donor plasmid that allows read-out using Tn-seq technology (transposon insertion site sequencing) was constructed. Mutant libraries of various sizes were prepared by standard in vitro mutagenesis protocols. To characterize the newly generated transposon mutant libraries, the Tn-seq technology has been established in the laboratory of Partner 3, which enabled us to determine the relative abundance of each transposon mutant within the library. The Tn-seq analysis showed the successful generation of thousands of unique transposon insertion mutants in C. jejuni isolates M1, 81-176, and 11168. This dataset was used to assess which genes in the C. jejuni genome are required for fitness of the bacterium; genes required for growth cannot be inactivated by a transposon element or insertion of a transposon element may result in reduced growth.

The M1 transposon mutant library pool of ~10,000 unique mutants was selected for screening in various in vivo and in vitro models. Screening of the M1 transposon mutant library in a chicken colonization model using 3-week old Ross 308 commercial broilers was done in collaboration with Partner 1. Six days post-infection, birds were sacrificed and the mutant library composition was analysed from the caecal content by Tn-seq. This was to identify the genes of which mutants were capable of colonizing the birds and which were negatively selected from the mutant library pool, i.e. less capable of colonising chickens. In addition, genes which when inactivated appear to be enriched in the mutant library population were also identified), these were not considered for further analysis. Among the genes of which mutants were less capable of colonising birds, we found a significant enrichment of motility genes. To validate the findings of the Tn-seq screen, a panel of defined gene deletion mutants were generated. As part of this work a rapid method for the construction of defined gene deletion mutants based on overlapping PCR was developed; joining left and right flanking regions of the gene of interest with an antibiotic resistance cassette. The importance for colonisation was confirmed for 7 out of the 17 tested defined gene deletion mutants. Six gene deletion mutants colonised at wild-type levels, whereas four of the tested mutants colonised significantly better than the wild-type C. jejuni M1 strain. The discrepancy found between the transposon mutant library screen and validation using defined gene deletion mutants could (at least partially) be the result of the screen being a competition-based experiment (thousands of transposon mutants compete during colonisation of the bird) and the validation experiments were conducted as single strain infection experiments. For the genes of which their role in chicken colonisation was confirmed, gene deletion mutants were genetically complemented by insertion of the gene of interest in a pseudogene region, thereby restoring its expression. For this a new genetic complementation system was developed.

In addition to the Tn-seq screen, the transcriptional adaptation of C. jejuni strain M1 to the chicken GI tract during colonization was monitored. Ross 308 commercial broilers (3-weeks old) were infected with the 'wild-type' M1 strain and sacrificed 6-days post-infection. RNA was isolated from the caecal contents of three birds and pooled. The C. jejuni colonization levels of birds selected for RNAseq analysis ranged from 2x106 to 4x107 CFU. The pooled RNA was used to construct an RNAseq sequencing library, and sequenced using paired-end sequencing on the Illumina MiSeq platform by Partner 3. This yielded ~80 million sequencing reads of which only ~72,000 (0.1%) mapped to the C. jejuni M1 genome, indicating that construction of comprehensive expression profiles would require many sequencing runs. Considering a recent publication reporting a detailed insight into the transcriptional adaptation of C. jejuni 11168H in a 1-day old chicken colonization model, we decided not to invest the large amount of time, money and animals in this part of the work as such data are available and also due to the very high cost in deriving the amount of sequencing runs required to improve on the published data available.

The adhesion and subsequent invasion of host epithelial cells is considered a prerequisite for persistent colonization and infection. To study this in more detail Partner 3 optimized models of epithelial cell adhesion and invasion using the human colon carcinoma cell line CACO-2. The use of the model confirmed that directed gene deletion mutants of flagella associated genes, known to be required for invasion, were severely attenuated for both adhesion and invasion of CACO-2 cells. Next, the M1 transposon mutant library pool of ~10,000 unique mutants was screened during adhesion and invasion of CACO-2 cells. Fifty-seven genes were found to be required for invasion of gut epithelial cells, whereas, the screen only detected two genes involved in adhesion. This suggests that other mutants in the library pool may facilitate adherence of mutants that in single infection would display attenuated adherence, however, this hypothesis remains to be investigated. Out of the 57 genes implicated in invasion of gut epithelial cells, 14 were selected for validation using defined gene deletion mutants. The role in invasion was confirmed for 13 out of 15 tested defined gene deletion mutants. Motility was the driving factor for adhesion and invasion. Interestingly, we found that semi-motile and motile mutants were also attenuated for both adhesion and invasion. Genetic complementation of mutants restored their adherent and invasive capacity.

Task 1.4.1: Exposure of C. jejuni to hostile environments: Partner 3 performed transposon mutant library screen (~10,000 unique mutants) to identify mechanisms of survival during exposure to cold (4°C) under different conditions, i.e. chicken juice (liquid obtained when thawing frozen chicken carcases purchased from supermarkets), nutrient-rich culture medium (Brain Heart Infusion broth), sterile water, tap water and rain water. The survival of mutants was analysed after 7 days at 4°C under different conditions. Overall, a relatively low number of genes were linked to survival during cold-shock, indicating that survival may be mediated by passive rather than gene-mediated mechanisms. A panel of 10 genes of which transposon mutants showed reduced survival under the various tested conditions were selected for validation using defined gene deletion mutants. Interestingly, survival of the M1 wild-type strain in chicken juice was lower compared to all other tested conditions. C. jejuni M1 gene deletion mutants were tested under all the conditions used for the transposon mutant library screens. A total of 6 out of 10 genes tested for validation were found to play a role in survival during cold-shock under the different conditions tested.

WP 2: INSECT VECTORS FOR CAMPYLOBACTER TRANSMISSION
PARTNER 3: Will analyse the TraDIS mutants from the fly infection studies and will determine C. jejuni gene expression in flies.
Task 2.2 C. jejuni gene expression through passage of the house fly: This task did not look at gene expression, but made use of the Tn-seq C. jejuni M1 library established by Cambridge (Partner 3) to investigate genes necessary for survival in flies. A protocol was produced in collaboration with Partner 8 for screening of the mutant library in the fly model. Pilot experiments were conducted by Partner 8 to establish the appropriate inoculation dose as well as appropriate incubation times in flies to achieve approximately a 1 log reduction in Campylobacter numbers. As a conclusion from the pilots, flies for the mutant library screening were inoculated with 106 CFU and incubated for 4 h at 20°C. Five groups of 10 flies were individually inoculated with 106 CFU of the M1 transposon mutant library (~10,000 unique mutants) in a 1 µl volume via their proboscis. After 4 h incubation at 20°C, flies were homogenized and transposon mutants were recovered from flies on agar plates. To identify the mutants that were unable or less able to survive in flies, the relative abundance of each mutant (inoculum versus recovered) was determined by Tn-seq. The screen identified 48 candidate genes linked to survival in the housefly. To validate these findings, a panel of 7 defined gene deletion mutants were tested for their survival in the fly. The role of 3 genes required for fly survival were confirmed.

3. How might the findings be taken forward and by whom?
Having discovered that the flagella/motility is the most important factor for the survival/colonisation/virulence of C. jejuni, in collaboration with a pharmaceutical company we applied for BBSRC-LINK funding to develop a biologic that targeted the flagella. Unfortunately, this research was not funded.
Exploitation Route Possibly by the pharmaceutical industry or in other research grants.
Sectors Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
 
Description Narrative Impact: non-academic impact of the findings One of the members of the team at the University of Cambridge has recently appeared on BBC Radio 4 Farming Today discussing Campylobacter, the problem and potential solutions; pieces from this interview have been used in other BBC Radio 4 programmes. A journalist picked-up on the piece on the radio and as a result, the same individual was featured in a 2-page spread in the Cambridge Independent newspaper, discussing the Campylobacter research that was on-going in the laboratory. As well as this, the individual has just written a 1,500-word piece for a University of Cambridge magazine that is distributed to alumni, benefactors and is made available to the public.
First Year Of Impact 2016
Sector Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Societal
 
Description BBSRC Pathfinder
Amount £11,980 (GBP)
Funding ID BB/S009817/1 
Organisation University of Cambridge 
Sector Academic/University
Country United Kingdom
Start 10/2018 
End 04/2019
 
Description Novel biotherapeutic strategies for the control of Campylobacter in chicken
Amount £25,987 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2020 
End 08/2020
 
Description Collaboration with Paul Everest 
Organisation University of Glasgow
Department School of Veterinary Medicine Glasgow
Country United Kingdom 
Sector Academic/University 
PI Contribution Provision of a Campylobacter jejuni transposon library for in vivo screening through pigs, followed by sequencing and bioinformatics data analysis
Collaborator Contribution Provision of a Campylobacter jejuni transposon library for in vivo screening through pigs, followed by sequencing and bioinformatics data analysis
Impact Paper in preparation
Start Year 2015
 
Description A double-page spread in a local newspaper detailing the studies into Campylobacter 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Media (as a channel to the public)
Results and Impact A double-page spread in a local newspaper detailing the studies into Campylobacter
Year(s) Of Engagement Activity 2017
 
Description Interview for BBC Radio 4 Farming Today 
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 One of the research team was interviewed on the BBC Radio 4 Farming Today programme about Campylobacter in the food chain and their work to understand and control the bacteria
Year(s) Of Engagement Activity 2016