IMMUNOLOGY, HEALTH, AND DISEASE
Efficacy of a bacteriophage isolated from chickens as a therapeutic agent
for colibacillosis in broiler chickens
G. L. Lau,* C. C. Sieo,*†1 W. S. Tan,*† M. Hair-Bejo,‡ A. Jalila,§ and Y. W. Ho†
*Department of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, †Institute of Bioscience,
‡Department of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine,
and §Department of Veterinary Clinical Studies, Faculty of Veterinary Medicine,
Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia
ABSTRACT The efficacy of bacteriophage EC1, a lytic
bacteriophage, against Escherichia coli O78:K80, which
causes colibacillosis in poultry, was determined in the
present study. A total of 480 one-day-old birds were
randomly assigned to 4 treatments groups, each with 4
pens of 30 birds. Birds from the control groups (groups
I and II) received PBS (pH 7.4) or 1010 pfu of bacteriophage EC1, respectively. Group III consisted of birds
challenged with 108 cfu of E. coli O78:K80 and treated
with 1010 pfu of bacteriophage EC1 at 2 h postinfection, whereas birds from group IV were challenged with
108 cfu of E. coli O78:K80 only. All the materials were
introduced into the birds by intratracheal inoculation.
Based on the results of the present study, the infection was found to be less severe in the treated E. colichallenged group. Mean total viable cell counts of E.
coli identified on eosin methylene blue agar (designated
EMB + E. coli) in the lungs were significantly lower in
treated, E. coli-challenged birds than in untreated, E.
coli-challenged birds on d 1 and 2 postinfection. The
EMB + E. coli isolation frequency was also lower in
treated birds; no E. coli was detectable in blood samples on any sampling day, and E. coli were isolated only
in the liver, heart, and spleen of treated chickens at a
ratio of 2/6, 1/6, and 3/6, respectively, at d 1 postinfection. The BW of birds from the E. coli-challenged group
treated with bacteriophage EC1 were not significantly
different from those of birds from both control groups
but were 15.4% higher than those of the untreated, E.
coli-challenged group on d 21 postinfection. The total
mortality rate of birds during the 3-wk experimental
period decreased from 83.3% in the untreated, E. colichallenged birds (group IV) to 13.3% in birds treated
with bacteriophage EC1 (group III). These results suggest that bacteriophage EC1 is effective in vivo and
could be used to treat colibacillosis in chickens.
Key words: bacteriophage, Escherichia coli, colibacillosis, broiler
2010 Poultry Science 89:2589–2596
doi:10.3382/ps.2010-00904
INTRODUCTION
Escherichia coli is commonly found in the avian gastrointestinal tract and other mucosal surfaces. Although
most of the strains are commensals, a separate group,
designated avian pathogenic E. coli, has the ability to
cause extraintestinal disease in poultry, collectively
called colibacillosis (Kariyawasam et al., 2006; Bonnet
et al., 2009). Serotypes O1, O2, and O78, and to some
extent O15 and O55, are the most common serotypes
associated with colibacillosis found in chickens (Gomis
et al., 1997; Raji et al., 2007). They commonly cause
airsacculitis, pericarditis, perihepatitis, peritonitis, salpingitis, and subsequently the most acute form, septi©2010 Poultry Science Association Inc.
Received May 20, 2010.
Accepted August 26, 2010.
1 Corresponding author: [email protected]
cemia, resulting in sudden death (Mellata et al., 2003;
Ask et al., 2006). The poultry industries worldwide suffer great financial losses every year because of the high
morbidity and mortality rates caused by colibacillosis.
Treatment strategies include the control of environmental factors and the use of antibiotics. However, concerns
exist regarding the emergence of antibiotic resistance
of normal microflora and pathogenic bacteria, which
may in turn threaten human health through transfer
of drug resistance genes to zoonotic bacteria (Food and
Agriculture Organization of the United Nations, World
Health Organization, and World Organization for Animal Health, 2003).
Bacteriophage therapy is one of the emerging methods used to overcome bacterial infections without creating antibiotic resistance. Lytic bacteriophages are nonhazardous viruses that can self-replicate within a host
and increase in number as they destroy the targeted
bacteria (Jamalludeen et al., 2009). The self-replicating
2589
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Lau et al.
and self-limiting nature of lytic bacteriophages makes
them a safe and attractive alternative to antibiotics for
the prevention and treatment of animal diseases (Huff
et al., 2006b). A single administration of bacteriophages may be enough for treatment, as opposed to other
antimicrobial agents, which may require several doses
per day for complete elimination of pathogens. Moreover, application of a bacteriophage is an easy process
because it can be delivered in food or drink, or even
topically (Clark and March, 2006). Although bacteriophages are ubiquitous in the environment (Goodridge
and Abedon, 2003), not all of them are suitable for
the in vivo prevention and treatment of diseases. The
inability of endogenous bacteriophages to control bacterial infection may be due to alterations in bacteriophage functions during infection caused by factors such
as an elevated body temperature (Smith et al., 1987),
an acidic environment in the gastrointestinal tract (Joerger, 2003), and the presence of naturally occurring
antibacteriophage antibodies (Merril et al., 1996). In
addition, inappropriate dosage of a bacteriophage, that
is, application of a phage at a high multiplication of
infection, may result in a phenomenon known as “lysis
from without.” In this condition, the bacteria are attacked by a very large number of phages, resulting in
premature lysis of the host bacteria without free phage
progeny being liberated for the next cycle of infection.
Lysis of the bacteria under such conditions could be
due to damage of the host cell membrane by the phage
rather than by the infection (Bach et al., 2003). Thus,
in vivo study of isolated bacteriophages is essential before the efficacy of the phage can be determined. In
this study, we isolated a lytic bacteriophage, EC1, from
chicken fecal samples for the treatment of colibacillosis
in broiler chickens caused by E. coli O78:K80. Although
many bacteriophages have been isolated for various infectious agents, very few in vivo applications of bacteriophages against colibacillosis have been reported. In
this study, the ability of bacteriophage EC1 to overcome an E. coli O78:K80 infection was evaluated by the
gross lesions present, the bacteria isolated, body and
organ weights, and the mortality rate of birds.
MATERIALS AND METHODS
Screening, Isolation, and Amplification
of Bacteriophage
The avian pathogenic E. coli O78:K80 strain, which
was supplied by the Veterinary Research Institute
(Ipoh, Malaysia), was used in this study. This isolate,
obtained from chickens at a local farm that presented
signs of colibacillosis, is a virulent strain causing high
mortality in chickens. A bacteriophage (designated
EC1) that produces a clear plaque was selected for
this study. It was isolated from chicken fecal samples
obtained from poultry farms by using the soft agar
overlay method (Adams, 1959). Amplification of bac-
teriophage EC1 was carried out by infecting 500 mL of
early log-phase E. coli O78:K80 (optical density at 600
nm of approximately 0.6) with the bacteriophage at a
multiplication of infection = 1. After 3 h of incubation
at 37°C and shaking at 180 rpm, the lysate was treated
with 0.2 μg/mL of DNase I (Vivantis Inc., Oceanside,
CA) and incubated for an additional 15 min under the
same conditions. Sodium chloride (12.5 g) was then
added, followed by incubation on ice for 1 h. After centrifugation of the lysate at 11,000 × g for 10 min at
4°C, the bacteriophage particles were precipitated from
the supernatant with 10% (wt/vol) polyethylene glycol
(PEG) 8000 (Sigma-Aldrich Co., Steinheim, Germany)
and incubated overnight at 4°C. Centrifugation was carried out again at 11,000 × g for 10 min at 4°C, and the
pellets were drained by inverting the bottles on paper
towels for several minutes. The pellets were then resuspended in 10 mL of sterile 10% (wt/vol) PEG 8000 solution (10 g of PEG 8000, 100 mL of 10 mM Tris-HCl,
pH 8.0, 1 mM EDTA) and the mixture was centrifuged
at 7,500 × g for 10 min at 4°C. The bacteriophages were
extracted from the precipitate by adding 2.5 mL of 1 M
NaCl/TE (1 M NaCl, 1 mM EDTA, 10 mM Tris-HCl;
pH 8.0) and centrifuged again at 7,500 × g for 10 min at
4°C. The supernatant that contained the bacteriophage
was purified by overlaying it on a CsCl (Ameresco Co.,
Solon, OH) step gradient in UltraClear tubes (Beckman
Coulter Inc., Fullerton, CA) and then centrifuging at
210,000 × g for 1 h at 10°C using a Beckman Optima
Max Ultracentrifuge (Beckman Instruments, Fullerton,
CA). The purified bacteriophages were dialyzed against
100 vol of dialysis buffer (0.1 M NaCl, 0.1 M Tris-HCl;
pH 8.0) for 1 h with a 10,000-Da pore size membrane.
Bacteriophage titer was determined using the soft agar
overlay method (Adams, 1959).
Experimental Animals and Treatments
A 3-wk experiment was conducted to determine the
efficacy of bacteriophage EC1 in treating respiratory
infection in birds caused by E. coli O78:K80. A total
of 480 one-day-old male broiler chicks (Ross 308) were
obtained from a commercial hatchery. The chicks were
assigned randomly to 4 treatment groups, each with 4
pens of 30 chicks per pen. Water and broiler feed (antibiotic free) were provided ad libitum throughout the experimental period. The 4 treatment groups were group
I (control), in which untreated, unchallenged birds were
administered 0.2 mL of PBS only (0.14 M NaCl, 0.0027
M KCl, 0.01 M Na2HPO4, 0.0018 M KH2PO4; pH 7.4);
group II (control), in which unchallenged birds were
treated with 0.2 mL of bacteriophage EC1 (1011 pfu/
mL); group III, in which birds were challenged with
0.2 mL of a 5-h-old E. coli O78:K80 culture (grown in
Luria-Bertani broth at 37°C and shaken at 180 rpm)
containing 109 cfu of bacterial cells/mL, followed by
0.2 mL of bacteriophage EC1 (1011 pfu/mL) at 2 h
postchallenge; and group IV, in which birds were chal-
BACTERIOPHAGE THERAPY AGAINST COLIBACILLOSIS
lenged with 0.2 mL of a 5-h-old E. coli O78:K80 culture containing 109 cfu of bacteria cells/mL only. The
time point at which to inoculate the bacteriophage (2
h postchallenge) was selected based on the results of
a preliminary trial showing that E. coli O78:K80 had
colonized the lungs and that the bacteria had spread
to other organs, such as the liver and heart, 2 h after
the birds were challenged with the pathogen (data not
shown). All the materials were inoculated directly into
the trachea of the 1-d-old chicks by using a feeding
needle in a farm setting.
The BW of live birds were taken weekly. Sampling
was carried out on d 0 (before inoculation of E. coli or
bacteriophage EC1), 1, 2, 3, 7, 14, and 21 from 3 of the
pens of each treatment group. The last pen was used
for the observation of mortality rate. On each sampling
day, 6 birds from each group (2 randomly selected from
each of the 3 sampling pens) were weighed and killed by
CO2 inhalation for laboratory examination. Birds that
died on the sampling day were also dissected and subjected to the same laboratory examinations. All animal
management and sampling procedures complied with
the guidelines of the Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching (Federation of Animal Science Societies, 1999).
Laboratory Examinations
Gross Lesion Examinations. Macroscopic examinations of the air sac, liver, and heart of slaughtered birds
were carried out. Opacity or thickening of the air sac
and the presence of tissue lesions or fibrinous exudates
on the liver and heart were considered indicative of airsacculitis, perihepatitis, and pericarditis, respectively.
Organ Weight. At necropsy, the lung, liver, heart,
and spleen were excised aseptically and weighed. The
weights of the organs were reported as the percentage
relative to BW (organ weight/BW × 100%; Huff et al.,
2006a).
Isolation of E. coli from Lungs (Quantitative
Analysis). The lungs of birds were removed aseptically, weighed, diluted 10× in Maximum Recovery Diluent (Merck KGaA, Darmstadt, Germany), and homogenized. The homogenates were then serially diluted
before plating on eosin methylene blue (EMB) agar
(Merck KGaA). The EMB agar plates were incubated
overnight at 37°C, after which the metallic green sheen
colonies of E. coli (designated EMB + E. coli) were
counted to determine the number of E. coli (cfu/g) colonizing the lungs. The populations of EMB + E. coli
in lung samples from birds in the different treatment
groups were then compared to determine the severity
of infection.
Isolation of E. coli from Organs and Blood. Blood
samples of birds were collected by cardiac puncture and
cultured on EMB agar. The liver, heart, and spleen of
each bird were cut open, and the inner parts of these
organs were swabbed 3 to 4 times with sterile cotton
2591
buds and plated directly on EMB agar. The plates were
then incubated at 37°C for 16 to 18 h, and the presence of E. coli colonies (designated EMB + E. coli) was
determined.
Statistical Analysis
The data were analyzed using 1-way ANOVA, followed by Duncan’s multiple range test. Fisher’s exact
tests were performed to determine significant differences between the untreated and treated E. coli-challenged
groups for isolation of EMB + E. coli from different
organs and the presence of gross lesions. A chi-squared
test was used to analyze the effect of bacteriophage
EC1 on the mortality of birds. All analyses were performed using SPSS software for Windows version 13
(SPSS Inc., Chicago, IL). A P-value of <0.05 was considered statistically significant.
RESULTS
Laboratory Examination
Gross Lesions. Intratracheal inoculation of E. coli
caused airsacculitis within 24 h postinfection (groups
III and IV) (Table 1). In the untreated, E. coli-challenged group (group IV), the condition of airsacculitis
was more severe than in the treated, E. coli-challenged
group (group III). In these birds, thickened air sacs and
cloudy membranes were observed. Fibrinous to caseous
yellow exudate was also seen in the air sac (data not
shown). At d 21 postinfection, airsacculitis was not observed in treated, E. coli-challenged birds (group III).
Pericarditis and perihepatitis were found only in birds
with severe airsacculitis, and the incidence was higher
in untreated, E. coli-challenged birds. In treated, E.
coli-challenged birds, pericarditis occurred only at d 2
to 3 postinfection in 3/6 and 1/6 of the birds, respectively. Perihepatitis was not observed in this group of
birds throughout the experimental period. In the untreated, E. coli-challenged group (group IV), pericarditis could still be detected in the birds at d 21 (3/6 of
the birds), and the number of birds with perihepatitis
was significantly higher than the number of birds in
group III at d 2 postinfection. Birds that died during
the experimental period presented similar pathological
lesions, with 100% showing airsacculitis. High incidences of pericarditis and perihepatitis were observed from
d 2 postinfection (Table 1).
Organ Weight. The relative organ weights of birds
are shown in Table 2. In comparison with the control
groups (groups I and II), enlargement of hearts of untreated, E. coli-challenged birds was observed throughout the experimental period beginning from d 2 postinfection. The lungs and livers of these birds were also
enlarged at d 2, 3, and 21 postinfection. Significantly
enlarged spleens of these birds were observed at d 2, 3,
and 7 postinfection.
2592
Lau et al.
Table 1. Effect of bacteriophage EC1 on airsacculitis, pericarditis, and perihepatitis of infected
birds
Time after inoculation of bacteriophage (d)
Item1
Airsacculitis2
(n birds positive/total)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
Pericarditis2 (n birds positive/total)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
Perihepatitis2 (n birds positive/total)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
0
1
2
3
7
14
21
0/6
0/6
0/6
0/6
—
—
0/6
0/6
4/6
5/6
4/4
6/6
0/6
0/6
6/6
6/6
4/4
5/5
0/6
0/6
6/6
4/6
2/2
5/5
0/6
0/6
3/6
3/6
—
1/1
0/6
0/6
2/6
3/6
1/1
—
0/6
0/6
0/6
3/6
—
—
0/6
0/6
0/6
0/6
—
—
0/6
0/6
0/6
1/6
1/4
3/6
0/6
0/6
3/6
5/6
2/4
4/5
0/6
0/6
1/6
3/6
2/2
5/5
0/6
0/6
0/6
3/6
—
1/1
0/6
0/6
0/6
2/6
1/1
—
0/6
0/6
0/6
3/6
—
—
0/6
0/6
0/6
0/6
—
—
0/6
0/6
0/6
0/6
0/4
1/6
0/6
0/6
0/6
4/6*
1/4
4/5
0/6
0/6
0/6
2/6
2/2
3/5
0/6
0/6
0/6
2/6
—
1/1
0/6
0/6
0/6
2/6
1/1
—
0/6
0/6
0/6
3/6
—
—
1Groups I and II served as controls, in which the birds were inoculated with PBS and bacteriophage, respectively; group III was challenged with Escherichia coli and treated with bacteriophage EC1; group IV was challenged
with E. coli without treatment; group III(D) consisted of dead bird(s) from group III; group IV(D) consisted of
dead bird(s) from group IV.
2Number of samples positive with signs vs. number of birds investigated are displayed; comparison between
groups III and IV was performed using Fisher’s exact test. A dash (—) indicates no dead birds.
*P < 0.05.
In the E. coli-challenged birds that were treated with
bacteriophage EC1, enlargement of organs was detected
in the lungs from d 1 to 3 postinfection. The sizes of the
heart, liver, and spleen were not significantly different
(P > 0.05) from those of birds in the control groups
throughout the experimental period.
Isolation of E. coli from Lungs (Quantitative Analysis). The results in Table 3 show that low numbers of
EMB + E. coli (3.31 to 4.58 log10 cfu/g) were consistently found in the lungs of control birds. In untreated,
E. coli-challenged birds (group IV), the number of EMB
+ E. coli increased by 4 log10 cfu/g (from 2.98 to 7.20
log10 cfu/g) at d 1 postinfection and then decreased
gradually to 4.46 log10 at d 7, when it was not significantly different from those of control birds. However, in
treated, E. coli-challenged birds, the number of EMB
+ E. coli increased by only approximately 3 log10 cfu/g
(from 2.82 to 5.87 log10 cfu/g) at d 1 postinfection. In
these birds, the number of EMB + E. coli remained at
approximately 5 log10 cfu/g for the first 3 d postinfection, after which (at d 7 postinfection) it was not significantly different from those of control birds. Birds from
groups III and IV that died during the experimental
period had significantly higher numbers of EMB + E.
coli in their lung samples compared with the numbers
found in live birds from all groups.
Isolation of E. coli from Different Organs and
Blood. To assess the dissemination of E. coli in various internal organs, isolation of the bacteria from the
blood, liver, heart, and spleen of birds was carried out.
The EMB + E. coli were isolated from test samples of
untreated, E. coli-challenged birds mostly at d 1 and 2
postinfection (Table 4). When bacteriophage EC1 was
applied (group III), the occurrence of EMB + E. coli
in the liver, heart, and spleen was reduced, and EMB
+ E. coli were totally absent from the blood samples
throughout the experimental period. The EMB + E.
coli were isolated from only 2/6 of liver, 1/6 of heart,
and 3/6 of spleen samples of these birds at d 1 postinfection. The EMB + E. coli were recovered in all blood
and organ samples of birds that died during the experimental period.
BW and Mortality
At d 7 postinfection, the BW of treated, E. coli-challenged birds (group III) and untreated, E. coli-challenged birds (group IV) were not significantly different
(P > 0.05) but were lower (P < 0.05) than the BW of
control birds (groups I and II; Table 5). The positive effect of bacteriophage treatment on the BW of birds was
observed only after d 14 postinfection, when, at d 14
and 21, the BW of E. coli-challenged birds treated with
bacteriophage EC1 (group III) were not significantly
different from those of the control groups (groups I
and II). The BW of untreated, E. coli-challenged birds
(group IV) remained the lowest throughout the experimental period. Treatment with bacteriophage EC1
2593
BACTERIOPHAGE THERAPY AGAINST COLIBACILLOSIS
Table 2. Effect of bacteriophage EC1 on relative weights of the lungs, heart, liver, and
spleen1
Time after inoculation of bacteriophage (d)
Relative
weight2
Lungs3 (%)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
Heart3 (%)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
Liver3 (%)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
Spleen3 (%)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
0
1
2
3
7
0.86
0.87
0.84
0.91
± 0.07a
± 0.04a
± 0.06a
± 0.06a
—
—
0.93
1.00
1.36
1.23
1.46
1.44
±
±
±
±
±
±
0.05c
0.02bc
0.08a
0.05ab
0.13a
0.09a
0.76
0.90
1.31
1.20
1.25
1.47
±
±
±
±
±
±
0.05c
0.05c
0.05ab
0.05b
0.07b
0.10a
0.73
0.80
1.10
1.19
1.57
1.25
±
±
±
±
±
±
0.06c
0.03c
0.08b
0.12b
0.27a
0.07b
0.61
0.71
0.83
0.81
0.87
0.87
0.87
0.79
± 0.05a
± 0.04a
± 0.05a
± 0.03a
—
—
0.89
0.89
0.94
0.95
0.98
1.05
±
±
±
±
±
±
0.05b
0.04b
0.04ab
0.07ab
0.05ab
0.04a
0.83
0.81
1.00
1.82
1.42
1.46
±
±
±
±
±
±
0.03c
0.04c
0.05c
0.12a
0.04b
0.12ab
0.89
0.71
0.84
1.26
1.57
2.34
±
±
±
±
±
±
0.03c
0.06c
0.05c
0.14b
0.24b
0.17a
0.75
0.82
0.82
1.59
3.77
4.18
3.87
3.71
± 0.49a
± 0.20a
± 0.27a
± 0.08a
—
—
4.20
4.64
5.36
5.05
6.49
5.57
±
±
±
±
±
±
0.16c
0.17bc
0.08ab
0.31ab
0.39abc
0.26a
5.12
5.95
6.41
7.24
6.57
6.44
±
±
±
±
±
±
0.22c
0.33bc
0.22ab
0.27a
0.49ab
0.50ab
5.67
5.69
6.16
7.38
7.74
9.35
±
±
±
±
±
±
0.42d
0.33d
0.26cd
0.46bc
0.96b
0.50a
5.20
5.08
4.76
6.33
0.047
0.053
0.052
0.040
± 0.010a
± 0.009a
± 0.004a
± 0.010a
—
—
0.052
0.052
0.083
0.084
0.082
0.089
±
±
±
±
±
±
0.005a
0.008a
0.021a
0.017a
0.004a
0.012a
0.062
0.073
0.099
0.193
0.162
0.175
±
±
±
±
±
±
0.005b
0.005b
0.006b
0.029a
0.022a
0.028a
0.058
0.063
0.116
0.186
0.216
0.358
±
±
±
±
±
±
0.004c
0.011c
0.010bc
0.025ab
0.054a
0.076a
0.097
0.085
0.096
0.234
14
21
± 0.04b
± 0.02ab
± 0.05a
± 0.08a
—
1.39
0.58 ± 0.05b
0.66 ± 0.03ab
0.68 ± 0.06ab
0.84 ± 0.11a
0.90
—
0.60
0.58
0.71
0.91
± 0.05b
± 0.03b
± 0.05b
± 0.06a
—
—
± 0.05b
± 0.03b
± 0.06b
± 0.29a
—
2.34
0.66 ± 0.04b
0.63 ± 0.05b
0.66 ± 0.03b
1.76 ± 0.52a
1.56
—
0.56
0.57
0.52
1.42
± 0.02b
± 0.02b
± 0.05b
± 0.49a
—
—
± 0.50a
± 0.21a
± 0.30a
± 0.79a
—
10.7
4.42 ± 0.18a
4.49 ± 0.27a
4.62 ± 0.12a
5.49 ± 1.10a
11.35
—
3.22
3.29
3.20
4.41
± 0.06b
± 0.07b
± 0.18b
± 0.60a
—
—
0.087 ± 0.007a
0.083 ± 0.010a
0.086 ± 0.009a
0.137 ± 0.041a
0.534
—
0.076
0.085
0.089
0.194
± 0.007a
± 0.006a
± 0.008a
± 0.074a
—
—
± 0.008b
± 0.007b
± 0.006b
± 0.081a
—
0.289
a–dValues
within a column followed by different superscripts are significantly different (P < 0.05).
represent the mean ± SEM of 3 replicate pens of 2 birds per pen.
2Groups I and II served as controls, in which the birds were inoculated with PBS and bacteriophage, respectively; group III was challenged with
Escherichia coli and treated with bacteriophage EC1; group IV was challenged with E. coli without treatment; group III(D) consisted of dead bird(s)
from group III; group IV(D) consisted of dead bird(s) from group IV.
3Relative weight of organs (%) = (organ weight/BW) × 100%. A dash (—) indicates no dead birds.
1Values
significantly (P < 0.05) reduced the mortality rate of
birds. At the end of the experimental period, the total
mortality rate of birds (based on 1 pen of 30 birds in
each group) challenged with 108 cfu of E. coli (group
IV) was 83.3% (25/30). The mortality rate of birds that
were challenged and treated with bacteriophage EC1
(group III) was reduced by 70 to 13.3% (4/30). No
mortality occurred in the control groups (groups I and
II) during the course of the experiment.
DISCUSSION
Colibacillosis is often lethal to poultry, particularly
broilers and turkeys. The causative agent, E. coli, gains
entry into the bloodstream from an infected site, primarily the respiratory tract, via translocation across
air capillary walls, causing bacteria spread to various
internal organs, resulting in septicemia and death of
the birds (Antão et al., 2008). In the present study, the
Table 3. Effect of bacteriophage EC1 on Escherichia coli counts on eosin methylene blue agar (EMB + E. coli counts) in the lung
samples of birds1
EMB + E. coli
counts/
concentration2
(log10 cfu/g)
Group
Group
Group
Group
Group
Group
I
II
III
IV
III(D)
IV(D)
a–dValues
Time after inoculation of bacteria (d)
0
3.36
3.31
2.82
2.98
± 0.09a
± 0.16a
± 0.11a
± 0.22a
—
—
1
3.92
3.32
5.87
7.20
7.11
8.83
±
±
±
±
±
±
2
0.04d
0.11d
0.13c
0.37b
0.29b
0.13a
3.47
3.85
5.54
6.25
8.75
9.22
±
±
±
±
±
±
3
0.08d
0.10d
0.19c
0.14b
0.24a
0.14a
4.04
3.83
5.52
5.90
6.92
8.90
±
±
±
±
±
±
7
0.07d
0.06d
0.21c
0.13c
0.14b
0.15a
± 0.05a
± 0.14a
± 0.10a
± 0.30a
—
8.48
4.20
4.22
4.22
4.46
14
4.56 ± 0.04a
4.14 ± 0.11a
4.31 ± 0.07a
4.62 ± 0.50a
4.77
—
21
4.58
4.50
4.13
4.35
± 0.08a
± 0.14a
± 0.08a
± 0.27a
—
—
within a column followed by different superscripts are significantly different (P < 0.05).
represent the mean ± SEM of 3 replicate pens of 2 birds per pen.
2Groups I and II served as controls, in which the birds were inoculated with PBS and bacteriophage, respectively; group III was challenged with E.
coli and treated with bacteriophage EC1; group IV was challenged with E. coli without treatment; group III(D) consisted of dead bird(s) from group
III; group IV(D) consisted of dead bird(s) from group IV. A dash (—) indicates no dead birds.
1Values
2594
Lau et al.
Table 4. Effect of bacteriophage EC1 on the isolation of Escherichia coli on eosin methylene blue
agar from the blood, liver, heart, and spleen
Time after inoculation of bacteriophage (d)
Item1
Blood2
(n birds positive/total)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
Liver2 (n birds positive/total)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
Heart2 (n birds positive/total)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
Spleen2 (n birds positive/total)
Group I
Group II
Group III
Group IV
Group III(D)
Group IV(D)
0
1
0/6
0/6
0/6
0/6
ND2
ND
0/6
0/6
0/6
3/6
ND
ND
0/6
0/6
0/6
0/6
—
—
2
3
7
14
21
0/6
0/6
0/6
5/6*
ND
ND
0/6
0/6
0/6
1/6
ND
ND
0/6
0/6
0/6
0/6
ND
ND
0/6
0/6
0/6
0/6
ND
ND
0/6
0/6
0/6
0/6
ND
ND
0/6
0/6
2/6
5/6
4/5
6/6
0/6
0/6
0/6
5/6*
4/4
6/6
0/6
0/6
0/6
1/6
1/2
6/6
0/6
0/6
0/6
2/6
—
1/1
0/6
0/6
0/6
1/6
1/1
—
0/6
0/6
0/6
1/6
—
—
0/6
0/6
0/6
0/6
—
—
0/6
0/6
1/6
4/6
4/5
6/6
0/6
0/6
0/6
5/6*
4/4
6/6
0/6
0/6
0/6
2/6
1/2
6/6
0/6
0/6
0/6
1/6
—
1/1
0/6
0/6
0/6
1/6
1/1
—
0/6
0/6
0/6
1/6
—
—
0/6
0/6
0/6
0/6
—
—
0/6
0/6
3/6
5/6
4/5
6/6
0/6
0/6
0/6
4/6*
4/4
6/6
0/6
0/6
2/6
2/6
1/2
6/6
0/6
0/6
0/6
0/6
—
1/1
0/6
0/6
0/6
1/6
1/1
—
0/6
0/6
0/6
1/6
—
—
1Groups I and II served as controls, in which the birds were inoculated with PBS and bacteriophage, respectively; group III was challenged with E. coli and treated with bacteriophage EC1; group IV was challenged with
E. coli without treatment; group III(D) consisted of dead bird(s) from group III; group IV(D) consisted of dead
bird(s) from group IV.
2Number of samples positive with signs vs. number of birds investigated are displayed; comparison between
groups III and IV was performed using Fisher’s exact test. A dash (—) indicates no dead birds, and ND indicates
not determined.
*P < 0.05.
efficacy of the bacteriophage EC1, originally isolated
from the feces of chickens, to combat E. coli O78:K80
infection in broiler chickens was evaluated. Overall, the
results of the present study suggest that bacteriophage
EC1 exhibited a beneficial therapeutic effect. Intratracheal inoculation of 108 cfu of virulent E. coli O78:K80
resulted in more severe disease and a higher mortality rate in untreated birds than in similarly challenged
birds that were treated once intratracheally with 1010
pfu of bacteriophage EC1 2 h after challenge.
The therapeutic effects on several variables were evident. Examination of gross lesions showed that lesions
occurred in at least 50% of the air sacs of untreated,
E. coli-challenged birds. Incidences of pericarditis and
perihepatitis, which indicated systemic infection (Ask
et al., 2006), were detected in untreated, E. coli-challenged birds. Although airsacculitis was also observed
in treated, E. coli-challenged birds, pericarditis was observed in only 3/6 and 1/6 birds at d 2 and 3 postinfection, respectively. Perihepatitis was not detected in
Table 5. Effect of bacteriophage EC1 on the BW (g) of birds1
Time after inoculation of bacteriophage (d)
BW2
Group
Group
Group
Group
0
I
II
III
IV
a,bValues
44.9
44.9
44.1
45.4
±
±
±
±
7
0.8a
0.7a
0.7a
0.6a
159.1
161.0
143.5
134.1
14
3.2b
2.8b
3.7a
±
±
±
± 11.4a
373.1
383.8
362.7
318.0
±
±
±
±
21
10.1b
8.1b
11.1b
50.3a
777.5
761.7
732.0
619.0
±
±
±
±
24.8b
13.8b
18.8b
104.5a
within a column followed by different superscripts are significantly different (P < 0.05).
represent the mean ± SEM of 3 replicate pens of 2 birds per pen.
2Groups I and II served as controls, in which the birds were inoculated with PBS and bacteriophage, respectively; group III was challenged with Escherichia coli and treated with bacteriophage EC1; group IV was challenged
with E. coli without treatment.
1Values
BACTERIOPHAGE THERAPY AGAINST COLIBACILLOSIS
these birds. The results indicated that E. coli invaded
and damaged respiratory tissues rapidly. Treatment
with bacteriophage EC1 at 2 h postinfection reduced
the severity of the infection. The lungs of untreated, E.
coli-challenged birds contained 7.20 log10 cfu of EMB
+ E. coli per gram of lung tissue at d 1 postinfection,
whereas the lungs of treated, E. coli-challenged birds
contained 5.87 log10 cfu of EMB + E. coli per gram of
lung tissue at the same sampling time. Tortora et al.
(2003) reported that at the onset of pathogen infection,
the host-defense system will release inflammatory mediators to the infection site and increase permeability
of the blood vessels, which causes exudation of plasma
protein, such as fibrin, into the inflamed tissue. These
processes subsequently result in swelling and edema
of the infected organs. The significantly higher lung
weights of untreated, E. coli-challenged birds at d 2,
3, and 21 postinfection and treated E. coli-challenged
birds at d 1 to 3 postinfection could be due to inflammation. The presence of E. coli in the blood of untreated E. coli-challenged birds indicated that systemic
infection occurred more frequently and that bacteria
spread transiently to other organs, such as the liver,
heart, and spleen. The enlarged organs (heart, liver,
and spleen) of these untreated, E. coli-challenged birds
showed the presence of E. coli, and the incidence was
particularly high at d 2 postinfection. The therapeutic
effect of bacteriophage EC1 was evident in that enlargements of the heart, liver, and spleen of treated, E. colichallenged birds were not observed. In contrast, Huff et
al. (2002) reported that bacteriophage treatment did
not show any consistent effects on the relative weights
of the liver, heart, spleen, and bursa of Fabricius of
birds that survived colibacillosis. Results of the present
study showed that E. coli was not detected in the blood
of treated birds during the entire experimental period,
and a lower incidence of E. coli isolation from various
organs was recorded on d 1 postinfection. According
to Brüssow (2005) and Górski and Weber-Dabrowska
(2005), bacteriophages can migrate through the mucosal surface and even across the blood-brain barrier,
thereby providing good protection to the host. The adsorption of a phage to its host bacteria is reported to
be more efficient in a highly fluid environment such
as blood, as compared with viscous and solid environments (Joerger, 2003).
In general, the BW gain in infected birds was lower.
As reported in previous studies, birds infected with E.
coli show growth retardation caused by anorexia (Dunnington et al., 1991; Gomis et al., 1997). Treatment
with bacteriophage EC1 successfully reduced the severity of the infection, and thus allowed the birds to regain
their growth by d 14 postinfection. In contrast, Huff
et al. (2003a,b) found that the BW of bacteriophagetreated birds was not significantly different from that
of untreated, E. coli-challenged birds. Treatment with
bacteriophage EC1 was also found to reduce the mortality rate of infected birds from 83.3% (group IV) to
13.3% (group III).
2595
In conclusion, the results of the current study demonstrated the potential use of bacteriophage EC1 as
a therapeutic agent for treatment of colibacillosis. Although phage therapy has been reported to be effective in reducing bacterial infections and bacterial loads
in a variety of animal and food studies, including E.
coli septicemia and meningitis-like infection in chickens
and calves (Barrow et al., 1998), E. coli O157:H7 contamination of beef (O’Flynn et al., 2004), Salmonella
spp. (Fiorentin et al., 2005; Atterbury et al., 2007) and
Campylobacter jejuni infections in chickens (Wagenaar
et al., 2005), bacterial disease in Penaeus monodon
shrimp larvae (Karunasagar et al., 2007), and many
others, more detailed studies need to be carried out
before the true potential of phage therapy can be determined.
ACKNOWLEDGMENTS
This research was supported by the Ministry of Science, Technology, and Innovation of Malaysia (Kuala
Lumpur, Malaysia). We thank the undergraduate students from Universiti Putra Malaysia, the internship
students from University College Sedaya International
(Kuala Lumpur, Malaysia), and all postgraduate students in the laboratory for their kind assistance in the
in vivo field trial experiment.
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