Prevalence of Clostridium difficile Toxin Genes in the Feces of Veal Calves and Incidence of Ground Veal Contamination

Abstract

A study was conducted in two parts to determine the prevalence of toxigenic Clostridium difficile in veal calves and retail meat. The first part of the study focused on the veal production continuum (farm to abattoir). Fifty calves from 4 veal herds (n=200) were followed for 18–22 weeks from the time of arrival on the veal farm to the time of slaughter. Fecal samples were collected from calves every 4–6 weeks. Half of the calves included in the study (n=100) were followed to the abattoir where carcass swabs were collected post slaughter. Fecal samples and carcass swabs were screened for genes encoding C. difficile toxins TcdA, TcdB, and CDT by using real-time polymerase chain reaction (PCR). Carcass swabs were also screened for toxigenic C. difficile by using traditional culture methods. In the second part of the study, ground veal products (n=50 samples) purchased from local grocery stores were examined for toxigenic C. difficile by using real-time PCR and traditional culture methods. Fecal samples from 56 of 200 (28%) calves tested positive for C. difficile toxin genes at least once over the course of the study. Calf age (p=0.011) influenced prevalence of C. difficile toxin genes in calf feces. Toxin genes of C. difficile were detected in one carcass swab by multiplex real-time PCR only. Toxigenic C. difficile was detected by PCR and culture in four (8%) and three (6%) ground veal samples, respectively. The findings of the study reveal that toxigenic C. difficile was most prevalent in veal calves (12%) just before slaughter, although viable toxigenic C. difficile was not recovered from veal carcasses. On the contrary, viable toxigenic C. difficle was recovered from 6% retail meat, thus suggesting that contamination occurs either during or after veal fabrication.

Introduction

F ood animals including cattle, pigs, and broiler chickens have been identified as carriers of toxigenic Clostridium difficile (TXCD) (Songer and Anderson, 2006; Jhung et al., 2008; Avbersek et al., 2009; Indra et al., 2009). A previous report stated that 27 out of 622 of animal stool samples contained TXCD (Lemee et al., 2004). Recent studies have shown that calves can act as asymptomatic carriers of TXCD (Rodriguez-Palacios et al., 2006; Hammitt et al., 2008). Researchers have observed genotypic similarity between C. difficile isolates of human and animal origin, thus suggesting that animals should be considered an important source of exposure to C. difficile relevant to public health (Rodriguez-Palacios et al., 2006; Rupnik, 2007; Jhung et al., 2008). C. difficile has been isolated from ground beef, veal, pork, and turkey meant for human consumption. These findings suggest that C. difficile may be considered a zoonotic agent disseminated from food animals to humans through direct contact or consumption of contaminated food animal products (Rupnik, 2007). Weese (2010) points out that a foodborne association may be difficult to establish in the case of C. difficile, if one does exist. It is possible that C. difficile infection may not occur until sometime after ingestion of the organism, thus making it difficult to determine a point of exposure. It is also possible that foodborne exposure is only significant if other risk factors are encountered, such as subsequent antibiotic therapy or use of proton pump inhibitors (Borriello, 1990; Dial et al., 2005; Weese, 2010).

C. difficile is a well-established human pathogen known to cause a disease state ranging from mild diarrhea to fulminant colitis, which may result in death. In the past, C. difficile was considered a nosocomial pathogen that mainly affected the elderly, the severely ill, and long-term hospitalized patients (CDC, 2005; Hookman and Barkin, 2007). Recently, an increase in community acquired C. difficile associated disease has been reported in populations that were previously considered at low risk of infection (CDC, 2005; Wilcox et al., 2008; Pituch, 2009).

C. difficile-associated disease is caused by two large clostridial toxins, TcdA and TcdB (Hookman and Barkin, 2007). Both toxins act on the Rho family of proteins through monoglucosylation (Just et al., 1995a, 1995b). Disruption of F-actin regulation leads to loss of cytoskeletal integrity, thus resulting in the loss of tight junctions between intestinal epithelial cells (Keel and Songer, 2006). Some toxigenic strains also produce a binary toxin, CDT, which acts through ADP-ribosylation of actin (Popoff et al., 1988). This toxin is expressed in two components, an enzymatic subunit (CDTA) and a binding subunit (CDTB).

To date, there are no reports on the dynamics of C. difficile prevalence and distribution in veal calves from production through slaughter. It is anticipated that the findings of this study will provide a better understanding of the prevalence of TXCD at different points of production and will provide vital information needed for implementing prevention and control of C. difficile in veal calf and veal processing operations.

Materials and Methods

Veal calves

Four veal calf operations that raise veal calves for a large veal production company participated in the study. Calves were routinely purchased throughout the northeast United States by the production company and sent to veal growers in central Pennsylvania at ∼1 week of age. A diet of milk replacer and routine veterinary care were provided by the production company and were identical on all veal grower farms. Four veal herds raised by three different veal growers were included in the study. Calves in three of the four herds (herds A, B, and D) were housed in separate stalls and confined to head ties. Calves in herd C were not tied and were housed two per stall; dividers in each stall separated the calves until ∼8 weeks of age when dividers were removed. Herds A and B were raised during the time period of October to March and April to August and by the same veal grower, respectively. Herd C was raised during the period of September to January. Herd D was raised during the time period of April to July. When the calves had reached sale weight (∼20–22 weeks of age), they were shipped to a common facility for slaughter. Finished veal product of the veal company used in this study is sold in retail grocery stores throughout Pennsylvania.

Sample collection

The prevalence of TXCD in veal calves was monitored from the time of arrival at the veal grower until slaughter. The four herds included in the study contained a total of 600 calves. A sample size of n=194 calves was determined by using power analysis with an expected prevalence of 11%. Therefore, 50 calves from each herd, for a total of 200, were included in the study. Fecal samples were rectally collected from calves every 4–6 weeks for a total of four to five samples from each calf. Calves (n=100) from herds B and D were followed to slaughter. The outside surface of carcasses were swabbed with a sterile 3×3 inch gauze pad at the abattoir immediately after slaughter (postevisceration) before and after the carcasses were washed with 2.5% citric acid as previously described (Dorsa et al., 1996). An effort was made to swab as much external carcass surface area as possible. One pound ground veal samples (n=50) produced by the veal operation participating in the study were purchased from two local grocery stores over a period of 4 months.

Sample analysis

All fecal samples (1 g) and carcass swabs were anerobically incubated in 9 and 20 mL cycloserine cefoxitin fructose broth, respectively, for 1 week at 37°C. All ground veal samples were cultured for C. difficile as previously described by Songer et al. (2009). Briefly, two 1 g aliquots of ground veal sample were homogenized in two different tubes containing 9 mL of prereduced brain heart infusion broth (BHI) supplemented with 0.5% yeast extract, 0.1% taurocholate, and 0.05% cysteine. One tube was heated to 80°C for 10 min for detection of spores. Both tubes were anerobically incubated at 37°C for 96 h. DNA was extracted from all fecal cultures by using the QIAamp DNA Stool Mini Kit (Qiagen) and from carcass swab and ground veal cultures by using the DNeasy Blood and Tissue Kit (Qiagen).

All DNA extracts from fecal samples and swabs were examined for C. difficile by using polymerase chain reaction (PCR) targeting tpi, a housekeeping gene, as previously described (Lemee et al., 2004). Samples positive for tpi were then screened for toxin genes by using the multiplex real-time PCR assay validated in a previous study (Houser et al., 2010). Samples positive for tpi and negative for all toxin genes were considered positive for nontoxigenic C. difficile.

To determine whether viable TXCD was present on carcasses and in ground veal, carcass swab and ground meat broth cultures were also streaked on cycloserine cefoxitin fructose agar containing 0.1% taurocholate and incubated up to 7 days or until growth was observed. Isolates showing characteristic morphology of C. difficile were subcultured, and DNA was extracted from pure culture by using standard methods (Pospiech and Neumann, 1995). To confirm an identification of C. difficile, DNA from all isolates was screened for tpi as just described (Lemee et al., 2004). Isolates carrying the tpi gene were then screened for toxin genes by using multiplex real-time PCR (Houser et al., 2010).

Data analysis

Descriptive statistics were used to calculate the overall prevalence of C. difficile toxin genes or the organism itself in veal calf feces over time, on veal carcasses, and in finished veal product. The chi-square test of independence (SPSS version 17.0) was used to determine whether the prevalence of C. difficile toxin genes in feces was influenced by calf age.

Results and Discussion

Prevalence of C. difficile in veal calves

The prevalence of C. difficile in diarrheic calves has been well reported, whereas there are only a few reports on the prevalence of C. difficile in healthy or asymptomatic calves (Rodriguez-Palacios et al., 2006; Hammitt et al., 2008). In this study, 200 healthy calves from four different herds (50 calves from each herd) were monitored for 18–22 weeks for prevalence of C. difficile toxin genes. A total of 850 fecal samples (four to five from each calf) were examined. Seventy-five fecal samples tested positive for tpi in this study; of these, 60 (80%) were positive for at least one toxin gene (Table 1). Seven different toxin gene profiles were observed among fecal samples positive for C. difficile toxin genes (Table 2). Among the 60 TXCD positive fecal samples, toxin genes tcdA, tcdB, cdtA, and cdtB were detected in 34 (56.7%), 28 (46.7%), 45 (75.0%), and 45 (75.0%) samples, respectively. These data suggest that healthy veal calves should be considered a reservoir for TXCD.

Table 1.

Prevalence of Clostridium difficile in Veal Calves

	TXCD prevalence in calves by weeks of age							Overall CD prevalence in fecal samples
Herd	n	<2	4–6	8–10	12–18	20–22	Total	n	NTCD	TXCD	Total
A	50 (%)	—	0 (0)	0 (0)	1 (2)	1 (2)	2 (4)	200 (%)	3 (1.5)	2 (1.0)	5 (2.5)
B	50 (%)	1 (2)	3 (6)	0 (0)	9 (18)	10 (20)	21 (42)^a	250 (%)	8 (3.2)	23 (9.2)	31 (12.4)
C	50 (%)	7 (14)	13 (26)	1 (2)	0 (0)	—	19 (38)^a	200 (%)	0 (0.0)	21 (10.5)	21 (10.5)
D	50 (%)	—	2 (4)	5 (10)	0 (0)	7 (14)	14 (28)	200 (%)	4 (2.0)	14 (7.0)	18 (9.0)
Total	200 (%)	8 (8)^b	18 (9)	6 (3)	10 (5)	18 (12)^b	56 (28)	850 (%)	15 (1.8)	60 (7.1)	75 (8.8)

Toxigenic Clostridium difficile was detected more than once in the same calf during the study period.

Values in parenthesis represent percentage of total calves sampled in respective age groups.

TXCD, toxigenic C. difficile, includes tpi positive fecal samples that tested positive for at least one toxin gene; CD, C. difficile, includes samples that tested positive for both toxigenic C. difficile and nontoxigenic C. difficile; NTCD, nontoxigenic C. difficile, includes tpi positive samples that tested negative for all toxin genes.

Table 2.

Clostridium difficile Toxin Profiles Observed in Veal Calf Feces

	Toxin profile				Number of samples by herd
Profile no.	tcdA	tcdB	cdtA	cdtB	A	B	C	D	Total
1	+	−	−	−	1	2	2	5	10
2	+	+	−	−	0	1	1	1	3
3	+	+	+	+	0	2	9	0	11
4	+	−	+	+	0	2	4	4	10
5	−	+	−	−	1	1	0	0	2
6	−	+	+	+	0	6	3	3	12
7	−	−	+	+	0	9	2	1	12
Total^a	34	28	45	45	2	23	21	14	60

Total number of samples that tested positive for toxin genes.

A high prevalence of genes encoding TcdA and TcdB has been previously reported in C. difficile isolated from calves. Hammitt et al. (2008) observed that all C. difficile isolated from diarrheic calves were PCR positive for tcdA and tcdB. In this study, the prevalence of C. difficile toxin genes in calf feces increased over time, and the highest prevalence was observed at the last sampling before slaughter for herds A, B, and D. For herd C, the highest prevalence was observed at the second sampling when calves were ∼4–6 weeks of age (Table 1).

Trend data for herds A, B, and D showed an overall increase in prevalence of TXCD throughout the 22 week period. Chi-square test of independence showed that the prevalence of C. difficile toxin genes in herds B (χ ²=14.20, p=0.007), C (χ ²=22.49, p=0.51e⁻⁴), and D (χ ²=8.91, p=0.03) was influenced by calf age. Prevalence was not shown to be significantly influenced by calf age for herd A (χ ²=3.89, p=0.27), although calf age was also found to influence fecal toxin gene prevalence when analyzing all herds collectively (χ ²=14.91, p=0.011). The highest overall prevalence was observed for calves 20–22 weeks of age (Table 1). This finding is significant, because the highest overall prevalence of TXCD was observed just before the calves were sent for slaughter. The increase in prevalence over time may be due to housing of calves in close proximity, which facilitates fecal-oral transmission of bacteria. Although calves were housed on grates, fecal material noticeably accumulated in barns on all farms over time. The overall reduction in the prevalence of C. difficile toxin genes in feces of calves in herd C may be a result of less stressful housing conditions than those used on the other farms.

Very few calves (n=4) tested positive for toxin genes more than once over the course of the study, thus suggesting that carriage of TXCD is transient. C. difficile is unable to colonize the gut and cause disease unless the gut microflora is disrupted (Borriello et al., 1987; Borriello, 1990). Normal gut flora does not allow sporulation and colonization of C. difficile by out-competing the pathogen for nutrients (Borriello, 1990). It is likely that C. difficile cells move rapidly through the digestive tract of healthy calves due to colonization resistance as described by other researchers (Wilson et al., 1985).

Incidence of veal carcass contamination

We observed a very low prevalence of C. difficile toxin genes in pre-wash carcass swabs collected at the abattoir. The C. difficile housekeeping gene, tpi, was detected by PCR on 4 of 100 precitric acid wash carcass swabs, 2 from herd B and 2 from herd D. One of the tpi positive carcass swabs from herd B was positive for the tcdA gene. No toxin genes were detected on postcitric acid wash carcass swabs. C. difficile was not isolated from any carcass sample, pre or postwash, by using enriched culture techniques.

These findings are not surprising given the advances that have been made in abattoir sanitation with the implementation of standard operating procedures for sanitation and hazard analysis critical control point plans for pathogen contamination reduction. Sanitation procedures used at veal processing plant that participated in this study included a hot acid carcass wash. Carcass washing has been validated as an effective method for reducing carcass contamination (Dormedy et al., 2000; Cutter and Rivera-Betancourt, 2006).

Incidence of ground veal contamination

Despite an extremely low prevalence of TXCD on veal carcasses, toxin genes of C. difficile were detected by multiplex real-time PCR in 4 out of 50 (8%) ground veal samples that originated from the veal processor that participated in the study. Genes encoding toxins TcdA, TcdB, and CDT were detected in three samples, whereas only genes encoding CDT were detected in the fourth positive sample. Toxigenic C. difficile isolates were recovered from three out of four PCR positive ground veal samples. Isolates from two culture positive samples encoded for all four toxin genes, the isolate from the third sample encoded for TcdA only.

These results suggest that either carcass washing does not completely eliminate TXCD contamination or TXCD contamination occurs during processing sometime after the carcass wash. It is well established that raw meat products can be cross-contaminated with foodborne pathogens including Campylobacter, Escherichia coli, and Salmonella through contact with contaminated processing plant surfaces and equipment (Bouvet et al., 2002; Gun et al., 2003; Malakauskas et al., 2006; Bertrand et al., 2010). Therefore, it is reasonable to infer that C. difficile spores are able to persist in the abattoir environment and may contaminate veal during postcarcass wash processing. In the case of ground meat, one of the most probable sources of postcarcass wash contamination is during the grinding process. Persistence of foodborne pathogens in the meat processing environment is strongly attributed to the formation of biofilms that can occur on virtually any surface (Kumar and Anand, 1998). Organic material from meat is absorbed by surfaces in the meat processing environment and forms a conditioning film, which allows attachment of microorganisms. Sanitization practices may be ineffective in completely removing organic material from processing equipment (Farrell et al., 1998). Residual organic material that remains on processing equipment allows subsequent adherence of vegetative cells and spores and facilitates cross-contamination of meat products (Carpentier and Cerf, 1993; Ronner and Lee Wong, 1993).

Weese et al. (2009) reported that 12% of Canadian retail ground beef and pork samples contained TXCD, although on enumeration, contamination levels were found to be low. In another study, C. difficile was detected in 20.8% and 14.3% of Canadian retail ground beef and veal, respectively, and most recovered isolates encoded for TcdA, TcdB, and CDT (Rodriguez-Palacios et al., 2007). A much higher incidence of C. difficile in retail ground meat was reported for Tucson, Arizona, where 42.4%, 41.3%, and 44.4% of beef, pork, and turkey were found to contain TXCD, respectively (Songer et al., 2009). Other studies report a much lower international prevalence of C. difficile in ground meat (Indra et al., 2009; von Abercron et al., 2009; Bouttier et al., 2010; Jobstl et al., 2010; Weese, 2010).

Public health significance

Although it is clear that raw meat is a source of community exposure to toxigenic C. difficile, the resulting risk to public health has not yet been established. Heat resistance of C. difficile spores results in survival of the microorganism in meat even after heating to proper cooking temperature. In fact, it has been reported that C. difficile spores are able to survive for up to 2 h in ground beef heated to 71°C (recommended cooking temperature) (Rodriguez-Palacios et al., 2007). Still, no cases of foodborne C. difficile infection have been reported (Weese et al., 2010).

Conclusions

The results reported here confirm that veal calves are asymptomatic carriers of TXCD, and the prevalence of C. difficile toxin genes in calf feces may vary with calf age. Toxigenic strains of C. difficile, possibly originating from veal calves, are able to contaminate veal carcasses at slaughter and can be found in finished ground veal product meant for human consumption. The practice of citric acid carcass wash may be effective in reducing vegetative forms of C. difficile on carcasses, although the presence of TXCD in ground veal suggests that other routes of contamination need to be assessed.

Footnotes

Disclosure Statement

No competing financial interests exist.

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