Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Comparison of sampling methods to detect Salmonella

infection of turkey flocks
D. Mueller-Doblies1, A.R. Sayers2, J.J. Carrique-Mas1 and R.H. Davies1
1 Food and Environmental Safety Department, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey, UK
2 Centre for Epidemiology and Risk Analysis, Veterinary Laboratories Agency Weybridge, New Haw, Addlestone, Surrey, UK

Keywords
detection, environment, Salmonella, sampling
methods, turkeys.
Correspondence
Robert H. Davies, Food and Environmental
Safety Department, Veterinary Laboratories
Agency Weybridge, New Haw, Addlestone,
Surrey, KT15 3NB, UK.
E-mail: r.h.davies@vla.defra.gsi.gov.uk
2008 ⁄ 2110: received 10 December 2008,
revised 10 December 2008 and accepted 5
January 2009
doi:10.1111/j.1365-2672.2009.04230.x
Abstract
Aims: To compare the efficiency of various sampling methods for detection of
Salmonella in turkey flocks.
Methods and Results: In a field study that compared various sampling
methods one pair of boot swabs taken from the whole turkey house provided
suitably sensitive results for fattening and rearing flocks and was no less
sensitive than two pairs, each from half the house, tested as a pooled sample.
The sensitivity was further enhanced by adding a dust sample. The dust sample
appeared to be particularly useful in flocks with a low prevalence, especially in
breeding flocks, and was more sensitive than a method which used five pairs of
boot swabs per flock. Combined incubation of a boot swab and a dust sample
showed no interference between the two sample types and a maximum sensitiv-
ity of detection. Litter samples and commercial sponge drag swabs provided a
lower level of detection.
Conclusions: A single pair of boot swabs taken from the whole house is recom-
mended for routine sampling of commercial rearing or fattening flocks. An
additional dust sample could be added to increase detection in flocks with a
low prevalence or in breeding flocks, but adding an additional pair of boot
swabs would not increase detection compared with a single pair.
Significance and Impact of the Study: This study demonstrates that significant
efficiencies can be made in sampling programmes for detection of Salmonella
in turkey flocks without detracting from the sensitivity. Similar studies are
recommended for other poultry sectors, particularly in chicken breeding flocks.

although a significant decrease was observed over the pre-

Introduction
Although the number of human cases of salmonellosis
has declined in the past decade, there were still 14 060
laboratory-confirmed cases of Salmonella infection
reported in the UK in 2006, thus ranking it among the
major causes of foodborne zoonoses. More than half of
the reported cases in England and Wales in 2006 were
attributable to S. Enteritidis (7157 cases, 56% of all
cases), and S. Typhimurium was the second most com-
mon serovar observed (1735 cases, 14% of all cases)
(Anonymous 2007; EFSA 2008c). In the EU, salmonellosis
was the second most commonly reported foodborne zoo-
nosis in 2006, accounting for 160 649 reported cases,
vious three years (EFSA 2008a).
Most cases of human salmonellosis are attributed to
the consumption of eggs and chicken meat (Humphrey
1990; Poppe et al. 1991; de Jong and Ekdahl 2006), but
very little is known about the role of turkeys in the infec-
tion chain, and there are few references to turkey meat as
a cause of human salmonellosis (Baggesen et al. 1996).
Relatively little research on the epidemiology and control
of Salmonella in turkeys has been done in Europe.
Between October 2006 and September 2007, a EU-wide
survey of turkey flocks (SANCO ⁄ 2083 ⁄ 2006) was carried
out, providing the first robust data on the prevalence of
Salmonella infections in turkey flocks across the EU

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Salmonella detection in turkey flocks
(EFSA 2008b). This survey found 29Æ3% of fattening
turkey flocks across the EU to be infected with Salmonella
spp., and the EU-wide prevalence was estimated as 30Æ7%,
with 3Æ8% being positive for either S. Typhimurium or
S. Enteritidis and 27Æ3% being positive for other serovars.
In the UK, the prevalence of Salmonella spp. in fattening
turkeys was estimated as 32Æ2%, with 4Æ6% being positive
for either S. Typhimurium or S. Enteritidis and 28%
being positive for serovars other than S. Typhimurium or
S. Enteritidis.
In the US, Salmonella spp. were reported to have
accounted for 42% of outbreaks and 51% of cases of gas-
troenteritis due to bacterial pathogens irrespective of the
food vehicle, and 4Æ6% of Salmonella outbreaks were
thought to be associated with consumption of turkey meat
between 1973 and 1987 (Bean and Griffin 1990). A recent
publication from the US found S. Kentucky to be the most
prevalent serovar in turkeys, followed by S. Senftenberg
and S. Muenster (Santos et al. 2007). In another study from
the US, Cox and co-authors sampled individual faecal
droppings and found 17Æ7% of female and 31Æ1% of male
turkeys positive for Salmonella (Cox et al. 2000).
As the estimation of prevalence and identification of
infected flocks in order to apply enhanced control mea-
sures depends on the sampling method, it is essential to
choose a sensitive method of sampling for all stages of
turkey production (Entis 2006; Fletcher 2006), and exten-
sive sampling is initially required to define parameters for
testing this (Davies et al. 1998; Nayak et al. 2003). At the
same time, the costs of the sampling regime have to be
kept as low as possible to make it feasible on a large scale.
There is also a need to define the sensitivity of different
sampling methods in relation to each other in order to be
able to provide advice to risk managers and the turkey
industry. Several sampling methods are currently in use
by the turkey industry and by researchers to detect
Salmonella in occupied turkey houses, but very little work
has been done to compare their performance. Sampling
individual birds using cloacal swabs has been widely used
by the UK turkey industry, but various other methods to
sample the environment of the birds are in use, such as
litter samples, boot swabs ⁄ sock swabs, drag swabs,
dust samples ⁄ dust swabs and pooled individual faecal
droppings.
The EU baseline survey of fattening and breeding
turkey flocks (October 2006–September 2007) was based
on sampling occupied houses using five pairs of
boot swabs, cultured as five separate pairs. Previous
VLA studies have shown that faeces samples alone are
insufficient to demonstrate infection in some commercial
egg laying flocks, and the addition of dust may improve
the sensitivity (Carrique-Mas et al. 2008). Most published
studies have compared Salmonella sampling methods in
636 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 635–645
D. Mueller-Doblies et al.
chicken houses (broiler, rearing and laying flocks) (Davies
and Wray 1996a; Skov et al. 1999; Gradel et al. 2002),
and the results may not necessarily be extrapolated to
turkeys.
In this study, we selected 106 turkey flocks in England
and compared the following sampling methods – (i) Full
EU survey protocol (i.e. five pairs of boot swabs cultured
as five separate pairs); (ii) one pair of boot swabs taken
from the whole house and cultured as a pair; (iii) two
pairs of boot swabs, each taken from half the house and
cultured together; (iv) a litter sample; (v) a commercial
drag swab sample and (vi) a dust sample. From 45 of the
106 flocks, an additional 60 individual faecal samples
were analysed. Results were entered onto a database and
statistical methods were used to assess which was the
most sensitive sampling methodology.
Material and methods
Farms and flocks
A total of 106 flocks on 24 farms across England were
sampled between February and October 2007. Three
farms were visited more than once. The study focused on
farms where positive flocks had been identified in the EU
turkey survey as well as through the companies’ own sur-
veillance systems. The flocks that were sampled in the
current study were the follow-on flocks for which the
Salmonella-status was unknown. The 106 flocks included
15 breeding flocks, 29 rearing flocks and 62 finishing
flocks. On average, three to four flocks per farm and visit
were tested.
Sampling
Boot swab method
For the sampling methods involving five (‘EU survey
method’, ‘BS5’) and two spun olefin-fabric boot swabs
(‘two-boot swab’ method, ‘BS2’), each house was divided
into five and two approximately equal sectors respectively.
All sections of the house were represented in the sampling
in a proportionate way. After walking through any boot-
dips and before putting on the boot swabs, a new pair of
plastic over boots was put on to avoid contact of the disin-
fectant with the boot swab. For each pair of boot swabs a
new pair of plastic over boots was used. The surface of the
boot swabs (‘Overshoes’, Bowman and Knights, Norwich,
UK) was moistened with sterile distilled water prior to use.
Within each chosen sector, at least 100 shuffling steps per
pair of boot swabs were taken, ensuring that all parts of
the sector were sampled. For the ‘BS1’ - method, at least
100 steps were walked covering a circuit of the whole
house. Boot swabs were put into sterile plastic pots.
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D. Mueller-Doblies et al.
Drag swab method
A commercially available sponge drag swab (5 · 10 cm
Polywipe Premoistened Sponge Swab with Draw Cord,
Medical Wire and Equipment, Corsham, UK) was used.
The drag swab sample was taken by walking around the
whole of the house (at least 100 steps) and dragging the
swab along. The drag swab was then put into a plastic pot.
Litter sampling method
Forty pinches of litter were collected from the whole
house into a sterile plastic pot.
Dust sampling method
Dust, representative of the whole house, was collected
into a sterile plastic pot using a gloved hand. Dust was
collected from horizontal surfaces such as ledges, pipes,
beams and partitions, but not from feeders or heaters.
Of the 106 visited flocks, 71 flocks were sampled with the
whole range of sampling methods, whereas for the remain-
ing 35 flocks, a reduced set of samples was taken. For all
production types (i.e. breeding, rearing or finishing units)
the same sampling approach was applied. For free range
flocks, samples were only collected inside the houses.
From 45 of the 106 flocks, 60 individual faecal samples
were collected in addition to the samples described above.
Sampling of individual faeces
Sixty fresh faeces from the whole house were collected
individually into sterile faeces specimen pots.
Combination sampling method
Sixty-eight flocks were sampled of which 22 proved to be
Salmonella-positive and 46 proved to be negative. One
pair of boot swabs was collected from the littered floor
area of each flock as described above. Dust was sampled
with a large gauze swab (Kleenex Readiwipe, Robinson
HealthCare, Chesterfield, UK), which had been premois-
tened with Buffered Peptone Water. The swab was swept
through dusty surfaces, typically beams and ledges, in at
least 10 different representative parts of the house until it
was liberally coated with dust.
Processing and culture of samples
Samples were normally transported to the laboratory
within 6 h and incubated in buffered peptone water
(BPW) at 37C for 18 h. In some cases, samples were
stored overnight at 4C before incubation was started.
Each pair of boot swab samples was incubated in
225 ml BPW, except for the two pairs from half houses,
which were incubated together in 450 ml BPW. Litter and
dust samples were divided and each half was incubated in
225 ml BPW. The drag swab sample was incubated in
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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 635–645
Salmonella detection in turkey flocks
90 ml BPW. The 60 individual faeces were each incubated
in 20 ml BPW.
The combined boot swab ⁄ dust swab sample was
incubated in 450 ml BPW.
Selective enrichment of Salmonella was carried out
using Modified Semi-solid Rappaport-Vassiliadis agar
(MSRV), followed by Rambach agar as described previ-
ously (Davies and Wray 1994).
Serotyping
Serotyping was performed according to the Kauffmann–
White–Scheme (Popoff 2001) and phage typing was
performed according to the HPA Colindale Scheme
(Anderson et al. 1977). One colony per plate, from the
end of the streak, was picked and serotyped.
Statistical analysis
The statistical analysis was done on two outcome measures
(i) whether or not Salmonella was detected at a flock visit
and (ii) the proportion of samples positive. The sampling
methods were compared using the GLMM (generalized
linear mixed models) procedure run in GENSTAT. The
farm ⁄ house ⁄ visit combinations were specified as random
effects and sampling method as a fixed effect.
Two models were fitted to the results from 106 flocks
using the six methods (BS1, BS2, BS5, litter sample, drag
swab, dust sample). The first (model 1) included these
methods individually and the second (model 2) combined
the BS1 and dust sample results into a single outcome,
regarding positive as either one or both methods positive.
A further model compared the proportions of flocks posi-
tive by the individual faecal sampling with the other
methods.
All pairs of methods were compared by dividing the
differences in the predicted means (on the logit scale) by
their standard errors and treating the results as a standard
normal deviate. The results are given as two-tailed
P-values. Because of the multiple comparisons, the
emphasis is placed on P-values <0Æ01.
Comparisons of the flock type prevalences detected by
BS5 were done by Fisher’s exact test.
Results
Detection of Salmonella in turkey flocks
The full set of samples was collected from 71 flocks, of
which 52 (73Æ2%) tested positive for Salmonella in at least
one sample. Of the remaining 35 flocks, where one or more
samples were not taken, 29 (82Æ9%) tested positive for
Salmonella. Therefore, we can regard the overall percentage
637
Salmonella detection in turkey flocks D. Mueller-Doblies et al.

Table 1 Number of Salmonella-positive houses in visited farms with Table 2 Results by serovar
more than one house
Number of Number of positive
Number of visits positive farms ⁄ positive
Serovar flocks companies
1 All houses negative 4Æ5%
3 One house positive 13Æ6% S. Typhimurium (STM) (including 16 8 farms
18 Two or more houses positive 81Æ8% all phagetypes and
14 All houses positive 63Æ6% untypeable isolates)
Total: 22 visits STM DT104 10 5 farms
STM DT120 1 1 farm
STM U302 2 1 farm
of 76Æ4% positive flocks as a lower limit for the percentage STM untypeable 2 1 farm
of positive flocks. Eight out of 15 breeding flocks (53%), 29 O rough:e,h:1,2 1 1 farm
O Rough:e,h:1,5 1 1 farm
of the 29 rearing flocks (100%) and 44 of the 62 finishing
O rough:i:1,2 1 1 farm
flocks (71%) were positive for Salmonella. Senftenberg 5 1 farm
To investigate whether it is likely that a farm where one 4, 12:d:- 1 1 farm
positive house was identified has one or more other posi- Kedougou 12 6 farms
tive houses, results from 97 flocks included in 22 visits were Derby 2 2 farms
analysed and are shown in Table 1. Farms with only one Indiana 2 1 farm
house and visits where only one house was sampled were Saintpaul 1 1 farm
Montevideo 2 1 farm
not included in the analysis. Of the 22 visits, only one farm
Kottbus 14 5 farms
(4Æ5%) had all houses negative for Salmonella. Three farms Newport 33 6 farms
(13Æ6%) had only one positive house; whereas the remain- (3 belonging to
ing 18 farms (81Æ8%) had two or more positive houses on same company)
the date of the visit. Fourteen out of the 22 sampled farms
(63Æ6%) had all houses positive for Salmonella.
Testing individual faeces did not add any more infor- two flocks negative, two flocks positive dust only, one
mation in terms of presence ⁄ absence of Salmonella, as all flock positive litter and dust only, one flock positive BS2
flocks, which had one or more positive individual faecal only; no individual faeces positive).
samples, also had at least one other positive sample (boot
swabs, litter, dust or drag swab) (data not shown).
Sensitivity of the BS1-method combined with a dust
sample
Serotyping of Salmonella isolates
Results from 65 eligible positive flocks were analysed to
From all visits, a total of 13 different serovars was see how many of the positive flocks were detected by the
isolated. Results are shown in Table 2. In 71 of the 81 combination of one pair of boot swabs combined with a
positive flocks (88%), only one serovar was detected, dust sample. Four flocks (6Æ2%) would have been missed
whereas in 10 flocks (12%) more than one serovar was if only these two samples had been taken. As the addition
detected. The most frequently isolated serovars were of a dust sample enhances the chance of detecting flocks
S. Typhimurium (eight farms), S. Kedougou (six farms), with a low prevalence, this method would be particularly
S. Newport (six farms) and S. Kottbus (five farms). suitable for monitoring of breeding flocks.

Sensitivity of the BS5 (EU survey) method
The results from 77 eligible positive flocks were analysed
to investigate the detection of positive flocks by the BS5
method compared to BS2, BS1, dust sample, drag swab
and litter sample. A flock was defined as positive if any
one of the samples was positive. If only the BS5 method
had been used, 9 of the 77 positive flocks (11Æ7%) would
have been missed. Interestingly, four out of the nine
flocks that would have been missed originated from the
same farm, and this particular farm was a breeding farm
with a low prevalence of Salmonella (six flocks sampled;
Sensitivity of the BS5 – method for different flock types
Eight of 15 breeding flocks (53%), 29 of the 29 rearing
flocks (100%) and 44 of the 62 finishing flocks (71%) were
positive for Salmonella, indicating a significantly lower
prevalence in breeding flocks than in rearing and finishing
flocks (P < 0Æ001). To further analyse whether different
sampling methods should be used to test different types of
flocks, we compared the number of BS5 samples that tested
positive for the different flock types and the number of
flocks from the different production groups (i.e. breeding,
rearing and finishing flocks) that would have been missed

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D. Mueller-Doblies et al. Salmonella detection in turkey flocks

100
100
All flocks (n = 77) Finishing flocks (n = 40)

40 60 80
40 60 80

Percent (%)
Percent (%)

20
20

0
0
0 1 2 3 4 5 0 1 2 3 4 5
No. pairs of boot swabs positive No. pairs of boot swab positive

100
100
Rearing flocks (n = 29) Breeding flocks (n = 8)

40 60 80
40 60 80

Percent (%)
Percent (%)

20
20

0
0

Figure 1 Number of BS5 samples that tested 0 1 2 3 4 5 0 1 2 3 4 5

positive for the different flock types. No. pairs of boot swabs positive No. pairs of boot swabs positive

by the BS5-method. Results are shown in Fig. 1. When all
flock types are analysed together, 53 of the 77 positive
flocks had all five pairs of boot swabs positive for Salmo-
nella. Only 9 of the 77 (11Æ7%) flocks were not detected by
any of the five pairs of boot swabs, but were positive by one
of the other sample types, as described above. However, if
the different types of flocks are analysed separately, results
vary markedly between the groups. Four out of the 40
(10%) positive finishing flocks were negative by any of the
five boot swabs, whereas only 1 of the 29 (3Æ4%) positive
rearing flocks were not detected by any of the five boot
swabs. In contrast to that, four of the eight (50%) of posi-
tive breeding flocks were missed by the five pairs of boot
swabs, indicating that the use of five pairs of boot swabs for
sampling might not be sufficiently sensitive to detect a rea-
sonable percentage of positive breeding flocks.
Individual faeces results
In 18 flocks (four breeding, two rearing and 12 finish-
ing flocks), Salmonella was not detected in individual
faeces samples. In the remaining 27 flocks (60%), where
Salmonella was detected, the within-flock prevalence
ranged from 1Æ7% to 96Æ7% with a mean of 42Æ9% and
a median of 26Æ7%.
Statistical comparisons of sampling methods
Samples that were included in the statistical analysis are
shown in Table 3.
Model 1
Salmonella detection
There were highly significant differences between the
sampling methods (Wald test, P £ 0Æ001). The predicted
means from the fitted model back-transformed to the
original scale (probabilities) and the P-values for the
method comparisons are given in Table 4a.
The five pairs of boot swabs (BS5) and the dust sample
were equally effective and significantly more sensitive than
the drag swab and the litter sample. The single pair of
boot swabs (BS1) was nearly as good as BS5 or dust and
also differed significantly from the drag swab at

Table 3 Samples included in the statistical

analysis Number of Number of
Number Number of % positive samples positive % positive
Type of visits positive visits visits (a) cultured samples samples (b)

BS1 102 60 58Æ8 102 60 58Æ8

BS2 100 56 56Æ0 100 56 56Æ0
BS5 102 68 66Æ7 510 294 57Æ6
Drag swab 93 38 40Æ9 93 38 40Æ9
Litter 95 50 52Æ6 207 110 53Æ1
Dust 86 56 65Æ1 172 100 58Æ1
All types 578 328 56Æ7 1184 578 48Æ8

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Salmonella detection in turkey flocks D. Mueller-Doblies et al.

Table 4 Model 1: (a) Salmonella detection and (b) proportion of samples positive: P-values for method comparisons*

P-values for method differences

Proportion of
Method flocks positive BS1 BS2 BS5 Drag swab Dust sample

(a)
BS1 0Æ590 *
BS2 0Æ556 0Æ420 *
BS5 0Æ668 0Æ052 0Æ006
Drag swab 0Æ439 <0Æ001 0Æ007 <0Æ001
Dust sample 0Æ671 0Æ055 0Æ008 0Æ941 <0Æ001
Litter sample 0Æ527 0Æ136 0Æ486 <0Æ001 0Æ046 0Æ001

P-values for method differences

Mean proportion
Method samples positive BS1 BS2 BS5 Drag swab Litter sample

(b)
BS1 0Æ590 *
BS2 0Æ554 0Æ456 *
BS5 0Æ578 0Æ747 0Æ522
Drag swab 0Æ445 0Æ003 0Æ027 <0Æ001
Dust sample 0Æ605 0Æ711 0Æ231 0Æ361 <0Æ001
Litter sample 0Æ493 0Æ046 0Æ150 0Æ003 0Æ267 0Æ002

*Bold values indicate a statistically significant result.

P < 0Æ001. The isolation rate from the single pair of boot Table 5 Model 2: Salmonella detection: P-values for method
swabs was greater than that from two pairs pooled (BS2), comparisons*

but not significantly so. P-values for method differences

Proportion of
Method flocks positive BS1+dust BS2 BS5 Drag swab
Proportion of samples positive
BS1 + dust 0Æ727 *
There were again highly significant differences between BS2 0Æ560 <0Æ001 *
the methods (Wald test, P < 0Æ001). The predicted means BS5 0Æ670 0Æ161 0Æ007
from the fitted model back-transformed to the original Drag swab 0Æ439 <0Æ001 0Æ006 <0Æ001
scale (probabilities) and the P-values for the method Litter sample 0Æ528 <0Æ001 0Æ462 <0Æ001 0Æ044

comparisons are given in Table 4b.
There was no evidence of differences among the boot
swab methods (BS1, BS2 and BS5) and the dust sample
and all were superior to the drag swab. BS5 and the
dust sample were also significantly greater than the litter
sample.
Model 2
Salmonella detection
There were highly significant differences between the
methods (Wald test, P £ 0Æ001). The predicted means
from the fitted model back-transformed to the original
scale (probabilities) and the P-values for the method
comparisons are given in Table 5.
The BS1+dust combination and BS5 were signifi-
cantly better than BS2 and the litter sample, which
gave similar results. The drag swab again had the worst
performance.
*Bold values indicate a statistically significant result.
Results for individual faecal samples
A comparison of all the individual methods using the
GLMM model showed that the proportion of flocks with
one or more positive individual faecal samples was signif-
icantly higher (P = 0Æ001) than the proportion positive
using the drag swab method (back-transformed means
0Æ64 and 0Æ44 respectively). It did not differ significantly
from any of the other methods,
Results for samples from breeding flocks
Table 6 shows the results of sampling 15 breeding
flocks. BS5 and BS1 provided equivalent detection
results but three flocks were only detected by dust
samples.

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D. Mueller-Doblies et al.
Results for combination sampling study
Table 7 shows the results of sampling 22 Salmonella-posi-
tive turkey flocks. Forty-six additional flocks were also
sampled with negative results. Salmonella was found in all
22 positive flocks where boot swabs and dust swabs were
combined as a single sample, compared with 20 flocks
where two pairs of boot swabs alone were used and 17
flocks were a dust swab only was used.
Discussion
Various sampling methods are currently used by the
turkey industry to monitor flocks for the presence of
Salmonella, but hardly any data are available on the
relative sensitivity of these methods. As most studies
comparing sampling methods have been carried out in
chicken houses, these data have to be interpreted with
care and only limited statements can be made about their
validity for turkey flocks. In small turkey companies or
independent farms, little monitoring is carried out, even
in breeding flocks, and sensitive as well as economic and
practical methods are required to optimize monitoring
programmes.
In the UK turkey industry, cloacal swabs have been
widely used as part of a routine monitoring scheme.
However, they have been shown to be relatively insensi-
tive, as they sample only a small amount of faeces (Funk
et al. 2000; Kotton et al. 2006), and a large number of
birds must be tested to compensate for intermittent shed-
ding and for a low within-flock prevalence (Cannon and
Roe 1982). Sensitivity may be further reduced when indi-
vidual samples from a number of animals are pooled
(Arnold et al. 2005, 2008), so alternative methods are
needed to get reliable information about the Salmonella-
status of a flock.
A widely-used approach is environmental sampling of
houses, which in general is regarded as more sensitive
than sampling a large number of individual birds, pro-
vided that appropriate samples are collected and tested
(Davies and Wray 1996a). The most common environ-
mental samples taken in poultry houses are faeces, litter
and dust. Salmonella-positive faeces provide indication of
a current infection of the flock, whereas dust may indi-
cate previous infection, which has passed its peak, result-
ing in a low within-flock prevalence at the time of
sampling.
Collecting individual faeces and culturing them indi-
vidually involves considerable work on the farm as well
as in the laboratory. Alternatively, individual faeces
could be cultured as pools, but it is likely that pooling
might result in a loss of sensitivity due to a dilution
effect, as described previously for pigs and cattle
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Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 635–645
Salmonella detection in turkey flocks
(Arnold et al. 2005, 2008). In our study, there was no
flock that was only identified as positive by taking
individual faecal samples, as all faeces-positive flocks
were also positive by at least one of the other methods
used.
Collecting and pooling pinches of litter from several
different locations in the house is another way of collect-
ing faecally-soiled material from a large number of birds,
but due to the different types of litter used (for example
straw or shavings) this method is difficult to standardize
and sampling of dry straw bedding is difficult. It has also
been shown, that direct litter sampling may be inferior to
drag swabs (Hayes et al. 2000), although this depends on
the sampling protocol employed.
Boot swabs are a convenient alternative to litter sam-
ples, as they collect faecal material from a larger num-
ber of birds than litter samples taken from a limited
number at point locations. Boot swabs can also be
easily posted to the laboratory and potentially provide a
more standardized sampling method than litter sam-
pling, which is more likely to be influenced by the dili-
gence of the sampler. These considerations as well as a
theoretical ability to detect a 1% within-flock prevalence
(Skov et al. 1999) were instrumental in the decision to
choose five pairs of boot swabs (cultured separately) as
a sampling method for the EU Salmonella Baseline
surveys of broilers, turkeys and laying hens on floor
systems (i.e., barns and free-range houses) (Anonymous
2004, 2005, 2006) and for the statutory monitoring of
chicken breeding flocks [Commission Regulation (EC)
No. 1003 ⁄ 2005)].
An alternative to the boot swabs is the sock model
(Gradel et al. 2002), and it was shown that two pairs
of socks, analysed as one sample, are at least as sensi-
tive as the collection of 60 individual broiler faeces,
analysed as one sample (Gradel et al. 2002). Another
study showed, that five pairs of socks appeared to be
as effective in detecting Salmonella in chicken flocks as
60 faecal pools (each consisting of five individual fae-
ces), whereas one pair of socks proved to be inferior to
60 pools of five, particularly in flocks with low preva-
lence (Skov et al. 1999). In another study, surgical shoe
covers resulted in better recovery of Salmonella than
traditional drag swabs (McCrea et al. 2005). In a UK
study, sock swabs were found to be more laborious
and less sensitive than boot swabs (Food Standards
Agency 2006).
Drag swabs are widely used in the poultry industry in
the USA, and slightly different models are in use. Most
models which have been described in the scientific litera-
ture consist of large gauze pads, which are dragged along
the floor of the poultry house picking up faeces from a
large number of birds. Different sizes of gauze pads
641
Salmonella detection in turkey flocks D. Mueller-Doblies et al.

have been reported (usually between 5 · 5 cm and 10 · Table 6 Breeding flocks tested for Salmonella
10 cm, constructed as a multiple swab assembly), and Farm House ID BS5 result BS1 result Dust result
studies have looked at different saturating ⁄ transport
media, such as skim milk, saline, buffered peptone water 1 A1 ) ) +
or chicken broth (Kingston 1981; Opara et al. 1992, 1994; 1 A2 ) ) +
1 B2 ) ) +
Davison et al. 1995; Byrd et al. 1997; Caldwell et al. 1998;
1 C1 ) ) )
Rolfe et al. 2000; Buhr et al. 2007). However, it is difficult 1 C2 ) ) )
to draw definitive conclusions from these studies as the 1 D* ) ) )
drag swab assemblies and the transport media vary 2 W1R ) ) )
between the different studies. Most of the drag swab 2 W1L ) ) )
models are also quite awkward to assemble, and use in 2 W2R + + )
densely-stocked poultry houses, especially turkey houses 2 W2L ) ) )
3 H4 + + Not sampled
where birds are less willing to move out of the paths of
3 H5 ) ) Not sampled
the sampler, can be difficult. 3 H6 + + Not sampled
In our study, we investigated a different type of drag 3 H7 + + Not sampled
swab which is marketed in the UK. There are no data 4 ) ) Not sampled
available comparing the performance of this type of drag
Positive flocks are marked in bold.
swab with the US style drag swab assemblies, but as it has
*Farm 1, house D was positive only by the BS2 sample.
a comparatively small surface and is also very light, it
may not achieve the same degree of contact with the
faeces as the gauze models and is therefore likely to be Table 7 Results of the comparison of sampling turkey houses using
less sensitive. boot swabs (BS), dust and combined samples – number of positive
Collection of dust is not as widely used as other samples

methods, but as it has been shown that Salmonella can BS+Dust

survive in dust for several months, this method pro- Flock ID BS2 Dust (combined) Serotype
vides a valuable tool for the detection of infections
1 + + + S Derby
which peaked prior to the time of sampling (Davies
2 + + + S Derby
and Wray 1996b). 3 + + + S Derby
Our results show that one pair of boot swabs taken 4 + + + S Derby
from the whole turkey house gives suitably sensitive results 5 + + + S Derby
for fattening and rearing flocks and is very cost-efficient. 6 + + + S Derby
The sensitivity can be further enhanced by adding a dust 7 + + + S Derby
sample, which is usually easy to collect from horizontal 8 + + + S Derby
9 + + + S Derby
surfaces such as partitions, beams and ledges at the same
10 + + + S Derby
time as carrying out the boot swabbing. The dust sample 11 + + + S Derby
seems to be particularly useful in flocks with a low preva- 12 + + + S Derby
lence, for example in breeding flocks, and our results 13 + + + S Derby
show that four of the eight (50%) of all Salmonella- 14 + + + S Derby
positive breeding flocks would have been missed by the 15 ) ) + S Derby
BS5 method, whereas the dust sample detected three 16 + ) + S Derby
17 + + + S Newport
out of four of those flocks that were negative by the BS5
18 + ) + S Derby
sample. 19 ) + + S Derby
Dust can easily be collected in houses with controlled 20 + ) + S Derby
ventilation where copious dust accumulates on extractor 21 + + + S Derby
fan baffles, however, it is not always present in some 22 + ) + S Derby
houses in sufficient amounts, especially in open (pole) total 20 17 22
barns or in houses of very young flocks, but the latter BS2 = Two pairs of boot swabs cultured together.
are not routinely sampled. In other cases, dust is pres-
ent only on high beams and is not easy to sample.
Also some farmers prefer to sample faeces since dust a large surface area using a moist fabric swab. Salmo-
sampling requires the wearing of a face mask and may nella appears to have a relative advantage in dust com-
require the use of goggles on some occasions. In such pared to other Enterobacteriaceae, which do not tend to
situations, the fine layer of dust can be gathered from survive in high numbers in dry conditions for such a

ª 2009 The Authors

642 Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 635–645
D. Mueller-Doblies et al.
long period (Haysom and Sharp 2003). This makes the
detection of Salmonella in dust easier than in faeces
samples where there is normally large, and potentially
overwhelming competitive flora, which must be effec-
tively suppressed by selective enrichment and plating
before Salmonella can be identified. As Salmonella has
been reported to survive in poultry houses for at least
53 weeks in dust (Davies and Wray 1996b), a dust
sample can provide an indication of an earlier infection
which has become undetectable by faecal monitoring.
However, faeces may provide a more accurate indica-
tion of substantial current infection, which may be
more relevant to contamination of carcasses at slaughter
and hence to public health.
Our data show further, that the drag swab and the lit-
ter sample are less effective for the detection of Salmo-
nella, and both methods only seem to be sensitive enough
for flocks with a relatively high prevalence. It is not clear
why the litter sample is less effective, but it might be due
to the effect of straw, which provides a sample with less
available surface area for faecal contamination than
wood-shavings, which provides more effective litter sam-
ples (data not shown).
The statistical analysis of our data showed that combin-
ing one pair of boot swabs (BS1) and a dust sample
detected more Salmonella-positive houses than any of the
individual methods, but the difference compared to BS5
was not statistically significant. However, the combination
of one pair of boot swabs and a dust sample proved the
easiest and fastest way to sample a house effectively, and
it was also cost-efficient, as only two cultures were
needed. The two samples can also be combined as shown
in the combination sampling study. Similar studies, which
indicate the possibility of combining sample types, have
also been carried out in broiler farms (Food Standards
Agency 2006). The high rate of isolation of Salmonella
from single pairs of boot swabs combined with dust in
flocks where dust samples were negative supports the
previous findings of equivalence of one pair and two pairs
of boot swabs.
Acknowledgements
The authors would like to thank the collaborating turkey
companies and farmers for their help in carrying out this
study; the Salmonella lab team at VLA Weybridge for
excellent technical assistance and help with farm visits;
staff from VLA regional labs for help with farm visits;
Felicity Clifton-Hadley (VLA Weybridge) for valuable
discussions and critical reading of the manuscript. This
work was funded by the Department for Environment,
Food and Rural Affairs (Defra) under project code
OZ0328.
ª 2009 The Authors
Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 107 (2009) 635–645
Salmonella detection in turkey flocks
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