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, NorwaySigrun Gumundsdottir, Icelandic Fisheries Lab (IFL), IcelandMarja-Liisa Hänninen, Helsinki University, Finland

http//www.CampyFood.org

CAMPYFOOD

A Molecular Safety Approach for Campylobacter

ActivitiesWe 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

CAMPYFOOD

Clostridium botulinum SalmonellaClostridium botulinum Salmonella

Virulence expression Rapid methods

I II

Food safety will be increased by the application of molecular-based

techniques

http//www.CampyFood.org

CAMPYFOOD

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 neurotoxinon chromosome: types A, B, E, Fon bacteriophage: types C, Don plasmid: type G

Clostridium Clostridium botulinumbotulinum

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

proteinVirulence

factorsmRNA

DNA

Virulence expression

Environmentalfactors

C. botulinum type B

Time (h)

0 5 10 15 20 25 30 35 40 45 50 55

OD

(62

0 nm

)

-0.2

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Re

lative

ex

pres

sio

n

0

2

4

6

8

10

12

Bo

NT

/B (n

g/m

l)

0

500

1000

1500

2000

2500

3000

<2h4h5h20h

Control in Control in foodsfoods

Preservative Effect CO2 Inhibition of growth,

Inhibition of outgrowth of spores

NaCl Inhibition of growth,

Inhibition of outgrowth of spores

NaNO2 Inhibition of growth

Effect of NaClEffect of NaCl

10% CO2

0% NaCl0 ppm NaNO2

10% CO2

2.5% NaCl0 ppm NaNO2

Time (h)

0 10 20 30 40

Relative exp

ression

0

2

4

6

8

10

12

14

OD

(620 nm

)0

1

2

3

4

5

Time (h)

0 10 20 30 40

Relative exp

ression

0

2

4

6

8

10

12

14

OD

(620 nm

)

0

1

2

3

4

5

27 ng*ml -1*OD-1*

47 ng*ml -1*OD-1

*

Effect of NaNOEffect of NaNO22

10% CO2

0% NaCl0 ppm NaNO2

10% CO2

0% NaCl75 ppm NaNO2

Time (h)

0 10 20 30 40

Relative exp

ression

0

2

4

6

8

10

12

14

OD

(620 nm

)

0

1

2

3

4

5

Time (h)

0 10 20 30 40 50 60

Relative exp

ression

0

2

4

6

8

10

12

14

OD

(620 nm

)

0

1

2

3

4

5

27 ng*ml -1*OD-1*

30 ng*ml -1*OD-1

*

Effect of CO2

10% CO2

0% NaCl0 ppm NaNO2

70% CO2

0% NaCl0 ppm NaNO2

Relative exp

ression

Time (h)

0 10 20 30 400

2

4

6

8

10

12

14

OD

(620 nm

)

0

1

2

3

4

5Time (h)

0 10 20 30 40

Relative exp

ression

0

2

4

6

8

10

12

14

OD

(620 nm

)

0

1

2

3

4

5

27 ng*ml -1*OD-1*

126 ng*ml -1*OD-

1

*

Effect of CO2, NaCl and NaNO2

Time (h)

0 5 10 15 20 25 30 35 40

OD

(620 nm

)

0

1

2

3

4

5

Rela

tive e

xpress

ion

0

2

4

6

8

10

12

14

10% CO2

0% NaCl0 ppm NaNO2

27 ng*ml -1*OD-1*

Time (h)

0 20 40 60 80 100 120

OD

(620 nm

)

0

1

2

3

4

5

Rela

tive e

xpress

ion

0

2

4

6

8

10

12

14

70% CO2

1.25% NaCl75 ppm NaNO2

154 ng*ml -1*OD-1

*

LPD= m =b0+b13*[CO2]*[NaNO2]+b22*[NaCl]2+e

log(RE)=b0+b11*[CO2]2+b22*[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 expressiona 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)

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).

SalmonellaSalmonella

Rapid methods

II

Day 0

Day 1

Day 3

Day 4

25 g feed + 225 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

QuantitativeSimplicity

-Specificity

RapidLaborious

Rapid Methods1. Cell counting methods

Flow cytometryDirect epifluorescent microscopy

2. Modified and automated conventional methodsSpiral plater

DipslidesChromogenic/fluorogenic media

3. Impedimetry4. Bioluminescence5. Immunological methods

ELISA

Immunocapture

6. Nucleic acid-based assaysHybridisation

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 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

The importance of DNA polymerase and PCR facilitators in

Diagnostic PCR

PCR amplification mixture1

Feed sample

Taq2 rTth, BSA3 Tth4 Tth, glycerol5

1. Fish meal - - + + 2. Rapeseed meal - - - - 3. Soybean - - + + 4. Soybean (acidified)

- - - +

5. Oats + + + + 6. Soybean meal - - + + 7. Wheat + + + + 8. Meat meal + + + + 9. Piglett pellets - + + + 10. Rapeseed - - - - 11. Palm kernel - - + + 12. Corn pellets - - - + 13. Corn glutelin - - + + 14. Drank - - (U.S) + 15. Whey - - - - 1 - : No amplification, + : Amplification of invA , U.S.: unspecific amplification, ND: Not determined. 2 Taq DNA polymerase (Roche Diagnostics Gmbh) 3 rTth DNA polymerase (Applied Biosystems), Facilitator:BSA (Bovine Serum Albumin) 4 Tth DNA polymerase (Roche Diagnostics Gmbh) 5 Tth DNA polymerase (Roche Diagnostics Gmbh), Facilitator: glycerol

Diagnostic PCR

1. Sampling

2. Sample preparation

3. DNA amplification

4. Detection of PCR products

DNA polymerases

PCR facilitatorsP

re-P

CR

Pro

cessin

g

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 h @ 37ºC (isolate obtained!)

• Samples withdrawn after shaking

• No DNA extr. or cell lysis!

• Tth DNA

polymerase

• Gel electrophoresis

Enrichment PCR method

0

0.2

0.4

0.6

0.8

1

-6 -4 -2 0 2 4 6 8 10 12

Salmonella Senftenberg (log CFU/25 g feed)

Det

ectio

n pr

obab

ility

A, B C

PCR Method

Detection limit

No of positive

samples Sample type No of

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

Evaluation of the developed diagnostic PCR protocol on natural

samples

Amplification

Molecular Methods

+Specificity

RapidAutomation

-Detection Limit

RobustnessAcceptance

Immunological methodsPolymerase Chain Reaction

Acknowledgements

Waleed Abu Al-Soud, 2000Ingrid Artin, ---

Halfdan Grage, 2002Oskar Hagberg, 2005

Rickard Knutsson, 2001Charlotta Löfström, 2005

Maria Lövenklev, 2003Petra Wolffs, 2004