# Patent

## Title
Bacteriophage and antibacterial composition comprising the same

## Abstract

Provided is a novel bacteriophage ΦCJ21 (KCCM11363P). In addition, the present invention relates to an antibacterial composition including the bacteriophage ΦCJ21 (KCCM11363P) as an active ingredient. Further, provided is a method of preventing and/or treating infectious diseases by Clostridium perfringens in animals except for humans using the bacteriophage ΦCJ21 (KCCM11363P) or the antibacterial composition containing the bacteriophage ΦCJ21 (KCCM11363P) as an active ingredient.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is the U.S. National Phase Application under 35 U.S.C. §371 of International Application No. PCT/KR2014/001592, filed Feb. 26, 2014, designating the U.S. and published as WO 2014/133323 A1 on Sep. 4, 2014 which claims the benefit of Korean Patent Application No. KR-10-2013-0021499, filed Feb. 27, 2013. Any and all applications for which a foreign and/or a domestic priority is claimed is/are identified in the Application Data Sheet filed herewith and is/are hereby incorporated by reference in their entirety under 37 C.F.R. §1.57.
REFERENCE TO SEQUENCE LISTING
This application incorporates by reference the sequence listing submitted as ASCII text filed via EFS-Web on Aug. 11, 2015, and updated by a file entitled “AIP22.011APC_REPLACEMENT_SEQLIST.txt” which is 62,240 bytes in size, created on Nov. 19, 2015, and last modified on Nov. 23, 2015.
TECHNICAL FIELD
The present invention relates to a novel bacteriophage having a specific bactericidal activity against pathogenic Clostridium perfringens and an antibacterial composition comprising the same. In addition, the present invention relates to a method of preventing or treating animal diseases using the novel bacteriophage or the antibacterial composition.
BACKGROUND ART
Clostridium perfringens (CP), which is gram-positive large obligatory anaerobic bacillus, has been known as a bacterium that does not have flagellum and forms a spore. Clostridium perfringens, which is a bacterium causing diarrhea, or the like, particularly, in domestic animals such as chicken, pig, etc., and the like, has been recognized as one of the important and fatal pathogenic bacteria in a livestock industry, as well as Salmonella causing fowl typhoid.
Currently, one of the diseases frequently generated in poultry and pork industries is Necrotic Enteritis by Clostridium perfringens. It is known that Necrotic Enteritis is frequently generated by co-infection of Clostridium perfringens and Coccidium, and as a main symptom of necrotic enteritis, there is bloody diarrhea due to severe necrotic lesions in a lower portion of small intestine of chickens, swines, or the like.
This necrotic enteritis generates dehydration symptom, periodic diarrhea, and the like, in an infected animal according to the disease severity, gradually debilitates a body of the animal, and causes growth retardation, and the like, such that necrotic enteritis has become a significant problem in the livestock industry. Further, since Clostridium perfringens is easily propagated through feces of animal, transmission between animals in a common breeding space may be easily generated by oral infection through soil or contaminated feed, or the like. Particularly, incidence in young animal is high, such that Clostridium perfringens has become a problem.
Meanwhile, bacteriophage is a specialized type of virus that infects and destroys only bacteria, and can self-replicate only inside host bacteria. The bacteriophage has strong host specificity as compared to antibiotics, and recently, a problem of emergence of strain resistant against antibiotics has become serious, such that an interest in practical use of the bacteriophage has increased (Non-Patent Documents 1 and 2).
Therefore, research concerning the bacteriophage has been actively conducted in various countries around the world, and in addition to a patent application for bacteriophage, an attempt to acquire Food and Drug Administration (FDA) approval for a composition containing the bacteriophage has been gradually increased.
As the prior art documents concerning the bacteriophage, a bacteriophage having a specific bactericidal activity against Clostridium perfringens has been disclosed in Patent Document 1, and a bacteriophage having a specific bactericidal activity against Staphylococcus aureus has been disclosed in Patent Document 2. Further, lytic protein derived from a bacteriophage specifically destroying peptidoglycan structure of bacterial cell membrane, and bacteria lysates by the lytic protein have been disclosed in Patent Document 3.
However, in spite of presence of the following prior arts, a technology associated with the bacteriophage for preventing and/or treating infectious diseases, particularly, necrotic enteritis by Clostridium perfringens that is a still important problem in the livestock industry including poultry and pork industries is still insufficient, such that a bacteriophage and a technology associated with the bacteriophage should be developed.
PRIOR ART DOCUMENT
Patent Document
(Patent Document 1) Korean Patent Laid-open Publication No. 10-2012-0076710 A
(Patent Document 2) Korea Patent Registration No. 10-0910961 B1
(Patent Document 3) Korean Patent Laid-Open Publication No. 10-2009-0021475 A
Non-Patent Document
(Non Patent Document 1) Cislo M, et al., Arch. Immunol. Ther. Exp. 2:175-183, 1987
(Non Patent Document 2) Sung Hoon Kim et al, Bacteriophage, novel alternative antibiotics, BioWave Vol. 7 No. 15, 2005, BRIC
DISCLOSURE
Technical Problem
The present inventors conducted studies in order to solve problems such as resistant bacteria occurring upon the use of antibiotics, antibiotics remaining in meat, and the like, and efficiently prevent and treat infectious diseases by Clostridium perfringens, and as a result, the present inventors isolated new bacteriophage ΦCJ21 (KCCM11363P) having a specific bactericidal activity against Clostridium perfringens from the nature.
In addition, the present inventors identified morphological, biochemical, and genetic characteristics of the novel bacteriophage and confirmed that the bacteriophage had excellent acid resistance, heat resistance, drought resistance, and the like, thereby developing an antibiotic, a disinfectant, a feed additive, and other compositions using the novel bacteriophage. Further, the present inventors developed a composition for preventing or treating infectious diseases by Clostridium perfringens and a method of preventing or treating the disease using the composition.
The present invention intends to provide a novel bacteriophage ΦCJ21 (KCCM11363P) having a specific bactericidal activity against Clostridium perfringens. 
In addition, the present invention intends to provide a composition for preventing and/or treating infectious diseases by Clostridium perfringens containing the bacteriophage ΦCJ21 (KCCM11363P) as an active ingredient.
Further, the present invention intends to provide an antibiotic, a feed additive, a drinking water additive, a disinfectant, or a cleaner containing the bacteriophage ΦCJ21 (KCCM11363P) as an active ingredient.
Furthermore, the present invention intends to provide a method of preventing and/or treating infectious diseases by Clostridium perfringens in animals except for humans using the bacteriophage ΦCJ21 (KCCM11363P) or a composition containing the same (KCCM11363P) as an active ingredient.
Technical Solution
According to an exemplary embodiment of the present invention, there is provided a novel bacteriophage ΦCJ21 (KCCM11363P) having a specific bactericidal activity against Clostridium perfringens. 
According to another exemplary embodiment of the present invention, there is provided a composition for preventing or treating an infectious disease caused by Clostridium perfringens, the composition containing the bacteriophage ΦCJ21 (KCCM11363P) as described above as an active ingredient.
According to another exemplary embodiment of the present invention, there are provided an antibiotic, a feed additive, a drinking water additive, a disinfectant or a cleaner containing the bacteriophage ΦCJ21 (KCCM11363P) as described above as an active ingredient.
According to another exemplary embodiment of the present invention, there is provided a method of preventing or treating an infectious disease caused by Clostridium perfringens, comprising administering the bacteriophage ΦCJ21 (KCCM11363P) or the composition containing the same as described above to animals except for humans.
Advantageous Effects
The bacteriophage ΦCJ21 (KCCM11363P) according to the present invention has an effect of specifically killing Clostridium perfringens. 
In addition, the bacteriophage ΦCJ21 (KCCM11363P) according to the present invention has excellent acid resistance, heat resistance, and drought resistance, such that the bacteriophage ΦCJ21 (KCCM11363P) may be used as a material for preventing or treating infectious diseases by Clostridium perfringens in various temperature or pH ranges, moisture conditions, and the like, and utilized as an antibiotic, a feed additive, a drinking water additive, a disinfectant, a cleaner, or the like.
Further, according to the present invention, infectious diseases caused by Clostridium perfringens may be prevented or treated by administering the bacteriophage ΦCJ21 (KCCM11363P) or a composition containing the same (KCCM11363P) as an active ingredient to animals except for human.



DESCRIPTION OF DRAWINGS
FIG. 1 is an electron microscope photograph of a novel bacteriophage ΦCJ21 (KCCM11363P, hereinafter, referred to as ‘ΦCJ21’).
FIG. 2 shows a pulsed field gel electrophoresis (PFGE) result of the novel bacteriophage ΦCJ21.
FIG. 3 shows a sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result of the novel bacteriophage ΦCJ21.
FIG. 4 is a graph showing a result of an acid resistance test of the novel bacteriophage ΦCJ21.
FIG. 5 is a graph showing a result of a heat resistance test of the novel bacteriophage ΦCJ21.
FIG. 6 is a graph showing a result of a drought resistance test of the novel bacteriophage ΦCJ21.



BEST MODE
Hereinafter, the present invention will be described in detail. Since contents that are not described in the present specification may be sufficiently recognized and inferred by those skilled in the art or similar art, a description thereof will be omitted.
In one embodiment, the present invention provides a novel bacteriophage ΦCJ21 (KCCM11363P) having a specific bactericidal activity against Clostridium perfringens (CP).
It is known that Clostridium perfringens, which is gram-positive large obligatory anaerobic bacillus, does not have a flagellum and forms a spore. Clostridium perfringens, which is a bacterium causing diarrhea, or the like, in animals, particularly, in domestic animals such as poultry, swine, and the like, has been recognized as one of the dangerous and fatal pathogenic bacteria in a livestock industry such as Salmonella causing fowl typhoid.
A bacteriophage is a bacteria-specific virus infecting specific bacteria to suppress and inhibit growth of the bacteria, and means a virus including single or double stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) as a genetic material.
A bacteriophage ΦCJ21 of the present invention, which is a bacteriophage having species-specificity of selectively infecting Clostridium perfringens has an isometric capsid and a non-contractile tail, and morphologically belongs to Siphoviridae (FIG. 1). Homology analysis data of nucleic acid sequences between bacteriophage ΦCJ21 and other bacteriophages is shown in table 1. Activity of bacteriophage ΦCJ21 was stable at the range from pH 4 to pH 9.8 (acid resistance, see FIG. 4). ΦCJ21 retained its activity for 2 hours when it was exposed at 60° C. (heat resistance, see FIG. 5), and its titer was decreased about 1/10 after drying (see FIG. 6). The nucleic acid sequence of bacteriophage ΦCJ21 is the same as SEQ ID NO: 1.
The bacteriophage ΦCJ21, which was a bacteriophage newly isolated by the present inventors, was deposited at Korean Culture Center of Microorganisms (361-221, Hongjedong, Seodaemun-gu, Seoul, Korea) as a deposition number KCCM11363P on Jan. 30, 2013.
In another embodiment, the present invention provides a composition for preventing or treating infectious diseases by Clostridium perfringens containing the bacteriophage ΦCJ21 as an active ingredient. As a preferable example of the composition, the present invention provides an antibiotic.
Since the bacteriophage ΦCJ21 has an antibacterial activity capable of specifically killing Clostridium perfringens, the bacteriophage ΦCJ21 may be used to prevent or treat diseases generated by infection of Clostridium perfringens. As a suitable example of the infectious disease caused by Clostridium perfringens capable of being treated using the bacteriophage ΦCJ21, there is Necrotic Enteritis, but the present invention is not limited thereto.
Necrotic Enteritis, which is one of the main infectious diseases caused by Clostridium perfringens, corresponds to a bacterial disease most frequently generated in domestic animals, particularly, poultry and causes significant damage. The disease may be generated in poultry, especially chickens substantially at all ages, but is mainly generated in chickens (2 to 5 weeks old) bred on the floor and also frequently generated in chickens (12 to 16 weeks old) bred in a cage.
As Clostridium perfringens is excessively proliferated in the small intestine, symptoms of Necrotic Enteritis are generated, and necrosis of gastrointestinal mucosa, sudden diarrhea, and the like, are caused. For example, in swines, in the case of very acute necrotic enteritis, after 1 to 2 days of occurrence, mortality of the swine is generated, and in the case of acute necrotic enteritis, after 2 to 3 days of bloody diarrhea, mortality of the swine is generated. Further, in the case of sub-acute necrotic enteritis, diarrhea (there is no bloody feces) proceeds for 5 to 7 days, and then weakness and dehydration are caused, and in the case of chronic necrotic enteritis, intermittent diarrhea is caused, and growth disorder may be generated.
The term “prevention” as used herein refers to all actions of providing the bacteriophage ΦCJ21 and/or the composition containing the same as the active ingredient to animals except for humans to suppress the corresponding disease or retard disease occurring.
The term “treatment” as used herein refers to all actions of providing the bacteriophage ΦCJ21 and/or the composition containing the same as the active ingredient to animals, except for humans, to thereby allow the symptom of the corresponding disease caused by infection to get better or be alleviated.
As an example of the infectious disease caused by Clostridium perfringens to which the bacteriophage ΦCJ21 and/or the composition containing the same as the active ingredient may be applied, there is necrotic enteritis, but the present invention is not limited thereto.
The composition for preventing or treating the infectious diseases caused by Clostridium perfringens according to the present invention may contain the bacteriophage ΦCJ21 at a content of preferably 5×106 to 5×1012 pfu/ml, more preferably, 1×106 to 1×1010 pfu/ml.
The composition for preventing or treating infectious diseases caused by Clostridium perfringens according to the present invention may further contain a pharmaceutically acceptable carrier, and be formulated together with the carrier to thereby be provided as food, a drug, a feed additive, a drinking water additive, and the like. The term “Pharmaceutically acceptable carrier” as used herein means a carrier or a diluent that does not stimulate living organism nor inhibit biological activity and properties of an administered compound.
A kind of carrier usable in the present invention is not particularly limited, and any carrier may be used as long as it is generally used in the art and is pharmaceutically acceptable. As a non-restrictive example of the carrier, there are saline, sterile water, Ringer's solution, buffered saline, an albumin injection solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, and the like. These can be used alone or as a mixture of at least two of these.
In addition, if necessary, another general additive such as an antioxidant, a buffer, and/or a bacteriostatic agent, etc., may be further added and used, and the composition may be formulated into an injection formulation such as an aqueous solution, suspension, emulsion, or the like, pills, capsules, granules, tablets, or the like by additionally adding a diluent, a dispersant, a surfactant, a binder, and/or a lubricant, etc., and then used.
An administration method of the composition for preventing or treating infectious diseases by Clostridium perfringens is not particularly limited, but any method generally used in the art may be used. As a non-restrictive example of the administration method, the composition may be orally or parenterally administered.
As a non-restrictive example of the formulation for oral administration, there are troches, lozenge, tablets, aqueous suspensions, oily suspensions, prepared powder, granules, emulsions, hard capsules, soft capsules, syrups, elixirs, or the like.
In order to formulate the composition according to the present invention into a formulation such as a tablet, a capsule, or the like, the formulation may further contain a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, gelatin; an excipient such as dicalcium phosphate, or the like; a disintegrant such as corn starch, sweet potato starch, or the like; a lubricant such as magnesium stearate, calcium stearate, sodium stearyl fumarate, polyethylene glycol wax, or the like. In the case of the capsule formulation, the formulation may additionally contain a liquid carrier such as fatty oil in addition to the above-mentioned materials.
As a parenteral administration method, an intravenous administration method, an intraperitoneal administration method, an intramuscular administration method, a subcutaneous administration method, or a local administration method, etc., may be used. In addition, a method of applying or spraying the composition onto a disease site may also be used, but the present invention is not limited thereto.
An example of the formulation for parenteral administration may include injection formulations for subcutaneous injection, intravenous injection, intramuscular injection, or the like; suppository formulations; spray formulations such as aerosol formulations capable of being inhaled through respiratory system, or the like, but the present invention is not limited thereto. In order to formulate the composition into the injection formulation, the composition according to the present invention may be mixed with a stabilizer or a buffer in water to thereby prepare a solution or suspension, and then, the prepared solution or suspension may be formulated in a unit dose for an ampoule or vial. In the case of formulating the composition into the spray formulation such as the aerosol formulation, or the like, a propellant, or the like, may be mixed together with an additive so that a water-dispersed condensate or wet powder is dispersed.
A suitable application, spray, or administration dose of the composition for preventing or treating infectious diseases by Clostridium perfringens may be variously determined depending on factors such as age, weight, sex, degree of symptom of disease, a kind of food, excretion rate of administration subject animals, or the like, as well as a method of formulating the composition, an administration method, an administration time and/or route. Generally, a veterinarian having ordinary skill in the art may easily determine and prescribe an effective dose for the desired treatment.
In another embodiment, the present invention may provide an antibiotic containing the bacteriophage ΦCJ21 as an active ingredient.
The term “antibiotic” as used herein means an agent capable of being provided to animals including humans in a drug form to thereby kill bacteria, and corresponds to a concept collectively indicating a preservative, a disinfectant, and an antibacterial agent.
The antibiotic containing the bacteriophage ΦCJ21 according to the present invention as the active ingredient may have high specificity to Clostridium perfringens as compared to an antibiotic according to the prior art to thereby not kill beneficial bacteria but kill specific pathogenic bacteria, and does not induce drug resistance, such that the antibiotic according to the present invention may be provided as a novel antibiotic having an elongated lifespan as compared to the antibiotic according to the prior art.
In another embodiment, the present invention may provide a feed additive and a drinking water additive containing the bacteriophage ΦCJ21 as an active ingredient.
The feed additive and the drinking water additive according to the present invention may be used in a manner in which the bacteriophage ΦCJ21 or the composition containing the smae is individually prepared in a feed additive or drinking water additive form and then mixed with a feed or drinking water, or in a manner in which the bacteriophage ΦCJ21 or the composition containing the same is directly added at the time of preparing the feed or the drinking water.
The bacteriophage ΦCJ21 or the composition containing the same used as the feed additive or drinking water additive according to the present invention may be in a liquid state or dried state, and preferably, in a dried powder form.
A drying method for preparing the feed additive and the drinking water additive according to the present invention in the dried powder form is not particularly limited, but a method generally used in the art may be used. As a non-restrictive example of the drying method, there is air drying method, natural drying method, a spray drying method, a freeze-drying method, or the like. One method of these methods may be used alone or at least two methods may be used together with each other.
Another non-pathogenic microbe may be additionally added to the feed additive or drinking water additive. A non-restrictive example of the microbe capable of being added may be selected from a group consisting of bacillus subtilis capable of producing protease, lipase, and/or sugar converting enzyme such as bacillus subtilis, or the like; Lactobacillus sp. having physiological activity and degradation activity for an organic material under anaerobic conditions such as stomach of cow; mold fungi having effects of increasing a weight of domestic animal, a milk yield, and digestibility of the feed such as Aspergillus oryzae, or the like; and yeasts such as Saccharomyces cerevisiae, or the like. These can be used alone or as a mixture of at least two of these.
The feed additive or the drinking water additive containing the bacteriophage ΦCJ21 according to the present invention as the active ingredient may further contain other additives, as needed. As a non-restrictive example of the usable additive, there are a binder, an emulsifier, a preservative, and the like, which are added in order to prevent quality of the feed or driving water from being deteriorated; amino acids, vitamins, enzymes, probiotics, flavoring agents, non-protein nitrogen compounds, silicates, buffers, coloring agents, extractants, oligosaccharides, and the like, which are added in order to increase utility of the feed or drinking water. Otherwise, the additive may further include a feed mixing agent, or the like. These can be used alone or as a mixture of at least two of these.
The feed additive may be contained at a content of 0.05 to 10, more preferably 0.1 to 2 parts by weight based on 100 parts by weight of the feed. The drinking water additive may be contained at a content of 0.0001 to 0.01, more preferably 0.001 to 0.005 parts by weight based on 100 parts by weight of the drinking water. The activity of the bacteriophage ΦCJ21 against Clostridium perfringens may be sufficiently exhibited in the above-mentioned range.
In another embodiment the present invention provides a feed or drinking water prepared by adding a feed additive or a drinking water additive containing the same as an active ingredient or directly adding the bacteriophage ΦCJ21.
The feed used in the present invention is not particularly limited, but any feed generally used in the art may be used. A non-restrictive example of the feed may include plant feeds such as grains, roots and fruit, food processing byproducts, algaes, fiber, pharmaceutical byproducts, fats, starches, cucurbitaceous, or grain byproducts; and animal feeds such as proteins, inorganic materials, fats, minerals, single cell proteins, animal planktons, or foods. These can be used alone or as a mixture of at least two of these.
The drinking water used in the present invention is not particularly limited, but any drinking water generally used in the present invention may be used.
In another embodiment, the present invention may provide a disinfectant or a cleaner containing the bacteriophage ΦCJ21 as an active ingredient. A formulation of the disinfectant or cleaner is not particularly limited, but the disinfectant or cleaner may be formulated into any formulation known in the art.
The disinfectant may be sprayed in order to remove Clostridium perfringens onto a region in which animals live, a slaughterhouse, a mortality rea, a cooking place or cooking equipment, or the like, but the present invention is not limited thereto.
The cleaner may be used to wash surfaces of the skin or each of the sites of bodies of animals, particularly, poultry or swines, exposed or to be exposed to Clostridium perfringens, but the present invention is not limited thereto.
In another embodiment, the present invention provides a method of preventing or treating infectious diseases caused by Clostridium perfringens by using the bacteriophage ΦCJ21 or the composition containing the same as an active ingredient. The infectious disease may be preferably necrotic enteritis, but the present invention is not limited thereto. The target of preventing or treating infectious disease caused by Clostridium perfringens may be a poultry or swine, but the present invention is not limited thereto.
In detail, the method of preventing or treating infectious diseases according to the present invention may include administering the bacteriophage ΦCJ21 or the composition containing the same as the active ingredient to targets infected by Clostridium perfringens or being at risk of infection of Clostridium perfringens except for humans in a pharmaceutically effective dose. It will be apparent to those skilled in the art that when the pharmaceutical composition is administered to patient, the suitable total daily dose may be determined by an attending physician or veterinarian within the scope of sound medical judgement.
A specific pharmaceutically effective dose of the bacteriophage ΦCJ21 or the composition containing the same as the active ingredient for a specific animal may be determined by considering an administration time and an administration route of the bacteriophage ΦCJ21 or the composition containing the same, a secretion rate of the composition, a therapy duration period, or the like, in addition to a kind and a degree of the desired response, an age, a weight, a general healthy state, sex, or diet of the corresponding individual. In addition, the pharmaceutically effective dose may be variously changed according to various factors such as ingredients of drugs or other compositions simultaneously or separately used and similar factors well known in a medical field.
The bacteriophage ΦCJ21 according to the present invention or the composition containing the same as the active ingredient may be administered as a pharmaceutical form (nasal spray) to animals or administered in a method of directly added to a feed or drinking water of the animals and then feeding the feed or drinking water. In addition, the bacteriophage ΦCJ21 or the composition containing the same may be mixed in a feed or drinking water in a form of a feed additive or drinking water additive and then administered.
The administration route and administration method of the bacteriophage ΦCJ21 according to the present invention or the composition containing the same as the active ingredient are not particularly limited, but any administration route and administration method may be used as long as the bacteriophage ΦCJ21 or the composition containing the same may arrive at the corresponding target tissue. That is, the bacteriophage ΦCJ21 or the composition containing the same as the active ingredient may be administered through various oral or parenteral routes. As a non-restrictive example of the administration route, oral, rectal, local, intravenous, intraperitoneal, intramuscular, intraarterial, subcutaneous, and nasal administration, or inhalation, etc., may be performed.
Hereinafter, the present invention will be described in detail through the Examples. However, these Examples are only to illustrate the present invention, and a scope of the present invention is not limited to these Examples.
Example 1 Isolation of Bacteriophage Infecting Clostridium perfringens 
Examples 1-1
Screening of Bacteriophage and Isolation of Single Bacteriophage
After 50 ml of a sample isolated from a feces sample of Samwhaw Gps. Breeding Agri. Inc., which is a chicken and pig farm in South Chungchong Province, was moved a centrifuge bottle and centrifuged at 4,000 rpm for 10 minutes, the supernatant was filtered with a 0.45 μm filter to prepare a sample solution, and then a soft agar overlay method was performed using the prepared sample solution. The soft agar overlay method is a method of observing a lysis action of bacteriophage using host cells growing in top agar (attached onto a solid medium using 0.7% agar).
In detail, 18 ml of sample filtrates was mixed with 150 μl of a shake culture solution (OD600=2) of Clostridium perfringens, (CP, BCCP 17-1) isolated at Animal and Plant Quarantine Agency and 2 ml of 10× Brain-heart infusion (hereinafter, ‘BHI’ medium) (composed so as to have a final volume of 1 L) and cultured at 37° C. for 18 hours. Then, the culture solution was centrifuged at 4,000 rpm for 10 minutes, and the supernatant was filtered using the 0.45 μm filter. Thereafter, after a mixture of 5 ml of 0.7% agar (w/v) and 150 μl of the shake culture solution (OD600=2) of Clostridium perfringens (BCCP 17-1) was poured and hardened into a BHI plate (BHI+0.2% sheep blood), 10 μl of the sample culture filtrate solution was dropped thereon, followed by culturing at 30° C. for 18 hours. Then, it was confirmed that a plaque was formed.
After the sample culture filtrate solution in which lysis was generated was appropriately diluted and mixed with 150 μl of the shake culture solution (OD600=2) of Clostridium perfringens (BCCP 17-1), the soft agar overlay method was performed, thereby obtaining a single plaque. Since it is considered that a single plaque is formed of a single bacteriophage, in order to purify and isolate the single bacteriophage, a single plaque was selected, put into 400 μl of a SM solution (NaCl 5.8 g/l; MgSO47H2O 2 g/l; 1 M Tris-Cl (pH 7.5), 50 ml; H2O, composed so as to have a final volume of 1 L), and left at room temperature for 4 hours, thereby purifying and isolating the single bacteriophage.
In order to secure a large amount of the isolated bacteriophage, 100 μl of a supernatant of a single bacteriophage solution was selected and mixed with 120 of 0.7% agar and 500 of the shake culture solution of Clostridium perfringens (BCCP 17-1), followed by performing the soft agar overlay method in a LB medium having a diameter of 150 mm. After pouring 15 ml of the SM solution into a plate in which lysis was completely generated, the plate was softly shaken at room temperature for 4 hours, thereby discharging the bacteriophage in the top-agar. The SM solution in which the bacteriophage was discharged was recovered, and chloroform was added thereto at an amount of 1% of the final volume and suitably mixed for 10 minutes, followed by centrifugation at 4,000 rpm for 10 minutes. The supernatant obtained as described above was filtered with a 0.45 μm filter and stored at a cold temperature.
Examples 1-2
Large Scale Culture and Purification of Bacteriophage
The selected bacteriophage was cultured at large scale using Clostridium perfringens (BCCP 17-1), and then the bacteriophage was purified therefrom.
In detail, 1% of the shake culture solution of Clostridium perfringens (BCCP 17-1) was inoculated into a liquid culture medium for mass production, and at the same time, the bacteriophage was put thereinto at multiplicity of infection (MOI) of 0.1, simultaneously with the inoculation of Clostridium perfringens (BCCP 17-1), thereby performing co-infection. Then, static culture was performed at 30° C. under anaerobic conditions.
Thereafter, centrifugation was performed at 4° C. and 12,000 rpm for 20 minutes, and then the supernatant was filtered with a 0.45 μm filter. After NaCl and polyethylene glycol (PEG) were added to the filtered supernatant so as to have final concentrations of 1M and 10% (w/v), respectively, and mixed with each other, the mixture was further left at 4° C. for 8 hours or more. Next, after centrifugation was performed at 4° C. and 12,000 rpm for 20 minutes, then the supernatant was removed, and the precipitates were obtained.
The obtained precipitate was resuspended using 5 ml of the SM solution and was left at room temperature for 20 minutes. Thereafter, the supernatant was filtered with a 0.45 μm filter, and ultracentrifugation (35,000 rpm, 1 hour, 4° C.) using a glycerol density gradient method (density: 40%, 5% glycerol) was performed, thereby purifying the bacteriophage ΦCJ21. After the purified ΦCJ21 was resuspended using 500 μl of the SM solution, a titer was measured.
The present inventor called the bacteriophage obtained by extracting the sample from feces and having the specific bacteriocidal activity against Clostridium perfringens “Bacteriophage ΦCJ21” and deposited the bacteriophage at Korean Culture Center of Microorganisms (361-221, Hongjedong, Seodaemun-gu, Seoul, Korea) as a deposition number KCCM11363P on Jan. 30, 2013.
Example 2
Morphology Observation of ΦCJ21
The purified bacteriophage ΦCJ21 was diluted in 0.01% gelatin solution and then fixed by 2.5% glutaraldehyde solution. The fixed bacteriophage was dropped onto a carbon-coated mica plate (ca. 2.5 mm×2.5 mm), adapted thereto for 10 minutes, and washed with sterile distilled water. A carbon film was mounted on a copper grid, stained with 4% uranyl acetate for 30 to 60 seconds, dried, and investigated using a transmission electron microscope (JEM-1011, 80 kV, magnification: X120,000 to X200,000) (FIG. 1).

FIG. 1 shows an electron microscope photograph of the bacteriophage ΦCJ21, and it may be appreciated that since the bacteriophage does not have an isometric capsid and a contractile tail, the bacteriophage morphologically belongs to Siphoviridae.

Example 3
Genomic DNA Size Analysis of ΦCJ21
Genomic DNA was extracted from the bacteriophage ΦCJ21 purified by the ultracentrifugation. In detail, ethylenediaminetetraacetic acid (EDTA, pH 8.0), proteinase K, and sodium dodecyl sulfate (SDS) were added to a culture solution of the purified bacteriophage ΦCJ21 so as to have final concentrations of 20 mM, 50 μg/ml, and 0.5% (w/v), respectively, and then, were in a stationary state at 50° C. for 1 hour. Thereafter, an equal volume of phenol (pH 8.0) was added thereto and stirred, followed by centrifugation at room temperature and 12,000 rpm for 10 minutes, thereby obtaining a supernatant.

The supernatant was mixed with an equal volume of PC (phenol:chloroform=1:1) and centrifuged at room temperature and 12,000 rpm for 10 minutes, thereby obtaining a supernatant. The supernatant was mixed with an equal volume of chloroform and centrifuged at room temperature and 12,000 rpm for 10 minutes, thereby obtaining a supernatant. The obtained supernatant was sequentially mixed with 10% (v/v) of 3M sodium acetate and a double volume of cold 95% ethanol, based on the total volume, and left at −20° C. for 1 hour. Subsequently, centrifugation was performed at 0° C. and 12,000 rpm for 10 minutes, and the precipitate was obtained by removing the supernatant. Then, 50 μl of Tris-EDTA (TE) buffer (pH 8.0) was added thereto to thereby dissolve the obtained precipitate. The extracted DNA was diluted 10 times, and a concentration was measured by measuring absorbance at OD260.

Next, 1 μg of DNA was loaded onto 1% pulse-field gel electrophoresis (PFGE) agarose gel, and electrophoresis was performed at room temperature for 20 hours using a BIORAD PFGE system program 7 (size range: 25-100 kb; switch time ramp: 0.4-2.0 seconds, linear shape; forward voltage: 180 V; reverse voltage: 120 V) (FIG. 2).

FIG. 2 is a pulsed field gel electrophoresis (PFGE) photograph of the genomic DNA of the bacteriophage ΦCJ21, and it may be confirmed that the genomic DNA of the bacteriophage ΦCJ21 has a size of about 56 kb.

Example 4
Protein Pattern Analysis of ΦCJ21
15 μl of purified bacteriophage ΦCJ21 solution (1010 pfu/ml titer) was mixed with 3 μl of a 5× SDS sample solution, and heated for 5 minutes. Thereafter, the total protein of the bacteriophage ΦCJ21 was expanded in 15% SDS-PAGE gel, and then the gel was stained at room temperature for 1 hour using a coomassie blue dye solution (FIG. 3).

FIG. 3 is an electrophoresis photograph showing a result of SDS-PAGE performed on the bacteriophage ΦCJ21, and main proteins having sizes of about 40 kDa, 51 kDa, 53 kDa, and 70 kDa were observed. In FIG. 3, M is a protein that becomes a standard for measuring a molecular weight.

Example 5
Gene Sequence Analysis of ΦCJ21
In order to confirm genetic characteristics of the purified bacteriophage ΦCJ21, DNA of the bacteriophage ΦCJ21 was analyzed using a FLX titanium sequencer (Roche), which is a gene analysis apparatus. Genes was assembled at Macrogen INC. using GS and de novo assembler software (Roche). Sequence analysis of an open reading frame was performed using GeneMArk.hmm, Glimmer v3.02, and FGENESB software. Identification of the open reading frame was performed using BLASTP and InterProScan program.

The genome sequence of the bacteriophage had various similarities with that of the existing reported bacteriophage, but it was confirmed that a bacteriophage of which all of the fractions were completely (100%) equal to those of the bacteriophage of the present invention did not exist. Therefore, it may be confirmed that the bacteriophage was a newly isolated bacteriophage.

Homology analysis data of nucleic acid sequence between bacteriophage ΦCJ21 and other bacteriophages is shown in table 1.










TABLE 1







Query
Subject
Identities














Name
Length
Start
End
Description
E-Value
Match/Total
Pct.(%)

















contig00001_orf00003
543
70
537
conserved hypothetical
3E−05
42/161
26






protein [Clostridium botulinum






BKT015925]


contig00001_orf00010
207
1
204
hypothetical protein
2E−12
32/68
47






phi34O_gp33 [Clostridium






phage phiCP34O]


contig00001_orf00011
543
19
496
hypothetical protein
5E−17
63/180
35






CbC4_4068 [Clostridium






botulinum BKT015925]


contig00001_orf00013
588
7
570
thymidine kinase [Clostridium
2E−55
109/190
57






butyricum E4 str. BoNT E






BL5262]


contig00001_orf00009
363
34
294
putative phage related protein
6E−09
34/87
39






[Selenomonas ruminantium






subsp. lactilytica TAM6421]


contig00001_orf00017
528
247
360
hypothetical protein
0.0003
17/38
44






[Pelotomaculum






thermopropionicum Si]


contig00001_orf00016
1974
133
1556
primase [Gordonia phage
3E−36
137/494
27






GTE2]


contig00001_orf00031
930
7
888
hypothetical protein [Gordonia
5E−13
 80/324
24






phage GTE2]


contig00001_orf00030
2442
226
2388
DNA polymerase 1 [Gordonia
2E−97
265/755
35






phage GTE2]


contig00001_orf00024
762
358
756
thymidylate synthase
8E−16
 45/134
33






[Clostridium acetobutylicum






DSM 1731]


contig00001_orf00035
513
1
273
hypothetical protein
9E−18
48/91
52






CbC4_7040 [Clostridium






botulinum BKT015925]


contig00001_orf00032
884
48
702
gp089 [Lactococcus phage
2E−06
 57/220
25






KSY1]


contig00001_orf00036
1452
54
1437
domain protein, SNF2 family
4E−56
150/475
31






[Bryantella formatexigens






DSM 14469]


contig00001_orf00026
163
18
180
hypothetical protein
6E−08
30/70
42






AC5_1713 [Clostridium






perfringens CPE str. F4969]


contig00001_orf00038
441
31
384
putative protein [Aquifex
1E−10
 54/152
35






aeolicus VF5]


contig00001_orf00044
1359
583
753
hypothetical protein
1E−05
31/57
54






PCC7424_5514 [Cyanothece






sp. PCC 7424]


contig00001_orf00045
1248
7
1239
endolysin [Clostridium
 3E−158
295/415
71






perfringens E str. JGS1987]


contig00001_orf00050
2913
2455
2904
hypothetical protein
6E−58
107/150
71






AC3_2545 [Clostridium






perfringens E str. JGS1987]


contig00001_orf00052
1059
28
1047
putative base plate assembly
4E−52
119/341
34






protein: putative PBSX






prophage protein [Bacillus






subtilis subsp. subtilis str.






168]


contig00001_orf00051
588
28
573
hypothetical protein
3E−15
 55/184
29






HMPREF0830_00205






[Eubacteriaceae bacterium






CM2]


contig00001_orf00048
150
1
135
hypothetical protein
9E−12
33/45
73






CLJ_B2512 [Clostridium






botulinum Ba4 str. 657]


contig00001_orf00047
213
1
207
bacteriocin uviB homolog
3E−22
47/69
68






[Clostridium perfringens C str.






JGS1495]


contig00001_orf00049
594
1
585
conserved hypothetical
1E−25
 75/198
36






protein [Clostridium






perfringens E str. JGS1987]


contig00001_orf00056
714
19
429
ZkdP [Bacillus sp. JS]
9E−19
 53/139
38


contig00001_orf00055
987
1
960
hypothetical protein
1E−41
110/328
33






2016_scalfold57_00038






[unidentified phage]


contig00001_orf00053
496
1
381
hypothetical protein
3E−14
 47/131
35






PlanB_05085 [Paenibacillus,






larvae-subsp. larvae B-3650]


contig00001_orf00057
2562
25
1239
phage tail tape measure
1E−88
194/410
47






protein. TP901 family






(Herpetosiphon aurantacus






DSM765]


contig00001_orf00059
420
88
417
hypothetical protein
3E−13
 40/111
30






BSSCB_30350 [Bacillus






subtilis subsp. subtilis str:






SC-8]


contig00001_orf00063
435
1
339
hypothetical protein putative
1E−07
 37/116
31






PBSX [Bacillus






amyfolique factens XH7]


contig00001_orf00064
534
43
522
hypothetical protein
1E−45
 89/170
52






B1NLASEDRAFT_3746:






[Bacillus sp. 1NLA3E]


contig00001_orf00065
563
13
336
hypothetical protein
8E−13
 45/108
41






B1NLA3EDRAFT_3747






[Bacillus sp. 1NLA3E]


contig00001_orf00060
432
10
420
hypothetical protein
6E−16
 47/139
33






RUMOBE_01063






[Ruminococcus obeum ATCC






29174]


contig00001_orf00069
1260
1
1089
hypothetical protein
2E−02
153/300
39






RUMOBE_01052






[Ruminococcus obeum ATCC






29174]


contig00001_orf00068
921
10
882
conserved hypothetical
4E−86
185/201
58






protein [Bacillus cereus






03BB108]


contig00001_orf00066
405
16
402
hypothetical protein
5E−10
 44/130
33






RUMOBE_01056






[Ruminococcus obeum ATCC






29174]


contig00001_orf00070
885
19
879
head morphogenesis protein
7E−50
118/300
39






SPP1 gp7 [Bacillus sp.






1NLA3E]


contig00001_orf00081
1332
1
1329
Phage tail sheath protein
1E−63
169/449
37






[Desulfosporosinus youngiae






DSM-17734]


contig00001_orf00072
398
1
372
hypothetical protein
4E−11
 59/135
43






2016_scalfold57_00101






[unidentified phage]


contig00001_orf00071
1467
46
1449
hypothetical protein
 5E−108
215/479
44






RUMOBE_01050






[Ruminococcus obeum ATCC






29174]


contig00002_orf00002
637
259
678
phage terminase, small
2E−25
 66/164
40






subunit/PBSX family






[Clostridium bolulinum [Ba4






str. 057]


contig00004_orf00001
408
1
357
CMP/dCMP deaminase zinc-
2E−23
 56/131
42






binding protein






[Methanphalobium






evestigatum Z-7303]


contig00003_orf00004
420
1
417
gp317 [Bacillus phage G]
5E−24
 67/154
43









A partial genome sequence of bacteriophage ΦCJ21 prepared in the above is the same to SEQ ID No: 1. The genome sequence was determined by genetic analyzer.
Example 6
Stability Test of ΦCJ21 Depending on pH In order to confirm stability of the bacteriophage ΦCJ21 in a low pH environment, stability test was performed over a wide pH range (pH 4.0, 5.5, 6.4, 6.9, 7.4, 8.2, 9.0, and 9.8).
For test, various pH solutions (Sodium acetate buffer (pH 4.0, pH 5.5, and pH 6.4), Sodium phosphate buffer (pH 6.9 and pH 7.4), and Tris-HCl solution (pH 8.2, pH 9.0, and pH 9.8)) were prepared at a concentration of 0.2 M, respectively.
After 90 μl of each of the pH solutions was mixed with 10 μl of bacteriophage solution having a titer of 1.0×109 pfu/ml so that a concentration of each of pH solution became 1 M, each of the pH solutions was left at room temperature for 30 minutes, 1 hour, and 2 hours. Then, the reaction solution was diluted step by step, 100 of the diluted solution at each step was dropped and cultured at 30° C. for 18 hours by a soft agar overlay method, and the titer was measured through the presence or absence of lysis (FIG. 4).
FIG. 4 shows a result of the acid resistance test of the bacteriophage ΦCJ21. As shown in FIG. 4, it may be confirmed that the bacteriophage ΦCJ21 did not lose its activity and was significantly stable in a pH range of 4.0 to 9.8 for up to 2 hours.
Example 7
Stability Test of ΦCJ21 Depending on Temperature
A test for confirming stability against heat generated during a formulating process of the bacteriophage in the case of using the bacteriophage as a feed additive formulation among formulations of the bacteriophage was performed.
In detail, 2000 of bacteriophage ΦCJ21 solution having a titer of 1.0×108 pfu/ml was left at 60° C. for 0, 10, 30, 60, and 120 minutes. Then, the solutions above were diluted step by step, 10 μl of each of the diluted solutions was dropped and cultured at 30° C. for 18 hours by a soft agar overlay method, and the titer was measured through the presence or absence of lysis (FIG. 5).
FIG. 5 shows a result of a heat resistance test of the bacteriophage ΦCJ21. As shown in FIG. 5, it may be appreciated that the activity was not significantly decreased until the bacteriophage ΦCJ21 was exposed at 60° C. for 2 hours.
Example 8
Stability Test of ΦCJ21 Against Drying
A test for confirming stability against drying conditions generated during a formulating process of the bacteriophage in the case of using the bacteriophage as a feed additive formulation among formulations of the bacteriophage was performed.
In detail, 100 μl of bacteriophage ΦCJ21 solution having a titer of 1.0×108 pfu/ml was dried at 60° C. for 120 minutes using a speed-vacuum (Speed-Vacuum Concentrator 5301, Eppendorf). The pellet obtained after drying was put and resuspended in a SM solution at an amount equal to that of an initial solution at 4° C. for one day.
Then, the solutions above were diluted step by step, 10 μl of the diluted solution at each step was dropped and cultured at 30° C. for 18 hours by a soft agar overlay method, and the titer was measured through the presence or absence of lysis (FIG. 6).
FIG. 6 shows a result of a drought resistance test of the bacteriophage ΦCJ21. As shown in FIG. 6, it may be appreciated that the titer of the bacteriophage ΦCJ21 after drying was decreased to about 1/10 of the titer before drying.
Example 9
Infection Spectrum Test of ΦCJ21 with Respect to Wild-Type Strains of Clostridium perfringens 
Whether or not the bacteriophage ΦCJ21 had a lytic activity was tested on 45 wild-type strains of Clostridium perfringens isolated by Animal and Plant Quarantine Agency and Kunkuk University other than Clostridium perfringens (BCCP 17-1) used in the experiment.
In detail, 10 μl of bacteriophage ΦCJ21 solution having a titer of 1.0×1010 pfu/ml and mixed with 150 μl of a shake culture solution (OD600=2) of each of the strains was dropped and cultured at 30° C. for 18 hours by a soft agar overlay method. Then, whether or not a plaque was formed was observed.
As a result of the experiment, among 45 wild-type strains of Clostridium perfringens, 44 strains were infected, such that an infection ratio was about 97.7% and a lysis ratio was about 97.7%. The results were shown in Tables 2 and 3.











TABLE 2







Origin of CP
CP strain
Infectivity









Kunkuk University
HLYS-1
+



Kunkuk University
HLYS-3
+++




JSH-1
+++




KCCM 40947T
+++




KJW-2
++




OYS-2
+++




KCCM 12098
+++





















TABLE 3





Origin of CP
CP strain
Infectivity
CP strain
Infectivity







Animal and
CP-KJW-1
+++
BCCP43-1
+/−


Plant


Quarantine


Agency


Animal and
CP-JSH-1
+++
BCCP44-3
+/−


Plant
CP-OYS-2
+++
BCCP47-2
+++


Quarantine
CP-BC-1
+++
BCCP48-3
+++


Agency
CP-BSW-4
+++
BCCP50-1-3
+



CP-HBM-2
+++
BCCP50-1-8
++



CP-HL
+++
BCCP51-1-1
+++



CP-KW-1
+++
BCCP51-1-5
+++



CP-BS-1
−
BCCP52-2-8
+++



CP-HL-1
+++
BCCP53-2-3
+++



CP-LJN-1
+++
BCCP54-3-8
+++



BCCP17-1
+++
BCCP55-3-1
+++



BCCP23-4
+++
SBCCP429-2
+++



BCCP37-2
+++(T)
SBCCP321
+++



BCCP38-1
+++
SBCCP343
+



BCCP39-1
+++
SBCCP361
+++



BCCP40-1
+
ELCCP Suksan
+++





Kim



BCCP41-3
+++
ELCCP6-1
+++





intestines



BCCP42-2
+++
appendix
++

## Claims

1. A method of preventing or treating an infectious disease caused by Clostridium perfringens, comprising administering, to an animal excluding human, a bacteriophage ΦCJ21 (KCCM11363P) having a bactericidal activity against Clostridium perfringens or a composition comprising the bacteriophage ΦCJ21 (KCCM11363P).

2. The method of claim 1, wherein the infectious disease is necrotic enteritis.

3. A method of preparing an animal feed composition, the method comprising:
providing a bacteriophage ΦCJ21 (KCCM11363P); and
mixing the bacteria with an animal feed base to provide the animal feed composition.

4. The method of claim 3, wherein the bacteriophage is in an amount of 0.05 to 10 by weight based on 100 parts by weight of the feed composition.

5. A method of feeding, the method comprising:
preparing the animal feed composition according to the method of claim 3; and
feeding the animal feed composition as a feed to an animal.

6. A method of preparing a drinking water composition, the method comprising:
providing a bacteriophage ΦCJ21 (KCCM11363P); and
mixing the bacteriophage with drinking water to provide the drinking water composition.

7. The method of claim 6, wherein the bacteriophage is in an amount of 0.0001 to 0.01 by weight based on 100 parts by weight of the drinking water.

8. A method of feeding, the method comprising:
preparing the drinking water composition according to the method of claim 6; and
feeding the drinking water composition as a feed to an animal.