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Journal of Clinical Microbiology, July 2005, p. 3033-3037, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3033-3037.2005
Copyright © 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 Danish Institute for Food and Veterinary Research (DFVF), DK-1790 Copenhagen, Denmark2


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INTRODUCTION
 
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.


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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 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.


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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 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).


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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 41.5°C ± 1°C (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 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 time-course studies under identical conditions are needed to clarify the appropriate nonselective preenrichment and selective enrichment times necessary for PCR testing.


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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).



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  		FIG. 1. Integrated approach to establishment of diagnostic PCR according to Hoorfar et al. (16). Adapted from reference 16 with permission of the publisher.

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-chloroform 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.


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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 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).


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CONCLUDING REMARKS AND RECOMMENDATIONS FOR STANDARDIZATION
 
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 real-time 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 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.


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


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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 "Experimentelle Analyze der Wirkungsmechanismen von Probiotika beim Schwein."

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


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FOOTNOTES
 
* Corresponding author. Mailing address: Danish Institute for Food and Veterinary Research (DFVF), Bülowsvej 27, DK-1790 Copenhagen, Denmark. Phone: 45-723-46 251. Fax: 45-723-46 001. E-mail: jho{at}dfvf.dk. Back


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Journal of Clinical Microbiology, July 2005, p. 3033-3037, Vol. 43, No. 7
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.7.3033-3037.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.




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