FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
“NOVEL BACTERIOPHAGE AND ANTIBACTERIAL
COMPOSITION COMPRISING THE SAME”
CJ CHEILJEDANG CORPORATION
500, Namdaemunno 5-ga, Jung-gu,
Seoul 100-749 Republic of Korea.
The following specification particularly describes the invention and the
manner in which it is to be performed.
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[DESCRIPTION]
[Invention Title]
Novel bacteriophage and antibacterial composition
comprising the same
[Technical Field]
The present invention relates to a novel bacteriophage,
more particularly, a bacteriophage that has a specific
bactericidal activity against Fowl Typhoid causing
Salmonella Gallinarum (SG) and Pullorum disease-causing
Salmonella Pullorum (SP). Further, the present invention
relates to a composition for the prevention or treatment of
infectious diseases caused by Salmonella Gallinarum or
Salmonella Pullorum, comprising the bacteriophage as an
active ingredient. Furthermore, the present invention
relates to feed and drinking water for poultry, sanitizers
and cleaners, comprising the bacteriophage as an active
ingredient.
[Background Art]
Salmonella is a genus of the family Enterobacteriaceae,
characterized as Gram-negative, facultatively anaerobic, non
spore-forming, rod-shaped bacteria, and most strains are
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motile by flagella. Salmonella has an average genome GC
content of 50-52%, which is similar to those of Escherichia
coli and Shigella. The genus Salmonella is a pathogenic
microorganism that causes infections in livestock as well as
in humans. Salmonella enterica, a species of Salmonella
bacterium, has a variety of serovars including Salmonella
Gallinarum, Salmonella Pullorum, Salmonella Typhimurium,
Salmonella Enteritis, Salmonella Typhi, Salmonella
Choleraesuis and Salmonella derby (Bopp CA, Brenner FW,
Wells JG, Strokebine NA. Escherichia, Shigella, Salmonella.
In Murry PR, Baron EJ, et al eds Manual of clinical
Microbiology. 7th ed. Washington DC American Society for
Microbiology 1999;467-74 ; Ryan KJ. Ray CG (editors) (2004)).
Of them, Salmonella Typhimurium and Salmonella Enteritis are
pathogenic for human and animals, Salmonella Typhi is a
human-adapted pathogen, Salmonella Choleraesuis and
Salmonella derby are swine-adapted pathogens, and Salmonella
Gallinarum and Pullorum are fowl-adapted pathogens. Each
serovar causes illness in that species, which results in
tremendous damage to farmers and/or consumers.
A disease of domestic birds caused by Salmonella
bacterium is Fowl Typhoid (FT), which is caused by a
pathogen, Salmonella Gallinarum (hereinbelow, designated as
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SG). Fowl Typhoid (FT) is a septicemic disease of domestic
birds such as chicken and turkey, and the course may be
acute or chronic with high mortality. Recently, it has been
reported that Fowl Typhoid frequently occurs in Europe,
South America, Africa, and South-East Asia, and damages are
increasing. Outbreaks of FT in Korea have been reported
since 1992 and economic losses coupled by FT in brown, egglaying
chickens are very serious (Kwon Yong-Kook. 2001
annual report on avian diseases. information publication by
National Veterinary Research & Quarantine Service. March,
2001; Kim Ae-Ran et al., The prevalence of pullorum diseasefowl
typhoid in grandparent stock and parent stock in Korea,
2003, Korean J Vet Res(2006) 46(4): 347~353). Pullorum
Disease is also caused by one of the Salmonella bacteria,
Salmonella Pullorum (hereinbelow, designated as SP).
Pullorum disease occurs in any age or season, but young
chicken is very susceptible to the disease. During the past
one century, it has been a serious disease to young chickens
at 1-2 weeks of age or younger by egg-transmitted infections
in the world and Korea. Since the 80’s, the occurrence has
greatly decreased. However, it has been growing in the
middle of the 90’s.
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In Korea, the outbreaks of Fowl Typhoid and Pullorum
disease have been increasing since the 90’s, inflicting
economic damages to farmers. For this reason, a live
attenuated SG vaccine has been used in broilers for the
prevention of Fowl Typhoid from 2004 (Kim Ae-Ran et al., The
prevalence of pullorum disease-fowl typhoid in grandparent
stock and parent stock in Korea, 2003, Korean J Vet
Res(2006) 46(4): 347~353), while its efficacy is doubtful,
and the live vaccine is not allowed to be used for layers
because of the risk of egg-transmitted infections.
Unfortunately, there are still no commercially available
preventive strategies against Pullorum disease, unlike Fowl
Typhoid. Thus, there is an urgent need for new ways to
prevent Fowl Typhoid and Pullorum disease.
A Bacteriophage is a specialized type of virus that
only infects and destroys bacteria, and can self-replicate
only inside a host bacteria. A Bacteriophage consists of
genetic material-the nucleic acid-single or double stranded
DNA or RNA surrounded by a protein shell. There are three
basic structural forms of bacteriophage: an icosahedral
(twenty-sided) head with a tail, an icosahedral head without
a tail, and a filamentous form. Bacteriophages are
classified based on their morphological structure and
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genetic material. Based on their tail structure,
bacteriophages having icosahedral head and double-stranded,
linear DNA as their genetic material are divided into three
families: Myoviridae, Siphoviridae, and Podoviridae, which
are characterized by contractile, long noncontractile, and
short noncontractile tails, respectively. Bacteriophages
having icosahedral head without a tail and RNA or DNA as
their genetic material are divided based on their head shape
and components, and the presence of shell. Filamentous
bacteriophages having DNA as their genetic material are
divided based on their size, shape, shell, and filament
components (H.W.Ackermann, Frequency of morphological phage
descriptions in the year 2000; Arch Virol (2001) 146:843-
857; Elizabeth Kutter et al. Bacteriophages biology and
application; CRC press). During infection, a bacteriophage
attaches to a bacterium and inserts its genetic material
into the cell. After this a bacteriophage follows one of
two life cycles, lytic or lysogenic. Lytic bacteriophages
take over the machinery of the cell to make phage components.
They then destroy or lyse the cell, releasing new phage
particles. Lysogenic bacteriophages incorporate their
nucleic acid into the chromosome of the host cell and
replicate with it as a unit without destroying the cell.
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Under certain conditions, lysogenic phages can be induced to
follow a lytic cycle (Elizabeth Kutter et al. Bacteriophages
biology and application, CRC press).
After the discovery of bacteriophages, a great deal of
faith was initially placed in their use for infectiousdisease
therapy. However, when broad spectrum antibiotics
came into common use, bacteriophages were seen as
unnecessary because of having a specific target spectrum.
Nevertheless, the misuse and overuse of antibiotics resulted
in rising concerns about antibiotic resistance and harmful
effects of residual antibiotics in foods (Cislo, M et al.
Bacteriophage treatment of suppurative skin infections. Arch
Immunol.Ther.Exp. 1987.2:175-183; Kim sung-hun et al.,
Bacteriophage; New Alternative Antibiotics. biological
research information center. BRIC). In particular,
antimicrobial growth promoter (AGP), added to animal feed to
enhance growth, is known to induce antibiotic resistance,
and therefore, the ban of using antimicrobial growth
promoter (AGP) has been recently introduced. In the
European Union, the use of all antimicrobial growth
promoters (AGPs) was banned from 2006. Korea banned the use
of some AGPs, and is considering restrictions on the use of
all AGPs.
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These growing concerns about the use of antibiotics
have led to a resurgence of interest in bacteriophage as an
alternative to antibiotics. 7 bacteriophages for control of
E.coli O157:H are disclosed in US Patent 6485902 (applied
for in 2002 - Use of bacteriophages for control of
Escherichia coli O157). 2 bacteriophages for control of
various microorganisms are disclosed in US Patent 6942858
(applied for by Nymox in 2005 - Compositions containing
bacteriophages and method of using bacteriophages to treat
infections). Many companies have been actively trying to
develop various products using bacteriophages. EBI food
system (Europe) developed a food additive for preventing
food poisoning caused by Listeria monocytogenes, named
Listex-P100, which is the first bacteriophage product
approved by the US FDA. A phage-based product, LMP-102 was
also developed as a food additive against Listeria
monocytogenes, approved as GRAS (Generally regarded as safe).
In 2007, a phage-based wash produced by OmniLytics was
developed to prevent E.coli O157 contamination of beef
during slaughter, approved by USDA’s Food Safety and
Inspection Service (FSIS). In Europe, Clostridium
sporogenes phage NCIMB 30008 and Clostridium tyrobutiricum
phage NCIMB 30008 were registered as a feed preservative
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against Clostridium contamination of feed in 2003 and 2005,
respectively. Such studies show that research into
bacteriophages for use as antibiotics against zoonotic
pathogens in livestock products is presently ongoing.
[Disclosure]
[Technical Problem]
In order to solve the problems generated by the use of
broad spectrum antibiotics, the present inventors isolated a
novel Salmonella bacteriophage from natural sources, and
identified its morphological, biochemical, and genetic
properties. The present inventors found that the
bacteriophage has a specific bactericidal activity against
Salmonella Gallinarum (SG) and Salmonella Pullorum (SP)
without affecting beneficial bacteria, and excellent acid-,
heat- and dry-resistance, and thus can be applied to various
products for the control of Salmonella Gallinarum (SG) and
Salmonella Pullorum (SP), including feed and drinking water
for poultry, sanitizers, and cleaners , in addition to a
composition for the prevention and treatment of infectious
diseases caused by Salmonella Gallinarum and Salmonella
Pullorum, in particular, Fowl Typhoid and Pullorum Disease,
thereby completing the present invention.
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[Technical Solution]
It is an object of the present invention to provide a
novel bacteriophage having a specific bactericidal activity
against Salmonella Gallinarum and Salmonella Pullorum.
It is another object of the present invention to
provide a composition for the prevention and treatment of
infectious diseases caused by Salmonella Gallinarum and
Salmonella Pullorum, comprising the bacteriophage as an
active ingredient.
It is still another object of the present invention to
provide a feed and drinking water for poultry, comprising
the bacteriophage as an active ingredient.
It is still another object of the present invention to
provide a sanitizer and cleaner, comprising the
bacteriophage as an active ingredient.
It is still another object of the present invention to
provide a method for preventing and treating infectious
diseases, Fowl Typhoid or Pullorum disease caused by
Salmonella Gallinarum or Salmonella Pullorum using the
composition comprising the bacteriophage as an active
ingredient.
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[Advantageous Effects]
The novel bacteriophage of the present invention has a
specific bactericidal activity against Salmonella Gallinarum
and Salmonella Pullorum, and excellent acid-, heat- and dryresistance,
and thus can be used for the prevention and
treatment of infectious diseases caused by Salmonella
Gallinarum or Salmonella Pullorum, including Fowl Typhoid
and Pullorum disease, and also used for the control of
Salmonella Gallinarum and Salmonella Pullorum.
[Description of Drawings]
FIG. 1 is an electron microscopy photograph of ΦCJ1, in
which ΦCJ1 belongs to the morphotype group of the family
Siphoviridae, characterized by isometric capsid and long
non-contractile tail;
FIG. 2 is the result of SDS-PAGE of the isolated
bacteriophage ΦCJ1, in which protein patterns of the
bacteriophage are shown, major proteins of 38 kDa and 49 kDa
and other proteins of 8 kDa, 17 kDa, 80 kDa, and 100 kDa
(See-blue plus 2 prestained-standard (Invitrogen) used as a
marker);
FIG. 3 is the result of PFGE of the isolated
bacteriophage ΦCJ1, showing the total genome size of
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approximately 61 kbp (5 kbp DNA size standard (Bio-rad) as a
size marker);
FIG. 4 is the result of PCR, performed by using each
primer set of ΦCJ1 genomic DNA, in which (A; PCR
amplification using primer set of SEQ ID NOs. 6 and 7, B;
PCR amplification using primer set of SEQ ID NOs. 10 and 11,
C; PCR amplification using primer set of SEQ ID NOs. 8 and 9,
D; PCR amplification using primer set of SEQ ID NOs. 12 and
13) each of A, B, C and D lanes had a PCR product of
approximately 660 bp, 1.3 kbp, 670 bp, and 1.8 kbp;
FIG. 5 is the result of a one-step growth experiment of
the bacteriophage ΦCJ1, in which (A; Salmonella Gallinarum,
B; Salmonella Pullorum) the bacteriophage had the burst size
of 102 or more in Salmonella Gallinarum and Salmonella
Pullorum;
FIG. 6 is the result of an efficiency test (clearing
assay) of the bacteriophage ΦCJ1, in which (A; Salmonella
Gallinarum, B; Salmonella Pullorum, MOI (multiply of
infection): the number of bacteriophage inoculated per host
cell) as the bacteriophage ΦCJ1 has higher efficiency
infection, it can effectively lyse host cells;
FIG. 7 is the result of an acid-resistance test on the
bacteriophage ΦCJ1, showing the number of surviving
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bacteriophage at pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 6.9,
7.4, 8.0, 9.0, in which the bacteriophage ΦCJ1 did not lose
its activity until pH 2.5, but completely lost its activity
at pH 2.1, as compared to the control;
FIG. 8 is the result of a heat-resistance test on the
bacteriophage ΦCJ1, showing the number of surviving
bacteriophage at 37, 45, 53, 60, 70, 80℃, and a time point
of 0, 10, 30, 60, 120 min, in which the bacteriophage ΦCJ1
did not lose its activity even after exposure at 70℃ for 2
hrs, and its activity was decreased after exposure at 80℃
for 10 min, but maintained constantly; and
FIG. 9 is the result of a dry-resistance test on the
bacteriophage ΦCJ1, performed at 60℃ for 120 min using a
speed vacuum dryer (SVD), in which changes in the titers
before and after drying were compared to examine the
relative stability, and its activity was decreased to 102.
[Best Mode]
In accordance with an aspect, the present invention
relates to a novel bacteriophage having a specific
bactericidal activity against Salmonella Gallinarum and
Salmonella Pullorum.
The bacteriophage of the present invention belongs to
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the morphotype group of the family Siphoviridae,
characterized by isometric capsid and long non-contractile
tail, and has a total genome size of 61 kbp and major
structural proteins with a size of 38 kDa and 49 kDa.
The bacteriophage of the present invention has the
capability of selectively infecting Salmonella Gallinarum
and Salmonella Pullorum, namely, species specificity.
Further, the bacteriophage of the present invention has
a total genome size of approximately 61 kbp, and may include
a nucleic acid molecule represented by SEQ ID NOs. 1 to 5
within the entire genome.
As used herein, the term “nucleic acid molecule”
encompasses DNA (gDNA and cDNA) and RNA molecules, and the
term nucleotide, as the basic structural unit of nucleic
acids, encompasses natural nucleotides and sugar or basemodified
analogues thereof.
Further, the bacteriophage of the present invention has
the biochemical properties of acid- and heat-resistance, in
which it can stably survive in a wide range of pH
environment from pH 2.5 to pH 9.0, and in a high temperature
environment from 37℃ to 70℃. In addition, the
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bacteriophage of the present invention has dry-resistance to
stably maintain even after high-temperature drying (at 60℃
for 120 minutes). Such properties of acid-, heat-, and
drying-resistance also allow application of the
bacteriophage of the present invention under various
temperature and pH conditions, upon the production of
prophylactic or therapeutic compositions for poultry
diseases caused by SG and SP, and other products comprising
the bacteriophage as an active ingredient.
The present inventors collected sewage samples at
chicken slaughterhouse, and isolated the bacteriophage of
the present invention that has a SG and SP-specific
bactericidal activity and the above characteristics, which
was designated as ΦCJ1 and deposited at the Korean Culture
Center of Microorganisms on October 24, 2008 under accession
number KCCM10969P.
In accordance with the specific Example of the present
invention, the present inventors collected sewage samples at
a chicken slaughterhouse to isolate bacteriophages that lyse
the host cell SP, and they confirmed that the bacteriophages
are able to lyse SG and SP, specifically. Further, they
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examined the bacteriophage (ΦCJ1) under electron microscope,
and found that it belongs to the morphotype group of the
family Siphoviridae (FIG. 1).
The protein patterns of the bacteriophage ΦCJ1 were
also analyzed, resulting in that it has major structural
proteins with a size of 38 kDa and 49 kDa.
The total genome size of the bacteriophage ΦCJ1 was
also analyzed, resulting in that it has a total genome size
of approximately 61 kbp. The results of analyzing its
genetic features showed that the bacteriophage includes a
nucleic acid molecule represented by SEQ ID NOs. 1 to 5
within the total genome. Based on these results, genetic
similarity with other species was compared. It was found
that the bacteriophage showed very low genetic similarity
with the known bacteriophages, indicating that the
bacteriophage is a novel bacteriophage. More particularly,
the ΦCJ1-specific primer set, e.g., SEQ ID NOs. 6 and 7, 10
and 11, 8 and 9, 12 and 13, 14 and 15, 16 and 17, and 18 and
19, were used to perform PCR. Each PCR product was found to
have a size of 660 bp, 1.3 kbp, 670 bp 1.8 kbp, 1 kbp, 1kbp
and 1kbp.
Further, when SG and SP were infected with ΦCJ1, the
phage plaques (clear zone on soft agar created by host cell
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lysis of one bacteriophage) showed the same size and
turbidity. The lysis of SG and SP by the bacteriophage ΦCJ1
was observed, showing its growth in inhibitory effects on SG
and SP.
Furthermore, the stability of ΦCJ1 was examined under
various temperature and pH conditions, resulting in that
ΦCJ1 stably maintains in a wide range of pH environments
from pH 2.5 to pH 9.0 and in a high temperature environment
from 37℃ to 70℃, and even after high-temperature drying
(at 60℃ for 120 minutes). These results indicate that the
bacteriophage ΦCJ1 of the present invention can be readily
applied to various products for the control of SG and SP.
In accordance with another aspect, the present
invention relates to a composition for the prevention or
treatment of Fowl Typhoid or Pullorum disease caused by
Salmonella Gallinarum or Salmonella Pullorum, comprising the
bacteriophage as an active ingredient.
The bacteriophage of the present invention has a
specific bactericidal activity against Salmonella Gallinarum
and Salmonella Pullorum, and thus can be used for the
purpose of preventing or treating diseases that are caused
by Salmonella Gallinarum and Salmonella Pullorum.
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Specifically, in the preferred embodiment, antibiotics may
be included.
As used herein, the term “prevention” means all of the
actions in which the disease is restrained or retarded by
the administration of the composition. As used herein, the
term “treatment” means all of the actions in which the
disease has taken a turn for the better or been modified
favorably by the administration of the composition.
Preferred examples of infectious diseases, to which the
composition of the present invention can be applied, include
Fowl Typhoid caused by Salmonella Gallinarum and Pullorum
disease caused by Salmonella Pullorum, but are not limited
thereto.
The composition of the present invention comprises ΦCJ1
of 5 x 102 to 5 x 1012 pfu/ml, preferably 1 x 106 to 1 x 1010
pfu/ml.
The composition of the present invention may
additionally include a pharmaceutically acceptable carrier,
and be formulated together with the carrier to provide foods,
medicines, and feed additives.
As used herein, the term “pharmaceutically acceptable
carrier” refers to a carrier or diluent that does not cause
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significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound.
For formulation of the composition into a liquid
preparation, a pharmaceutically acceptable carrier which is
sterile and biocompatible may be used such as saline,
sterile water, Ringer’s solution, buffered physiological
saline, albumin infusion solution, dextrose solution,
maltodextrin solution, glycerol, and ethanol. These
materials may be used alone or in any combination thereof.
If necessary, other conventional additives may be added such
as antioxidants, buffers, bacteriostatic agents, and the
like. Further, diluents, dispersants, surfactants, binders
and lubricants may be additionally added to the composition
to prepare injectable formulations such as aqueous solutions,
suspensions, and emulsions, or oral formulations such as
pills, capsules, granules, or tablets.
Examples of the oral dosage forms suitable for the
composition of the present invention include tablets,
troches, lozenges, aqueous or emulsive suspensions, powder
or granules, emulsions, hard or soft capsules, syrups, or
elixirs. For formulation such as tablets and capsules,
useful are a binder such as lactose, saccharose, sorbitol,
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mannitol, starch, amylopectin, cellulose or gelatin, an
excipient such as dicalcium phosphate, a disintegrant such
as corn starch or sweet potato starch, a lubricant such as
magnesium stearate, calcium stearate, sodium stearylfumarate,
or polyethylene glycol wax. For capsules, a liquid carrier
such as a lipid may be further used in addition to the
above-mentioned compounds.
For non-oral administration, the composition of the
present invention may be formulated into injections for
subcutaneous, intravenous, or intramuscular routes,
suppositories, or sprays inhalable via the respiratory tract,
such as aerosols. Injection preparations may be obtained by
dissolving or suspending the composition of the present
invention, together with a stabilizer or a buffer, in water
and packaging the solution or suspension in ampules or vial
units. For sprays, such as aerosol, a propellant for
spraying a water-dispersed concentrate or wetting powder may
be used in combination with an additive.
In accordance with still another aspect, the present
invention relates to an antibiotic, comprising the
composition for the prevention or treatment of Fowl Typhoid
caused by Salmonella Gallinarum or Pullorum disease caused
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by Salmonella Pullorum.
As used herein, the term “antibiotic” means any drug
that is applied to animals to kill pathogens, and used
herein as a general term for antiseptics, bactericidal
agents and antibacterial agents. The bacteriophage of the
present invention, unlike the conventional antibiotics, has
a high specificity to Salmonella Gallinarum and Salmonella
Pullorum to kill the specific pathogens without affecting
beneficial bacteria, and does not induce resistance so that
its life cycling is comparatively long.
In accordance with still another aspect, the present
invention relates to a poultry feed and drinking water for
poultry, comprising the bacteriophage as an active
ingredient.
The bacteriophage of the present invention may be
separately prepared as a feed additive, and then added to
the animal feed, or directly added to the animal feed. The
bacteriophage of the present invention may be contained in
the animal feed as a liquid or in a dried form. The drying
process may be performed by air drying, natural drying,
spray drying, and freeze-drying, but is not limited thereto.
The bacteriophage of the present invention may be added as a
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powder form in an amount of 0.05 to 10% by weight,
preferably 0.1 to 2% by weight, based on the weight of
animal feed.
The feed comprising ΦCJ1 of the present invention may
include plant-based feeds, such as grain, nut, food
byproduct, seaweed, fiber, drug byproduct, oil, starch, meal,
and grain byproduct, and animal-based feeds such as protein,
mineral, fat, single cell protein, zooplankton, and food
waste, but is not limited thereto.
The feed additive comprising ΦCJ1 of the present
invention may include binders, emulsifiers, and
preservatives for the prevention of quality deterioration,
amino acids, vitamins, enzymes, probiotics, flavorings, nonprotein
nitrogen, silicates, buffering agents, coloring
agents, extracts, and oligosaccharides for the efficiency
improvement, and other feed premixtures, but is not limited
thereto.
Further, the supply of drinking water mixed with the
bacteriophage of the present invention can reduce the number
of Salmonella Gallinarum or Salmonella Pullorum in the
intestine of livestock, thereby obtaining Salmonella
Gallinarum or Salmonella Pullorum-free livestock.
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In accordance with still another aspect, the present
invention relates to a sanitizer and a cleaner, comprising
the bacteriophage as an active ingredient.
In order to remove Salmonella Gallinarum and Salmonella
Pullorum, the sanitizer comprising the bacteriophage as an
active ingredient can be used in the poultry barns,
slaughterhouses, contaminated areas, and other production
facilities, but is not limited thereto.
Further, the cleaner comprising the bacteriophage as an
active ingredient can be applied to the contaminated skin,
feather, and other contaminated body parts of living poultry,
in order to remove Salmonella Gallinarum and Salmonella
Pullorum.
In accordance with still another aspect, the present
invention relates to a method for preventing or treating
infectious diseases, Fowl Typhoid or Pullorum disease caused
by Salmonella Gallinarum or Salmonella Pullorum using the
composition for the prevention or treatment of Fowl Typhoid
caused by Salmonella Gallinarum or Pullorum disease caused
by Salmonella Pullorum.
The composition of the present invention may be
administered into animals in a pharmaceutical formulation or
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as a component of the animal feed or in their drinking water,
preferably administered by mixing into the animal feed as a
feed additive.
The composition of the present invention may be
administered in a typical manner via any route such as oral
or parenteral routes, in particular, oral, rectal, topical,
intravenous, intraperitoneal, intramuscular, intraarterial,
transdermal, intranasal, and inhalation routes.
The method for treating the diseases of the present
invention includes administration of a pharmaceutically
effective amount of the composition of the present invention.
It will be obvious to those skilled in the art that the
total daily dose should be determined through appropriate
medical judgment by a physician. The therapeutically
effective amount for patients may vary depending on various
factors well known in the medical art, including the kind
and degree of the response to be achieved, the patient’s
condition such as age, body weight, state of health, sex,
and diet, time and route of administration, the secretion
rate of the composition, the time period of therapy,
concrete compositions according to whether other agents are
used therewith or not, etc.
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Hereinafter, the present invention will be described in
more detail with reference to the following Examples.
However, these Examples are for illustrative purposes only,
and the invention is not intended to be limited by these
Examples.
[Mode for Invention]
Example 1: Salmonella Bacteriophage Isolation
1-1. Bacteriophage screening and single bacteriophage
isolation
50 ml of a sample from the chicken slaughterhouse and
sewage effluent was transferred to a centrifuge tube, and
centrifuged at 4000 rpm for 10 minutes. Then, the
supernatant was filtered using a 0.45 ㎛ filter. 18 ml of
sample filtrate was mixed with 150 ㎕ of SP shaking culture
medium (OD600=2) and 2 ml of 10 X Luria-Bertani medium
(Hereinbelow, designated as LB medium, tryptone 10 g; yeast
extract 5 g; NaCl 10 g; final volume to 1 L). The mixture
was cultured at 37℃ for 18 hours, and the culture medium
was centrifuged at 4000 rpm for 10 minutes. The supernatant
was filtered using a 0.2 ㎛ filter. 3 ml of 0.7% agar (w/v)
and 150 ㎕ of SP shaking culture medium (OD600=2) were mixed,
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and plated onto an LB plate, and changed to a solid medium.
10 ㎕ of culture filtrate was spread thereon, and cultured
for 18 hours at 37℃ (0.7% agar was used as “top-agar”, and
the titration of phage lysate was performed on the top-agar,
called soft agar overlay method).
The sample culture medium containing the phage lysate
was diluted, and mixed with 150 ㎕ of SP shaking culture
medium (OD600=2), followed by the soft agar overlay method,
so that single plaques were obtained. Since a single plaque
represents one bacteriophage, to isolate single
bacteriophages, one plaque was added to 400 ul of SM
solution (NaCl, 5.8 g; MgSO47H2O, 2 g; 1 M Tris-Cl (pH7.5),
50 ml; H2O, final volume to 1 L), and left for 4 hours at
room temperature to isolate single bacteriophages. To purify
the bacteriophage in large quantities, 100 ㎕ of supernatant
was taken from the single bacteriophage solution, and mixed
with 12 ml of 0.7% agar and 500 ㎕ of SP shaking culture
medium, followed by the soft agar overlay method on an LB
plate (150 mm diameter). When lysis was completed, 15 ml of
SM solution was added to the plate. The plate was gently
shaken for 4 hours at room temperature to elute the
bacteriophages from the top-agar. The solution containing
the eluted bacteriophages was recovered, and chloroform was
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added to a final volume of 1%, and mixed well for 10 minutes.
The solution was centrifuged at 4000 rpm for 10 minutes.
The obtained supernatant was filtered using a 0.2 ㎛ filter,
and stored in the refrigerator.
1-2. Large-scale culture of bacteriophage
The selected bacteriophages were cultured in large
quantities using SP. SP was shaking-cultured, and an
aliquot of 1.5 X 1010 cfu (colony forming unit) was
centrifuged at 4000 rpm for 10 minutes, and the pellet was
re-suspended in 4 ml of SM solution. The bacteriophage of
7.5 X 107 pfu (plaque forming unit) was inoculated thereto
(MOI: multiplicity of infection=0.005), and left at 37℃ for
20 minutes. The solution was inoculated into 150 ml of LB
media, and cultured at 37℃ for 5 hours. Chloroform was
added to a final volume of 1%, and the culture solution was
shaken for 20 minutes. DNase I and RNase A were added to a
final concentration of 1 ㎍/ml, respectively. The solution
was left at 37℃ for 30 minutes. NaCl and PEG (polyethylene
glycol) were added to a final concentration of 1 M and 10%
(w/v), respectively and left at 37℃ for an additional 3
hours. The solution was centrifuged at 4℃ and 12000 rpm for
20 minutes to discard the supernatant. The pellet was re-
27 -
suspended in 5ml of SM solution, and left at room
temperature for 20 minutes. 4ml of chloroform was added
thereto and mixed well, followed by centrifugation at 4℃
and 4000 rpm for 20 minutes. The supernatant was filtered
using a 0.2 ㎛ filter, and ΦCJ1 was purified by glycerol
density gradient ultracentrifugation (density: 40%, 5%
glycerol at 35,000 rpm and 4℃ for 1 hour). The purified
ΦCJ1 was re-suspended in 300 ㎕ of SM solution, followed by
titration.
Example 2: Examination on ΦCJ1 Infection of Salmonella
To examine the lytic activity of the selected
bacteriophages on other Salmonella species as well as SP,
cross-infection attempts with other Salmonella species were
made. As a result, ΦCJ1 did not infect ST (Salmonella
enterica Serotype Typhimurium), SE (Salmonella enterica
Serotype Enteritis), SC (Salmonella enterica Serotype
Choleraesuis), SD (Salmonella enterica Serotype Derby), SA
(Salmonella enterica subsp. Arizonae), SB (Salmonella
enterica subsp. Bongori), but specifically infected SG and
SP (see Example 13). The results are shown in the following
Table 1. The bacteriophages ΦCJ1 produced using SG as a
host cell showed the same plaque size and plaque turbidity,
- 28 -
and the same protein patterns and genome size as those
produced using SP as a host cell.
[Table 1]
<ΦCJ1 infection of Salmonella>
Sero
type
Strain
name
Plaque
formation
Sero
type
Strain
name
Plaque
formation
SG
SGSC
2293
O SE
SGSC
2282
X
SP
SGSC
2294
O SC
ATCC
13312
X
SGSC
2295
O
SD
SCSG
2467
X
ST
ATCC
14028
X
SGSC
2468
X
LT2 X SA
ATCC
13314
X
UK1 X SB
ATCC
43975
X
* ATCC: The Global Bioresource Center
* SGSC: salmonella genetic stock center
Example 3: Morphology Examination of Bacteriophage ΦCJ1
- 29 -
The purified ΦCJ1 was diluted in 0.01% gelatin solution,
and then fixed in 2.5% glutaraldehyde solution. After the
sample was dropped onto a carbon-coated mica plate (ca.2.5 X
2.5 mm) and adapted for 10 minutes, it was washed with
sterile distilled water. Carbon film was mounted on a
copper grid, and stained with 4% uranyl acetate for 30-60
seconds, dried, and examined under a JEM-1011 transmission
electron microscope (80kV, magnification of X 120,000 ~ X
200,000). As a result, the purified ΦCJ1 had morphological
characteristics including an isometric capsid and a long
non-contractile tail, as shown in , indicating that
it belongs to the morphotype group of the family
Siphoviridae.
Example 4: Protein Pattern Analysis of Bacteriophage
ΦCJ1
15 ㎕ of purified ΦCJ1 solution (1011 pfu/ml titer) was
treated with 3 ㎕ of 5X SDS sample solution, and heated for 5
minutes. The total protein of ΦCJ1 was run in 4-12% NuPAGE
Bis-Tris gel (Invitrogen), and then the gel was stained with
coomassie blue for 1 hour at room temperature. As shown in
, the protein patterns showed that 38 kDa and 49 kDa
bands were observed as major proteins, and 8 kDa, 17 kDa, 80
- 30 -
kDa, and 100 kDa bands were also observed.
Example 5: Total Genomic DNA Size Analysis of
Bacteriophage ΦCJ1
Genomic DNA was isolated from the purified ΦCJ1 by
ultracentrifugation. Specifically, to the purified ΦCJ1
culture medium, EDTA (ethylenediaminetetraacetic acid
(pH8.0)), proteinase K, and SDS (sodium dodecyl sulfate)
were added to a final concentration of 20 mM, 50 ㎍/ml, and
0.5% (w/v), respectively and left at 50℃ for 1 hour. An
equal amount of phenol (pH8.0) was added and mixed well,
followed by centrifugation at 12000 rpm and room temperature
for 10 minutes. The supernatant was mixed well with an
equal amount of PC (phenol:chloroform=1:1), followed by
centrifugation at 12000 rpm and room temperature for 10
minutes. The supernatant was mixed well with an equal
amount of chloroform, followed by centrifugation at 12000
rpm and room temperature for 10 minutes. Again, to the
supernatant, added were 1/10 volume of 3 M sodium acetate
and two volumes of cold 95% ethanol, and left at -20℃ for 1
hour. After centrifugation at 0℃ and 12000 rpm for 10
minutes, the supernatant was completely removed, and the DNA
pellet was dissolved in 50 ㎕ TE (Tris-EDTA (pH 8.0)). The
- 31 -
extracted DNA was diluted 10-fold, and its absorbance was
measured at OD260. After loading 1 ㎍ of total genomic DNA in
1% PFGE (pulse-field gel electrophoresis) agarose gel,
electrophoresis was performed using a BIORAD PFGE system
program 7 (size range 25-100 kbp; switch time ramp 0.4-2.0
seconds, linear shape; forward voltage 180 V; reverse
voltage 120 V) at room temperature for 20 hours. As shown
in , ΦCJ1 had a genomic DNA size of approximately 61
kbp.
Example 6: Genetic Analysis of Bacteriophage ΦCJ1
To analyze genetic features of the purified ΦCJ1, 5 ㎍
of genomic DNA of ΦCJ1 was treated with the restriction
enzymes, EcoR V and Sca I. The vector, pBluescript SK+
(Promega) was digested with EcoR V, and treated with CIP
(calf intestinal alkaline phosphatase). The digested
genomic DNA and vector were mixed in a ratio of 3:1, and
ligated at 16℃ for 5 hours. The ligation product was
transformed into E.coli DH5α. The transformed cells were
plated on an LB plate containing ampicillin and X-gal (5-
bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) for
blue/white selection, so as to select two colonies. The
selected colony was shaking-cultured in a culture medium
- 32 -
containing ampicillin for 16 hours. Then, plasmids were
extracted using a plasmid purification kit (Promega).
The cloning of the plasmids was confirmed by PCR using
a primer set of M13 forward and M13 reverse, and insert
fragments of 1 kbp or more were selected, and their base
sequence was analyzed using the primer set of M13 forward
and M13 reverse. The results were shown in SEQ ID NOs. 1 to
5. Sequence similarity was analyzed using a NCBI blastx
program, and the results are shown in Table 2.
As shown in Table 2, ΦCJ1 showed 36% sequence
similarity with Dcm of bacteriophage TLS in the forward
sequence of SEQ ID NO. 1, and 31% sequence similarity with
DR0530-like primase of Burkholderia cepacia phage BcepNazgul
in the backward sequence. The backward sequence of SEQ ID
NO. 1 showed 34% sequence similarity with tail spike protein
of salmonella phage Det7. The comparison with proteins of
the known bacteriophages showed low similarity, and the base
sequence analysis of SEQ ID NOs. 1 to 5 by the NCBI blastn
program resulted in no sequence similarity. These results
indicate that ΦCJ1 is a novel bacteriophage.
[Table 2]

organism protein
Accessi
on
number
Subject
locatio
n
Query
locatio
n
identiti
es
E
value
1
Bacterioph
age TLS
Dcm
AAR0930
5
34 -
211
827 -
1351
66/181
(36%)
4e-28
Burkholder
ia cepacia
phage
BcepNazgul
DR0530-like
primase
AAQ6337
5
591 -
799
6 - 635
70/220
(31%)
2e-18
2
Salmonella
phage Det7
tailspike
protein
CAO7873
8
194 -
700
1926 -
430
178/510
(34%)
4e-76
Salmonella
phage
SETP3
tail
component
protein
ABN4733
2
1 - 357
469 -
584
38/119
(31%)
5e-09
3
Pseudomona
s phage D3
Orf65
NP_0615
61.1|
67-151 826-587
32/89
(35%)
0.010
Pseudomona
s phage
PA11
hypothetica
l protein
ORF003
YP_0012
94596.1
61-92 826-732
18/32
(56%)
0.039
4 Pseudomona putative YP_0012 403-519 4-357 43/118 6e-14
- 34 -
s phage 73 DNA
polymerase
93408 (36%)
Bacillus
cereus
E33L
GIY-YIG
catalytic
domain -
containing
protein
YP_0850
11
2-109 440-769
38/110
(34%)
2e-10
5
Phage
phiJL001
gp77
YP_2240
01.1|
38-130 627-328
36/100
(36%)
3e-06
Pseudomona
s phage 73
hypothetica
l protein
ORF019
YP_0012
93426.1
53-132 988-770
32/80
(40%)
7e-07
Example 7: Construction of ΦCJ1-specific primer
sequence
To identify ΦCJ1, ΦCJ1-specific primers were
constructed. The PCR primer sets of SEQ ID NOs. 6 and 7,
and SEQ ID NOs. 10 and 11 were constructed on the basis of
SEQ ID NO. 1. In addition, the PCR primer sets of SEQ ID
NOs. 8 and 9, and SEQ ID NOs. 12 and 13 were constructed on
the basis of SEQ ID NO. 2. Further, each primer set of SEQ
ID NOs. 14 and 15, SEQ ID NOs. 16 and 17, and SEQ ID NOs. 18
and 19 was constructed on the basis of SEQ ID NOs. 3, 4 and
- 35 -
5. PCR was performed using each primer set of SEQ ID NOs. 6
and 7, SEQ ID NOs. 10 and 11, SEQ ID NOs. 8 and 9, SEQ ID
NOs. 12 and 13, SEQ ID NOs. 14 and 15, SEQ ID NOs. 16 and 17,
and SEQ ID NOs. 18 and 19. 0.1 ㎍ of genomic DNA of
bacteriophage and 0.5 pmol of primer were added to a pre-mix
(Bioneer), and the final volume was adjusted to 20 ㎕. PCR
was performed with 30 cycles of denaturation; 94℃ 30 sec,
annealing; 60℃ 30 sec, and polymerization; 72℃, 1 min. When
SEQ ID NOs. 3 and 4, and SEQ ID NOs. 7 and 8 were used as
primer sets, PCR products of approximately 660 bp and 1.3
kbp were obtained, respectively. In addition, when SEQ ID
NOs. 5 and 6, and SEQ ID NOs. 9 and 10 were used as primer
sets, PCR products of approximately 670 bp and 1.8 kbp were
obtained, respectively. The results are shown in .
In addition, when SEQ ID NOs. 14 and 15, SEQ ID NOs. 16 and
17, and SEQ ID NOs. 18 and 19 were used as primer set, PCR
products of approximately 1 kbp were obtained (data not
shown).
Example 8: Test on Infection Efficiency of
Bacteriophage
To test the infection efficiency of the bacteriophage
ΦCJ1, a one-step growth experiment was performed.
- 36 -
50 ml of SG culture medium (OD600=0.5) was centrifuged
at 4000 rpm for 10 minutes, and resuspended in 25 ml of
fresh LB medium. The purified bacteriophage (MOI=0.0005)
was inoculated thereto, and left for 5 minutes. The
reaction solution was centrifuged at 4000 rpm for 10 minutes,
and the cell pellet was re-suspended in fresh LB medium.
While the cells were cultured at 37℃, two samples of cell
culture medium were collected every 10 minutes, and
centrifuged at 12000 rpm for 3 minutes. The obtained
supernatant was serially diluted, and 10 ㎕ of each diluted
sample was cultured at 37℃ for 18 hours by the soft agar
overlay method, and the titration of phage lysates was
performed. ΦCJ1 is a bacteriophage that is able to infect
SG and SP, simultaneously. Thus, the same experiment was
performed on SP. The result of the one-step growth
experiment on SG and SP showed the burst size of 102 or more.
The results are shown in .
Example 9: Examination of Bacteriophage Efficiency
To examine the efficiency of ΦCJ1 in a liquid medium on
SG, a clearing assay was performed under various conditions.
35 ml of LB medium was added to a 250 ml flask, and the
bacteriophage single plaque and the SG cell were inoculated
- 37 -
thereto in a cell ratio of 1:1, 1:100, and 1:10000, and then
cultured at 37℃ and 200 rpm. The changes in OD600 were
monitored at each time interval. As a result, the
inhibitory effects of ΦCJ1 on SG and SP growth were observed.
The results are shown in .
Example 10: pH Stability Test on Bacteriophage
To test the stability of ΦCJ1 in a low-pH environment
like a chicken stomach, the stability test was performed in
a wide range of pH levels (pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5,
6.4, 6.9, 7.4, 8.2, 9.0). Various pH solutions (Sodium
acetate buffer (pH 2.1, pH 4.0, pH 5.5, pH 6.4)), Sodium
citrate buffer (pH 2.5, pH 3.0, pH 3.5), Sodium phosphate
buffer (pH 6.9, pH 7.4), Tris-HCl (pH 8.2, pH 9.0)) were
prepared at a concentration of 2 M. 100 ㎕ of a pH solution
was mixed with an equal amount of bacteriophage solution
(1.0 X 1011 pfu/ml) to the concentration of each pH solution
to 1 M, and left at room temperature for 1 hour. The
reaction solution was serially diluted, and 10 ㎕ of each
diluted sample was cultured at 37℃ for 18 hours by the soft
agar overlay method, and the titration of phage lysates was
performed. Changes in the titers according to pH difference
were compared to examine the relative stability. The
- 38 -
results showed that the bacteriophage did not lose its
activity and maintained stability until pH 2.5. However, it
lost its activity at pH 2.1. The results are shown in .
Example 11: Heat Stability Test on Bacteriophage
To test the stability of the bacteriophage to heat
generated during the formulation process when used as a feed
additive, the following experiment was performed. 200 ㎕ of
ΦCJ1 solution (1.0 X 1011 pfu/ml) was left at 37℃, 45℃, 53℃,
60℃, 70℃, and 80℃ for 0 min, 10 min, 30 min, 60 min, and
120 min, respectively. The solution was serially diluted,
and 10 ㎕ of each diluted sample was cultured at 37℃ for 18
hours by soft agar overlay method, and the titration of
phage lysates was performed. Changes in the titers
according to temperature and exposure time were compared to
examine the relative stability. The results showed that the
bacteriophage did not lose its activity at 70℃ until the
exposure time of 2 hours. However, the bacteriophage
rapidly lost its activity at 80℃ or higher after exposure
for 10 minutes, but the activity was constantly maintained.
The results are shown in .
- 39 -
Example 12: Dry Stability Test on Bacteriophage
To test the stability of the bacteriophage under the
dry conditions during the formulation process when used as a
feed additive, the following experiment was performed. On
the basis of the results of the heat stability test, the
experiment was performed under high-temperature drying
conditions (at 60℃ for 120 min). 200 ㎕ of ΦCJ1 solution
(1.0 X 1011 pfu/ml) was dried using a Speed vacuum (Speed -
Vacuum Concentrator 5301, Eppendorf). The obtained pellet
was completely re-suspended in an equal amount of SM
solution at 4℃ for one day. The solution was serially
diluted, and 10 ㎕ of each diluted sample was cultured at 37℃
for 18 hours by the soft agar overlay method, and the
titration of phage lysates was performed. Changes in the
titers before and after drying were compared to examine the
relative stability. The results showed that its activity
was decreased to 102. The results are shown in .
Example 13: Examination of Bacteriophage Infection of
Wild-Type SG/SP
The lytic activity of bacteriophage ΦCJ1 was tested for
15 and 5 strains of the Korean wild-type SG and SP, isolated
by Laboratory of Avian Diseases, College of Veterinary
- 40 -
Medicine, Seoul National University, in addition to SG (SG
RKS4994) and SP(SP RKS2242) used in the present invention.
150 ㎕ of each strain shaking culture medium (OD600=2) was
mixed, and 10 ㎕ of ΦCJ1 solution (1010 pfu/ml) was cultured
at 37℃ for 18 hours by the soft agar overlay method, and
the plaque formation was examined. It was found that the
bacteriophage ΦCJ1 showed 100% lytic activity on the wildtype
SG and SP. The results are shown in .
[Table 3]

Sero
type
Strain
name
ΦCJ1 plaque
formation
Sero
type
Strain
name
ΦCJ1 plaque
formation
SG SNU SG1 O SG SNU SG11 O
SNU SG2 O SNU SG12 O
SNU SG3 O SNU SG13 O
SNU SG4 O SNU SG14 O
SNU SG5 O SNU SG15 O
SNU SG6 O SP SNU SP1 O
SNU SG7 O SNU SP 4 O
SNU SG8 O SNU SP 5 O
SNU SG9 O SNU SP 8 O
SNU O SNU SP O
- 41 -
SG10 11
* SG/SP source: Laboratory of Avian Diseases, College
of Veterinary Medicine, Seoul National University
Example 14: Toxicity test on Bacteriophage
A toxicity test on the bacteriophage ΦCJ1 for the
prevention of Fowl Typhoid was performed by evaluation of
its safety, residual amount, and eggs in layer chickens.
The layer chickens are divided into three groups to perform
a pathogenicity test and egg test, and to examine the
presence of clinical signs and phage content in the cecal
feces.
For the pathogenicity test, 13 brown layer chickens
were divided into a ΦCJ1-treated group with 8 layers and a
control group with 5 layers. The ΦCJ1-treated group was fed
with a mixture of feed and ΦCJ1 (108 pfu or more per feed
(g)) and the control group was fed with feed only, and egg
production rate and clinical signs were examined for 3 weeks
after phage treatment. As shown in , the ΦCJ1-
treated group and the control group showed about 42% and 50%
egg production rates, respectively. In addition, clinical
signs after phage treatment were examined, resulting in that
respiratory and digestive lesions were not observed for 24
- 42 -
days after ΦCJ1 treatment, and abnormal activity was not
observed. The results indicate that ΦCJ1 treatment does not
generate adverse effects.
For the egg test, 10 eggs were collected on day 3, 6,
and 9 after ΦCJ1 treatment, and the egg surface was washed
with 70% ethanol and broken out. Egg yolk and egg white
were mixed, and 5 ml of the mixture was diluted with 45 ml
of PBS by 10-1, 10-2, and 10-3. 106 cfu of SNUSG0197 was added
to 25 ml of each diluted solution, and incubated at 37℃ for
3 hours, and cells were isolated by centrifugation. 500 ㎕
of supernatant and 100 ㎕ of SNUSG0197 (109 cfu/ml) were
mixed with each other, and plated on a tryptic soy agar
plate by the top-agar overlay technique. After incubation
at 37℃ for 18 hrs, the number of plaque was counted to
calculate the number of phage per 1 ml of egg. As shown in
, no ΦCJ1 was found in 17 eggs that were collected
on day 3, 6, and 9.
Next, the presence of clinical signs and ΦCJ1 content
in the cecal feces were examined after ΦCJ1 treatment. At 3
weeks after ΦCJ1 treatment, the test layer chickens were
euthanatized, and necropsy was performed to examine gross
lesions in the liver, spleen, kidney and ovary. The liver
sample was aseptically collected with sterile cotton swab,
- 43 -
and plated on a Mac Conkey agar plate to examine the
presence of Salmonella Gallinarum. The cecal feces were
also collected to measure the ΦCJ1 content in the individual
chickens. Briefly, 1 g of cecal feces was suspended in 9 ml
of PBS, and centrifuged at 15000 g for 30 min. 1 ml of
supernatant was diluted with PBS by 10-1 to 10-4, and 500 ㎕
of the dilution and 100 ㎕ of SG0197 (109 cfu/ml) were mixed
with each other, and plated on a 10x tryptic soy agar plate
by the top-agar overlay technique. After incubation at 37℃
for 18 hrs, the number of plaque was counted to calculate
the number of phage per cecal feces (g), taking into account
the serial dilution. As a result, no abnormal clinical
signs were observed during the examination period, and about
3.7 x 104 pfu of ΦCJ1 per cecal feces (g) was measured,
indicating survival of the bacteriophage in the intestine
after passing through the stomach.
Bacteriophage distribution in the organs was examined.
Briefly, 10 SPF chicks (11 day-old) were divided into two
groups with 5 chicks each. For 3 days, the treated group
was fed with feed supplemented with 108 pfu of ΦCJ1 (per g),
and the control group was fed with feed only. The chicks
were sacrificed to collect the liver, kidney and cecal feces,
and the presence of ΦCJ1 was examined. Each of the
- 44 -
collected liver, kidney and cecal feces was emulsified with
an equal volume of PBS. 1 ml of the liver and the whole
quantity of the kidney and cecal feces were transferred into
1.5 ml tubes, followed by centrifugation at 15,000 rpm for
15 min. 1 ml of supernatant was diluted with PBS by 10-1 to
10-4, and 500 ㎕ of the dilution and 100 ㎕ of SG0197 (109
cfu/ml) were mixed with each other, and plated on a 10x
tryptic soy agar plate by the top-agar overlay technique.
After incubation at 37℃ for 18 hrs, the number of plaque was
counted to calculate the number of bacteriophage per cecal
feces (g), taking into account the serial dilution. As
shown in , ΦCJ1 was not observed in the liver and
kidney, but in the cecal feces.

ΦCJ1 control
1 day Feeding day
2 day
3 day 7 5
6 day 8 7
7 day 3 3
8 day 6 4
- 45 -
9 day 2 1
10 day 3 3
13 day 11 8
14 day
15 day 10 7
16 day
17 day 6 6
20 day 11 7
21 day
22 day 9 4
23 day
24 day 6 3
Egg
production
rate
42.2% 50.8%

Collection
day
ΦCJ1 control
3 day 0/7 0/5
6 day 0/8 0/7
- 46 -
9 day 0/2 0/1
Total 0/17 0/13

Test
chicken
ΦCJ1-treated group Control group
liver kidney cecal
feces
liver kidney cecal
feces
1 - - + - - -
2 - - + - - -
3 - - + - - -
4 - - + - - -
5 - - + - - -
Example 15: Efficacy test on Bacteriophage
In order to evaluate the efficacy of ΦCJ1 on the
prevention and treatment of SG, an efficacy test was
performed in chickens.
20 brown layers (1-day-old) were divided into 10 test
groups with 10 layers each (ΦCJ1 treated group + non-treated
challenged group 1). For 1 week, the test chicks were fed
with feed supplemented with 107 pfu of ΦCJ1 (per g) and
drinking water supplemented with 107 pfu of ΦCJ1 (per ml).
At 1 week, 106 cfu of SG0197 (per chick) and 107 pfu (MOI=10)
of phage was mixed with 500 ㎕ of TSB, and left in ice for 1
hour or less, followed by oral administration. The
- 47 -
mortality rate was examined for 2 weeks. The surviving
chicks were subjected to necropsy and examined for gross
lesions, and the bacteria were isolated. As shown in , it was found that the ΦCJ1-treated group showed a
significantly higher protection rate (P<0.05) than the nontreated
group.

ΦCJ1-treated
challenged
group
Non-treated
challenged
group
Survival 8 3
Mortality
rate 20% 70%
Clinical
signs 1/8 1/3
SG
reisolation 0/8 0/3
Protection
rate 70% 20%
- 48 -
[Industrial Applicability]
The bacteriophage of the present invention has a
specific bactericidal activity against Salmonella Gallinarum
and Salmonella Pullorum without affecting beneficial
bacteria, and excellent acid-, heat- and dry-resistance, and
thus can be used for the prevention and treatment of Fowl
Typhoid and Pullorum disease that are caused by Salmonella
Gallinarum and Salmonella Pullorum, and also applied to
control Salmonella Gallinarum and Salmonella Pullorum. For
example, the bacteriophage is used for a composition for the
prevention or treatment of infectious diseases caused by
Salmonella Gallinarum or Salmonella Pullorum, feed and
drinking water for poultry, sanitizers and cleaners, as an
active ingredient.
- 49 -
[CLAIMS]
[Claim 1]
A bacteriophage that has a specific bactericidal
activity against Salmonella Gallinarum and Salmonella
Pullorum, belonging to the morphotype group B1 of the family
Siphoviridae, characterized by isometric capsid and long
non-contractile tail, and having a total genome size of 61
kbp and major structural proteins with a size of 38 kDa and
49 kDa.
[Claim 2]
The bacteriophage according to claim 1, wherein the
bacteriophage has the morphology depicted in FIG. 1.
[Claim 3]
The bacteriophage according to claim 1, wherein the
bacteriophage include one or more nucleic acid molecules
selected from the group consisting of SEQ ID NOs. 1, 2, 3, 4,
and 5 within the entire genome.
[Claim 4]
The bacteriophage according to claim 1, wherein the
bacteriophage has each PCR product of approximately 660 bp,
670 bp, 1.3 kbp, 1.8 kbp, 1 kbp, 1 kbp, and l kbp when PCR
was performed using one or more primer sets selected from
the group consisting of SEQ ID NOs. 6 and 7, SEQ ID NOs. 8
- 50 -
and 9, SEQ ID NOs. 10 and 11, SEQ ID NOs. 12 and 13, SEQ ID
NOs. 14 and 15, SEQ ID NOs. 16 and 17, and SEQ ID NOs. 18
and 19.
[Claim 5]
The bacteriophage according to claim 1, wherein the
bacteriophage has acid-resistance in a pH range from pH 2.5
to pH 9.0, heat-resistance in a temperature range from 37℃
to 70℃, and dry-resistance at 60℃ for 120 minutes.
[Claim 6]
The bacteriophage according to claim 1, wherein the
bacteriophage is identified by accession number KCCM10969P.
[Claim 7]
A composition for the prevention or treatment of
infectious diseases caused by Salmonella Gallinarum or
Salmonella Pullorum, comprising the bacteriophage of claim 1
as an active ingredient.
[Claim 8]
The composition according to claim 7, wherein the
infectious disease caused by Salmonella Gallinarum is Fowl
Typhoid, and the infectious disease caused by Salmonella
Pullorum is Pullorum disease.
[Claim 9]
The composition according to claim 7, wherein the
- 51 -
composition is used as an antibiotic.
[Claim 10]
An animal feed or drinking water, comprising the
bacteriophage of any one of claims 1 to 6 as an active
ingredient.
[Claim 11]
A sanitizer or cleaner, comprising the bacteriophage of
any one of claims 1 to 6 as an active ingredient.
[Claim 12]
A method for treating infectious diseases caused by
Salmonella Gallinarum or Salmonella Pullorum in animals,
except for human, using the bacteriophage of claim 1 or the
composition of claim 7.
Dated this 07th day of September 2009
G. ARUN KUMAR
OF K & S PARTNERS
AGENT FOR THE APPLICANT