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


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Documents

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