Bacteriophage and antibacterial composition comprising the same

23 Pages·2014·2.19 MB·English

US008771936B2
(12) United States Patent (10) Patent N0.: US 8,771,936 B2
Kang et a1. (45) Date of Patent: *Jul. 8, 2014
(54) BACTERIOPHAGE AND ANTIBACTERIAL (58) Field of Classi?cation Search
COMPOSITION COMPRISING THE SAME None
See application ?le for complete search history.
(71) Applicant: CJ Cheiljedang Corporation, Seoul
(KR) (56) References Cited
(72) Inventors: In Hye Kang, Suwon-si (KR); Min Tae U.S. PATENT DOCUMENTS
Park, Seoul (KR); Young Wook Cho,
6,485,902 B2 11/2002 Waddell et a1.
Seoul (KR); $00 An Shin, Seoul (KR);
6,942,858 B1 9/2005 Ghanbari et al.
Hyang Choi, Anyang-si (KR)
8,288,146 B2 * 10/2012 Kang et a1. ............... .. 435/235.1
2004/0208853 A1 10/2004 Sulakvelidze et al.
(73) Assignee: CJ Cheiljedang Corporation, Seoul
OTHER PUBLICATIONS
(KR)
O’Flynn, et al. Journal oprplied Microbiology, 2006, 101 :25 1-259.
( * ) Notice: Subject to any disclaimer, the term of this Barrangou, et al. Applied and Environmental Microbiology, 2002,
patent is extended or adjusted under 35 68(11):5452-5458.
U.S.C. 154(b) by 0 days. Goto, et al. J. Food Hyg. Soc. Japan, 2004, 45(1):25-28.
This patent is subject to a terminal dis * cited by examiner
claimer.
Primary Examiner * Bao Li
(21) App1.No.: 13/621,730 (74) Attorney, Agent, or Firm * Seed IP Law Group PLLC
(22) Filed: Sep. 17, 2012 (57) ABSTRACT
The present invention relates to a novel bacteriophage, more
(65) Prior Publication Data
particularly, a bacteriophage that has a speci?c bactericidal
US 2013/0022579 A1 Jan. 24, 2013 activity against Salmonella typhimurium, Salmonella galli
narum, or Salmonella pullorum, a composition for the pre
vention or treatment of infectious diseases including salmo
Related US. Application Data
nellosis and Salmonella food poisoning caused by
(63) Continuation-in-part of application No. 12/560,263, Salmonella typhimurium, Fowl typhoid caused by Salmo
?led on Sep. 15, 2009, noW Pat. No. 8,288,146. nella gallinarum, and Pullorum disease caused by Salmo
nella pullorum, Which comprises the bacteriophage as an
(30) Foreign Application Priority Data active ingredient, and an animal feed, drinking water, cleaner,
and sanitizer Which comprise the bacteriophage as an active
Dec. 24, 2008 (KR) ...................... .. 10-2008-0133909 ingredient. The present invention also provides important
insights into prevention and control strategies against Salmo
(51) Int. Cl. nella infection and suggests that the use of bacteriophage can
C12Q 1/70 (2006.01) be a novel, safe, and effectively plausible alternative to anti
A61K 39/112 (2006.01) biotics for the prevention of Salmonella infection in poultry.
(52) US. Cl.
USPC ............................................ .. 435/5; 424/936 2 Claims, 5 Drawing Sheets
US. Patent Jul. 8, 2014 Sheet 1 0f5 US 8,771,936 B2
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US. Patent Jul. 8,2014 Sheet 2 0f5 US 8,771,936 B2
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US. Patent Jul. 8,2014 Sheet 5 0f5 US 8,771,936 B2
FIGUREIO
100 +Group1
30 (SG+/phage+)
g -E—Group1
E0’ 60 (SG-lphage+)
.g 40 “ +Group2
é (SG+/phage-)
20 —I—Group2
(SG-lphage-)
0
1 3 5 7 9 111315171921
Days after infection
US 8,771,936 B2
1 2
BACTERIOPHAGE AND ANTIBACTERIAL the past century, Pullorum disease as an egg-transmitted
COMPOSITION COMPRISING THE SAME infection has seriously affected young chickens at 1-2 weeks
of age or younger in the world and Korea. In the 1980’s,
CROSS-REFERENCE TO RELATED disease occurrence greatly decreased. However, incidence
APPLICATIONS began increasing in the mid 1990’s (Kwon Yong-Kook. 2000
annual report on avian diseases. Information publication by
The present application is a continuation-in-part of and National Veterinary Research & Quarantine Service. March,
claims priority to co-pending US. patent application Ser. No. 2001; KimAe-Ran et al., The prevalence ofp ullorum disease
12/5 60,263, entitled “Novel Bacteriophage and Antibacterial fowl typhoid in grandparent stock and parent stock in Korea,
Composition Comprising the Same,” ?led in the US. Patent 2003, Korean J Vet Res, 2006, 46(4): 347-353).
and Trademark Of?ce on Sep. 15, 2009 and having a common In Korea, outbreaks of Fowl Typhoid and Pullorum disease
inventor as the present document which claims priority to KR have been increasing since the 1990’s, in?icting economic
10-2008-0133909, ?led Dec. 24, 2008. The above-referenced damages on farmers. For this reason, a live attenuated SG
applications are incorporated herein by reference. vaccine has been used in broilers for the prevention of Fowl
Typhoid from 2004 (Kim Ae-Ran et al., The prevalence of
TECHNICAL FIELD pullorum disease-fowl typhoid in grandparent stock and par
ent stock in Korea, 2003, Korean J Vet Res, 2006, 46(4):
The present invention relates to a novel bacteriophage, a 347-353), even though its ef?cacy is doubtful, and the live
composition comprising the bacteriophage, and a method for vaccine is not allowed to be used for layers because of the risk
preventing infectious diseases caused by Salmonella using 20 of egg-transmitted infections. Unfortunately, there are still no
the bacteriophage. commercially available preventive strategies against
Pullorum disease, unlike Fowl Typhoid. Thus, there is an
BACKGROUND ART urgent need for new ways to prevent Fowl Typhoid and
Pullorum disease.
Salmonella is a genus of the family Enterobacteriaceae, 25 Meanwhile, Salmonella ljxphimurium (hereinbelow, des
characterized as Gram-negative, facultatively anaerobic, non ignated as ST) is a zoonotic pathogen which shows no host
spore-forming, rod-shaped bacteria, and most strains are speci?city, unlike SG or SP (Zoobises Report; United King
motile by ?agella. Salmonella has an average genome GC dom 2003).
content of 50-52%, which is similar to those of Escherichia ST is a cause of salmonellosis in poultry, pigs, and cattle,
coli and Shigella. The genus Salmonella is a pathogenic 30 etc. Salmonellosis is caused by Salmonella bacteria, an acute
microorganism that causes infections in livestock as well as in or chronic infection of the digestive tract in livestock, and
human. Salmonella enierica, a species ofSalmonella bacte shows the major symptoms of fever, enteritis, and septicemia,
rium, has a variety of serovars including Gallinarum, occasionally pneumonia, arthritis, abortion, and mastitis. Sal
Pullorum, Dphimurium, Enlerilidis, Dphi, Choleraesuis, monellosis occurs worldwide, and most frequently during the
and derby (Bopp C A, Brenner F W, Wells J G, Strokebine N 35 summer months (T. R. Callaway et al., J. Anim. Sci. 86:
A. Escherichia, Shigella, Salmonella. In Murry P R, Baron E E163-E172, 2008). In cattle, typical symptoms include loss of
J, et al., eds Manual of clinical Microbiology. 7th ed., Wash appetite, fever, dark brown diarrhea or bloody mucous stool.
ington DC. American Society for Microbiology 1999; 467 Acute infection in calves leads to rapid death, and infection
74; Ryan K J. Ray C G (editors), 2004, Sherris Medical during pregnancy leads to fetal death due to septicemia,
Microbiology (4th ed). McGraw Hill. ISBN 0-8385-8529-9). 40 resulting in premature abortion. In pigs, salmonellosis is
Of them, Salmonella Gallinarum and Pullorum are fowl characterized clinically by three major syndromesiacute
adapted pathogens, Salmonella Dphi is a human-adapted septicemia, acute enteritis, and chronic enteritis. Acute sep
pathogen, Salmonella Choleraesuis and derby are swine ticemia occurs in 2-4 month-old piglets, and death usually
adapted pathogens, and Salmonella Enteritis and Typhimu occurs within 2-4 days after onset of symptoms. Acute enteri
rium are pathogenic for both human and animals. Each sero 45 tis occurs during the fattening period, and is accompanied by
var causes illness in that species, resulting in serious negative diarrhea, high fever, pneumonia, and nervous signs. Discol
effects for farmers or consumers. oration of the skin may occur in some severe cases. Chronic
A disease of domestic birds caused by Salmonella bacte enteritis is accompanied by continuing diarrhea.
rium is Fowl Typhoid (FT), which is caused by a pathogen, Once an outbreak of salmonellosis occurs in poultry, pigs,
Salmonella Gallinarum (hereinbelow, designated as SG). 50 and cattle, it is dif?cult to halt with therapeutic agents. The
Fowl Typhoid (FT) is a septicemic disease of domestic birds reasons are that Salmonella bacteria exhibit a strong resis
such as chicken and turkey, and the course may be acute or tance to various drugs and live in cells being impermeable to
chronic with high mortality. Recently, it has been reported antibiotics. Up to now, there have been no methods for effec
that Fowl Typhoid frequently occurs in Europe, South tively treating salmonellosis caused by ST, including antibi
America, Africa, and South-East Asia, and damages are 55 otics.
increasing. Outbreaks of FT in Korea have been reported In addition to livestock, ST causes infections in human via
since 1992 and economic losses from FT in brown, egg livestock products, leading to Salmonella food poisoning.
laying chickens are very serious (Kwon Yong-Kook. 2000 Consumption of infected, improperly cooked livestock prod
annual report on avian diseases. Information publication by ucts (e.g., meat products, poultry products, eggs and by
National Veterinary Research & Quarantine Service. March, 60 products) is a cause of human infection. Salmonella food
2001 ; KimAe-Ran et al., The prevalence ofp ullorum disease poisoning in human usually involves the prompt onset of
fowl typhoid in grandparent stock and parent stock in Korea, headache, fever, abdominal pain, diarrhea, nausea, and vom
2003, Korean J Vet Res, 2006, 46(4): 347-353). iting. The symptoms commonly appear within 6-72 hours
Pullorum disease is also caused by one of Salmonella bac after the ingestion of the organism, and may persist for as long
Zeria, Salmonella Pullorum (hereinbelow, designated as SP). 65 as 4-7 days or even longer (NSW+HEALTH. 2008 Jan. 14).
Pullorum disease occurs in any age or season, but young According to a report by the CDC (The Centers for Disease
chickens are particularly susceptible to the disease. During Control and Prevention, USA), 16% of human food poisoning
US 8,771,936 B2
3 4
outbreaks between 2005 and 2008 attributed to Salmonella their head shape and components, and the presence of a shell.
bacteria, and 18% of them were ST (Salmonella Typhimu Filamentous bacteriophages having DNA as their genetic
rium). With respect to Salmonella food poisoning in human material are divided based on their size, shape, shell, and
between 1973 and 1984, the implicated food vehicles of ?lament components (H. W. Ackermann. Frequency of mor
transmission were reportedly chicken (5%), beef (19%), pork phological phage descriptions in the year 2000; Arch Virol,
(7%), dairy products (6%), and turkey (9%). In 1974-1984, 2001, 146: 843-857; Elizabeth Kutter et al. Bacteriophages
the bacterial contamination test on broilers during the slaugh biology and application; CRC press).
ter process showed 35% or more of Salmonella incidence. In During infection, a bacteriophage attaches to a bacterium
1983, Salmonella was isolated in 50.6% ofchicken, 68.8% of and inserts its genetic material into the cell. After this a
turkey, 60% of goose, 11.6% of pork, and 1.5% of beef. bacteriophage follows one of two life cycles, lytic or
Further, a survey carried out in 2007 reported that salmonella lysogenic. Lytic bacteriophages take over the machinery of
was found in 5.5% of raw poultry meat and 1.1% of raw pork. the cell to make phage components. They then destroy or lyse
In particular, it was revealed that ST commonly originated the cell, releasing new phage particles. Lysogenic bacterioph
from contaminated pork, poultry meat, and beef (Centers for ages incorporate their nucleic acid into the chromosome of
Disease Control and Prevention, CDC). A risk assessment the host cell and replicate with it as a unit without destroying
conducted by FAO and WHO in 2002 noted that the human the cell. Under certain conditions, lysogenic phages can be
incidence of salmonellosis transmitted through eggs and induced to follow a lytic cycle (Elizabeth Kutter et al. Bacte
poultry meat appeared to have a linear relationship to the riophages biology and application. CRC press).
observed Salmonella prevalence in poultry. This implies that, After the discovery of bacteriophages, a great deal of faith
by reducing the prevalence of Salmonella in poultry, the 20 was initially placed in their use for infectious -disease therapy.
incidence of salmonellosis in humans will correspondingly However, when broad spectrum antibiotics came into com
fall (Salmonella control at the source; World Health Organi monuse, bacteriophages were seen as unnecessary because of
zation. International Food Safety Authorities Network (IN having a speci?c target spectrum. Nevertheless, the misuse
FOSAN) Information Note No. 03/2007). Recently, fears and overuse of antibiotics resulted in rising concerns about
about food safety have been spurred by outbreaks of Salmo 25 antibiotic resistance and harmful effects of residual antibiot
nella from products as varied as peanuts, spinach, tomatoes, ics in foods (Cislo, M et al. Bacteriophage treatment of sup
pistachios, peppers and, most recently, cookie dough (Jane purative skin infections. Arch Immunol. Ther. Exp. 1987, 2:
Black and Ed O’Keefe. Overhaul of Food Safety Rules in the 175-183; Kim sung-hun et al., Bacteriophage; New Altema
Works. Washington Post Staff Writers Wednesday, Jul. 8, tive Antibiotics. Biological Research Information Center,
2009). 30 BRIC). In particular, antimicrobial growth promoters
For these reasons, Salmonella infections must be reported (AGPs), added to animal feed to enhance growth, is known to
in Germany (§6 and §7 of the German law on infectious induce antibiotic resistance, and therefore, a ban on the use of
disease prevention, Infektionsschutzgesetz). Between 1990 AGPs has been recently introduced. In the European Union,
and 2005 the number of of?cially recorded cases decreased the use of all antimicrobial growth promoters (AGPs) was
from approximately 200,000 cases to approximately 50,000. 35 banned from 2006. Korea has banned the use of some AGPs
It is estimated that every ?fth person in Germany is a carrier from 2009, and is considering restrictions on the use of all
of Salmonella. In the USA, there are approximately 40,000 AGPs by 2013-2015.
cases of Salmonella infection reported each year. These growing concerns about the use of antibiotics have
Therefore, there is an urgent need to control ST, which led to a resurgence of interest in bacteriophage as an altema
causes salmonellosis in livestock and human. The collabora 40 tive to antibiotics. 7 bacteriophages for control of E. coli
tive efforts of USDA and FDA have led to the development of O157:H are disclosed in US. Pat. No. 6,485,902 (applied in
a number of effective strategies to prevent salmonellosis that 2002-Use of bacteriophages for control of Escherichia coli
causes over 1 million cases of foodborne illness in the United 0157). 2 bacteriophages for control of various microorgan
States. In Denmark, conservative estimates from a cost ben isms are disclosed in US. Pat. No. 6,942,858 (applied by
e?t analysis comparing Salmonella control costs in the pro 45 Nymox in 2005). Many companies have been actively trying
duction sector with the overall public health costs of salmo to develop various products using bacteriophages. EBI food
nellosis suggest that Salmonella control measures saved the system (Europe) developed a food additive for preventing
Danish society US$ 14.1 million inthe year 2001 (Salmonella food poisoning caused by Listeria monocytogenes, named
control at the source. World Health Organization. Interna Listex-P100, which is the ?rst bacteriophage product
tional Food Safety Authorities Network (INFOSAN) Infor 50 approved by the US FDA. A phage-based product, LMP-102
mation Note No. 03/ 2007). was also developed as a food additive against Listeria mono
Meanwhile, a bacteriophage is a specialized type of virus cytogenes, approved as GRAS (Generally regarded as safe).
that only infects and destroys bacteria, and can self-replicate In 2007, a phage-based wash produced by OmniLytics was
only inside a host bacteria. A bacteriophage consists of developed to prevent E. coli Ol57 contamination of beef
genetic material being single or double-stranded DNA or 55 during slaughter, approved by USDA’s Food Safety and
RNA surrounded by a protein shell. There are three basic Inspection Service (FSIS). In Europe, Clostridium sporo
structural forms of bacteriophage: an icosahedral (twenty genes phage NCIMB 30008 and Clostridium tyrobutiricum
sided) head with a tail, an icosahedral head without a tail, and phage NCIMB 30008 were registered as a feed preservative
a ?lamentous form. Bacteriophages are classi?ed based on against Clostridium contamination of feed in 2003 and 2005,
their morphological structure and genetic material. Based on 60 respectively. Such studies show that research into bacterioph
their tail structure, bacteriophages having an icosahedral head ages for use as antibiotics against zoonotic pathogens in live
and double-stranded, linear DNA as their genetic material are stock products is presently ongoing.
divided into three families: Myoviridae, Siphoviridae, and However, most of the phage biocontrol studies have
Podoviridae, which are characterized by contractile, long focused on the control of E. coli, Listeria, and Clostridium.
noncontractile, and short noncontractile tails, respectively. 65 Salmonella is also a zoonotic pathogen, and damages due to
Bacteriophages having an icosahedral head without a tail and this pathogen are signi?cant. As mentioned above, since ST
RNA or DNA as their genetic material are divided based on has a multiple drug resistance, nationwide antimicrobial
US 8,771,936 B2
5 6
resistance surveillance has been conducted in Korea under an rium, Salmonella gallinarum, and Salmonella pullorum, and
Enforcement Decree of the Act on the Prevention of Conta excellent acid-, heat- and dry-resistance. Thus, it can be used
gious Disease (Executive Order 16961), an Enforcement for the prevention or treatment of infectious diseases caused
ordinance of the Act on the Prevention of Contagious Disease by Salmonella Zyphimurium, Salmonella gallinarum or Sal
(Ministry of Health and Welfare’s Order 179), and Organiza
monella pullorum, including salmonellosis, Salmonella food
tion of the National Institute of Health (Executive Order
poisoning, Fowl typhoid or Pullorum disease, and also used
17164). Accordingly, there is a need for the development of
for the control of Salmonella typhimurium, Salmonella galli
bacteriophages to control Salmonella.
narum and Salmonella pullorum.
DISCLOSURE
DESCRIPTION OF DRAWINGS
Technical Problem
FIG. 1 is an electron microscopy photograph of (IJCJ2, in
In order to solve the problems including antibiotic resis which CI>CJ2 belongs to the morphotype of the family
tance due to the misuse and overuse of antibiotics, harmful Siphoviridae, characterized by isometric capsid and long
effects of residual antibiotics in foods, and the problems
non-contractile tail;
generated by the use of broad spectrum antibiotics, the
FIG. 2 is the result of SDS-PAGE of the isolated bacte
present inventors isolated from natural sources a novel Sal
riophage CDC] 2, in which protein patterns of the bacterioph
monella bacteriophage having a speci?c bactericidal activity
against Salmonella which causes major diseases in livestock, age are shown, including a major protein of approximately 40
and identi?ed its morphological, biochemical, and genetic 20 kDa and other proteins of approximately 65 kDa, 17 kDa, and
properties. The present inventors found that the bacterioph 15 kDa (See-blue plus 2 prestained-standard (Invitrogen)
age has a speci?c bactericidal activity against Salmonella used as marker);
Zyphimurium (ST), Salmonella gallinarum (SG) and Salmo FIG. 3 is the result of PFGE of the isolated bacteriophage
nellapullorum (SP) without affecting bene?cial bacteria, and (IJCJ2, showing the total genome size of approximately 43
excellent acid-, heat- and dry-resistance, and thus can be
25 kbp (5 kbp DNA size standard (Bio-rad) as size marker);
applied to the compositions that can be used for the preven FIG. 4 is the result of PCR, performed using each primer
tion or treatment of livestock salmonellosis caused by Salmo
set of CI>CJ2 genomic DNA, in which (A; PCR ampli?cation
nella lyphimurium, Salmonella food poisoning caused by
using primer set of SEQ ID NOs. 4 and 5, B; PCR ampli?ca
contaminated livestock products, and infectious diseases
tion using primer set of SEQ ID NOs. 6 and 7, C; PCR
caused by Salmonella gallinarum or Salmonellapullorum, in
ampli?cation using primer set of SEQ ID NOs. 8 and 9) each
particular, Fowl typhoid or Pullorum disease, and to various
of A, B, and C lanes shows a PCR product of approximately
products to control Salmonella, such as animal feed additive
1 kbp;
and drinking water for livestock, barn sanitizers, and cleaners
for meat products, thereby completing the present invention. FIG. 5 is the result of acid-resistance test on the bacterioph
age CDC] 2, showing the number of surviving bacteriophage at
Technical Solution 35 pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 6.9, 7.4, 8.0, and 9.0, in
which the bacteriophage CI>CJ2 retains its infectivity to pH
It is an object of the present invention to provide a novel 2.5, but entirely loses its infectivity at pH 2.1, as compared to
bacteriophage having a bactericidal activity against Salmo control;
nella Zyphimurium, Salmonella gallinarum or Salmonella FIG. 6 is the result of heat-resistance test on the bacterioph
pullorum. 40 age CDC] 2, showing the number of surviving bacteriophage at
It is another object of the present invention to provide a 37, 45, 53, 60, 70, and 80° C. and a time point of0, 10, 30, 60,
composition for the prevention or treatment of infectious 120 min, in which the bacteriophage CI>CJ2 retains its infec
diseases caused by Salmonella lyphimurium, Salmonella
tivity even after incubation at 60° C. for 2 hrs, but entirely
gallinarum or Salmonella pullorum, comprising the bacte
loses its infectivity after incubation at 70° C. for 10 min;
riophage as an active ingredient, in particular, a composition
45 FIG. 7 is the result of dry-resistance test on the bacterioph
for the prevention or treatment of the infectious diseases
age (IDCJ2, performed at 60° C. for 120 min using a speed
which is used as an antibiotic.
vacuum dryer (SVD), in which changes in viral titers before
It is still another object of the present invention to provide
and after drying were compared to examine the relative sta
an animal feed and drinking water, comprising the bacte
bility, and the phage’ s infectivity was shown not to decrease;
riophage as an active ingredient.
It is still another object of the present invention to provide 50 FIG. 8 is the result of single dose oral toxicity study of
CDC] 2 in rats, showing changes in body weight (0; male
a sanitizer and cleaner, comprising the bacteriophage as an
active ingredient. control group treated with the mixed solution of 20 mM
It is still another object of the present invention to provide Tris-HCl and 2 mM MgC12, O; male test group treated with
a method for preventing or treating livestock salmonellosis 1><1012 pfu of (IJCJ2, B; female control group treated with the
caused by Salmonella lyphimurium or Salmonella food poi 55 mixed solution of 20 mM Tris-HCl and 2 mM MgC12, I;
soning caused by contaminated livestock products, using the female test group treated with 1><1012 pfu of CDC] 2), in which
composition that comprises the bacteriophage as an active no signi?cant changes in body weight were observed even
ingredient. after 14 days; and
It is still another object of the present invention to provide FIG. 9 is the result of single dose intravenous toxicity study
a method for preventing or treating infectious diseases, Fowl 60 of CI>CJ2 in rats, showing changes in body weight (0; male
Typhoid or Pullorum disease caused by Salmonella galli control group treated with the mixed solution of 20 mM
narum and Salmonella pullorum. Tris-HCl and 2 mM MgC12, O; male test group treated with
1><1012 pfu of (IJCJ2, B; female control group treated with the
Advantageous Effect mixed solution of 20 mM Tris-HCl and 2 mM MgC12, I;
65 female test group treated with 1><1012 pfu of CDC] 2), in which
The novel bacteriophage of the present invention has a no signi?cant changes in body weight were observed even
speci?c bactericidal activity against Salmonella lyphimu after 14 days.
US 8,771,936 B2
7 8
FIG. 10 is the results of comparing efficacy of CI>CJ2 on In accordance with the speci?c Example of the present
survival rate of SG challenged and contact chickens. Six invention, the present inventors collected sewage samples at
week-old chickens, each challenged with 5><108 CFUs of SG, chicken slaughterhouses to isolate bacteriophages that lyse
cohabited with contact chickens treated with 106 PFU/kg of the host cell ST, and they con?rmed that the bacteriophages
CI>CJ2 prepared in feed additives for 7 days before, and 21 are able to lyse SG and SP, speci?cally (Table 1). Further, they
days after challenge with SG. Mortality was observed for 3 examined the bacteriophage (CIJCJ2) under electron micro
week after challenge. Asterisk (*) indicates signi?cant differ scope, and found that it belongs to the morphotype of the
family Siphoviridae (FIG. 1).
ence (P<0.05) between (IJCJ2-treated and untreated contact
Further, the protein patterns of the bacteriophage (CIDCJ2
chickens.
were also analyzed, revealing a major structural protein with
a size of approximately 40 kDa (FIG. 2).
BEST MODE
Furthermore, the total genome size of the bacteriophage
CDC] 2 was also analyzed, revealing that it has a total genome
In accordance with an aspect, the present invention relates
size of approximately 43 kbp (FIG. 3). The results of analyz
to a novel bacteriophage having a speci?c bactericidal activ
ing its genetic features showed that the bacteriophage
ity against Salmonella Zyphimurium, Salmonella gallinarum
includes nucleic acid sequences represented by SEQ ID NOs.
or Salmonella pullorum.
l to 3 within the total genome (Example 6). Based on these
The bacteriophage of the present invention belongs to the
results, genetic similarity with other species was compared. It
morphotype of the family Siphoviridae, characterized by iso
was found that the bacteriophage showed very low genetic
metric capsid and long non-contractile tail, and has a total 20 similarity with the known bacteriophages, indicating that the
genome size of 43 kbp and a major structural protein with a bacteriophage is a novel bacteriophage (Table 2). More par
size of 40 kDa. ticularly, the (IJCJ2-speci?c primer set, in particular, SEQ ID
Speci?cally, the bacteriophage of the present invention has NOs. 4 and 5, SEQ ID NOs. 6 and 7, and SEQ ID NOs. 8 and
the capability of selectively infecting Salmonella lyphimu 9, was used to perform PCR. Each PCR product was found to
rium, Salmonella gallinarum and Salmonella pullorum, 25 have a size of approximately 1 kbp (FIG. 4).
namely, species speci?city. Further, when SG and SP were infected with (IJCJ2, the
The bacteriophage of the present invention genetically has phage plaques (clear zone on soft agar created by host cell
a total genome size of 43 kbp, and may include one or more lysis of one bacteriophage) showed the same size and turbid
nucleic acid molecules selected from the group consisting of ity.
SEQ ID NOs. l, 2, and 3 within the entire genome, preferably 30 Furthermore, the stability of CI>CJ2 was examined under
nucleic acid molecules represented by SEQ ID NOs. l to 3 various temperature and pH conditions, revealing that CI>CJ2
within the entire genome. stably maintains its infectivity in a wide range of pH environ
When the bacteriophage of the present invention is sub ments from pH 2.5 to pH 9.0 (FIG. 5) and in a high tempera
jected to PCR using one or more primer sets selected from the ture environment from 370 C. to 600 C. (FIG. 6), and even
group consisting ofSEQ ID NOs. 4 and 5, SEQ ID NOs. 6 and 35 after high-temperature drying at 600 C. for 120 minutes (FIG.
7, and SEQ ID NOs. 8 and 9, each PCR product has a size of 7). These results indicate that the bacteriophage CI>CJ2 of the
approximately 1 kbp. Preferably, when PCR is performed present invention can be readily applied to various products
using all of the primer sets, each PCR product has a size of for the control of Salmonella.
approximately 1 kbp. In accordance with another aspect, the present invention
As used herein, the term “nucleic acid molecule” encom 40 relates to a composition for the prevention or treatment of
passes DNA (gDNA and cDNA) and RNA molecules, and the infectious diseases caused by one or more selected from the
term nucleotide, as the basic structural unit of nucleic acids, group consisting of Salmonella typhimurium, Salmonella
encompasses natural nucleotides and sugar or base-modi?ed gallinarum and Salmonella pullorum, comprising the bacte
analogues thereof. riophage as an active ingredient.
The genome of the bacteriophage of the present invention 45 In one preferred embodiment, the therapeutic composition
encodes a major structural protein with a size of 40 kDa. may include an antibiotic.
Further, the bacteriophage of the present invention has the The bacteriophage of the present invention has a speci?c
biochemical properties of acid- and heat-resistance, in which bactericidal activity against Salmonella lyphimurium, Salmo
it can stably retain its infectivity in a wide range of pH nella gallinarum and Salmonella pullorum, and thus can be
environments from pH 2.5 to pH 9.0, and in a high tempera 50 used for the purpose of preventing or treating diseases that are
ture environment from 37° C. to 60° C. In addition, the caused by these bacteria. Preferably, examples of the diseases
bacteriophage of the present invention has dry-resistance to caused by Salmonella typhimurium may include salmonello
stably maintain its infectivity even after high-temperature sis or Salmonella food poisoning, examples of the diseases
drying. Such properties of acid-, heat-, and drying-resistance caused by Salmonella gallinarum may include Fowl typhoid,
allow application of the bacteriophage of the present inven 55 and examples of the diseases caused by Salmonella pullorum
tion under various temperature and pH conditions upon the may include Pullorum disease, but are not limited thereto.
production of prophylactic or therapeutic compositions for As used herein, the term “salmonellosis” refers to symp
livestock diseases caused by ST, SG and SP or human dis toms caused by Salmonella infection, including fever, head
eases caused by the contaminated livestock. ache, diarrhea, and vomiting, namely, diseases caused by
The present inventors collected sewage samples at chicken 60 bacteria of the genus Salmonella, which is de?ned two clini
slaughterhouses, and isolated therefrom the bacteriophage of cal formsian acute septicemic form that resembles typhoid
the present invention having a speci?c bactericidal activity fever and an acute gastroenteritis, including enteritis, food
against ST, SG and SP and the above characteristics, which poisoning, and acute septicemia.
was designated as Bacteriophage CI>CJ2 and deposited at the As used herein, the term “prevention” means all of the
Korean Culture Center of Microorganisms (361-221, Honje 65 actions in which the disease is restrained or retarded by the
l, Sudaemun, Seoul) on Dec. 17, 2008 under accession num administration of the composition. As used herein, the term
ber KCCM10976P. “treatment” means all of the actions in which the disease has