Effects of Seasonality and a Diet of Brassicas on the Shedding of Escherichia coli O157 in Sheep

Abstract

Sheep flocks were tested for Escherichia coli O157 from pooled fecal samples while they grazed on pasture in winter, brassicas in spring, and on pasture during the summer. The winter pasture study reported an average individual prevalence of 3.1% (95% confidence interval [CI]: 0.6–5.6%) and an average farm-level prevalence of 10.4% (95% CI: 2.1–18.8%) over the 3-year study period. The spring brassica study reported a prevalence of 0% and the summer pasture study had an individual prevalence of 6.3% (95% CI: 2.1–12.1%) and a farm prevalence of 36.8% (95% CI: 15.8–57.8%). Analysis showed significant differences between the shedding of E. coli O157 in sheep grazing on brassicas in spring when compared to sheep grazing on pasture in the summer (p<0.01) and in winter (p=0.044; odds ratio [OR]=0.106). Furthermore, sheep excreted a lower prevalence of E. coli O157 in winter while grazing on pasture (p=0.017; OR=0.199). E. coli O157 isolates were characterized using polymerase chain reaction for the presence of known virulence factors; all carried the eae and stx2 gene and 10/11 positive flocks possessed the stx2c gene, suggesting that sheep are a potential source of human infection.

Introduction

S higa toxin–producing Escherichia coli (STEC) O157 is a pathogen posing a major risk to human health. Symptoms of STEC infection include bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (Gyles, 2007), which can result in death (Griffin and Tauxe, 1991). Strains of STEC produce two major types of Shiga toxins referred to as stx1 and stx2 (Paton and Paton, 1998).

Ruminants are the primary reservoir of E. coli O157, where it colonizes the intestine, causing the pathogen to be shed in the feces (Beutin et al., 1993; Prendergast et al., 2011; Vande Walle et al., 2011). Routes of human infection include the consumption of contaminated foods (meat products, fruit, vegetables, and dairy products) and water, environmental transmission, and direct transfer through animal-to-human contact and person-to-person transmission (Ferens and Hovde, 2011; Prendergast et al., 2011).

Compared with other regions of the United Kingdom and Europe, Scotland exhibits higher rates of E. coli O157 infection, with 4.8 cases per 100,000 population (Locking et al., 2012). Northeast (NE) Scotland is of particular concern, as it routinely exhibits one of the highest regional rates at 7.8 cases per 100,000 population in 2011 (Locking et al., 2012).

Previous prevalence studies in NE Scotland have reported that sheep grazing on pasture during the summer have an individual prevalence of 6.5% (with 34.1% of these animals shedding high concentrations [>10⁴/g] E. coli O157) and a farm prevalence of 40% (Ogden et al., 2005). A winter sheep study (January–March) in the same region reported that sheep have an individual prevalence of 5.8% and a group prevalence of 42.8% (Solecki et al., 2009). Solecki et al. (2009) also reported that the strains found in sheep grazing on brassicas (root crops; e.g., turnips and swedes; all routinely used by farmers as a feed for sheep during the winter/spring) during winter/early spring lacked both the stx₁ and stx₂ toxin genes.

Ruminants that are colonized by E. coli O157 are asymptomatic (Beutin et al., 1993); hence, livestock keepers are unable to identify animals that may be shedding the organism. Due to the possibility that humans may become exposed to and infected by ruminant-derived E. coli O157, there is a need to develop a means to limit the colonization of livestock by the organism. A number of methods have been evaluated, including vaccination (Snedeker et al., 2012) and various dietary manipulations. None of these as yet has proved sufficiently effective for widespread use. The finding that feeding brassica to sheep may influence the E. coli O157 status of sheep (Solecki et al., 2009) is therefore of considerable interest and potential importance to the ruminant livestock sector.

The aim of this study was to investigate whether season and diet have an effect on the shedding of E. coli O157 in sheep flocks kept in a farm environment. This was addressed by investigating the E. coli O157 status on sheep farms in winter (while sheep were grazing on pasture) and in spring (while sheep were grazing on brassicas). Results were compared to the shedding of E. coli O157 during the summer (while sheep were grazing on pasture). The virulence genes present in E. coli O157 isolates were also determined.

Materials and Methods

Farm sampling

Farmers in NE Scotland were contacted from a list of all farms in the region and were selected for the study on the basis of having sheep grazing outside on pasture over the winter and using brassicas as a feedstuff in spring. Flocks consisted of ewes, yearlings, pregnant ewes, and ewes with lambs. Farms were widespread, within a 25-mile radius of Aberdeen (NE Scotland). A power calculation was used to determine the sample size required to have 80% power to detect a reduction of 50% in prevalence (i.e., final flock prevalence of 20%). The number of flocks needed to be tested would be 40. The calculation was done by Monte Carlo simulation using @RISK 4.5 (Palisade) add-in to Microsoft Excel. A total of 48 flocks were sampled while sheep grazed on pasture, and a total of 41 flocks were sampled while they were grazing on brassicas.

Winter and spring study

Sixteen farms were recruited to the winter sheep study, with flock sizes ranging from 100 to 600 sheep. Flocks were tested for E. coli O157 twice: while they were grazing pasture in winter and then again when grazing on brassicas in spring (Table 1). The sheep were either strip grazed on brassicas or the brassicas were lifted into the field (diet consisted of ∼70% brassica). The same 16 sheep farms were revisited during the next 2 years while sheep were grazing on pasture and on brassicas (Table 1). Only nine of the 16 farms were sampled while the sheep were grazing on brassicas in the final year of the study. The number of fecal samples/flock collected was 50. Over the 3 years, 4450 fresh fecal grab samples were collected from the ground, ensuring that only one fecal sample was collected per sheep. Samples were collected in sterile plastic bags and stored at 4°C during transport to the laboratory and tested as pools of five grabs in 890 bags.

Table 1.

Farm Positivity of Escherichia coli O157 Isolates Found in Sheep Feces in Northeast Scotland Sheep Farms During Winter and Spring

Farm no.	Date sampled	Presence/ absence of O157 (+/–)Grazing on pasture	Date sampled	Presence/ absence of O157 (+/–) Grazing on brassicas	Date sampled	Presence/ absence of O157 (+/–) Grazing on pasture	Date sampled	Presence/ absence of O157 (+/–) Grazing on brassicas	Date sampled	Presence/ absence of O157 (+/–)Grazing on pasture	Date sampled	Presence/ absence of O157 (+/–) Grazing on brassicas
S1	11/30/2009	−	03/29/2010	−	1/10/2011	−	03/21/2011	−	11/14/2011	−	03/12/2012	−
S2	11/30/2009	−	04/27/2010	−	1/18/2011	−	04/03/2011	−	01/16/2012	−	NA	NA
S3	11/30/2009	−	03/09/2010	−	10/26/2010	−	03/21/2011	−	01/16/2012	−	NA	NA
S4	11/30/2009	−	03/29/2010	−	10/26/2010	−	03/21/2011	−	11/14/2011	−	03/12/2012	−
S5	11/30/2009	−	03/29/2010	−	10/26/2010	+	03/21/2011	−	11/14/2011	−	03/12/2012	−
S6	12/07/2009	−	03/17/2010	−	11/22/2010	+	03/07/2011	−	11/21/2011	+	03/25/2012	−
S7	12/07/2009	−	03/22/2010	−	11/01/2010	−	03/07/2011	−	11/14/2011	−	NA	NA
S8	12/07/2009	−	04/27/2010	−	01/10/2010	−	04/03/2011	−	12/05/2011	−	NA	NA
S9	12/07/2009	−	04/19/2010	−	12/12/2010	+	04/03/2011	−	12/05/2011	−	NA	NA
S10	12/07/2009	−	04/19/2010	−	11/15/2010	−	03/07/2011	−	11/21/2011	−	03/25/2012	−
S11	12/14/2009	−	04/05/2010	−	11/08/2010	+	03/28/2011	−	11/28/2011	−	03/19/2012	−
S12	12/14/2009	−	04/05/2010	−	12/12/2010	−	03/28/2011	−	11/28/2011	−	03/19/2012	−
S13	12/14/2009	−	04/05/2010	−	12/12/2010	−	03/28/2011	−	11/28/2011	−	03/19/2012	−
S14	12/14/2009	−	04/19/2010	−	12/12/2010	−	03/07/2011	−	12/05/2011	−	03/25/2012	−
S15	11/01/2010	−	03/17/2010	−	11/22/2010	−	03/13/2011	−	01/16/2012	−	NA	NA
S16	01/25/2010	−	04/27/2010	−	01/10/2011	−	04/11/2011	−	01/15/2012	−	NA	NA

NA, not applicable.

Summer study

Nineteen separate farms with sheep grazing on pasture were recruited in the summer study. These farms did not use brassicas as a feed; also, flocks varied in terms of age, breed, and gender. These flocks were sampled once between June 28, 2010 and September 27, 2010. Nine hundred and fifty feces (as above) were collected and pooled together into 190 bags of five fecal samples.

Isolation of E. coli O157

Pooled fecal samples were homogenized and 25 g was dispersed into 225 mL of buffered peptone water (Oxoid CM509, Basingstoke, UK) supplemented with 8 mg/L of vancomycin and enriched at 42°C for 6 h. Immunomagnetic separation (IMS; KingFisher, Thermoelectron, Basingstoke, UK) was performed after incubation. The beads were recovered, resuspended in phosphate-buffered saline, and 0.1 mL was equally plated onto Harlequin^™ SMAC BCIG agar (LabM, IDG, Bury, UK), agar supplemented with cefixime (0.05 mg/L), and potassium tellurite (2.5 mg/L) and onto sorbitol MacConkey agar (Oxoid CM813, Basingstoke, UK) supplemented with cefixime and potassium tellurite, as before. The plates were incubated at 37°C for 18–24 h and any nonsorbitol fermenting and beta-glucuronidase-negative colonies were plated onto plain agar and confirmed as positive targets by E. coli O157 latex agglutination (Oxoid DR620, Basingstoke, UK).

DNA extraction

Positive colonies were plated onto nutrient agar (Oxoid, Basingstoke, England) and one colony was transferred into 1 mL of phosphate-buffered saline prepared with DNAse-, RNAse-free water (Sigma Aldrich Company Ltd., Irvine, Ayrshire, UK), and boiled for 10 min in a heating block.

Virulence profiling

Polymerase chain reaction (PCR) was used to detect the presence of virulence genes: stx1, stx2, stx2c, stx2d, stx2e, and eae in each of the E. coli O157 isolates. The primers and PCR conditions used for eae, stx1, and stx2 genes were described by Paton and Paton (1998), while the PCR for the stx2c, stx2d, and stx2e genes was carried out by the methods of Wang et al. (2002).

Statistical analysis

Data from the winter pasture study and the spring brassica study were compared with the summer pasture study using an Excel add-in for a one-tailed Fisher's exact test (www.obertfamily.com/software/fisherexact.html) to determine whether shedding of E. coli O157 while sheep grazed different diets was statistically significant. Odds ratios (OR) with 95% confidence intervals (CI) were calculated using Excel to define the odds of sheep shedding E. coli O157 in the winter while grazing on pasture and spring grazing on brassicas compared to sheep grazing on pasture in the summer. Farm prevalence and estimated individual prevalences from pooled samples were calculated and confidence levels were obtained using Monte Carlo simulation using poptools add-in (http://www.cse.csiro.au/poptools/) to Microsoft Excel. To estimate individual prevalences, the original data was resampled with replacement, and simulation of 10,000 iterations of the model using Monte Carlo analysis was used to determine the estimated number of positive samples in each pooled fecal sample.

Results

Winter and spring study

From the first-stage fecal sampling, 160 bags of five pooled feces were analyzed, all of which tested negative for E. coli O157. The same farms were re-sampled while the sheep were grazing on brassicas (160 bags of five pooled feces were analyzed). These were all negative for E. coli O157. The farm study was repeated the following year. Four of the farms that had previously tested negative were found to have sheep that were positive for E. coli O157 while they were grazing on pasture (Table 1). Of the 160 bags of feces analyzed (five feces pooled), four bags were positive for E. coli O157. This showed an individual prevalence of 2.5% (95% CI: 0.6–5%) and a farm prevalence of 25% (95% CI: 6.3–50%). Although there was E. coli O157 present, the number of organisms per gram was low, inferred from low target numbers postenrichment and IMS. All 16 of the sheep farms (including the four that had previously tested positive while the sheep were at pasture) tested negative, while the sheep were grazing on brassicas. Prevalence concurred with the previous year at 0%. The third and final year of the farm study found that one farm was positive for E. coli O157 (individual prevalence of 0.63% and a farm prevalence of 6.25%) while sheep were grazing on pasture, and nine farms sampled of the 16 farms were negative for E. coli O157 while sheep were grazing on brassicas. Over the 3-year study period, the winter pasture study reported an average individual prevalence of 3.1% (95% CI: 0.6–5.6%) and an average farm-level prevalence of 10.4% (95% CI: 2.1–18.8%). A one-tailed Fisher's test was used to compare the association between the shedding of E. coli O157 in sheep grazing on pasture in winter and shedding from sheep grazing on brassicas in spring; results showed that there were significantly fewer flocks on brassicas shedding E. coli O157 (p=0.044; OR=0.106; Table 2).

Table 2.

Association Between Escherichia coli O157 Found in Sheep Feces and Diet

Diet and season	No. of positive farms	No. of negative farms	OR	95% CI	p-Value
Pasture in summer	7	12	1^a
Pasture in winter	5	43	0.199	0.054–0.742	0.017
Brassicas in spring	0.5^b	40.5^b	0.021	0.001–0.400	0.0002
Pasture in winter	5	43	1^a
Brassicas in spring	0.5^b	40.5^b	0.106	0.005–2.006	0.044

Reference category.

Since zero farms were positive, it is usual to use 0.5 as a conservative approach (Whitehead, 2002).

OR, odds ratio; CI, confidence interval.

Summer study

Of the 19 farms sampled, seven were positive for E. coli O157, giving an individual prevalence of 6.3% (95% CI: 2.1–12.1%) and a farm prevalence of 36.8% (95% CI: 15.8–57.8%). When the farm-level prevalence data of the summer pasture study were compared to the results of the winter pasture study, it was found that there was a significant difference between shedding of E. coli O157 in winter and summer (p=0.017; OR=0.199, Table 2). Furthermore, there was also a significant difference between the shedding of E. coli O157 in sheep during the summer compared with sheep grazing on brassicas in spring (p=0.0002; OR=0.021). One farm (S23) probably had at least one animal shedding high numbers (confluent target growth post IMS), while the others exhibited low numbers of E. coli O157 (Table 3).

Table 3.

Farm Positivity of Escherichia coli O157 Isolates Found in Sheep Feces in Northeast Scotland Sheep Farms During the Summer

Farm no.	Date sampled	Presence/absence of O157 (+/–)
S17	07/19/2010	+
S18	07/12/2010	−
S19	07/19/2010	−
S20	07/19/2010	−
S21	07/19/2010	−
S22	07/26/2010	−
S23	07/26/2010	+
S24	08/02/2010	+
S25	08/03/2010	−
S26	08/03/2010	−
S27	08/09/2010	−
S28	08/09/2010	−
S29	08/09/2010	−
S30	08/09/2010	−
S31	09/06/2010	+
S32	09/06/2010	+
S33	09/13/2010	+
S34	09/20/2010	+
S35	09/20/2010	−

Virulence profiles

From the winter study, all E. coli O157 isolates collected carried the eae, stx2, and stx2c genes. Furthermore, none of these isolates carried the stx1 or the stx2d and stx2e gene. From the summer study, sheep from two farms (S17 and S23) carried the eae, stx1, stx2, and stx2c gene while the remaining five samples were negative for stx1. Sheep from farm S24 was the only one negative for the stx2c gene.

Discussion

The average farm prevalence of E. coli O157 in the winter study was 10.4% (95% CI: 2.1–18.8%) when sheep were grazing on pasture. Seasonality can affect the prevalence of E. coli O157, as increased shedding of the organism has been reported in spring and summer during warmer temperatures, which may promote the growth and survival of E. coli O157 in the environment (Money et al., 2010). This may explain why the prevalence of the summer sheep study was higher than the winter sheep study here. According to the Health Protection Agency (2011), the majority of human infections from E. coli O157 occur in the warmer months between July and September. Also, cattle shed higher concentrations of E. coli O157 during the summer months in Alberta (Stanford et al., 2005) and Sweden (Albihn et al., 2003), and in Italy, 17% of cattle carry E. coli O157 in warmer months compared to 2.9% in winter (Bonardi et al., 1999).

The prevalence of E. coli O157 in the spring study was 0% when sheep were grazing on brassicas in all 3 years of the study. The same sheep flocks that were positive when grazing on pasture in the winter were re-sampled on brassicas in the spring; however, feces sampled from these flocks were negative when sheep were moved onto brassicas. A 15-month survey of a cattle farm in the United Kingdom showed that the highest prevalence was between May and July, then no E. coli O157 was detected between December and May (Mechie et al., 1997). This concurs with the results from the spring study, with no E. coli O157 detected between March and April, suggesting that seasonality may affect the prevalences. Studies suggest that higher numbers of E. coli O157 are shed by cattle in warmer months (Chapman et al., 1997; Money et al., 2010). However, there was a decrease in the shedding of E. coli O157 in the spring when temperatures were an average of 5°C higher than in winter (see Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/fpd). These findings suggest that diet rather than season may have an influence on the shedding of E. coli in sheep.

When the brassica study was compared to the summer pasture study, the results were highly significant (p<0.01), indicating that the feeding of brassicas could reduce the shedding of E. coli O157 in sheep. Furthermore, the odds ratio was 0.021, which indicates that E. coli O157 is less likely to be shed while sheep are feeding on brassicas in spring than on pasture in the summer. Previous studies in the same geographical area reported that sheep fed on brassicas during the winter shed lower concentrations of E. coli O157; furthermore, these strains were atoxigenic (Solecki et al., 2009).

Brassicas contain a range of internal compounds called glucosinolates (Liang et al., 2006). Glucosinolates are hydrolyzed by the enzyme myrosinase during ruminant mastication, releasing oxazolidithione, isothiocyanates, nitrile, and thiocyanates (Tripathi and Mishra, 2007). Isothiocyanates produced as a result of this reaction have been reported to have antimicrobial properties (Tripathi and Mishra, 2007), with studies reporting that allyl isothiocyanate present in mustard flour can eradicate E. coli O157:H7 from minced beef during refrigeration and storage (Nadarajah et al., 2005a; Nadarajah et al., 2005b). Although only low numbers of E. coli O157 were detected in sheep prior to the change in diet, it can be hypothesized that the prolonged grazing on brassicas during the spring could have an impact on the levels of E. coli O157 shed due to the potentially inhibitory dietary compounds. If this observation were true, the livestock industry could benefit. In Scotland, lambs are routinely fed on brassicas before slaughter. Therefore, increasing the practice of feeding livestock brassicas could reduce the shedding of E. coli O157. This study design, however, prevents controlling for confounding by season. Future work should include a prevalence study where flocks are split and fed a diet of brassicas or pasture in the same season. Furthermore, the farms were only sampled once per diet change; therefore, longitudinal studies of the flocks are needed to provide more convincing evidence as the low prevalence may be caused by a confounding variable other than diet.

Other studies have reported that diet can influence the level of E. coli O157 shed in ruminants (Callaway et al., 2009). In Scotland, feeding distillers grain has been shown to increase the shedding of E. coli O157 (Synge et al., 2003). Also, phenolic acids found in forage plants have been shown to decrease the survival of E. coli O157 in cattle feces (Wells et al., 2005).

Solecki et al. (2009) reported an individual prevalence of 5.8% and a farm prevalence of 42.8% in sheep during the winter compared to our findings of an average individual prevalence of 3.1% and a farm prevalence of 10.4%. Both of these farm studies were carried out in the same geographical region. In our study, 12 farms always tested negative for E. coli O157 and one farm tested positive for E. coli O157 twice. To control for repeated measures within farms, the probability of detecting farms that were always positive for E. coli O157 and always negative for E. coli O157 were calculated. The probability of 12 or more farms always being negative for E. coli O157 is 0.39, which suggests that we are not selecting for farms that are always negative. Furthermore, the probability of one or more farms being positive by chance is 0.44. Since only one farm tested positive twice, this suggests that we have not detected an effect of a particular farm remaining positive. Recent results for the summer study (individual prevalence of 6.3% and farm prevalence of 36.8%), however, were similar to results reported in a summer study of sheep grazing pasture with an individual prevalence of 6.5% and a farm prevalence of 40% (Ogden et al., 2005). Reported prevalences of E. coli O157 in sheep are variable; however, they ranged from 0% to 7.3% (Chapman et al., 1997; Heuvelink et al., 1998; Paiba et al., 2002; Battisti et al., 2006; Lenahan et al., 2007; Milnes et al., 2008; Oporto et al., 2008), which are similar to the yearly prevalences reported in this study.

The virulence genes present in the sheep isolates were similar to those in previous sheep studies (Ogden et al., 2005), with all isolates possessing the eae and stx2 genes and the majority of isolates negative for the stx1 gene. The uniformity of the stx profiles in this study suggests that the ecology of E. coli O157 among sheep may not be subject to frequent immigration of novel strains of E. coli O157. These isolates therefore have the potential to cause human infection as most clinical isolates in the UK are stx2 positive (Ogden et al., 2004).

Conclusions

E. coli O157 infection in Scotland remains a problem, with NE Scotland exhibiting one of the highest rates of human infection from this pathogen. The present study analysis demonstrated that there was less E. coli O157 shed in sheep during the winter compared with during the summer, suggesting that season has an effect on the shedding of E. coli O157 in sheep. Intervention strategies at slaughter are in place to reduce the levels of contamination on meat. Developing strategies that can limit the carriage of E. coli O157 in sheep on the farm could have the significant potential to reduce contamination and future human illness. This study reported that the prevalence of E. coli O157 was 0% while sheep grazed on brassicas in spring. Additional statistical analysis reported that E. coli O157 is less likely to be shed on brassicas in spring than on pasture in summer and winter. The temperatures in spring when sheep were grazing on brassicas were higher than those in the winter therefore; it can be hypothesized that brassicas can affect the shedding of E. coli O157 in sheep. Further testing of this hypothesis is needed as well as a demonstration of its feasibility as a practical approach to reduce E. coli O157. Future work should include a summer prevalence study where sheep flocks are split into two groups and fed either a diet of brassicas or pasture, as this will essentially control the effect of season on the shedding of E. coli O157.

Footnotes

Acknowledgments

We would like to thank the Food Standards Agency Scotland, National Farmers Union Scotland, Quality Meat Scotland, and the University of Aberdeen for funding this study. We offer thanks to Professor Charlotte Maltin (Quality Meat Scotland) for suggestions regarding the farm studies and for commenting on the draft manuscript. We also thank the farmers who allowed their sheep to be sampled.

Disclosure Statement

No competing financial interests exist.

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