Combined live/dead discrimination and real-time PCR approach for the quantitative risk assessment of viable Campylobacter applicable in international control strategies (CAMPY-TRACE)
This third-party funded project is conducted in the framework of the BfR research programme on exposure estimation and assessment of biological risks.
BMBF grant number: 031B0054A
Project homepage: -
Campylobacter is an internationally underestimated food-borne pathogen. Although far less known by the public than Salmonella, campylobacteriosis is currently the most frequently reported bacterial food-borne illness in humans worldwide, with broiler meat comprising the crucial source of infections. In Vietnam, pioneer studies suggested that Campylobacter might contribute to child morbidity and poultry products were accordingly proposed to constitute the main source of infection.
The main objective of this collaborative project is the establishment, validation and implementation of a cultivation-independent direct quantification method by a real-time PCR approach for effective control of the poultry food chain. These methods have to be improved in order to sufficiently discriminate between live and dead bacteria with suitable reference standards (in particular, an internal sample process control, ISPC), enhancing the informative value of CFU (colony forming unit), currently accepted to be the “gold standard” in food control. For this purpose, the BfR in cooperation with the Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit recently proposed a new term of “intact and potentially infectious units (IPIU)”, corresponding to all viable bacteria with an intact membrane, including those that are transiently metabolically inactive.
Parallel to the establishment and validation of the method, the research cooperation will contribute to broaden our understanding of quantitative dissemination of this important pathogen, taking into account the different climatic situations of the two cooperating countries and various steps from primary production to retail. The project aims at uncovering possible critical contamination sources along the production and distribution chain of poultry. Filling in the gaps for reliable quantitative detection of Campylobacter will allow more targeted intervention strategies and overall reduction in the transmission of the pathogen, especially regarding the internationally rising demand and global trade of chicken products.
BfR parts of the project:
The BfR is the coordinator of the project and responsible for following work packages (WP):
WP2 Development of an Internal Sample Process Control (ISPC) for quantification of Campylobacter by live/dead discriminatory real-time PCR: the BfR in cooperation with Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit already showed in previous work that the signal reduction from dead Campylobacter depends on various parameters. The development and implementation of an internal sample process control (ISPC) for quantification of viable Campylobacter in a given sample is therefore essential.
WP6 Conservation of the membrane-permeability defined reference standard for distribution to collaborating laboratories: The goal of this work package is to establish methods to conserve the ISPC developed in WP2 in order to render it distributable as reference standard for collaborating laboratories. Different conservation media will be tested. Lyophilisation will be one of the preferred methods for maintenance of chromosomal number in a DNase-resistant but intercalator-accessible state. Homogeneity and stability of the reference standard as well as variability of membrane permeability (i. e. accessibility of intercalating dyes to DNA) have to be defined, which will be realised in close cooperation with LGL-DE.
WP9 Third stage - quantitative detection of Campylobacter on retail chicken in Germany and Vietnam: A fairly strong correlation between CFU and IPIU is expected for fresh samples from the slaughterhouse. On retail products, where Campylobacter has been exposed to stress conditions, the correlation is expected to be much weaker. The previous “gold standard” CFU is misleading and does not reflect the proper quantitative risk of infection (see also state-of-the-art). The ISPC-controlled viable/dead discriminating real-time PCR will, therefore, be used in WP9 for proof-of-principle and to fill in quantitative knowledge gaps.
WP11 Comparison of German and Vietnamese data from all stages of the poultry food chain and from human stool samples and identification of intervention strategies: The goal of WP11 is to integrate all obtained data in order to identify crucial reduction strategies for Campylobacter control. The whole project was designed to focus on the acquisition of respective relevant data (using appropriate specific detection) on Campylobacter with respect to the physiology of the pathogen, and to develop and validate a culture-independent quantification method for detection of viable Campylobacter.
- Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany
- National Institute of Hygiene and Epidemiology, Vietnam
- National Institute of Veterinary Research, Vietnam
- Vietnam Food Administration, Vietnam