1304

Journal of Food Protection, Vol. 73, No. 7, 2010, Pages 1304–1312

Copyright G, International Association for Food Protection

Oral Delivery Systems for Encapsulated Bacteriophages Targeted

at Escherichia coli O157:H7 in Feedlot Cattle
K. STANFORD,1* T. A. MCALLISTER,2 Y. D. NIU,2 T. P. STEPHENS,1 A. MAZZOCCO,3 T. E. WADDELL,4 AND
R. P. JOHNSON3

1Alberta Agriculture and Rural Development, Agriculture Centre, Lethbridge, Alberta, Canada T1J 4V6; 2Agriculture and Agri-Food Canada, Lethbridge

Research Centre, Lethbridge, Alberta, Canada T1J 4B1; 3Public Health Agency of Canada, Guelph, Ontario, Canada N1G 3W4; and 4Pro-Lab Diagnostics,
Richmond Hill, Ontario, Canada L4B 1K3

MS 09-493: Received 20 November 2009/Accepted 11 April 2010

ABSTRACT
Bacteriophages are natural predators of bacteria and may mitigate Escherichia coli O157:H7 in cattle and their environment.
As bacteriophages targeted to E. coli O157:H7 (phages) lose activity at low pH, protection from gastric acidity may enhance
efficacy of orally administered phages. Polymer encapsulation of four phages, wV8, rV5, wV7, and wV11, and exposure to
pH 3.0 for 20 min resulted in an average 13.6% recovery of phages after release from encapsulation at pH 7.2. In contrast,
untreated phages under similar conditions had a complete loss of activity. Steers (n ~ 24) received 1011 CFU of naladixic acid–
resistant E. coli O157:H7 on day 0 and were housed in six pens of four steers. Two pens were control (naladixic acid–resistant E.
coli O157:H7 only), and the remaining pens received polymer-encapsulated phages (Ephage) on days 21, 1, 3, 6, and 8. Two
pens received Ephage orally in gelatin capsules (bolus; 1010 PFU per steer per day), and the remaining two pens received Ephage
top-dressed on their feed (feed; estimated 1011 PFU per steer per day). Shedding of E. coli O157:H7 was monitored for 10 weeks
by collecting fecal grab and hide swab samples. Acceptable activity of mixed phages at delivery to steers was found for bolus and
feed, averaging 1.82 and 1.13 | 109 PFU/g, respectively. However, Ephage did not reduce shedding of naladixic acid–resistant
E. coli O157:H7, although duration of shedding was reduced by 14 days (P , 0.1) in bolus-fed steers as compared with control
steers. Two successful systems for delivery of Ephage were developed, but a better understanding of phage–E. coli O157:H7
ecology is required to make phage therapy a viable strategy for mitigation of this organism in feedlot cattle.

Methods for both preharvest and postharvest control of
Escherichia coli O157:H7 have been widely investigated,
but after more than 20 years of study, development of a
simple, universally effective mitigation strategy remains
elusive (53). Much of the difficulty in controlling E. coli
O157:H7 stems from the low infectious dose in humans (as
low as 10 cells) (6), asymptomatic shedding by cattle (28),
and a lack of clarity regarding the ecology of this organism.
Shedding of E. coli O157:H7 by cattle is often intermittent
and seasonal (27, 47), and proposed underlying mechanisms
have included day length (17), housing (35), stress (14), and
diet (22). Recently, Niu et al. (33) demonstrated that
seasonal fluctuations in the abundance of E. coli O157:H7
in the feces and environment of feedlot cattle were inversely
correlated with levels of endemic bacteriophages that were
active against E. coli O157:H7. Although the role of
bacteriophages in the ecology of E. coli O157:H7 requires
further study, the potential exists for bacteriophages to at
least partially mitigate shedding of E. coli O157:H7.
In vitro, bacteriophages targeted to E. coli O157:H7
(phages) have often shown promise as a mitigation strategy
for E. coli O157:H7 (7, 32, 34, 49). In vivo results of phage
* Author for correspondence. Tel: 403-381-5150; Fax: 403-382-4526;
E-mail: kim.stanford@gov.ab.ca.
therapy have been more mixed, reducing shedding of E. coli
O157:H7 in some cases (5, 11, 45, 51), while having a
negligible effect in others (7, 42, 48). Some of the variability
in vivo is likely due to conditions in the digestive tract,
which may substantially lower phage activity, including low
pH and the presence of bile and digestive enzymes (25).
Protection from acid digestion was proposed by Smith
et al. (46) in order to ensure delivery of active phages to the
large intestine. A variety of materials have been used to
protect probiotic bacteria from the digestive tract (4), but
information regarding the encapsulation of viruses is more
limited (25). Consequently, the present study had three
objectives: (i) to develop a method for encapsulation that
would protect phages at low pH and release active phages at
pH . 7.0; (ii) to evaluate two oral delivery systems, bolus
and direct fed, for the encapsulated phages; and (iii) to
monitor the effectiveness of the two delivery systems in
feedlot cattle inoculated with E. coli O157:H7.
MATERIALS AND METHODS
Encapsulation of phages and acid tolerance. Bacteriophag-
es rV5, wV7, wV8, and wV11 from the collection of the Public
Health Agency of Canada Laboratory for Foodborne Zoonoses,
previously described by Waddell et al. (51), were propagated on
late-log-phase cultures of susceptible, verotoxin-negative, non-
J. Food Prot., Vol. 73, No. 7 ENCAPSULATED BACTERIOPHAGES FOR E. COLI O157:H7
O157 E. coli strain EC19990806 (for rV5, wV7, and wV11) or
strain EC19990779 (for wV8). The phages were cultured in 5-liter
volumes of Phytone Peptone (Becton Dickinson, Franklin Lakes,
NJ) broth containing 5 mM MgSO4 and were incubated at 37uC for
16 to 20 h, with stirring at 250 rpm using an Innova 40 shaking
incubator (New Brunswick Scientific, Edison, NJ). Lysates were
filtered with 0.45-mm Acrodisc syringe filters (Pall Corp.,
Mississauga, Ontario, Canada) to remove viable bacteria, and the
resulting filtrates were titrated on their respective hosts and stored
at 4uC.
Each phage was then encapsulated in methacrylate polymer
(Eudragit S100, Röhm Pharma, Weiterstadt, Germany) which
releases at pH . 7.0 and is generally regarded as safe (52). In
short, the filtered phage lysates were added separately to 10 volumes
of an aqueous methacrylate polymer solution containing a stabilizer,
then spray dried to a powder with approximately 4% moisture
content by using a Niro 7 co-current spray dryer (Niro Atomizer,
Hudson, WI) fitted with a rotary atomizer. Resulting encapsulated
bacteriophages (Ephage) were then stored at 20uC. The concentra-
tions, PFU per gram of active phages in Ephage, were determined by
titration of samples dissolved in 10 mM phosphate-buffered saline
(PBS), pH 7.2, on their corresponding non-O157 hosts and an E. coli
O157:H7 phage type 14 host strain EC19990298.
Acid tolerance of Ephage and representative strains of phage
were determined by adding 2 ml of 5 mM citric acid to 500 mg of
phages or Ephage to attain pH 3.0. From this mixture, a 20-ml
aliquot was immediately added to 180 ml of lambda diluent
(10 mM Tris buffer, 8 mM MgSO4?7H2O; Difco, Becton
Dickinson, Sparks, MD) and filtered (0.2-mm syringe filters).
Diluted filtrate (100 ml) was added to 100 ml of E. coli O157:H7
strain R508N and incubated for 20 min at 37uC. Molten 0.7%
UltraPure agarose (3.5 ml) was added, tubes were mixed by
inversion, and then they were poured onto prehardened, modified
nutrient agar (MNA; 8.5g/liter NaCl, 1 mM CaCl2, 1 mM FeCl3,
and 4 mM MgSO4) plates by using the soft-agar overlay technique
(43). Initial concentrations of active phages were then determined
from plates with 30 to 300 plaques. After 20 min at pH 3.0, 4 ml of
carbonate-bicarbonate buffer (pH 9.6) was added to bring the pH of
the solution to 7.0, and three 20-ml aliquots were titrated by using
the soft-agar overlay technique.
Experimental animals and Ephage delivery systems.
Twenty-four Angus cross yearling steers (average weight of 391
¡ 24 kg) were randomly assigned to three treatments (two pens of
four animals per treatment), with replicate pens sharing a water
bowl. An empty pen was used to prevent contact between
treatment groups, and a separate handling system was used for
collection of samples from individual animals in control (E. coli
O157:H7 only) pens. Cattle were cared for according to guidelines
established by the Canadian Council on Animal Care (13). Steers
were adapted to their environment and started on a typical feedlot
finishing diet consisting of 10% barley silage, 85% barley grain,
and 5% protein supplement for 4 weeks prior to initiation of the
study. Fecal grab samples, hide swabs, and samples of feed, water,
and fecal pats were collected on a weekly basis during this
adaptation period to verify the absence of E. coli O157:H7 strains
and bacteriophages specific for E. coli O157:H7 in the experi-
mental animals and their environment. On day 0, all cattle received
an oral inoculation of 1011 CFU of a five-strain mixture of
naladixic acid–resistant E. coli O157:H7. One day prior to
inoculation with E. coli O157:H7 and on days 1, 3, 6, and 8
after inoculation, steers in the Ephage treatment groups received
either an oral bolus of Ephage (bolus) or an Ephage-barley-silage
mixture top-dressed on the feed bunk (feed). To ensure complete
1305
consumption of feed, cattle in all treatment groups were fed at 95%
of ad libitum intake.
For the bolus treatment, 1 g of each type of Ephage was added
to a gelatin capsule (size 10, Torpac, Inc., Fairfield, NJ), and the
remainder of the capsule filled with 1.8 g of purified cornstarch.
Total weight of the bolus was 7.4 g. For the feed treatment, 16 g of
Ephage (1 g of each type of phage per animal in the pen) was added
to 1.1 kg of rolled barley in a self-sealing plastic bag. Barley and
Ephage were mixed by rolling the bag for 30 s. Barley silage
(0.55 kg) was then added to the bag and blended for an additional
1 min to reach a pH of 5.6. A 150-g subsample of feed was removed
from each bag to verify phage titer, and feed was then evenly
distributed over the finishing diet along the length of the feed bunk.
Titer of the feed was verified after 1.5 h at 20uC. Ten grams of
the mixture was weighed into a stomacher bag, 40 ml of citric acid
(pH 2.9) added, and the bag mixed at 230 rpm for 1 min in a
Stomacher 400 Circulator (Seward Medical, London, UK).
Twenty-two milliliters of carbonate-bicarbonate buffer (pH 9.6)
was then added to the stomacher bag to bring the pH to 7.1, and
phage titer was verified with the soft-agar overlay technique
described previously.
Daily individual animal doses in Ephage treatment groups
were estimated at 1010 PFU (bolus, Table 1) and 1011 PFU (feed,
Table 2) for mixed phages. Levels of E. coli O157:H7 and phages
were measured in fecal grab samples from all cattle. Presence of E.
coli O157:H7 was monitored in environmental samples (water,
feed, fecal pats, and hide swabs), while the presence of phages was
monitored in water, feed, and fecal pats at intervals throughout the
subsequent 10 weeks after inoculation with E. coli O157:H7.
Collection of samples. Fecal samples and hide swabs were
collected weekly during the 4-week adaptation period; on days 0,
2, 7, 8, 9, 14, and 16, after E. coli O157:H7 inoculation; and
subsequently twice per week for the 10-week experimental period.
Fecal samples (,10 g) were collected from each steer by digital
rectal retrieval and placed in 120-ml polypropylene containers
(Fisher Scientific, Nepean, Ontario, Canada). Latex gloves were
used during collection and were changed between each animal.
Samples were then transported to the laboratory for analysis. Hide
swabs were collected from each animal by using a sterile
SpongeSicle (Med-Ox Diagnostics, Inc., Ottawa, Ontario, Canada)
hydrated with 10 ml of PBS, and by swabbing an area 1,600 cm2
on the mediolateral axis of the ventrum.
Feed, water, and fecal pat samples were taken once a week
during the adaptation period, on Ephage inoculation days (feed
samples only), and once weekly for the 10-week experimental
period. At each sampling of feed from the bunk, five 20-g samples
from different areas of the feed bunk were collected and pooled 3 h
after morning feed delivery. From this larger sample, a 10-g
subsample was used for analysis. An area 100 cm2 of the water
trough at the water–water trough interface was swabbed with a
25-cm2 sterile gauze pad and added to a 120-ml sample of thoroughly
mixed water from the trough. A 10-g fecal pat sample was taken from
a pooled collection of three pats (20 g) from three different locations
in the pen. All samples were placed individually in 120-ml
polypropylene containers for analysis within 1 h after collection.
Isolation and enumeration of E. coli O157:H7 from feces.
Fecal samples collected from steers during the adaptation period
were assessed for the presence of nalidixic acid–resistant bacteria
and naturally occurring strains of E. coli O157:H7 by enrichment
in modified E. coli broth (mEC) for 6 h at 37uC, and then by
immunomagnetic separation (IMS) by using Dynabeads anti–E.
coli O157 (Invitrogen Corp., Carlsbad, CA), according to
1306 STANFORD ET AL. J. Food Prot., Vol. 73, No. 7

TABLE 1. Titers of individual and encapsulated bacteriophages targeted to E. coli O157:H7 pre- and postexposure to pH 3.0 for 20 min,
and after storage for 1 year at 20uC
Phage preencapsulation Encapsulated phagea

Initial titer Titer after acid Initial titer Titer after acid exposure Titer after storage
Phage (PFU/g | 1010) exposureb (PFU/g | 109) (PFU/g | 109)c (PFU/g | 108)d

rV5 2.5 0 1.06 1.82 0.18

wV7 0.4 NAe 0.02 0.09 0.14
wV8 1.0 0 8.31 7.99 1.92
wV11 4.0 NA 0.69 0.93 0.03
Mean of individual phage 2.0 0 2.56 2.71 0.57
Mean of mixed phage in bolusf 2.0 NA 1.80 1.82 0.25
a
All titers for encapsulated phages determined at pH 7.2.
b
Titer after 5 min at pH 3.0.
c
Twenty minutes at pH 3.0.
d
Storage for 1 year at 20uC.
e
NA, not available.
f
Bolus included 4 g of mixed phages and 1.8 g of cornstarch, for a delivery of an estimated 1.05 | 1010 PFU per bolus.

manufacturer’s instructions, and plating onto sorbitol MacConkey
agar supplemented with 2.5 mg/liter potassium tellurite and
0.05 mg/liter cefixime (CT-SMAC).
For enumeration of E. coli O157:H7 from fecal samples after
inoculation, fecal material was serially diluted (1:10) in PBS,
mixed by vortex, and duplicate 100-ml aliquots of a range of
dilutions were spread plated onto CT-SMAC with 50 mg/ml
naladixic acid. Plates were incubated for 18 to 24 h at 37uC prior to
counting colonies, with only those plates having 30 to 300 colonies
used in determination of bacterial populations. When E. coli
O157:H7 was no longer detectable by standard dilution plating,
enrichment in mEC for 6 h at 37uC and IMS with Dynabeads anti-
E. coli O157 was performed. After IMS, a 50-ml aliquot of the
antibody-coated bead suspension was plated onto a single plate of
CT-SMAC with 50 mg/ml naladixic acid, and the plate was
incubated for 18 to 24 h at 37uC. A single non–sorbitol-fermenting
colony from each plate was tested for the presence of the O157
antigen by using an Oxoid E. coli O157 latex kit (Oxoid, Nepean,
Ontario, Canada). Multiplex PCR assays (18) were then used to
verify the presence of vt, eaeA, and fliC (H7) genes. For analysis of
hide swabs, 45 ml of mEC was added and the swab incubated
TABLE 2. Mean titer (equal mixture of polymer-encapsulated
bacteriophages wV8, rV5, wV7, and wV11) recovered from a 166-
g subsample of feed a
Feed phages Feed phages delivered per animal
Dayb (PFU | log 109 g21) (PFU | 1011)c
21 1.07 4.04
1 1.50 7.26
3 0.52 1.98
6 1.11 4.20
8 1.47 5.58
Mean 1.13 4.30
a
A 166-g subsample of feed consisted of 4 g of each type of
encapsulated phage per steer in the pen added to 1.1 kg of
rolled barley and 0.55 kg of barley silage at a pH of 5.6, with
delivery of 1,516 g of feed to feed bunks in pens containing four
steers.
b
Day before (21) or after inoculation with 1011 CFU E. coli
O157:H7.
c
Assuming each steer received an equal dose of phages.
overnight at 37uC. IMS with Dynabeads anti–E. coli O157 was
then performed as described previously.
Isolation of E. coli O157:H7 from environmental samples.
Water samples (120 ml) were filtered through disposable 100-ml
MicroFunnel SP 0.2-mm filter units (Pall Life Sciences, Mis-
sissauga, Ontario, Canada), and the filters were then removed and
placed into 90 ml of mEC. For feed and fecal pat samples, 10-g
aliquots were placed into 90 ml of mEC, and the feed samples were
mixed thoroughly for 30 s at 230 rpm in the stomacher. The water
filter and feed samples were incubated for 18 to 24 h at 37uC, and
fecal pat samples were incubated for 6 h at 37uC prior to IMS and
selective plating, as described previously.
Detection and enumeration of bacteriophages. Samples of
feces or feed were mixed in 9 ml of PBS, and 1.8-ml aliquots were
centrifuged at 9,100 | g for 10 min. The supernatant was then
passed through a 0.2-mm syringe filter to remove bacterial cells.
The soft agar overlay assay previously described (43) was used for
enumeration of phages by using 40 ml of filtrate in 360 ml of
lambda diluent that was incubated for 20 min with 100 ml of E. coli
O157:H7 (R508N). Additional dilutions in lambda diluent were
performed as required to produce 30 to 300 plaques per MNA
plate. For detection of phages at low concentrations, 450 ml of the
filtered supernatant (feces or feed) was incubated with 50 ml of E.
coli O157:H7 strain R508N, under rotation for 60 min at 37uC.
After incubation, four 20-ml aliquots were spotted on an MNA
plate and incubated for 16 h at 37uC before the presence of plaques
could be observed. For water samples, 15 ml of filtrate was
concentrated in a Centriprep-30 (molecular weight cutoff of
30,000; Millipore, Billerica, MA) to approximately 3.0 ml by
centrifugation at 1,500 | g for 15 min. The concentrated water
(450 ml) was then used for detection of phages, as previously
outlined. During the adaptation phase (prior to inoculation with E.
coli O157:H7 and during the 10-week experimental period), fecal
samples were also evaluated for the presence of endemic, large-
plaque bacteriophages (42) on the same MNA plates that were used
to enumerate phages.
Statistical methods. The MIXED procedure of the SAS
software (44) was used to compare numbers of E. coli O157:H7
shed over time in the treatment groups. Orthogonal contrasts and
odds ratios within the GENMOD procedure of SAS were used to
J. Food Prot., Vol. 73, No. 7 ENCAPSULATED BACTERIOPHAGES FOR E. COLI O157:H7
compare effect of the two delivery systems for Ephage on the
incidence of E. coli O157:H7 in samples collected from individual
animals and from the feedlot environment. As the fecal counts of
E. coli O157:H7 were not normally distributed, the NLIN
procedure of SAS was used to compare the E. coli disappearance
curves among treatment groups and to evaluate inflection points. A
three-parameter, single-exponential decay model was used:
CFU ~ AA z B({c|d)
CFU is that of E. coli O157:H7 (log), AA is the curve asymptote, B
is the slope of the curve, c the fractional rate of disappearance of
CFU, and d is the time in days after inoculation with the organism.
RESULTS AND DISCUSSION
In vitro activity of encapsulated phages. Polymer
encapsulation successfully protected phages from a pH of
3.0, releasing an average of 2.71 | 109 PFU of active
phages after the pH was increased to 7.2 (Table 1). In
contrast, rV5 and wV8 were not active after 5 min of
exposure to pH 3.0 prior to encapsulation. As acid tolerance
of wV7 and wV11 would be similar to that of rV5 and wV8,
respectively (23), acid tolerance of wV7 and wV11 prior to
encapsulation was not directly evaluated.
Spray drying during the encapsulation process caused
an expected 1-log reduction in activity, similar to that
reported by O’Riordan et al. (38), as exposure to heat is
known to reduce phage activity (31). As each bolus included
1.8 g of cornstarch along with 4 g of Ephage, activity of the
mixed phages recovered from bolus was similar to that for
the average of individual Ephage (Table 1). The approxi-
mately 1.5-log reduction in activity after 1 year of storage at
20uC in the present study would be comparable to that
reported for encapsulation by using chitosan-alginate
microspheres (25), which lost 1.5 log of bacteriophage
activity over 6 weeks of storage at 22uC. Storage of Ephage
with desiccants to protect the integrity of the polymer matrix
or under refrigeration (38) might improve long-term activity
of Ephage and will be addressed in future studies, although
the 1-year stability demonstrated for Ephage should be
sufficient for most applications.
Encapsulation to provide protection from acid digestion
requires a precise balance. Insufficient protection may lead
to acid damage (38), while overly protected Ephage would
pass through the digestive tract without active phages being
released from the encapsulation matrix into the intestine. As
pH of the bovine digestive tract is often highest at the ileum,
ranging between 7.4 and 7.9 (15) and dropping in the large
intestine and feces because of hindgut fermentation of starch
(8), release of Ephage likely occurred in the ileum. In this
specific application, it was critical that active phages be
released prior to the rectal-anal junction, where the majority
of E. coli O157:H7 colonization occurs (29).
Measurement of ileal pH in the present study would
have verified release of phages. However, as measurement
of ileal pH requires surgical insertion of a cannula, with
disruption of the integrity of the digestive tract, these data
were not collected. The compromise chosen in the present
study favored acid protection of phages, and release of
phages in the ileum of the majority of animals receiving
barley-based diets (16, 41). Although none of the cattle in
1307
our study exhibited clinical symptoms of acidosis, there is
the possibility that the lower intestinal pH exhibited in
acidotic cattle might preclude the complete release of
phages from the encapsulation matrix (26). As pH of the
intestinal tract may vary greatly in individual cattle even in
the absence of acidosis (9, 16), selection of a pH-dependant
encapsulation medium that ensures complete release and
delivery of active phages to all areas of the digestive tract
colonized by E. coli O157:H7 is likely not feasible.
Delivery of phages per day in the feed treatment is
shown in Table 2. As consumption of the Ephage-treated
feed by cattle could not be regulated, mean daily dosages of
active phages were 2 log higher for feed than for the bolus
treatment (Table 1) in order to compensate for possible
inequities in feed consumption. Using silage as a carrier kept
pH of feed to a mean of 5.6, a strategy that was used to avoid
premature release of phages from the encapsulated matrix. As
the feed delivery system for phages did not require animal
handling, the feed system would likely be more readily
adopted by the beef industry for mitigation of E. coli
O157:H7 (39), although the bolus method of administration
ensured a uniform dosage of phages among steers.
Shedding of E. coli O157:H7, phages, and endemic
bacteriophages. The two Ephage treatments did not differ
from the control in the prevalence of fecal grab samples
positive for E. coli O157:H7 (Table 3), although more
positive hide swabs were present in the feed treatment than in
the control (P , 0.05). Similarly, levels of E. coli O157:H7
shed did not differ among treatment groups (Fig. 1). The
most notable affect of Ephage was that the bolus treatment
reduced the shedding period for E. coli O157:H7 by 14 days
(P , 0.10) as compared with that of the control (Table 4).
Part of the lack of efficacy of phage treatment in an earlier
study (42) was attributed to unintentional but rapid
transmission of phages to control animals. However, in the
present study, inoculated strains of phages were never
detected in the feces of control animals, although phages
were detected in environmental samples from control pens
(Table 5), possibly due to airborne transmission.
Limited influence of the Ephage treatments on
incidence or shedding of E. coli O157:H7 was not because
of a failure in establishing populations of phages. Shedding
of phages did not differ by treatment among the Ephage
cohorts (Fig. 2) and persisted until day 42 in the feed group,
although shedding of phages in both feed- and bolus-treated
steers was intermittent. The level of phages shed in the
present study was lower than that reported by Rozema et al.
(42), likely due in part to the previously discussed loss of
phage activity after encapsulation. However, active phages
were recovered in the feces of all phage-treated steers, with
the exception of one in the feed and two in the bolus
treatments (data not shown). Possibly, the steer in the feed
treatment that did not shed phages was a submissive animal
that did not gain access to feed, as these animals often
consume the majority of their diet in off-peak times such as
the middle of the night (19). Reasons for the lack of
establishment of phages in the two bolus-treated cattle are
unknown, but shedding of E. coli O157:H7 by these animals
1308 STANFORD ET AL. J. Food Prot., Vol. 73, No. 7

TABLE 3. Prevalence of positive samples and odds ratios comparing each treatment with the control (E. coli O157:H7 inoculation only)
for detection of bacteriophages and E. coli O157:H7 from feces and from hide swabs, for 10 weeks after inoculation
Treatment group:

Control Bolus Feed

a b c
Evaluation Positive OR Significance Positive OR Significance Positive OR Significance

Feces O157:H7 102/164 1.08 NS 98/157 1.06 NS 100/165 0.98 NS

Hide O157:H7 83/160 0.91 NS 76/151 1.00 NS 95/160 1.10 *
Phages (inoculated strains) in feces 0/164 0.60 *** 14/156 1.29 * 13/165 1.29 *
Phages (endemic, large plaque) in
feces 1/164 1.03 NS 5/156 1.03 NS 1/165 0.99 NS
a
Positive, number of positive samples/number of samples collected.
b
OR, odds ratio.
c
Significance: NS, not significant; *, P , 0.05; ***, P , 0.001.

was similar to that of other cattle within the treatment.
Prestudy tests of the gelatin capsule indicated that it
consistently dissolved after 7 min of contact with rumen
fluid (data not shown). Possibly, these two animals may
have had an intestinal pH that was not optimal for complete
release of phages, although such data could not be collected
without cannulation, as discussed previously.
In a previous challenge study, we isolated endemic
bacteriophages of the family Siphoviridae, which exhibited
a morphology that was different from our therapeutic
phages (42). Endemic bacteriophages are also routinely
present in commercial feedlots in southern Alberta (33).
Accordingly, in the present study, endemic bacteriophages
exhibiting a morphology similar to that detected by Rozema
et al. (42) were isolated from all treatment groups in the first
10 days after inoculation with E. coli O157:H7 (Table 3).
As both the endemic and inoculated phages use E. coli
O157:H7 as a host, the endemic bacteriophages may
interfere with replication and establishment of the experi-
mental phages through competitive interference (11).
Endemic bacteriophages were isolated only when popula-
tions of E. coli O157:H7 exceeded 103 CFU/g. Conse-
quently, high levels of host bacteria likely triggered rapid
proliferation of a small but apparently resilient and
ubiquitous population of endemic bacteriophages within
the digestive tract of steers. Similarly, Oot et al. (37)
detected a high (97%) prevalence of endemic bacteriophag-
es in feedlot cattle after a 12-h enrichment of fecal samples.
Possibly, endemic bacteriophages outcompeted the
therapeutic phages for E. coli O157:H7, as our therapeutic
phages were not originally isolated from the digestive tract
of cattle or within their immediate environment (51). Others
have proposed that isolation from the environment to which
they are going to be therapeutically introduced might
increase the efficacy of phage therapy for mitigating E. coli
O157:H7 (11). However, as members of the Siphoviridae
family may be lysogenic as well as lytic (2), the endemic
bacteriophages isolated in our study would require extensive
characterization before implementation in bacteriophage
therapy. Accordingly, most studies on the use of bacterio-
phage therapy to control E. coli O157:H7 have employed
lytic bacteriophages from the Myoviridae family (1, 40, 42).

Effect of phage treatment on the environment. As

FIGURE 1. Fecal shedding of E. coli O157:H7 (log CFU per
gram), measured by dilution plating without enrichment for cattle
in control, bolus, and feed treatment groups after inoculation with
1011 CFU of naladixic acid–resistant E. coli O157:H7 on day 0.
expected, fecal pats were superior (P , 0.001) to water and
feed samples for detection of both E. coli O157:H7 and
phages (Table 5). As phages were detected in feed only
once, the very limited detection of phages in samples of
mixed diet retrieved 3 h after delivery of feed confirmed
consumption of feed by the cattle. The silage carrier for feed
appeared to be preferentially consumed by the cattle and
was not evident within 15 min of delivery. In accordance
with previous studies (42, 47), the chlorinated water
supplied to the Lethbridge Research Centre did not promote
growth of E. coli O157:H7, although a single water sample
positive for phages was collected. Similar to samples
collected from individual cattle, environmental samples
did not differ in detection of E. coli O157:H7 among
treatment groups. Experimental phages were detected in two
environmental samples from the control group, attesting to
the ease of transmission of phages between open pens of
feedlot cattle (42), even though animals in different
treatment groups were strictly segregated. As well, phages
J. Food Prot., Vol. 73, No. 7 ENCAPSULATED BACTERIOPHAGES FOR E. COLI O157:H7 1309

TABLE 4. Patterns of E. coli O157:H7 shedding by cattle within treatment groups after inoculation with 1011 CFU on day 0
Treatmenta Days of shedding E. coli O157:H7b Inflection point in shedding curve (d)c Log CFU at inflection point

d
Control 46.0 ¡ 5.7 B 29.5 ¡ 3.6 b 0.2 ¡ 0.3
Bolus 31.9 ¡ 6.1 A 17.0 ¡ 4.1 a 1.1 ¡ 0.3
Feed 41.2 ¡ 5.7 AB 24.6 ¡ 3.2 ab 0.7 ¡ 0.3
a
Treatment with 1011 PFU per steer (feed treatment) or 1010 PFU per steer (bolus treatment) bacteriophages targeted to E. coli O157:H7
occurred on days 21, 1, 3, 6, and 8.
b
E. coli O157:H7 detected by direct plating or immunomagnetic separation.
c
Inflection point is the day when log CFU began to increase after continual decline postinoculation.
d
Means with different capital letters differ (P , 0.10); means within a column with different lowercase letters differ (P , 0.05).

exhibited environmental resiliency, as we were able to
isolate phages from our research feedlot after the pens were
left empty for a period of 6 months (data not shown).
Similarly, Smith et al. (46) detected bacteriophages in their
research facility for a year after study completion. As this
study was conducted in the winter and early spring,
temperatures .20uC with high humidity, which were
determined by Niu et al. (33) to be optimal for environ-
mental proliferation of endemic bacteriophages, would not
have occurred, and the effect of phages under optimal
environmental conditions should be further addressed.
Relationship among levels of E. coli O157:H7 and
phage efficacy. As phages require contact with their hosts
to initiate the steps leading to lysis, the chance of such an
encounter decreases as the number of phages or hosts
present within the intestinal tract decline. Consequently, a
theoretical threshold exists at which point low numbers of E.
coli O157:H7 would reduce the likelihood of host–phage
contact and efficacy of phage therapy. In the present study,
fecal concentrations of log 1.77 CFU or greater of E. coli
O157:H7 were positively correlated with presence of phages
(P , 0.05), while concentrations of log 1.71 CFU or less E.
coli O157:H7 per g of feces were not correlated to phage
presence (data not shown). Greer (21) proposed that
bacterial populations from 3 to 5 log CFU/g were required
for replication of bacteriophages in food, while Rozema et
al. (42) found phages most effective when E. coli O157:H7
populations were .4 log CFU/g of feces.
In common with other inoculation studies (11, 47), the
population of E. coli O157:H7 followed an initial rapid
decline in all treatment groups before inflection points were
reached at which transitory increases occurred in shedding
of E. coli O157:H7 (Fig. 1). For the bolus treatment, the
inflection point in the shedding curve occurred more rapidly
(P , 0.05) than it did for control steers (Table 4) and was
possibly linked to a decline in E. coli O157:H7 populations
below the threshold required for effective phage replication.
The inflection point in the shedding curve of cattle receiving
feed was later than it was for bolus-treated animals and did
not differ from that of the control, likely because of the
limited influence of the feed treatment on shedding
dynamics of E. coli O157:H7. Due to the predator-prey
relationship between phages and host, alternating cycles of
population expansion and contraction of E. coli O157:H7
may occur (12).
Levels of phages and phage efficacy. Over the first
10 days of the study, steers in the bolus and feed treatments
shed an average of 1.25 | 1010 PFU of phages per g of
feces (Fig. 2). As steers weighing 400 kg would produce an
estimated 4.0 to 5.0 kg of feces on a daily basis (3), an
average steer in the bolus and feed treatments would have
excreted 5.62 | 1013 PFU phages per day over this period.
Compared with dosing of Ephage (1010 and 1011 PFU per
day for the bolus and feed treatments, respectively) and
considering the 1-log loss in phage activity because of the
encapsulation process (Table 1), these results provide
evidence for replication of phages in the digestive tracts
of the steers.
The limited effect of the feed treatment on shedding of
E. coli O157:H7 was unexpected, as this treatment delivered

TABLE 5. Prevalence of positive environmental samples and odds ratios comparing fecal pats with the other two sample types or control
(E. coli O157:H7 inoculation only) with the other two bacteriophage treatments for detection of bacteriophages and E. coli O157:H7 in the
environment, for 10 weeks after inoculation
Sample type Positive for E. coli O157a ORb Significancec Positive for bacteriophages OR Significance

Fecal pat 10/60 5.53 *** 9/60 1.29 ***

Feed 0/54 0.40 *** 1/54 0.88 **
Water 0/39 0.45 *** 1/39 0.88 **
Control 17/48d 1.03 NS 2/148d 0.90 NS
Bolus 17/48 1.00 NS 5/148 1.06 NS
Feed 16/57 1.02 NS 4/148 1.04 NS
a
Number of positive samples/number of samples collected.
b
OR, odds ratio.
c
Significance: ***, P , 0.001; **, P , 0.01; NS, not significant.
d
Total positive environmental samples (fecal pat, feed, and water)/number of samples collected per treatment.
1310 STANFORD ET AL.
FIGURE 2. Fecal shedding of mixed experimental bacteriophag-
es (log PFU per gram) of cattle after treatment with 1010 PFU
(bolus treatment) and 1011 PFU (feed treatment) on days 21, 1, 3,
6, and 8.
an average of 2 log PFU more phages per animal per day of
treatment than did the bolus treatment, and shedding of
phages was detected longer for feed animals (42 days) than
it was for bolus-treated steers (35 days). Callaway et al. (11)
found a 1:1 ratio of phage and E. coli O157:H7 was most
effective for control of E. coli O157:H7. Consequently, it is
possible that excess phages to hosts in the feed treatment
interfered with phage replication, similar to the competitive
interference described by Kutter and Sulakvelidze (24),
which may have also occurred between the endemic and
experimental bacteriophages in our study. Other factors
reducing efficacy of the feed treatment are unknown, but
could possibly include desiccation and exposure to ambient
temperatures from 220 to 4uC, although cattle were
observed to rapidly consume the feed after delivery to the
bunk, and activity of bacteriophages by using E. coli as a
host was not reduced by 48 h of refrigeration (36).
Other factors influencing efficacy of Ephage. As the
polymer encapsulation did not release phages until pH of
digesta was greater than 7.0, it is likely that Ephage
bypassed regions anterior to the ileum within the upper
digestive tract, which may have also contained populations
of E. coli O157:H7. Although the terminal rectum has been
identified as a major site of colonization with E. coli
O157:H7 in feedlot cattle (29, 30), this organism is also
routinely recovered from the oral cavity (47), rumen (11),
and duodenum (20). Consequently, efficacy of Ephage for
controlling E. coli O157:H7 in these more acidic regions of
the digestive tract may be questionable.
Treatment with phages is most likely to be undertaken
immediately prior to shipment of cattle to slaughter (12),
when cattle are receiving high-grain finishing diets. A
number of dietary factors such as level of grain processing
(16), type of cereal grain (i.e., barley versus corn versus
wheat (8, 9)), use of other byproduct feeds (50), and level of
forage in the finishing diet (10) influence pH of digesta and
may influence the efficacy of Ephage for control of E. coli
J. Food Prot., Vol. 73, No. 7
O157:H7. The Ephage system was developed to be
applicable for the majority of barley-fed animals having
10% or more forage in their diet. Alternate encapsulation
systems may be required for use in feedlot diets that lower
pH in the large intestine, as is the case when corn is the
primary cereal grain in the diet (8).
Polymer encapsulation in combination with the bolus
and feed delivery systems developed in the present study
successfully released active phages at pH .7.0, and in
theory should have been a useful mitigation for E. coli
O157:H7 in the majority of cattle receiving barley-based
finishing diets. In practice, neither the bolus nor the feed
delivery systems effectively controlled shedding of E. coli
O157:H7. Prior to future studies of Ephage therapy in
feedlot cattle, further work is required to characterize the
relationships between endemic and experimental phages,
effective doses of phages relative to E. coli O157:H7, and
the relative importance of colonization of multiple regions
of the gastrointestinal tract on shedding of E. coli O157:H7.
ACKNOWLEDGMENTS
Many thanks are extended to the technical team involved in this study,
including Jenilee Graham, Jessica Hoffarth, Chelsea Agopsowicz, Ho-
mayoun Zahiroddini, and Geoff Wallins. The work of Merlin Anderson and
Brant Baker in care of animals and assistance in sample collection is also
much appreciated. Funding from the Food Safety Initiative of Alberta
Agriculture and Rural Development is gratefully acknowledged.
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