Evaluation of Bacteriophage Application to Cattle in Lairage at Beef Processing Plants to Reduce Escherichia coli O157:H7 Prevalence on Hides and Carcasses

Abstract

Escherichia coli O157:H7 is a major food safety concern for the beef industry. Several studies have provided evidence that cattle hides are the main source of beef carcass contamination during processing and that reductions in the E. coli O157:H7 load on the hides of cattle entering processing facilities will lead to reductions in carcass contamination. Bacteriophages have been proposed as a novel preharvest antimicrobial intervention to reduce the levels of E. coli O157:H7 on cattle hides. The objective of this study was to evaluate a commercialized phage application administered in the lairage area of commercial beef processing plants for the ability to reduce E. coli O157:H7 contamination of cattle hides and carcasses. Cattle lots either received phage spray treatment (n = 289) or did not (n = 301), as they entered the lairage environments in two separate experiments at two different commercial beef processing plants. Hide and carcass samples were collected and analyzed for E. coli O157:H7 prevalence and concentration. Cattle hides receiving phage treatment had an E. coli O157:H7 prevalence of 51.8%, whereas untreated hides had a prevalence of 57.6%. For carcass samples, the E. coli O157 prevalence in treated and untreated samples was 17.1% and 17.6%, respectively. The results obtained from these experiments demonstrated that the treatment of cattle hides with bacteriophages before processing did not produce a significant reduction of E. coli O157:H7 on cattle hides or beef carcasses during processing.

Introduction

E scherichia coli O157:H7 is a major food safety concern for the beef industry. Several studies (Barkocy-Gallagher et al., 2001; Nou et al., 2003; Bosilevac et al., 2005) have provided convincing evidence that cattle hides are the main source of beef carcass contamination during processing and that reductions in the E. coli O157:H7 load on the hides of cattle entering processing facilities will lead to reductions in carcass contamination. The prevalence of E. coli O157:H7 on cattle hides during the summer months in feedlot production settings frequently approaches 100% (Arthur et al., 2009; Kalchayanand et al., 2009). In addition, transportation from the feedlot and temporary holding at the processing plant have been shown to contribute to E. coli O157:H7 hide contamination (Arthur et al., 2007, 2008; Dewell et al., 2008), indicating that interventions applied to the live animal at the processing plant would be valuable additions to the multi-hurdle antimicrobial intervention schemes employed by commercial beef processing plants.

An alternative to pre-harvest interventions is the application of interventions at the pre-harvest/post-harvest boundary, which is the lairage environment at beef processing plants. Such an application would have the benefit that all animals would be treated and treated in a consistent manner, removing the variation in treatment caused by differing management practices among producers. Although multiple chemical processes are approved for use on cattle hides post-harvest, these chemicals cannot be used on live animals due to animal welfare concerns. The application of bacteriophages (phages) to cattle hides does not cause deleterious animal health issues and is approved for use before slaughter, but a determination of efficacy is lacking.

Bacteriophages (phages) are viruses that infect and potentially kill bacteria. Lytic phages have long been considered ideal biocontrol agents for bacterial pathogens due to their role as naturally occurring bacterial predators, specificity for a particular host, and the fact that they are functionally inert when interacting with eukaryotic cells (Abedon, 2009). Hence, phage application has been developed as a novel preharvest antimicrobial intervention to reduce the levels of E. coli O157:H7 on cattle hides before processing to reduce that pathogen load transferred to the carcass and to increase the food safety of the finished product. Coffey et al. (2011) reported a 1.5 log colony-forming unit (CFU)/cm² reduction in E. coli O157:H7 inoculated onto pieces of cattle hide when treated with bacteriophages and given a 1-h dwell timing. Phages are FDA approved for use in or on live cattle for control of E. coli O157:H7 associated with cattle. However, there are little to no definitive data demonstrating the efficacy of phage treatments in reducing the concentration or prevalence of pathogenic bacteria colonizing a live animal. The objective of this study was to evaluate a commercialized phage application administered in the lairage area of beef processing plants for the ability to reduce E. coli O157:H7 contamination of cattle hides and carcasses.

Materials and Methods

Study design

Two experiments were performed for this project. In both experiments, phages were applied to cattle hides at commercial beef processing plants through a spray application system according to the manufacturer's instructions (Finalyse^®; Elanco Animal Health, Greenfield, IN). Briefly, the phage solution, containing a proprietary mixture of phages, was applied at a dose of ∼3 × 10¹⁰ phage/head of cattle in one gallon of water/head with a dwell timing of at least 1 h before harvest. The spray applications were administered in the lairage environment to cattle lots shortly after cattle were unloaded from delivery trucks. Individual cattle lots either received phage application or did not; at no time were subsets of lots treated.

Experiment 1

Sample collection

Cattle hide samples were collected both before and after a phage or control application at a commercial beef processing company. Control animals (n = 140) received a water spray that did not contain Finalyse, whereas cattle (n = 120) in the treated group received a water spray containing Finalyse. To prevent carryover of phage via the application system, the system was thoroughly rinsed after application to treated animals. BEFORE samples were collected as the animals exited the transport trailer or shortly after arrival into the processing plant lairage area. AFTER samples were collected at least 1-h post phage/water application, immediately after the exsanguinated carcass was shackled and placed on the processing line. Both sample types were obtained by using a sterile sponge (Biotrace International, Inc., Bothell, WA) that was premoistened with 20 mL of buffered peptone water (Becton Dickenson, Sparks, MD), and swabbing an area of ≈1000 cm² on the rump. Animals were sampled over five sampling days, with 20–70 cattle being sampled per day (250 total). Animals were marked so that the BEFORE and AFTER samples were matched to an individual animal. An approximately equal number of treated and control samples were collected on each sampling day, packed on ice, and shipped to the laboratory. On arrival at the lab, the samples were analyzed for concentration and prevalence of E. coli O157:H7.

Experiment 2

Sample collection

A total of 340 carcasses were sampled over 3 days. Hide and pre-evisceration carcass samples were collected from 10 carcasses per lot from 34 lots. Ten lots were sampled per day on Day 1 and Day 2, and 14 lots were sampled on Day 3. Each day, half of the lots did not receive bacteriophage treatment on arrival into the lairage environment holding area and were considered CONTROL lots. The remaining lots did receive bacteriophage treatment on entry into the lairage environment and were considered TREATED lots. Bacteriophage application and dwell timing were administered by processing plant personnel according to standard operating procedures and the manufacturer's instructions. Hide sponge samples were collected after exsanguination and shackling. Hide sponge samples consisted of swabbing a 1000-cm² area on the front shoulder. Carcass samples were taken immediately after hide removal, but before the application of any antimicrobial interventions to the carcass surface. Carcass sponge samples were collected by swabbing a 4000-cm² area following the carcass midline from the navel to brisket and included the foreshank (Arthur et al., 2004). No attempt was made to sample the hides and carcasses of the same animals. Samples were packed on ice and shipped to the laboratory for processing. On arrival at the lab, the samples were analyzed for concentration and prevalence of E. coli O157:H7.

Enumeration

E. coli O157:H7 was enumerated from hide samples by using a Spiral Plater (Spiral Biotech, Norwood, MA) and following previously described methods (Brichta-Harhay et al., 2007). The sponge samples were homogenized by hand massage, after which 500 μL of the homogenate was transferred to a microfuge tube. After vortexing and a 3-min holding period to allow the particulates to settle, 50 μL aliquots were spiral plated on ntChromagar (Chromagar O157 [DRG International; Mountainside, NJ] supplemented with novobiocin [5 mg/L; Sigma, St. Louis, MO] and potassium tellurite [2.5 mg/L; Sigma]) plates. After incubating the plates overnight at 42°C, the plates were counted with suspect colonies confirmed by PCR. PCR was used to confirm that each E. coli O157:H7 isolate harbored genes for the O157 antigen, H7 flagella, γ-intimin, and at least one of the Shiga toxins (Hu et al., 1999). The limit of detection in the enumeration assay was 40 CFU/100 cm².

Sample processing for prevalence

Samples were processed according to methods previously described (Barkocy-Gallagher et al., 2002, 2005; Nou et al., 2006), with slight modifications. After removing the 500 μL aliquot for enumeration, the sponge samples were enriched with 80 mL of Tryptic Soy Broth (Becton Dickinson Microbiology Systems, Sparks, MD) and incubated at 25°C for 2 h, 42°C for 6 h and then held at 4°C overnight. After incubation, 1 mL from each enrichment was subjected to anti-O157 immunomagnetic separation. Enrichments (1 mL) received 20 μL of anti-O157 beads (Thermo Fisher Scientific, Inc., Waltham, MA). Thereafter, the beads were extracted from enrichment samples and washed three times in phosphate-buffered saline-Tween 20 (Sigma) by using an automated magnetic particle processor (KingFisher 96; Thermo Fisher Scientific, Inc.). The final bead-bacteria complexes were spread-plated onto ntChromagar and incubated at 37°C overnight. After incubation, up to three suspect colonies were picked for confirmation. PCR was used to confirm that each E. coli O157:H7 isolate harbored genes for the O157 antigen, H7 flagella, γ-intimin, and at least one of the Shiga toxins (Hu et al., 1999).

Statistical analysis

Statistical analysis was performed by using Stata/SE 13.1 (StataCorp, College Station, TX). The unit of statistical analysis was the individual animal. The effect of phage application on the prevalence of E. coli O157:H7 on the cattle hides (experiment 1), and the hides and carcasses (experiment 2) was modeled with generalized estimating equations (GEE) accounting for the clustering of observations by lots. In both experiments, sampling day was included in the GEE model as a fixed effect. In experiment 1, sampling point (before and after phage application) was included as a fixed effect to account for the repeated measurements. Model-adjusted prevalence for E. coli O157:H7 was obtained from the marginal predicted values. Significance was declared at a p < 0.05.

Results and Discussion

A considerable amount of research and development has resulted in highly effective post-harvest antimicrobial interventions, which are currently utilized in the beef processing industry. These interventions are applied in a multi-hurdle approach to sequentially reduce bacterial contamination that is transferred from cattle hides to beef carcasses during processing (Arthur et al., 2004; Callaway et al., 2004; Koohmaraie et al., 2005; Brichta-Harhay et al., 2007). However, conditions continue to arise where the incoming contamination load on cattle hides exceeds the reducing capacity of the post-harvest interventions, resulting in finished product contamination (Vosough Ahmadi et al., 2007; Arthur et al., 2014). Therefore, effective pre-harvest interventions are needed to aid in reducing the incoming contamination load on cattle hides.

To date, multiple pre-harvest technologies have been developed (vaccines, direct fed microbials, and ingestible phage inoculants) for use in cattle production to reduce the shedding of E. coli O157:H7 in the feces (Fairbrother and Nadeau, 2006; Sargeant et al., 2007; Berry and Wells, 2010). By reducing the prevalence and concentrations of E. coli O157:H7 in the feces, it would be possible to reduce the amount of E. coli O157:H7 contamination on cattle hides (Cobbold and Desmarchelier, 2000; Chase-Topping et al., 2008; Arthur et al., 2009). However, although several studies have found these applications promising for future development, none have shown efficacy in consistently reducing E. coli O157 shedding in the feces of cattle (Greer, 2005; Van Donkersgoed et al., 2005; Callaway et al., 2008; Moxley et al., 2009; Goodridge and Bisha, 2011; Wileman et al., 2011; Cull et al., 2012). Also, additional contamination is acquired on the hide after cattle leave production settings when they are transported to and held at beef processing plant lairage environments (Arthur et al., 2007, 2008; Dewell et al., 2008).

Prevalence of E. coli O157:H7 in hide samples collected before and after phage application

In Experiment 1, the E. coli O157:H7 prevalence on cattle hides as they were unloaded at the processing plant lairage area was 6.4% and 13.3% for the control and treated groups, respectively (Table 1). The prevalence of E. coli O157:H7 increased significantly (p < 0.001) for both control and treated cattle from the time they entered lairage and phage was applied to the point where the animals were stunned, exsanguinated, and shackled. The resulting E. coli O157:H7 prevalence on the hides was 42.0% and 38.7% for the control and treated groups, respectively. Although the treated group did not have as large of an increase in E. coli O157:H7 prevalence as did the control group, phage application in the lairage environment did not have a significant (p = 0.547) effect on the prevalence of E. coli O157:H7 on the hide samples (Table 1).

Table 1.

Prevalence (%) of Escherichia coli O157:H7 on the Hides of Beef Cattle Before and After Phage Application (Exp I)

	Control					Treated
Sampling day	Before	n^a	After	n	p-value ^b	Before	n	After	n	p-value
1	0^c (0–30.8)	10	36.4 (10.9–69.2)	11	0.09	0 (0–30.8)	10	10 (0.3–44.5)	10	1
2	7.5 (1.6–20.4)	40	80 (61.4–92.3)	30	<0.001	30 (14.7–49.4)	30	69.0 (49.2–84.7)	29	0.004
3	0 (0–11.6)	30	46.7 (28.3–65.7)	30	<0.001	10 (1.2–31.7)	20	50 (27.2–72.8)	20	0.014
4	3.3 (0.08–17.2)	30	23.3 (9.9–42.3)	30	0.052	0 (0–11.6)	30	26.7 (12.3–45.9)	30	0.005
5	16.7 (5.6–34.7)	30	20 (7.7–38.6)	30	1	16.7 (5.6–34.7)	30	23.3 (9.9–42.3)	30	0.748
Overall	6.4 (3.0–11.9)	140	42.0 (33.4–50.9)	131	<0.001	13.3 (7.8–20.7)	120	38.7 (29.9–48.0)	119	<0.001

n represents the number of cattle sampled.

The p-values compare the prevalence of Escherichia coli O157:H7 between “before” and “after” samples within each group (control and treated).

Prevalences were presented as point estimate (95% confidence interval).

Previous research has described the potential for hide contamination to increase as cattle progress through processing plant lairage environments (Arthur et al., 2007, 2008; Dewell et al., 2008). This occurs when large numbers of cattle pass through common spaces and deposit fecal matter laden with bacterial pathogens. As subsequent cattle pass through these spaces, they acquire additional contamination on their hides through splashing or lying down on contaminated surfaces. Due to this potential to accumulate hide contamination after arrival at the processing plant, it was determined that hide sampling both before and after phage application in the lairage environment was not a useful evaluation of the phage application efficacy. It was determined that a better evaluation of the intervention efficacy would be attained by comparing the E. coli O157:H7 prevalence and concentrations on the hides and carcasses of treated cattle post-phage application and not collecting any BEFORE samples.

Effect of phage application on the prevalence of E. coli O157:H7 on hide and carcass samples

In Experiment 2, the E. coli O157:H7 prevalence for the first two sampling days was numerically slightly lower on treated cattle hides when compared with control cattle hides, but not on the third sampling day (Table 2). Overall, phage treatment did not have any significant (p = 0.644) effect on the prevalence of E. coli O157:H7 in hide samples. Similarly, the E. coli O157:H7 prevalence for the first two sampling days was numerically slightly lower on carcasses from treated cattle when compared with control cattle carcasses, but not on the third sampling day (Table 3). Again, phage treatment did not have any significant (p = 0.928) effect on the overall prevalence of E. coli O157:H7 in carcass samples.

Table 2.

Prevalence (%) of Escherichia coli O157:H7 on Hides of Cattle With or Without Phage Treatment (Exp II)

	Control		Treated
Sampling day	n^b	Prevalence ^a (95% CI)	n	Prevalence (95% CI)	p-value ^c
1	50	98 (89.4–99.9)	50	86 (73.6–94.2)	0.208
2	50	62 (47.2–75.3)	50	48 (33.7–62.6)	0.430
3	70	25.7 (16.0–37.6)	70	30 (19.6–42.1)	0.753
Overall	170	57.6 (49.8–65.2)	170	51.8 (44.0–59.5)	0.644

Prevalences were presented as point estimate (95% confidence interval).

n represents the number of hides sampled.

The p-values compare the prevalence of E. coli O157:H7 between the control and treated groups.

Table 3.

Prevalence (%) of Escherichia coli O157:H7 on Beef Carcasses With or Without Phage Treatment (Exp II)

	Control		Treated
Sampling day	n^b	Prevalence ^a (95% CI)	n	Prevalence (95% CI)	p-value ^c
1	50	16 (7.2–29.1)	50	8 (2.2–19.2)	0.272
2	50	38 (24.7–52.8)	50	34 (21.2–48.8)	0.712
3	70	4.3 (0.9–12.1)	70	11.4 (5–21.3)	0.161
Overall	170	17.6 (12.2–24.2)	170	17.1 (11.7–23.6)	0.928

Prevalences were presented as point estimate (95% confidence interval).

n represents the number of carcasses sampled.

The p-values compare the prevalence of E. coli O157:H7 between the control and treated groups.

Effect of phage application on concentration of E. coli O157:H7 on cattle hides and carcasses

For Experiment 1, there were no BEFORE hide samples harboring concentrations of E. coli O157:H7 greater than the lower limit of detection (40 CFU/100 cm²) for the enumeration assay used in this study. There were seven AFTER samples (four treated and three control) harboring enumerable E. coli O157:H7 (data not shown). From Experiment 2, there were numerically more samples with E. coli O157:H7 concentrations ≥40 CFU/100 cm² on the control hides as opposed to the treated hides (25 vs. 16), but the difference was not significant (p = 0.133). For the carcass samples, there were more treated carcass samples with enumerable E. coli O157:H7 than control carcass samples (4 vs. 2), but this difference was not statistically significant (p = 0.685).

Previous applications of phage for the purposes of reducing E. coli O157:H7 associated with cattle have focused on killing the pathogen in the bovine gastrointestinal (GI) tract to reduce the concentrations of E. coli O157:H7 shed in the feces. These applications, which typically use either orally or rectally inoculated phage or a combination of both, have achieved only modest reductions in E. coli O157:H7 shedding. Sheng et al. (2006) employed a dual phage cocktail via multiple rectal applications and a continuous supply of phage to the drinking water to reduce the concentrations of E. coli O157:H7 being shed by inoculated calves. This treatment was not successful in completely eliminating the pathogen, as most animals continued to shed E. coli O157:H7 at low levels at the end of the study. Because E. coli O157:H7 have been shown to colonize the recto-anal junction (Naylor et al., 2003; Low et al., 2005; Cobbold et al., 2007; Fox et al., 2008), it has been speculated that optimal application for a phage-based intervention would bypass the bovine rumen and target the lower GI tract. Stanford et al. (2010) developed a polymer encapsulation technology that would protect the orally inoculated phage formulation from the microbial milieu that exists in the rumen and release the phages in a viable state as they entered the lower GI tract. This application failed to reduce E. coli O157:H7 shedding overall, but it did shorten the duration of shedding (Stanford et al., 2010). Although several hypotheses have been given (improper phage:host ratios, inability of phage to penetrate mucous layers, etc), it is not known why susceptible host strains are not substantially reduced by phage-based interventions in the GI tract.

One potential reason for the lack of efficacy when phages are applied to cattle hides is the large amount of organic matter and pathogen contamination that cattle encounter as they move through the lairage environment. Because the phage treatment is applied before the cattle are exposed to the majority of the lairage environment contamination, it may be that the phages are simply overwhelmed by additional contamination. Further research is needed to determine whether moving the point of application closer to the exit of lairage would increase the efficacy. In addition, it may be beneficial to develop a phage treatment that is applied to carcasses immediately after dehiding to minimize the organic load and have a higher phage-to-target ratio.

To conclude, many studies have shown that during beef processing cattle hides represent the most significant source of bacterial pathogen contamination of the carcass (Barkocy-Gallagher et al., 2001; Nou et al., 2003; Bosilevac et al., 2005). The beef industry would benefit from having additional antimicrobial interventions that can be applied before the cattle entering the processing plant facility. The application of phage to cattle hides in the lairage environment was seen as a potential intervention technology that could be used on the live animal for the improvement of food safety. However, through the experiments conducted here, it was demonstrated that the treatment of cattle hides with bacteriophages before passing through the lairage environment and before processing did not produce a significant reduction in the levels or prevalence of E. coli O157:H7 on hides or beef carcasses during processing; hence, it is doubtful that this application is improving the food safety of the beef supply.

Footnotes

Acknowledgments

The authors thank Trent Ahlers, Julie Dyer, Bruce Jasch, and Frank Reno for their technical support and Jody Gallagher for her secretarial support.

Disclosure Statement

No competing financial interest exist.

References

Abedon

. Kinetics of phage-mediated biocontrol of bacteria. Foodborne Pathog Dis, 2009; 6:807–815.

Arthur

, Bono

, Kalchayanand

. Characterization of Escherichia coli O157:H7 strains from contaminated raw beef trim during “high event periods”. Appl Environ Microbiol, 2014; 80:506–514.

Arthur

, Bosilevac

, Brichta-Harhay

, Guerini

, Kalchayanand

, Shackelford

, Wheeler

, Koohmaraie

. Transportation and lairage environment effects on prevalence, numbers, and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing. J Food Prot, 2007; 70:280–286.

Arthur

, Bosilevac

, Brichta-Harhay

, Kalchayanand

, King

, Shackelford

, Wheeler

, Koohmaraie

. Source tracking of Escherichia coli O157:H7 and Salmonella contamination in the lairage environment at commercial U.S. beef processing plants and identification of an effective intervention. J Food Prot, 2008; 71:1752–1760.

Arthur

, Bosilevac

, Nou

, Shackelford

, Wheeler

, Kent

, Jaroni

, Pauling

, Allen

, Koohmaraie

. Escherichia coli O157 prevalence and enumeration of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various steps in commercial beef processing plants. J Food Prot, 2004; 67:658–665.

Arthur

, Keen

, Bosilevac

, Brichta-Harhay

, Kalchayanand

, Shackelford

, Wheeler

, Nou

, Koohmaraie

. Longitudinal study of Escherichia coli O157:H7 in a beef cattle feedlot and role of high-level shedders in hide contamination. Appl Environ Microbiol, 2009; 75:6515–6523.

Barkocy-Gallagher

, Arthur

, Siragusa

, Keen

, Elder

, Laegreid

, Koohmaraie

. Genotypic analyses of Escherichia coli O157:H7 and O157 nonmotile isolates recovered from beef cattle and carcasses at processing plants in the Midwestern states of the United States. Appl Environ Microbiol, 2001; 67:3810–3818.

Barkocy-Gallagher

, Berry

, Rivera-Betancourt

, Arthur

, Nou

, Koohmaraie

. Development of methods for the recovery of Escherichia coli O157:H7 and Salmonella from beef carcass sponge samples and bovine fecal and hide samples. J Food Prot, 2002; 65:1527–1534.

Barkocy-Gallagher

, Edwards

, Nou

, Bosilevac

, Arthur

, Shackelford

, Koohmaraie

. Methods for recovering Escherichia coli O157:H7 from cattle fecal, hide, and carcass samples: Sensitivity and improvements. J Food Prot, 2005; 68:2264–2268.

10.

Berry

, Wells

. Escherichia coli O157:H7: Recent advances in research on occurrence, transmission, and control in cattle and the production environment. Adv Food Nutr Res, 2010; 60:67–117.

11.

Bosilevac

, Nou

, Osborn

, Allen

, Koohmaraie

. Development and evaluation of an on-line hide decontamination procedure for use in a commercial beef processing plant. J Food Prot, 2005; 68:265–272.

12.

Brichta-Harhay

, Arthur

, Bosilevac

, Guerini

, Kalchayanand

, Koohmaraie

. Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and faecal samples using direct plating methods. J Appl Microbiol, 2007; 103:1657–1668.

13.

Callaway

, Anderson

, Edrington

, Genovese

, Bischoff

, Poole

, Jung

, Harvey

, Nisbet

. What are we doing about Escherichia coli O157:H7 in cattle?. J Anim Sci, 2004; 82 E-Suppl:E93–E99.

14.

Callaway

, Edrington

, Brabban

, Anderson

, Rossman

, Engler

, Carr

, Genovese

, Keen

, Looper

, Kutter

, Nisbet

. Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts. Foodborne Pathog Dis, 2008; 5:183–191.

15.

Chase-Topping

, Gally

, Low

, Matthews

, Woolhouse

. Super-shedding and the link between human infection and livestock carriage of Escherichia coli O157. Nat Rev Microbiol, 2008; 6:904–912.

16.

Cobbold

, Desmarchelier

. A longitudinal study of Shiga-toxigenic Escherichia coli (STEC) prevalence in three Australian diary herds. Vet Microbiol, 2000; 71:125–137.

17.

Cobbold

, Hancock

, Rice

, Berg

, Stilborn

, Hovde

, Besser

. Rectoanal junction colonization of feedlot cattle by Escherichia coli O157:H7 and its association with supershedders and excretion dynamics. Appl Environ Microbiol, 2007; 73:1563–1568.

18.

Coffey

, Rivas

, Duffy

, Coffey

, Ross

, McAuliffe

. Assessment of Escherichia coli O157:H7-specific bacteriophages e11/2 and e4/1c in model broth and hide environments. Int J Food Microbiol, 2011; 147:188–194.

19.

Cull

, Paddock

, Nagaraja

, Bello

, Babcock

, Renter

. Efficacy of a vaccine and a direct-fed microbial against fecal shedding of Escherichia coli O157:H7 in a randomized pen-level field trial of commercial feedlot cattle. Vaccine, 2012; 30:6210–6215.

20.

Dewell

, Simpson

, Dewell

, Hyatt

, Belk

, Scanga

, Morley

, Grandin

, Smith

, Dargatz

, Wagner

, Salman

. Impact of transportation and lairage on hide contamination with Escherichia coli O157 in finished beef cattle. J Food Prot, 2008; 71:1114–1118.

21.

Fairbrother

, Nadeau

. Escherichia coli: On-farm contamination of animals. Rev Sci Tech, 2006; 25:555–569.

22.

Fox

, Shi

, Nagaraja

. Escherichia coli O157 in the rectoanal mucosal region of cattle. Foodborne Pathog Dis, 2008; 5:69–77.

23.

Goodridge

, Bisha

. Phage-based biocontrol strategies to reduce foodborne pathogens in foods. Bacteriophage, 2011; 1:130–137.

24.

Greer

. Bacteriophage control of foodborne bacteriat. J Food Prot, 2005; 68:1102–1111.

25.

, Zhang

, Meitzler

. Rapid and sensitive detection of Escherichia coli O157:H7 in bovine faeces by a multiplex PCR. J Appl Microbiol, 1999; 87:867–876.

26.

Kalchayanand

, Brichta-Harhay

, Arthur

, Bosilevac

, Guerini

, Wheeler

, Shackelford

, Koohmaraie

. Prevalence rates of Escherichia coli O157:H7 and Salmonella at different sampling sites on cattle hides at a feedlot and processing plant. J Food Prot, 2009; 72:1267–1271.

27.

Koohmaraie

, Arthur

, Bosilevac

, Guerini

, Shackelford

, Wheeler

. Post-harvest interventions to reduce/eliminate pathogens in beef. Meat Sci, 2005; 71:79–91.

28.

Low

, McKendrick

, McKechnie

, Fenlon

, Naylor

, Currie

, Smith

, Allison

, Gally

. Rectal carriage of enterohemorrhagic Escherichia coli O157 in slaughtered cattle. Appl Environ Microbiol, 2005; 71:93–97.

29.

Moxley

, Smith

, Luebbe

, Erickson

, Klopfenstein

, Rogan

. Escherichia coli O157:H7 vaccine dose-effect in feedlot cattle. Foodborne Pathog Dis, 2009; 6:879–884.

30.

Naylor

, Low

, Besser

, Mahajan

, Gunn

, Pearce

, McKendrick

, Smith

, Gally

. Lymphoid follicle-dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7 in the bovine host. Infect Immun, 2003; 71:1505–1512.

31.

Nou

, Arthur

, Bosilevac

, Brichta-Harhay

, Guerini

, Kalchayanand

, Koohmaraie

. Improvement of immunomagnetic separation for Escherichia coli O157:H7 detection by the PickPen magnetic particle separation device. J Food Prot, 2006; 69:2870–2874.

32.

Nou

, Rivera-Betancourt

, Bosilevac

, Wheeler

, Shackelford

, Gwartney

, Reagan

, Koohmaraie

. Effect of chemical dehairing on the prevalence of Escherichia coli O157:H7 and the levels of aerobic bacteria and Enterobacteriaceae on carcasses in a commercial beef processing plant. J Food Prot, 2003; 66:2005–2009.

33.

Sargeant

, Amezcua

, Rajic

, Waddell

. Pre-harvest interventions to reduce the shedding of E. coli O157 in the faeces of weaned domestic ruminants: A systematic review. Zoonoses Public Health, 2007; 54:260–277.

34.

Sheng

, Knecht

, Kudva

, Hovde

. Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants. Appl Environ Microbiol, 2006; 72:5359–5366.

35.

Stanford

, McAllister

, Niu

, Stephens

, Mazzocco

, Waddell

, Johnson

. Oral delivery systems for encapsulated bacteriophages targeted at Escherichia coli O157:H7 in feedlot cattle. J Food Prot, 2010; 73:1304–1312.

36.

Van Donkersgoed

, Hancock

, Rogan

, Potter

. Escherichia coli O157:H7 vaccine field trial in 9 feedlots in Alberta and Saskatchewan. Can Vet J, 2005; 46:724–728.

37.

Vosough Ahmadi

, Frankena

, Turner

, Velthuis

, Hogeveen

, Huirne

. Effectiveness of simulated interventions in reducing the estimated prevalence of E. coli O157:H7 in lactating cows in dairy herds. Vet Res, 2007; 38:755–771.

38.

Wileman

, Thomson

, Olson

, Jaeger

, Pacheco

, Bolte

, Burkhardt

, Emery

, Straub

. Escherichia coli O157:H7 shedding in vaccinated beef calves born to cows vaccinated prepartum with Escherichia coli O157:H7 SRP vaccine. J Food Prot, 2011; 74:1599–1604.