Kim O. Gradel Danmarks Veterin æ rinstitut, Å rhus Reng ø ring og Desinfektion af Stalde
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Transcript of Kim O. Gradel Danmarks Veterin æ rinstitut, Å rhus Reng ø ring og Desinfektion af Stalde
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.