Kim Oren Gradel Department of Infectious Diseases Aalborg Hospital Aarhus University Hospital

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


Main infection sources in humans

Danish Zoonosis Centre: Annual report on Zoonoses in Denmark 2006.


Table of contents

• Salmonella control programmes in chickens

• Salmonella in chicken houses• Salmonella in humans


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


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)


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)


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!


Table of contents

• Salmonella control programmes in chickens

• Salmonella in chicken houses• Salmonella in humans


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


Questionnaire-based field study


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


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)


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)


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


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”


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


Heat laboratory tests


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).


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)


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 + + + + + + - - - -


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


Heat laboratory tests: gold standard for field studies

• No bacteria were detected at 60 oC and 100% RH after 24 hours of heating


Heat field tests


Heat field tests: principles I


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



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


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


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


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


Resistance to disinfectants


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


Resistance to disinfectants:adaptation and de-adaptation

MIC-testsMIC-tests


MICs for isolates from Danish chicken houses

”Non-persistent” serotypes

”Persistent” serotypes

”Danish” disinfectants ”English” disinfectants

Higher MICs


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


Surface disinfection tests


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


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


Surface disinfection tests:concrete flag with feed


Surface disinfection tests:feed chain links with feed


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.


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


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


• Salmonella control programmes in chickens• Salmonella in chicken houses

• Salmonella in humans


Interest of study group

• Zoonotic Salmonella and Campylobacter

• Bacteremia, ”all” micro-organisms

• Patient-related factors :– age– chronic diseases– medicine

• Registry-based research


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



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)


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

”




Magnitude of non-typhoid salmonellosis: prognosis


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


Magnitude of bacteremia: prognosis


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



Antibiotic prescription rates in non-typhoid Salmonella

.51

1.5

22

.5O

dds

ratio

(9

5% C

I)

213263952Week


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


! תודה רבה

Mange tak!

Kim Oren Gradel Department of Infectious Diseases Aalborg Hospital Aarhus University Hospital

Documents

danish veterinary laboratory

salmonella infected

zoonosis directive samples

iestable egg houses

chicken housessalmonella

humansdanish zoonosis

feed heat

main infection sources