Quantification of Campylobacter and Salmonella in Cattle Before,During,and After the Slaughter Process

Abstract

Salmonella and Campylobacter cause a significant number of human illnesses globally, most of which are food related. Cattle can be asymptomatic carriers of both of these pathogens. The objective of this study was to determine the association between the concentration of Salmonella and Campylobacter pre- and postharvest in cattle. Samples were collected from each of 98 individually identified cattle during the periharvest and postharvest period. For each animal, four different phases were sampled: on farm (fecal sample), poststunning and exsanguination (hide sponge and rectal content sample [lairage]), prechilling (carcass sponge), and final product (ground meat). Salmonella and Campylobacter were cultured and quantified at each stage by using the direct dilution and most probable number (MPN) method. Salmonella was not isolated from any sample. The proportion (%) of samples that were Campylobacter positive was 77, 82, 97, 55, and 12 for farm, rectal content, hide, carcass, and meat samples respectively. The mean Campylobacter concentration for each sample was as follows: fecal sample from farm, 3.7×10⁴ cfu/g; rectal content sample from lairage, 1.6×10⁵ cfu/g; hide sponge, 0.9 cfu/cm²; carcass sponge, 8.7 cfu/half carcass; and meat, 1.1 cfu/g. There were no associations between Campylobacter concentrations for any two sample types. This lack of association could indicate that there is an environmental reservoir that can contaminate the final meat product, or since the majority of animals were positive entering the slaughter process, that the process itself reduces the load of Campylobacter regardless of the initial concentration. In addition, contamination of beef may be more strongly associated with periharvest practices than animal carriage rates.

Introduction

S almonella and Campylobacter cause a significant number of human illnesses globally, most of which are food related (Scallan et al., 2011). Campylobacter has been isolated from healthy and diarrheic animals, which may lead to fecal contamination of meat during processing. (Blaser et al., 1980; Prescott and Bruin-Mosch, 1981; Munroe et al., 1983). Quantitative measures of food safety risk are rarely reported and represent a critical data gap for development of quantitative risk assessments. The objective of this study was to determine the association between the concentration of Salmonella and Campylobacter in bovine samples collected before and during slaughter with concentrations on meat. Our hypothesis was that there was a positive association between pathogen concentrations from samples taken during the harvest process and concentrations on meat.

Methods

Animals

A convenience sample of 98 steers was utilized for this project. The angus cross steers were part of another dietary study (Cernicchiaro et al., 2010). The steers were reared at the Ohio Agricultural Research and Development Center in Wooster, Ohio. The steers were shipped to two different slaughter facilities. Both slaughter facilities (Plants A and B) processed cattle and swine. Plant B also processed sheep. One slaughter facility was privately owned (Plant A) and the other was owned and run by the Ohio State University (OSU, Plant B). Cattle slaughtered at Plant A (N=60) were processed in groups of ten steers on each of six sampling days (April 3, 10, 17, 24 and May 1, 8, 2006). Plant B processed steers (N=38) on six separate dates; on three occasions (April 4, 25, and May 9) steers were processed in groups of four, on two occasions in groups of 8 (April 18 and May 2), and once as a group of 10 steers (April 11, 2006).

Sample collection

Within 48 hours before transport to the slaughter facility, a fresh fecal sample (∼20 g) was obtained from individually identified steers. A rectal content sample (∼20 g) was obtained from the rectum of each steer immediately postmortem. The area of the hide that was most visually contaminated with feces and dirt (flank and rump region in all cattle) was sampled immediately postmortem in two different areas of ∼910 cm² with sponges (hydrated-sponge; 3M, Saint Paul, MN). The entire surface of the hot carcass was sampled postwashing and prechilling with a total of 10 sponges (3M), five sponges per half carcass. Each sponge was swabbed over a distinct region of each carcass half. These five surface areas were bounded by the following anatomical markers: external carcass surface (three sponges); (1) the proximal pelvis to the tarsus, (2) the proximal pelvis to the caudal sternum, and (3) the caudal sternum to the proximal end of the carcass and the internal carcass surface (two sponges) which was divided into two regions by the diaphragm (regions 4 and 5). Five sponges from one-half carcass were pooled for Salmonella culture procedures and the other five were pooled for Campylobacter culture procedures. One-week post-slaughter, trim from each intact carcass was obtained and grounded (1 pound/carcass). Sponges were sterile and premoistened in 10 mL of buffered peptone water (BPW). Samples were transported to the lab on ice and processed within 4 hours of collection.

Salmonella most probable number

The fecal and rectal content samples were quantitatively cultured using a three-tube most probable number (MPN) method. The MPN was calculated based on the spreadsheet and recommendations of Garthright and Blodgett (2003). Briefly, 4 g of feces was added to 36 mL of tetrathionate broth (TTB; Becton Dickenson, Sparks, MD). The dilutions were conducted by taking 4 mL from the 10⁻¹ dilution, which was then mixed in the next tube with 36 mL of TTB to make the 10⁻² dilution and was continued to a final dilution of 10⁻⁴. The fecal and rectal content samples from farm and lairage were incubated at 37°C for 24 hours. A 100 μL aliquot from each tube was plated onto xylose lysine tergitol 4 (XLT4; Becton Dickenson) agar plates and incubated for 18–24 hours at 37°C. Typical Salmonella colonies morphology on XLT4 agar produces hydrogen sulfide, resulting in black colony formation for the target organism.

Hide sponge samples were processed in 30 mL of TTB. Ten milliliters of the mixture was added to three tubes and 1 mL was taken from the 10⁻¹ dilution and added to 9 mL of TTB to make the next dilution and so on for the 3 tube, ×4 dilution MPN. The hide samples then followed the same protocol as the fecal samples. The five sponges from the carcass were pooled and mixed with 150 mL of BPW. A 30 mL aliquot of the pooled carcass sample was added to each of three tubes and 3 mL was added to 27 mL to make a 3 tube, ×4 dilution MPN. The tubes were incubated for 24 hours at 37°C. One-hundred microliters from each tube was then added to 10 mL of Rappaport-Vassiliadis R10 (Becton Dickenson) broth and incubated at 42°C for 18–24 hours. An aliquot of 100 μL from each tube was plated onto XLT4 agar plates and incubated for 18–24 hours at 37°C.

Ten grams of meat was mixed with 90 mL of BPW and 10 mL of this mixture was added to 90 mL of BPW to make the 3 tube, ×4 dilution MPN. The samples were processed by the same procedures as the carcass sponges.

Campylobacter direct dilution and MPN

The fecal and rectal content samples were processed by adding 1 g of feces to 9 mL of BPW. Three replicates were made by taking 1 mL from the initial 10⁻¹ dilution and adding to 9 mL of BPW, continuing for each dilution to a maximum dilution of 10⁻⁴. One-hundred microliters from each dilution was plated in duplicate on Campy-Cefex (Oyarzabal et al., 2005) plates and incubated under microaerophilic conditions with the use of GasPak EZ Campy Container System and GasPak EZ Campy Container Sachets (Becton Dickenson) for 48 hours at 42°C. Campylobacter suspect colonies were counted.

The hide samples were processed by mixing the sponge with 30 mL of Bolton broth (Oxoid, Hampshire, United Kingdom). Ten milliliters of the mixture was added to three tubes and four 10⁻¹ serial dilutions were performed for each tube. The tubes were incubated under microaerophilic conditions for 48 hours at 42°C. One-hundred microliters from each dilution was plated onto Campy-Cefex plates and incubated under microaerophilic conditions for 48 hours at 42°C.

The five sponges from the carcass were pooled and mixed with 90 mL of Bolton broth. A 30 mL aliquot of the pooled carcass sample was added to each of three tubes and 3 mL from the 10⁻¹ dilution was added to 27 mL of Bolton broth to make a 3 tube, ×4 dilution MPN. The samples then followed the protocol for the hide sponges.

Ten grams of ground meat was mixed with 90 mL of Bolton broth and 10 mL of this mixture was added to 90 mL of Bolton broth for a 3 tube, ×4 dilution MPN. The samples then followed the protocol for the hide and carcass sponges. For each presumptive positive sample, a composite of suspect Campylobacter colonies was stored at −80°C in Brucella broth (Becton Dickenson) and 10% glycerol for further processing.

Campylobacter confirmation and speciation

Campylobacter suspect colonies were revived from the freezer onto Mueller Hinton (Becton Dickinson) plates. Catalase (Becton Dickenson) and oxidase (Becton Dickenson) tests were performed and all colonies that were positive for both in addition to colonies that were positive for oxidase only were grown in brucella (Becton Dickenson) broth for 72 hours at 42°C for DNA extraction using the DNeasy Tissue kit (Qiagen, Valencia, CA). Polymerase chain reaction (PCR) was performed on the extracted DNA targeting the hipO gene for Campylobacter jejuni and the glyA gene for Campylobacter coli (LaGier et al., 2004). C. jejuni subsp. jejuni ATCC 33560 and C. coli ATCC 49941 were used as positive controls for the PCR.

Statistical analysis

Descriptive statistics were performed for prevalence, mean, and median concentration using the presumptive counts. Spearman's Rank Correlation Coefficient was calculated to ascertain correlations of Campylobacter concentrations between meat samples and each other sample type (feces, rectal content, hide, and carcass). Odds ratios were calculated to determine the odds of a meat sample being positive when fecal samples, rectal content samples, hide sponge, or carcass sponge samples were positive. All statistics were performed using STATA (Intercooled STATA 9; StataCorp, College Station, TX).

Results

Salmonella prevalence

No Salmonella was isolated from any sample.

Campylobacter concentrations

The proportion (%) of samples that were Campylobacter positive was 77, 82, 97, 55, and 12 for fecal samples, rectal content samples, hide sponges, carcass sponges, and meat samples, respectively (Fig. 1). The mean Campylobacter concentration for each sample type was as follows: fecal sample, 3.7×10⁴ cfu/g; rectal content sample, 1.6×10⁵ cfu/g; hide sponge, 0.9 cfu/cm²; carcass sponge 8.7 cfu/half carcass; and meat 1.1 cfu/g (Fig. 2). The median Campylobacter concentration for each sample type was as follows: fecal, 3.0×10³ cfu/g; rectal content sample, 9.8×10³ cfu/g; hide sponge, 1.2×10² cfu/cm²; carcass sponge, 1.2×10³ cfu/half carcass; and meat, 0 cfu/g.

FIG. 1.

The prevalence of Campylobacter sp. isolated from the different sample types (farm, lairage, hide, carcass, and meat) in cattle N=98. The dark gray bars represent plant A and the light gray bars represent plant B.

FIG. 2.

The mean value of the quantitative count of Campylobacter sp. from all samples (farm, lairage, hide, carcass, and meat) using direct dilution for farm and lairage and most probable number for hide, carcass, and meat from cattle. The dark gray bars represent plant A and the light gray bars represent plant B.

Speciation of Campylobacter

Of the 351 putative Campylobacter isolates, 242 (69%) were catalase and oxidase positive, 80 (23%) were negative for oxidase, and 29 (8%) were not recovered from cryopreservation. Of the 242 isolates with biochemical reactions consistent with Campylobacter, 93 (38%) were C. jejuni, 88 (36%) were not C. jejuni or C. coli, 71 (29%) were C. coli, and 18 (7%) were positive for both C. jejuni and C. coli. There was a shift from predominantly C. jejuni recovered from the fecal samples to only C. coli recovered from the meat samples (Fig. 3).

FIG. 3.

Species results of Campylobacter determined by polymerase chain reaction (hipO–Campylobacter jejuni, glyA–Campylobacter coli) for all sample types (farm, lairage, hide, carcass, and meat) from cattle. The light gray bars represent C. coli and the dark gray bars represent C. jejuni.

Statistical analysis

There was no correlation between the concentration of Campylobacter in “upstream” samples and concentration in meat samples (Table 1). Campylobacter contamination of the hide was associated with a lower odds of Campylobacter contamination in ground beef (odds ratio=0.06, p=0.04; Table 2). No other associations were identified (Table 2).

Table 1.

Association Between Concentrations of Campylobacter in Meat Compared to Farm, Lairage, Hide, and Carcass Samples from Cattle (Spearman's Rank Correlation Coefficient)

	Spearman's R	p-Value
Fecal sample from farm	−0.008	0.94
Rectal content sample from lairage	0.0092	0.93
Hide sponge	−0.1367	0.19
Carcass sponge	−0.0456	0.66

Table 2.

Univariate Odds Ratios for the Risk of Campylobacter-Positive Meat Sample with the Following Exposures (Farm Positive, Lairage Positive, Hide Positive, or Carcass Positive) from Cattle

Risk for Campylobacter-positive meat samples
Exposure	OR	95% CI	p-Value
Fecal sample from farm	3.08	0.37–25.5	0.45
Rectal content sample from lairage	2.6	0.34–20.65	0.69
Hide sponge	0.06	0.005–0.71	0.04
Carcass sponge	0.77	0.23–2.59	0.76

OR, odds ratio; CI, confidence interval.

Discussion

Reports of fecal prevalence of Salmonella in cattle range from 0.08% to 46% (Fedorka-Cray et al., 1998; Van Donkersgoed et al., 1999; Barham et al., 2002; Beach et al., 2002). The relatively small sample size of our study coupled with the cattle from one farm may have contributed to our inability to isolate Salmonella.

Reported Campylobacter prevalence on cattle farms varies widely, ranging from 0.8% to 84% [0.8% (Rosef et al., 1983), 5% (Hoar et al., 2001), 19.5% (Cabrita et al., 1992), 31.1% (Hakkinen et al., 2007), 46.7% (Giacoboni et al., 1993), 53.9% (Pezzotti et al., 2003), 58% (Bailey et al., 2003), 58.9% (Oporto et al., 2007), 83.7% (Inglis et al., 2003)].

The prevalence of Campylobacter on cattle hides that we have observed in this study differed considerably from a previous report where Campylobacter was not recovered (Reid et al., 2002). However, we sampled an area of the hide that was about nine times larger than Reid et al. (2002) sampled. Reid et al. (2002) sampled the hide in three areas (rump, flank, and brisket) using one swipe within a 10×10 cm template. In this study, the area on the hide that had the highest contamination of dirt and feces was sampled, which was on the rump and/or flank areas. Reid et al. (2002) described their cattle as being “visually clean” meaning there was no visible mud/feces on the hides and the hides were dry, which was not what we observed for the cattle in our study.

We found a higher proportion of Campylobacter-positive carcasses than has been reported in a number of previous studies [0% (Madden et al., 2001), 0.8% (Vanderlinde et al., 1998), 3.3% (Ghafir et al., 2007), 3.5% and 4% (McNamara, 1995; Hakkinen et al., 2007), 32% (Bolton et al., 1982), 66% (Christensen et al., 1994)]. One explanation is that we sampled more surface area, that is, an entire half carcass, than had been sampled previously. This difference in sampling technique may have contributed to the difference in prevalence, with our increased sampling effort resulting in increased sensitivity for detection. Because of using MPN methodology, we made multiple dilutions of each sample that may have increased the sensitivity for detection.

The prevalence of Campylobacter in ground beef that we observed is comparable to other studies that reported prevalence ranging from 0% to 23.6% in various retail beef products [0% (Madden, 1998), 0% (Ono and Yamamoto, 1999), 0.5% (Zhao et al., 2001), 0.6% (Ghafir et al., 2007), 1.2% (Hong et al., 2007), 1.3% (Pezzotti et al., 2003), 2% (Osano et al., 1999), 3.2% (Whyte et al., 2004), 3.5% (Wong et al., 2007), 10% (Korsak et al., 1998), 10% (Taremi et al., 2006), 20% (Cloak et al., 2001), 23.6% (Fricker et al., 1989)].

Although C. coli has been detected in retail beef (Cloak et al., 2001; Whyte et al., 2004; Wong et al., 2007; Hannon et al., 2009), the shift in Campylobacter species from feces (90% C. jejuni) to meat (100% C. coli) was unexpected. One potential explanation for the shift in Campylobacter species is that these slaughter facilities process multiple species, including swine. C. coli is the most common species isolated from swine (Horrocks et al., 2009). In both plants, if swine were slaughtered on the same day, they were done prior to the cattle, with cleaning and disinfection of the facilities occurring between species. Another possible explanation for this observed change may be a result of the fact that the culture methods we used for the fecal samples (including rectal contents) and the hide, carcass, and meat differed. The fecal samples were direct plated and were not enriched in Bolton broth prior to plating, as were the hide, carcass, and ground beef samples. According to the manufacturers of the different media that we used, Bolton broth is recommended for all thermotolerant Campylobacter and Campy-cefex is recommended for C. jejuni, C. coli, and C. lari. We found no reports of Campy-cefex or Bolton broth preferentially selecting for one species over another. It is unclear whether the media we used played a role in the species of Campylobacter we recovered. Further investigations comparing different culture methods and Campylobacter species selection may help to clarify this issue.

One limitation of this study is that 80 isolates (23%) were negative for oxidase and were therefore not considered to be Campylobacter. Since the confirmation was completed after recovery following regrowth from freezer stock, the Campylobacter might not have survived and a contaminant was evaluated instead. As discussed by Garthright and Blodgett (2003) there are inherent limitations regarding the MPN methodology, which would also apply to quantifying bacterial concentrations via direct dilution methods. Although several representative colonies were selected for biochemical confirmation, the population of colonies present on a plate may not be accurately represented by the colonies selected. There is an inherent challenge in culture-based bacterial quantification methods in balancing accuracy of target colony identification with pragmatic consideration of laboratory resource limitations to verify all colonies present. In this study, there is uncertainty of how well the selected colonies represented the population present in the sample that may explain both the isolates that were ultimately not considered Campylobacter as well as a lack of association between concentrations found on each sample type. To the best of our knowledge, there is no description of the population distribution of bacterial species that would demonstrate typical Campylobacter morphology across these different sample types. We had 88 putative Campylobacter isolates (36%) that were neither C. jejuni nor C. coli based on PCR of the hipO gene or glyA gene. Other studies have identified C. fetus subsp. fetus, C. lanienae, C. hyointestinalis in addition to C. jejuni and C. coli in cattle (Manser et al., 1985; Inglis et al., 2003; Pezzotti et al., 2003; Hakkinen et al., 2007). The unidentified isolates could be another species of Campylobacter or another organism that is oxidase positive and can grow on Campy-cefex agar. There was no obvious clustering of these isolates by animal, date of collection, or sample type.

Relatively few studies have quantified Campylobacter in cattle. A study of beef cattle going to slaughter found an average MPN per g of 6.1×10² in the small intestine (Stanley et al., 1998). A United States–based study found a mean C. jejuni concentration of 0.1 MPN/cm² on carcasses (McNamara, 1995). In studies analyzing meat contamination, one reported that only one of four minced beef samples contained C. jejuni above the detection level of 5–10 MPN/g (Cloak et al., 2001). Another study found that 8/230 samples of beef were contaminated at a level of 0.3 MPN/g (Wong et al., 2007). These studies quantifying Campylobacter on the carcass and in the meat are consistent with our results (McNamara, 1995).

The decreased odds of contamination of ground beef if the hide was culture positive is biologically difficult to explain. One possibility for this relationship is that almost all hide samples were positive so the high number of positives may have impacted the statistical interpretation. We believe that this result is not biologically significant, because the idea that a contaminated hide leads to less contamination of the meat is not a biologically plausible explanation.

Ingestion of only 500 cells of Campylobacter can lead to clinical illness in humans (Robinson et al., 1979; Deming et al., 1987; Black et al., 1988). Such a low infectious dose shows the importance of reducing Campylobacter contamination in retail beef samples. Based on the average concentration from this study, an individual would need to consume 500 g of raw ground beef to receive an infectious dose. Cross-contamination through the transfer of Campylobacter cells from raw meat to kitchen surfaces is also possible. Further research to discern this risk is recommended.

Conclusions

The overall objective of this study was to determine if there was any association with the final meat product and the other samples collected “upstream.” No biologically significant associations were found. One explanation is that there are no associations between the concentration or prevalence of Campylobacter in the feces, on the hide, or carcass and the final retail meat product. This explanation implies that there is an environmental reservoir, or since the majority of animals were positive entering the slaughter process, that the process itself reduces the load of Campylobacter regardless of the initial concentration. Contamination of the meat may be more influenced by periharvest practices than by animal carriage rates. The shift from C. jejuni to C. coli may impact these associations. The different culture methods also could account for the lack of association, by having different selection pressures and allowing different sets of Campylobacter to be cultured. The individual animal also might not be the best level to look at these associations. We recommend future studies to evaluate these associations at the group and/or herd level.

Footnotes

Acknowledgments

This research was funded by the Food Safety Research and Response Network USDA CSREES Special Research Grant #2003–34475–13066. We gratefully thank the employees of the processing facilities for their cooperation. We also acknowledge the following laboratory personnel for their assistance: Jamie Berning, Katie Collins, Mallory Cordle, Shawna Guffey, Bekah Harvey, Luke Heider, Kristen Hinebaugh, Katie Kleinhenz, Dixie Mollenkopf, Heidi Schultz, Laura Stephan, Miranda Vieson, James White, Meagan Williams, and Jillian Yarnell.

Disclosure Statement

No competing financial interests exist.

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