presentation of results from wp2 dealing with egg and … wp2 egg… · presentation of results...

Department of Veterinary disease biology

Presentation of results from WP2 dealing with egg and egg products

John Elmerdahl Olsen

University of Copenhagen

Products considered in research


Salmonellaa. Number two zoonosis in Europe

Dias 2

a. Number two zoonosis in Europeb. 40 % of cases egg related


15-09-2013

Dias 3

� Estimation of Salmonella prevalence on eggshells in seropositive flocks

11+/28 : 39.3 % 44+/4200 : 1.05 % IC: 0.78 – 1.14

0.6 % (1/150) – 8.6 % (13/150)

Results belong to ANSES, France

Day of trial post infection versus number of

laying hens versus number of eggs

20

25

30

35

Results confirmed by RT-PCR

0

5

10

15

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Day of trial post infection

number of laying hens number of eggs number of positive egg shell

Data from VetFac – to be repeated

Impact of egg pooling on Salmonella detection

S. EnteritidisConfirmationPFGE

*

+

+

+

+

+

+

1+9

1+8

1+7

1+6

1+5

1+4

S. Enteritidis

DETECTION

ISO 6579

PFGE

*15 repeats per pooling were tested

Results belong to University of Bologna

qPCR

Eggshell pooling – Results ISO METHOD

Contamination level

N°°°° eggs per pool

N°°°° positive pools*

N°°°° eggs tested

neggs-95%

10 cfu/pool

10 6 150

9 2 135

8 2 120

7 0 105

6 1 90

5 0 75

10 5 150

16 pools of 10 eggs are needed to be 95 % sure not to release

cfu/egg

*15 repeats per pooling were tested

100 cfu/pool

10 5 150

9 7 135

8 7 120

7 5 105

6 7 90

5 7 75

1000 cfu/pool

10 12 150

9 11 135

8 13 120

7 13 105

6 12 90

5 12 75Data belongs to Universities of Bologna & Cordoba

be 95 % sure not to release contaminated eggs

cfu/egg

cfu/egg

How to improve sampling plans ?

Location of

sampling

Numberof

samples

Frequency of sampling

Sample

unit

Analytical method

x x x x

�Increasing number of samples?�Increasing frequency of sampling or sample size?�Using a validated alternative method with highersensitivity and shorter time of analysis?

What if we use Real-Time PCR?

Real-Time PCR

DNA Extraction

Enrichment Results in 1-2 daysExtraction

Master Mix preparation and amplification

1-2 days

Higher sensitivity

qPCR - Salmonella on table eggs

20

22

24

26

28

30

32

34

36

38

40

Ct

va

lue

Ct

Lineare (Ct)

Inoculated CFU/eggshell

N°°°° of tested egg

N°°°° of positive with ISO method

N of positive with Real-time PcR

104-105 10 10 10

Quantification by qPCR

Detection

20

0 1 2 3 4 5

Inoculated Salmonella (log10 CFU/g of eggshell)

10 10 10 10 10

103-104 10 10 10

102-103 10 10 10

101-102 10 8 10

100-101 10 6 10

UniBo

171819202122232425262728293031

cT v

alu

e

standard

stress

Quantification of S. Tennessee in pasteurized egg yolk


101112131415161718

0.00 1.00 2.00 3.00 4.00 5.00 6.00

Lg CFU/ml

stress

Standart curves produced of ten-fold serial dilutions of initial CFU/mlpasteurized egg yolk. Stressed cells – 48 °C for 30 min. DNA wasextracted using PrepMan Ultra after 8 hours of pre-enrichment.

UCPH

Results quantification

Comparison between qPCR and MPN quantification

R² = 0,770

1,00

1,50

2,00

2,50

3,00

3,50

4,00

MP

N (

log

10

MP

N/e

gg

she

ll)

Serie1

Lineare (Serie1)

6

0,00

0,50

0,00 1,00 2,00 3,00 4,00 5,00

MP

N (

log

10

MP

N/e

gg

she

ll)

Inoculated Salmonella (log10 CFU/eggshell)

R² = 0,916

0

1

2

3

4

5

0 2 4 6

qP

CR

(lo

g1

0 C

FU

/eg

gsh

ell

)

Inoculated Salmonella (log10 CFU/eggshell)

qPCR quantification

log CFU

Lineare ( qPCR

quantification log

CFU)

UniBo

MonteCarlo simulation results on the number of pooled egg samplesto be tested to detect Salmonella with 95% certainty by Real-TimePCR and ISO 6579

n. of eggshells per

pool

n. of pools to be sampled

ISO 6579 Real-Time PCR

Detection of Salmonella spp. on pools of eggshells

eggshells per pool ISO 6579 Real-Time PCR

10 16 169 54 88 56 127 995 9826 125 565 929 33

CONCLUSIONS

Real-Time PCR for detection of Salmonella spp. ineggs is a good alternative to ISO 6579

Because�Results in 1-2 days

How?

�Results in 1-2 days� High sensitivity and specificity� Confirmatory Tests not required

Following ISO 22119 recommendations


15-09-2013

Dias 15

In silico sampling of powdered eggs –

1st model – assumption: localised contamination

c

1 - c

Bruxelles - 17-18 October 2012

w-sample = weight of the sample (g)w-batch = weight of the batch (g)c = proportion of the contaminated fraction (0-1)K-batch = microbial cells in the batch (cfu/batch)K* = microbial concentration expected in collected samples (cfu/sample)N = number of samples taken from the batchNc = number of samples drawn from the contaminated part of the batch*= Nc follows a binomial distribution

Sampling simulation: Powdered egg

Sample size Lot weight

17

Number of samples

collected

Contaminated part of

the lot

Concentration in

contaminated

samples

Probability of making a mistake

n w (g) p N (g)

c(cfu/g

) Pacc Prej

20 50 0,01 10000 17 0,8179 0,1821

20 50 0,05 10000 17 0,4341 0,5659

20 50 0,1 10000 17 0,3075 0,6925

Practical example. Powdered egg

18

20 50 0,01 1000 17 0,8179 0,1821

20 50 0,01 100000 17 0,8915 0,1085

50 20 0,01 10000 17 0,6153 0,3847

20 50 0,01 10000 776 0,8179 0,1821

20 50 0,05 10000 776 0,3585 0,6415

20 50 0,1 10000 776 0,1216 0,8784

20 50 0,01 1000 776 0,8179 0,1821

20 50 0,01 100000 776 0,8179 0,1821

50 20 0,01 10000 776 0,605 0,395

a) Low contamination vs high contamination (17 – 776 cfu/g)

Sampling is more effective as c increases, since Pacc decreases.

b) Increasing proportion of the contaminated part of the lot (from 0.01 to 0.1)

Practical example. Powdered egg

19

Sampling is more effective as p increases, especially from 0.01 to 0.05

c) Lot size effect (1000, 10000, 100000 g)

No significant effect on Pacc

d) Combining number of samples and sample size (n and w)

Increasing n and decreasing w is more effective to detect positives, regardless of

the microbial contamination


15-09-2013

Dias 20

Commission Regulation (EC) No 2073/2005 of 15 November 2005on microbiological criteria for foodstuffs

Food safety Criteria

Compliance criteria

Food category Micro-organism

Sampling plan

Limits Analytical reference method

Stage where the criterion applies

n c

Meat and egg products*

Salmonella 5 0 Absence in 25 or 10 g

EN/ISO 6579 Retail

*Food categories 1,4; 1,5; 1,6; 1,7; 1,8; 1,9; 1,14**Food category 1,2**Food category 1,2

However…

• 0.3 % pasteurized egg products were positive for Salmonella in 2011 in USA (FSIS).

• 1.7 % pasteurized egg products were positive in Japan (Hara-Kudo & Takatory, 2009)

Outbreaks in Europe include


Outbreaks in Europe include

a) from pasteurized egg white and yolk used for chocolate mousse, pasteurized egg white for desert and salmon mousse in 2007

b) from pasteurized egg white (ready to drink) 2012

Dias 22

Growth and survival of Salmonella

strains in egg contents and pasteurized products at 20°°°°C

20/04-2012Department of Veterinary disease biology

Main Effects Plot for µ

0,07

0,12

0,17

0,22

0,27

µ

Results S. Enteritidis and growth in pasteurized egg

-0,03

0,02

0,07

T°cSel

LyzozymepH

Results belong to Anses

Temperature Salt Lysozyme pH

Main Effects Plot for µ

0,8

S. E. growth in sweet matrix

0

0,2

0,4

0,6

µ

T°Csucre

lysozymepHTemperature Sucker Lysozyme pH

Inoculum size


S. EnteritidisPasteurized liquid egg. 24°C

Dias 26

Addapted from Sakha & Fujikawa, 2012. Biocontrol Science.

liquid egg. 24°C

Growth of S. Enteritidis for DNA microarray in whole egg and LB

Dias 27

Log10 CFU/m

l

6

7

8

9

10

WE1 SE 6/12-12

WE2 SE 06/12-12

WE3 SE 06/12-12

LB SE 6/12-12

LB SE 06/12-12


Time, h

3

4

5

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38

LB SE 06/12-12

LB SE 06/12-12

Timepoint of RNA extraction

Genes upregulated in whole egg compared to LB

Dias 28



Thank you for your attention

Dias 29

presentation of results from wp2 dealing with egg and … wp2 egg… · presentation of results...

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