Heat as a disinfection method for poultry houses persistently infected with Salmonella – an outline of

methods and results

Kim O. GradelDanmarks Veterinærinstitut, Århus

Rengøring og Desinfektion af StaldeHotel Australia, Vejle, 11.-12. November 2003

Background

In general, heat rather than chemical disinfection of contaminated items is recommended whenever possible.

It is difficult to clean animal houses and cover all areas by surface disinfection.

Hypothesis: heat can be evenly distributed and it can penetrate organic matter without damaging materials.

Environmental considerations.

Disposition

Laboratory tests Materials and methods Results Conclusions

Field tests Materials and methods Results Conclusions

Heat tests related to general disinfection factors

Micro-organism

Disinfectant Surroundings

Type”State/condition”

Organic matterTemperaturepHBiofilm

TypeConcentration

Heat project

Laboratory tests

Aims: To find a temperature-humidity-time

treatment that kills Salmonella. Investigate correlations between

susceptibility of spiked Salmonella and naturally occurring E. coli.

Principles: Simulation of real-life conditions. Worst-case scenarios. Full factorial design.

Laboratory tests: factors

FactorSalmonella (S. Enteritidis, PT8, S. Typhimurium, DT110, S. Infantis)Escherichia coli (naturally occurring in faeces)

Organic matter (pelleted feed for layers, chicken faeces)

Drying during 10-day period prior to heating (yes, no)

Humidity at heating (low = 16-30%; high = 100%)

Heating temperature (50, 55, 60, 65, 70 oC)

Laboratory tests:test protocol

1. Thirty gram test material (feed or faeces) were put in beakers, yielding a layer 2-2.5 cm thick.

2. Spiked with high numbers of Salmonella.3. Beakers at 20 oC and 30% RH during 10-day

period, lidded (no drying) or un-lidded (drying).4. 10-day samples checked microbiologically,

other samples moved to cooled incubator.5. Other samples checked microbiologically as for

10-day samples after varying time periods, cf. next slide.

Laboratory tests: overview of tests

10-daysamples

0-hoursamples

24-hoursamples

48-hoursamples

72-hoursamples

20 oC

1 oC per hour

Final heatingtemperature

20 oC

Laboratory tests: samples prior to heating

Laboratory tests: temperature control

programme

Laboratory tests: heating of high humidity

samples

Laboratory tests:microbiological procedures

From each beaker, traditional Salmonella procedures: pre-enrichment, selective enrichment, plating on Rambach agar.

From all beakers with faeces: plating from pre-enrichment broth on MacConkey agar (presence/absence of E. coli).

Laboratory tests:replicate tests

For the ”gold” standard, i.e. a temperature-humidity-time scheme that killed all bacteria, replicate tests were made.

Only S. Enteritidis was used, as there were few differences between the three Salmonella serotypes.

Only samples dried in the 10-day period were used. Crumbled feed and egg yolk as well as pelleted

feed and faeces were used. All tests were repeated four times. Three different selective enrichment procedures

were applied.

Laboratory tests: overview of traditional

Salmonella 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 + + + + + + - - - -

Laboratory tests:correlations between results

for Salmonella and E. coli

E. coli detected?

Salmonella spp. detected on Rambach agar?

SUM

Yes No

Pure culture of

Salmonella

Salmonella- and non-

Salmonella

Growth of non-

Salmonella Sterile

Yes 8 90 10 8 116

No 40 8 3 222 273

SUM 48 98 13 230 389

Laboratory tests:conclusions

Humidity, both before and during heating, was an important factor in the bacterial killing.

In general, there was a higher survival in feed than in faeces.

There were high correlations between the survival of spiked Salmonella and naturally occurring E. coli.

No bacteria were detected at 60 oC and 100% RH after 24 hours of heating (i.e. ”gold” standard for field studies), including in replicate tests.

Field tests

Aim: To test if the gold standard (60 oC & 100% RH during 24 hours) was valid in the field.

Principles: Salmonella samples before and after heating.

Salmonella results per se. Non-sterile Rambach agar plates (i.e. coliforms as

possible indicator bacteria). Challenge samples, placed at sites where

temperature was measured.

Field tests:principles I

Field tests:principles II

Field tests:Overview of farms,

houses and treatments

FARM HOUSEHOUSE TYPE

TREATMENT

No. of Salmonella

samples Challenge

samplesBefore

After

A A1 Barn Steam without F

287 288 Yes

A2 Battery Steam without F

302 303 Yes

B B1 Barn Steam with F 100 102 Yes

B2 Barn Pulse fogging 100 96 No

B3 Barn Surface dis. 100 100 No

C C1 Battery Steam with F 298 308 Yes

D D1 Battery Steam with F 289 290 Yes

E E1 Battery Steam with F 308 308 Yes

F F1 Battery Steam with F 150 150 No

Field tests:results for Salmonella

samples

HOUSE

TREATMENT

Salmonella (positive/sum)

Non-sterile Rambach

(positive/sum)R SBefor

eAfter Before After

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

B

B2 Pulse fog. 6/100 0/96 58/100 33/96 1.7 C

B3 Surface 5/100 0/100 68/100 8/100 8.5 CD

C1 Steam, +F 37/298

0/308 180/298 2/308 92.9

E

D1 Steam, +F 1/289 0/290 78/289 1/290 79.4

F

E1 Steam, +F 40/308

0/308 224/308 9/308 25.1

D

F1 Steam, +F 16/150

0/150 146/150 3/150 48.7

Field tests:Salmonella ”hot” sites

SITEPOSITIVE/SUM

% POSITIVE

Beams/ledges 9/74 12.2

Droppings belts 5/48 10.4

Drip channels 12/51 23.5

Feed chain ”corner wheels”

6/33 18.2

Egg equipment 14/93 15.1

Floor related samples

103/507 20.3

Field tests:correlations between

Salmonella and coliforms on house level

R2 = 0.6137

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0.0 5.0 10.0 15.0 20.0 25.0

% Salmonella-positive samples

% n

on

-ste

rile

Ram

bac

h a

gar

pla

tes

Field tests:correlations between

Salmonella and coliforms on site level

R2 = 0.0072

R2 = 0.3842

0

10

20

30

40

50

60

70

80

90

100

110

120

0 5 10 15 20 25 30 35 40 45 50 55

% Salmonella-positive samples

% n

on

-ste

rile

Ram

bac

h a

gar

pla

tes

Field tests:results for challenge samples

Feed Faeces

E. faecalis E. coli Enterococci E. coli

Mean tempe-rature (oC)

Without F

With F

Without F

With F

Without F

With F

Without F

With F

> 62.5 0/4 0/11 0/4 0/11 0/4 0/11 0/4 0/11

60-62.5 0/8 0/8 1/8 0/8 2/8 1/7 0/8 0/8

57.5-60 1/3 0/9 0/3 0/9 1/3 2/9 0/3 0/9

55-57.5 2/4 1/2 1/4 1/2 2/4 1/2 0/4 0/2

52.5-55 2/5 0/5 1/5 0/5

50-52.5 0/1 0/3 0/1 0/3 0/1 0/3 0/1 0/3

47.5-50 1/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1

45-47.5 2/2 0/2 2/2 1/2 2/2 1/2 0/2 0/2

42.5-45

40-42.5 1/1 1/1 1/1 0/1

37.5-40 1/1 1/1 1/1 1/1

35-37.5

32.5-35

? 32.5 1/1 3/3 1/1 1/3 1/1 1/3 1/1 0/3

Field tests:”traditional” Danish heating

procedure

Practised for years in Danish poultry houses.

Principle: a short steam and formaldehyde heating until the temperature reaches ca. 60 oC, after which the heating ceases.

The house remains sealed for ca. 24 hours.

Field tests:”traditional” Danish heating: a typical temperature curve

6164_ 009 (TF)

0

10

20

30

40

50

60

T i d

LAV

HØJ

Field tests:”traditional” Danish heating:

challenge samples

E. Faecalis

Feed

E. coliFeed

Enterococci

Faeces

E. coliFaeces

Barn

13 ppm

Cage

22 ppm

Par.30ppm

Barn

13 ppm

Cage

22 ppm

Par.30ppm

Barn

13 ppm

Cage

22 ppm

Par.30ppm

Barn

13 ppm

Cage

22 ppm

Par.30ppm

Pos/

sum

H 3/6 6/6 4/6 1/6 5/6 5/6 0/6 5/6 2/6 0/6 1/6 0/6

L 4/6 6/6 2/6 3/6 6/6 2/6 3/6 6/6 1/6 0/6 0/6 2/6

S 7/12

12/12

6/12

4/12

11/12

7/12

3/12

11/12

3/12

0/12

1/12

2/12

Field tests:”traditional” heating:

challenge samples and peak temperatures

Peak tempe-rature

E. Faecalis

Feed

E. ColiFeed

Enterococci

Faeces

E. ColiFaeces

60-65 8/13 5/13 4/13 1/13

55-60 11/14 11/14 8/14 0/14

28-55 6/9 6/9 5/9 2/9

Field tests:Conclusions

In tight houses, 60 oC and 100% RH was achieved minimum 10 cm above floor level within one hour and was easily maintained during 24 hours.

60 oC and 100% RH during 24 hours seemed effective in eliminating Salmonella and putative indicator bacteria.

The addition of 30 ppm formaldehyde at the beginning of the process seemed to lower the lethal temperature by 2-5 oC.

The occurrence of coliforms could be a guidance for the efficacy against Salmonella, although no cut-off values could be predicted.

The ”traditional” Danish steam and formaldehyde procedure is ineffective.

Future perspectives

Certification of heating procedures Possible model: Danish Technological

Institute has certified a heat treatment against Dry Rot in buildings. Companies that can document a time-temperature scheme with validated equipment are approved by this system. It also includes insurance against re-occurrence of Dry Rot. See www.vks-udvalget.dk

Acknowledgments Kirsten Holm, DVI, Århus, Denmark.

Kirsten Christensen, DVI, Århus, Denmark.

Staff at Landskontoret for Fjerkrærådgivning, Århus, Denmark.

Staff at Danish Meat Research Institute, Roskilde, Denmark.

Participating farmers.