JOURNAL OF CLINICAL MICROBIOLOGY, July 2005, p.

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0095-1137/05/$08.000 doi:10.1128/JCM.43.7.30333037.2005
Copyright 2005, American Society for Microbiology. All Rights Reserved.

Vol. 43, No. 7

MINIREVIEW
Toward Standardization of Diagnostic PCR Testing of Fecal Samples:
Lessons from the Detection of Salmonellae in Pigs
B. Malorny1 and J. Hoorfar2*
Federal Institute for Risk Assessment (BfR), D-12277 Berlin, Germany,1 and Danish Institute for Food and
Veterinary Research (DFVF), DK-1790 Copenhagen, Denmark2
are the main sample matrix taken for Salmonella monitoring
programs in swine herds, since pigs shed the pathogen through
feces, which can be easily collected from individual animals.
The detection of Salmonella in feces is done mainly by using
traditional bacteriological methods, despite the availability of
rapid, cost-effective, and reliable PCR-based methods developed within the last few years. Here, an automated PCR testing system for monitoring Salmonella in swine herds could
screen thousands of feces samples in a short period to obtain
substantial data for qualitative and quantitative risk assessments. The expected high proportion of negative samples
could then be discarded, whereas the positive samples could be
cultured for the isolation of strains useful for further epidemiological studies. However, although the technology is available,
such a system has not yet been implemented. The main reason
for not using PCR testing more widely could be the lack of
standardized protocols directed towards the detection and
quantification of pathogens in such a difficult material as feces.
Although substantial PCR standardization efforts have been
done on food samples (26), important primary samples such as
feed and feces have not yet received sufficient attention.

Why fecal samples? The International Organization for

Standardization and the European Committee for Standardization have recently decided to include the standardization of
fecal testing in their agenda. The aim is to prepare documents
describing standardized protocols for the detection of epidemiologically important zoonotic agents, which can often be found
in low numbers in fecal samples in food production animals. The
effort is to provide standard protocols for primary samples as
part of the farm-to-fork approach. Samples taken from the
primary production herds have been rather neglected in the
standardization efforts compared to food or clinical samples.
The work will include both conventional and PCR-based
methods in order to improve the detection limit, particularly in
subclinically infected herds. The present review with recommendations is the first of three reviews which will pave the way
for standard protocols, facilitating the comparison of epidemiological data and providing quantitative methods for microbiological risk assessments.

WHAT DO WE KNOW ABOUT SALMONELLA IN PIGS?

Salmonella spp. are some of the most common food-borne
pathogens of humans (34). The estimated costs associated with
human salmonellosis are nearly $1 billion (range, $0.6 to $3.5
billion) annually in the United States (8). Pork is, in addition to
beef, dairy, poultry, and seafood, a major vehicle for the transmission of Salmonella from animals to humans. The reduction
of human salmonellosis within communities would save substantial clinical and economic resources. One major key to
reducing the economic burden is the reduction of the level of
Salmonella in livestock animals (37). For example, in 1995
Denmark introduced a nationwide control program of controlling Salmonella in pork by monitoring the whole food chain
from feed to food. The program successfully reduced the
level of Salmonella in pork from 3.5% in the year 1993 to 0.7%
in the year 2000 and saved the Danish state $25.5 million (27).
However, the eradication of Salmonella in swine herds can
be difficult because of the continual nature of the animal production system and, therefore, a control strategy should focus
on reducing the infection pressure at the herd level (37). Feces

PREVALENCE AND IMPACT OF SALMONELLA

IN PIG FECES
The detection of Salmonella in pigs is difficult, because infection does not commonly result in clinical symptoms. However, subclinical Salmonella infections in pigs are an important
food safety problem because of the transmission route of Salmonella through the food chain to humans.
Salmonella in pigs can be divided in two groups. The first
group consists of the host-adapted serotype Choleraesuis and
is usually associated with acute septicemia and enterocolitis.
The second group consists of all other serotypes, which have
broader host ranges and are associated with systemic, enteric,
or unapparent symptoms. In 1995, the USDA National Animal
Health Monitoring System conducted a national study of U.S.
pork producers in 16 states. Salmonella was found in 17.5% of
the 988 pens sampled (35). Some of the more frequent Salmonella enterica serotypes from nonclinical finishing swine were
serotype Derby, serotype Agona, serotype Typhimurium, serotype Brandenburg, and serotype Mbandaka. Serotype Choleraesuis was, after serotype Derby, the second-most-isolated serotype from clinical swine. In Europe, the predominant

* Corresponding author. Mailing address: Danish Institute for Food

and Veterinary Research (DFVF), Bu
lowsvej 27, DK-1790 Copenhagen, Denmark. Phone: 45-723-46 251. Fax: 45-723-46 001. E-mail:
jho@dfvf.dk.
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MINIREVIEW

serotype isolated from pigs is serotype Typhimurium, followed

by serotype Derby (13). A national survey in Great Britain
conducted over a 12-month period beginning in 1999 revealed
that Salmonella was present in 23% of cecal samples (11). Half
of these salmonellae were serotype Typhimurium, mainly
phage types DT104, DT193, DT208, and U302, all of which are
resistant to a number of antibiotics. In Denmark, 6.2% of cecal
samples were found positive in a study investigating 13,468 pigs
with a high prevalence of serotype Typhimurium (6). Nearly
half of the strains belonged to the phage type DT12.
Shedding of Salmonella by an asymptomatic carrier pig is
intermittent and often in small numbers (14). This makes the
detection of Salmonella very difficult, and sensitive methods for
detection are needed. The duration of shedding in asymptomatic Danish swine herds has been estimated to be on average
around 18 to 26 days (22). The occurrences of Salmonella can
vary between and within age groups within herds, and shedding
was found on more than one occasion. Individual animals may
remain carriers for up to 36 weeks (39).
Pigs infected with Salmonella and showing clinical symptoms
often shed the pathogen in large numbers in their feces. A
number of studies have shown that during acute disease, pigs
will shed up to 106 serotype Choleraesuis organisms (32) or 107
serotype Typhimurium organisms per gram of feces (15, 39).
Pigs infected experimentally with 108 CFU of a serotype Typhimurium DT104 strain via the oral route had shedding rates
up to 109 CFU during acute disease within the first days of
infection (unpublished data).
Further background information on the epidemiology, transmission, pathogenesis, disease, and measures for control of
Salmonella in pigs is reviewed by Fedorka-Cray et al. (13).
TRADITIONAL VERSUS PCR-BASED
DETECTION TESTS
Nearly all studies investigating the prevalence of Salmonella
in pig feces use reference culture methods. Samples from herds
with peak clinical salmonellosis can be easily identified directly
and without any enrichment by plating on selective agars,
whereas samples from chronically infected pigs or from the
environment always require preenrichments and selective enrichments. Various selective media have been used in epidemiological studies (13), and some of them fail to isolate the
host-adapted serotype Choleraesuis (10). For the isolation of
Salmonella from poultry feces material, many studies have
shown that modified semisolid Rappaport-Vassiliadis (MSRV)
medium leads to higher sensitivities after 48 h of incubation at
41.5C 1C (36). It is obvious that MSRV also could be more
appropriate than liquid Rappaport-Vassiliadis medium for the
examination of swine feces. However, the large microbial load
of feces, with highly competing background floras, can hamper
the identification of Salmonella on the agar plates. Furthermore, MSRV is intended for the detection of motile salmonellae and is less appropriate for the detection of nonmotile
salmonellae.
Surprisingly, few reports exist on PCR-based methods for
the detection of Salmonella in swine feces (31, 33), and comprehensive comparisons of culture and PCR on swine feces
have not yet been published. The detection of Salmonella at
low levels by PCR methods still requires an enrichment culture

J. CLIN. MICROBIOL.

step for the multiplication of the cells prior to the PCR assay
(to a level of approximately 103 to 104 cells per ml of enriched
broth). Thus, careful consideration should be given to the
enrichment strategies for Salmonella cells in swine feces in
combination with subsequent PCR testing. An optimal enrichment should inhibit the growth of background flora but simultaneously recover and multiply sublethally damaged Salmonella cells.
Often, buffered peptone water (BPW) is used as the nonselective broth, although it confers the risk of the overgrowth of
Salmonella colonies by a high level of background flora. The
addition of novobiocin (0.1%) in the preenrichment step using
BPW followed by plating on MSRV could facilitate the detection of Salmonella (19). Novobiocin causes a reduction in the
number of gram-positive competitive background floras, leading to a higher Salmonella/non-Salmonella ratio. Enrichment
culture prior to PCR was also successfully performed by using
a pooled culture broth of tetrathionate, selenite, and Rappaport-Vassiliadis medium (31). After 5 days of selective incubation, there was a consistent detection by PCR compared to
the cultural method. Of 67 swine fecal samples tested, 41 were
positive by both methods. The PCR method detected two additional fecal samples as Salmonella positive. This indicates
that a longer selective incubation could be useful to increase
the sensitivity of the method. However, more careful timecourse studies under identical conditions are needed to clarify
the appropriate nonselective preenrichment and selective enrichment times necessary for PCR testing.
SAMPLE TREATMENT OR DNA PURIFICATION?
It is known that feces contain large amounts of phenolic and
metabolic compounds and polysaccharides that are inhibitory
for PCR. Common inhibitors are DNases, polysaccharides, and
proteases (38). Thus, sample treatment should be assessed
before evaluating the primer selectivities on target and nontarget strains (Fig. 1) (16). Much effort has been spent to
neutralize such substances by using effective DNA purification
protocols or PCR facilitators. Amplification facilitators are
substances that can enhance the efficiency of PCR. For feces,
it was shown that the addition of bovine serum albumin (BSA)
is most effective for overcoming PCR-inhibitory substances (2).
Many authors have used 0.4% (wt/vol) BSA in the PCR (2, 29):
this concentration allowed DNA amplification by Taq polymerase in the presence of 4% instead of 0.4% (vol/vol) feces. The
purity of the BSA seems to play a minor role (29). However,
acetylated BSA in high concentrations could inhibit the PCR
(23), and it is highly recommended that proteinase-free BSA
fractions are used. Another strategy to overcome PCR inhibition is to use a polymerase that is more resistant than Taq. For
the rTth and Pwo polymerases it was found that a 0.4% fecal
homogenate in the PCR was not inhibitory in comparison to
Taq and other polymerases (1). A multifactorial design experiment would help to investigate all aforementioned parameters
in a single experimental setup (21).
DNA purification methods can, in addition to removing inhibitors, concentrate total genomic DNA, especially from bacteria. A study has shown, using pig fecal samples, that the
commercially available QIAmp stool kit yielded higher, and
less degraded, DNA than did the conventional phenol-chloro-

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MINIREVIEW

3035

detection of different concentrations of Salmonella is usually

done after the first selective enrichment step. Based on the
presence or absence of Salmonella in the enriched broth, the
MPN can be exactly calculated as applied for the culture-based
MPN (7). A semiquantitative strategy using real-time PCR
could be the calculation of the proportionality of cycle threshold values to the primary sample material; that is, a sample
with a higher initial Salmonella load may result in lower cycle
threshold values (reverse correlation). This has been shown for
Campylobacter spp. in chicken rinse (20) but could be interesting to evaluate for feces, which have much higher levels of
background flora than does chicken rinse. An important issue
here is the calculation of the detection probability (21), in
particular with a logistic regression model to accurately evaluate the detection limit of PCR (16).
CONCLUDING REMARKS AND RECOMMENDATIONS
FOR STANDARDIZATION
FIG. 1. Integrated approach to establishment of diagnostic PCR
according to Hoorfar et al. (16). Adapted from reference 16 with
permission of the publisher.

form extraction method commonly used (18). Many studies

which isolated food-borne pathogens directly from feces also
used this kit, with good reproducibilities and sensitivities (17,
29). Silica membrane columns provide a convenient method
for purification of DNA which is relatively free of inhibitors.
Spin columns can, however, result in cross-contamination during the handling steps. Therefore, high safety standards are
needed in the laboratory to avoid cross-contamination.
QUANTITATIVE PCR DATA
It is prerequisite to estimate the levels of Salmonella contamination in a swine herd or at the slaughterhouse in order to
assess the risk of hygiene failure and provide appropriate risk
management options. The culture-based most probable number (MPN) test (7) is particularly useful for the determination
of low concentrations of Salmonella in feces. Higher levels of
Salmonella (500 CFU/g feces) can be determined by direct
plating on selective agar, such as xylose lysine deoxycholate.
However, high levels of background flora can disturb the
growth of Salmonella. Meanwhile, the use of real-time PCR
allows a faster and reliable method for the quantification of
Salmonella in pig feces. Similar to what is seen with direct
plating, higher levels could be readily detected directly without
an enrichment step. We developed a pre-PCR processing protocol based on the QIAmp stool kit (QIAGEN), followed by a
real-time PCR assay originally used (25) for the detection of
Salmonella in food. This assay, including a reference standard
DNA and an internal amplification control, showed an accurate standard curve over a 5-log-unit linear range, enabling
accurate detection down to 100 to 200 CFU/g of feces.
However, the small-volume (1 ml) sample taken for an individual PCR-based test may not facilitate the direct detection
of low numbers of Salmonella. An MPN-PCR strategy could
therefore be employed (especially attractive as an alternative
to the labor-intensive culture-based MPN method). Here, the

Various gaps and recommendations for standardization

have been identified and should be considered in future.
(i) BPW broths used for a preenrichment step can come
from different producers, and they can contain minor differences in composition (sometimes between batches) which can
affect the growth rate of Salmonella and its compatibility with
the subsequent PCR assay. Furthermore, the time necessary
for preenrichment has historically been optimized for selective
culturing, not for PCR. There are some indications that a
shorter preenrichment time (e.g., 6 h) may improve the PCR
detection limit because of the consequent low number of background floras (9, 12, 24).
(ii) It is necessary to develop pre-PCR sample treatment
methods that are specifically designed for the feces.
(iii) The use of nonproprietary DNA purification methods
should be encouraged in order to avoid the use of certain
commercial kits in proficiency trials. However, manufacturers
could provide kits that have performances similar to those of
noncommercial reference methods and that comply with the
standard requirements.
(iv) Available DNA polymerase enzymes and buffers can
vary substantially, with some being more prone to inhibition by
the harsh inhibitors of feces than others. Special attention
should be given to this issue in standardized protocols.
(v) In real-time PCR, the interaction of a probe dye with
feces needs to be investigated in order to remove any possible
quenching effect of the matrix on the fluorescence activity of
the probes.
(vi) The aforementioned issues should be considered in light
of the newly standardized procedures for PCR testing, which
recommend the inclusion of internal amplification control, the
processing of positive and negative controls (3), a consensus on
the determination of the cutoff level in real-time PCR (5), and
the use of statistical calculations for determining detection
probability (24).
The availability of more-advanced but user-friendly realtime PCR provides us with a cost-effective alternative to culture-based detection and quantification methods. It remains,
however, to investigate the fate of sublethally injured Salmonella cells as well as the interference by dead Salmonella cells
in pig feces. PCR based on DNA detection is not able to

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J. CLIN. MICROBIOL.

MINIREVIEW

TABLE 1. Recommendations for the PCR-based detection of

Salmonella in feces
Recommendation no.

Recommendation

1 ...........................A pre-PCR cultural enrichment step should be

carefully selected. For highly sensitive detection a short preenrichment step in BPW
(56 h) followed by selective enrichment on
MSRV for 2448 h is advantageous.
2 ...........................To eliminate PCR inhibitors, a DNA purification step should be performed. DNA purification kits based on silica-membrane based
columns result in PCR-compatible DNA.
Other non-spin-column-based methods, such
as that using magnetic beads, should be
used only if the efficiency and specificity are
documented.
3 ...........................BSA in a concentration of 0.51 g/l should
be always added to the PCR reaction mix as
a PCR facilitator. A dilution of the DNA
preparation can overcome PCR inhibitors in
an extract. Be aware of the consequent
lower detection probability.
4 ...........................Only validated real-time PCR assays should be
used, and they must include an internal amplification control. The detection probability
of the assay should be described.
5 ...........................Always analyze a fecal sample in duplicate. If
at least one replicate is positive, the sample
should be considered as positive.
6 ...........................Quantification can be performed with realtime PCR. The preprocessing step should be
well documented and describe the efficiency
of the isolation. A standard curve should
contain at least 4 2 or 6 1 data points.
7 ...........................The cultural isolation for comparison of PCR
results should follow the new recommendations (4) of the International Organization
for Standardization for feces samples: (i)
BPW incubation at 37C 1C for 18 h
2 h, (ii) on MSRV at 41.5C 1C for 24 h
3 h and, if negative, for a further incubation for 24 h 3 h; or (iii) on xylose lysine
deoxycholate at 37C 1C for 24 h 3 h.

discriminate between dead and viable cells. However, the sampling of feces freshly taken directly from the ceca of pigs
minimizes the detection of dead cells. Stressed cells must be
considered as a risk, as they often do not multiply in selective
cultural media but are still detected by PCR. In future, methods should be developed to discriminate among vital, sublethally injured, and dead cells. A promising application is the
ethidium monoazide (EMA)-PCR. EMA can selectively enter
cells with damaged membranes and subsequently be covalently
bound to DNA, inhibiting the PCR (28). Recently, it was
shown that EMA-PCR is a tool valuable for the quantitative
distinction between viable and dead Campylobacter cells in
poultry (30).
Practical recommendations for the use of PCR-based methods to detect Salmonella in swine feces are given in Table 1.
ACKNOWLEDGMENTS
The work was supported in part by the European Union through the
Food-PCR 2 research project as part of the Network of Excellence
MED-VET-NET (FOOD-CT-2004-506122) under the 6th RTD Framework and by the Deutsche Forschungsgemeinschaft project Experi-

mentelle Analyze der Wirkungsmechanismen von Probiotika beim

Schwein.
The authors thank Nigel Cook of CSL for the critical reading.
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