Journal of Applied Microbiology 2000, 89, 169ÿ177

Comparison of methods for isolating Salmonella bacteria from
faeces of naturally infected pigs
P.R. Davies1, P.K. Turkson1*, J.A. Funk1, M.A. Nichols1 S.R. Ladely2 and P.J. Fedorka-Cray2
1
Department of Farm Animal Health and Resource Management, College of Veterinary Medicine, North Carolina State
University, Raleigh, and 2USDA-ARS, Russell Research Center, Athens, GA, USA

99/12/99: received 20 December 1999, revised 13 March 2000 and accepted 15 March 2000

P.R. DAVIES, P.K. TURKSON, J.A. FUNK, M.A. NICHOLS, S.R. LADELY AND P.J. FEDORKA-CRAY.
2000.A series of experiments was conducted using faecal samples collected from commercial
swine farms to evaluate the effects of variation in methods used for the detection of
Salmonella bacteria. The primary objective of the studies was to compare the protocols
routinely used in two laboratories in the USA. The studies included ®ve experiments
comparing the enrichment protocols used routinely in the respective laboratories (Method
1: 10 g faecesÐbuffered peptone water (BPW) pre-enrichmentÐselective enrichment in
Rappaport/Vassiliadis (RV) broth; Method 2: 01g faecesÐprimary enrichments in
tetrathionate and Hajna GN brothsÐsecondary enrichment in RV broth). The effects of
enrichment temperatures (37 vs 42  C) using RV broth (two experiments) and delayed
secondary enrichment (four experiments) were also evaluated. Direct comparison of Method
1 and Method 2 indicated comparable results. However, when compared using faecal
samples of equal weight, the Method 2 enrichment protocol was more sensitive for
detecting Salmonella bacteria than the Method 1 protocol. Enrichment in RV at 42  C was
superior to 37  C, particularly for samples that were pre-enriched in BPW. Delayed
secondary enrichment increased detection of Salmonella bacteria in swine faeces. These
results highlight the imperfect sensitivity of culture methods, and the need for researchers
to consider the sensitivity of bacteriological methods in the design and interpretation of the
results of epidemiologic studies based on faecal culture

INTRODUCTION programme by the swine industry in Denmark, the leading
pork exporting nation, has also increased the importance of
Public concerns about food safety have drawn attention to
Salmonella control to the US swine industry, which has
the desirability of reducing the prevalence of food-borne
recently become a net exporter of pork products (Davies
pathogens in food animal populations. Salmonellosis was
1997; Mousing et al. 1997).
ranked by the United States Department of Agriculture to
Despite considerable advances in newer diagnostic meth-
be the most important food-borne disease in the USA
ods, conventional bacteriology remains the foundation of
linked to red meat and poultry products (Anon. 1995). A
epidemiological studies of Salmonella bacteria (Lax et al.
general conclusion of that review was that there is inade-
1995). It has been suggested that there may be more media
quate knowledge relating to the epidemiology of food-
and culture methods for the isolation of Salmonella bacteria
borne agents in animal production, and the need for a bet-
than for any other bacteria (Waltman 1998). A plethora of
ter understanding of the epidemiology of infections caused
studies have compared microbiological techniques for iso-
by Salmonella has been speci®cally emphasized (Tauxe
lating Salmonella bacteria from diverse materials, and con-
1991). The implementation of a national Salmonella control
¯icting ®ndings among studies abound (Harvey and Price
1981; D'aoust 1989; Busse 1995). The scope of this metho-
dological dilemma was highlighted in a survey of US veter-
Correspondence to: P. Davies, EpiCentre, Institute of Veterinary, Animal and inary laboratories culturing poultry tissue and
Biomedical Sciences, Wool Building, Massey University, Private Bag 11222,
Palmerston North, New Zealand (e-mail:p.r.davies@massey.ac.nz).
environmental samples for Salmonella bacteria. No two of
*Present address: Animal Science Department, University of Cape Coast, the 74 respondent laboratories used identical protocols, and
Ghana. the authors reported the use of 17 different selective
= 2000 The Society for Applied Microbiology

two experiments studies by the authors' respective groups. For the TTB samples. Faecal samples were swine in the USA published recently. In 1995. for 24 Salmonella bacteria in earlier studies. so that results from different studies slants. 1997). serotyping. including at Four experiments were performed to compare the two pre- least one culture-positive faecal sample on 24 (83%) farms enrichment/enrichment protocols. and a framework broth. into each of three culture tubes containing 9 ml GN Hajna going epidemiological studies in the USA. or quantify differences with triple sugar iron (TSI) and Christensen's urea agar among procedures. A 01 ml aliquot was transferred to 99 ml RV broth lected from individual pigs on commercial swine farms in and incubated at 37  C (for the Hajna and TTB samples). for some standardization of laboratory protocols. A series of 11 experiments was performed: four experi- Further impetus to conduct the methodological studies ments compared the pre-enrichment/enrichment protocols reported here came from disparate estimates of the preva. enrichment media or combinations of enrichment media. which were incubated overnight at 37  C. North Carolina. 2000). In the second laboratory. 1996). when sam. All sam- ples (excluding those in TTB) were incubated overnight at MATERIALS AND METHODS 37  C. and having previously demonstrated a may have contributed to the varied ®ndings. 1998) and are outlined schematically in media (Waltman and Mallinson 1995). and the authors argued there was a critical need Veterinary Services Laboratories (NVSL). respectively. Additional objectives collected from 459 pigs on four commercial farms (one were to compare temperatures for enrichment in farm per experiment) in North Carolina (96±121 pigs per Rappaport±Vassiliadis broth (37 vs 42  C) and to evaluate farm). The standard protocols used in the respective labora- considerable variation in the duration and temperature of tories have been described elsewhere (Davies et al. 1. Among other differences in the design ence in faecal sample weight (1 g vs 10 g) in the two stan- of these studies. and of faecal shedding of Salmonella bacteria by ®nishing hogs four experiments evaluated delayed secondary enrichment on 152 farms in 16 states found 60% of 6655 individual in RV broth. 1999). the use of different laboratory protocols dard protocols. = 2000 The Society for Applied Microbiology. The farms used for a h. Within experiments. all comparisons of plate with morphology consistent with Salmonella was isolation methods were made on paired samples collected screened biochemically. used instead of swabs to place small volume samples in It is hoped that these ®ndings will provide some basis for tubes. 1998. The fourth method involved tocols for researchers. The implications Fig. peptone water. USA. Because of the differ- (Davies et al. All farms had been shown to have or at 42  C (for the loopful and 10 g BPW samples). One colony per gical studies. Similarly. R . of the respective methods. the incubation period was All experiments were conducted using swine faeces col. IA. One loopful of faeces from each sample was placed more meaningful interpretation of earlier and other on. ples were to be collected as part of concurrent epidemiolo. and the use of 14 different plating Fedorka-Cray et al. 1997). Small animal faecal loops were faecal samples (Niet®eld et al. Laboratories used conventional biochemical screen- for interpreting data from different laboratories are ing of isolates prior to serotyping by the National obvious. D A V I E S E T A L . In this laboratory. In contrast. samples and 38% of farms had Salmonella bacteria (Anon. 25% of 2288 faecal samples were found to contain Salmonella bacteria. 89. Samples were placed into sterile plastic bags and further delayed secondary enrichment (DSE) with swine processed the same day. or collected from pigs ®ed as Salmonella bacteria were submitted to NVSL for observed defaecating. one experiment compared the lence of Salmonella bacteria in ®nishing pigs reported in two isolation methods in both laboratories. 48 h. 169ÿ177 . of Salmonella in Pork Production in 1996 (Anon. tergitol-4 agar and brilliant green sulphur agar plates. 1998. selective enrichment. and isolates presumptively identi- per rectum from individual pigs. Ames. in a study of 29 ®nishing farms in Comparison of enrichment protocols North Carolina in 1994/95. isolates were screened biochemically the need to standardize procedures. and were also serogrouped sessions at the First International Meeting on the Ecology before being sent to NVSL. USA. an additional protocols routinely used in the two laboratories which have treatment was included to enable comparison of the proto- collectively participated in most studies of Salmonella in cols using samples of equal weight. Each RV broth culture was streaked on xylosine±lysine± given experiment were selected by convenience. isolates were screened with can be compared was a common conclusion from workshop TSI and lysine-iron agar (LIA). diluting approximately 10 g faeces in 2% BPW solution in a 1:9 ratio by weight in plastic bags (Method 1). Journal of Applied Microbiology. a survey compared enrichment of RV broth at 37 and 42  C. The primary marked effect of sample weight on detection of Salmonella objective of the studies reported here was to compare the bacteria using Method 1 (Funk et al.170 P . sodium tetrathionate broth (TTB) or 2% buffered for developing standardized or comparable laboratory pro. O'Carroll et al.

Paired faecal samples were collected from 96 pigs at a ®n- ishing farm in North Carolina. In the second experiment (96 ®nishing pigs). 89. Transfer methods and but included an additional treatment using 37  C incuba- volumes into RV broth were as described above for the tion (air incubator) as well as 42  C incubation in a water- respective methods. The ®rst experiment (128 Athens. 1 Schematic outline of routine methods used in the respective laboratories to isolate Salmonella from swine faeces Comparison of Method 1 and Method 2 in both with current standard procedures). USA. Journal of Applied Microbiology. GA. Broths from Method 1 were streaked bath. while broths from Method 2 were pre-enrichment of 10 g faeces in BPW (as in Method 1) streaked on both XLT4 and BGS plates (in accordance and enrichment of a loopful of faeces in TTB for 48 h = 2000 The Society for Applied Microbiology. 10 boars at one farm) followed the Method 1 protocol isolation methods at each laboratory. where paired sets were either stored overnight at 4  C (NC sam. The samples were processed with both sows. 169ÿ177 . both on XLT4 plates only. SALMONELLA IN SWINE FAECES 171 Fig. Two experiments were conducted to compare enrichment ples) or transported on ice to the participating laboratory in in RV broth at 37 and 42  C. No attempt was made laboratories to standardize procedures for colony selection and con®r- mation. The samples were trans- Comparison of 37 and 42  C enrichment in RV broth ported to this laboratory at ambient temperature.

copenhagen. 4 and 8 between individual experiments. the proportion of all positive pigs detected with perature (20±22  C) for 4 days. {var. For both BPW and TTB. in two experiments. 847%) was very similar to Method 2 was pipetted to 99 ml RV broth and incubated in a 42  C (91. 820%). As the 4 day delay ment. both enrichment in compared . For individual samples across the three water-bath for 24 h (DSE). Statistical analysis Enrichment in TTB alone yielded 902% of positive results Comparisons of the proportion of positive samples were with Method 2. excluding Hajna broth) preceded RV (37 and Agreement between results of methods and laboratories 42  C) enrichment. on XLT4 agar plates and processed in an identical manner the two protocols showed excellent agreement (89% agree- to the primary enrichment samples. 4). R . When compared on sam- days before secondary enrichment was performed were also ples of equal weight (faecal loops). Salmonella bacteria were not isolated in experiment 3. one of which has been reported sepa. (80±98%) and kappa (059±088) varied considerably delays of 0 (secondary enrichment without delay). Journal of Applied Microbiology. after which a 01 ml aliquot Method 1 (94 of 111. These samples were streaked experiments in which Salmonella bacteria were detected. 01 ml broth was evaluated using the kappa statistic (Fleiss 1981). TTB (74%) and in Hajna (48%) alone yielded a higher proportion (P < 0001) of positive cultures than did pre- enrichment in BPW (23%). kappa ˆ 072). (Method 2. Plating was performed on XLT4 agar. Table 1 Number of culture-positive samples for four culture protocols in four experiments (n ˆ number of faecal samples evaluated) Experiment 1 Experiment 2 Experiment 3 Experiment 4 Total Treatment n ˆ 121 n ˆ 121 n ˆ 96 n ˆ 121 n ˆ 459 Method 1: BPW-RV Loop of faeces 9 6 0 11 26 10 g faeces* 14 8 0 72 94 Method 2: TTB/Hajna ± TTB-RV Loop of faeces ±TTB 13 10 0 59 82 Loop of faeces ±Hajna 8 6 0 39 53 Loop±Hajna or TTB{ 15 10 0 66 91 Positive (%) on any culture 20 (165) 10 (83) 0 (0) 81 (669) 111 (242) Serotypes isolated typhimurium typhimurium{ typhimurium derby typhimurium{ new brunswick brandenburg heidelberg 4. 1999).12:i monophasic *Standard method in Davies Laboratory. In all experiments. Salmonella bacteria were cul- Delayed secondary enrichment tured from 111 (242%) of the 459 faecal samples (Table Delayed secondary enrichment (DSE) was evaluated in 1). Overall. compared with 57% from enrichment in tested using McNemar's chi-square test for paired samples. D A V I E S E T A L . = 2000 The Society for Applied Microbiology. RV tubes teria ranged from 0% (experiment 3) to 65% (experiment from the primary enrichments were stored at room tem. Comparison of enrichment protocols Among the four experiments. 89. However. Hajna broth was not included because of logistic constraints and the fact that TTB enrichment usually contributes the majority RESULTS of isolates obtained with Method 2. percentage of agreement used in the initial study was arbitrary. Faecal sample weight had a marked effect on results with the Method 1 protocol. {Standard method in Cray Laboratory (samples positive with either TTB or Hajna enrichment). 169ÿ177 . was transferred to 99 ml RV broth by pipette. and the prevalence of samples containing Salmonella bac- rately (O'Carroll et al. GN Hajna. four experiments.172 P .

give. of which the individual laboratories ium var. organisms. Salmonella bacteria were isolated each identi®ed Salmonella bacteria in 37 pigs (81% agree- from more samples (76 of 192. untypable Lab. typhimurium (39) and Salm. from which Salmonella bac- teria were isolated on either XLT4 or BGS agars. 169ÿ177 . This qualitative difference in the growth of 061). Using the kappa statistic as a mea- enriched samples incubated in RV at 37  C had moderate sure of agreement. Of these. 4 and 8 days before secondary enrichment. Serotypes isolated were Salm. from enrichment in GN Hajna. Combining the two experiments which compared delays of 0. the same serotypes were isolated in all cate that sensitivity for detecting positive samples would be instances. and typical colonies were not present on Delayed secondary enrichment BGS plates of 37 (114%) samples. copenhagen. The data indi- both temperatures. Salmonella enrichment without delay (0 days). A total of nine serotypes were identi- than at 37  C (65 of 192. Journal of Applied Microbiology. compared with four of the seven single enrichment detected 784% of positive samples serotypes isolated following 37  C incubation. typhimurium. bietri the predominant serotype among marily attributable to the samples pre-enriched in BPW (44 isolates from both laboratories. = 2000 The Society for Applied Microbiology. P < 005) rather isolated from slightly more samples using Method 1 (37) than samples enriched in TTB (32 vs 28 samples positive. 396%) incubated at 42  C ment. copenhagen (15). heidelberg. whereas the between laboratories within methods (kappa ˆ 055. copenhagen Lab. 50 were culture-positive for Salmonella In experiment 1. respectively. but the difference was not statistically P ˆ 010). RV enrichment at 37 and 42  C follow. For the four (positive by either SE or DSE). experiments in which DSE after a 4-d delay was evaluated. 059) paired 42  C samples had much less growth of competing and between methods within laboratories (kappa ˆ 045. 2 33 26 37 bietri. while DSE alone detected samples from which Salmonella bacteria were isolated at 197 (817%) of positive samples (Table 3). Pooling the data from 70 samples in this experiment and 255 samples in the four experiments described above. Salmonella bacteria were isolated from a total of 71 of 178 (40%) faecal Comparison of 37 and 42  C enrichment in RV broth samples. ples incubated at 42  C. heidelberg. 46 after secondary were isolated from 19 of 136 faecal samples. kappa ˆ 060). This difference was pri- ®ed. Combining results from both laboratories. typhimurium var. 1 or 2 38 35 46 *Two serotypes were isolated from individual pigs in laboratory 1 and from four pigs in laboratory 2. and serotypes identi®ed at the two laboratories Method 1 Method 2 Method 1 or 2 Serotypes* Lab. 339%). compared with 47% (26 of 55) organisms seen at both temperatures. typhimur- sampled (Table 2). Comparison of Methods 1 and 2 in both laboratories resulted in detection of Salmonella bacteria in 50 of 96 (52%) faecal samples. with Salm. kentucky. All seven serotypes were isolated from sam. on those samples that were culture-negative after primary ing either BPW pre-enrichment or TTB enrichment enrichment. improved by approximately 25% if DSE were performed In experiment 2. with relatively light growth of competing results with Method 2. 1 24 29 37 bietri. seven serotypes of Salmonella bacteria bacteria after primary enrichment alone. Combining results from the four 37  C (seven). enrich- competing organisms was not evident for the TTB ment in TTB alone yielded 82% (45 of 55) of positive enriched cultures. typhimurium. 37 positive at 37  C. Salm. derby. SALMONELLA IN SWINE FAECES 173 Table 2 Number of cultures (pigs) found to contain Salmonella bacteria using Method 1 and Method 2. Many of the XLT4 plates from BPW pre- signi®cant (P ˆ 049). Salmonella bacteria were cultured from 46 of the pigs muenchen (87). typhimurium var. typical Salmonella colonies were not present on XLT4 plates of 28 (86%) samples. were culture-positive for Salmonella bacteria after 4 and 8 bated in RV broth at 42  C (16) than samples incubated at day delays. than Method 2 (35). and 56 and 51 samples bacteria were isolated from more samples (P ˆ 002) incu. 89. derby. Salmonella bacteria were positive at 42  C. mbandaka. give. the results indicated similar agreement to heavy growth of competing organisms.

89. Bager and Petersen 1991. However. All these studies of positive samples (i. (ii) pre-enrich. likely faeces. Hoofar and Baggessen Davies et al. The range of options that might be evaluated in to have low numbers of Salmonella bacteria that may have methodological studies of Salmonella isolation is over. D'aoust 1989. including (i) weight of what empirically with swine faecal samples. 04±07 indicates reasonable agreement. contaminated materials may be counterproductive (Aho tories indicates that differences in laboratory procedures 1992). positive on any culture) detected compared RV broth with Muller±Kaufmann tetrathionate by any one method ranged from 55 to 74%. and researchers restricted to the use teria and competing organisms in the source material of small samples (e. and re¯ects the Salmonella bacteria after single enrichment (SE) or delayed generally unreliable nature of bacteriological culture of secondary enrichment (DSE) with a 4 day delay individual faecal samples. and studies faecal sample (10 g vs approximately 1 g). Based on kappa statistics. values greater than 07 indicate excellent agreement (Fleiss Vassiliadis et al. relatively few studies have evaluated Pre-enrichment is conventionally recommended for methods for isolating Salmonella bacteria from swine materials. and (iv) plating on XLT4 agar alone vs plating on BPW pre-enrichment with faecal samples may be linked to XLT4 and BGS agars. the two studies that found RV broth to = 2000 The Society for Applied Microbiology. effort on the methods used in the respective laboratories The case for pre-enrichment of faecal samples is less clear. it is the popularity of RV enrichment broth. Table 3 Numbers of 742 faecal cultures found to contain (O'Carroll et al. or high inoculum ratios (Harvey were comparable with inter-laboratory differences as and Price 1980). DISCUSSION The likely effects of varying sample weights need to be considered when interpreting studies that employ different Enrichment in selective media is a standard procedure for sample weights for comparing enrichment methods isolation of Salmonella bacteria from contaminated materi. pre-enrichment of swine faecal samples are unlikely to account for differences in the results has become a common practice. citing the example of pig and other ®elds. D A V I E S E T A L . studies of its ef®cacy (Anon. (iii) incubation of RV broth at 42  C vs impact of pre-enrichment on the results obtained. and Method 2 was markedly superior to Method 1 when compared on samples of equal weight.174 P . such as food and environmental samples. 2000). comparing enrichment broths for isolating Salmonella bac- ment in BPW in Method 1 vs direct enrichment into TTB teria from swine faeces have failed to address the potential and Hajna broths. and reported con¯icting ®ndings (Skovgaard et al. for Salmonella bacteria. 1985. 2000). It is notable among these studies that the proportion Cherrington and Huis in't Veld 1993).g. 1993. for recent epidemiological studies in swine. 1997). or freezing and thawing (D'aoust 1989). Pre-enrichment appears to have been adopted some- methods have notable differences. (Skovgaard et al. which requires inferred that differences between the respective methods small inoculum volumes. This result is serendipitous because the 1998). This effect was again evident using Method 1 in this study. 169ÿ177 . Use of 37  C. Interestingly. The conventional interpretation of A relatively small number of previous studies comparing kappa is that values less than 04 indicate poor agreement enrichment broths with swine faecal (or caecal) samples has between tests. The kappa statistic is widely used to need for high inoculum ratios to be an advantage when assess agreement between observations in clinical medicine pre-enrichment is used but. 1987. 1997. Harvey and Price (1981) considered the sources of variation. but no previous example has been found faeces. in studies of piglets) should not use (Jameson 1962.e. 1981). and the approach for this study was to focus osmotic shock. The effect of faecal sample weight on detection of SE ‡ SE ± Total Salmonella bacteria has been largely ignored. 1999. In comparison the Method 1 protocol. with food samples. R . a potential liability if direct inoculation with large of the use of this statistic for comparing culture methods amounts of material is desirable. 1985). Further studies of the effect of fae- als. and some also included selenite broth to estimates of relative sensitivity observed in other studies (SB). Results with 10 g faecal samples in Method 1 were compar- able with results from approximately 1 g samples in Method 2. and the relative performance of isolation methods is cal sample weight using direct enrichment of swine faeces affected by the relative concentrations of Salmonella bac. However. This is similar broth (MKTB). been stressed or injured by factors such as thermal or whelming. The similarity and it has been suggested that pre-enrichment of heavily of the results obtained with the two methods and labora. Funk et al. Busse 1995). Journal of Applied Microbiology. a DSE ‡ 145 52 197 marked increase in Salmonella detection has been demon- DSE± 41 504 545 strated with increasing sample weights ranging from rectal Total 189 556 742 swabs (estimated 05 g) to 25 g faeces (Funk et al. in TTB are indicated. despite an apparent lack of reported in previous studies by our groups (Anon.

attributable to the period of room temperature storage sequently. plating on XLT4 agar alone detected over 90% of samples gested that the apparent contradictions in other previous found to contain Salmonella bacteria using double plating studies are attributable to the use of BPW pre-enrichment on both XLT4 and brilliant green agars. indicating that single 1985. earlier ®ndings (Davies et al. This observation is consistent agar compared with brilliant green agar is consistent with with some previously published work (Skovgaard et al. The present data. 169ÿ177 . rather than 37  C. compared with 24 h Salmonella bacteria. Kim et al. The favourable performance of XLT4 followed by RV enrichment. These results sup. It would be imprudent to recommend direct enrichment or pre-enrichment. 1997). tended to be reduced if pre-enrichment in BPW was used. 1987. the prevalent serotypes in the material. the two studies reporting and other sources. 1991. SALMONELLA IN SWINE FAECES 175 give inferior results used direct inoculation of faeces into Delayed secondary enrichment has also been effective in MKTB and selenite broth. Waltman et al. an inescapable conclusion is that increased diag- Recently. 1985. prior to enrichment in tetrathionate broths (Bager and In reviewing the literature on detection of Salmonella Petersen 1991. Design of epidemiological studies generally Studies with a range of materials have commonly found entails some compromise due to logistic and ®nancial con- RV to be superior to tetrathionate broths when compared straints. described here. The present of results from different studies. Vassiliadis et al. Cherrington and Huis in't Veld 1993). were recognized to standardized bacteriological procedures for studies that are be factors likely to in¯uence the results of studies compar. data also support incubation at 42  C. and the use of of their studies. However. increased diagnostic effort against the expected marginal Rhodes and Quesnel 1986). and the present data using (TTB/RV or Hajna/RV) yielded signi®cantly superior RV broth indicate a comparable increase in sensitivity of detection than did non-selective pre-enrichment in BPW the order of 25%. D'aoust 1989). Such a framework would provide ¯exibility port the respective practices employed in our laboratories for all researchers yet maintain some basis for comparison when enriching in RV (24 h) or TTB (48 h). it should be pointed out that due to the use of a water-bath ACKNOWLEDGEMENTS for the 42  C incubation instead of an air incubator (37  C). The for- ing the duration of enrichment (prolonged enrichment). potential benchmark for other swine researchers in the Trichopoulos et al. but did not signi®cantly increase detection some assessment of their relative sensitivity for detecting of Salmonella bacteria in RV broth. Cherrington and Huis poultry (Rigby and Pettit 1980. The authors acknowledge the co- advantage seen at 42  C. or by secondary respective laboratories yielded similar results provides a enrichment (Jameson 1962. 1972. operation of the individual pork producers who facilitated = 2000 The Society for Applied Microbiology. 1993). The observation that second- BPW for all samples (Vassiliadis et al. Salmonella bacteria from swine faeces enriched in TTB will yield increased detection (Harvey and Price 1967. Journal of Applied Microbiology. In contrast. 1970.1993) in't Veld 1993). indicate that double selective enrichment Waltmann et al. likely to have different aims and constraints. In a study using USA. and BPW pre-enrichment for increasing detection of Salmonella bacteria in samples from RV samples (Skovgaard et al. bacteria. of RV broth from samples pre-enriched in BPW. The nature of the source mate- gain in sensitivity of detection. there has been no previous effort to reconcile rather than to a second enrichment step. 1991. Curiously. in relation to the objectives rial. the more recent studies (Bager lowest recovery of Salmonella bacteria is consistent with and Petersen 1991. ing enrichment media (Harvey and Price 1981). superior results with RV broth used pre-enrichment in 1998. or tuitous result that the different methods used in our by using two enrichment steps in series. be it through more intensive sampling or the use of multiple enrichment broths or plating media. Most previous these contradictory ®ndings. prolonged Salmonella bacteria from swine faeces which differ from enrichment (48 h) was advantageous when using MKTB those evaluated in this study should be accompanied by and SB broths. However. (1999) reported that recovery of nostic endeavour. It is suggested that adoption of methods for isolating faecal samples from naturally infected pigs. a more rapid attainment of enrichment temperature This work was supported by a grant from the National and a more stable temperature may have contributed to the Pork Producers Council. D'aoust 1989). on samples studies of DSE have used TTB (Rigby and Pettit 1980. 1999). Investigators need to weigh the marginal cost of subsequent to pre-enrichment (Harvey and Price 1981. O'Carroll et al. Cherrington and Huis in't Veld 1993) the view that the bene®cial effects reported with DSE are did not cite the preceding studies identi®ed here and con. 89. Bager and ary enrichment without delay (0 days delay) yielded the Petersen 1991). of equal weight. including swine faeces (Niet®eld et al. reconciliation of ®ndings from different studies. Cherrington and Huis in't Veld 1993) and it is sug. compared with one of the methods enrichment (Bager and Petersen 1991). haphazard adoption by different investigators of the almost While enrichment for a period of 24 h is common. countless methodological options does present a barrier to reports indicate that detection can be improved by increas.

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