Summer and Winter Prevalence of Shiga Toxin–Producing Escherichia coli (STEC) O26,O45,O103,O111,O121,O145,and O157 in Feces of Feedlot Cattle

Abstract

The United States Department of Agriculture Food Safety and Inspection Service has declared seven Shiga toxin–producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, nonintact beef products. The objective of this study was to determine the prevalence of these seven serogroups and the associated virulence genes (Shiga toxin [stx1, stx2], and intimin [eae]) in cattle feces during summer (June–August 2013) and winter (January–March 2014) months. Twenty-four pen floor fecal samples were collected from each of 24 cattle pens, in both summer and winter months, at a commercial feedlot in the United States. Samples were subjected to culture-based detection methods that included enrichment, serogroup-specific immunomagnetic separation and plating on selective media, followed by a multiplex polymerase chain reaction for serogroup confirmation and virulence gene detection. A sample was considered STEC positive if a recovered isolate harbored an O gene, stx1, and/or stx2, and eae genes. All O serogroups of interest were detected in summer months, and model-adjusted prevalence estimates are as follows: O26 (17.8%), O45 (14.6%), O103 (59.9%), O111 (0.2%), O121 (2.0%), O145 (2.7%), and O157 (41.6%); however, most non-O157 isolates did not harbor virulence genes. The cumulative model-adjusted sample-level prevalence estimates of STEC O26, O103, O145, and O157 during summer (n=576) were 1.0, 1.6, 0.8, and 41.4%, respectively; STEC O45, O111, and O121 were not detected during summer months. In winter, serogroups O26 (0.9%), O45 (1.5%), O103 (40.2%), and O121 (0.2%) were isolated; however, no virulence genes were detected in isolates from cattle feces collected during winter (n=576). Statistically significant seasonal differences in prevalence were identified for STEC O103 and O157 (p<0.05), but data on other STEC were sparse. The results of this study indicate that although non-O157 serogroups were present, non-O157 STEC were rarely detected in feces from the feedlot cattle populations tested in summer and winter months.

Introduction

Shiga toxin–producing Escherichia coli (STEC) are foodborne pathogens of public health importance. Cattle serve as a principal reservoir of STEC and shed these bacteria in their feces (Bettelheim, 2000). Human STEC infections are acquired through direct or indirect fecal–oral contact with human or animal feces (Evans and Evans, 1996), and can lead to illnesses including mild to severe diarrhea, hemorrhagic colitis, and potentially life-threatening complications such as hemolytic uremic syndrome or thrombotic thrombocytopenia purpura (Siegler and Oakes, 2005; Kuter et al., 2014). Several foods, including beef, have been implicated in STEC outbreaks in humans in North America (Scallan et al., 2011; Painter et al., 2013). As a result, the United States Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) has declared STEC of seven serogroups (O26, O45, O103, O111, O121, O145, and O157) as adulterants in raw, nonintact beef products.

Although there has been extensive research on the epidemiology and ecology of STEC O157 in cattle, information on the prevalence and distribution of non-O157 STEC in the cattle reservoir and their production environment is limited. Periharvest commercial beef operations are a critical node in the beef system continuum, as they represent the origin of fecally originated microbial contamination to animal hides, which then can subsequently disseminate to preintervention carcasses during slaughter, and potentially, beef products during further stages of beef processing. A strong seasonal pattern has been identified for E. coli O157:H7, where fecal prevalence in cattle peaks during summer months and greatly decreases in winter months (Barkocy-Gallagher et al., 2003; Smith et al., 2005; Edrington et al., 2006). In a mathematical model, prevalence of E. coli O157:H7 in cattle was correlated with ground beef prevalence and human illnesses attributed to E. coli O157:H7 (Williams et al., 2010). Frequency of human disease attributed to STEC O157 has declined since 2000; however, illnesses due to non-O157 STEC in the United States have increased (Gould et al., 2013), with the majority (58%) of outbreaks occurring during summer months (Luna-Gierke et al., 2014). The recent apparent increase in the incidence of non-O157 STEC human illness may be overstated due to a recent increase in the development and application of detection methods (Gould et al., 2013; Crim et al., 2014). However, accounting for the under-reporting of foodborne illness, it is estimated that more non-O157 STEC infections occur annually compared to STEC O157 in the United States (Scallan et al., 2011).

Recently, fecal prevalence of non-O157 serogroups and virulence genes in cattle and their environment has been studied in the United States. Non-O157 serogroup and STEC fecal prevalence estimates reported in cattle range from 0.0 to 90.7% and 0.9 to 16.9%, respectively, in the United States (Paddock et al., 2012; Cernicchiaro et al., 2013; Dargatz et al., 2013; Baltasar et al., 2014; Ekiri et al., 2014). However, only a few studies have assessed seasonality of non-O157 STEC shedding in cattle (Barkocy-Gallagher et al., 2003; Alexa et al., 2011; Tanaro et al., 2012). Often publications report combined prevalence estimates for all non-O157 STEC collectively, rather than reporting serogroup-specific prevalence estimates. Collective prevalence estimates may inaccurately represent the prevalence of the non-O157 STEC serogroups of regulatory or public health importance. Therefore, the objective of this study was to determine the prevalence of seven STEC O serogroups (O26, O45, O103, O111, O121, O145, and O157) and their virulence genes (stx1, stx2, and eae) in feces of preharvest commercial feedlot cattle during summer and winter months.

Materials and Methods

Study population and sample collection

Pens of crossbred beef cattle from a large commercial feedlot in the central United States were sampled weekly for 12 weeks in summer (June–August 2013) and 10 weeks in winter (January–March 2014). Cattle were managed following standard operating procedures of the feedlot. This study followed a repeated cross-sectional design. During summer months, 24 pen floor fecal samples from each of 2 pens of finishing cattle, within 24 h of harvest, were sampled each week. Twenty-four pen floor fecal samples from each of up to four pens of cattle, within 2 weeks of harvest, were sampled weekly during winter months. Samples were collected from 24 cattle pens in summer and 24 cattle pens in winter months. Sample sizes were determined using prevalence estimates from a previous study (Cernicchiaro et al., 2013) to detect seasonal differences as low as 5%, with a power of 80%, and Type I error of 5%. Approximately 10 g of freshly voided feces were collected in individual plastic bags (WHIRL-PAK^®; Nasco, Fort Atkinson, WI) using plastic spoons, placed in a cooler on ice, and transported to the Pre-harvest Food Safety Laboratory at Kansas State University for processing within 36 h.

Culture-based detection

Approximately 2 g of feces were added to 18 mL of E. coli broth (EC; Difco, ThermoFisher, Lenexa, KS), vortexed, and incubated for 6 h at 37°C. Postenrichment, immunomagnetic separation (IMS) procedures were performed in a Kingfisher Flex Magnetic Particle Processor (Thermo Scientific, Waltham, MA). An aliquot of 980 μL of enriched fecal suspension was added to 20 μL serogroup-specific IMS beads (Abraxis^®; Abraxis LLC., Warminister, PA), for each of the E. coli serogroups of interest (O26, O45, O103, O111, O121, O145, and O157); a total of 7 IMS runs were completed for each sample tested. The IMS method consisted of a 30-minute binding step, three 2-min washes with 1000 μL of phosphate-buffered saline (PBS) Tween 20, and a 1-min elution step using 100 μL PBS Tween 20. Following IMS, 50 μL of IMS bead suspension was spread-plated onto either Sorbitol MacConkey agar with cefixime and potassium tellurite (CT-SMAC) for O157, or Modified Possé (MP) (Possé et al., 2008) agar for non-O157 beads. The modification included reduction of novobiocin and potassium tellurite concentrations to 5.0 mg L^–1 and 0.5 mg L^–1, respectively. After the 20–24-h incubation, 6 chromogenic colonies were chosen from each of the 6 MP plates, and 6 non-sorbitol fermenting colonies were chosen from the CT-SMAC plate. These colonies were streaked onto blood agar plates and incubated for 24 h at 37°C.

PCR serogroup confirmation and virulence gene detection

Putative O157 colonies grown on blood agar were tested for the O157 antigen by latex agglutination and, if positive, then tested for indole production. Isolates positive by latex agglutination and indole assays were then confirmed by a multiplex PCR (six-plex) for detection of the six major genes (rfbE_O157, fliC_H7, eae, stx1, stx2, and ehxA) of E. coli O157 as outlined in Bai et al. (2010).

Putative non-O157 colonies grown on blood agar were pooled and tested by a multiplex PCR (seven-plex) targeting serogroup-specific genes (O26, O45, O103, O111, O121, and O145; Paddock et al., 2012). If the colony pool tested positive for one of the six non-O157 serogroups, then each colony was individually tested by a multiplex PCR (Bai et al., 2012) targeting six non-O157 serogroups and three virulence genes (stx1, stx2, and eae).

Samples were considered serogroup positive if the individual isolate tested positive for an O gene of interest by the multiplex PCR for non-O157 serogroups, or for the rfbE_O157 gene by the six-plex PCR. A sample was considered serogroup negative if colony pools tested negative for all O genes of interest by the seven-plex PCR; for serogroup O157, samples were considered negative if colonies tested negative to latex agglutination, indole production, or six-plex PCR assays. Samples were considered STEC positive if a serogroup-positive (O157 or non-O157) isolate carried at least one Shiga toxin gene (stx1 and/or stx2), and the intimin (eae) gene. Samples were classified as positive if at least one isolate tested positive to the respective serogroup and STEC definitions.

Statistical analyses

Crude cumulative pen-level prevalence estimates and 95% CIs were computed for the serogroup case definitions for each season. Prevalence at the pen level was calculated as the number of positive samples per pen divided by the total number of samples tested per pen.

Model-adjusted cumulative pen-level prevalence estimates and their 95% CIs were estimated from model intercepts using generalized linear mixed models. Monthly STEC prevalence estimates were also obtained by including the variable month (June, July, and August) as a fixed effect. Statistical models were fitted in Proc Glimmix (SAS 9.3; SAS Institute Inc., Cary, NC) using a binomial distribution, logit link, residual pseudo-likelihood estimation technique, Kendward-Roger degrees of freedom approximation, and a random effect of pens within weeks to account for the clustering effect of pens nested within sampling week. Nonparametric permutation tests were used to compare the within-pen mean number of positive samples between seasons, using the exact option for hypothesis testing (Proc Npar1way command in SAS 9.3). P-values<0.05 were deemed significant.

Results

Study population

Pens of healthy cattle closest to harvest, identified weekly by the feedlot manager, were sampled; consequently, study pens were sampled only once. A total of 48 pens housing 17,511 cattle were sampled. During summer months, 24 pens housing a total of 6473 steers were sampled. Each pen sampled between June and August 2013 housed an average of 270 cattle (range=121–299 cattle, median=283 cattle). In winter months, 24 pens were sampled, housing a total of 11,038 cattle (22 pen of steers [n=10,036] and 2 pens of heifers [n=1,002]). In winter, each pen housed an average of 460 cattle (range=189–627 cattle, median=544 cattle). A total of 1152 pen-floor fecal samples were collected from 48 pens (576 samples from 24 pens in both summer and winter months).

Summer prevalence

Serogroup O103 was the most prevalent serogroup isolated from this cattle population. In summer months, serogroup O103 was detected in 340 (59.0%) fecal samples; cumulative crude prevalence for serogroups O26, O45, O111, O121, O145, and O157 was 22.0, 16.5, 0.2, 2.1, 3.1, and 43.1%, respectively (Table 1). All seven serogroups were identified during summer months; however, serogroup O111 was isolated only in a single sample. Although non-O157 serogroups were detected during summer, non-O157 isolates rarely harbored stx and eae genes in our study population; however, nearly all O157 isolates harbored stx and eae genes.

Table 1.

Distribution of Positive Samples for Escherichia coli O Serogroups and Virulence Genes Isolated in Summer and Winter Months from Cattle Feces

	Summer
Serogroup	O gene n (%)	O gene+ stx1 n (%)	O gene+ stx2 n (%)	O gene+ stx1 and stx2 n (%)	O gene+ stx1 and/or stx2 n (%)	O gene+ stx1 and/or stx2+eae n (%)	Winter ^a O gene n (%)
O26	127 (22.0)	7 (1.2)	0 (0.0)	0 (0.0)	7 (1.2)	7 (1.2)	7 (1.2)
O45	95 (16.5)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	11 (1.9)
O103	340 (59.0)	9 (1.6)	1 (0.1)	0 (0.0)	10 (1.7)	10 (1.7)	236 (41.0)
O111	1 (0.2)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)
O121	12 (2.1)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	3 (0.5)
O145	18 (3.1)	4 (0.7)	2 (0.3)	0 (0.0)	6 (1.0)	6 (1.0)	0 (0.0)
O157	248 (43.1)	95 (16.5)	246 (42.7)	93 (16.7)	248 (43.1)	247 (42.9)	0 (0.0)

Proportions were calculated over the total number of fecal samples tested in winter and summer (n=576), respectively.

No isolates with virulence genes were detected in winter months.

The most common STEC isolated in summer months was STEC O157 (Table 1). Shiga toxin–producing E. coli O157 samples predominately tested positive for stx2 (42.7%) versus stx1 (16.5%) genes; however, 16.7% of samples harbored isolates that tested positive for both stx1 and stx2 genes. Shiga toxigenic E. coli O26, O103, and O145 were isolated in summer months; however, STEC O45, O111, and O121 were not detected during this period (Table 1). In contrast to STEC O157, the Shiga toxin gene most commonly associated with non-O157 STEC was stx1 (87%), while stx2 was present in only a few samples (13%), and no isolates harbored both genes. Serogroup and STEC model-adjusted cumulative prevalence estimates are presented in Table 2.

Table 2.

Model-Adjusted Cumulative Prevalence Estimates ^a of O Serogroups and Shiga Toxin–Producing Escherichia coli (STEC) for Summer and Winter Months

	Serogroup prevalence, % (95% CI)		STEC prevalence, % (95% CI)
O gene	Summer	Winter	Summer	Winter
O26	17.8 (15.1, 20.4)	0.9 (0.0, 4.1)	1.0 (0.0, 4.2)	0.0
O45	14.6 (12.2, 17.0)	1.5 (0.0, 4.5)	0.0	0.0
O103	59.9 (57.5, 62.2)	40.2 (37.9, 42.5)	1.6 (0.0, 4.4)	0.0
O111	0.2 (0.0, 5.5)	0.0	0.0	0.0
O121	2.0 (0.0, 4.7)	0.2 (0.0, 5.6)	0.0	0.0
O145	2.7 (0.0, 5.4)	0.0	0.8 (0.0, 4.1)	0.0
O157	41.6 (39.2, 44.0)	0.0	41.4 (39.0, 43.8)	0.0

Denominator for summer and winter is 576 fecal samples.

Estimates and 95% CIs from generalized linear mixed models using binomial distribution, logit link, and a random intercept of pens within week to estimate prevalence by season.

Pen-level crude prevalence values varied greatly between pens of cattle; pen-level crude prevalence of serogroups O26, O45, O103, O111, O121, O145, and O157 ranged from 0 to 95.8, 0 to 79.2, 16.7 to 91.7, 0 to 4.2, 0 to 12.5, 0 to 16.7, and 0 to 79.2%, respectively (Table 3). Similarly, variability in pen-level prevalence was also observed for STEC shedding among pens during summer months; the crude pen-level prevalence of STEC O26, O103, O145, and O157 ranged from 0 to 8.3, 0 to 12.5, 0 to 12.5, and 0 to 79.2%, respectively (pen-level data not shown).

Table 3.

Crude Pen-Level and Cumulative Prevalence ^a of O Serogroups by Pen and Sampling Week During Summer Months (June–August 2013)

Pen	Date	No. of cattle	O26 % (n)	045 % (n)	0103 % (n)	0111 % (n)	0121 % (n)	0145 % (n)	0157 % (n)
1	6/9/2013	284	25.0 (6)	16.7 (4)	45.8 (11)	0.0 (0)	0.0 (0)	4.2 (1)	50.0 (12)
2	6/9/2013	278	95.8 (23)	12.5 (3)	54.2 (13)	0.0 (0)	0.0 (0)	16.7 (4)	33.3 (8)
3	6/16/2013	281	25.0 (6)	12.5 (3)	41.7 (10)	0.0 (0)	0.0 (0)	0.0 (0)	0.0 (0)
4	6/16/2013	283	25.0 (6)	12.5 (3)	25.0 (6)	0.0 (0)	0.0 (0)	12.5 (3)	0.0 (0)
5	6/23/2013	284	83.3 (20)	25.0 (6)	41.7 (10)	0.0 (0)	0.0 (0)	4.2 (1)	70.8 (17)
6	6/23/2013	279	50.0 (12)	37.5 (9)	54.2 (13)	0.0 (0)	4.2 (1)	0.0 (0)	12.5 (3)
7	6/30/2013	284	16.7 (4)	8.3 (2)	62.5 (15)	0.0 (0)	4.2 (1)	4.2 (1)	58.3 (14)
8	6/30/2013	281	33.3 (8)	16.7 (4)	16.7 (4)	0.0 (0)	4.2 (1)	0.0 (0)	33.3 (8)
9	7/7/2013	273	25.0 (6)	8.3 (2)	66.7 (16)	0.0 (0)	4.2 (1)	0.0 (0)	50.0 (12)
10	7/7/2013	283	12.5 (3)	12.5 (3)	66.7 (16)	0.0 (0)	8.3 (2)	0.0 (0)	54.2 (13)
11	7/14/2013	284	25.0 (6)	29.2 (7)	83.3 (20)	0.0 (0)	0.0 (0)	0.0 (0)	58.3 (14)
12	7/14/2013	291	20.8 (5)	16.7 (4)	83.3 (20)	0.0 (0)	0.0 (0)	8.3 (2)	45.8 (11)
13	7/21/2013	277	8.3 (2)	16.7 (4)	83.3 (20)	0.0 (0)	0.0 (0)	0.0 (0)	58.3 (14)
14	7/21/2013	298	12.5 (3)	4.2 (1)	70.8 (17)	0.0 (0)	0.0 (0)	0.0 (0)	29.2 (7)
15	7/28/2013	292	4.2 (1)	4.2 (1)	50.0 (12)	0.0 (0)	0.0 (0)	12.5 (3)	75.0 (18)
16	7/28/2013	123	0.0 (0)	12.5 (3)	41.7 (10)	0.0 (0)	4.2 (1)	4.2 (1)	79.2 (19)
17	8/4/2013	299	20.8 (5)	79.2 (19)	91.7 (22)	0.0 (0)	12.5 (3)	0.0 (0)	33.3 (8)
18	8/4/2013	299	0.0 (0)	0.0 (0)	70.8 (17)	0.0 (0)	0.0 (0)	0.0 (0)	54.2 (13)
19	8/11/2013	279	4.2 (1)	4.2 (1)	62.5 (15)	4.2 (1)	0.0 (0)	0.0 (0)	37.5 (9)
20	8/11/2013	282	4.2 (1)	20.8 (5)	45.8 (11)	0.0 (0)	4.2 (1)	0.0 (0)	45.8 (11)
21	8/18/2013	294	4.2 (1)	20.8 (5)	66.7 (16)	0.0 (0)	0.0 (0)	4.2 (1)	41.7 (10)
22	8/18/2013	284	16.7 (4)	4.2 (1)	87.5 (21)	0.0 (0)	0.0 (0)	0.0 (0)	29.2 (7)
23	8/25/2013	240	8.3 (2)	16.7 (4)	66.7 (16)	0.0 (0)	0.0 (0)	4.2 (1)	33.3 (8)
24	8/25/2013	121	8.3 (2)	4.2 (1)	37.5 (9)	0.0 (0)	4.2 (1)	0.0 (0)	50.0 (12)
Pen prevalence range, %			0.0–95.8	0.0–79.2	16.7–91.7	0.0–4.2	0.0–12.5	0.0–16.7	0.0–79.2
Crude cumulative prevalence, %			22.0	16.5	59.0	0.2	2.1	3.1	43.1
95% CI, %			18.7–25.7	13.6–19.8	54.9–63.1	0.0–1.0	1.1–3.6	1.9–4.9	39.0–47.2

The number of positive samples divided by the total number of samples tested expressed as a percentage. The denominator (n=24) was the same for all pens.

Model-adjusted fecal prevalence of STEC O157 significantly differed (p<0.01) between study months: June (32.3%; 95% CI=25.7–39.7%), July (56.3%; 95% CI=48.7–63.5%), and August (40.1%; 95% CI=33.0–47.6%). Significant differences in STEC O157 fecal prevalence were established between June and July (p<0.01) and between July and August (p<0.01); however, June and August were not significantly different. Sampling month was not statistically significant for STEC O26, O103, and O145. Monthly model-adjusted prevalence estimates (and 95% CI) for STEC O26, O103, and O145 in June were 3.1% (1.3–7.1%), 3.6% (1.7–7.8%), and 2.6% (1.0–6.4%), respectively; in July, monthly estimates for STEC O26, O103, and O145 were 0.5% (0.1–4.0%), 1.6% (0.5–5.1%), and 0.5% (0.1–4.0%), respectively. In August, STEC O26, O103, and O145 were not detected. The effect of month on fecal prevalence was not tested for STEC O45, O111, and O121 as these STEC were not detected in this group of samples during summer months.

Winter prevalence

Serogroup O103 was the most prevalent serogroup isolated from this cattle population in winter. Cumulative crude prevalence for serogroups O26, O45, O103, and O121 was 1.2, 1.9, 41.0, and 0.5%, respectively (Table 1). Serogroups O111, O145, and O157 were not isolated during this period. Model-adjusted cumulative serogroup prevalence estimates for winter are presented in Table 2. Crude prevalence estimates remained variable among pens; however, they were at lower proportions than observed in summer months (Table 4). In winter, serogroup O26, O45, O103, and O121 shedding ranged from 0 to 12.5, 0 to 12.5, 8.3 to 79.3, and 0 to 12.5%, respectively. Notably, serogroup O103 shedding was highly variable between study pens and was detected throughout all pens sampled during winter.

Table 4.

Crude Pen-Level and Cumulative Prevalence ^a of O Serogroups by Pen and Sampling Week During Winter Months (January–March 2014)

Pen ^b	Date	No. of cattle	026 % (n)	045 % (n)	0103 % (n)	0121 % (n)
25	1/6/2014	275	0.0 (0)	0.0 (0)	33.3 (8)	0.0 (0)
26^b	1/6/2014	461	0.0 (0)	0.0 (0)	20.8 (5)	0.0 (0)
27	1/6/2014	280	0.0 (0)	0.0 (0)	25.0 (6)	0.0 (0)
28	1/13/2014	189	0.0 (0)	0.0 (0)	41.7 (10)	0.0 (0)
29	1/13/2014	565	0.0 (0)	0.0 (0)	58.3 (14)	0.0 (0)
30	1/13/2014	553	0.0 (0)	4.2 (1)	58.3 (14)	12.5 (3)
31	1/13/2014	623	0.0 (0)	4.2 (1)	75.0 (18)	0.0 (0)
32	1/20/2014	289	4.2 (1)	0.0 (0)	25.0 (6)	0.0 (0)
33	1/20/2014	560	0.0 (0)	0.0 (0)	54.2 (13)	0.0 (0)
34	1/20/2014	557	0.0 (0)	12.5 (3)	54.2 (13)	0.0 (0)
35^b	1/27/2014	541	0.0 (0)	4.2 (1)	29.2 (7)	0.0 (0)
36	1/27/2014	560	0.0 (0)	0.0 (0)	50.0 (12)	0.0 (0)
37	2/3/2014	294	0.0 (0)	0.0 (0)	79.2 (19)	0.0 (0)
38	2/3/2014	624	4.2 (1)	0.0 (0)	33.3 (8)	0.0 (0)
39	2/10/2014	561	0.0 (0)	0.0 (0)	45.8 (11)	0.0 (0)
40	2/10/2014	627	0.0 (0)	0.0 (0)	12.5 (3)	0.0 (0)
41	2/17/2014	443	0.0 (0)	0.0 (0)	25.0 (6)	0.0 (0)
42	2/17/2014	546	0.0 (0)	0.0 (0)	45.8 (11)	0.0 (0)
43	2/24/2014	554	8.3 (2)	0.0 (0)	62.5 (15)	0.0 (0)
44	2/24/2014	530	0.0 (0)	8.3 (2)	37.5 (9)	0.0 (0)
45	3/3/2014	281	0.0 (0)	12.5 (3)	8.3 (2)	0.0 (0)
46	3/3/2014	564	12.5 (3)	0.0 (0)	20.8 (5)	0.0 (0)
47	3/10/2014	284	0.0 (0)	0.0 (0)	25.0 (6)	0.0 (0)
48	3/10/2014	277	0.0 (0)	0.0 (0)	62.5 (15)	0.0 (0)
Pen prevalence range, %			0.0–12.5	0.0–12.5	8.3–79.2	0.0–12.5
Crude cumulative prevalence, %			1.2	1.9	41.0	0.5
95% CI, %			0.5–2.5	1.0–3.4	36.9–45.1	0.1–1.5

Serogroups O111, O145, and O157 were not detected in winter months.

The number of positive samples divided by the total number of samples tested expressed as a percentage. The denominator (n=24) was the same for all pens.

Indicates the sex of the pen is heifers, the remaining pens comprise steers.

Although serogroups (O26, O45, O103, and O121) were isolated during winter months, no virulence genes were detected in those isolates. None of the samples collected during winter months tested positive for any of the seven STEC of interest.

Statistical comparison between summer and winter prevalence

Significant seasonal differences (p<0.05) were identified for pen-level prevalence of serogroups O26, O45, O103, O145, and O157. Statistically significant differences (p<0.05) were also observed for pen-level fecal prevalence of STEC O103 and O157 between study seasons; however, no significant differences were observed for the STEC isolates that were less commonly identified.

Discussion

The prevalence of seven STEC serogroups (O26, O45, O103, O111, O121, O145, and O157) and their associated major virulence genes was determined in feces of commercial feedlot cattle prior to harvest in both summer and winter months. Although a few studies have addressed the detection of non-O157 STEC in cattle feces, data regarding the frequency of non-O157 STEC serogroups in preharvest cattle in the Unites States are still limited (Cernicchiaro et al., 2013; Dargatz et al., 2013; Baltasar et al., 2014; Ekiri et al., 2014). The prevalence of non-O157 STEC organisms obtained in our study was lower than what has been reported in previous studies (Barkocy-Gallagher et al., 2003; Cernicchiaro et al., 2013; Ekiri et al., 2014) but comparable to others (Cobbold et al., 2004). Different case definitions, study populations, and detection protocols may explain differences in prevalence estimates obtained across studies.

Diagnostic methods employed for detection and isolation of serogroups and virulence genes influence prevalence estimates (Cernicchiaro et al., 2013). Methods for culturing and confirming STEC O157 have been well established; however, various non-O157 diagnostic methods and their impact on detection specificity and sensitivity are still being explored. The current lack of standardized and well-established detection methods for non-O157 STEC in cattle feces poses a challenge when comparing prevalence estimates across studies. The use of IMS techniques has been shown to increase the sensitivity of detection of STEC O157 compared to direct plating culture methods (Chapman et al., 1994). Recently, the use of IMS in culture-based detection methods has been shown to increase the apparent prevalence of non-O157 serogroups detected in feces, when compared to direct PCR (Cernicchiaro et al., 2013). Despite the recent development of increasingly sensitive molecular methodologies, subjecting samples to culture methods may still be needed to obtain isolates for further characterization and accurate linkage of O serogroup and virulence genes from bacterial isolates.

Although O157 was the most commonly detected STEC serogroup, among all seven O groups tested, O103 was the most commonly isolated serogroup from these cattle populations in both seasons. In winter months, serogroup O103 remained prevalent throughout all study pens, unlike other serogroups of interest. We hypothesize that serogroup O103 may have different microbial properties allowing the bacterium to endure harsher conditions and outcompete other microbiota, potentially allowing for longer survival in the cattle reservoir and subsequent environment. In another United States study, using similar detection methods, serogroup O103 also was the most frequently isolated non-O157 serogroup among cattle (Ekiri et al., 2014).

Previous research has demonstrated that non-O157 IMS bead specificity is variable and nonspecific binding of beads occurs (Cernicchiaro et al., 2013). This issue needs to be further evaluated as diagnostic sensitivity and specificity directly impact prevalence estimates. Other potential reasons for the low or no detection of some serogroups and virulence genes in either summer or winter may be related to insufficient samples per pen or study design limitations. Although repeated sampling from pens provides a perspective of how prevalence varies within pens close to harvest, the potential intermittent nature of fecal shedding can impact sample-level detection. In the present study, different pens of cattle were tested each week, which provide useful data pertaining to the STEC prevalence in cattle at harvest. However, point-in-time estimates may not be representative of the prevalence that exists in pens of cattle over time, if as for STEC O157, fecal shedding of non-O157 organisms is intermittent. (Sargeant et al., 2000; Renter et al., 2002; Chase-Topping et al., 2008; Menrath et al., 2010). Furthermore, because we measured prevalence of STEC, we cannot separate factors related to new animals shedding bacteria (i.e., incidence) and those associated with duration of shedding. Regardless, measures targeted at reducing STEC prevalence in preharvest cattle will be beneficial to potentially decrease microbial transmission to other penmates and the environment, as well as to reduce contamination of hides and subsequent transfer to carcass surfaces as those animals are harvested.

Seasonality of E. coli O157 has been well established in the bovine reservoir and in human illnesses (Barkocy-Gallagher et al., 2003; Rangel et al., 2005; Smith et al., 2005; Williams et al., 2010). An increase in non-O157 STEC human illness outbreaks has been observed in the United States for the warmer months (Luna-Gierke et al., 2014); however, the seasonality of non-O157 STEC shedding in cattle has not yet been established. The increased recognition of the importance of non-O157 STEC human infections, and the seasonality associated with human illness outbreaks, emphasizes the need for non-O157 STEC data in the bovine reservoir, particularly prior to harvest. In the present study, significant seasonal differences between summer and winter months were identified for serogroups O26, O45, O103, O145, and O157, as well as STEC O103 and O157. Cobbold et al. reported that STEC were more prevalent in cattle feces in the fall than in winter (Cobbold et al., 2004). Similarly, another study showed that cattle shed non-O157 STEC more frequently in spring and fall than in summer and winter (Barkocy-Gallagher et al., 2003). In our study, we did not detect non-O157 STEC in winter months. Future research is needed to generate data on prevalence of non-O157 STEC in other seasons (i.e., fall and spring), as well as in other feedlot operations, geographic regions, and cattle types (e.g., cull beef, fed dairy, cull dairy).

In conclusion, our results indicate that non-O157 STEC in feedlot cattle were rarely detected in summer and were undetected in winter months. The most commonly identified serogroups were O26, O103, and O157 during summer; and O26, O45, and O103 in winter. Conversely, the most common STEC detected in this cattle population in summer was STEC O157 followed by STEC O103, O26, and O145 in much lower frequency. These findings contribute to filling the data gaps regarding prevalence of non-O157 STEC in cattle prior to entering the food supply, in two seasons. These estimates will be used to populate quantitative microbial risk assessment models that are crucial to understanding the risk of human illnesses due to STEC that are attributed to cattle and subsequent beef products.

Footnotes

Acknowledgments

Thanks are given to the participating feedlot, field crews, and laboratory personnel. Authors would also like to acknowledge STEC-CAP student interns involved on the project: Megan Spencer, Shelby Miller, Diane Larson, Rachel Henderson, Cody Baumgartner, and David Hanks. Research was supported by the Agriculture and Food Research Initiative Grant No. 2012-68003-30155 from the USDA National Institute of Food and Agriculture, Prevention, Detection and Control of Shiga Toxin–Producing Escherichia coli (STEC CAP) from Pre-Harvest Through Consumption of Beef Products Program–A410 and by the College of Veterinary Medicine, Kansas State University.

Disclosure Statement

No competing financial interests exist.

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