Case study of contamination by Listeria
monocytogenes in raw goat milk cheese:
development of a quantitative risk
assessment model of the production chain
KORSAK Nicolas, DAUBE Georges &
DELHALLE Laurent
University of Liège, Faculty of Veterinary Medicine,
Department of Food Science, Sart-Tilman, B43bis, 4000 Liege, Belgium Tel: +32 4 366 40 40, fax: +32 4 366 40 44
Plan of the presentation
• Introduction
– Risk analysis and Listeria monocytogenes
– Milk products and Listeria monocytogenes
• Contamination of goat milk products
– Anamnestic elements
– Building-up of a risk assessment model
– Main results of the model
– What-if scenarios
Risk analysis
Codex Alimentarius (WTO)
Risk Assessment
Assessment of likelihood of an event and the severity of effects on human health, following exposure.
Risk
assessment
(
EFSA
)
Risk
management
(
executive
)
Risk
Communication
(
EFSA
)
1. Hazard Identification
3. Hazard Characterization
2. Exposure assessment
4. Risk Characterization
Risk Analysis
Listeria monocytogenes
• Ubiquitous, short rod, catalase-neg,
Gram pos.
• Special motility at 25 °C
• Intracellular pathogen
• High-risk population : > 60y,
immuno-compromised, pregnant women
• Long incubation period : 3 to 60d
• Mandatory surveillance in EU
Listeria monocytogenes:
reported incidence in the EU in 2009
EU summary report on trends and sources of zoonoses an d zoonotic agents and food-borne outbreaks 2009
2.2 Zoonoses and item-specific summaries
The importance of a zoonosis as a human infection is not dependent on incidence in the population alone. The severity of the disease and case fatality are also important factors affecting the relevance of the disease.
For instance, despite the relatively low number of cases caused by VTEC, Listeria, Echinococcus, Trichinella
and Lyssavirus (rabies), compared to the number of human campylobacteriosis and salmonellosis cases,
these infections are considered important due to the severity of the illness and higher case fatality rate.
Figure SU1. Reported notifica tion rates of zoonoses in confir med human cases in EU, 2009
Note: Total number of confirmed cases is indicated at the end each column. * Data from 2008 ( 1 ) ( 1 1 5 ) ( 4 0 1 ) ( 7 4 8 ) ( 7 9 0 ) ( 1 , 6 4 5 ) ( 1 , 9 8 7 ) ( 1 , 2 5 9 ) ( 3 , 5 7 3 ) ( 7 , 5 9 5 ) ( 1 0 8 , 6 1 4 ) ( 1 9 8 , 2 5 2 ) 0 5 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 5 0 R a b i e s T u b e r c u l o s i s c a u s e d b y M . b o v i s * B r u c e l l o s i s T r i c h i n e l l o s i s E c h i n o c o c c o s i s L i s t e r i o s i s Q f e v e r T o x o p l a s m o s i s V T E C Y e r s i n i o s i s S a l m o n e l l o s i s C a m p y l o b a c t e r i o s i s
No tif ication rate p er 100,000 p o p ulatio n
Zo on os es 5 EFSA
Listeria monocytogenes :
reported incidence in Belgium
2006 2007 2008 2009 52 54 56 58 60 62 64 66 68
Isolated strains in Belgium in 2008:
clustering by age and serovar
1-5 d 6d - 1y 2 - 10y 11 - 20y 31 - 40y 41 - 50y 51 - 60y 61 - 70y 71 - 80y 81 - 90y 0 2 4 6 8 10 12 14 16 18 1/2a 1/2b 1/2c 4b serogroup 4 total
Listeria monocytogenes and milk products
• % of outbreaks
implicating milk
products: from 2 to
6 %
(De Buyser et al.,
2001)
• % of outbreaks
implicating milk and
milk products
(France,
1988-1997, n=177)
:
Salmonella; 19.18% S. Aureus; 58.74% C. Perfringens; 1.70% other agents; 5.09% unknow aetiology; 15.28%Listeriosis outbreaks linked to milk products (1/2)
year
Country of
origin
Milk product
Type of milk
Cases (deaths)
1983-1987
Switzerland
Soft cheese (Vacherin
Mont d’Or)
Unpasteurized
122 (33)
1983
USA
Milk
Pasteurized
49 (14)
1985
USA
Mexican-style soft
cheese
pasteurized
milk
142 (48)
1987
USA
Butter
unspecified
11
1989
Luxembourg
Camembert
unspecified
2
1989-1990
Denmark
Hard and blue cheese
unspecified
26 (6)
Listeriosis outbreaks linked to milk products (2/2)
year
Country of
origin
Milk product
Type of milk
Cases (deaths)
1995
France
Brie de Meaux
raw
37 (11)
1997
France
Livarot, Pont-l’évêque
raw
14
1988-1999
Finland
Butter
Pasteurized
18 (4)
2000
USA
Mexican-style soft
cheese
Raw milk
13
2001
Sweden
Soft cheese
unspecified
33
Case of listeria contamination
in cheese made raw goat milk – anamnestic elements
• Herd: 350 goats, Alpine breed
• Births from October to March
• 2.3 kids / female
• Culling after 5 lactations periods
(3.11 l/goat/day)
• Feed: hay and herb silage
• Historic of clinical animal listeriosis in
the herd (nervous signs, abortions…)
Herd of goats
3. Mixing of milk
4. Adjunction of
starter to milk
5. Adjunction of
rennet to milk
6. Draining off
of curdles
7. Salting 1
and 2
8. Cooled
storage and
wrapping
1. Storage of the
evening milk
2. Storage of the
morning milk
Process of cheese making
pH
a
wSalt content (%)
Fresh goat milk
6.63
1
0
Goat milk with adjunction of ferment and rennet (24h)
6.58
1
0
Curdles (24h) not drained off
4.41
0.987
0
birth of the 22 Feb 04 no L. monocytogenes in the products 20 March 05 Dropping of the “shedder” goat March 05 11 July 05
New analyses upon request
in an external laboratory.
Lm in the products (in 25g). The herd is then blocked and the sale. The
farmer himself performs the recall of products.
12 July 05 Numbers of
Lm in different
final products.
fresh cheese not ripened: 4.3.10³ cfu/g ;
goat cheese ripened: 6.5.10³ cfu/g
goat cheese ripened and coated with charcoal: 5.1.10³ cfu/g
Time line: case of Lm contamination (2/3)
pools of 20 samples of milk from goats directly after milking. 15-18 July 05 Transfer of the goat to the faculty veterinary medicine.19 July 05 End July 05
Re-start of the fabrication process with mandatory analyses 14-22 Sept July 05 Milk samples taken on the hospitalized goat 4.3.10² cfu L. monocytogenes/ml for
the right part. Absence of
L. monocytogenes in 25 ml
for the left part of the mammary gland Only 1 pool positive + only one clearly excreting goat in the herd. No clinical signs observed 5 samples on the 1st batch of the final
products, then 1 sample each [for the next] 5 batches of final products – both ripened goat
Time line: case of Lm contamination (3/3)
Strain sent to the (NRL) in Brussels. Meanwhile, another strain (from the 4 Oct 05 Serotyping, antibiogram and PFGE on both strains and human strains 11 Oct – 3 Nov 05 Sept-Dec 05 Request of surveillance of final products in the herd at a frequency of 1analysis/15 days of production
From January 06
Request of surveillance of final products in the herd at a frequency of 1 analysis/6 months of production (with the
following parameters: Listeria, Salmonella, E. coli and coagulase – positive staphylococci). confirmation of serotype 1/2a, with a β-hemolysis no L. monocytogenes
found in the products no L. monocytogenes found in the products
To sum up (1/2)
• Analyses in cheese
– fresh cheese not ripened: 4.3.10³ Lm (3.63 log) cfu/g
– goat cheese ripened: 6.5.10³ Lm (3.81 log) cfu/g
– goat cheese ripened and coated with charcoal: 5.1.10³ Lm
(3.7 log) cfu/g
• Analyses of milk from the hospitalized goat
– 4.3.10² cfu Lm/ml for the right part
– Absence of Lm in 25 ml for the left part of the mammary
gland
To sum up (2/2)
• Strains isolated from milk and
cheese
– Sensitive to the 10 antimicrobials
tested
– Sensitive to Arsenic and Cadmium
– Serotype 1/2a with a β-hemolysis
– PFGE : Pulsovar A, no matching
with the 40 human strains
collected at the NRL in 2005
(16
strains of serovar 1/2a, of which 7
sensitive to As and Cd)
C h e e se C h e e se m ilk m ilkBuilding-up of the model
• Principles of the modular process risk model
– On the 8 modules: (1) storage of the evening milk, (2) storage of
the morning milk, (3) mixing of the morning and evening milk,
(4) adjunction of the starter to the milk, (5) adjunction of rennet
to the milk, (6) draining off of curds, (7) salting at ambient
temperature and (8) cooled storage.
– On fresh unripened
cheese
• Characterization
1
L. monocytogenes / matrices
characteristics Parameter Description Values / Distributions Units References
L. monocytogenes characteristics
Tmin Minimum temperature for growth (°C) N(-1.8;0.72) °C
(Augustin, 1999; Ellouze and Augustin, 2010; ICMSF, 1996)
Tref Reference temperature for growth (°C) N(38.2;0.76) °C
pHmin Minimum pH for growth N(4.19;0.12) pH units
pHmax Maximum pH for growth 9.61 pH units
aw min Minimum aw for growth N(0.922;0.009) aw units
Growth characteristics in milk
µref.milk Reference growth rate in milk N(0.75; 0.13)
h-1 (Augustin et al., 2005; Rosenow and Marth., 1987)
Nmax Maximum concentration in milk 7 log cfu
pHref.milk Reference pH in milk 6.60 pH units
pHmilk pH of the milk 6.63 pH units (measured)
aw milk aw of the milk 1 aw units (measured)
Growth characteristics in cheese
µref.cheese Reference growth rate in cheese NT(0.21;0.19;0.02) h-1 (Rosenow et al., 1987)
Nmax Maximum concentration in cheese 7 log cfu
pHref.cheese Reference pH in cheese 4.80 pH units
pHcheese pH of the cheese 4.28 pH units (measured)
Baseline results of the exposure assessment and the risk characterization modules
1
Modules Item Acronym Percentiles Unit
5th 50th 95th
Milking Concentration in a tank before storage over night of the evening milk
Ctank evening milk -5 0 0.47 log cfu/ml
Storage of the evening milk
Concentration in the tank after storage over night of the evening
milk Ctank night stor -3.3 2.0 2.8 log cfu/ml Adjunction of
ferment and rennet
Concentration before draining off
the curdles Cferment,rennet -2.1 3.3 4.8 log cfu/ml
Draining off the
curds Number of cheese L. monocytogenes per Ncheese 0.2 5.7 7.4 log cfu/cheese
Cooled storage and wrapping
Concentration of
L. monocytogenes in a serving of
cheese Cserving -1.8 3.7 5.4 log cfu/g Number of L. monocytogenes per
serving of cheese Nserving -0.1 5.4 7.1 log cfu/serving
Human effect
Risk of human listeriosis (normal
population) Rhealthy 0 0 0
Risk of human listeriosis
