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A Multi-Pathogen Pre-Screening Tool for Safer Pork Products

Final Report Summary - PASSPORK (A Multi-Pathogen Pre-Screening Tool for Safer Pork Products)

Executive Summary:
Foodborne pathogens in fresh and processed meat products pose a serious threat to the health of consumers, as well as an additional burden on healthcare systems. In today’s meat supply chain, products may reach consumers in geographically dispersed markets within two to three days, thus making rapid and accurate pathogen detection technologies are key to protecting public health and ensuring “business as usual” in the meat industry.
Within the European meat sector the pork industry is crucial both in terms of consumption and in terms of its commercial importance; pork is the most produced and consumed meat in Europe (51%), as well as the most exported. According to FaoStat, pig meat supply in the EU in 2007 was estimated at over 21 million tonnes, with pig meat export reaching a total value of over 10 billion Euros in 2009. Despite these positive figures, pork is also responsible for a higher number of verified outbreaks per year, comparing to beef or bovine meat. These outbreaks are due to certain specific pathogens or serotypes, the most important of which are Salmonella, Yersinia, Listeria and Campylobacter which represent virtually all verified pork-associated bacterial infections reported in 2007.
The purpose of this project is to develop a device for identifying particular key pathogens in pork meat, some of which have been relatively overlooked by research. The nominated solution will be an analyzer that will use a combination of recent advances in immuno-chromatography and optical scanner technologies, together with specific monoclonal antibodies with high affinity to the target pathogens (Salmonella, Yersinia, Listeria and Campylobacter), to allow the simultaneous, rapid and accurate detection of the key pathogens in the pork industry. This combination of technologies will improve limit of detection. This will allow meat producers, such as abattoirs and meat-processors, to immediately take appropriate remedial action, significantly reducing risk of cross contamination and the possibility of pathogens entering the food chain. During the project development E.Coli was also included as fifth pathogen, due to its presence in the pork industry.

Project Context and Objectives:
There is a clear need for an affordable solution for reliable pathogen detection which can be applied in HACCP in small businesses such as abattoirs and meat processors. Concerned SMEs in the pork sector are therefore keen to develop new methodologies that will quickly and economically identify the key pathogens reoccurring in pork meat, adding value to their product and increasing SME competitiveness in the market. Such a device should be accurate, rapid, handheld and easy-to-use and, above all, tailored to the specific needs of SMEs in the pork industry. However, while the device is expected to be essential to SMEs, it will also be of great use for other companies in the pork supply chain.
The proposed solution will be applied at key specific processing points to augment the HACCP procedures mandatory in meat production. The analyser will not be a replacement system, but offer pre-screening and rapid detection of potential pathogens within the processing system. This will allow meat producers, such as abattoirs and meat-processors, to immediately take appropriate remedial action, significantly reducing risk of cross contamination and the possibility of pathogens entering the food chain.
Immuno-chromatography assays, or lateral flow tests, are widely considered robust and cost effective “front line” diagnostic techniques and have been certified for the diagnosis of animal diseases by the World Organisation for Animal Health8. These tests are based in the recognition of a target analyte by antibodies that react specifically with the analyte. The most common set-up for a lateral device is a double antibody sandwich assay, which requires two different antibodies against each target species . One of the antibodies is immobilised onto a membrane and the second one is labelled with a tag (usually latex or gold beads, enzymes, fluorophores, etc). Once the antigen (sample) binds to the immobilised antibody, it is then recognized by the labelled antibody and a detectable signal is produced (e.g. colorimetric, fluorescent). Optical scanner technologies, fully integrated with the lateral flow device (LFD), allow for the detection of the produced signal. With FORSITE and ATEKNEA in the consortium, PassPork proposed to bring standard colorimetric LFD beyond the state-of-the-art by utilising fluorescence and an optical scanner, thus significantly improving the sensitivity and the LOD of the device.
This technology was identified by the RTD performers as suitable in order to fulfil the initial key traits specified by the SMEs for the development of an affordable pathogen analyser:
1. A multi-pathogen analyser, able to detect the major pathogens of concern in pork, thus eliminating the need for applying several tests and offering a “complete” solution;
2. A rapid device allowing real-time decisions to be made and reducing cross contamination;
3. A durable device that can be used in the abattoir and on the processing floor, thus allowing rapid process control testing;
4. A technology that is highly reliable and capable of significantly reducing interpretation errors and the frequency of false-negatives.
The main objective of the PASSPORK project was to develop, validate and test an affordable, durable, rapid and reliable multi-pathogen detector for use by non-technical staff in the pork industry.

Scientific and Technological Objectives:
1. Identification, screening and production of specific antibodies for Salmonella, Yersina, Listeria and Campylobacter to be used in lateral flow assays. Currently available polyclonal antibodies do not have the required specificity in order to produce reliable detection. DTU will therefore develop monoclonal antibodies for the 4 pathogens of interest.
2. Design of a compatible meat sampling method and treatment protocol adapted to field conditions. Many state-of-the-art methods require a sample enrichment stage aiming to increase the concentration of the target analyte. In order to save the time-associated with the enrichment stage, DTU will propose a sampling protocol that is both easy to use and capable of producing high concentrations.
3. Development of a reliable detection system using fluorescence labelling which will not only signal the presence of a pathogen but will also indicate whether the concentration detected poses a health risk
4. Development of a rapid and robust fluorescence optical reader, corresponding to lateral flow assays for the detection of four pathogens of importance in the porcine industry. The system, developed by ATEKNEA and FORSITE, will consist of a set of 2 lateral flow assays, each detecting a pair of pathogens, which will be plugged into the reader sequentially.
5. Development of simple and fully-integrated device featuring a user-friendly interface based on a touch screen that will allow the storage and visualization of performed measurements as well as serial communication for easy data transfer to external computers.
6. Lab validation and field testing of the final prototype in order to verify the device indeed corresponds to the needs of SMEs and fully delivers system specifications and performance targets.
Strategic objectives:
The project delivers the following strategic objectives:
• Secure property rights and generate a viable business plan as a means of increasing the competitiveness of SMEs in the early stages of the pork meat supply chain by reducing their cost of compliance with strict regulations and preventing cross-contamination, thus enabling SMEs to compete with bigger businesses.
• Improving safety and quality of meat products entering the European meat-supply chain. Food safety is a major issue for the European agro-food industry and for European consumers. In line with this the EU has drawn up extensive legislation in the area of food and feed safety6. The realisation and implementation of such legislation represents an enormous challenge for all companies involved at various levels of the food chain, particularly SMEs and those from the New Member States. The key objective of this proposal, that of enabling rapid pathogen detection in meat, will level the playing field between European meat trading SMEs and larger enterprises and will assist SMEs throughout the supply chain to meet these new regulations.
• Addressing common problems and challenges associated with raising the standards of meat safety across Europe and ensuring that rapid, cost effective and easy to use tools are implemented on a European-wide scale to safeguard the health and safety of European consumers.
• Restoring and improving consumer trust in the European meat industry, by enabling the production of safer products, and thus also creating fierce competition for imported meat products from leading markets such as Brazil, Argentina, New Zealand, etc.

Project Results:
The project was divided into 9 WPs, WP1-WP6 related to R&D activities, WP7 DEMO, WP8 OTHER and WP9 MGT.

The consortium partners OLOT, ISLAND, QLAB, JCB, DTU and COVALAB assisted the work made by ATEKNEA (WP1) in the preparation of a market survey (done by means of a questionnaire) to determine the demands of the European meat sector for pathogen detection methods in terms of price, pathogens and disadvantages of the current methods. The questionnaire has been translated to all partner languages and sent to approximately 1500 companies involved in meat sector across Europe. The questionnaire has also been published online at the webpage and was available on the website throughout the entire project period. To create the questionnaire the online survey tool SurveyMonkey has been used. The analysis of the questionnaire answers shows the main pathogen problems, the price sensitivity of the market and the features to improve in a new pathogen detection tool.
From the questionnaire answers it is clear that European companies would improve their competitiveness if they could improve their pathogen detection methods, especially in the detection of Salmonella. This improvement would ameliorate their image and their benefits/costs ratio. Two features of the current methods require further improvement: the cost of the analyses and the time required to obtain the results. The PASSPORK technology would not require the expensive reagents and equipment required for the culture-based methods. The price of the consumables of this system should thus allow a price per analysis inferior to 5 euros. Such a price would really be advantageous in the market. The time to obtain results is a consequence of the basis of the technology chosen. In the case of classical culture methods, the basis is the selective growth of specific species. Hence, the time to results in these methods depends on the bacterial growth rate and the duration of the lag phase. It can take 12-24 hours to form detectable colonies. The expected time to obtain results with the PASSPORK equipment should be much shorter than with culture-based methods. The results show that the companies using external laboratories can have important delays in the reception of the results. These companies are probably interested in using any new method that reduces these waiting times if the use of the new method does not imply the construction of an expensive in-house laboratory. The characteristics of the PASSPORK are close to such a method. Most of the answering companies had not staff working only on pathogen detection tasks. One could wonder whether these staff members have been specifically trained for pathogen detection tasks. The development of the PASSPORK system should take into account that non-specialised staff may use the equipment. All the information gathered has then been used during the specification definition of the PASSPORK system.

RTD DTU, with the cooperation of ATEKNEA, QLAB and FORSITE and the advice of OLOTMEATS and ISLAND, worked in the comparative evaluation of several sampling methods (WP2) to establish which one yields the highest concentration of pathogens. Methods were compared on the basis of preparation time of the sample, skill level required and cost. Four sampling techniques were investigated. Three non destructive swabbing methods were compared to the destructive sampling, excision of skin tissue. The microbiological results from the testing can straight away be transformed into the areas used according to the EU regulation. Although tissue skin sampling is more sensitive than swab sampling, when testing the same area, the swab sampling method was favoured for the LFD analysis. The non-destructive methods do not damage the skin in any way and it is easy to vary the sampling area, if necessary. Of course, there are limitations by use of these methods as well. As this experiment showed, the level of recovery is lower and it is hard to make the pressure uniform when sampling.
Overall, all procedures and equipment used adhere to the requirements for providing a simple and cheap sampling and sample preparation method, which can be performed by non-specialist personnel in food processing SMEs such as pig slaughterhouses and without the need for expensive and technically complicated equipment. Finally, a prototype of the sample preparation kit has been developed in the task to serve as an auxiliary component of the final system (Objective 2).

On the other hand, DTU, with the support of ATEKNEA, FORSITE and COVALAB, undertook the development of the antibodies for identifying the selected pathogens (WP3). A highly specific antibody against Salmonella was delivered to FORSITE along with the antigen. Further, mice were immunized with bacterial surface lipoproteins extracted from different serotypes. For Yersinia and Campylobacter, hybridomas were subcloned and promising clone candidates were prepared. The SMEs decided to include also E. coli in the panel and mice have been immunized and tests for specificity to E. coli and cross-reactivity within E. coli serotypes is ongoing. A Listeria mAb from COVALAB was tested in flow cytometry but was not functional in this assay.

The aim of WP4, lead by FORSITE, was to develop two multiplexed lateral flow devices for the detection of food pathogens in pork carcass wash samples; one for E.coli/Salmonella and the other for Campylobacter/E.coli. These devices would be tested with samples taken using the protocol developed by DTU and would be made using specifically developed reagents. Unfortunately, due to unforeseen developments none of the antibody reagents produced by DTU were suitable for use with the fluorescent latex so further sources were explored. 14 commercial antibodies were identified and investigated to ascertain which, if any, would be suitable for use in the PASSPORK LFD. Out of these 14 only three were considered suitable for further development and their use in the detection of Campylobacter, E.coli and Salmonella was ascertained by the development of gold-based proof-of-principle tests. All three antibodies produced tests which detected the following: Campylobacter to around 10 x 10-5 CFU/ml, E.coli to around 10 x 10-6 CFU/ml and Salmonella to 10 x 10-1 CFU ml. On the basis of these results the antibodies were tested for suitability for conjugation to fluorescent particles.

Using the Campylobacter, E.coli and Salmonella antibodies identified during the PoC work above, three methods for antibody conjugation to fluorescent particles were investigated; the method of choice being covalent coupling to Dragon Green latex. This method was utilized to produce a multiplexed E.coli/Salmonella lateral flow device which was optimised along with staff from Ateknea using the PASSPORK fluorescent reader to determine ideal assay parameters. Due to antibody limitations it was not possible to transfer the Campylobacter device. Initial work on the multiplexed test showed a lower limit of quantitation (LLOQ) of 1 x 10-9 CFU/ml for E.coli and 1x 10-6 CFU/ml for Salmonella. Sensitivity testing showed that the tests were limited in detecting some serotypes of both E.coli and Salmonella, detecting around 50% of Salmonella strains and about 29% of E.coli out of the samples tested. This was due to antibody limitations. The PASSPORK test was able to successfully detect E.coli and Salmonella bacteria spiked into high levels of other bacterial species (Yersinia, Campylobacter and Serratia) and detected E.coli and Salmonella in blind testing studies using pork carcass wash samples taken from slaughterhouses.

The objective of WP5 was to develop a low-cost fluorescence analyser to detect the pathogens using lateral flow assays enabling this technology to be used on-site for rapid multi-pathogen detection.
The optomechanical system of the fluorescence analyser was developed, as well as the analogue electronics for the light excitation source and the signal conditioning was also developed (PCBs) and the firmware to control the complete system. A user-friendly interface based on a touch screen display was developed, taking into account the final end-user does not necessary have technical skills. Several issues were faced along the project regarding firmware and the user interface, however all those problems were solved and a functional reader was provided to the partners. The developed reader constituted a robust and low-cost alternative to existing fluorescence laboratory systems enabling this technology to be used on-site for rapid multi-pathogen detection.

Once the previous WPs were completed, the system had to be integrated and validated under lab conditions before starting the field trials under real conditions by the end-users of the project.

WP6 dealt with the system integration and initial lab validation. In order to ensure that all components worked as expected, a validation protocol was created by the RTD (ATEKNEA, FORSITE and DTU) where the sample preparation protocol, the LFDs preparation and the reader manual was presented. The reader performance validation was done in terms of specificity, sensitivity, accuracy and precision. LFDs acceptance criteria was also included in the Validation protocol.
The system was then validated in the lab using complex samples with known presence of the different pathogens and using unknown samples and comparing the results with other laboratory detection methods. The results of this validation were presented in a report (deliverable 6.2).
Results of the lab analysis were used to define the dynamic range, thresholds of positive and negatives, as well as to fix bugs that appeared during the experiments.
The last two months of the project, the consortium worked on the field validation of the PASSPORK technology including the sampling protocol. Q-LAB and OLOT supported by the RTDs tested real samples and compared the results to the standard laboratory methods. The detection limit was determined after this validation phase and compared to the detection limit found out during the preliminary test: detection limits for E. coli was 1.50E+9 and 1.00E+7 CFU/mL for the LFD reader and lamp, respectively. For Salmonella the detection limits were 8.30E+6 and 5.70E+5 CFU/mL for the LFD reader and UV lamp, respectively. Our test trial confirmed partially these detection limits, but further testing would be needed to completely validate the system and even improve the sensitivity and/or specificity. Optimization of the LFD reader lead to a 10 fold increase in the detection limit for Salmonella.

A training plan was produced to ensure that SMEs assimilate the results of the project. All members of the consortium were actively involved under the coordination of the Exploitation Manager QLAB.
The project website ( was created and it will serve as vehicle of communication and dissemination of the project and its results. All consortium members have been actively involved in dissemination activities. All members of the consortium also created a draft of the Plan for Use and Dissemination of Foreground generated during the project.

A productive working atmosphere, with fluent and regular communication, has been established between the SMEs and the RTD performers, and the project is advancing with small deviation respect to the work plan.

Potential Impact:
PassPork is a portable, robust, easy-to-use and fast analyser able to detect simultaneously E.Coli and Salmonella in porkmeat.

As demonstrated by the recent E. coli VTEC O104 outbreak in Europe, the spread of pathogens in the food chain is an area with huge gaps in knowledge. Most significantly was the time that it took for the source of the outbreak to be identified, with additional infections occurring and damaging the consumption of certain products due to fear provoking the destruction of certain markets.

PassPork technology is specially designed for pork meat applications. Despite the analysis required by regulations, pork is responsible for several outbreaks per year. In fact, recent studies place pork products as the second source of human salmonellosis, after laying hens. Due to globalisation, an infected meat may be distributed worldwide in few days posing in danger the human health and dramatically damaging the brand of the producer company. Following the EU regulations aiming at maintaining food security and to prevent the reduction of sales an outbreak would cause, companies perform tests to detect the most common pathogens in meat. Although several methods exist, they are time consuming and expensive, and need specialised technicians to carry out the analyses. Therefore, SMEs, which represent the 94% of the sector, cannot afford this equipment and only perform mandatory tests. PassPork will help them by offering a tool to make more screening tests and being more competitive compared to large enterprises.

PassPork’s market are mainly abattoirs and meat processing plants which will benefit from performing quick screening tests allowing rapid decision-making to avoid cross-contamination and putting into the market infected products. Thus, society as a whole will result benefited from the use of this new technology as only safe products will reach the consumers.

SME competitiveness will be enhanced in three ways:
a) Benefit to SMEs holding intellectual property rights: as holders of IPR, the SMEs in the consortium will economically benefit from the commercialization of the final system, as well as any component which will be deemed independently marketable at project wrap-off.
b) Benefits to end-users: the use of the developed device at slaughterhouses (such as ISLAND) and other businesses in the meat supply chain will directly decrease their cost of compliance with food safety regulations and increase their profit margin, thus stabilizing businesses from a financial point-of-view and closing gaps with competitors in the industry.
c) Benefits to lead-users: SMEs in the position of lead-users (QLAB and JCB) expect to integrate the developed device as part of the product or service offer. Thus, the project will secure these companies an additional income channel, also providing them a first mover advantage in a new market niche of low-cost pathogen detection methods. QLAB will distribute the product and JCB will produce the analyzer. The rest of SMEs will benefit from royalties.

When the project was coming to an end, SME partners agreed to jointly own all the outcomes of the project. The main exploitable results are the reader and the consumables which SME partners plan to commercialise basically through distributors. Partner QLAB will act as a distributor mainly in Germany, Belgium and the Netherlands whereas OLOTMEATS will distribute it in Spain. The technical SME partner JCB will produce the analyser on demand. With expected market price of €1370/device and €13 for the two tests, the consortium estimates sales of approx. 3,5M€ in the next 5 years, with an estimated pay-back of 1.12 years.

The importance of this new detection system is proven by the high number of online publications that echoed the project, for example, La Vanguardia, El Mundo, an R&D supplement from El Comercio, specialized online media about meat industry (Ediporc, Eurocarne) or agrofood (Agromeat) or the Veterinary Association of Cáceres (Spain). PassPork project was presented in the Catalan Cluster of Innovation at the Pork Industry (INNOVACC) and in BizBarcelona 2014 (the biggest business and entrepreneurs event in Catalonia).

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Contact details – Project Coordinator
Ateknea Solutions
Telephone: +34 93 204 99 22
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