floor swab culture
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veterinarymicrobiology
Veterinary Microbiology 50 (1996) 117-127
Determination of an effective sampling regime to
detect salmonella enteritidis in the environment of
poultry units
Robert H. Davies * Clifford Wray
Bacteri ology Department, Cent ral Veterinary Laboratory , New Haw , Addl estone, Surrey K Tl5 3NB, UK
Received 11 October 1995; accepted 5 February 1996
Abstract
A study of the dissemination of Salmonella enteritidis in the poultry breeder industry in the
UK showed that the choice of sites for sampling the environment of occupied houses and emptyhouses which had been disinfected after depopulation had a significant influence on the outcome.
Increased isolation rates could be achieved by sampling nest box floors and dust in open slave
feed hoppers in occupied poultry houses. Nest box floors were the most sensitive sites for
detection of residual environmental contamination in poultry houses where enrofloxacin treatment
had been used. Floor sweepings, nest box floors, slave feed hoppers, hydrated wall fabric
junctions and high beams and pipes were the most sensitive sample sites in cleansed and
disinfected poultry houses. The use of universal disinfectant neutralisers gave good results in
laboratory trials but appeared to reduce the isolation rate from field samples.
Keyw ords: Salmonell a enterit idi s; Chicken; Cleansing and disinfection; Sampling methods
1 Introduction
Persistent environmental contamination of housing is an important factor in mainte-
nance of Salmonella enteritidis infection in poultry flocks (Kradel and Miller, 1991;
Baggesen et al., 1992). Shell eggs (Humphrey, 1990) and poultry meat products (Poppe
et al., 1991) are regarded as important sources of human salmonellosis so it is essential
to reduce the level of contaminated material entering the human food chain to avoid
* Corresponding author, Tel.: 01932-347487, Fax: 01932-347046.
0378-1135/96/ 15.00 0 1996 Elsevier Science B.V. All rights reserved
PII SO378-1135(96)00031-4
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118 RaberrH. Danvies,C. Wray/ Vet erinar y M it t- abiol ogy 50 t /996) 117-127
food borne infections and consequent economic loss ((Roberts, 1988, Todd, 1989,
Bender and Ebel, 1992, Bailey, 1993).
The effective decontamination of housing of salmonella infected flocks beforerepopulation is a highly important consideration in a HACCP approach for poultry
breeder units (Anon, 1991). It is therefore extremely important that an effective
monitoring protocol for the detection of residual salmonella contamination after cleans-
ing and disinfection is used to assess the success of the operation.
More effective techniques for sampling poultry houses combined with maximising
the sensitivity of salmonella culture (Davies and Wray, 1994) will also increase the
chance of detecting infected flocks at an early stage and enable action to be taken to
reduce further dissemination of the infection.
2. Materials and methods
2 I Sampling methods
Solid samples such as litter or dust were collected directly into jars of sterile buffered
peptone water (BPW, Oxoid CM509). Environmental swabs were taken by vigorous
swabbing of 0.5 1.0 m* surface area using a bunch of six gauze surgical swabs
(Robinson Healthcare, Chesterfield, UK: No. 63024) which had been autoclaved within
jars of BPW. The swabs were returned to the jars after swabbing.
Sweepings samples were gathered using autoclaved stiff deck brooms. Each samplewas estimated to provide 15-25 g of material which was swept up into the jar of BPW
using the moist gauze swabs. Wall fabric junctions were hydrated by pouring 200 ml of
BPW over junctions between boarding and concrete parts of the wall. Surface swabs
were then taken after 30-60 min.
2.2, Salmonella culture methods
These were as described in Davies and Wray (1994) briefly: samples were pre-en-
riched in BPW at 37°C for 18 h followed by selective enrichment in Semi-solid
Rappaport Medium (MSRV, LabM; Lab 150) for 48 h at 41.5”C. Subcultures werestreaked on to Rambach agar (Merck 7500) which was incubated at 37°C for 24 h.
Suspect salmonella colonies were confirmed by full serotyping.
2.3. Universal disinfectant neutraliser
This was prepared according to the following formula:
BPW
Sodium thiosulphate
0.25 N Phosphate buffer
Tween 80L-Histadine
Lecithin
Ammonium chloride
960 ml
5g10 ml
30 ml
lg
3g
1Og
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RobertH . Davies, C. Wray / Veteri nary M icrobiol ogy 50 (19 ) I 1 7-l 27
2.4. Definition of terms used to describe poultry house equipment
119
Slave feed hoppers - these are open short-term feed reservoirs which house and feedthe motor units which power the chain feeder system.
Chain feeder system - this is a system of metal channels arranged to convey feed
around the poultry house by means of a moving chain within the channel.
Nest box front beam - this is the wooden plank at the front of the nest boxes which
retains litter.
Perches - these are wooden rails fixed to nest box frames on which birds perch to
gain entry to nest boxes.
2.5. Statistical methods
Probability values were obtained with the Chi-Square test (Yates Correction) using
2 X 2 tables on EPI-INFO Statcalc programme. Results shown in each table were pooled
for analysis as described in the Results section.
3. Results
3.1. Detection of salmonella in occupied poultry houses
Comparative results of sampling in occupied poultry houses are shown in Table 1.
There was a threefold higher salmonella isolation rate from nest box floors and dust on
in-house slave feed hoppers ( p < 0.01) than from drinkers, chain feeders, slats, perches
Table 1
Detection of 5. enterifidis in occupied poultry houses - SalmaneEZa isolation from various sample sites
Poultry Type of unit Slave feed hopper Nest boxes Other sites
unit
code
No. of No. positive No. of No. positive No. of No. positive
samples for Salmonella a samples for Salmonella a samples for Salmonella a
A Broiler breeder 4 1 (25.0) 5 1 (20.0) 20 0
B Turkey breeder ’ 12 2 (16.7) 6 0 26 0
C Broiler breeder 1 1(100.0) 4 0 24 l(4.1)
D Layer 4 3 (75.0) NA - 32 l(3.1)
E Layer 4 1 (25.0) NA - 38 0
F Broiler breeder 3 1 (33.3) 9 7 (77.8) 24 1 (4.7)
G Broiler breeder 4 4 (100.0) 16 11 (68.7) 64 33 (51.6)
H Broiler breeder 2 2 (50.0) 16 8 (50.0) 64 9 (21.8)
(sep. feed bin
in ante-room)
Totals 34 15 (44.1) 56 27 (48.2) 292 45 (15.4)
a % of samples positive for S. enteritidis in brackets.
b S. heidelberg infection.
NA = Not applicable.
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12 RobertH. Daoies. C. Wray/ Veterinav Microbiology 50 (1996) I1 7-127
and dust on beams and ventilation ducts. In broiler breeder houses salmonella was
isolated from all the egg sorting tables and 75% of the egg collection trolleys sampled.
3.2. Detection of S. enteritidis contamination of poultry houses after antibiotic treatment
of the flock
Table 2 shows results of sampling in occupied broiler breeder houses where the flock
had recently received antimicrobial (Enrofloxacin) treatment in the water supply. In
these cases there was a general reduction (pre-treatment sampling data not shown) in the
prevalence of salmonella in environmental samples after treatment which was less
marked on nest box floors, whereas, there was a substantial reduction in the prevalence
of salmonella in other sample sites. The slave hoppers were not a sensitive sample site
following antibiotic treatment.
3.3. Detection of persistent S. enteritidis by periodic sampling of a trial poultry house
after cleansing and disinfection
The results of sampling a breeder house at 1, 2, 6 and 12 weeks after cleansing and
disinfection following depopulation of a broiler breeder flock which was naturally
infected with S. enteritidis are shown in Table 3. A sampling regime comprising floor
sweepings, in-house feed hoppers and hydrated wall fabric junctions successfully
detected persistent salmonella contamination on each occasion compared with swabbing
of floors, walls, beams and drinkers which gave negative results (p < 0.05). Liquid
vacuum suction, using a BPW filled domestic vacuum/carpet cleaner which had been
sterilised by fumigation with ethylene oxide, failed to detect salmonella despite collect-
ing a much larger volume of dust from the floor than was obtained by sweeping.
3.4. Detection of persistent S. enteritidis infection in commercial poultry breeder houses
after cleansing and disinfection
Table 4 compares salmonella isolation rates obtained from various sample sites in
commercial broiler or layer breeder houses after cleansing and disinfection. Floor
sweepings and nest box floors were the most sensitive sample sites followed by slave
feed hoppers, hydrated wall fabric junctions and high beams and pipes p < 0.01).
Samples from wall comers, floor expansion joints, fan ventilation shafts, chain feeders,
drinkers, perches and nest box front beams were relatively insensitive with 15/ 16 (94%)
of the houses being identified as salmonella negative when these sample sites were used
in a simulated standard sampling regime of 23 sites per house. The most useful other
sample sites were egg collection buggies and egg handling tables. Where dead mice or
mouse droppings were present these too usually had a high prevalence of salmonella.
3.5. Use of universal disinfectant neutraliser for enhanced detection of salmonella ondisinfected sur aces
Table 5 shows the results of a small study using universal disinfectant neutraliser
added to the BPW used for swabbing and culture.
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124 RobrrtH. Dacirs. C. Wru,v / Veterina~ Microbiology 50 (19961 I 17-127
Table 5
Use of universal disinfectant neutraliser for Isolation of Mrrwnellu on disinfected surfaces
No. of Positive for Salmonellu Positive for Salmonellusample\ by standard culture using neutraliser
No. ‘Z NO. c/r
Laboratory floor surface5 133 52 39. I 14 55.6
Poultry house environmental -1x -I6 95.X 39/w 72.5
SUd2KY?
Laboratory floor surfaces which had been accidentally contaminated with salmonella
were disinfected with various chemical disinfectants. Swabbing after 2-8 h contact timeproduced a significantly increased salmonella isolation rate ( p < 0.01) when the disin-
fectant neutraliser was used This contrasted with the situation in a naturally contami-
nated poultry house where the isolation rate was reduced ( p < 0.01) when disinfectant
neutraliser was used.
4. Discussion
Environmental monitoring of the poultry house has been shown to be a highly
effective means of detecting salmonella infection in the flock (Kradel and Miller, 1991,
Poppe et al., 1992). A well designed environmental monitoring programme can therefore
be a useful adjunct to regimes designed to detect infection in faeces, post mortem tissues
and meconium. Monitoring regimes commonly also involve pooling of samples for
culture, but this has been shown to reduce detection sensitivity (Giessen et al., 1991) so
it is best to culture as many individual samples as economically possible.
The object of these studies was to define specific sampling sites within poultry
houses for enhanced detection of salmonella. particularly after cleansing and disinfec-
tion. In occupied poultry houses swabs taken from nest box floors and slave feed
hoppers gave higher isolation rates than litter, dust and drinker samples. Nest boxes are
kept relatively clean so there is little competitive “litter effect” which leads to reduction
of salmonella as a result of direct competition for space and nutrients and toxic effects
of metabolites produced by the action of other bacteria in the littered areas of the house
(Opara et al.. 1992). Thus salmonellas excreted by birds during the early stages of
infection may persist preferentially in nest boxes, despite the widespread use of
paraformaldehyde prills or powder as a nest box litter disinfectant. The body heat of the
birds may also lead to multiplication of salmonellas within nest boxes.
In-house slave feed hoppers in broiler breeder houses or spilled feed from caged layer
flock conveyor feeding systems were also highly sensitive sample sites. It is likely that
the organic matter in feed dust supplies a protective nutritive matrix to enhance the
survival of salmonellas. We have demonstrated survival of S. enteritidis inoculated into
poultry feed at 10” organisms/100 g feed, for at least two years (unpublished data).
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RobertH . Dauies, C. Wray/ Veteri nary M icrobiology 50 (1996) I1 7-127 125
The most sensitive of the other sample sites in occupied breeder houses were the egg
sorting tables and egg collection buggies. Swabs taken from slats, perches and the front
boards of nest boxes were not very useful unless a high level of general salmonellacontamination was present.
When antibiotic treatment is used in salmonella infected flocks there may be a
subsequent effect on the level of environmental contamination through excretion of the
antibiotic and its metabolites by the birds and suppression of ongoing excretion of
salmonella in faeces. When enrofloxacin has been used the reduction of environmental
contamination makes environmental monitoring less useful but nest box floors were still
a sensitive sampling site. Interestingly the isolation rate of salmonella from slave feed
hoppers dropped dramatically after treatment. This suggests that feed dust in slave feed
hoppers may enhance detection of current excretion of salmonellas which is reduced
after antibiotic treatment.
The objective of sampling poultry houses for salmonella after cleansing and disinfec-
tion is to ensure that decontamination has been successful. After cleansing and disinfec-
tion we investigated the use of various sampling techniques and sample sites. Large
gauze swabs were used to maximise the surface area which could be sampled and to
provide abrasive properties for vigorous rubbing of cleaned surfaces. Even using these
large swabs it was not possible to isolate salmonella during periodic sampling of floor
and wall surfaces in a disinfected poultry house whereas S. enteritidis was found on
swabs taken from open feed hoppers, hydrated wall fabric junctions and floor sweep-
ings. Interestingly liquid vacuum suction of large areas of floor surfaces did not enhancesalmonella detection. This may be because the organisms were intimately attached to
floor surfaces in established biofilms and vacuum suction has little abrasive effect. It has
been shown that sampling such biofilms may give false negative results (Kim et al.,
1993, Wirtanen and Mattila-Sandholm, 1993) and that vigorous physical abrasion such
as brushing is the best means of detaching organisms from the biofilms (Carpentier and
Cerf, 1993). It is also possible to quickly sample a much larger surface area of cleaned
floor surface by sweeping than by swabbing.
Hydration of wall fabric junctions was successful in isolating salmonella but this was
not true of cracks and expansion joints in the floor. During field trials sampling such
cracks after disinfection was similarly unrewarding despite large quantities of residual
organic matter of&en being present. It may be that floor cracks remain wet for long
periods after disinfection lending to anaerobic conditions favouring competitive bacteria.
Also disinfectant activity is likely to be better in floor cracks because of pooling of the
liquid compared with rapid run-off and evaporation from vertical wall surfaces,
Field studies of poultry houses after cleansing and disinfection confirmed the value of
sampling floor sweepings, slave feed hoppers and hydrated wall fabric junctions. In a
few cases there had been recontamination of the environment by S. enteritidis in
droppings from a residual infected mouse population. Sweepings samples and samples
from slave feed hoppers were more likely to detect mouse droppings which may not benormally noticed unless supplementary bright lighting is used to assist inspection of the
building.
There was also a high isolation rate from nest box floors, which were often not
disinfected as thoroughly as other fittings even though they are the most highly
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126 RohrrtH. Da s. C. Wruy / Veterinap Microhivlvg~ 50 II9961 I 7- 127
contaminated item in a breeder house. High beams and pipes were also worthwhile
sample sites with salmonella persisting largely through inconsistent cleansing and
application of disinfectant to less accessible areas.Swab samples from floor expansion joints, wall comers, fan boxes, slats and perches.
drinkers, chain feeders and nest box front beams were very poor for isolation of
salmonella and only the most contaminated house was identified when these samples
were used
It was interesting that no salmonellas were isolated from water header tanks in this
study even though in some cases the tanks were not covered and had been poorly
cleaned. Some bacteria are capable of long-term persistence in biofilms in water tanks
and pipes (Manz et al., 1993). It is uncertain whether failure to detect salmonella in this
work reflects true absence of the organism or the presence of a dormant non-culturable
state (Roszak et al., 1983). We have observed an increased isolation rate of S. erzteritidis
from open drinkers and dust samples taken from occupied poultry houses when small
pieces of cellulose sponge are used during pre-enrichment culture as a protective matrix
(unpublished data). This suggests that dormancy and poor culturability of salmonella
may occur in aqueous and dry poultry house environmental samples.
Universal disinfectant neutralisers have been recommended for use in environmental
sampling after cleansing and disinfection (F. Sellars and G.P. Wilding, personal
communications).
Our laboratory based studies confirmed an increase in salmonella isolations in swabs
taken from disinfected surfaces (Table 5) but the opposite occurred with samples takenin a disinfected poultry house where sub-lethal concentrations of disinfectant had
inadvertently been used. In disinfected poultry houses it is common to find single figure
levels of salmonella against a background of 4-8 log ,(I levels of other coliforms. It may
be that ingredients such as lecithin and histidine promote overgrowth of salmonella by
competitive organisms during pre-enrichment culture of field samples. Similarly, a low
environmental salmonella isolation rate in disinfected hatcheries produced by swabbing
using disinfectant neutraliser has been shown to produce a underestimate of the extent of
contamination compared with use of BPW for swabbing (unpublished data). More work
is required to evaluate the use of disinfectant neutralisers under controlled conditions in
the field.
It is difficult to evaluate the hazard posed by survival of salmonella in poultry houses
after cleansing and disinfection. The presence of salmonella contamination may not
always result in infection of the flock (Bailey, 19931 but it is difficult to carry out risk
assessment studies in the field because disinfection is normally repeated when persistent
environmental contamination has been demonstrated. This was true of this study except
for houses A. B and F (Table 4) where there was insufficient time for redisinfection
before restocking. In houses A and F there was a rapid re-infection of the new broiler
breeder flocks and in house B, which was on the same site as house A re-infection
occurred later and so S. enteritidis may have spread from the flock in house A.
This work shows that improvements in choice of sampling sites and techniques for
detection of salmonella on poultry units can be made. Although much work remains to
be done. sampling strategies based on the methods use in this study, particularly
swabbing nest box floors, egg handling equipment and slave feed hoppers, and the use
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RobertH . Davi es, C. Wray / Veteri nar?, M i crobi ol ogy 50 (1996) 117-127 127
of floor sweepings samples, should enhance control measures for S, enteritidis in the
poultry industry.
Acknowledgements
This work was funded by the Ministry of Agriculture, Fisheries and Food. The
authors are grateful to Mrs S. Bedford and Mrs K. McIntosh for technical assistance, and
would also like to thank colleagues in the Animal Health (Zoonoses) Division and State
Veterinary Service for their assistance with arranging field work.
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