Koret School of Veterinary Medicine, 8 April 2008 1
Salmonella in chickens, their houses, and consumers: excerpts from the Danish
farm-to-fork chain
Kim Oren Gradel Department of Infectious Diseases
Aalborg HospitalAarhus University Hospital
Aalborg, Denmark
Koret School of Veterinary Medicine, 8 April 2008 2
Main infection sources in humans
Danish Zoonosis Centre: Annual report on Zoonoses in Denmark 2006.
Koret School of Veterinary Medicine, 8 April 2008 3
Table of contents
• Salmonella control programmes in chickens
• Salmonella in chicken houses• Salmonella in humans
Koret School of Veterinary Medicine, 8 April 2008 4
Broiler production
Import of day-old chicks
Central rearing
Parent flocks
Hatchery
Broiler farms
Feed millsFeed mills:
All feed heat treated to 81 oC as from the late 80’ies
Broiler farms: Sampled at 3 weeks of age as from 1989
Central rearing: Zoonosis directive samples as from 1994
Parent flocks: Zoonosis directive samples as from 1994
Central rearing: Salmonella control programme as from 1996
Parent flocks: Salmonella control programme as from 1996
Hatchery: Salmonella control programme as from 1996
Koret School of Veterinary Medicine, 8 April 2008 5
Table egg productionImport of day-old chicks
Central rearing
Parent flocks
Hatchery
Commercial rearing
Feed mills
Table egg houses
Feed mills:All feed heat
treated to 81 oC as from the late 80’ies
Table egg houses: Voluntary programme (cloacal swabs before slaughter) as from 1992
Central rearing: Zoonosis directive samples (1994)
Parent flocks: Zoonosis directive samples (1994)
Central rearing: Salmonella control programme (1996)
Parent flocks: Salmonella control programme (1996)
Hatchery: Salmonella control programme (1996)
Commercial rearing: Salmonella control programme (1996)
Table egg houses: Salmonella control programme (1996)
Koret School of Veterinary Medicine, 8 April 2008 6
Two DVMs employed at the Danish Veterinary Laboratory in 1996
• Principal tasks:– Overview of Salmonella situation (databases)– Tracking infection sources (databases,
enquiries, visits to farms) – Advising Salmonella infected farmers (visits to
farms)
Koret School of Veterinary Medicine, 8 April 2008 7
Combating Salmonella in poultry
• Reduction principles:– Vaccination (not allowed in DK)– Antibiotics (should not be an option)– Competitive exclusion (variable results)
• Elimination principles (Scandinavia):– Culling of infected flocks– Cleaning and disinfection of infected premises
• Which is why: you’ll hear little about Salmonella in chickens, but more about Salmonella in chicken houses!
Koret School of Veterinary Medicine, 8 April 2008 8
Table of contents
• Salmonella control programmes in chickens
• Salmonella in chicken houses• Salmonella in humans
Koret School of Veterinary Medicine, 8 April 2008 9
Salmonella sources in broiler flocks (1/11/96-31/10/99)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
No
v-9
6
De
c-9
6
Jan
-97
Fe
b-9
7
Ma
r-9
7
Ap
r-9
7
Ma
y-9
7
Jun
-97
Jul-
97
Au
g-9
7
Se
p-9
7
Oct
-97
No
v-9
7
De
c-9
7
Jan
-98
Fe
b-9
8
Ma
r-9
8
Ap
r-9
8
Ma
y-9
8
Jun
-98
Jul-
98
Au
g-9
8
Se
p-9
8
Oct
-98
No
v-9
8
De
c-9
8
Jan
-99
Fe
b-9
9
Ma
r-9
9
Ap
r-9
9
Ma
y-9
9
Jun
-99
Jul-
99
Au
g-9
9
Se
p-9
9
Oct
-99
Month/year
No
. o
f fl
ocks
Unknown source
Other animals
Exotic, not PI
Ent/Typ, unknown source
Hatchery
PI-houses
Persistently Salmonella-infected broiler houses
Gradel KO, unpublished results
Koret School of Veterinary Medicine, 8 April 2008 10
Questionnaire-based field study
Koret School of Veterinary Medicine, 8 April 2008 11
Questionnaire-based field study: overview
Parent flock infected with
S. Enteritidis, PT8 (SE8)
Parent flock infected with
S. Typhimurium, PT66 (ST66)
May-June 1997:First timeoccurrenceof these twoSalmonellatypes in theDanish broilersector!
Hatchery
51 farms, comprising 84 broiler houses,
had SE8 and/or ST66 detected
Koret School of Veterinary Medicine, 8 April 2008 12
Questionnaire-based field study: Methods
• 78 broiler houses (93%) on 42 farms (82%) were visited
• Each visit:– assessment of rodents, beetles, equipment, cleaning standard– the farmer was asked about routine procedures
• Broiler houses with Salmonella in ≥2 crops (multiple-infection houses) were compared with broiler houses with Salmonella in 1 crop (single-infection houses)
Koret School of Veterinary Medicine, 8 April 2008 13
Main results: factors that decreased the risk of multiple-infection houses
• ST66 rather than SE8• Lower number of positive samples (1-5) in the
first crop• Antiseptic soap and water in the ante-room • Equipment for removal of dead birds did not
cross the hygiene barriers• Gravel alongside the broiler house• Systematic check of indoor rodent-bait depots• Combined surface and pulse fog disinfection (in
contrast to separate use of each of these)
Koret School of Veterinary Medicine, 8 April 2008 14
Interpretations and practical implications
• Many variables!• Two interpretations:
– Significant factors have a specific impact on Salmonella persistence
– Significant factors illustrate the advantage of general order and system
Koret School of Veterinary Medicine, 8 April 2008 15
Disinfection projects under the Salmonella control programme
• 2000 (three years after the implementation of the Salmonella control programme): money allocated to research
• Focus: persistently Salmonella infected premises
• Two projects:– “Heat disinfection project”– “Chemical disinfection project”
Koret School of Veterinary Medicine, 8 April 2008 16
Background for disinfectant studies
• Most reported disinfection tests are suspension tests:– Impressive results (best case scenarios)– Therefore widely used by the disinfectant
companies!– Extrapolation to realistic conditions is very
difficult
• Very little research on disinfection of animal buildings
Koret School of Veterinary Medicine, 8 April 2008 17
Heat laboratory tests
Koret School of Veterinary Medicine, 8 April 2008 18
Heat laboratory tests: aim
• Finding a time-temperature-humidity gold standard that eliminates Salmonella and relevant indicator bacteria under worst-case scenario conditions (i.e., poorly cleaned poultry houses).
Koret School of Veterinary Medicine, 8 April 2008 19
Heat laboratory tests: factors
Factor
Salmonella or Escherichia coli (naturally occurring in faeces)
Organic matter (feed, chicken faeces)
Drying before heating (yes, no)
Humidity at heating (16-30%, 100%)
Heating temperature (50, 55, 60, 65, 70 oC)
Koret School of Veterinary Medicine, 8 April 2008 20
Heat laboratory tests: results
LOW HUMIDITY HIGH HUMIDITY TE OM DR 10d 0 24 48 72 10d 0 24 48 72 50 Faec Yes - + + + + + - - - - 50 Faec Yes - + + - - 50 Faec Yes + - - - - 50 Faec No + - - - - + - - - - 50 Feed Yes + + + + + + + + + + 50 Feed Yes + + + + + 50 Feed Yes + + + + - 50 Feed No + + + + + + + + - - 60 Faec Yes + + + + + + - - - - 60 Faec Yes - - + + + + - - - - 60 Faec No + - - - - + - - - - 60 Feed Yes + + + + + + + - - - 60 Feed Yes + + + + + + + - - - 60 Feed No + + + + + + - - - - 70 Faec Yes + + + + + + - - - - 70 Faec Yes + - - - - 70 Faec No - - - - - + - - - - 70 Feed Yes + + + + + + - - - - 70 Feed Yes + - - - - 70 Feed No + + + + + + - - - -
Koret School of Veterinary Medicine, 8 April 2008 21
Heat disinfection tests: correlations between Salmonella and E. coli
Salmonella on Rambach agar?
Yes No
E. Coli on MacConkey agar?
Pure culture
With non-Salmonella
Non-Salmonella
Sterile
Yes 8 90 10 8
No 40 8 3 222
Koret School of Veterinary Medicine, 8 April 2008 22
Heat laboratory tests: gold standard for field studies
• No bacteria were detected at 60 oC and 100% RH after 24 hours of heating
Koret School of Veterinary Medicine, 8 April 2008 23
Heat field tests
Koret School of Veterinary Medicine, 8 April 2008 24
Heat field tests: principles I
Koret School of Veterinary Medicine, 8 April 2008 25
Heat field tests: principles II
Heat field tests: sampling
• 300 Salmonella samples taken before and after heat treatment
• Challenge samples in each house at the 12 sites where temperature was logged every 5 minutes:– Feed: Enterococcus faecalis, E. coli– Faeces: Enterococci, E. coli
Koret School of Veterinary Medicine, 8 April 2008 26
Koret School of Veterinary Medicine, 8 April 2008 27
Heat field tests: overview of farms, houses and treatments
Farm House House type TreatmentChallenge
samples
A A1 Barn Steam/no formaldehyde Yes
A2 Battery Steam/no formaldehyde Yes
B B1 Barn Steam/ formaldehyde Yes
B2 Barn Pulse No
B3 Barn Surface No
C C1 Battery Steam/ formaldehyde Yes
D D1 Battery Steam/ formaldehyde Yes
E E1 Battery Steam/ formaldehyde Yes
Koret School of Veterinary Medicine, 8 April 2008 28
Heat field tests: results for Salmonella samples
House TreatmentSalmonella Non-sterile
RR SBef Aft Bef Aft
A1 Steam/no F 36/287 6/288 278/287 94/288 3.0 A
A2 Steam/no F 65/302 0/303 284/302 114/303 2.5 A
B1 Steam/F 0/100 0/102 41/100 3/102 14.1 BC
B2 Pulse 6/100 0/96 58/100 33/96 1.7 A
B3 Surface 5/100 0/100 68/100 8/100 8.5 B
C1 Steam/F 37/298 0/308 180/298 2/308 92.9 C
D1 Steam/F 1/289 0/290 78/289 1/290 79.4 C
E1 Steam/F 40/308 0/308 224/308 9/308 25.1 BC
Koret School of Veterinary Medicine, 8 April 2008 29
Heat field tests: conclusions
• In tight houses: 60 oC and 100% RH achieved minimum 10 cm above floor level within one hour and was easily maintained
• 60 oC and 100% RH during 24 hours: effective in eliminating Salmonella and putative indicator bacteria
• 30 ppm formaldehyde seemed to lower the lethal temperature by 2-5 oC
• Today, 5-6 years later, Salmonella has not been detected in any of the heat treated layer houses
Koret School of Veterinary Medicine, 8 April 2008 30
Heat field tests: implications
• Steam and formaldehyde implemented in other places:– Visit by DVMs from Agricola Tre Valli, Italy (Europes 3rd biggest
poultry company)– Building a steam generator in Japan
• Enquiries from Spain and Sweden• Economically feasible:
– Farmers’ expenditures due to persistent Salmonella infections: $100,000-200,000 or more
– The owner of Denmark’s biggest table layer farm went bankrupt after spending $2,200,000 on Salmonella before heat treatment was documented
– Cost of heat treating poultry house: $10,000
Koret School of Veterinary Medicine, 8 April 2008 31
Resistance to disinfectants
Koret School of Veterinary Medicine, 8 April 2008 32
Resistance to disinfectants
• Hypothesis:There is an association between persistence of
Salmonella in poultry houses and the common use of a few types of disinfectants in these
• Aims:– To see if minimum inhibitory concentrations (MICs)
against five commonly used disinfectants could be related to Salmonella persistence or use of disinfectants in Danish broiler houses.
– To see if resistance against the five disinfectants could be introduced and maintained in the laboratory
Koret School of Veterinary Medicine, 8 April 2008 33
Resistance to disinfectants:adaptation and de-adaptation
MIC-testsMIC-tests
Koret School of Veterinary Medicine, 8 April 2008 34
MICs for isolates from Danish chicken houses
”Non-persistent” serotypes
”Persistent” serotypes
”Danish” disinfectants ”English” disinfectants
Higher MICs
Koret School of Veterinary Medicine, 8 April 2008 35
Resistance to disinfectants:conclusions
• No associations between MICs and use of disinfectants in the preceding download period
• No associations between MICs and Salmonella persistence
• Adaptation or de-adaptation did not alter any MICs beyond one doubling dilution, i.e., within normal biological variation
Koret School of Veterinary Medicine, 8 April 2008 36
Surface disinfection tests
Koret School of Veterinary Medicine, 8 April 2008 37
Surface disinfection tests: general principles
• Worst-case scenario surface disinfection tests simulating conditions and disinfection procedures encountered in badly cleaned poultry houses, especially at low temperatures
Koret School of Veterinary Medicine, 8 April 2008 38
Surface disinfection tests: factors
• Isolates: S. Enteritidis, S. Senftenberg, Enterococcus faecalis
• Poultry house materials: Concrete, rusty metal, wood, jute
• Organic matter: Feed, fats, egg yolk.• Disinfectants: formalin, glutaraldehyde/benzalkomium
chloride, oxidising compound, water (control)• Temperatures before and after disinfection:
6/11/20/30 and 6/11/30 oC, respectively• Disinfection time: 5, 15, 30 minutes
Koret School of Veterinary Medicine, 8 April 2008 39
Surface disinfection tests:concrete flag with feed
Koret School of Veterinary Medicine, 8 April 2008 40
Surface disinfection tests:feed chain links with feed
Koret School of Veterinary Medicine, 8 April 2008 41
Surface disinfection tests: results I
From: Gradel, K.O.: Disinfection of empty animal houses - scientific evidence for applied procedures.In Kurladze, G.V. (ed.): Environmental Microbiology Research Trends. New York, USA: Nova SciencePublishers, Inc., 2007, pp. 59-98. ISBN 978-1-60021-939-9.
Koret School of Veterinary Medicine, 8 April 2008 42
Surface disinfection tests: results II
• S. Senftenberg was more susceptible than S. Enteritidis in tests with the oxidising compound and water, in spite of higher MICs for S. Senftenberg
• In general, Enterococcus faecalis was more recalcitrant than the two Salmonella isolates. Thus, it is a putative indicator bacterium in field trials
Koret School of Veterinary Medicine, 8 April 2008 43
Future recommendations
• More worst-case scenario laboratory studies with various conditions (different types of organic matter, temperature, disinfectants)
• Field intervention studies• Standardized monitoring programmes (e.g.,
Hazard Analysis Critical Control Points)• Useful for many other micro-organisms than
Salmonella
Koret School of Veterinary Medicine, 8 April 2008 44
• Salmonella control programmes in chickens• Salmonella in chicken houses
• Salmonella in humans
Koret School of Veterinary Medicine, 8 April 2008 45
Interest of study group
• Zoonotic Salmonella and Campylobacter
• Bacteremia, ”all” micro-organisms
• Patient-related factors :– age– chronic diseases– medicine
• Registry-based research
Koret School of Veterinary Medicine, 8 April 2008 46
Salmonella bacteremia
Koret School of Veterinary Medicine, 8 April 2008 47
Salmonella bacteremia
• Prognostic study for 111 patients• Possible prognostic factors, e.g.:
– Age– Chronic diseases– Salmonella serotype– Antibiotic treatment– Leukocytes– C-reactive protein– Albumin– Haemoglobin– Creatinine
Koret School of Veterinary Medicine, 8 April 2008 48
Salmonella bacteremia
Factor 30-day
mortality rate ratio
180-day
mortality rate ratio
Age (years)
0-15 Not valid Not valid
16-64 1 (reference) 1 (reference)
65-80 1.6 (0.7-3.5) 1.8 (0.8-3.8)
>80 2.4 (1.0-5.6) 3.5 (1.5-8.3)
Charlson comorbidity
0 points 1 (reference) 1 (reference)
1-2 points 3.7 (1.5-8.9) 4.1 (1.7-9.8)
>2 points 6.5 (2.5-16.8) 8.5 (3.3-22.0)
Koret School of Veterinary Medicine, 8 April 2008 49
Seasonal variation of salmonellosis
Koret School of Veterinary Medicine, 8 April 2008 50
Seasonal variation of salmonellosis
• Background: the ”typical” seasonal variation was less evident for bacteremia patients
• Hypothesis:
Non-hospitalized
Hospitalized
BacteremiaEnd
ogen
ous
fact
ors:
The
sea
sona
l va
riatio
n ”m
inim
izes
”
Exo
geno
us
fact
ors:
The
sea
sona
l va
riatio
n ”m
axim
izes
”
Koret School of Veterinary Medicine, 8 April 2008 51
Seasonal variation of salmonellosis
Koret School of Veterinary Medicine, 8 April 2008 52
Magnitude of non-typhoid salmonellosis: prognosis
Koret School of Veterinary Medicine, 8 April 2008 53
Magnitude of non-typhoid salmonellosis: prognosis
0.00
0.25
0.50
0.75
1.00
Sur
viva
l
0 30 180 365
Days since receipt date
Salmonella in 0 bottlesSalmonella in 1 bottle
Salmonella in 2 bottlesSalmonella in 3 bottles
220 patients
Koret School of Veterinary Medicine, 8 April 2008 54
Magnitude of bacteremia: prognosis
Koret School of Veterinary Medicine, 8 April 2008 55
Magnitude of bacteremia: prognosis
0
10
20
30
40
50
% m
orta
lity
07 30 365Days after first-time bacteremia
Bacteria in 3 bottlesBacteria in 2 bottlesBacteria in 1 bottle
6,406 patients
Antibiotic prescription rates in non-typhoid Salmonella
Koret School of Veterinary Medicine, 8 April 2008 56
Koret School of Veterinary Medicine, 8 April 2008 57
Antibiotic prescription rates in non-typhoid Salmonella
.51
1.5
22
.5O
dds
ratio
(9
5% C
I)
213263952Week
Koret School of Veterinary Medicine, 8 April 2008 58
Human studies: overall conclusions
• Zoonotic infections are generally more severe in debilitated patient groups
• Patient related factors may be more important in the acquisition of zoonotic infections than previously considered
• Magnitude of the infection is a plausible method of assessing the prognostic impact of the infection per se, independently of patient factors
Koret School of Veterinary Medicine, 8 April 2008 59
! תודה רבה
Mange tak!