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Molecular Safety Approach for Campylobacter C.AMPYFOOD

Strengthening the bridge between the Nordic food industry, research institutes, and universities in the field of microbiological food safety, with a special focus on Campylobacters and molecular techniques.

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Molecular Safety Approach for Campylobacter C.AMPYFOOD

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  1. A Molecular Safety Approach for Campylobacter C AMPYFOOD http//www.CampyFood.org 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

  2. C AMPYFOOD 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//www.CampyFood.org

  3. C AMPYFOOD http//www.CampyFood.org Food safety will be increased by the application of molecular-based techniques II I Virulence expression Rapid methods Clostridium botulinum Salmonella

  4. 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

  5. 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

  6. Virulence expression DNA Virulence factors protein mRNA Environmental factors

  7. 3000 2500 2000 BoNT/B (ng/ml) 1500 1000 500 0 V V V V 20h 5h 4h <2h C. botulinum type B 1.2 12 1.0 10 0.8 8 0.6 OD (620 nm) Relative expression 6 0.4 4 0.2 2 0.0 -0.2 0 0 5 10 15 20 25 30 35 40 45 50 55 Time (h)

  8. Control in foods

  9. * 27 ng*ml -1*OD-1 47 ng*ml -1*OD-1 * Effect of NaCl 14 5 12 4 10% CO2 0% NaCl 0 ppm NaNO2 10 3 8 OD (620 nm) Relative expression 6 2 4 1 2 0 0 0 10 20 30 40 Time (h) 14 5 12 4 10% CO2 2.5% NaCl 0 ppm NaNO2 10 3 8 OD (620 nm) Relative expression 6 2 4 1 2 0 0 0 10 20 30 40 Time (h)

  10. * 27 ng*ml -1*OD-1 30 ng*ml -1*OD-1 * Effect of NaNO2 14 5 12 4 10% CO2 0% NaCl 0 ppm NaNO2 10 3 8 OD (620 nm) Relative expression 6 2 4 1 2 0 0 0 10 20 30 40 Time (h) 14 5 12 4 10 10% CO2 0% NaCl 75 ppm NaNO2 3 8 Relative expression OD (620 nm) 6 2 4 1 2 0 0 0 10 20 30 40 50 60 Time (h)

  11. * * 27 ng*ml -1*OD-1 126 ng*ml -1*OD-1 Effect of CO2 14 5 12 4 10 10% CO2 0% NaCl 0 ppm NaNO2 3 8 OD (620 nm) 6 Relative expression 2 4 1 2 0 0 0 10 20 30 40 Time (h) 14 5 12 4 10 70% CO2 0% NaCl 0 ppm NaNO2 3 8 OD (620 nm) Relative expression 6 2 4 1 2 0 0 0 10 20 30 40 Time (h)

  12. Effect of CO2, NaCl and NaNO2 5 14 * 12 4 27 ng*ml -1*OD-1 10 10% CO2 0% NaCl 0 ppm NaNO2 5 14 3 8 OD (620 nm) Relative expression 12 6 2 4 4 10 1 2 3 8 0 0 OD (620 nm) 0 5 10 15 20 25 30 35 40 6 Relative expression 2 Time (h) 4 1 2 154 ng*ml -1*OD-1 * 0 0 70% CO2 1.25% NaCl 75 ppm NaNO2 0 20 40 60 80 100 120 Time (h)

  13. Traditional food preservatives (CO2, NaCl and NaNO2) stimulates the neurotoxin formation increasing the risk for food borne botulism LPD= m =b0+b13*[CO2]*[NaNO2]+b22*[NaCl]2+e log(RE)=b0+b11*[CO2]2+b22*[NaCl]2+e

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

  15. EU 6th FP. Area: Food Quality and Safety (5.4.4 Area: Traceability processes along the production chain) T5.4.4.1 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).

  16. II Rapid methods Salmonella

  17. Conventional analysis of Salmonella 25 g feed + 225 ml BPW Day 0 Pre-enrichment Day 1 Enrichment RVS Day 2 Day 3 Selective agar plates Day 4 Confirmation

  18. 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)

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

  20. 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

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

  22. PCR Inhibitor Mechanism Ref. Proteinases Degr. of Polym. Powell et al. 1994 IgG Binding to DNA Abu Al-Soud et al. 2000 Polysaccharides Binding to Polym. Monteiro et al. 1997 Lactoferrin Release of iron ions Abu Al-Soud, Rådström 2001 Calcium ions Competing with Mg2+ Bickley et al. 1996 Phenol Denatur. of Polym. Katcher, Schwartz 1994 EDTA Chelation of Mg2+ Rossen et al. 1992 Heparin Binding to DNA Satsangi et al. 1994 Taq DNA polymerase

  23. The importance of DNA polymerase and PCR facilitators in Diagnostic PCR

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

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

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

  27. Detection limit PCR Method

  28. Evaluation of the developed diagnostic PCR protocol on natural samples No of positive No of Sample type samples samples NMKL PCR Faeces and intestines 22 0 0 Fish meal 4 0 1 Maize gluten 1 0 1 Meat meal 1 0 0 Mixed feed 24 0 0 Rape meal 8 0 0 Soya 59 3 3 Soya, acidified 36 1 7 Total 155 4 12

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

  30. 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

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