Abstract:
A composition includes an isolated bacteriophage Str-PAP-1 having the ability to kill  Streptococcus parauberis  cells specifically by infecting the same, and may be used to prevent and treat  Streptococcus parauberis  infections. The bacteriophage Str-PAP-1 that is an active ingredient of the composition has the ability to kill  Streptococcus parauberis  cells and characteristically has the genome represented by nucleotide sequence of SEQ. ID. NO: 1.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims the benefit of and priority to Korean Patent Application No. 10-2014-0051211, filed Apr. 29, 2014, the entire disclosure of which is incorporated by reference herein. 
       BACKGROUND 
       [0002]    The present application relates to a composition usable for preventing or treating  Streptococcus parauberis  infections comprising an isolated bacteriophage that is able to infect and kill  Streptococcus parauberis  cells, and a method for preventing and treating  Streptococcus parauberis  infections using the composition. More particularly, the present application relates to the bacteriophage characteristically having the genome represented by nucleotide sequence of SEQ. ID. NO: 1 that is able to kill specifically  Streptococcus parauberis  cells, a composition comprising the bacteriophage for preventing and treating  Streptococcus parauberis  infections and a method for preventing and treating  Streptococcus parauberis  infections using the composition. 
         [0003]    The importance of aquaculture production in the fishery industry increases continuously. Because the wild fish captured are not sufficient to meet global fish demand, the supply and demand of aquaculture products are expected to increase continuously. Aquaculture production is expected to remain an important part of fishery industry. 
         [0004]    In aquaculture production, disease outbreaks are considered to be a key constraint in the fish farming sector, resulting in significant losses. Disease outbreaks cause the economic damage and reduced productivity attributed to the increased general expense of fish culture resulted from the increase of drug use and management costs thereby. In addition, antibiotics residue has been another sensitive social issue, so the production of high quality safe aquatic products seems to be in doubt. Therefore, a method for preventing and treating the disease of cultured fish has been a major interest not just domestically, but internationally. 
         [0005]      Streptococcus parauberis  is well known as one of the most representative causative pathogen of streptococcosis in cultured fish. In addition to  Streptococcus parauberis, S. iniae, S. difficilis, S. shiloi , and  Lactococcus garvieae  are also known as pathogenic bacteria that cause streptococcosis. Among them,  Streptococcus parauberis  is the most frequently reported causative pathogen of streptococcosis in fish. 
         [0006]    In terms of external signs, a sea fish having streptococcosis shows darkened body color, exophthalmos and hyperemia, abdominal distension, and hernia. Internally, such symptoms as ascites, abdominal wall hemorrhage, and heart abscess were observed. In some cases, only gill erosion is observed without any other symptoms. Streptococcosis usually occurs in adult fish rather than a juvenile fish, indicating that economic damage is comparatively higher than other bacterial diseases. 
         [0007]    Outbreak of streptococcosis can be influenced by several factors of deterioration of environmental conditions, inappropriate culture methods, and inappropriate feeds, etc. Streptococcosis is commonly found when water temperatures are warm, but may be common in the winter when water temperatures are low. The damage in the aquaculture production by such  Streptococcus parauberis  infections is getting bigger, so that a method to prevent the infections and also to treat the infections efficiently is urgently requested. 
         [0008]    A variety of antibiotics have been used for the prevention or treatment of  Streptococcus parauberis  infections. However, according to the increase of antibiotic-resistant bacteria, another way of treating the infections is urgently requested. To control the infections caused by  Streptococcus parauberis , a vaccine has been developed. However, the variety of vaccines cannot catch up with the variety of diseases. In addition, to control the multiple diseases broken at the same time, a combined control method to treat them along with a vaccine has to be prepared. 
         [0009]    Recently, the use of bacteriophages has drawn our attention as a new way of treating bacterial infections. Particularly, the reason of our high interest in bacteriophages is because bacteriophage-based treatment is a nature-friendly method. Bacteriophages are an extremely small microorganism that infects bacteria, which is called phage in short. Once it infects bacteria, the bacteriophage is proliferated in the inside of the bacterial cell. After full proliferation, the progenies destroy the bacterial cell wall to escape from the host, suggesting that the bacteriophage has bacteria killing ability. The bacteriophage infection is characterized by high specificity, so that a certain bacteriophage infects only a specific bacterium. That is, the bacterium that can be infected by certain bacteriophage is limited, suggesting that bacteriophage can kill only a specific bacterium and cannot harm other bacteria. 
         [0010]    Bacteriophage was first found out by an English bacteriologist Twort in 1915 when he noticed that  Micrococcus  colonies melted and became transparent by something unknown. In 1917, a French bacteriologist d′Herelle found out that  Shigella disentriae  in the filtrate of dysentery patient feces melted by something, and further studied about this phenomenon. As a result, he identified bacteriophage independently, and named it as bacteriophage which means a bacteria killer. Since then, bacteriophages specifically acting against such pathogenic bacteria as  Shigella, Salmonella typhi , and  Vibrio cholerae  have been continuously identified. 
         [0011]    Owing to the unique capability of bacteriophage to kill bacteria, bacteriophages have been studied and anticipated as a method to treat bacterial infections. However, after penicillin was found by Fleming, studies on bacteriophages had been only continued in some of Eastern European countries and the former Soviet Union because of the universalization of antibiotics. After the year of 2000, the merit of the conventional antibiotics faded because of the increase of antibiotic-resistant bacteria. So, bacteriophages are once again spotlighted as a new anti-bacterial agent that can replace the conventional antibiotics. 
         [0012]    According to the recent regulation of use of antibiotics by the government, the interest on bacteriophages increases more and more. 
         [0013]    Thus, the present inventors tried to develop a composition for preventing or treating  Streptococcus parauberis  infections by using an isolated bacteriophage that is able to kill  Streptococcus parauberis  cells specifically and to establish a method for preventing or treating  Streptococcus parauberis  infections using the composition. As a result, the present inventors secured the nucleotide sequence of the genome that can distinguish this bacteriophage from other bacteriophages and then developed a composition comprising the isolated bacteriophage as an active ingredient, and further confirmed that this composition could be efficiently used for the prevention and treatment of  Streptococcus parauberis  infections. 
       SUMMARY 
       [0014]    It is an object of the exemplary embodiment disclosed herein to provide a novel bacteriophage that has the capability to kill  Streptococcus parauberis  cells specifically. 
         [0015]    It is another object of the exemplary embodiment disclosed herein to provide a composition for preventing  Streptococcus parauberis  infections comprising the bacteriophage having the capability to kill  Streptococcus parauberis  cells by infecting  Streptococcus parauberis  cells, and a method for preventing  Streptococcus parauberis  infections using the said composition. 
         [0016]    It is also an object of the exemplary embodiment disclosed herein to provide a composition for treating  Streptococcus parauberis  infections comprising the bacteriophage having the capability to kill  Streptococcus parauberis  cells by infecting  Streptococcus parauberis  cells, and a method for treating  Streptococcus parauberis  infections using the said composition. 
         [0017]    It is further an object of the exemplary embodiment disclosed herein to provide a bath (immersion) treatment agent for preventing and treating  Streptococcus parauberis  infections using the said composition. 
         [0018]    It is also an object of the exemplary embodiment disclosed herein to provide a feed additive for preventing and treating  Streptococcus parauberis  infections using the said composition. 
         [0019]    To achieve the above objects, the exemplary embodiment disclosed herein provides a composition comprising an isolated bacteriophage that has the capability to kill  Streptococcus parauberis  cells by infecting the same as an active ingredient, and a method for preventing and treating  Streptococcus parauberis  infections by using the composition. 
         [0020]    The isolated bacteriophage included in the composition of the exemplary embodiment disclosed herein as an active ingredient is the bacteriophage Str-PAP-1 having the DNA genome represented by nucleotide sequence of SEQ. ID. NO: 1. The bacteriophage Str-PAP-1 isolated by the present inventors was deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Apr. 7, 2014 (Accession No: KCTC 12568BP). 
         [0021]    The exemplary embodiment disclosed herein also provides a bath treatment agent and a feed additive for the prevention and treatment of  Streptococcus parauberis  infections. 
         [0022]    The bacteriophage Str-PAP-1 included in the composition of the invention can kill  Streptococcus parauberis  cells efficiently, so that it displays the preventive or therapeutic effect on streptococcosis caused by  Streptococcus parauberis . Therefore, the composition of the exemplary embodiment disclosed herein can be used for the prevention and treatment of streptococcosis caused by  Streptococcus parauberis . In this description, streptococcosis indicates all the symptoms accompanied by  Streptococcus parauberis  infections. 
         [0023]    In this description, the term “treatment” or “treat” indicates (i) to suppress streptococcosis caused by  Streptococcus parauberis ; and (ii) to relieve streptococcosis caused by  Streptococcus parauberis.    
         [0024]    In this description, the term “isolation” or “isolated” indicates all the action to separate the bacteriophage by using diverse experimental techniques and to secure the characteristics that can distinguish this bacteriophage from others, and further includes the action of proliferating the bacteriophage via bioengineering techniques so as to make it useful. 
         [0025]    The bacteriophage of the exemplary embodiment disclosed herein includes the bacteriophage Str-PAP-1 and its variants. The “variants” herein indicate the bacteriophages that have minor variations in genomic sequence or polypeptide coding genetic information but have the same genotypic and phenotypic characteristics as the bacteriophage Str-PAP-1 of the invention. The said variants include polymorphic variants as well. It is preferred for those variants to have the same or equivalent biological functions as the bacteriophage Str-PAP-1 of the invention. 
         [0026]    The pharmaceutically acceptable carrier included in the composition of the exemplary embodiment disclosed herein is the one that is generally used for the preparation of a pharmaceutical formulation, which is exemplified by glucose, maltodextrin, lactose, dextrose, sucrose, sorbitol, mannitol, starch,  acacia  rubber, calcium phosphate, alginate, gelatin, calcium silcate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, but not always limited thereto. The composition of the exemplary embodiment disclosed herein can additionally include lubricants, wetting agents, sweeteners, flavors, emulsifiers, suspending agents, and preservatives, in addition to the above ingredients. 
         [0027]    In the composition of the exemplary embodiment disclosed herein, the bacteriophage Str-PAP-1 or the variants thereof are included as an active ingredient. At this time, the bacteriophage Str-PAP-1 or the variants thereof are preferably included at the concentration of 1×10 1  pfu/mL˜1×10 30  pfu/mL or 1×10 1  pfu/g˜1×10 30  pfu/g, and more preferably at the concentration of 1×10 4  pfu/mL˜1×10 15  pfu/mL or 1×10 4  pfu/g˜1×10 15  pfu/g. 
         [0028]    The composition of the exemplary embodiment disclosed herein can be formulated by the method that can be performed by those in the art by using a pharmaceutically acceptable carrier and/or excipient in the form of unit dose or in a multi-dose container. The formulation can be in the form of solution, suspension or emulsion in oil or water-soluble medium, extract, powder, granule, tablet or capsule. At this time, a dispersing agent or a stabilizer can be additionally included. 
         [0029]    The composition of the exemplary embodiment disclosed herein can be prepared as a bath treatment agent or a feed additive according to the purpose of use, but not always limited thereto. 
       ADVANTAGEOUS EFFECT 
       [0030]    The composition of the exemplary embodiment disclosed herein and the method for preventing and treating  Streptococcus parauberis  infections using the composition has the advantage of high specificity to  Streptococcus parauberis , compared with the conventional chemical or synthetic antibiotics. That means, the composition of the exemplary embodiment disclosed herein can be used for preventing or treating  Streptococcus parauberis  specifically without affecting other useful bacteria in vivo, and accordingly has less side effects. In general, when chemical materials such as antibiotics are used, the general beneficial bacteria are also damaged to weaken immunity in animals with carrying various side effects. In the meantime, the composition of the exemplary embodiment disclosed herein uses the isolated bacteriophage as an active ingredient, so that it is very nature-friendly. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0031]    The application of the preferred embodiments is best understood with reference to the accompanying drawings, wherein: 
           [0032]      FIG. 1  is an electron micrograph showing the bacteriophage Str-PAP-1. 
           [0033]      FIG. 2  is a photograph illustrating the capability of the bacteriophage Str-PAP-1 to kill  Streptococcus parauberis  cells. 
       
    
    
     DETAILED DESCRIPTION 
       [0034]    Practical and presently preferred embodiments are illustrative as shown in the following Examples. It is to be understood that the present disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims. 
         [0035]    However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention. The detailed description of the present disclosure is divided into various sections only for the reader&#39;s convenience and disclosure found in any section may be combined with that in another section. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. 
         [0036]    The following Examples are intended to further illustrate certain embodiments of the disclosure and are not intended to limit its scope. 
       Example 1 
     Isolation of Bacteriophage Capable of Killing  Streptococcus parauberis  Cells 
       [0037]    Samples were collected from the nature or animals to screen the bacteriophage having the capability to kill  Streptococcus parauberis  cells. The strains of  Streptococcus parauberis  used for the bacteriophage isolation herein were the one that had been already separated by the present inventors and identified as  Streptococcus parauberis.    
         [0038]    The isolation process of the bacteriophage is described in more detail hereinafter. The collected sample was added to the TSB (Tryptic Soy Broth) medium (casein digest, 17 g/L; soybean digest, 3 g/L; dextrose, 2.5 g/L; NaCl, 5 g/L; dipotassium phosphate, 2.5 g/L) inoculated with  Streptococcus parauberis  culture at the ratio of 1/1000, followed by shaking culture at 30° C. for overnight. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes and supernatant was collected. The recovered supernatant was filtered by using a 0.45 μm filter. The obtained filtrate was screened by spot assay to investigate whether or not the bacteriophage that can kill  Streptococcus parauberis  cells was included therein. 
         [0039]    Spot assay was performed as follows; TSB medium was inoculated with  Streptococcus parauberis  culture at the ratio of 1/1000, followed by shaking culture at 30° C. overnight. 3 mL (OD 600 =2.0) of the  Streptococcus parauberis  culture broth prepared above was spread on the TSA (Tryptic Soy Agar; casein digest, 15 g/L; soybean digest, 5 g/L; NaCl, 5 g/L; agar, 15 g/L) plate. The plate stood in a clean bench for 20 minutes to dry the culture broth. 
         [0040]    After the medium was dried, 10 μl of the prepared filtrate was dropped on the plate on which  Streptococcus parauberis  had been spread. The plate stood for about 20 minutes to dry the medium, followed by standing-culture at 30° C. for a day. The next day, the plate was observed to see whether or not a clear zone was generated on the spot where the filtrate was dropped. If a clear zone was generated where the filtrate was dropped, it could be judged that the bacteriophage that could kill  Streptococcus parauberis  cells was included in the filtrate. Through the above procedure, the filtrate containing the bacteriophage having the capability to kill  Streptococcus parauberis  cells could be obtained. 
         [0041]    Then, the bacteriophage was isolated from the filtrate confirmed above to have the bacteriophage capable of killing  Streptococcus parauberis  cells. The conventional plaque assay was used for the bacteriophage isolation. Particularly, a plaque formed in the course of the plaque assay was picked up by using a blade, which was then added to phage buffer (10 mM Tris-HCl (pH7.5), 10 mM MgSO4, NaCl 4 g/L). The buffer stood for 4 hours. Centrifugation was performed at 5,000 rpm for 15 minutes to obtain supernatant. The obtained supernatant was filtered by using a 0.45 μm filter. The obtained supernatant was used for plaque assay again. In general, the pure bacteriophage isolation is not completed by one-time process, so the above procedure was repeated. At least 5 times of repeated procedure, the solution containing the pure bacteriophage was obtained. The procedure for the isolation of the pure bacteriophage is generally repeated until the generated plaques become similar in sizes and morphologies. The final pure bacteriophage isolation was confirmed by the observation under electron microscope. Until the pure bacteriophage isolation was confirmed under electron microscope, the above process was repeated. The observation under electron microscope was performed by the conventional method. Briefly, the solution containing the pure bacteriophage was loaded on copper grid, followed by negative staining with 2% uranyl acetate. After drying thereof, the morphology was observed under transmission electron microscope. 
         [0042]    The solution containing the pure bacteriophage confirmed above proceeded to purification.  Streptococcus parauberis  culture broth was added to the solution containing the pure bacteriophage at the volume of 1/10 of the total volume of the bacteriophage solution, followed by culture for 4-5 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. The said procedure was repeated 5 times to obtain a solution containing enough numbers of the bacteriophage. The supernatant obtained from the final centrifugation was filtered by a 0.45 μm filter, followed by the conventional polyethylene glycol (PEG) precipitation. Particularly, PEG and NaCl were added to 100 mL of the filtrate (10% PEG 8000/1 M NaCl), which stood at 4° C. for overnight. Centrifugation was performed at 8,000 rpm for 30 minutes to obtain the bacteriophage precipitate. The obtained bacteriophage precipitate was resuspended in 5 mL of buffer (10 mM Tris-HCl, 10 mM MgSO4, 0.1% Gelatin, pH 8.0). This solution was called the bacteriophage suspension or bacteriophage solution. 
         [0043]    As a result, the purified pure bacteriophage was obtained, which was named as the bacteriophage Str-PAP-1 and then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology in Apr. 7, 2014 (Accession No: KCTC 12568BP). The electron micrograph of the bacteriophage Str-PAP-1 is presented in  FIG. 1 . From the morphological observation, the bacteriophage Str-PAP-1 was identified as belonging to the family Siphoviridae. 
       Example 2 
     Separation of the Bacteriophage Str-PAP-1 Genome and Sequencing Thereof  
       [0044]    The genome of the bacteriophage Str-PAP-1 was separated as follows. The genome was separated from the bacteriophage suspension obtained in Example 1. First, in order to eliminate DNA and RNA of  Streptococcus parauberis  host bacterial cells included in the suspension, DNase I and RNase A were added 200 U each to 10 mL of the bacteriophage suspension, which stood at 37° C. for 30 minutes. 30 minutes later, to remove the DNase I and RNase A activity, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added thereto, which stood for 10 minutes. The suspension stood at 65° C. for 10 minutes and then added with 100 μl of proteinase K (20 mg/mL) to break the outer wall of the bacteriophage, followed by incubation at 37° C. for 20 minutes. 500 μl of 10% sodium dodecyl sulfate (SDS) was added thereto, followed by incubation at 65° C. for 1 hour. 10 ml of the mixture of phenol:chloroform:isoamylalcohol (25:24:1) was added thereto, followed by mixing well. The mixture was centrifuged at 13,000 rpm for 15 minutes to separate each layer. The upper layer was obtained, to which isopropyl alcohol was added at the volume of 1.5 times the volume of the upper layer, followed by centrifugation at 13,000 rpm for 10 minutes to precipitate the genome of bacteriophage. After collecting the precipitate, 70% ethanol was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was vacuum-dried and then dissolved in 100 μl of water. The said process was repeated to obtain the bacteriophage Str-PAP-1 genome in a large scale. 
         [0045]    The nucleotide sequence of the genome of the bacteriophage Str-PAP-1 obtained above was analyzed by Next Generation Sequencing (NGS) at National Instrumentation Center for Environmental Management, Seoul National University. Briefly, DNA fragment was fixed on the slide, followed by bridge amplification to form DNA fragment cluster. Then, SBS (Sequence by Synthesis), that was a nucleotide synthesis reaction, was performed by using the cluster as a template along with four different fluorescent-labeled nucleotides. This method is unique by the following characteristics: wherein DNA sequence is not amplified in a reaction solution like other methods but amplified on the slide where DNA is fixed with DNA bending to form sequence clusters. The formed cluster proceeded to sequencing group by group, and the obtained results are converted into each read sequence information, followed by examination. A contig map was prepared by using the obtained gene sequence by the conventional method. The nucleotide sequence of the total genome in the size of 36,595 bp was analyzed by primer walking. The total genomic sequence of the bacteriophage Str-PAP-1 was represented by nucleotide sequence of SEQ. ID. NO: 1. 
         [0046]    Similarity of the genomic sequence of the bacteriophage Str-PAP-1 obtained above with the previously reported bacteriophage genome sequences was investigated by using BLAST on Web (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, the genomic sequence of the bacteriophage Str-PAP-1 had a similarity with only sequence of  Lactococcus  bacteriophage phi31 (GenBank Accession No. AJ292531.2), but the similarity is very low (1%). It suggests that the bacteriophage Str-PAP-1 as disclosed herein was clearly distinguished from the previously reported bacteriophages. 
         [0047]    From the above results, it was confirmed that the bacteriophage Str-PAP-1 was a novel bacteriophage that was completely different from the previously reported bacteriophages. 
       Example 3 
     Investigation of  Streptococcus parauberis  Killing Ability of the Bacteriophage Str-PAP-1 
       [0048]    The  Streptococcus parauberis  killing ability of the isolated bacteriophage Str-PAP-1 was investigated. To do so, the formation of clear zone was first observed by the spot assay in the same manner as described in Example 1. Those  Streptococcus parauberis  bacteria strains used for the killing ability investigation totaled 55 strains which were separated and identified as  Streptococcus parauberis  strains previously by the present inventors. The bacteriophage Str-PAP-1 demonstrated the ability to kill 35 strains of those  Streptococcus parauberis  bacteria. The representative results of this investigation are presented in  FIG. 2 . In the meantime, the activity of the bacteriophage Str-PAP-1 to kill  Edwardsiella tarda, Vibrio anguillarum, Vibrio ichthyoenteri, Lactococcus garvieae , and  Streptococcus iniae  was also investigated. As a result, the bacteriophage Str-PAP-1 did not have the activity of killing these microorganisms. 
         [0049]    Therefore, the results above indicate that the bacteriophage Str-PAP-1 of the present invention can be efficiently used as an active ingredient of a composition for preventing and treating  Streptococcus parauberis  infections. 
       Example 4 
     Preventive Effect of Bacteriophage Str-PAP-1 on  Streptococcus parauberis  Infections 
       [0050]    100 μl of the bacteriophage Str-PAP-1 solution (1×109 pfu/mL) was added to a tube containing 9 mL of TSB. To another tube containing 9 mL of TSB, 100 μl of TSB was added.  Streptococcus parauberis  culture was added to each tube to prepare bacterial suspensions of OD 600 =0.5. Then, the tubes were transferred in a 30° C. incubator, followed by shaking-culture, during which the growth of  Streptococcus parauberis  was observed. As presented in Table 1, the growth of  Streptococcus parauberis  was inhibited in the tube added with the bacteriophage Str-PAP-1 solution, while the growth of  Streptococcus parauberis  was not inhibited in the tube not added with the bacteriophage Str-PAP-1 solution. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Inhibition of  Streptococcus parauberis  growth 
               
             
          
           
               
                   
                 OD600 
               
             
          
           
               
                   
                 0 min. 
                 15 min. 
                 60 min. 
               
               
                   
                   
               
             
          
           
               
                   
                 (−) bacteriophage solution 
                 0.5 
                 0.62 
                 0.94 
               
               
                   
                 (+) bacteriophage solution 
                 0.5 
                 0.60 
                 0.32 
               
               
                   
                   
               
             
          
         
       
     
         [0051]    The above results indicate that the bacteriophage Str-PAP-1 not only inhibits the growth of  Streptococcus parauberis  cells but also can kill the bacteria. Therefore, the bacteriophage Str-PAP-1 can be used as an active ingredient of a composition for preventing  Streptococcus parauberis  infections. 
       Example 5 
     Therapeutic Effect of Bacteriophage Str-PAP-1 on  Streptococcus parauberis  Infections 
       [0052]    Therapeutic effect of the bacteriophage Str-PAP-1 on the olive flounder having  Streptococcus parauberis  infection was investigated. Particularly, two groups of juvenile olive flounder (10 fish per group, body length 6˜9 cm) at 4 months old were prepared, which were cultured separately in different water tanks for 14 days. Surrounding environment of the water tanks was controlled. The temperature and humidity in the laboratory where the water tanks stayed were also controlled. On the 7th day of the experiment,  Streptococcus parauberis  suspension (100 μl) was administered to the fish via intraperitoneal injection. The  Streptococcus parauberis  suspension was prepared as follows:  Streptococcus parauberis  was cultured in TSB medium at 30° C. for 18 hours. The bacterial cells were collected, which were prepared at the concentration of 1×108 CFU/mL by using saline (pH 7.2). From the next day of the  Streptococcus parauberis  challenge, the fish were intraperitoneally administered with 1×108 pfu/rite of the bacteriophage Str-PAP-1 (100 μl) once a day. The control group fish (bacteriophage solution not-treated) were not treated with anything. Feeds were equally provided to both the control and experimental groups. After the challenge of  Streptococcus parauberis , all the test fish were examined to see if streptococcosis was developed or not. The outbreak of streptococcosis was assessed by monitoring the extent of darkened body color. The measurement of dark coloration score (0: normal, 1: light dark coloration, 2: heavy dark coloration) was performed. The results are shown in Table 2. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Dark coloration score 
               
             
          
           
               
                   
                 Days after the  Streptococcus parauberis  challenge 
               
             
          
           
               
                   
                 0 
                 1 
                 2 
                 3 
                 4 
                 5 
                 6 
               
               
                   
                   
               
             
          
           
               
                 Control group 
                 1.0 
                 1.5 
                 1.5 
                 1.25 
                 1.1 
                 1.25 
                 1.25 
               
               
                 (−bacteriophage 
               
               
                 solution) 
               
               
                 Experimental group 
                 1.0 
                 0.5 
                 0.25 
                 0.25 
                 0.1 
                 0 
                 0 
               
               
                 (+bacteriophage 
               
               
                 solution) 
               
               
                   
               
             
          
         
       
     
         [0053]    From the above results, it was confirmed that the bacteriophage Str-PAP-1 as disclosed herein could be very efficient to treat  Streptococcus parauberis  infections. 
       Example 6 
     Preparation of Feed Additives and Feeds 
       [0054]    Feed additives were prepared by adding the bacteriophage Str-PAP-1 solution at the concentration of 1×109 pfu/g to the feed additives. The preparation method thereof was as follows: Maltodextrin (40 weight %) and trehalose (10 weight %) were added to the bacteriophage solution. After mixing well, the mixture was freeze-dried. Lastly, the dried mixture was ground into fine powders. The drying process above can be replaced with vacuum-drying, drying at warm temperature, or drying at room temperature. To prepare the control feed additive for comparison, feed additive that did not contain the bacteriophage but contained buffer (10 mM Tris-HCl, 10 mM MgSO4, 0.1% Gelatin, pH 8.0) were prepared. 
         [0055]    The above two kinds of feed additives were mixed with live fish meals at the volume of 250 times the volume of feed additive, resulting in two kinds of final feeds. 
       Example 7 
     Preparation of a Bath Treatment Agent 
       [0056]    A bath treatment agent containing 1×10 9  pfu/mL of bacteriophage Str-PAP-1 was prepared. The preparation method was as follows: 1×10 9  pfu of the bacteriophage Str-PAP-1 was added to 1 mL of buffer, which was well mixed. To prepare the control bath treatment agent, the buffer itself that was the same as the one used for the mixture of the bacteriophage solution was prepared. 
         [0057]    The prepared two kinds of bath treatment agents were diluted with water at the ratio of 1:1,000, resulting in the final bath treatment agents for the experiment. 
       Example 8 
     Effect on Fish Farming  
       [0058]    The effect of the feeds and the bath treatment agents prepared in Example 6 and Example 7 on olive flounder farming was investigated. Particularly, the investigation was focused on mortality. A total of 20 fish were split into two groups, 10 fish for each groups, which proceeded to the following experiment (group A; fed with feeds, group B; treated with bath treatment agents). Each group was divided by two sub-groups again, group of 5 fish each (sub-group-{circle around (1)}: treated with the bacteriophage Str-PAP-1, sub-group-{circle around (2)}: not-treated with the bacteriophage Str-PAP-1). The fish used for this experiment were the juvenile olive flounder at 4 months old. Each sub-group fish were cultured in separate water tanks placed at a sufficient distance from each other. Each sub-group was distinguished and named as shown in Table 3. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Sub-groups of feeding experiment 
               
             
          
           
               
                   
                 Sub-group 
               
             
          
           
               
                   
                 Treated with the 
                 Not-treated with the 
               
               
                   
                 bacteriophage Str-PAP-1 
                 bacteriophage Str-PAP-1 
               
               
                   
                   
               
             
          
           
               
                 Fed with feeds 
                 A-{circle around (1)} 
                 A-{circle around (2)} 
               
               
                 Treated with bath 
                 B-{circle around (1)} 
                 B-{circle around (2)} 
               
               
                 treatment agents 
               
               
                   
               
             
          
         
       
     
         [0059]    Feeds were provided according to the conventional feed supply method as presented in Table 3 with the feeds prepared in Example 6. The treatment of bath treatment agents was also performed by the conventional method according to Table 3 with the bath treatment agents prepared in Example 7. The results are shown in Table 4. 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 4 
               
               
                   
                   
               
               
                   
                 Group 
                 Mortality (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 A-{circle around (1)} 
                 0 
               
               
                   
                 A-{circle around (2)} 
                 20 
               
               
                   
                 B-{circle around (1)} 
                 0 
               
               
                   
                 B-{circle around (2)} 
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         [0060]    The above results indicate that the feeds prepared by the exemplary embodiment disclosed herein and the bath treatment agents prepared according to the exemplary embodiment disclosed herein are effective in reducing fish mortality. Therefore, it can be concluded that the composition of the exemplary embodiment disclosed herein can be efficiently applied for the improvement of productivity of fish farming. 
         [0061]    Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the exemplary embodiment disclosed herein. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended Claims.