31 participants will strengthen the bridge between the Nordic food industry, research institutes and universities in the field of microbiological food safety with special focus on campylobacters and/or molecular techniques Project Manager: Peter Rådström, Lund University, Sweden (Coordinator) Country coordinators: Jeffrey Hoorfar, DFVF, Denmark (Deputy coordinator) Elisabeth Borch, SIK, Sweden Knut Rudi, MATFORSK, Norway Sigrun Gumundsdottir, Icelandic Fisheries Lab (IFL), Iceland Marja-Liisa Hänninen, Helsinki University, Finland http// C AMPY F OOD A Molecular Safety Approach for Campylobacter

Activities We will strengthen ongoing research at the participating laboratories in order to ensure synergy and transfer the technology to the food industry *mobility of research personnel *a project homepage *hands-on demonstrations *newsletters *workshops http// C AMPY F OOD

Clostridium botulinum Salmonella Clostridium botulinum Salmonella Virulence expression Rapid methods I II Food safety will be increased by the application of molecular-based techniques http// C AMPY F OOD

Botulism – rare but deadly An intoxication in which 30ng neurotoxin can be lethal Consumption 0.1g contaminated food can result in botulism High fatality rate (~10% cases) 7 serotypes of the neurotoxin on chromosome: types A, B, E, F on bacteriophage: types C, D on plasmid: type G Clostridium botulinum Toxin, 150 kDa

Neurotoxin Formation Relative expression and quantification of bontB (mRNA) qRT-PCR BoNT/B production (protein) ELISA Biological activity of BoNT/B (active toxin) Mouse Bioassay

protein Virulence factors mRNA DNA Virulence expression Environmental factors

C. botulinum type B Time (h) OD (620 nm) Relative expression BoNT/B (ng/ml) <2h4h5h20h

Control in foods Preservative Effect CO 2 Inhibition of growth, Inhibition of outgrowth of spores NaClInhibition of growth, Inhibition of outgrowth of spores NaNO 2 Inhibition of growth

Effect of NaCl 10% CO 2 0% NaCl 0 ppm NaNO 2 10% CO 2 2.5% NaCl 0 ppm NaNO 2 Time (h) Relative expression OD (620 nm) Time (h) Relative expression OD (620 nm) ng*ml -1 *OD -1 * 47 ng*ml -1 *OD -1 *

Effect of NaNO 2 10% CO 2 0% NaCl 0 ppm NaNO 2 10% CO 2 0% NaCl 75 ppm NaNO 2 Time (h) Relative expression OD (620 nm) Time (h) Relative expression OD (620 nm) ng*ml -1 *OD -1 * 30 ng*ml -1 *OD -1 *

Effect of CO 2 10% CO 2 0% NaCl 0 ppm NaNO 2 70% CO 2 0% NaCl 0 ppm NaNO 2 Relative expression Time (h) OD (620 nm) Time (h) Relative expression OD (620 nm) ng*ml -1 *OD -1 * 126 ng*ml -1 *OD -1 *

Effect of CO 2, NaCl and NaNO 2 Time (h) OD (620 nm) Relative expression % CO 2 0% NaCl 0 ppm NaNO 2 27 ng*ml -1 *OD -1 * Time (h) OD (620 nm) Relative expression % CO % NaCl 75 ppm NaNO ng*ml -1 *OD -1 *

LPD= m =b 0 +b 13 *[CO 2 ]*[NaNO 2 ]+b 22 *[NaCl] 2 +e log(RE)=b 0 +b 11 *[CO 2 ] 2 +b 22 *[NaCl] 2 +e Traditional food preservatives (CO2, NaCl and NaNO2) stimulates the neurotoxin formation increasing the risk for food borne botulism

FOOD MICROB HUMAN Virulence expression a step towards formulating new strategies for food preservation, predictive modelling and risk assessment.

EU 6th FP. Area: Food Quality and Safety (5.4.4 Area: Traceability processes along the production chain) T Origin and development of unintended micro- organisms in the food and feed chains (IP) The objective is to develop and improve methods for tracing the origin of biological agents contaminating (including as the result of a criminal act) food (also bottled or canned drinking water) and animal feed and to model their development (growth, proliferation and toxicogenesis) as a function of ambient (e.g. temperature and relative humidity) and processing conditions, and their point of entry into the food chain (including the home environment).

Salmonella Rapid methods II

Day 0 Day 1 Day 3 Day 4 25 g feed ml BPW Pre-enrichment Enrichment RVS Selective agar plates Confirmation Day 2 Conventional analysis of Salmonella

Why do we need new methods? *Low detection limit (less than one pathogen per 25 gram) *High specificity and accuracy (no false-negative/-positive results) *High robustness (inter-lab reproducibility) *High Rapidity (at-line and on-line analysis) *Acceptance (validation and standardisation) *Low cost (number of test) *Simplicity (user-friendly and automation) *Sample matrix flexibility (no interference) *Quantitative analysis (food spoilage micro-organisms)

Amplification Growth-based Viable Counts + Detection Limit Quantitative Simplicity - Specificity Rapid Laborious

Rapid Methods 1. Cell counting methods Flow cytometry Direct epifluorescent microscopy 2. Modified and automated conventional methods Spiral plater Dipslides Chromogenic/fluorogenic media 3. Impedimetry 4. Bioluminescence 5. Immunological methods ELISA Immunocapture 6. Nucleic acid-based assays Hybridisation Amplification methods (PCR) Restriction fragment length polymorphism (RFLP) Random amplified polymorfic DNA (RAPD) RiboPrinter

Challenges with Diagnostic PCR Risk of inhibition from biological samples Low concentration of target Reduce the size of the heterogeneous bulk sample to a homogeneous PCR sample

PCR InhibitorMechanismRef. ProteinasesDegr. of Polym.Powell et al IgGBinding to DNAAbu Al-Soud et al PolysaccharidesBinding to Polym.Monteiro et al LactoferrinRelease of iron ionsAbu Al-Soud, Rådström 2001 Calcium ionsCompeting with Mg 2+ Bickley et al PhenolDenatur. of Polym.Katcher, Schwartz 1994 EDTAChelation of Mg2+Rossen et al HeparinBinding to DNASatsangi et al Taq DNA polymerase

The importance of DNA polymerase and PCR facilitators in Diagnostic PCR

Diagnostic PCR 1. Sampling 2. Sample preparation 3. DNA amplification 4. Detection of PCR products DNA polymerases PCR facilitators Pre-PCR Processing

Internal control 284 bp Salmonella (invA gene) 150 bp Internal control Rahn et al. 1992

1. Sampling 2. Sample preparation 3. DNA amplification 4. Detection of PCR products Feed in BPW 1:10 Homogenisation Pre-enrichment for 18 37ºC (isolate obtained!) Samples withdrawn after shaking No DNA extr. or cell lysis! Tth DNA polymerase Gel electrophoresis Enrichment PCR method

PCR Method Detection limit

No of positive samples Sample type No of samples NMKL PCR Faeces and intestines Fish meal Maize gluten Meat meal Mixed feed Rape meal Soya Soya, acidified Total Evaluation of the developed diagnostic PCR protocol on natural samples

Amplification Molecular Methods + Specificity Rapid Automation - Detection Limit Robustness Acceptance Immunological methods Polymerase Chain Reaction

Acknowledgements Waleed Abu Al-Soud, 2000 Ingrid Artin, --- Halfdan Grage, 2002 Oskar Hagberg, 2005 Rickard Knutsson, 2001 Charlotta Löfström, 2005 Maria Lövenklev, 2003 Petra Wolffs, 2004