Enumeration of Salmonella in Feces of Naturally Infected Pigs

Abstract

Quantification of Salmonella in asymptomatic pigs can be used to institute control measures and to assess risk of carcass contamination during slaughter. The objective of this study was to quantify the fecal concentration of Salmonella in naturally infected pigs. Individual fecal samples (positive [n=443], negative [n=1225] determined by microbiological culture) were submitted for direct quantitative real-time polymerase chain reaction (q-PCR). Direct q-PCR categorized 99.6% (1220/1225) of culture negative samples as negative. For culture positive samples, 15.4% (68/443) were detected by q-PCR, but only 3.4% (15/443) were within the direct q-PCR quantifiable range (≥10³ colony-forming units [CFU]/g of feces). Of these latter samples, the concentration range was 1.06×10³ to 1.73×10⁶ CFU/g feces. Of the 15 samples with high Salmonella concentrations, seven were collected from one pig and three samples were collected from its penmates. Direct q-PCR may be an alternative to traditional culture-dependent methods for detection of pigs with high fecal concentrations of Salmonella, but not for detection of pigs shedding low concentrations of Salmonella, which represented the majority of pigs in this study. When high shedding was detected it was clustered within a single pig and its penmates. These data contribute to quantitative risk assessments of the association between concentrations of Salmonella shed by pigs during the finishing phase and risk of carcass contamination at slaughter.

Introduction

S almonella species are one of the major causes of foodborne diseases in the United States and worldwide (Greig and Ravel, 2009; Scallan et al., 2011). A significant number of human cases of salmonellosis (1% to 6%) have been related to consumption of pork and pork products in North America (Miller et al., 2005; Ravel et al., 2009; CDC, 2010, 2013; Guo et al., 2011). Swine may be asymptomatic reservoirs for Salmonella and may shed intermittently in their feces (Wood and Rose, 1992; Scherer et al., 2008), which can be a source of carcass contamination (Baptista et al., 2010; van Hoek et al., 2012) and subsequent transmission to humans.

Since Salmonella is a ubiquitous organism, eradication as a control measure is not viable (Doyle and Erickson, 2012). Decreasing the concentration of Salmonella shed by swine may represent a more achievable disease-control target. Enumeration of bacterial load can be used to identify contamination pressure and to identify effective control measures to reduce contamination in swine herds (Fravalo et al., 2003). In addition, data are needed for quantitative microbial risk assessments and for modeling transmission patterns of Salmonella (EFSA, 2010). Most of what is known about the concentration of Salmonella shed in pig feces comes from experimental infection studies (Wood and Rose, 1992; Osterberg and Wallgren, 2008; Scherer et al., 2008; Osterberg et al., 2009; Rostagno et al., 2011). A limited number of studies have quantified Salmonella concentration in feces of naturally infected swine. These were either cross-sectional studies (Fravalo et al., 2003; Fablet et al., 2006; van Hoek et al., 2012) or estimates of pen contamination in lairage (O'Connor et al., 2006; Boughton et al., 2007).

Traditionally, quantification of Salmonella in fecal samples has been based on culture-dependent methodologies (Fravalo et al., 2003; Fablet et al., 2006; O'Connor et al., 2006; Boughton et al., 2007; Osterberg and Wallgren, 2008; Osterberg et al., 2009). Quantitative methods based on culture are time consuming (3–7 days), labor intensive, and costly and therefore are not practical for use in studies with a large number of samples. Culture-independent methods such as direct quantitative real-time polymerase chain reaction (q-PCR) assays have been used to quantify Salmonella in food matrices (Cheng et al., 2009; Elizaquivel et al., 2011), pork carcass swabs (Guy et al., 2006; Löfström et al., 2011), chicken rinses (Wolffs et al., 2006), and swine fecal material (Harris et al., 2007; Abley, 2011). The objective of this study was to use q-PCR assay to quantify the fecal concentration of Salmonella in naturally infected pigs.

Materials and Methods

Sample collection and processing

The samples were collected from a longitudinal study on a multisite farrow-to-finish production system (three finishing sites with six cohorts each) located in the Midwestern United States. Criteria used for the selection of the production system, finishing sites, pig selection, sample collection, and laboratory isolation of Salmonella were described previously (Pires et al., 2012). Fecal samples (10 g) were processed and cultured using standard methods described previously (Pires et al., 2012).

An aliquot of each fecal sample (200 mg) was stored at −80°C for use in q-PCR. A random selection of culture-negative samples (n=1225) and all culture-positive samples (n=443) were submitted for direct q-PCR. A list of culture negative samples was generated by simple random sampling of negative samples using a commercial statistical software package (Proc surveyselect; SAS 9.3; SAS Institute, Cary, NC). The sample size of culture-negative samples was based on a Bayesian approach for sample size calculations for surveys to substantiate freedom from an infectious agent (Johnson et al., 2004) and using software available online (Bayesfreecalc2; Available online at http://www.epi.ucdavis.edu/diagnostictests/module02.html). Assumptions for calculation of sample size for culture negative samples for q-PCR evaluation were as follows: <5% of the culture-negative samples would be false positive by direct q-PCR; microbiological culture sensitivity >0.6 and mode of 0.7; and microbiological culture specificity >0.95 and a mode of 0.99. Based on these assumptions, a sample size of 1225 provided at least 92.8% confidence that the true prevalence was zero.

Template DNA preparation and q-PCR assay

The DNA was extracted from 200 mg of feces using the QIAamp Stool Mini Kit (Qiagen, Valencia, CA) according to manufacturer instructions with slight modification. Final DNA was eluted in 75 μL of buffer AE provided in the kit. Steam-sterilized fecal samples that were culture-negative for Salmonella were spiked with serial 10-fold dilutions of Salmonella culture isolated from a pig in the current study. The DNA was extracted from 200 mg of the spiked fecal samples as described above and used for generation of standard calibration curves. Aliquots of the serial 10-fold dilutions of Salmonella culture were plated overnight in duplicate on bacterial culture plates. The colony-forming unit (CFU) of the culture plates was used to calculate the starting concentration of the Salmonella in the spiked fecal samples.

Standard curve-based qPCR was performed on an ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA). The PCR primers and probe (Intergrated DNA Technologies Inc, Coralville, IA) targeting 119 base pairs of the invA gene were described previously (Hoorfar et al., 2000). Each 20-μL PCR reaction contained 10 μL of PerfeCTa qPCR SuperMix (Quanta Biosciences, Gaithersburg, MD), 0.5 μM of each primer, 0.125 μM of the probe, and 5 μl of extracted DNA. The PCR was performed at 95°C for 10 min, followed by 45 cycles of 95°C for 15 s and 55°C for 60 s. All samples and the standard calibration were performed in triplicate reactions in 96-well plates.

Performance of the q-PCR assay was experimentally evaluated with the standard calibration curve. The coefficient of correlation (R ²) of the q-PCR was 0.994 and PCR efficiency was calculated to be 98.85%. The q-PCR assay had an analytical sensitivity that was down to a single copy of invA gene in the reaction tube. The q-PCR had a linear dynamic range from 8.3 to 8.3×10⁵ copies of invA gene per reaction. The upper limit of the linear dynamic range was not determined due to the limitation of the concentration of our stock culture. This linear quantification range was determined to be equivalent to 9.17×10² to 9.17×10⁷ CFU/g of feces (Fig. 1). At the PCR cutoff, the assay could detect Salmonella at concentrations as low as 100 CFU/g of feces.

FIG. 1.

Standard calibration curves generated from spiked samples with known concentration of Salmonella samples ranging from 9.17×10² to 9.17×10⁷ colony-forming units/g of feces. C-q, quantification cycle.

The quantification cycle (Cq) value from the real time q-PCR was used as a proxy measure of fecal bacterial load. The Cq value is inversely proportional to the amount of bacteria load in fecal sample; the lower the Cq value, the higher the fecal concentration. The Cq values were recorded for all tested samples, and a Cq average was obtained from duplicate or triplicate Cq of the sample. Bacterial load was estimated using the standard calibration curve performed on the same PCR plates as copy numbers of invA gene/g of feces.

A pig was considered Salmonella positive if the fecal sample tested positive by culture method at each sample period. The Cq values for all tested samples were used to determine a diagnostic Cq cutoff for the q-PCR assay, based on the Younden index of diagnostic accuracy (sensitivity+specificity−1) that optimizes the sensitivity and specificity (Greiner et al., 2000). Individual fecal samples were re-categorized into negative versus positive based on diagnostic Cq cutoff of q-PCR.

Descriptive statistics of Salmonella concentrations were presented based on grouping of test results on both methods and as copy numbers of invA gene/g of feces. Individual fecal samples were classified into five groups based on q-PCR and culture results. These groups were (1) culture negative and q-PCR negative; (2) culture positive and q-PCR negative; (3) culture positive and q-PCR positive but not within quantifiable range; (4) culture positive and q-PCR positive within the quantifiable range; and (5) culture negative and q-PCR positive not within quantifiable range.

Results

Of the 443 culture positive samples, 69 (15.6%) were detected by q-PCR assay, with a median Cq of 35.5 (95% confidence interval [CI] 35.4–35.7, range=24.6–38.6). Twenty-five culture negative samples (2.0%; 25/1225) were detected as positive using q-PCR, with a median Cq of 38.4 (95% CI 38.1–39.4, range=34.7–42.8). Of the culture-positive samples, only 3.4% (15/443) of the samples were detected in triplicate and within the quantifiable range (≥917 copies of the invA gene).

Using the culture method as criterion standard, the optimal diagnostic cutoff of Cq value was determined to be 37.52, which maximized clinical sensitivity (15.4%; 95% CI 12.1–19.0%), specificity (99.6%; 95% CI 99.1–99.9%), and corresponds to the maximum Youden index (0.149). The distribution of samples into various groups based on the culture and q-PCR results was as follows. A total of 1220 samples were negative for Salmonella by both test methods (group 1); 375 Salmonella cultured-positive samples were not detected by q-PCR assay (group 2); 53 Salmonella cultured-positive samples were detected by q-PCR below the quantifiable range (<1000 CFU/g feces) (group 3) and only 15 Salmonella cultured-positive samples were detected and could be quantified by q-PCR assay (group 4). Five of the Salmonella cultured-negative samples were deemed positive below quantifiable range by q-PCR assay (group 5). In group 2, 204 samples (54.5%) were from six cohorts of site B; 113 (30.1%) were from six cohorts of site A; and 58 samples (15.5%) were from 5 cohorts of site C. In group 3, 26 samples (49%) were from six cohorts of site A; 25 (47%) were from five cohorts of site B; and two samples (4%) were from two cohorts of site C.

For the 15 quantifiable q-PCR positive samples in group 4, the Salmonella concentration ranged from 1.06×10³ to 1.73×10⁶ copies of invA gene/g feces (median=2.97×10⁵ and standard deviation=6.16×10⁵) (Table 1). These 15 samples were collected from a total of nine pigs, and seven of those fecal samples were collected from the same pig, with the highest concentration of Salmonella shed (range 4.08×10⁵ to 1.73×10⁶ copies of the invA gene/g feces).

Table 1.

Concentration of Salmonella in Fecal Samples of Nine Pigs, Belonging to Group 4 (Culture-Positive and Quantitative Real-Time Polymerase Chain Reaction Positive in the Quantifiable Range, >10³ Colony-Forming Units/g)

Pig ID	Site ^a	Cohort (pen ID) ^b	Total Salmonella positive samples ^c	Pig age ^d	Copy of invA gene/g feces
1	A	1 (27)	6	12	1.06×10³
2	A	2 (22)	2	10	1.19×10⁴
3	B	2 (4)	3	24	2.97×10⁵
4	B	2 (4)	7	10	4.08×10⁵
4	B	2 (4)	7	12	1.71×10⁶
4	B	2 (4)	7	14	7.99×10⁵
4	B	2 (4)	7	16	1.73×10⁶
4	B	2 (4)	7	18	6.40×10⁵
4	B	2 (4)	7	20	1.15×10⁶
4	B	2 (4)	7	22	8.01×10⁵
5	B	2 (4)	3	12	1.13×10³
6	B	2 (4)	3	10	4.33×10³
7	B	4 (5)	4	10	2.81×10³
8	B	6 (2)	3	10	6.40×10⁴
9	B	6 (6)	4	10	4.81×10³

Three sites (A, B, C).

Six cohorts in each site. Pen of each pig was located.

Total Salmonella positive samples per pig during the study period (each pig was sampled every 2 weeks for 16 weeks, eight total sample periods per cohort).

Pig age at the respective sampling period.

Discussion

There are significant knowledge gaps regarding quantitative risk for Salmonella shedding on farms and risk of carcass contamination. It is intuitive that the concentration of bacteria shed in feces is related to both transmission dynamics on farms, as well as risk for carcass contamination, but data are limited. This is likely due to many factors, not the least of which is the challenge of quantifying Salmonella concentrations in complex matrices such as feces.

This study evaluates the potential application of direct q-PCR to detect and quantify shedding of Salmonella in pig feces. This approach removes the impediments of both logistics for labor and the challenges of interpretation of concentration after enrichment by traditional culture-dependent methods. The robustness of direct q-PCR allows enumeration of Salmonella in a large number and variety of samples, with an efficient turnaround time, lower cost, and potential to be performed in automated way (Malorny et al., 2008; Löfström et al., 2011). There was a strong positive correlation between the sample concentrations and q-PCR results. The q-PCR assay used in this study was shown to have excellent specificity, analytical sensitivity, and a wide linear dynamic range in detection of Salmonella present in swine fecal samples. The detection limit of this PCR assay is in agreement with other reports of 10³ to 10⁴ gene copies per gram of swine feces (Malorny and Hoorfar, 2005; Harris et al., 2007; Malorny et al., 2008; Abley, 2011). Our result showed that the assay was sensitive to detect Salmonella at about 100 CFU/g of feces and determine fecal load in swine shedding high concentrations of Salmonella (>1000 CFU/g feces).

One of the potential applications of this methodology is for identification of high shedders (>10³ CFU/g). This is particularly important at the lairage, since carrier pigs with low and intermittent shedding at the farm might re-excrete the bacteria in high concentrations after periods of potential stress such as handling and transportation to the slaughterhouse and lairage (Hurd et al., 2002; Lo Fo Wong et al., 2002). This might be of particular use to identify high shedders in order to apply control measures, such as segregation of pigs during transportation and harvest.

The majority of the Salmonella cultured-positive pigs in this study were shedding low concentrations (<10³ CFU/g) of Salmonella, which were below the quantitative limit of q-PCR. These results are in agreement with those using other diagnostic tests for quantification such as the mini-Modified Semi-Solid Rappaport-Vassiliadis (MRSV)–most probable number (MPN) and MPN techniques in naturally infected pigs (Fravalo et al., 2003; Fablet et al., 2006; O'Connor et al., 2006; Boughton et al., 2007; van Hoek et al., 2012) and experimentally challenged pigs (Scherer et al., 2008). In addition, data from these culture dependent studies confirm the tendency observed herein: few pigs shed high concentrations during the finishing phase.

It is known that some Salmonella strains (Salmonella Senftenberg and Salmonella Litchfield) have natural deletions within the Salmonella pathogenicity island 1 involving the inv, spa, and hil loci (Ginocchio et al., 1997). Although the absence of the invA gene could potentially contribute to a high percentage of culture-positive samples undetectable by the q-PCR assay, we believe this to be unlikely. The assay used in the current study has been shown to detect 110 Salmonella strains (Hoorfar et al., 2000); among those are the most common serovars found in swine (Salmonella Typhimurium, Salmonella Heidelberg, Salmonella Agona, S almonella Derby, etc). In addition, Malorny et al. (2003) tested 242 Salmonella strains. Only one serovar (Salmonella Saintpaul) tested negative and Salmonella Senftenberg and Salmonella Litchfield were identified; the authors reported that the absence of invA gene in Salmonella appears to be rare. A follow-up study to serotype these isolates is pending.

Overall, the q-PCR showed very high specificity using culture as the criterion standard (Hoorfar et al., 2000). A few Salmonella cultured-negative samples were detected as positive by q-PCR. This could have been due to the presence of nonviable Salmonella, which would not have been detected by culture.

To the best of our knowledge, this is first study to quantify the fecal concentration of Salmonella in repeated sampling of individual, naturally infected finishing pigs. Despite intensive longitudinal sampling, the few fecal samples identified with high concentrations of Salmonella in the feces were clustered within pig and pen. Quantitative risk assessment studies have suggested that most exposures of swine to Salmonella are at doses below the infectious dose (EFSA, 2010). Doses >10³ CFU increase the probability of infection in swine (Osterberg and Wallgren, 2008; EFSA, 2010). There are likely interactions between risk of infection with both concentration shed as well as the number of animals shedding. These data may provide insight into comparison of intervention strategies targeted at control of pigs that shed high concentrations, for perhaps long periods of time, as compared to interventions more generally targeted at control of prevalence.

The importance of high shedders for risk of contamination at the slaughterhouse is unknown. Quantitative risk-assessment studies have reported that interventions to reduce Salmonella cases in humans due to pork-related products includes reducing slaughter pig prevalence by reducing the number of infected pigs with high infection/contamination loads entering the slaughterhouse (EFSA, 2010). Identification and removal of high shedders in a timely manner may be more effective to prevent carcass contamination. The robustness and rapidity of direct q-PCR assay such as the assay described in this study can be a very useful screening tool for removal of high shedder at lairage, and reduced the risk of carcass contamination at the processing line.

Footnotes

Acknowledgments

The authors thank the participating pork producers and their staff for collaborating in the investigation, and staff and students at Michigan State University for their technical support. This work was supported by USDA-NRI, Epidemiologic Approaches to Food Safety Grant 2007–01775.

Disclosure Statement

No competing financial interests exist.

References

Abley

. Tracking, quantifying, phenotyping and genotyping of Campylobacter in cattle and pigs across the farm to fork continuum. Graduate Program in Veterinary Preventive Medicine Volume Doctor of Philosophy. Columbus, OH: The Ohio State University, 2011; 186.

Baptista

, Dahl

, Nielsen

. Factors influencing Salmonella carcass prevalence in Danish pig abattoirs. Prev Vet Med, 2010; 95:231–238.

Boughton

, Egan

, Kelly

, Markey

, Leonard

. Quantitative examination of Salmonella spp. in the lairage environment of a pig abattoir. Foodborne Pathog Dis, 2007; 4:26–32.

Cheng

, Chi

, Lin

, Chou

, Huang

. Rapid quantification of Salmonella Typhimurium inoculated to meat products by real-time PCR. Acta Vet Hung, 2009; 57:25–38.

[CDC] Center for Disease Control and Prevention. Surveillance for foodborne disease outbreaks—United States, 2009–2010. Morb Mortal Wkly Rep, 2013; 62:41–47.

[CDC] Surveillance for foodborne disease outbreaks—United States. 2007. Morbid Mortal Wkly Rep, 2010; 59:973–979.

Doyle

, Erickson

. Opportunities for mitigating pathogen contamination during on-farm food production. Int J Food Microbiol, 2012; 152:54–74.

[EFSA] European Food Safety Authority. Quantitative microbiological risk assessment on Salmonella in slaughter and breeder pigs: Final Report. 2010. http://www.efsa.europa.eu/en/supporting/doc/46e.pdf. 2012 March 12.

Elizaquivel

, Gabaldon

, Aznar

. Quantification of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 in non-spiked food products and evaluation of real-time PCR as a diagnostic tool in routine food analysis. Food Control, 2011; 22:158–164.

10.

Fablet

, Robinault

, Jolly

, Collet

, Chemaly

, Labbe

, Madec

, Fravalo

. Salmonella enterica level in French pig farms effluents: Experimental and field data. Livestock Sci, 2006; 102:216–225.

11.

Fravalo

, Hascoet

, Fellic

, Queguiner

, Petton

, Salvat

. Convenient method for rapid and quantitative assessment of Salmonella enterica contamination: The mini-MSRV MPN technique. J Rapid Methods Automat Microbiol, 2003; 11:81–88.

12.

Ginocchio

, Rahn

, Clarke

, Galan

. Naturally occurring deletions in the centisome 63 pathogenicity island of environmental isolates of Salmonella spp. Infect Immun, 1997; 65:1267–1272.

13.

Greig

, Ravel

. Analysis of foodborne outbreak data reported internationally for source attribution. Int J Food Microbiol, 2009; 130:77–87.

14.

Greiner

, Pfeiffer

, Smith

. Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests. Prev Vet Med, 2000; 45:23–41.

15.

Guo

, Hoekstra

, Schroeder

, Pires

, Ong

, Hartnett

, Naugle

, Harman

, Bennett

, Cieslak

, Scallan

, Rose

, Holt

, Kissler

, Mbandi

, Roodsari

, Angulo

, Cole

. Application of bayesian techniques to model the burden of human salmonellosis attributable to U.S. food commodities at the point of processing: Adaptation of a Danish model. Foodborne Pathog Dis, 2011; 8:509–516.

16.

Guy

, Kapoor

, Holicka

, Shepherd

, Horgen

. A rapid molecular-based assay for direct quantification of viable bacteria in slaughterhouses. J Food Prot, 2006; 69:1265–1272.

17.

Harris

DLH

, Harris

, Dickson

, Gaul

, Bosworth

, Feldman

. Quantification of Salmonella in Transport and Lairage to Assess Interventions for Reduction of Cross-Contamination in Pigs. Des Moines, IA: National Pork Board: Pork Checkoff, 2007; 1–11.

18.

Hoorfar

, Ahrens

, Radstrom

. Automated 5' nuclease PCR assay for identification of Salmonella enterica. J Clin Microbiol, 2000; 38:3429–3435.

19.

Hurd

, McKean

, Griffith

, Wesley

, Rostagno

. Salmonella enterica infections in market swine with and without transport and holding. Appl Environ Microbiol, 2002; 68:2376–2381.

20.

Johnson

, Su

C-L

, Gardner

, Christensen

. Sample size calculations for surveys to substantiate freedom of populations from infectious agents. Biometrics, 2004; 60:165–171.

21.

Lo Fo Wong

DMA

, Hald

, van der Wolf

, Swanenburg

. Epidemiology and control measures for Salmonella in pigs and pork. Livest Prod Sci, 2002; 76:215–222.

22.

Löfström

, Schelin

, Norling

, Vigre

, Hoorfar

, Rådström

. Culture-independent quantification of Salmonella enterica in carcass gauze swabs by flotation prior to real-time PCR. Int J Food Microbiol, 2011; 145:S103–S109.

23.

Malorny

, Hoorfar

. Toward standardization of diagnostic PCR testing of fecal samples: Lessons from the detection of salmonellae in pigs. J Clin Microbiol, 2005; 43:3033–3037.

24.

Malorny

, Lofstrom

, Wagner

, Kramer

, Hoorfar

. Enumeration of Salmonella bacteria in food and feed samples by real-time PCR for quantitative microbial risk assessment. Appl Environ Microbiol, 2008; 74:1299–1304.

25.

Malorny

, Hoorfar

, Bunge

, Helmuth

. Multicenter validation of the analytical accuracy of Salmonella PCR: Towards an international standard. Appl Environ Microbiol, 2003; 69:290–296.

26.

Miller

, Liu

, McNamara

, Barber

. Influence of Salmonella in pigs pre-harvest and during pork processing on human health costs and risks from pork. J Food Prot, 2005; 68:1788–1798.

27.

O'Connor

, Gailey

, McKean

, Hurd

. Quantity and distribution of Salmonella recovered from three swine lairage pens. J Food Prot, 2006; 69:1717–1719.

28.

Osterberg

, Lewerin

, Wallgren

. Patterns of excretion and antibody responses of pigs inoculated with Salmonella Derby and Salmonella Cubana. Vet Rec, 2009; 165:404–408.

29.

Osterberg

, Wallgren

. Effects of a challenge dose of Salmonella Typhimurium or Salmonella Yoruba on the patterns of excretion and antibody responses of pigs. Vet Rec, 2008; 162:580–586.

30.

Pires

AFA

, Funk

, Bolin

. Longitudinal study of Salmonella shedding in naturally infected finishing pigs. Epidemiol Infect, November 13, 2012, 10.1017/S0950268812002464.

31.

Ravel

, Greig

, Tinga

, Todd

, Campbell

, Cassidy

, Marshall

, Pollari

. Exploring historical Canadian foodborne outbreak data sets for human illness attribution. J Food Prot, 2009; 72:1963–1976.

32.

Rostagno

, Eicher

, Lay

DC.

Jr . Immunological, physiological, and behavioral effects of Salmonella enterica carriage and shedding in experimentally infected finishing pigs. Foodborne Pathog Dis, 2011; 8:623–630.

33.

Scallan

, Hoekstra

, Angulo

, Tauxe

, Widdowson

, Roy

, Jones

, Griffin

. Foodborne illness acquired in the United States—Major pathogens. Emerg Infect Dis, 2011; 17:7–15.

34.

Scherer

, Szabo

, Rosler

, Appel

, Hensel

, Nockler

. Time course of infection with Salmonella Typhimurium and its influence on fecal shedding, distribution in inner organs, and antibody response in fattening pigs. J Food Prot, 2008; 71:699–705.

35.

van Hoek

, de Jonge

, van Overbeek

, Bouw

, Pielaat

, Smid

, Malorny

, Junker

, Lofstrom

, Pedersen

, Aarts

HJM

, Heres

. A quantitative approach towards a better understanding of the dynamics of Salmonella spp. in a pork slaughter-line. Int J Food Microbiol, 2012; 153:45–52.

36.

Wolffs

PFG

, Glencross

, Thibaudeau

, Griffiths

. Direct quantitation and detection of salmonellae in biological samples without enrichment, using two-step filtration and real-time PCR. Appl Environ Microbiol, 2006; 72:3896–3900.

37.

Wood

, Rose

. Populations of Salmonella Typhimurium in internal organs of experimentally infected carrier swine. Am J Vet Res, 1992; 53:653–658.