16AGRITECHCAT5: A whole supply chain hurdle approach to control Campylobacter

Lead Research Organisation: University of Liverpool
Department Name: Institute of Infection and Global Health

Abstract

Campylobacter is responsible for 14% of all human diarrheal cases worldwide, with >280,000 food poisoning cases in the UK per year and c. 100 deaths. It costs the UK economy >£580m per year, much of this imposed on the NHS (Strachan et al 2010, Lancet, 376, 665-7). It is an extremely serious issue and the FSA now regularly monitor Campylobacter levels from retail purchased chickens and publish the results. Given the social impacts, Campylobacter is a primary risk threatening the UK poultry industry. An industry that contributes £3.6bn GVA to the UK economy. Defeating Campylobacter is a clear industry and policy aim, any techniques which reduces the impact of this disease will underpin both the economic and social sustainability of the industry and food security of consumers. Given the impact of Campylobacter on human consumers, the control of the disease has been the subject of considerable research, and a number of key interventions are currently deployed by the industry. These include increased emphasis on the biosecurity of flock, hygiene through the supply chain, novel animal feeds, transportation systems, cold chain systems, handling and processing techniques. However, in nearly all instances the impact of different interventions are typically studied in isolation. We still do not fully understand where Campylobacter enters and exits the supply chain, how interventions may select for the survivability of different strains, the impact of a range of different interventions applied in series through the supply chain and the resilience of these treatments with varying flocks and seasons. Given this lack of understanding it is very difficult to develop an integrated approach to defeat the pathogen through the supply chain. However, modern molecular techniques have also progressed at pace and very recent studies suggest whole genome sequencing (WGS) can be applied to effectively track and trace Campylobacter through a supply chain. This enables a hitherto impossible examination of the organism as it moves through a supply chain. We now have the ability to show where a strain entered and exited a supply chain, the specific impact of interventions and how they might be strain selective. In this project, we will apply modern molecular approaches to track and trace Campylobacter through the supply chain. Furthermore, we will examine how a series of interventions through a supply chain (the hurdle approach) can be optimised to help defeat the organism. The interventions studied will be the effects of changes to animal feeds, 2 thermal interventions applied in the factory and the impact of a novel blast chilling and modified atmosphere packaging system. This is a comprehensive study across an entire supply chain. The highly novel combination of a hurdle technology approach with molecular epidemiology will help underpin the poultry supply chain. We are aware of no other similar and wide ranging approach to control Campylobacter in the global poultry industry. WGS can help optimise a supply chain system, but is not a simple tool to apply in industry post project. Therefore, within the project we will also develop a novel quantitative PCR (qPCR) which can rapidly measure Campylobacter level through the supply chain. qPCR systems are available for Campylobacter but they have not yet been optimised to detect only live, rather than dead, cells.

Technical Summary

We will rigorously study a novel holistic hurdle technology approach comprising a consecutive series of interventions, underpinned by the largest molecular study of Campylobacter in a supply chain conducted to date. We will test the effect of region and season on Campylobacter populations, and then the efficacy of four novel interventions, including interactions between them. These are: 1) novel antimicrobial dietary Campylobacter control on farm; 2) a novel thermal intervention system; 3) the synergy of a further thermal invention; and 4) a novel packaging method. We will evaluate the effect of interventions, and their interactions on total bacterial communities and Campylobacter genetic variance and track and trace the fate of individual strains and sub-populations of Campylobacter.

Due to cost, previous studies sequenced genomes of a relatively few individual isolates from samples: this prevents any meaningful insight into populations. It is well described that Campylobacter is very genetically diverse in the UK. Sequencing a sufficient number of individuals from samples is not yet economically viable for large-scale studies such as this, where we will analyse populations from 392 samples. Instead we will use a novel pooled sequencing approach to circumvent this massive cost, but retain power. We will combine together 100 individuals from each sample and extract and then sequence this pooled DNA. The trade-off is we cannot track individual strains. The massive cost saving means we can meaningfully statistically compare populations to objectively evaluate if and how populations are affected by novel interventions. Informed by data from stage 1, we will then use targeted genome sequencing of individual Campylobacter to detail changes at the strain and sub-population level to evaluate if and how Campylobacter populations and specific strains are affected by intervention hurdles, and if populations differ in space and time.

Planned Impact

Campylobacter is responsible for 14% of all human diarrheal cases worldwide, with >280,000 food poisoning cases in the UK per year and c. 100 deaths. It costs the UK economy >£580m per year, much of this imposed on the NHS (Strachan et al 2010, Lancet, 376, 665-7). It is an extremely serious issue and the FSA now regularly monitor Campylobacter levels from retail purchased chickens and publish the results. Given the social impacts, Campylobacter is a primary risk threatening the UK poultry industry. An industry that contributes £3.6bn GVA to the UK economy. Defeating Campylobacter is a clear industry and policy aim, any techniques which reduces the impact of this disease will underpin both the economic and social sustainability of the industry and food security of consumers. Given the impact of Campylobacter on human consumers, the control of the disease has been the subject of considerable research, and a number of key interventions are currently deployed by the industry. These include increased emphasis on the biosecurity of flock, hygiene through the supply chain, novel animal feeds, transportation systems, cold chain systems, handling and processing techniques. However, in nearly all instances the impact of different interventions are typically studied in isolation. We still do not fully understand where Campylobacter enters and exits the supply chain, how interventions may select for the survivability of different strains, the impact of a range of different interventions applied in series through the supply chain and the resilience of these treatments with varying flocks and seasons. Given this lack of understanding it is very difficult to develop an integrated approach to defeat the pathogen through the supply chain. However, modern molecular techniques have also progressed at pace and very recent studies suggest whole genome sequencing (WGS) can be applied to effectively track and trace Campylobacter through a supply chain. This enables a hitherto impossible examination of the organism as it moves through a supply chain. We now have the ability to show where a strain entered and exited a supply chain, the specific impact of interventions and how they might be strain selective. In this project, we will apply modern molecular approaches to track and trace Campylobacter through the supply chain. Furthermore, we will examine how a series of interventions through a supply chain (the hurdle approach) can be optimised to help defeat the organism. The interventions studied will be the effects of changes to animal feeds, 2 thermal interventions applied in the factory and the impact of a novel blast chilling and modified atmosphere packaging system. This is a comprehensive study across an entire supply chain. The highly novel combination of a hurdle technology approach with molecular epidemiology will help underpin the poultry supply chain. We are aware of no other similar and wide ranging approach to control Campylobacter in the global poultry industry. WGS can help optimise a supply chain system, but is not a simple tool to apply in industry post project. Therefore, within the project we will also develop a novel quantitative PCR (qPCR) which can rapidly measure Campylobacter level through the supply chain. qPCR systems are available for Campylobacter but they have not yet been optimised to detect only live, rather than dead, cells.

Publications

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Description This work was undertaken by the University of Liverpool (UoL) and a molecular assay company that produce 'off the shelf' and 'lab in the box' PCR assays which aimed to detect and quantify Campylobacter a major gastrointestinal zoonotic pathogen. Campylobacter is associated with livestock production, especially poultry meat production so the aim of the project was to validate assays that had been developed by the company on a range of samples relevant to the poultry industry, so they have a method which can help detect 'hotspots' or areas with greater contamination by Campylobacter and thus inform where interventions could be applied. An additional aim was to develop a method which allowed the user to determine whether the DNA giving the signal in the molecular assay derived from live or 'viable' bacterial cells, rather than dead cells, as the PCR method is highly sensitive and can pick up any target DNA regardless of whether it originated in live or dead cells. However, it is important to know for the industry and if the method is to replace traditional standard culture which requires a number of days, that the DNA being amplified in the assay is from live cells which present a public health risk.
Validation work involved testing 218 Campylobacter species isolates from the UoL culture collection from diverse sources with two assays, one that individually tested the main two species (C. jejuni and C. coli) and a assay which detected both. The assays were found to perform well with a very low false positive and false negative rate. For the combined assay which was the assay of choice for taking forward, the false negatives included a small number of isolates (n=4), which were isolated from the wider environment and represented unique sequence types which have not been reported in poultry, or human infections. The use of the primers which detected both species also meant that this assay could be undertaken in one of their low cost real-time machines and therefore, constitute a 'lab in the box' approach with all reagents provided for extraction and running the assays for easy access by industry.
Propidium monoazide (PMA) is a dye that can covalently bind to DNA and can be used as a pre-treatment with samples to allow it to enter dead cells and following UV treatment bind to DNA and therefore prevent amplification of DNA by PCR. This pre-treatment of samples with PMA therefore allows differentiation between live and dead cells. The qPCR could detect C. jejuni and C. coli in spiked meat juice samples kept at 4oC samples, including after PMA treatment and indicated that both species were still viable, and even after 24 weeks both bacteria were still detected at relatively high numbers by qPCR with a 2log reduction in detection when pre-treating samples with PMA, indicating a loss in viability of proportion of the population. When this experiment was repeated in broth to enable standard enumeration by culture, there was also good correlation between the qPCR and conventional enumeration by culture for C. coli. However, for the C. jejuni strain this could not be cultured at 24 weeks, but was detected by qPCR and similar values were detected with and without PMA treatment, indicating the presence of viable cells, even though they could not be cultured. This might indicate a point at which the cells are viable but non-culturable (VNBC) that has been reported previously for Campylobacter, but that the cells are not sufficiently damaged as to take up the PMA stain and therefore, amplification of DNA is possible despite treatment with exposure to UV. The VBNC state has only been demonstrated in the laboratory and it is not known whether this occurs naturally within the poultry meat supply chain, or the public health threat that such cells pose and requires further investigation.
Further validation of PMA treatment and the qPCR assay on artificially contaminated chicken skin, to mimic neck skin which is used in the poultry industry for determining the level of contamination, as well as a survey on Halal poultry meat, which also included conventional enumeration was undertaken. There was good correlation between the inoculum levels of Campylobacter added to the poultry skin and that which was detected by qPCR with and without PMA treatment. However, when the samples were stored for longer periods, a reduction in detection was found in the PMA treated samples, compared to no treatment indicating again a reduction in viability of cells in those samples. There were also some erroneous results with the PMA treatment that could not be explained, therefore there was concern about whether the PMA pre-treatment is a reliable method to use on all sample types and this was also observed when the method was applied to caecal samples collected at slaughter and boot sock samples taken on poultry farms. However, the results do indicate that the qPCR is a reliable method for detection of Campylobacter with higher sensitivity than conventional culture.
Exploitation Route qPCR assays represent an attractive alternative to conventional culture and enumeration, which for Campylobacter species requires specific expertise and equipment for culture, given that Campylobacter is a microaerophilic organism that does not grow under normal oxygen levels and which has long incubation times. Such off the shelf assays with internal controls and which can be provided with low cost real time machines can provide an alternative to culture, especially for laboratories with limited microbiological expertise. Furthermore, the qPCR assay validated in this study was found to work well with neck skin samples which are examined across the industry to determine the levels of contamination on retail product and therefore, could be used to assess the impact of interventions put in place by industry.
The results around detection through culture and qPCR with and without the PMA pre-treatment suggested that whilst the bacteria could not be cultured on agar even using very sensitive techniques, the PMA treatment indicated that these bacteria were still alive. The VNBC state still requires further investigation as to whether these bacteria can revert back to a viable state which is capable of infecting people and therefore, is still a public health threat, or whether such bacteria are not capable of infection and therefore do not pose a threat.
Sectors Agriculture, Food and Drink

 
Description Invitation to sit on a restatement panel on the Campylobacter hosted by the University of Oxford 
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 I was invited to take part in a restatement workshop on Campylobacter which involved contributing to a restatement paper. This paper will be sent to stakeholders for further opinion and the subsequent paper published and used as a briefly document for policymakers.
Year(s) Of Engagement Activity 2017
 
Description Meet the Scientists event at Liverpool World Museum 
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 'Meet the scientists' was a free large organised event at the Liverpool World Museum featuring many different research groups presenting their work and in total 1900 visitors were recorded for the event. We had a stand called "Chicken or egg" and we highlighted all aspects of commercial chicken and egg production, accompanied by lots of interactive activities, as well as providing information on our research, including our Campylobacter work and how Campylobacter maybe transmitted on through the food chain and in the domestic kitchen.
Year(s) Of Engagement Activity 2018
 
Description Presentation about Campylobacter control at the 7th Pan Commonwealth Veterinary Conference, Bangalore March 2019 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Invited talk on the infection biology and control of Campylobacter in broilers and the broiler meat chain.
~70 people attended from India and other commonwealth countries
Year(s) Of Engagement Activity 2019
 
Description Trust Me I'm A Vet, BBC programme filming 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact I took part in filming of a BBC programme 'Trust me I'm a vet' which featured our laboratory and the related work we were doing on the current trend for feeding raw meat diets to household dogs and the risks this may contribute to, in terms of contamination of the household environment with food borne enteric pathogens, such as Campylobacter and the importance of good hygiene. We also highlighted the increased risk of faecal shedding by dogs of enteric pathogens, including Campylobacter.
Year(s) Of Engagement Activity 2017