DOI: 10.1111/jpn.

12513

ORIGINAL ARTICLE

Effects of dietary supplementation of bacteriophages against

enterotoxigenic Escherichia coli (ETEC) K88 on clinical symptoms
of post-weaning pigs challenged with the ETEC pathogen
C. Y. Lee1,*, S. J. Kim2,*, B. C. Park3 and J. H. Han2
1 Regional Animal Industry Center, Gyeongnam National University of Science and Technology, Jinju, Korea
2 College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon, Korea, and
3 Graduate School of International Agricultural Technology, Seoul National University, Pyeongchang, Korea

Summary
The present study was performed to investigate the effects of dietary supplementation of bacteriophages (phages)
against enterotoxigenic Escherichia coli (ETEC) K88 as a therapy against the ETEC infection in post-weaning pigs.
Two groups of post-weaning pigs aged 35 days, eight animals per group, were challenged with 3.0 9 1010 colony
forming units of ETEC K88, a third group given the vehicle. The unchallenged group and one challenged group
were fed a basal nursery diet for 14 days while the remaining challenged group was fed the basal diet supple-
mented with 1.0 9 107 plaque forming units of the phage per kg. Average daily gain (ADG), goblet cell density
and villous height:crypt depth (VH:CD) ratio in the intestine were less in the challenged group than in the
unchallenged group within the animals fed the basal diet (p < 0.05); the reverse was true for rectal temperature,
faecal consistency score (FCS), E. coli adhesion score (EAS) in the intestine, serum interleukin-8 (IL-8) and
tumour necrosis factor-a (TNF-a) concentrations and digesta pH in the stomach, caecum and colon. The ETEC
infection symptom within the challenged animals was alleviated by the dietary phage supplementation
(p < 0.05) in ADG, FCS, EAS in the jejunum, serum TNF-a concentration, digesta pH in the colon, goblet cell
density in the ileum and colon and VH:CD ratio in the ileum. Moreover, the infection symptom tended to be alle-
viated (p < 0.10) by the phage supplementation in rectal temperature, EAS in the ileum and caecum, and VH:
CD ratio in the duodenum and jejunum. However, EAS in the colon, digesta pH in the stomach and caecum, and
goblet cell density in the jejunum did not change due to the dietary phage. Overall, results indicate that the
phage therapy is effective for alleviation of acute ETEC K88 infection in post-weaning pigs.
Keywords weaned pig, bacteriophage, dietary supplement, enterotoxigenic Escherichia coli, diarrhoea, intestine

Correspondence J. H. Han, College of Veterinary Medicine and Institute of Veterinary Science, Kangwon National University, Chuncheon 24341,
Korea. Tel: +82 33 250 8657; Fax: +82 33 259 5625; E-mail: hanjh@kangwon.ac.kr

*Equally contributed to this work.

Received: 24 September 2015; accepted: 12 March 2016

ETEC secretes endotoxins causing diarrhoea (Zhang

Introduction
Post-weaning diarrhoea or colibacillosis inflicting a
significant economic damage in swine production is
mostly caused by enterotoxigenic Escherichia coli
(ETEC; Pluske et al., 1997; Kim et al., 2012; Heo
et al., 2013). The ETEC causing porcine colibacillosis
adhere to the intestinal mucosa by binding to ente-
rocyte receptors using a number of types of fim-
briae, of which the F4 (K88) antigen is the most
prevalent serotype in post-weaning pigs (Cox and
Houvenaghel, 1993; Osek, 1999; Zhang et al.,
2007). After colonization at the intestinal mucosa,
et al., 2007; Dubreuil, 2012), impairs the integrity of
the absorptive structure (Nabuurs et al., 1994; Kwon
et al., 2014), and stimulates the immune response
characterized by enhanced secretion of pro-inflamma-
tory cytokines (Fossum, 1998). These pathological
effects of ETEC result in suboptimal growth perfor-
mance of the infected animals.
Colibacillosis can be prevented or treated by antibi-
otics and ‘pharmacological’ zinc oxide (ZnO; 0.2–
0.4%), the effects of which are additive (Hill et al.,
2001; Kim et al., 2012; Heo et al., 2013). However,
use of antibiotics as dietary additives has been banned

88 Journal of Animal Physiology and Animal Nutrition 101 (2017) 88–95 © 2016 Blackwell Verlag GmbH
C. Y. Lee et al.
in the EU since 2006 because of concerns about the
emergence of antibiotic-resistant pathogens within
the animals and humans through prolonged exposure
to the in-feed antibiotics and their residuals in animal
products respectively (Aarestrup, 1999; Endersen
et al., 2014). Furthermore, pharmacological in-feed
ZnO is prohibited in the EU and could be prohibited in
non-European countries as well in the future to limit
the excretion of unabsorbed Zn in faeces that could
potentially result in environmental pollution. It is
thus necessary to find prophylactic agents against col-
ibacillosis that can substitute antibiotics and/or phar-
macological ZnO.
Lytic bacteriophages or simply phages, which
replicate and lyse the bacterial host cell immediately
after infection without entering into the non-lytic
lysogenic cycle, have lately received renewed atten-
tion as alternatives to antibiotics (Joerger, 2003;
Matsuzaki et al., 2005; Johnson et al., 2008). How-
ever, only limited information is available as to the
therapeutic or prophylactic effects for phages in por-
cine colibacillosis, although it has been reported
that in-feed phages alleviated the severity of experi-
mental ETEC diarrhoea in newborn (Smith and
Huggins, 1983) and post-weaning (Jamalludeen
et al., 2009; Cha et al., 2012) piglets and also in
naive piglets (Yoo et al., 2013). The present study
was therefore conducted to investigate the effects of
dietary supplementation of phages against ETEC
K88, the most prevalent pathogen causing porcine
colibacillosis (Cox and Houvenaghel, 1993; Osek,
1999; Zhang et al., 2007).
Materials and methods
Animals
The experimental protocol for the present study was
approved by the Institutional Animal Care and Use
Committee of Kangwon National University. The
phage used in the present study (L86 Phage; Intron,
Sungnam, Korea) was isolated from the pig manure
of a commercial farm on the ETEC K88 ‘bacterial
lawn’ and was subsequently identified as a member
of the Myoviridae family. The isolated phage was
diluted in a modified SM buffer (0.2 М Tris, pH 7.5,
containing 0.1 М NaCl, 1 mМ MgSO4, and 0.01%
gelatin) and spray-dried before used as a dietary
supplement.
Twenty-four (Landrace 9 Yorkshire) 9 Duroc-bred
castrated male piglets, which had been confirmed to
be ETEC K88 antigen-negative as determined by K88
fimbrial gene-specific real-time polymerase chain
reaction (PCR; Kim et al., 2015), were purchased from
Journal of Animal Physiology and Animal Nutrition 101 (2017) 88–95 © 2016 Blackwell Verlag GmbH
Effects of dietary phages in ETEC K88-challenged pigs
a commercial swine farm at weaning at 28 days of
age. The animals were allotted randomly to three
groups in three 1-m2 pens, with eight animals per
group/pen, each of which was equipped with a feeder
and a nipple waterer. Two groups were fed ad libitum
an anti-microbial additive-free basal nursery diet, the
composition of which had been reported previously
(Jang et al., 2014; Kwon et al., 2014); the remaining
group were fed ad libitum the same diet supplemented
with 1.0 9 107 plaque forming units of the ETEC
K88-selective phage per kg. On day 0 of the experi-
ment after 7 days of adaptation to the designated diet,
two groups on the basal diet and the phage-supple-
mented diet, respectively, were challenged orally with
3.0 9 1010 colony forming units of ETEC K88 in 3 ml
of phosphate-buffered saline, with the remaining
eight animals on the basal diet given the same volume
of vehicle. The animals were placed on the feeding
trial for 14 days.
Faecal consistency of each animal was scored
according to a 4-notch scale (Marquardt et al., 1999)
as described previously (Kwon et al., 2014; Kim et al.,
2015): 0, normal; 1, soft faeces; 2, mild diarrhoea; 3,
severe diarrhoea. Rectal temperature was measured at
the time of faecal consistency scoring. Blood was
obtained from the jugular vein on days 0, 2, 7 and 14
post-challenge, and serum was harvested and stored
at 70 °C until used for determination of cytokine
concentrations; faeces was obtained from the rectum
on days 1, 3, 7 and 14 to measure the excretion of
E. coli.
Necropsy
All animals were killed by electric stunning at termi-
nation of the feeding trial, after which the entire gas-
trointestinal (GI) tract was removed as described
previously (Lee et al., 2011). Cross-sectional segments
of the duodenum, jejunum, ileum, colon and caecum
were taken for determination of relative counts of
E. coli in these tissues as well as for histomorphological
examination of their mucosae as described (Kwon
et al., 2014; Park et al., 2015). The pH of the digesta
along the GI tract was measured using the litmus
paper (Thomas Scientific, Swedesboro, NJ, USA).
Scoring of faecal shedding and intestinal adhesion of
E. coli
A 0.2 mg native faeces equivalent of the faeces starter
culture in tryptic soy broth was incubated on the
E. coli-selective eosin methylene blue (EMB) agar at
37 °C for 24 or 72 h as described previously (Kwon
89
Effects of dietary phages in ETEC K88-challenged pigs
et al., 2014). One gram of intestinal tissue was
homogenized in 10 ml of phosphate-buffered saline,
after which 0.2 ml of the homogenate was cultured
on the EMB agar as for the faeces starter culture. The
abundance of E. coli was scored subjectively according
to the relative occupancy of the bacterial colonies on
the agar plate also as described (Heo et al., 2009;
Kwon et al., 2014): 0, no occupancy (no growth) after
72 h; 1, sparse occupancy after 72 h; partial conflu-
ency after 24 h; and 3, confluency after 24 h.
Determination of cytokines
Serum concentration of interleukin-8 (IL-8) was
determined using a porcine IL-8/CSCL8 enzyme-
linked immunosorbent assay (ELISA) kit (R & D Sys-
tem, Minneapolis, MN, USA). Serum tumour necrosis
factor (TNF-a) concentration was determined using a
homologous TNF-a ELISA kit (Pierce Biotechnology,
Rockford, IL, USA).
Histomorphological examination
The intestinal tissue was fixed, embedded, sliced,
mounted on the glass slide and stained as described
previously (Lee et al., 2011; Kwon et al., 2014; Park
et al., 2015). The goblet cell density, villous height
(VH) and crypt depth (CD) were determined micro-
scopically using the Diagnostic Insight visual analysis
program (Olympus, Tokyo, Japan) also as described
(Lee et al., 2011; Kwon et al., 2014).
Statistical analysis
All data were analysed using the GLM procedure
included with SAS/STAST Software for PC (Release 9.2;
SAS Inst., Cary, NC, USA). The GLM model included
the treatment only in all analyses except for those
involving repeated measurements where the day of
the experiment as well as its interaction with the
treatment was added to the model. The effects of the
treatment and the day including its interaction with
the treatment were tested using the piglet and the
day 9 piglet (treatment) as error terms respectively.
Means were separated using the probability difference
(PDIFF) option. The ‘significance’ and ‘tendency’ were
defined as p < 0.05 and 0.05 < p < 0.10 respectively.
Results
Growth performance
Average daily gain (ADG) of the piglets challenged
with the ETEC K88 and fed the basal diet (Chal/Basal
90
C. Y. Lee et al.
group) was much lower (p < 0.05) than that of the
unchallenged animals fed the same diet (Unchal/Basal
group; Table 1). The depressed weight gain of the ani-
mals caused by the challenge was increased (p < 0.05)
by dietary supplementation of the phage (Chal/
+Phage group), but ADG of the Chal/+Phage group
was still less than half that of the Unchal/Basal group.
Clinical measurements
The Unchal/Basal group maintained a steady level of
rectal temperature throughout the 14-day trial,
whereas the Chal/Basal and Chal/+Phage groups
exhibited transiently elevated temperatures
(p < 0.05) on days 1 and 2 and on day 2 post-chal-
lenge, respectively, above those on day 0 (Fig. 1, left).
The mean rectal temperature was greater in the Chal/
Basal group vs. Unchal/Basal group, tending to be less
(p = 0.097) in the Chal/+Phage vs. Chal/Basal group
(38.90, 39.15, and 39.00 °C for the Unchal/Basal,
Chal/Basal and Chal/+Phage groups respectively;
SEM = 0.04 °C).
The faecal consistency score (FCS) did not change
throughout the experiment in the Unchal/Basal
group (Fig. 1, right). The FCS increased above that
of the day-0 level on days 1 through 5 (p < 0.05)
before returning to the day-0 level by day 6 in the
Chal/Basal group, whereas in the Chal/+Phage
group, FCS increased only on day 1 post-challenge
and returned to the day-0 level by day 2. Overall,
mean FCS was greatest in the Chal/Basal group fol-
lowed sequentially by the Chal/+Phage and Unchal/
Basal groups (0.33, 1.25, and 0.79 the Unchal/Basal,
Table 1 Growth performance of post-weaning pigs challenged with
enterotoxigenic Escherichia coli (ETEC) K88: effects of the ETEC K88-spe-
cific bacteriophage
Challenged*
Unchallenged*
Item Basal† Basal† +Phage† SEM p value
Initial BW (kg) 11.38 10.13 10.58 0.46 0.18
Final BW (kg) 16.43a 11.51b 13.19c 0.52 <0.01
Average 361a 98b 186c 21 <0.01
daily gain (g)
Means with no common letter (a–c) within a row differ (p < 0.05).
*Post-weaning pigs aged 35 days received an oral administration of
3.0 9 1010 colony forming units of ETEC K88 or vehicle (Unchallenged)
on day 0 of the experiment.
†The animals were group-fed a nursery diet containing no phage (Basal)
or 1.0 9 107 plaque forming units of the ETEC K88-selective bacterio-
phage per kg diet (+Phage). Data are means of eight animals. Average
daily feed intakes were 841, 745 and 753 g in the Unchallenged/Basal,
Challenged/Basal and Challenged/+Phage groups respectively.
Journal of Animal Physiology and Animal Nutrition 101 (2017) 88–95 © 2016 Blackwell Verlag GmbH
C. Y. Lee et al. Effects of dietary phages in ETEC K88-challenged pigs

Fig. 1 Changes of rectal temperature (left) and faecal consistency score (right) of post-weaning pigs after an oral administration of enterotoxigenic
Escherichia coli (ETEC) K88: effects of the ETEC K88-specific bacteriophage. Thirty-five-day-old post-weaning pigs were challenged with 3.0 9 1010 col-
ony forming units of ETEC K88 or vehicle (unchallenged) on day 0 and fed either a basal diet or the same diet supplemented with 1.0 9 107 plaque
forming units of the ETEC K88-selective bacteriophage per kg diet. Left, the p value for the dietary treatment was <0.01, with SEM = 0.04 °C; the p val-
ues for the day and day 9 treatment were <0.01 and 0.21 respectively. Right, faecal consistency was scored subjectively: 0, normal; 1, soft faeces; 2,
mild diarrhoea; 3, severe diarrhoea. All the p values for the treatment, day and day 9 treatment were <0.01, with the treatment SEM = 0.09.

Chal/Basal and Chal/+Phage groups respectively;
SEM = 0.09).
The Unchal/Basal group excreted a low level of
E. coli throughout the experiment when the faecal
E. coli shedding was subjectively scored following
incubation of the faeces sample on the E. coli-specific
EMB agar plate (Table 2). The faecal shedding score
(FSS) in the Chal/Basal group, which was greater than
that in the Unchal/Basal group on day 1 (p < 0.05),
increased transiently on day 3 (p < 0.05) before
declining to the day-1 level by day 7. The FSS in the
Chal/+Phage group was reduced throughout the
experiment compared with that in the Chal/Basal
group, even though FSS in the former was greater
than that of the Unchal/Basal group on days 3 and 7.
Overall mean FSS was less in the Chal/+Phage group
than in the Chal/Basal group, but it was greater in the
former vs. the Unchal/Basal group.
The E. coli adhesion score (EAS), determined as in
FSS, increased due to the challenge along the jeju-
num, ileum, caecum, and colon in the piglets fed the
basal diet (Table 2). The EAS decreased in the jeju-
num (p < 0.05) and tended to decrease in the ileum
and caecum (p = 0.064 and 0.084 respectively) in
response to the dietary supplementation of the phage.
In the colon, however, EAS was not influenced by
dietary phage supplementation.
The serum IL-8 concentration did not change dur-
ing the experiment in the Unchal/Basal group
(Table 2). In the Chal/Basal and Chal/+Phage groups,
the IL-8 concentration increased to the peak level
between days 0 and 2. The IL-8 concentration
declined between days 2 and 7 and further declined
between days 7 and 14 to the day-0 level in the Chal/
Basal group, whereas in the Chal/+Phage group, it
declined to the day-0 level between days 2 and 7.
Moreover, the IL-8 concentration on day 2, which
was greater in the Chal/Basal and Chal/+Phage groups
than in the Unchal/Basal group, tended to be less
(p < 0.10) in the Chal/+Phage vs. Chal/Basal group,
although IL-8 concentration did not differ among the
three groups on day 14 as well as day 0. Overall mean
IL-8 concentration was greater in the Chal/Basal and
Chal/+Phage groups than in the Unchal/Basal group,
not being different between the former two groups.
The serum TNF-a concentration also did not change
during the experiment in the Unchal/Basal group,
whereas in the Chal/Basal and Chal/+Phage groups, it
increased between days 0 and 2 and declined to the
day-0 level by day 14. The mean TNF-a concentration
was reduced in the Chal/+Phage group compared with
that of the Chal/Basal group although it was greater in
the Chal/+Phage group as well as Chal/Basal group
than in the Unchal/Basal group.
Acidity of the digesta and morphology of the GI tract
The digesta pH value increased due to the challenge in
the stomach, caecum and colon in the animals fed the
basal diet, but in duodenum, jejunum and ileum, this
variable did not change due to the challenge or dietary
phage supplementation (Table 3). The increased
digesta pH value caused by the challenge did not
change in response to the dietary treatment in the
stomach or caecum, whereas in the colon, the digesta
pH value of the Chal/+Phage group decreased to an

Journal of Animal Physiology and Animal Nutrition 101 (2017) 88–95 © 2016 Blackwell Verlag GmbH 91
Effects of dietary phages in ETEC K88-challenged pigs C. Y. Lee et al.

Table 2 Effects of dietary supplementation of bacteriophages against Table 3 Effects of dietary supplementation of the enterotoxigenic
enterotoxigenic Escherichia coli (ETEC) K88 on faecal shedding and Escherichia coli (ETEC) K88-selective bacteriophage on digesta pH, gob-
intestinal adhesion of E. coli and circulating concentrations of pro- let cell density and morphology of the intestinal tract of post-weaning
inflammatory cytokines in post-weaning pigs challenged with the patho- pigs challenged with the pathogen
gen
Challenged*
Challenged* Unchallenged*
Unchallenged* p Item Basal† Basal† +Phage† SEM p value
Item Basal† Basal† +Phage† SEM value
Digesta pH at necropsy
Faecal shedding of E. coli‡ Stomach 2.86b 4.74a 4.41a 0.20 <0.01
Day 1 0.25b,x 1.50a,y 0.63b,y 0.25§ Duodenum 6.15 6.55 6.44 0.16 0.22
Day 3 0.25c,x 2.50a,x 1.50b,x Jejunum 6.15 6.39 6.60 0.18 0.22
c,x a,xy
Day 7 0.13 2.13 1.38b,x Ileum 6.38 6.81 6.79 0.15 0.08
Day 14 0.25b,x 1.75a,y 0.88b,xy Caecum 6.25b 7.58a 7.38a 0.18 <0.01
Overall¶ 0.22c,x 1.97a 1.09b 0.16 <0.01 Colon 6.30c 7.43a 6.91b 0.16 <0.01
Adhesion of E. coli to the intestinal mucosa‡ Goblet cell density (cells/mm2)
Jejunum 0.25b 1.88a 0.75b 0.29 <0.01 Duodenum
Ileum 0 b
1.88 a
1.00a 0.32 <0.01 Villous 350a 173b 219b 20 <0.01
Caecum 0.13 b
1.25 a
0.50ab 0.29 0.04 Crypt 318a 188b 233b 16 <0.01
Colon 0 1.13 0.50 0.32 0.06 Jejunum
Serum interleukin-8 (pg/ml) Villous 283a 161b 198b 16 <0.01
Day 0 56.4a,x 45.6a,z 61.4a,y 14.8§ Crypt 265a 158b 184b 15 <0.01
Day 2 54.3b,x 184.5a,x 145.1a,x Ileum
Day 7 56.4b,x 103.5a,y 82.5ab,y Villous 350a 150c 240b 14 <0.01
Day 14 53.9a,x 76.5a,yz 66.6a,y Crypt 361a 171c 254b 18 <0.01
Overall** 55.2 b,x
102.5 a
88.9a 6.7 <0.01 Colon 741a 319c 463b 29 <0.01
Serum tumour necrosis factor-a (pg/ml) Mucosal morphology
Day 0 58.0a,x 45.8a,z 48.3a,z 12.1§ Duodenum
Day 2 45.3 c,x
177.4 a,x
122.9b,x VH‡ (lm) 359a 295b 320b 11 <0.01
Day 7 54.8b,x 123.4a,y 95.3a,xy CD‡ (lm) 248b 297a 274ab 10 <0.01
Day 14 55.8 a,x
69.1 a,z
63.8a,yz VH:CD 1.46a 1.00b 1.17b 0.07 <0.01
Overall** 53.4c 103.9a 82.5b 7.2 <0.01 Jejunum
VH (lm) 346a 265b 292b 11 <0.01
Means with no common letter (a–c) within a row differ (p < 0.05). CD (lm) 245b 306a 293a 9 <0.01
Means with no common letter (x–z) within a column differ (p < 0.05). VH:CD 1.44a 0.87b 1.01b 0.06 <0.01
*Post-weaning pigs were challenged with 3.0 9 1010 colony forming Ileum
units of ETEC K88 or vehicle (Unchallenged) on day 0 of the experiment. VH (lm) 339a 265c 298b 11 <0.01
†The animals were fed either a basal diet or the same diet supple- CD (lm) 270c 325a 299b 9 <0.01
mented with 1 9 107 plaque forming units of the ETEC K88-selective VH:CD 1.26a 0.82c 1.00b 0.04 <0.01
bacteriophage. Data are means of eight animals.
‡Portions of faeces starter culture and mucosal homogenate corre- Means with no common letter (a–c) within a row differ (p < 0.05).
sponding to 0.2 mg native faeces and 0.18 g tissue, respectively, were *The animals were challenged orally with 3.0 9 1010 colony forming
cultured on the EMB agar plate selective for E. coli at 37 °C. Faecal units of ETEC K88 or vehicle (Unchallenged) on day 0 of the experiment.
shedding and adhesion of E. coli were scored according to the relative †The animals received a nursery diet containing no phage (Basal) or
occupancy of the bacterial colonies on the agar: 0, no occupancy (no 1.0 9 107 plaque forming units of the ETEC K88-selective bacteriophage
growth) after 72 h; 1, sparse occupancy after 72 h; partial confluency per kg diet (+Phage). Data are means of eight animals.
after 24 h; and 3, confluency after 24 h. ‡VH, villous height; CD, crypt depth.
§Applies to all day 9 treatment combinations.
¶The p values for the day and day 9 treatment were 0.02 and 0.38
respectively.
response to the dietary phage supplementation in the
**The p values for the day and day 9 treatment were <0.01 in both
cytokines. crypt of the duodenum (p = 0.058). In the ileal villous
and crypt and the colon, the cell density did increase
owing to the dietary phage to an intermediate level
intermediate level between those for the Unchal/Basal between those for the Unchal/Basal and Chal/Basal
and Chal/Basal groups. groups. However, the cell density was not responsive
The goblet cell density decreased consistently due to to the dietary phage in the villous of the duodenum or
the ETEC K88 challenge in the villous and crypt of the either the villous or crypt of the jejunum.
duodenum, jejunum and ileum as well as in the colon The VH and VH:CD ratio decreased and CD
(Table 3). The goblet cell density tended to increase in increased due to the challenge in the duodenum,

92 Journal of Animal Physiology and Animal Nutrition 101 (2017) 88–95 © 2016 Blackwell Verlag GmbH
C. Y. Lee et al.
jejunum and ileum in the animals fed the basal diet
(Table 3). The VH and CD did not change in response
to the phage supplementation in the duodenum or
jejunum, but the VH:CD ratio tended to increase in
response to the dietary phage in both intestinal seg-
ments (p < 0.10). In the ileum, the VH and VH:CD
ratio increased and CD decreased owing to the dietary
phage to an intermediate level between those for the
Unchal/Basal and Chal/Basal groups.
Discussion
The post-weaning pigs challenged with ETEC K88
exhibited the known infection symptoms including
the increases in rectal temperature, FCS, FSS, EAS
and serum IL-8 and TNF-a concentrations and
decreases in weight gain, goblet cell density and VH:
CD ratio (Chandler and Mynott, 1998; Fossum, 1998;
Bosi et al., 2004; Carroll et al., 2004; Lee et al., 2012;
Kwon et al., 2014). These infection symptoms were
alleviated or at least tended to be alleviated by dietary
supplementation of the phage accompanied by
decreased intestinal and faecal E. coli scores. The ther-
apeutic effects of the present phage in FCS and faecal
E. coli shedding during the initial 3 days post-chal-
lenge were comparable to those in post-weaning pigs
challenged with the O149:H10:F4 serotype of E. coli in
the study of Cha et al. (2012). These effects of the
dietary phage as well as those on weight gain and
integrity of intestinal morphology observed in the pre-
sent study, as a whole, were also comparable to those
of 120 ppm of in-feed apramycin (Kim et al., 2015) or
3125 ppm of ZnO (Kwon et al., 2014) in ETEC K88-
challenged post-weaning pigs. Moreover, the thera-
peutic effects of the dietary phage on weight gain and
FCS in the present study were greater than those in
unchallenged na€ıve piglets reported by Yoo et al.
(2013). However, the therapeutic effects of the pre-
sent phage were less dramatic than those in newborn
piglets challenged with E. coli P433 in Smith and Hug-
gins (1983) where four out of seven untreated piglets
died while the other seven piglets recovered from sev-
ere diarrhoea following oral administration of P433-
specific lytic phages.
It was noticeable that bacterial colonies were
detected in the E. coli-selective EMB agar plates for
the faeces and intestinal tissue homogenates of the
Unchal/Basal group even though only ETEC K88 anti-
gen-negative piglets were selected for the present
experiment. These colonies, however, are likely to
have originated from E. coli strains other than ETEC
K88 for the following reasons. First, E. coli are com-
mensal bacteria of both healthy and diseased pigs
Journal of Animal Physiology and Animal Nutrition 101 (2017) 88–95 © 2016 Blackwell Verlag GmbH
Effects of dietary phages in ETEC K88-challenged pigs
(Osek, 1999; Schierack et al., 2006; Heo et al., 2013).
Secondly, E. coli were detected only at low abundance
in the faeces and tissue homogenates with no tempo-
ral variation in the former in the Unchal/Basal group,
whereas in the challenged animals, high levels of
E. coli were detected in both types of samples with
apparent peaks at d 3 post-challenge in faecal E. coli
shedding. As such, the bacterial colonies on the EMB
agar plates for the challenged animals are likely to
have originated mostly from ETEC K88 which had
been orally administered at the outset of the experi-
ment.
The increased digesta pH value resulting from the
ETEC challenge in the stomach, caecum and colon
was partially consistent with the previous results of
our study where this variable increased due to the
ETEC K88 challenge in the jejunum and ileum in
addition to the stomach, caecum and colon (Kwon
et al., 2014). However, these results are different from
those of Wellock et al. (2008) and Slade et al. (2011)
where the ETEC challenge did not influence the pH
value of the GI tract. More studies are therefore neces-
sary to explain this discrepancy in different studies as
well as biological significance of decreased colon pH
due to the in-feed phage observed in the present
study. An increased pH value in the GI tract, in gen-
eral, is known as a negative indicator of GI well-being
in the pig for the following grounds (Nyachoti et al.,
2006). The increased pH value in the stomach seem-
ingly resulting from decreased gastric secretion is asso-
ciated with increased ETEC proliferation accompanied
by an increased incidence of diarrhoea (Owusu-
Asiedu et al., 2003), although it is unknown how
intestinal ETEC colonization impairs gastric secretion.
Furthermore, increased gut pH is also associated with
proliferation of coliform bacteria (Smith and Jones,
1963; Nagy and Fekete, 1999).
The goblet cell secretes mucins which subserve a
gut barrier function by binding the pathogenic
microbes to prevent them from binding to their ente-
rocyte receptors prior to colonization (Dean-Nystrom
and Samuel, 1994; Willing et al., 2013). The consis-
tent decrease in the goblet cell density in all segments
of the small intestine and colon due to the challenge
thus indicates that the gut barrier function was some-
how damaged by the ETEC infection. Conversely, the
increase or tendency of increase in goblet cell density
in response to dietary supplementation of the phage
in the intestinal tract was apparently associated with
decreased E. coli scores in the intestinal segments. In
line with this, there were significant negative correla-
tions between the goblet cell densities and E. coli
scores in the villous and crypt of the jejunum as well
93
Effects of dietary phages in ETEC K88-challenged pigs C. Y. Lee et al.

as ileum ( 0.59 < r < 0.45; p < 0.05) although the
correlation was not significant in the colon
(r = 0.32; p = 0.13). These results are thus inter-
preted to suggest that the dietary phage used in the
present study may also prevent the loss of goblet cells
by alleviating the ETEC infection, thereby enhancing
the impaired barrier function of the infected animals.
The VH and CD are commonly used indicators of
the morphological integrity and absorptive capacity
of the intestinal brush-border architecture, the VH:
CD ratio being regarded as a better indicator than
either of the two metrics (Pluske et al., 1997; Heo
et al., 2013; Xun et al., 2015). It is also known that
natural or artificial ETEC infection causes an
increased rate of cell loss or renewal of the villous
as well as increased crypt cell production resulting
in decreased VH and increased CD respectively. As
such, the consistently decreased VH and VH:CD
ratio as well as increased CD in the Chal/Basal vs.
Unchal/Basal group in all the examined intestinal
segments indicated that the absorptive architecture
was severely damaged by the artificial ETEC
infection. Likewise, the present phage therapy was
seemingly effective for amelioration of the structural
damage of the absorptive architecture caused by
ETEC infection, which was suggested by the
changes of all the three variables to intermediate
values between those of the Unchal/Basal and
Chal/Basal groups in the ileum, as well as the ten-
dency of increased VH:CD ratio in the duodenum
and ileum, in response to the in-feed phage.
In conclusion, the present results indicate that diet-
ary supplementation of the phage is effective for alle-
viation of colibacillosis due to acute ETEC infection in
post-weaning pigs. More studies are needed, however,
to evaluate the usefulness of the present phage as a
prophylactic feed additive in young pigs not infected
with ETEC.
Acknowledgements
The present study was supported financially by Col-
lege of Veterinary Medicine and Institute of Veteri-
nary Science, Kangwon National University.

References
Aarestrup, F. M., 1999: Association
between the consumption of antimicro-
bial agents in animal husbandry and the
occurrence of resistant bacteria among
food animals. International Journal of
Antimicrobial Agents 12, 279–285.
Bosi, P.; Casini, L.; Finamore, A.; Cremo-
kolini, C.; Merialdi, G.; Trevisi, P.;
Nobili, F.; Mengheri, E., 2004: Spray-
dried plasma improves growth perfor-
mance and reduces inflammatory status
of weaned pigs challenged with entero-
toxigenic Escherichia coli K88. Journal of
Animal Science 82, 1764–1772.
Carroll, J. A.; Fangman, T. J.; Hambach, A.
K.; Wiedmeyer, C. E., 2004: The acute
phase response in pigs experimentally
infected with Escherichia coli and treated
with systemic bactericidal antibiotics.
Livestock Production Science 85, 35–44.
Cha, S. B.; Yoo, A. N.; Lee, W. J.; Shin, M.
K.; Jung, M. H.; Shin, S. W.; Cho, Y. W.;
Yoo, H. S., 2012: Effect of bacteriophage
in enterotoxigenic Escherichia coli
(ETEC) infected pigs. Journal of Veteri-
nary Medical Science 78, 1037–1039.
Chandler, D. S.; Mynott, T. L., 1998:
Bromelain protects piglets from diarrhea
caused by oral challenge with K88 posi-
tive enterotoxigenic Escherichia coli. Gut
43, 196–202.
Cox, E.; Houvenaghel, A., 1993: Com-
parison of the in vitro adhesion of
K88, K99, F41 and P987 positive
Escherichia coli to intestinal villi of 4-
to 5-week-old pigs. Veterinary Microbiol-
ogy 34, 7–19.
Dean-Nystrom, E. A.; Samuel, J. E., 1994:
Age related resistance to 987P fimbria
mediated colonization correlates with
specific glycolipid receptors in intestinal
mucus in swine. Infection and Immunity
62, 4789–4794.
Dubreuil, J. D., 2012: The whole shebang:
the gastrointestinal tract, Escherichia coli
enterotoxins and secretion. Current Issues
in Molecular Biology 14, 71–82.
Endersen, L.; O’Mahony, J.; Hill, C.; Ross,
R. P.; McAuliffe, O.; Coffey, A., 2014:
Phage therapy in the food industry.
Annual Review of Food Science and Technol-
ogy 5, 327–349.
Fossum, C., 1998: Cytokines as markers
for infections and their effect on growth
performance and well-being in the pig.
Domestic Animal Endocrinology 15, 439–
444.
Heo, J. M.; Kim, J. H.; Hansen, C. F.; Mul-
lan, B. P.; Hampson, D. J.; Pluske, J. R.,
2009: Feeding a diet with decreased
potein content reduces indices of pro-
tein fermentation and the incidence of
postweaning diarrhea in weaned pigs
challenged with an enterotoxigenic
strain of Escherichia coli. Journal of Ani-
mal Science 87, 2833–2843.
Heo, J. M.; Opapeju, F. O.; Pluske, J. R.;
Kim, J. C.; Hampson, D. J.; Nyachoti, C.
M., 2013: Gastrointestinal health and
function in weaned pigs: a review of
feeding strategies to control post-wean-
ing diarrhoea without using in-feed
antimicrobial compounds. Journal of
Animal Physiology and Animal Nutrition
97, 207–237.
Hill, G. M.; Mahan, D. C.; Carter, S. D.;
Cromwell, G. L.; Ewan, R. C.; Harrold,
R. L.; Lewis, A. J.; Miller, P. S.; Shurson,
G. C.; Veum, T. L., 2001: Effect of phar-
macological concentrations of zinc oxide
with or without the inclusion of an
antibacterial agent on nursery pig per-
formance. Journal of Animal Science 79,
934–941.
Jamalludeen, N.; Johnson, R. P.; Shewen,
P. E.; Gyles, C. L., 2009: Evaluation of
bacteriophages for prevention and treat-
ment of diarrhea due to experimental
enterotoxigenic Escherichia coli O149
infection of pigs. Veterinary Microbiology
136, 135–141.
Jang, I.; Kwon, C. H.; Ha, D. M.; Jung, D.
Y.; Kang, S. Y.; Park, M. J.; Han, J. H.;
Park, B.-C.; Lee, C. Y., 2014: Effects of a
lipid-encapsulated zinc oxide supplement
on growth performance and intestinal
morphology and digestive enzyme

94 Journal of Animal Physiology and Animal Nutrition 101 (2017) 88–95 © 2016 Blackwell Verlag GmbH
C. Y. Lee et al.
activities in weanling pigs. Journal of Ani-
mal Science and Technology 56, 29.
Joerger, R. D., 2003: Alternatives to
antibiotics: bacteriocins, antimicrobial
peptides and bacteriophages. Poultry
Science 82, 640–647.
Johnson, R. P.; Gyles, C. L.; Huff, W. E.;
Ojha, S.; Huff, G. R.; Rath, N. C.; Dono-
ghue, A. M., 2008: Bacteriophages for
prophylaxis and therapy in cattle, poul-
try and pigs. Animal Health Research
Reviews 9, 201–215.
Kim, J. C.; Hansen, C. F.; Mullan, B. P.;
Pluske, J. R., 2012: Nutrition and
pathology of weaner pigs: nutritional
strategies to support barrier function in
the gastrointestinal tract. Animal Feed
Science and Technology 173, 3–16.
Kim, S. J.; Kwon, C. H.; Park, B. C.; Lee,
C. Y.; Han, J. H., 2015: Effects of a lipid-
encapsulated zinc oxide dietary supple-
ment, on growth parameters and intesti-
nal morphology in weanling pigs
artificially infected with enterotoxigenic
Escherichia coli. Journal of Animal Science
and Technology 57, 4.
Kwon, C. H.; Lee, C. Y.; Han, S. Y.; Kim, S.
J.; Park, B. C.; Jang, I.; Han, J. H., 2014:
Effects of dietary supplementation of
lipid-encapsulated zinc oxide on col-
ibacillosis, growth and intestinal mor-
phology in weaned piglets challenged
with enterotoxigenic Escherichia coli.
Animal Science Journal 85, 805–813.
Lee, C. Y.; Lim, J.-W.; Ko, Y. H.; Kang, S.
Y.; Park, M. J.; Ko, T.; Lee, J. H.; Hyun,
Y.; Jeong, K. S.; Jang, I. S., 2011: Intesti-
nal growth and development of wean-
ling pigs in response to dietary
supplementation of antibiotics, phyto-
genic products and brewer’s yeast plus
Bacillus spores. Journal of Animal Science
and Technology 53, 227–235.
Lee, J. S.; Awji, E. G.; Lee, S. J.; Tassew, D.
D.; Park, Y. B.; Park, K. S.; Kim, M. K.;
Kim, B.; Park, S. C., 2012: Effect of Lac-
tobacillus plantarum CJLP243 on the
growth performance and cytokine
response of weanling pigs challenged
with enterotoxigenic Esherichia coli.
Journal of Animal Science 90, 3709–3717.
Marquardt, R. R.; Jin, L. Z.; Kim, J.-W.;
Fang, L.; Frohlich, A. A.; Baidoo, S. K.,
1999: Passive protective effect of egg-
yolk antibodies against enterotoxigenic
Journal of Animal Physiology and Animal Nutrition 101 (2017) 88–95 © 2016 Blackwell Verlag GmbH
Effects of dietary phages in ETEC K88-challenged pigs
Escherichia coli K88+ infection in neona-
tal and and early-weaned piglets. FEMS
Immunology and Medical Microbiology 23,
283–288.
Matsuzaki, S.; Rashel, M.; Uchiyama, J.;
Sakura, S.; Ujihara, T.; Kuroda, M.;
Ikeuchi, M.; Tani, T.; Fujieda, M.; Waki-
guchi, H.; Imai, S., 2005: Bacteriophage
therapy: a revitalized therapy against
bacterial infectious diseases. Journal of
Chemotherapy 11, 211–219.
Nabuurs, M. J. A.; Hoogendoom, A.; van
Zijderveld, F. G., 1994: Effects of wean-
ing and enterotoxigenic Escherichia coli
on net absorption in the small intestine
of pigs. Research in Veterinary Science 56,
379–385.
Nagy, B.; Fekete, P. Z., 1999: Enterotoxi-
genic E. coli (ETEC) in farm animals.
Veterinary Research 30, 259–284.
Nyachoti, C. M.; Omogbenigun, F. O.;
Rademacher, M.; Blank, G., 2006: Per-
formance responses and indicators of
gastrointestinal health in early-weaned
pigs fed low-protein amino acid-supple-
mented diets. Journal of Animal Science
84, 125–134.
Osek, J., 1999: Prevalence of virulence
factors of Escherichia coli strains isolated
from diarrheic and healthy piglets after
weaning. Veterinary Microbiology 68,
209–217.
Owusu-Asiedu, A.; Nyachoti, C. M.; Mar-
quardt, R. R., 2003: Response of early-
weaned pigs to an enterotoxigenic
Escherichia coli (K88) challenge when fed
diets containing spray-dried porcine
plasma or pea protein isolate plus egg
yolk antibody, zinc oxide, fumaric acid,
or antibiotic. Journal of Animal Science
81, 1790–1798.
Park, B. C.; Jung, D. Y.; Kang, S. Y.; Ko, Y.
H.; Ha, D. M.; Kwon, C. H.; Park, M. J.;
Han, J. H.; Jang, I.; Lee, C. Y., 2015:
Effects of dietary supplementation of a
zinc oxide product encapsulated with
lipid on growth performance, intestinal
morphology, and digestive enzyme
activities in weanling pigs. Animal Feed
Science and Technology 200, 112–117.
Pluske, J. R.; Hampson, D. J.; Williams, I.
H., 1997: Factors influencing the struc-
ture and function of the small intestine
in the weaned pigs: a review. Livestock
Production Science 51, 215–236.
Schierack, P.; Steinruck, H.; Kleta, S.; Vah-
jen, W., 2006: Virulence factor gene fro-
files of Escherichia coli isolates from
clinically healthy pigs. Applied and Envi-
ronmental Microbiology 72, 6680–6686.
Slade, R. D.; Kyriazakis, I.; Carroll, S. M.;
Reynolds, F. H.; Wellock, I. J.; Broom,
L. J.; Miller, H. M., 2011: Effect of rear-
ing environment and dietary zinc oxide
on the response of group-housed
weaned pigs to enterotoxigenic Escheri-
chia coli O149 challenge. Animal 5,
1170–1178.
Smith, H. W.; Huggins, M. B., 1983: Effec-
tiveness of phages in treating experi-
mental Escherichia coli diarrhoea in
calves, piglets and lambs. Journal of Gen-
eral Microbiology 129, 2659–2675.
Smith, H. W.; Jones, J. E. T., 1963: Obser-
vations of the alimentary tract and its
bacterial flora in healthy and diseased
pigs. Journal of Pathology and Bacteriology
86, 387–412.
Wellock, I. J.; Fortomaris, P. D.; Houdijk,
J. G. M.; Kyriazakis, I., 2008: Effects of
dietary protein supply, weaning age and
experimental enterotoxigenic Escherichia
coli infection on newly weaned pigs:
health. Animal 2, 834–842.
Willing, B. P.; Malik, G.; Van Kessel, A. G.,
2013: Nutrition and gut health. In: L. I.
Chiba (ed.), Sustainable Swine Nutrition.
John Wiley & Sons, Oxford, pp. 197–
213.
Xun, W.; Shi, L.; Zhou, H.; Hou, G.; Cao,
T.; Zhao, C., 2015: Effects of curcumin
on growth performance, jejunal muco-
sal membrane integrity, morphology
and immune status in weaned piglets
challenged with enterotoxigenic Escheri-
chia coli. International Immunopharmacol-
ogy 27, 46–52.
Yoo, A.; Cha, S. B.; Shin, M. K.; Park, H.
T.; Seo, H. S.; Kim, J. W.; Yoo, H. S.,
2013: Field evaluation of enterotoxi-
genic Escherichia coli-specific bacterio-
phage (ФCJ19) as a feed additive.
Korean Journal of Veterinary Research 53,
83–88.
Zhang, W.; Zhao, M.; Ruesch, L.; Omot,
A.; Francis, D., 2007: Prevalence of viru-
lence genes in Escherichia coli strains
recently isolated from young pigs with
diarrhea in the US. Veterinary Microbiol-
ogy 123, 145–152.
95