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Veterinary Microbiology 136 (2009) 135–141

Contents lists available at ScienceDirect

Veterinary Microbiology
journal homepage: www.elsevier.com/locate/vetmic

Evaluation of bacteriophages for prevention and treatment of diarrhea

due to experimental enterotoxigenic Escherichia coli O149 infection
of pigs
Nidham Jamalludeen a, Roger P. Johnson b, Patricia E. Shewen a, Carlton L. Gyles a,*
a
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada
b
Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario N1G 3W4, Canada

A R T I C L E I N F O A B S T R A C T

Article history: The objective of this study was to determine the efficacy of selected phages individually
Received 4 September 2008 and in combination in prevention and treatment of diarrhea due to experimental
Received in revised form 16 October 2008 O149:H10:F4 enterotoxigenic Escherichia coli (ETEC) in weaned pigs. For prophylaxis, the
Accepted 21 October 2008
phages were administered orally shortly after challenge, and for therapeutic use, were
given 24 h after challenge, following the onset of diarrhea. The parameters used to assess
Keywords: outcomes were weight change, duration of diarrhea, severity of diarrhea, composite
Phages against O149 ETEC
diarrhea score, and extent of shedding of the challenge ETEC over 6 days.
Phages
Six phages that were tested individually in a prophylactic mode were effective as
Postweaning diarrhea
Enterotoxigenic E. coli determined by a significant change in each of the parameters, although the phages were
not present at titres greater than 103 PFU/g of feces. A modified protocol involving pre-
treatment of the pigs with florfenicol and oral administration of sodium bicarbonate prior
to the ETEC challenge and phage administration resulted in high levels of phages in the
feces. Using this protocol, a combination of three phages that was tested in the
prophylactic mode significantly reduced the severity of diarrhea and the composite
diarrhea score. A mixture of two phages given therapeutically significantly improved each
of the outcome parameters, without perturbation of the total fecal E. coli flora.
Enumeration of phages in feces after treatment indicated that the phages were replicating
to high titres in the intestinal tract of ETEC infected pigs within 1–2 days before declining
progressively. These findings indicate that the selected phages were effective in
moderating the course of experimental O149:H10:F4 ETEC diarrhea in weaned pigs
when given prophylactically or therapeutically.
ß 2008 Elsevier B.V. All rights reserved.

1. Introduction therapeutic use of antimicrobials in pigs and threaten

There are concerns that increasing antimicrobial
resistance of normal flora and enterotoxigenic Escherichia
coli (ETEC) in pigs (Dunlop et al., 1998; Noamani et al.,
2003; Fairbrother et al., 2005) may compromise the
* Corresponding author.
E-mail addresses: njamallu@uoguelph.ca (N. Jamalludeen),
cgyles@uoguelph.ca (C.L. Gyles).
human health through transfer of drug resistance genes to
zoonotic pathogens. There is therefore a need for safe and
practical alternatives to antimicrobials for prophylaxis and
therapy in pigs.
Bacteriophages (phages) are non-hazardous self-repli-
cating agents that increase their numbers as they destroy
target bacteria (Sulakvelidze et al., 2001; Huff et al., 2005).
In the 1980s, excellent studies on phage therapy were
carried out by Smith and colleagues, using E. coli infection
in mice and farm animals (Smith and Huggins, 1982, 1983;

0378-1135/$ – see front matter ß 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetmic.2008.10.021
 Author's personal copy
136 N. Jamalludeen et al. / Veterinary Microbiology 136 (2009) 135–141
Smith et al., 1987a, 1987b). Treatment of infected mice
with phages was more effective than treatment with
antibiotics, and phages were effective when they were
administered before or after infection (Smith and Huggins,
1982). The protective effect of phages was also shown
against E. coli diarrhea in neonatal pigs and calves (Smith
and Huggins, 1983; Smith et al., 1987b). Recently, the
effectiveness of phages against Salmonella Typhimurium
(Berchieri et al., 1991; Fiorentin et al., 2005), and
Escherichia coli in poultry has also been reported (Huff
et al., 2004, 2005). Development of bacterial resistance to
therapeutic phages and inactivation of orally administered
phages are concerns, but there are strategies to circumvent
these problems (Smith et al., 1987b; Sulakvelidze et al.,
2001).
In vitro screening of phages for efficacy and rapid
multiplication is valuable in selection of therapeutic
phages (Smith and Huggins, 1982). We therefore used in
vitro tests to characterize seven phages that lyse porcine
ETEC of O group 149, the most common cause of post-
weaning ETEC diarrhea in pigs, worldwide (Jamalludeen
et al., 2007). The objective of the current study was to
determine the efficacy of these phages individually and in
combination in prevention and treatment of experimental
O149 ETEC diarrhea in weaned pigs.
2. Materials and methods
2.1. Media
Brain Heart Infusion Broth (BHIB), and MacConkey
(MAC) agar were purchased from Difco Laboratories
(Detroit, MI) and prepared according to the manufacturer’s
instructions. Blood agar was purchased from Fisher
Scientific, Nepean, ON, Canada. LB broth, LB agarose, and
LB top agarose were prepared as described previously
(Jamalludeen et al., 2007). SM buffer was as described by
Sambrook et al. (1989).
2.2. Bacterial strains
The challenge strain for experimental infection of pigs
was O149:H10:F4 ETEC strain JG280, a hemolytic E. coli
with genes for STa, STb, LT, and EAST-I (Noamani et al.,
2003). This strain was isolated in 1999 from an Ontario pig
with post-weaning diarrhea (PWD) and was obtained
from Gallant Custom Laboratory, Cambridge, Ontario. It
was also used for detection of phages in pig feces, together
with O149:H43:F4 ETEC strain 3220, a 1983 Ontario
isolate which is hemolytic and carries genes for LT, STb,
and EAST-1. The organisms were streaked on blood agar,
checked for purity, tested for agglutination in O149
antiserum, and then aliquots were frozen at 70 8C in a
milk-based freezing solution (Harris, 1954). As required,
frozen stock cultures were plated overnight on MAC agar
at 37 8C and single colonies were cultured in BHIB for 18–
20 h at 37 8C with shaking at 150 rpm. For experimental
infections, BHIB cultures of ETEC strain JG280 were
sedimented by centrifugation at 5000  g, then resus-
pended in 0.01 M phosphate-buffered saline, pH 7.2 (PBS)
and adjusted spectrophotometrically to an OD600 of 1.4,
equivalent to approximately 1010 CFU/ml. The concentra-
tions of the bacterial suspensions were confirmed by
standard plate counts.
2.3. Phages
Seven lytic phages, designated GJ1–GJ7 (Jamalludeen
et al., 2007), were evaluated in this study. Phages GJ1–GJ6
were previously found to be highly effective in vitro against
ETEC O149:H10:F4 strain JG280, whereas phage GJ7 was
most effective against ETEC O149:H43:F4 strain 3220
(Jamalludeen et al., 2007). The phages were propagated in
E. coli strain JG280 or strain 3220 in LB top agarose, and
prepared as previously described (Jamalludeen et al.,
2007), yielding approximately 109 PFU/ml in SM buffer.
2.4. Animals
Pigs (n = 101) were obtained from the University of
Guelph swineherd, weaned at 3 weeks of age, transferred
to the Animal Isolation Facility at the Ontario Veterinary
College, and allowed to acclimatize for 2 days before
commencement of experiments. They were housed in
groups of up to five pigs, fed a standard non-medicated
ration for post-weaning pigs and had water ad libitum.
They were weighed at the commencement and end of the
experiments. Fecal samples were collected from the
rectum prior to and at intervals after infection and
treatment. At the end of the experiments, or earlier if
required for humane purposes, the pigs were euthanized
and necropsied. Tissues were collected at necropsy for the
enterocyte adhesion test (see below), which determines
the presence or absence of the F4 ETEC receptor, and hence
susceptibility to ETEC infection. Results for pigs whose
enterocytes failed to bind the challenge strain, GJ280, were
removed from the trial data and analyses. All animal care
and procedures complied with the requirements of the
Canadian Council on Animal Care.
2.5. Experimental trials
Of the five trials described below, Trials 1 and 3
evaluated the efficacy of the selected phages in preventing
development of diarrhea when given shortly after expo-
sure to ETEC (i.e. prophylactic use). In Trial 1, six phages
were evaluated individually, and in Trial 3, a mixture of
three phages was evaluated for prophylactic use. Trial 2 did
not include treatment of ETEC-infected pigs with phages
but was conducted to determine if treatment of pigs with
an antibiotic and an antacid prior to oral inoculation with
ETEC or the phages would increase the diarrheal response
to ETEC infection, and improve the survival of phages in the
gastrointestinal tract. All subsequent trials included these
modifications. Trials 4 and 5 evaluated a mixture of two
phages for alleviating diarrhea that developed after
experimental ETEC infection (i.e. therapeutic use).
2.5.1. Trial 1: prophylactic efficacy of individual phages
Twenty-eight pigs were allocated into six groups of four
or five for challenge and treatment with individual phages
GJ1–GJ6, and another 15 pigs were challenged but not
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N. Jamalludeen et al. / Veterinary Microbiology 136 (2009) 135–141
treated. The pigs were weighed prior to challenge and just
before euthanasia. Each pig was inoculated orally by
syringe with approximately 1010 CFU of O149:H10:F4
ETEC strain JG280, suspended in 5 ml of PBS. Fifteen
minutes later, pigs in the six treatment groups were
inoculated orally by syringe with 1010 PFU of phages GJ1–
GJ6 in 2 ml of SM buffer, one phage per group. The pigs
were monitored for 6 days, during which the occurrence,
duration and severity of diarrhea were recorded daily, and
fecal samples were collected daily for detection and
estimation of the levels of shedding of the challenge strain
and phages, as described below. The pigs were then
euthanized and necropsied for collection of samples for the
enterocyte adhesion test. The severity of diarrhea was
scored daily by a method similar to that described by
Jensen et al. (2006). A score of 0 was assigned when the
feces were firm and normally shaped, a score of 1 was
given when the feces were soft but able to retain some
shape, a score of 2 was given when the feces were brown
and liquid, and a score of 3 was assigned when there was
frequent passage of watery feces. Pigs with severe illness
resulting in death or euthanasia on humane grounds were
given the maximum score of 18 (score of 3 for 6 days).
These daily scores were used to calculate individual mean
diarrhea scores (sum of daily scores divided by the number
of days with diarrhea), and composite diarrhea scores
(mean diarrhea score multiplied by the duration of
diarrhea in days), and group means for these parameters,
where appropriate. Assessment of the effect of treatment
also included weight change during the study, and the
levels of shedding of the challenge ETEC and phages.
Challenge ETEC recovered from the pigs were tested for
susceptibility to the phage that had been administered.
2.5.2. Trial 2: modifications to the challenge and treatment
protocols
This trial was designed to determine if antibiotic (Van
der Stede et al., 2003) and antacid treatments (Smith et al.,
1987b) prior to inoculation with ETEC strain GJ280 would
result in a more severe diarrheal response in ETEC-
challenged pigs and improved phage survival in the
gastro-intestinal tract of pigs given only the phages. All
11 pigs were given 0.5 ml of florfenicol (300 mg/ml,
Schering Canada Inc, Pointe-Claire, Quebec) intramuscu-
larly on two consecutive days, followed by 1 day without
antibiotic treatment. The next day, all pigs were given
60 ml of 1.4% sodium bicarbonate orally, followed 15 min
later by oral inoculation with either ETEC strain GJ280 only
(five pigs) or phages GJ1–GJ6 only (one pig per phage).
Inoculations with bacteria or phages, monitoring, scoring
and other aspects of the trial were as described for Trial 1.
2.5.3. Trial 3: prophylactic efficacy of a mixture of three
phages
Twenty pigs were challenged orally with 1010 CFU of
ETEC strain GJ280, as described in Trial 1, after the
florfenicol and antacid pre-treatments described in Trial 2.
Fifteen minutes after challenge, 10 pigs were each
inoculated orally as described in Trial 1 with a mixture
of phages GJ1, GJ2 and GJ7, each at 109 PFU. The criteria
used for assessing diarrhea, weight change, excretion of
137
phage and challenge ETEC and the enterocyte adherence
test were as described for Trial 1.
2.5.4. Trials 4 and 5: therapeutic efficacy of a mixture
of two phages
To evaluate the therapeutic, as opposed to prophylactic
use of phage therapy, the phages were not administered
until 24 h after challenge, after the onset of diarrhea. Ten
pigs were challenged orally with 1010 CFU of ETEC strain
GJ280 as described in Trial 1 and following the florfenicol
and antacid pre-treatments described in Trial 2. At 24 h
after challenge, the severity of diarrhea was scored as
described in Trial 1, and fecal samples were taken to detect
and enumerate the ETEC strain GJ280 and total E. coli (see
below). The pigs were divided randomly into two groups of
five pigs, one in which pigs received no treatment and the
other in which pigs were treated with phages. Phage
treatment comprised of an oral inoculation as described in
Trial 1 with a mixture of phages GJ1 and GJ6, each at a dose
of 108 PFU, three times at 6 h intervals. Sodium bicarbo-
nate was administered once, prior to the first inoculation
with phage. The pigs were monitored daily for 5 days after
treatment, and then euthanized. As in other trials, daily
clinical observations, diarrhea scores, shedding of ETEC
strain GJ280 and phages, weight change during the
experiment, and the results of the enterocyte adherence
tests were used to assess the effectiveness of phage
treatment. Trial 5 was a repeat of Trial 4, with nine
challenged and untreated control pigs and eight chal-
lenged pigs treated with a mixture of phages GJ1 and GJ6,
as in Trial 4.
2.6. Detection and quantitation of bacteria in feces
Fecal samples were collected by swab from the rectum,
stored on ice and processed within 2 h of collection.
Samples collected during the adaptation period were
tested for the presence of hemolytic O149 ETEC by mixing
1 g of feces in 9 ml of PBS, plating the mixture on MAC agar
and blood agar and incubating the plates overnight at
37 8C. If hemolytic colonies were present, five were tested
for agglutination with O149 antiserum. After challenge, the
levels of the challenge strain GJ280 in feces were estimated
as percentage recovery of the hemolytic, O149-positive
challenge strain relative to other E. coli. Fecal samples were
diluted 10-fold in PBS and selected dilutions were plated
on blood agar and MAC agar plates. The plates were
incubated overnight at 37 8C, and examined for hemolytic
and non-hemolytic E. coli-like colonies. Hemolytic E. coli-
like colonies on blood agar were tested for agglutination
with O149 antiserum. The percentage recovery of the
challenge strain was calculated by using the total numbers
(per plate) of hemolytic, O149-positive E. coli-like colonies
on blood agar, and of E. coli-like colonies on MAC agar.
Daily estimates for individual animals were used to
calculate the individual and group mean percentage
recoveries of the challenge strain over the 6 days of the
experiments. Total fecal E. coli counts, determined in Trials
4 and 5, were estimated by counts of E. coli-like colonies
from serial 10-fold dilutions of feces on MAC agar. Selected
O149-positive E. coli isolated from the pigs after challenge
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138 N. Jamalludeen et al. / Veterinary Microbiology 136 (2009) 135–141
were tested by a spot test (Kutter and Sulakvelidze, 2005)
for their susceptibility to phages GJ1 and GJ6.
2.7. Detection and quantitation of phages in feces
A 1-g quantity of feces was added to 9 ml of sterile
PBS and 0.2 ml of chloroform was added. The mixture
was held for 15 min at room temperature, centrifuged at
4000  g for 10 min and then filtered through a 0.45 mm
membrane filter (Fisher Scientific). For pre-treatment
samples, the filtrate was tested for phages lytic for ETEC
strains GJ280 and 3220 by using a spot test (Kutter and
Sulakvelidze, 2005), with these two bacteria as hosts. For
post-treatment samples, the filtrate was diluted tenfold
(10 1 to 10 8) in PBS. A lawn of host bacteria was spread
on an LB agarose plate and allowed to dry for about
5 min and then 10 ml of the filtrate dilutions were
dropped on quadrants of the plate and allowed to dry.
The theoretical detection limit of this assay was 103 PFU/
g of feces. The plates were incubated at 37 8C for 6–8 h
then were examined for lysis in the areas on which the
drops of filtrate had been placed. The phage titre was
determined by counting the number of plaques in the
highest dilution in which plaques could be observed. A
negative value was recorded when there were no
plaques in the 10 1 dilution, representing a titre of less
than 103 PFU/g.
2.8. Enterocyte adhesion test
Immediately after euthanasia, 6 days post-challenge,
a sample of small intestine was collected to determine
whether the F4+ challenge E. coli could adhere to
enterocytes from each pig (Python, 2003). A 5-cm
portion of small intestine approximately 20 cm from
the ileo-cecal junction was removed, opened long-
itudinally, and immersed in ice-cold PBS, pH 6.8,
containing EDTA (3.72 g/l). Epithelial cells were
removed by scraping the mucosal surface with a
microscope slide and added to 40 ml of PBS containing
2% formaldehyde. Large fragments were allowed to
sediment, and cells in the supernatant were washed
twice by centrifuging for 10 min at 200  g and
resuspended in 10 ml PBS. The final suspension was
diluted to approximately 105–106 cells/ml in PBS con-
taining 2% D-mannose. A 1-ml volume of enterocyte
suspension was mixed with 1 ml of a broth culture of
ETEC strain JG280, that had been diluted 1:30 from an
overnight culture then incubated for 4 h at 37 8C to reach
an OD600 of 0.9. The mixture of enterocytes and bacteria
was incubated with gentle shaking in a water bath at
37 8C for 45 min. Following incubation, 20 ml of the
suspension were placed on a slide, allowed to dry, fixed
with 95% methanol, stained with Giemsa, then examined
microscopically. A pig was considered positive for the
binding of the challenge ETEC, and therefore susceptible
to infection, if more than 15% of the cells bound at least
five bacteria (Python, 2003). Pigs with negative results in
this test were considered not susceptible to the
challenge ETEC and their data were not included with
the results.
2.9. Statistical analysis
The general linear mixed (GLM) procedure and ANOVA
using SAS 9.1.3 (SAS Institute Inc., Cary, NC, USA) were
used to compare the parameters for response in the
treatment groups with those in the control group and the
association between parameters of the phage-treated
groups as compared with the control group in each trial.
ANOVA was used for analysis of shedding of E. coli and
phage, whereas GLM was used for all other analysis. A
difference was considered significant when the P value was
less than or equal to 0.05.
3. Results
For all the trials, hemolytic colonies were sometimes
found on pre-challenge culture of feces from the pigs. In no
case did the hemolytic colonies agglutinate in O149
antiserum. Phages active against the O149 ETEC were not
detected in any of the pigs prior to the trials. The outcome of
the enterocyte adherence assays resulted in exclusion of the
data from two pigs in trial 1 (one control and one treated
with phage JG2) and one treated pig in trial 5.
3.1. Prophylactic efficacy of individual phages (Trial 1)
ETEC JG280-infected pigs treated prophylactically with
individual phages GJ1–GJ6 had significantly lower group
mean weight changes (GLM, P  0.0001–0.0101), and group
mean composite diarrhea scores (GLM, P  0.0001–0.02)
compared to infected but untreated pigs (Fig. 1). Analysis by
the GLM revealed that treatment with individual phages
GJ1, GJ2 and GJ5 also resulted in significant reductions in
group duration of diarrhea (P = 0.0114–0.0329) and group
mean diarrhea scores (P = 0.0146–0.0264), and that pigs
treated with phage GJ6 had lower group mean diarrhea
Fig. 1. Effect of prophylactic treatment with individual phages GJ1–GJ6 on
diarrheal disease in weaned pigs after oral challenge with O149:H10:F4
ETEC strain JG280 (Trial 1). The phages were given orally 15 min after
challenge. Bars show group mean values for weight change in kg (black
bars), duration of diarrhea in days (white bars), diarrhea score (checkered
bars), and composite diarrhea score (speckled bars) over 6 days in
untreated control pigs (group C, n = 14) and pigs treated with individual
phages GJ1 (n = 5) GJ2 (n = 4), GJ3 (n = 4), GJ4 (n = 5), GJ5 (n = 4) or GJ6
(n = 5). See text for description of diarrhea scores. Bars = +1 S.E. Asterisks
indicate parameters with values significantly different from those of the
control group.
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N. Jamalludeen et al. / Veterinary Microbiology 136 (2009) 135–141
Table 1
Diarrheal disease in pigs treated with florfenicol by the I.M. route for 2 days and with sodium bicarbonate orally just prior to oral challenge with O149 ETEC
strain JG280 (Trial 2).
Pig # Weight change (kg) Duration of Meana diarrhea score
diarrhea (days)
1 1.77 3 3
2c 1.38 6 3
3 +2.05 3 1.3
4 +1.05 2 1.5
5 1.64 4 2.25
Mean 0.34 3.6 2.21
a
Mean diarrhea score = sum of daily diarrhea score/number of days with diarrhea.
b
Composite diarrhea score = average diarrhea score times duration of diarrhea.
c
Died on day 3 and assigned the maximum score 18.
score (P = 0.0191) but not a shorter mean duration of
diarrhea (Fig. 1). The mean percentage excretion of the
challenge ETEC within all phage-treated groups (range of
group means, 2.75–37.26%,) was significantly lower
(ANOVA, P < 0.0001 for GJ1–GJ5 and 0.0002 for phage
GJ6) than that of the infected but untreated controls (group
mean, 83.31%). Phages that were lytic for the challenge ETEC
were not detected in fecal samples from any of the pigs
that had received phages. The challenge bacteria that
were recovered from the feces of the pigs that received
phages remained susceptible to the phages that had been
administered.
3.2. Modifications to the challenge and treatment
protocols (Trial 2)
Administration of florfenicol to the pigs for 2 days prior
to ETEC challenge and sodium bicarbonate orally 15 min
before challenge did not alter the clinical outcome of
challenge with ETEC GJ280 (Table 1). Group means of
weight change, duration of diarrhea, diarrhea score and
composite diarrhea score of challenged pigs (Table 1) were
not significantly different (P value = 0.48, 0.72, 0.4, and
0.47, respectively) from those of the control group in trial 1
(Fig. 1). Also, shedding of the challenge ETEC strain (group
mean, 77.32%, Table 1) was similar to that of the control
group in trial 1 (group mean, 83.31%, above). However, the
modified protocol appeared to influence phage survival. In
contrast to the results of trial 1, in which the phages were
not detectable in the feces of treated pigs, all six phages
were detectable for 1–3 days in the feces of all six treated
pigs that received the phages but no ETEC, at levels ranging
from 1.5  106 on day 1 to 7  103 on day 3.
3.3. Prophylactic efficacy of a mixture of three phages (Trial 3)
Following the modified challenge and treatment pro-
tocol described in trial 2, one group of 10 pigs received
ETEC GJ280 alone and the other group of 10 received ETEC
GJ280 and a mixture of three phages (GJ1, GJ2, and GJ7).
The mixture of phages was effective in significantly
reducing the mean duration of diarrhea, mean diarrhea
score and the mean composite diarrhea score (GLM,
P  0.0001, 0.0020 and 0.0020, respectively) (Fig. 2), but
the difference in shedding of the challenge ETEC was not
significant (63.45% control group and 37.8% treated group,
139
Compositeb diarrhea score % of total E. coli
that was JG280
9 100
18 100
4 44.2
3 42.4
9 100
8.6 77.32
P value = 0.14). Phages were detected in the feces of all the
pigs that had received the phage mixture. The phages were
detected for 1–6 days in most pigs at titres of 104–
1011 PFU/g, with the highest mean titres on days 2 and 3.
3.4. Therapeutic efficacy of a mixture of two phages
(Trials 4 and 5)
In trial 4, all 10 pigs developed diarrhea by 24 h after
challenge and were divided into two groups of five pigs each.
In trial 5, all nine pigs in the control group and seven of the
eight pigs in the phage treatment group developed diarrhea
at 24 h post-ETEC challenge. The one pig which did not
develop diarrhea by 24 h, or at any time, gave negative
results in the enterocyte adhesion test, and was excluded
from the analysis. The severity of diarrhea in pigs assigned to
the treated and untreated groups, based on their diarrhea
scores on day 1, 24 h after challenge but before treatment,
was not significantly different in either trial (ANOVA,
P = 0.74) Analysis of the combined data for trials 4 and 5
(Fig. 3) showed that pigs given the three treatments with the
mixture of phages at 6 h intervals beginning 24 h after
challenge had significantly improved mean weight change,
mean duration of diarrhea, mean diarrhea score, and mean
composite diarrhea score (GLM, P < 0.0001). Also, the
average percentage of the challenge ETEC JG280 shed in
Fig. 2. Effect of prophylactic treatment with a combination of phages GJ1,
GJ2 and GJ7 on diarrheal disease in weaned pigs after oral challenge with
O149 ETEC strain JG280 (Trial 3). The phages were given orally 15 min
after challenge. Bars show group mean values for the designated
parameters over 6 days in 10 untreated control pigs (black bars) and
10 treated pigs (striped bars). See text for description of diarrhea scores.
Bars = +1 S.E. Asterisks indicate parameters with values in treated pigs
significantly different from those of the control group.
 Author's personal copy
140 N. Jamalludeen et al. / Veterinary Microbiology 136 (2009) 135–141
Fig. 3. Effect of therapeutic use of a combination of phages GJ1 and GJ6 on
diarrheal disease in weaned pigs following the onset of diarrhea after oral
challenge with O149 ETEC strain JG280 (Trials 4 and 5). The phages were
administered orally on day 1, 24 h after challenge. Bars show group mean
values for the designated parameters over 5 days after the onset of
diarrhea in 14 untreated control pigs (black bars) and 12 treated pigs
(white bars). See text for description of diarrhea scores. Bars = +1 S.E.
Asterisks indicate parameters with values in treated pigs significantly
different from those of the control group.
Fig. 4. Daily mean concentrations of total E.coli in the feces of 14
untreated pigs (black bars) and 12 pigs treated therapeutically with a
mixture of phages GJ1 and GJ6 (white bars) following oral challenge with
O149 ETEC strain JG280 (Trials 4 and 5). The phages were administered
orally on day 1, 24 h after challenge and after the onset of diarrhea.
Bar = +1 S.E.
feces over days 2–6 was significantly lower (ANOVA,
P < 0.0001) in the treated pigs (mean = 20.97  2.34%) than
in the controls (mean = 79.81  7.32). Mean total fecal E. coli
counts of the treated and control groups were very similar on
day 0, and increased similarly in both groups on day 1, 24 h
after challenge (Fig. 4). Thereafter, the total E. coli counts of the
treated pigs fell to day 0 levels by days 3, 4, 5 and 6, but in the
untreated control pigs remained significantly higher than day
0 levels (ANOVA, P = 0.35, 0.49, 0.14 and 0.12 for the treated
group and <0.0001 for the untreated group, respectively). The
phages were detected in the feces of the treated pigs for 1–5
days, at progressively declining mean titres ranging from
1011 PFU/g on day 2 to 103 PFU/g on day 5 (Fig. 5).
4. Discussion
This is the first report on the use of phages for the
prevention and treatment of experimental E. coli-induced
diarrhea in weaned pigs, although similar studies have
been reported for neonatal pigs (Smith and Huggins, 1983;
Fig. 5. Daily mean concentrations of O149 phages in the feces of 12 pigs
treated therapeutically with a mixture of phages GJ1 and GJ6 (black bars)
following oral challenge with O149 ETEC strain JG280 (Trials 4 and 5). The
phages were administered orally on day 1, 24 h after challenge and after
the onset of diarrhea. Bar = +1 S.E.
Smith et al., 1987a). The successes reported in the present
study were similar to those reported previously for
neonatal pigs. Because both the challenge ETEC and the
phages may be susceptible to low pH in the stomach and
upper small intestine, the model of experimental ETEC
diarrhea was modified by the pre-challenge oral admin-
istration of sodium bicarbonate. We also treated the pigs
with florfenicol prior to the challenge in an attempt to
reduce competing intestinal flora and enhance the chances
for the challenge ETEC to colonize. Cox et al. (1991) and
Van der Stede et al. (2003) have used this protocol in
several studies of experimental ETEC diarrhea in weaned
pigs. With this protocol, all six phages were detected in the
feces of pigs that had received them. Administration of the
phages shortly after feeding also is another strategy for
protecting them from exposure to low pH in the stomach
(Brussow, 2005).
The combination of phages tested for prophylaxis
included GJ1 and GJ2, selected based on their performance
against O149:H10 ETEC, and GJ7, which was active
primarily against O149:H43 ETEC (Jamalludeen et al.,
2007). The latter was expected to have only a moderate
effect on the O149:H10 challenge ETEC, but was included
as it may be valuable in field tests in which some
O149:H43 ETEC are expected to be present. Using the
modified protocol, the cocktail of phages proved to be
effective. However, although phages were detected in the
feces for 6 days, at titres as high as l011 PFU/g on days 1 and
2, reflecting replication of the phages, the results were not
as impressive as those with the individual phages. In part
this perhaps reflects the lower phage dose used in Trial 3
(109 PFU of each phage), compared to Trial 1 (1010 PFU),
and greater survival of the challenge strain with the
modified protocol. The advantage in using more than one
phage is that if resistant mutants arise to one phage other
phages to which the strain is susceptible would be
available. This approach was successfully used with ETEC
diarrhea in calves (Smith et al., 1987a), Salmonella
Enteritidis PT4 in broilers’ caeca (Fiorentin et al., 2005),
and Salmonella from poultry carcass rinses (Higgins et al.,
2005).
 Author's personal copy
N. Jamalludeen et al. / Veterinary Microbiology 136 (2009) 135–141
A mixture of phages GJ1 and GJ6 was selected to
determine effectiveness in a therapeutic mode. Previous
studies (Jamalludeen et al., 2007) showed that an
O149:H10:F4 ETEC mutant resistant to phage GJ1 was
still susceptible to phage GJ6 and a mutant resistant to
phage GJ6 was still susceptible to phage GJ1. The findings
that there were significant differences in diarrhea, weight
change, and excretion of the challenge ETEC in phage-
treated pigs compared with the control pigs indicate that
this combination of phages is suitable for further tests to
optimize doses, preservation and storage, and evaluate
efficacy in field studies.
After the onset of diarrhea the opportunity for phage to
contact ETEC may be more limited, as most ETEC will be
associated with the epithelial lining of the small intestine
(Moon et al., 1977; Kasman, 2005) and rapid excretion of
the fluid intestinal contents may promote removal of the
phage particles. For the therapeutic application, the phages
were therefore administered in three doses over an 18-h
period to reduce the chances that the entire phage
inoculum would be rapidly washed through the intestine.
The dose of phage was reduced compared to the dose of
approximately 109 PFU that was used in the prophylactic
mode because the expectation was that during ETEC
diarrhea there would be a heavy growth of the challenge E.
coli strain in the intestine of the pigs, and this would permit
considerable phage replication. The data on monitoring of
total E. coli in the feces are interpreted to mean that phage-
treatment had a minimal effect on the total E. coli fecal
flora. Similarly, there are reports that there was no decline
in commensal E. coli gut biota in mice that were orally
administered a four-phage cocktail (Chibani-Chennoufi
et al., 2004) or in human volunteers orally exposed to
phage T4 (Bruttin and Brussow, 2005).
In conclusion, all six phages that were tested indivi-
dually showed significant prophylactic activity against
diarrhea and shedding of the challenge ETEC following
experimental oral infection of pigs with an O149:H10:F4
ETEC. A combination of three phages also moderated the
course of diarrhea. In the therapeutic mode, a combination
of two phages significantly reduced the development of
diarrhea and the number of challenge bacteria in feces
without an apparent reduction in the normal E. coli flora.
Acknowledgements
This work was supported by the NSERC Canadian
Research Network on Bacterial Pathogens of Swine and the
Ontario Ministry of Agriculture, Food and Rural Affairs. We
are grateful to William Sears for assistance in statistical
analysis and to staff at the University Arkell swine facility
and the Animal Isolation Unit for invaluable assistance.
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