JOURNAL OF CLINICAL MICROBIOLOGY, July 2005, p. 3033–3037 Vol. 43, No. 70095-1137/05/$08.00�0 doi:10.1128/JCM.43.7.3033–3037.2005Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Why fecal samples? The International Organization forStandardization and the European Committee for Standard-ization have recently decided to include the standardization offecal testing in their agenda. The aim is to prepare documentsdescribing standardized protocols for the detection of epide-miologically important zoonotic agents, which can often be foundin low numbers in fecal samples in food production animals. Theeffort is to provide standard protocols for primary samples aspart of the farm-to-fork approach. Samples taken from theprimary production herds have been rather neglected in thestandardization efforts compared to food or clinical samples.

The work will include both conventional and PCR-basedmethods in order to improve the detection limit, particularly insubclinically infected herds. The present review with recom-mendations is the first of three reviews which will pave the wayfor standard protocols, facilitating the comparison of epidemi-ological data and providing quantitative methods for microbi-ological risk assessments.

WHAT DO WE KNOW ABOUT SALMONELLA IN PIGS?

Salmonella spp. are some of the most common food-bornepathogens of humans (34). The estimated costs associated withhuman salmonellosis are nearly $1 billion (range, $0.6 to $3.5billion) annually in the United States (8). Pork is, in addition tobeef, dairy, poultry, and seafood, a major vehicle for the trans-mission of Salmonella from animals to humans. The reductionof human salmonellosis within communities would save sub-stantial clinical and economic resources. One major key toreducing the economic burden is the reduction of the level ofSalmonella in livestock animals (37). For example, in 1995Denmark introduced a nationwide control program of control-ling Salmonella in pork by monitoring the whole food chainfrom “feed to food.” The program successfully reduced thelevel 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 canbe difficult because of the continual nature of the animal pro-duction system and, therefore, a control strategy should focuson reducing the infection pressure at the herd level (37). Feces

are the main sample matrix taken for Salmonella monitoringprograms in swine herds, since pigs shed the pathogen throughfeces, which can be easily collected from individual animals.

The detection of Salmonella in feces is done mainly by usingtraditional bacteriological methods, despite the availability ofrapid, cost-effective, and reliable PCR-based methods devel-oped within the last few years. Here, an automated PCR test-ing system for monitoring Salmonella in swine herds couldscreen thousands of feces samples in a short period to obtainsubstantial data for qualitative and quantitative risk assess-ments. The expected high proportion of negative samplescould then be discarded, whereas the positive samples could becultured for the isolation of strains useful for further epidemi-ological studies. However, although the technology is available,such a system has not yet been implemented. The main reasonfor not using PCR testing more widely could be the lack ofstandardized protocols directed towards the detection andquantification of pathogens in such a difficult material as feces.Although substantial PCR standardization efforts have beendone on food samples (26), important primary samples such asfeed and feces have not yet received sufficient attention.

PREVALENCE AND IMPACT OF SALMONELLAIN PIG FECES

The detection of Salmonella in pigs is difficult, because in-fection does not commonly result in clinical symptoms. How-ever, subclinical Salmonella infections in pigs are an importantfood safety problem because of the transmission route of Sal-monella through the food chain to humans.

Salmonella in pigs can be divided in two groups. The firstgroup consists of the host-adapted serotype Choleraesuis andis usually associated with acute septicemia and enterocolitis.The second group consists of all other serotypes, which havebroader host ranges and are associated with systemic, enteric,or unapparent symptoms. In 1995, the USDA National AnimalHealth Monitoring System conducted a national study of U.S.pork producers in 16 states. Salmonella was found in 17.5% ofthe 988 pens sampled (35). Some of the more frequent Salmo-nella enterica serotypes from nonclinical finishing swine wereserotype Derby, serotype Agona, serotype Typhimurium, sero-type Brandenburg, and serotype Mbandaka. Serotype Choler-aesuis was, after serotype Derby, the second-most-isolated se-rotype from clinical swine. In Europe, the predominant

* Corresponding author. Mailing address: Danish Institute for Foodand Veterinary Research (DFVF), Bulowsvej 27, DK-1790 Copenha-gen, Denmark. Phone: 45-723-46 251. Fax: 45-723-46 001. E-mail:jho@dfvf.dk.

3033

serotype isolated from pigs is serotype Typhimurium, followedby serotype Derby (13). A national survey in Great Britainconducted over a 12-month period beginning in 1999 revealedthat Salmonella was present in 23% of cecal samples (11). Halfof these salmonellae were serotype Typhimurium, mainlyphage types DT104, DT193, DT208, and U302, all of which areresistant to a number of antibiotics. In Denmark, 6.2% of cecalsamples were found positive in a study investigating 13,468 pigswith a high prevalence of serotype Typhimurium (6). Nearlyhalf of the strains belonged to the phage type DT12.

Shedding of Salmonella by an asymptomatic carrier pig isintermittent and often in small numbers (14). This makes thedetection of Salmonella very difficult, and sensitive methods fordetection are needed. The duration of shedding in asymptom-atic Danish swine herds has been estimated to be on averagearound 18 to 26 days (22). The occurrences of Salmonella canvary between and within age groups within herds, and sheddingwas found on more than one occasion. Individual animals mayremain carriers for up to 36 weeks (39).

Pigs infected with Salmonella and showing clinical symptomsoften shed the pathogen in large numbers in their feces. Anumber of studies have shown that during acute disease, pigswill 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 Ty-phimurium DT104 strain via the oral route had shedding ratesup to 109 CFU during acute disease within the first days ofinfection (unpublished data).

Further background information on the epidemiology, trans-mission, pathogenesis, disease, and measures for control ofSalmonella in pigs is reviewed by Fedorka-Cray et al. (13).

TRADITIONAL VERSUS PCR-BASEDDETECTION TESTS

Nearly all studies investigating the prevalence of Salmonellain pig feces use reference culture methods. Samples from herdswith peak clinical salmonellosis can be easily identified directlyand without any enrichment by plating on selective agars,whereas samples from chronically infected pigs or from theenvironment always require preenrichments and selective en-richments. Various selective media have been used in epide-miological studies (13), and some of them fail to isolate thehost-adapted serotype Choleraesuis (10). For the isolation ofSalmonella from poultry feces material, many studies haveshown that modified semisolid Rappaport-Vassiliadis (MSRV)medium leads to higher sensitivities after 48 h of incubation at41.5°C � 1°C (36). It is obvious that MSRV also could be moreappropriate than liquid Rappaport-Vassiliadis medium for theexamination of swine feces. However, the large microbial loadof feces, with highly competing background floras, can hamperthe identification of Salmonella on the agar plates. Further-more, MSRV is intended for the detection of motile salmo-nellae and is less appropriate for the detection of nonmotilesalmonellae.

Surprisingly, few reports exist on PCR-based methods forthe detection of Salmonella in swine feces (31, 33), and com-prehensive comparisons of culture and PCR on swine feceshave not yet been published. The detection of Salmonella atlow levels by PCR methods still requires an enrichment culture

step for the multiplication of the cells prior to the PCR assay(to a level of approximately 103 to 104 cells per ml of enrichedbroth). Thus, careful consideration should be given to theenrichment strategies for Salmonella cells in swine feces incombination with subsequent PCR testing. An optimal enrich-ment should inhibit the growth of background flora but simul-taneously recover and multiply sublethally damaged Salmo-nella cells.

Often, buffered peptone water (BPW) is used as the nonse-lective broth, although it confers the risk of the overgrowth ofSalmonella colonies by a high level of background flora. Theaddition of novobiocin (0.1%) in the preenrichment step usingBPW followed by plating on MSRV could facilitate the detec-tion of Salmonella (19). Novobiocin causes a reduction in thenumber of gram-positive competitive background floras, lead-ing to a higher Salmonella/non-Salmonella ratio. Enrichmentculture prior to PCR was also successfully performed by usinga pooled culture broth of tetrathionate, selenite, and Rappa-port-Vassiliadis medium (31). After 5 days of selective incu-bation, there was a consistent detection by PCR compared tothe cultural method. Of 67 swine fecal samples tested, 41 werepositive by both methods. The PCR method detected two ad-ditional fecal samples as Salmonella positive. This indicatesthat a longer selective incubation could be useful to increasethe sensitivity of the method. However, more careful time-course studies under identical conditions are needed to clarifythe appropriate nonselective preenrichment and selective en-richment times necessary for PCR testing.

SAMPLE TREATMENT OR DNA PURIFICATION?

It is known that feces contain large amounts of phenolic andmetabolic compounds and polysaccharides that are inhibitoryfor PCR. Common inhibitors are DNases, polysaccharides, andproteases (38). Thus, sample treatment should be assessedbefore evaluating the primer selectivities on target and non-target strains (Fig. 1) (16). Much effort has been spent toneutralize such substances by using effective DNA purificationprotocols or PCR facilitators. Amplification facilitators aresubstances 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 polymer-ase in the presence of 4% instead of 0.4% (vol/vol) feces. Thepurity 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 BSAfractions are used. Another strategy to overcome PCR inhibi-tion is to use a polymerase that is more resistant than Taq. Forthe rTth and Pwo polymerases it was found that a 0.4% fecalhomogenate in the PCR was not inhibitory in comparison toTaq and other polymerases (1). A multifactorial design exper-iment would help to investigate all aforementioned parametersin a single experimental setup (21).

DNA purification methods can, in addition to removing in-hibitors, concentrate total genomic DNA, especially from bac-teria. A study has shown, using pig fecal samples, that thecommercially available QIAmp stool kit yielded higher, andless degraded, DNA than did the conventional phenol-chloro-

3034 MINIREVIEW J. CLIN. MICROBIOL.

form extraction method commonly used (18). Many studieswhich isolated food-borne pathogens directly from feces alsoused this kit, with good reproducibilities and sensitivities (17,29). Silica membrane columns provide a convenient methodfor purification of DNA which is relatively free of inhibitors.Spin columns can, however, result in cross-contamination dur-ing the handling steps. Therefore, high safety standards areneeded in the laboratory to avoid cross-contamination.

QUANTITATIVE PCR DATA

It is prerequisite to estimate the levels of Salmonella con-tamination in a swine herd or at the slaughterhouse in order toassess the risk of hygiene failure and provide appropriate riskmanagement options. The culture-based most probable num-ber (MPN) test (7) is particularly useful for the determinationof low concentrations of Salmonella in feces. Higher levels ofSalmonella (�500 CFU/g feces) can be determined by directplating on selective agar, such as xylose lysine deoxycholate.However, high levels of background flora can disturb thegrowth of Salmonella. Meanwhile, the use of real-time PCRallows a faster and reliable method for the quantification ofSalmonella in pig feces. Similar to what is seen with directplating, higher levels could be readily detected directly withoutan enrichment step. We developed a pre-PCR processing pro-tocol based on the QIAmp stool kit (QIAGEN), followed by areal-time PCR assay originally used (25) for the detection ofSalmonella in food. This assay, including a reference standardDNA and an internal amplification control, showed an accu-rate standard curve over a 5-log-unit linear range, enablingaccurate detection down to 100 to 200 CFU/g of feces.

However, the small-volume (1 ml) sample taken for an in-dividual PCR-based test may not facilitate the direct detectionof low numbers of Salmonella. An MPN-PCR strategy couldtherefore be employed (especially attractive as an alternativeto the labor-intensive culture-based MPN method). Here, the

detection of different concentrations of Salmonella is usuallydone after the first selective enrichment step. Based on thepresence or absence of Salmonella in the enriched broth, theMPN can be exactly calculated as applied for the culture-basedMPN (7). A semiquantitative strategy using real-time PCRcould be the calculation of the proportionality of cycle thresh-old values to the primary sample material; that is, a samplewith a higher initial Salmonella load may result in lower cyclethreshold values (reverse correlation). This has been shown forCampylobacter spp. in chicken rinse (20) but could be interest-ing to evaluate for feces, which have much higher levels ofbackground flora than does chicken rinse. An important issuehere is the calculation of the detection probability (21), inparticular with a logistic regression model to accurately eval-uate the detection limit of PCR (16).

CONCLUDING REMARKS AND RECOMMENDATIONSFOR STANDARDIZATION

Various gaps and recommendations for standardizationhave been identified and should be considered in future.

(i) BPW broths used for a preenrichment step can comefrom different producers, and they can contain minor differ-ences in composition (sometimes between batches) which canaffect the growth rate of Salmonella and its compatibility withthe subsequent PCR assay. Furthermore, the time necessaryfor preenrichment has historically been optimized for selectiveculturing, not for PCR. There are some indications that ashorter preenrichment time (e.g., 6 h) may improve the PCRdetection limit because of the consequent low number of back-ground floras (9, 12, 24).

(ii) It is necessary to develop pre-PCR sample treatmentmethods that are specifically designed for the feces.

(iii) The use of nonproprietary DNA purification methodsshould be encouraged in order to avoid the use of certaincommercial kits in proficiency trials. However, manufacturerscould provide kits that have performances similar to those ofnoncommercial reference methods and that comply with thestandard requirements.

(iv) Available DNA polymerase enzymes and buffers canvary substantially, with some being more prone to inhibition bythe harsh inhibitors of feces than others. Special attentionshould be given to this issue in standardized protocols.

(v) In real-time PCR, the interaction of a probe dye withfeces needs to be investigated in order to remove any possiblequenching effect of the matrix on the fluorescence activity ofthe probes.

(vi) The aforementioned issues should be considered in lightof the newly standardized procedures for PCR testing, whichrecommend the inclusion of internal amplification control, theprocessing of positive and negative controls (3), a consensus onthe determination of the cutoff level in real-time PCR (5), andthe use of statistical calculations for determining detectionprobability (24).

The availability of more-advanced but user-friendly real-time PCR provides us with a cost-effective alternative to cul-ture-based detection and quantification methods. It remains,however, to investigate the fate of sublethally injured Salmo-nella cells as well as the interference by dead Salmonella cellsin pig feces. PCR based on DNA detection is not able to

FIG. 1. Integrated approach to establishment of diagnostic PCRaccording to Hoorfar et al. (16). Adapted from reference 16 withpermission of the publisher.

VOL. 43, 2005 MINIREVIEW 3035

discriminate between dead and viable cells. However, the sam-pling of feces freshly taken directly from the ceca of pigsminimizes the detection of dead cells. Stressed cells must beconsidered as a risk, as they often do not multiply in selectivecultural media but are still detected by PCR. In future, meth-ods should be developed to discriminate among vital, sub-lethally injured, and dead cells. A promising application is theethidium monoazide (EMA)-PCR. EMA can selectively entercells with damaged membranes and subsequently be covalentlybound to DNA, inhibiting the PCR (28). Recently, it wasshown that EMA-PCR is a tool valuable for the quantitativedistinction between viable and dead Campylobacter cells inpoultry (30).

Practical recommendations for the use of PCR-based meth-ods to detect Salmonella in swine feces are given in Table 1.

ACKNOWLEDGMENTS

The work was supported in part by the European Union through theFood-PCR 2 research project as part of the Network of ExcellenceMED-VET-NET (FOOD-CT-2004-506122) under the 6th RTD Frame-work and by the Deutsche Forschungsgemeinschaft project “Experi-

mentelle Analyze der Wirkungsmechanismen von Probiotika beimSchwein.”

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

REFERENCES

1. Al-Soud, W. A., and P. Rådstrom. 1998. Capacity of nine thermostable DNApolymerases to mediate DNA amplification in the presence of PCR-inhib-iting samples. Appl. Environ. Microbiol. 64:3748–3753.

2. Al-Soud, W. A., and P. Rådstrom. 2000. Effects of amplification facilitatorson diagnostic PCR in the presence of blood, feces, and meat. J. Clin. Mi-crobiol. 38:4463–4470.

3. Anonymous. 2003. Microbiology of food and animal feeding stuffs—poly-merase chain reaction (PCR) for the detection of food-borne pathogens—general method specific requirements (ISO 22174:2005). International Or-ganization for Standardization, Geneva, Switzerland.

4. Anonymous. 2005. ISO 6579—microbiology of food and animal feedingstuffs—horizontal method for the detection of Salmonella (doc ISO/TC34 SC9 N 681, annex D: detection of Salmonella spp. in animal faeces and insamples of the primary production stage). International Organization forStandardization, Geneva, Switzerland.

5. Anonymous. 2005. Microbiology of food and animal feeding stuffs—real-time polymerase chain reaction (PCR) for the detection of food-bornepathogens—general requirements and definitions (CEN/TC 275/WG 6/TAG3 N 0110:2005). European Committee for Standardization, AFNOR, Paris,France.

6. Baggesen, D. L., H. C. Wegener, F. Bager, H. Stege, and J. Christensen. 1996.Herd prevalence of Salmonella enterica infections in Danish slaughter pigsdetermined by microbiological testing. Prev. Vet. Med. 26:201–213.

7. Blodgett, R. 2001. Most probable number determination from serial dilu-tions. Appendix 2. FDA’s bacteriological analytical manual. [Online.] http://www.cfsan.fda.gov/�ebam/bam-toc.html.

8. Buzby, J. C., T. Roberts, C. T. J. Lin, and J. M. MacDonald. 1996. Bacterialfoodborne disease—medical costs and productivity losses. USDA EconomicResearch Service, Washington, D.C.

9. Croci, L., E. Delibato, G. Volpe, D. De Medici, and G. Palleschi. 2004.Comparison of PCR, electrochemical enzyme-linked immunosorbent assays,and the standard culture method for detecting Salmonella in meat products.Appl. Environ. Microbiol. 70:1393–1396.

10. Davies, P. R., W. E. Morrow, F. T. Jones, J. Deen, P. J. Fedorka-Cray, andI. T. Harris. 1997. Prevalence of Salmonella in finishing swine raised indifferent production systems in North Carolina, USA. Epidemiol. Infect.119:237–244.

11. Davies, R. H., R. Dalziel, J. C. Gibbens, J. W. Wilesmith, J. M. Ryan, S. J.Evans, C. Byrne, G. A. Paiba, S. J. Pascoe, and C. J. Teale. 2004. Nationalsurvey for Salmonella in pigs, cattle and sheep at slaughter in Great Britain(1999–2000). J. Appl. Microbiol. 96:750–760.

12. Ellingson, J. L., J. L. Anderson, S. A. Carlson, and V. K. Sharma. 2004.Twelve hour real-time PCR technique for the sensitive and specific detectionof Salmonella in raw and ready-to-eat meat products. Mol. Cell. Probes18:51–57.

13. Fedorka-Cray, P. J., J. T. Gray, and C. Wray. 2005. Salmonella infection inpigs, p. 191–207. In C. Wray and A. Wray (ed.), Salmonella in domesticanimals. CABI Publishing, Oxon, United Kingdom.

14. Gray, J. T., T. J. Stabel, and P. J. Fedorka-Cray. 1996. Effect of dose on theimmune response and persistence of Salmonella choleraesuis infection inswine. Am. J. Vet. Res. 57:313–319.

15. Gutzmann, F., H. Layton, K. Simkins, and H. Jarolmen. 1976. Influence ofantibiotic-supplemented feed on occurrence and persistence of Salmonellatyphimurium in experimentally infected swine. Am. J. Vet. Res. 37:649–655.

16. Hoorfar, J., P. Wolffs, and P. Radstrom. 2004. Diagnostic PCR: validationand sample preparation are two sides of the same coin. APMIS 112:808–814.

17. Inglis, G. D., and L. D. Kalischuk. 2003. Use of PCR for direct detection ofCampylobacter species in bovine feces. Appl. Environ. Microbiol. 69:3435–3447.

18. Jensen, A. N., and J. Hoorfar. 2002. Optimal purification and sensitivequantification of DNA from fecal samples. J. Rapid Meth. Automat. Micro-biol. 10:231–244.

19. Jensen, A. N., G. Sørensen, D. L. Baggesen, R. Bødker, and J. Hoorfar. 2003.Addition of novobiocin in pre-enrichment step can improve Salmonella cul-ture of modified semisolid Rappaport-Vassiliadis. J. Microbiol. Methods55:249–255.

20. Josefsen, M. H., N. R. Jacobsen, and J. Hoorfar. 2004. Enrichment followedby quantitative PCR both for rapid detection and as a tool for quantitativerisk assessment of food-borne thermotolerant campylobacters. Appl. Envi-ron. Microbiol. 70:3588–3592.

21. Knutsson, R., C. Lofstrom, H. Grage, J. Hoorfar, and P. Rådstrom. 2002.Modeling of 5� nuclease real-time responses for optimization of a high-throughput enrichment PCR procedure for Salmonella enterica. J. Clin. Mi-crobiol. 40:52–60.

22. Kranker, S., L. Alban, J. Boes, and J. Dahl. 2003. Longitudinal study ofSalmonella enterica serotype Typhimurium infection in three Danish farrow-to-finish swine herds. J. Clin. Microbiol. 41:2282–2288.

TABLE 1. Recommendations for the PCR-based detection ofSalmonella in feces

Recommen-dation no. Recommendation

1...........................A pre-PCR cultural enrichment step should becarefully selected. For highly sensitive detec-tion a short preenrichment step in BPW(5–6 h) followed by selective enrichment onMSRV for 24–48 h is advantageous.

2...........................To eliminate PCR inhibitors, a DNA purifica-tion step should be performed. DNA purifi-cation kits based on silica-membrane basedcolumns result in PCR-compatible DNA.Other non-spin-column-based methods, suchas that using magnetic beads, should beused only if the efficiency and specificity aredocumented.

3...........................BSA in a concentration of 0.5–1 �g/�l shouldbe always added to the PCR reaction mix asa PCR facilitator. A dilution of the DNApreparation can overcome PCR inhibitors inan extract. Be aware of the consequentlower detection probability.

4 ...........................Only validated real-time PCR assays should beused, and they must include an internal am-plification control. The detection probabilityof the assay should be described.

5...........................Always analyze a fecal sample in duplicate. Ifat least one replicate is positive, the sampleshould be considered as positive.

6 ...........................Quantification can be performed with real-time PCR. The preprocessing step should bewell documented and describe the efficiencyof the isolation. A standard curve shouldcontain at least 4 � 2 or 6 � 1 data points.

7 ...........................The cultural isolation for comparison of PCRresults should follow the new recommenda-tions (4) of the International Organizationfor Standardization for feces samples: (i)BPW incubation at 37°C � 1°C for 18 h �2 h, (ii) on MSRV at 41.5°C � 1°C for 24 h� 3 h and, if negative, for a further incuba-tion for 24 h � 3 h; or (iii) on xylose lysinedeoxycholate at 37°C � 1°C for 24 h � 3 h.

3036 MINIREVIEW J. CLIN. MICROBIOL.

23. Kreader, C. A. 1996. Relief of amplification inhibition in PCR with bovineserum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62:1102–1106.

24. Lofstrom, C., R. Knutsson, C. E. Axelsson, and P. Radstrom. 2004. Rapidand specific detection of Salmonella spp. in animal feed samples by PCRafter culture enrichment. Appl. Environ. Microbiol. 70:69–75.

25. Malorny, B., E. Paccassoni, P. Fach, C. Bunge, A. Martin, and R. Helmuth.2004. Diagnostic real-time PCR for the detection of Salmonella in food.Appl. Environ. Microbiol. 70:7046–7052.

26. Malorny, B., P. T. Tassios, P. Rådstrom, N. Cook, M. Wagner, and J.Hoorfar. 2003. Standardization of diagnostic PCR for the detection of food-borne pathogens. Int. J. Food Microbiol. 83:39–48.

27. Nielsen, B., L. Alban, H. Stege, L. L. Sorensen, V. Mogelmose, J. Bagger, J.Dahl, and D. L. Baggesen. 2001. A new Salmonella surveillance and controlprogramme in Danish pig herds and slaughterhouses. Berl. Muench. Tieraer-ztl. Wochenschr. 114:323–326.

28. Nogva, H. K., S. M. Dromtorp, H. Nissen, and K. Rudi. 2003. Ethidiummonoazide for DNA-based differentiation of viable and dead bacteria by5�-nuclease PCR. BioTechniques 34:804–813.

29. Rudi, K., H. K. Hoidal, T. Katla, B. K. Johansen, J. Nordal, and K. S.Jakobsen. 2004. Direct real-time PCR quantification of Campylobacter jejuniin chicken fecal and cecal samples by integrated cell concentration and DNApurification. Appl. Environ. Microbiol. 70:790–797.

30. Rudi, K., B. Moen, S. M. Dromtorp, and A. L. Holck. 2005. Use of ethidiummonoazide and PCR in combination for quantification of viable and deadcells in complex samples. Appl. Environ. Microbiol. 71:1018–1024.

31. Sibley, J., B. Yue, F. Huang, J. Harding, J. Kingdon, M. Chirino-Trejo, and

G. D. Appleyard. 2003. Comparison of bacterial enriched-broth culture,enzyme linked immunosorbent assay, and broth culture-polymerase chainreaction techniques for identifying asymptomatic infections with Salmonellain swine. Can. J. Vet. Res. 67:219–224.

32. Smith, H. W., and J. E. T. Jones. 1967. Observations on experimental oralinfection with Salmonella dublin in calves and Salmonella choleraesuis in pigs.J. Pathol. Bacteriol. 93:141–156.

33. Stone, G. G., R. D. Oberst, M. P. Hays, S. McVey, and M. M. Chengappa.1994. Detection of Salmonella serovars from clinical samples by enrichmentbroth cultivation-PCR procedure. J. Clin. Microbiol. 32:1742–1749.

34. Thorns, C. J. 2000. Bacterial food-borne zoonoses. Rev. Sci. Tech. Off. Int.Epizoot. 19:226–239.

35. United States Department of Agriculture. 1997. Shedding of Salmonella byfinisher hogs in the U.S. [Online.] http://www.aphis.usda.gov/vs/ceah/ncahs/nahms/swine/swine95/sw95gap.htm.

36. Voogt, N., M. Raes, W. J. Wannet, A. M. Henken, and A. W. van de Giessen.2001. Comparison of selective enrichment media for the detection of Sal-monella in poultry faeces. Lett. Appl. Microbiol. 32:89–92.

37. Wegener, H. C., T. Hald, W. D. L. Fo, M. Madsen, H. Korsgaard, F. Bager,P. Gerner-Smidt, and K. Molbak. 2003. Salmonella control programs inDenmark. Emerg. Infect. Dis. 9:774–780.

38. Wilson, I. G. 1997. Inhibition and facilitation of nucleic acid amplification.Appl. Environ. Microbiol. 63:3741–3751.

39. Wood, R. L., and R. Rose. 1992. Populations of Salmonella typhimurium ininternal organs of experimentally infected carrier swine. Am. J. Vet. Res.53:653–658.

VOL. 43, 2005 MINIREVIEW 3037