WO2016126009A1 - Novel edwardsiella tarda bacteriophage edw-tap-1 and use thereof for inhibiting proliferation of edwardsiella tarda - Google Patents

Abstract

The present invention relates to Myoviridae bacteriophage Edw-TAP-1 (accession No. KCTC 12740BP) isolated from nature, characterized by holding an ability to specifically kill Edwardsiella tarda and having the genome represented by SEQ ID NO: 1, and to a method for preventing and treating the infection with Edwardsiella tarda using a composition containing the same as an active ingredient.

Description

Novel Edd Siela Tarda Bacteriophage Edb-TAP-1 and its Edd Siela Tarda Suppression

The present invention prevents the infection of Edward siela tarda using a composition containing the bacteriophage isolated from nature capable of killing Edward siela tarda and killing Edward siela tarda and an active ingredient. And it relates to a method of treatment, more specifically myobidae isolated from nature characterized in that it has a genome represented by SEQ ID NO: 1 having the ability to specifically kill Eddsiella tarda bacteria Bacteriophage Edw-TAP-1 (Accession No. KCTC 12740BP), and a method for preventing and post-infection treatment of Ed Ward siella tarda bacteria using the composition comprising the bacteriophage as an active ingredient.

Edwardsiella tarda ) is a Gram-negative bacillus belonging to Enterobacteriaceae, and has cell-like antigens (Somatic, O-antigens) and flagella antigens (Flagella, H-antigens) of lipopolysaccharides (LPS) on the cell wall. Combinations of these antigens can be divided into various serotypes.

Edward Siela tarda is a major causative agent of Edwardsiellosis and is widely known to infect marine and freshwater fish such as flounder, salmon, catfish, eel, and tilapia. Fish infected with Edward disease are visually observed with blackening, abdominal bloating, and hernia, and anatomical findings include nodules in the liver and kidneys, and turbidity and odor in the presence of ascites. Occasionally, a combination of streptococcal disease and the infection occurs, and in some cases muscle abscesses. In particular, fish with Edward disease often die of gangrene and septicemia, causing serious economic losses in the aquaculture industry.

Edward disease is prevalent in high water temperatures, but recent developments suggest that it is not necessarily due to water temperature or seasonal effects. In addition, there are few cases of death in a short period of time, so once the onset of death is a long-term mortality is often economic damage. In most cases, bacterial infections alone or mixed infections are treated with antibiotics. However, these methods can cause problems such as resistant bacteria and environmental pollution due to the abuse of antibiotics. Therefore, it is necessary to develop a more natural way.

Recently, bacteriophage has received a great deal of attention as an environmentally friendly bacterial countermeasure. Bacteriophages are tiny microorganisms that infect bacteria, often called phage. Bacteriophages have the ability to infect bacteria and multiply inside bacterial cells, and kill off bacteria by destroying the cell wall of the host bacteria when the progeny bacteriophages come out of the bacteria. Bacteriophage bacterial infections are highly specific, so there are only a few types of bacteriophages that can infect certain bacteria. That is, certain bacteriophages can infect only a specific category of bacteria, so that certain bacteriophages kill only certain bacteria and do not affect other bacteria.

The bacteriophage was discovered in 1915 by a British bacteriologist Twort, who studied the phenomenon of colonization of the micrococcus colony transparently by something. In 1917, French bacteriologist d'Herelle discovered that some of the filtrates of foreign patients had a function of dissolving Shigella disenteriae , and through this study, they independently discovered bacteriophages and consumed them. In the sense, they named it bacteriophage. Since then, bacteriophages have been found for many pathogenic bacteria such as dysentery, typhoid, and cholera.

Because of its special ability to kill bacteria, bacteriophages have been expected to be an effective way to combat bacterial infections since their discovery and many studies have been done. However, after the discovery of penicillin by Flemming, the prevalence of antibiotics has led to studies of bacteriophages only limited to some Eastern European countries and the former Soviet Union. However, since 2000, due to the increase of antibiotic-resistant bacteria, the limit of existing antibiotics appears, and as the possibility of developing an alternative to the existing antibiotics is highlighted, bacteriophage is attracting attention as an anti-bacterial agent.

In particular, with the recent tightening of government-wide regulations on the use of antibiotics, interest in bacteriophages is increasing and industrial use cases are increasing.

Accordingly, the present inventors have developed a composition that can be used to prevent or treat an infection of Edwardela tarta bacteria by using bacteriophage isolated from nature capable of selectively killing Edwardela tarta bacteria. After trying to develop a method for preventing or treating the infection of Ed C. tarda by using the composition, it is possible to isolate suitable bacteriophages from nature and to distinguish them from other bacteriophages. By securing a gene sequence of the genome so as to develop a composition comprising the bacteriophage as an active ingredient, and then confirming that the composition can be effectively used to prevent and treat the infection of Ed C. Was completed.

Accordingly, an object of the present invention is Myoviridae bacteriophage Edw- isolated from nature, characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill Eddsiella tarda. TAP-1 (Accession No. KCTC 12740BP).

It is still another object of the present invention to provide an active ingredient of an isolated bacteriophage Edw-TAP-1 capable of infecting Edwadsiela tarda and killing Edwadsiela tarda. It is to provide a composition that can be used to prevent infection and a method for preventing infection of Eddsiella tarda bacteria using the composition.

It is still another object of the present invention to provide an active ingredient of an isolated bacteriophage Edw-TAP-1 capable of infecting Edwadsiela tarda and killing Edwadsiela tarda. The present invention provides a composition that can be used to treat an infection, and a method for treating infection of Eddsiella tarda using the composition.

Still another object of the present invention is to provide a medicament for the purpose of preventing and treating Eddsiella tarda infection using the compositions.

Another object of the present invention is to provide a feed additive for the purpose of providing a specification effect through the prevention and treatment of Ed ward siella tarda infection using the compositions.

The present invention is a Myobiridae bacteriophage Edw-TAP-1 (accession number), characterized in that it has a genome represented by SEQ ID NO: 1 having the ability to specifically kill the Edwadsiella tarda bacteria KCTC 12740BP), and a method for preventing and treating infection with Eddsiella tarda using the composition comprising the same as an active ingredient.

Bacteriophage Edw-TAP-1 was isolated by the inventors and deposited in the Korea Institute of Biotechnology and Biotechnology Center on January 8, 2015 (Accession No. KCTC 12740BP).

The present invention also provides a bath and feed additive comprising bacteriophage Edw-TAP-1 as an active ingredient that can be used to prevent or treat the infection of Ed C.

Bacteriophage Edw-TAP-1 included in the composition of the present invention effectively kills Eddsiella tarda bacteria, thus preventing (infecting) or treating Edward disease caused by Eddsiella tarda bacteria (infection treatment) ) To effect. Therefore, the composition of the present invention can be used for the purpose of prevention and treatment of Ed Ward disease caused by Ed Ward Siella tarda.

As used herein, the term "treatment" or "treatment" refers to (i) suppression of Edward disease caused by Eddsiella tarda; And (ii) alleviation of Edward disease caused by the Eddwardella tarda bacterium.

As used herein, “isolated” or “isolated” refers to the separation of bacteriophages from the natural state using various experimental techniques, and to securing specific characteristics that can be distinguished from other bacteriophages. This includes growing the bacteriophages for industrial use.

Pharmaceutically acceptable carriers included in the compositions of the present invention are those commonly used in the formulation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate , Microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. no. The composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives and the like in addition to the above components.

The composition of the present invention includes bacteriophage Edw-TAP-1 as an active ingredient. The bacteriophage Edw-TAP-1 included at this time is included as 1 × 10 ¹ pfu / ㎖ 1 × 10 ³⁰ pfu / ㎖ or 1 × 10 ¹ pfu / g to 1 × 10 ³⁰ pfu / g, preferably 1 × 10 ⁴ pfu / ml to 1 × 10 ¹⁵ pfu / ml or 1 × 10 ⁴ pfu / g to 1 × 10 ¹⁵ pfu / g.

The compositions of the present invention may be prepared in unit dosage form by being formulated with pharmaceutically acceptable carriers and / or excipients, according to methods which may be readily practiced by those skilled in the art. It may also be prepared by incorporation into a multi-dose container. The formulations here may be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.

The composition of the present invention, depending on the mode of utilization, but is not limited to this, may be implemented as a bath and feed additives.

Infection prevention and treatment method of Ed Ward siella tarda using a composition comprising the bacteriophage Edw-TAP-1 of the present invention as an active ingredient Ed Wad Siela compared to conventional methods based on chemicals such as antibiotics It can provide the advantage of very high specificity for tar. This means that it can be used for the purpose of preventing or treating Edwadsiella tarda without influencing other useful flora, and the side effects of its use are very small. In general, the use of chemicals, such as antibiotics will also damage the common flora bacteria, resulting in a decrease in the immunity of animals, resulting in various side effects. On the other hand, the present invention can also provide the advantage of being very natural because it is used as an active ingredient of the composition to separate the bacteriophage already present in nature.

1 is an electron micrograph of the bacteriophage Edw-TAP-1.

Figure 2 is an experimental result showing the killing ability of Ed Wd Siela Tarda bacteria of bacteriophage Edw-TAP-1. The transparent part is the lysate plaque formed by lysis of bacteria.

Hereinafter, although an Example demonstrates this invention more concretely, these Examples are only illustrations of this invention, The scope of the present invention is not limited to these Examples.

Example  One: Edwad Siela  Isolation of Bacteriophage Can Kill Tarda

For the screening of bacteriophages capable of killing Edwardsiella tarda, samples obtained from the natural environment were used. On the other hand, Edwadsiella tarda bacteria used for bacteriophage separation have been previously identified by the present inventors and identified as Edwadsiella tarda bacteria.

More specifically the bacteriophage separation process, Ella necessarily the collected sample and the Ed tar is inoculated with the bacteria in TSB 1/1000 ratio (T S oy ryptic roth B) medium (Casein Digest, 17 g / L; Soy bean Digest, 3 g / L; dextrose, 2.5 g / L; NaCl, 5 g / L; dipotassium phosphate, 2.5 g / L) together and shaken at 37 ° C. for 3-4 hours. After incubation, the supernatant was recovered by centrifugation at 8,000 rpm for 20 minutes. Edwad siella tarda was inoculated in the recovered supernatant at a rate of 1/1000 and then shaken again for 3-4 hours at 37 ℃. When the bacteriophage was included in the sample, the process was repeated five times in order to increase the number of bacteriophages (Titer). After five repetitions, the culture was centrifuged at 8,000 rpm for 20 minutes. After centrifugation, the recovered supernatant was filtered using a 0.45 μm filter. It was examined whether there was a bacteriophage capable of killing Eddsiella tarda by the usual spot assay using the obtained filtrate.

The drip experiment was conducted as follows. EdBd siella tarda was inoculated in TSB medium at a rate of 1/1000 and then shaken at 37 ° C. for one night. Thus prepared ed and eat Ella tar is a culture solution of bacteria 3 ㎖ (OD ₆₀₀ is 1.5) TSA (T ryptic S oy A gar) plate medium (Casein Digest, 15 g / L; Soy bean digest, 5 g / L; Spreading in NaCl, 5 g / L; agar, 15 g / L). The plated flat medium was left in a clean bench for about 30 minutes to allow the smear to dry. After drying, 10 μl of the filtrate prepared above was dropped onto a plate medium plated with Eddsiella tarda. It was left to dry for 30 minutes. After drying, the plated medium was incubated for one day at 37 ° C., and then a clear zone was formed at the position where the filtrate was separated. It can be determined that the filtrate in which the transparent ring is formed contains bacteriophages capable of killing Eddsiella tarda. Through this investigation, it was possible to obtain a filtrate including bacteriophage having the ability to kill Edwardsiella tarda.

Separation of pure bacteriophages was carried out using a filtrate in which the presence of bacteriophages with killing ability against Edward siella tarda was confirmed. Separation of pure bacteriophage was carried out using a conventional Plaque assay. To explain this in detail, one of the lytic plaques formed in the lytic plaque assay was recovered using a sterile tip, and then added to the Eddsiella tarda bacterial culture and incubated together at 37 ° C. for 4-5 hours. After incubation, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. To the supernatant obtained, the Edd Siella tarda bacteria culture solution was added at a volume of 50/50, and then incubated at 37 ° C for 4-5 hours. In order to increase the number of bacteriophages, this procedure was performed at least five times, and finally, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. Using the obtained supernatant, lysis plate analysis was performed again. Since the separation of the pure bacteriophage is not usually achieved in one step of the above process, the previous step was repeated again using the lysate formed. This procedure was carried out at least five times to obtain a solution containing pure bacteriophage. Typically, separation of pure bacteriophage was repeated until both the size and shape of the lysate formed were similar. And finally, the electron microscope confirmed the pure separation of bacteriophage. The procedure described above was repeated until pure separation was confirmed by electron microscopy analysis. Electron microscopic analysis was performed according to a conventional method. This is briefly described as follows. The solution containing pure bacteriophage was buried in a copper grid, subjected to reverse staining and drying with 2% uranyl acetate, and the form was photographed through a transmission electron microscope. Electron micrographs of purely isolated bacteriophages are shown in FIG. 1. Judging from the morphological features, the newly acquired bacteriophage belonged to the Myoviridae bacteriophage.

The solution containing pure bacteriophage identified in this way was subjected to the following purification process. Edwadsiella tarda culture medium was added to a volume of 1/50 of the total volume of the solution including the pure bacteriophage, and then incubated again at 37 ° C. for 4-5 hours. After incubation, the supernatant was obtained by centrifugation at 8,000 rpm for 20 minutes. This procedure was repeated a total of five times to obtain a solution containing a sufficient number of bacteriophages. The supernatant obtained by the final centrifugation was filtered using a 0.45 μm filter followed by a conventional polyethylene glycol (PEG) precipitation process. Specifically, PEG and NaCl were added to 100 ml of the filtrate to be 10% PEG 8000 / 0.5 M NaCl, and then allowed to stand at 4 ° C. for 2-3 hours, followed by centrifugation at 8,000 rpm for 30 minutes to obtain a bacteriophage precipitate. The bacteriophage precipitate thus obtained was suspended in 5 ml of buffer (Buffer; 10 mM Tris-HCl, 10 mM MgSO ₄ , 0.1% Gelatin, pH 8.0). This is called bacteriophage suspension or bacteriophage solution.

In this way, purified pure bacteriophage was secured, and this bacteriophage was named as bacteriophage Edw-TAP-1, and deposited on January 8, 2015 at the Korea Research Institute of Bioscience and Biotechnology (Accession Number KCTC 12740BP).

Example  2: bacteriophage Edw Genome Isolation and Sequence Analysis of TAP-1

The genome of bacteriophage Edw-TAP-1 was isolated as follows. Bacteriophage suspension obtained in the same manner as in Example 1 was used for dielectric separation. First, in order to remove DNA and RNA of Eddsiella tarda, which may be included in the suspension, 200 U of each of DNase I and RNase A was added to 10 ml of the bacteriophage suspension, followed by standing at 37 ° C for 30 minutes. In order to remove the activity of DNase I and RNase A after 30 minutes, 500 μl of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added and allowed to stand for 10 minutes. The mixture was left at 65 ° C. for 10 minutes, and then 100 μl of proteinase K (20 mg / ml) was added to disintegrate the bacteriophage outer wall, followed by reaction at 37 ° C. for 20 minutes. Thereafter, 500 μl of 10% sodium dodecyl sulfate (SDS) was added thereto, followed by reaction at 65 ° C. for 1 hour. After the reaction for 1 hour, 10 ml of a mixture of phenol (Phenol): chloroform (Isoamylalcohol) having a composition ratio of 25: 24: 1 was added to the reaction solution, and the mixture was mixed well. After centrifugation at 13,000 rpm for 15 minutes to separate the layers, take the upper layer from the separated layers, add 1.5 volume ratio of Isopropyl alcohol, and centrifuge at 13,000 rpm for 10 minutes. Precipitated. After recovering the precipitate, 70% ethanol (Ethanol) was added to the precipitate, followed by centrifugation at 13,000 rpm for 10 minutes to wash the precipitate. The washed precipitate was recovered, dried in vacuo and dissolved in 100 μl of water. The process was repeated to secure a large amount of the genome of bacteriophage Edw-TAP-1.

Using the obtained genome, genome of bacteriophage Edw-TAP-1 through the next generation sequencing analysis using illumina Mi-Seq instrument at the National Instrumentation Center for Environmental Management at Seoul National University. Sequencing was performed. Finally, the analyzed bacteriophage Edw-TAP-1 genome has a size of 86,001 bp and the entire genome sequence is set forth in SEQ ID NO: 1.

Based on the obtained genome sequence of bacteriophage Edw-TAP-1, homology with previously known bacteriophage genome sequence using BLAST on the web (http://www.ncbi.nlm.nih.gov/BLAST/) I checked. BLAST investigation revealed no bacteriophage sequence with more than 50% homology.

Based on this fact, bacteriophage Edw-TAP-1 could be considered as a novel bacteriophage that has not been reported previously.

Example  3: bacteriophage Edw Of TAP-1 Edwad Siela  Against tar Death  Research

The killing ability of the isolated bacteriophage Edw-TAP-1 against Edward siella tarda was investigated. The killing ability was investigated by the drop test in the same manner as in Example 1 to determine the production of transparent rings. Ed ward siela tarda bacteria used for killing ability investigation were separated by the present inventors and 12 species were identified as ed ward siela tarda bacteria. Bacteriophage Edw-TAP-1 had the ability to kill against 9 species of Edward sidella tarda. Representative experimental results are shown in FIG. 2. On the other hand, bacteriophage Vibrio Anguilla room (Vibrio anguillarum) of Edw-TAP-1, V. ripening thio yen Terry (Vibrio ichthyoenteri), Vibrio para Molly's Tea Syracuse (Vibrio parahaemolyticus), Streptococcus para Ube lease (Streptococcus parauberis), Streptococcus am a (Streptococcus iniae), and Lactococcus (Lactococcus a non-teaching garvieae ) was also investigated, and as a result, bacteriophage Edw-TAP-1 had no killing ability against these species.

As a result, the bacteriophage Edw-TAP-1 has a specific killing ability against Ed Ward siella tarda, and it can be confirmed that it can exert an antimicrobial effect against a number of Ed Ward siella tarda. This means that the bacteriophage Edw-TAP-1 can be used as an active ingredient of the composition for preventing and treating Eddsiella tarda.

Example  4: bacteriophage Edw Of TAP-1 Edwad Siela  About prevention of infection of tar Experimental Example

100 μl of 1 × 10 ⁸ pfu / ml of bacteriophage Edw-TAP-1 solution was added to one tube containing 9 ml TSB medium, and the same amount of TSB medium was added to the other tube containing 9 ml TSB medium. Additionally added. Then, each tube was added with a culture medium of Eddsiella tarda so that the absorbance was about 0.5 at 600 nm. After adding Ed ward siella tarda bacteria, the tubes were transferred to a 37 ° C. incubator and shaken to observe the growth state of Ed ward siella tarda bacteria. As shown in Table 1, the growth inhibition of Eddsiella tarda was observed in the tube to which the bacteriophage Edw-TAP-1 solution was added, whereas in the tube without the bacteriophage solution, the Edwadsiella tarda bacterium was added. No growth inhibition was observed.

Inhibition of the growth of Edwardsiella tarda division OD ₆₀₀ absorbance value Incubation 0 minutes 60 minutes after incubation 120 minutes after incubation No bacteriophage solution added 0.50 1.14 1.92 Add bacteriophage solution 0.50 0.35 0.20

From this result, it can be confirmed that the bacteriophage Edw-TAP-1 of the present invention has the ability to inhibit the growth and kill the Edwadsiella tarda, and from this, the bacteriophage Edw-TAP-1 is Edwad City. It was concluded that it can be used as an active ingredient of a composition for the purpose of preventing the infection of Ella tarda.

Example  5: bacteriophage Edw With TAP-1 Edwad Siela  Tarda Infection Disease Procedure

The treatment effect of bacteriophage Edw-TAP-1 in halibut induced by Edward sickle tarda bacteria was investigated. Four-month-old halibut fry (6-9 cm) was divided into two groups, which were divided into two groups, and then separated and bred in a tank for 14 days. The environment of the bath was controlled and the temperature of the laboratory containing the bath was kept constant. The feed containing Edward sidella tarda bacteria was fed twice a day by the usual feed method at a level of 1 × 10 ⁸ cfu / g for 3 days from the 5th day from the start of the experiment. In both tanks, individuals with clinical symptoms of Edward disease were identified from the last day of feed containing the Edwardsiella tarda. From the day after the feeding of the feed containing Edddingella tarda for three days (the eighth day from the start of the test), the flounder in the experimental group (bacteriophage treated group) was 1 × 10 ⁸ pfu / g of bacteriophage Edw-. Feed containing TAP-1 was fed according to a conventional feed feeding method. On the other hand, the flounders of the control group (not administered bacteriophage) were fed with the same composition without the bacteriophage Edw-TAP-1. From the 8th day from the start of the test, all test animals were examined for the incidence of Ed disease. Ed Ward's disease status was investigated by measuring the body color blackening index. The body color blackening index was measured by measuring the dark coloration (DC) score (normal: 0, light blackening: 1, dark blackening: 2) which is commonly used. The results were shown in Table 2.

Body color blackening index measurement results (average) date D8 D9 D10 D11 D12 D13 D14 Control group (not administered bacteriophage) 1.00 1.36 1.64 1.68 1.72 1.52 1.36 Experimental group (bacteriophage administration) 1.00 0.76 0.24 0.16 0.16 0.08 0.00

From these results, it was confirmed that the bacteriophage Edw-TAP-1 of the present invention is very effective in the treatment of an infectious disease caused by Eddwardella tarda.

Example  6: Feed additives and preparation of feed

A feed additive was prepared using bacteriophage Edw-TAP-1 solution to include 1 × 10 ⁸ pfu of bacteriophage Edw-TAP-1 per g of feed additive. The method of preparing a feed additive was prepared by adding maltodextrin to the bacteriophage solution (40%, w / v) and adding trihalose to a total of 10%, followed by lyophilization. Finally, it was ground to a fine powder form. The drying process in the manufacturing process may be replaced by reduced pressure drying, warming drying, room temperature drying. For the control experiment, the feed additive without bacteriophage also used the buffer used to prepare the bacteriophage solution (Buffer; 10 mM Tris-HCl, 10 mM MgSO ₄ , 0.1% Gelatin, pH 8.0) instead of the bacteriophage solution. It was prepared by.

Each of the two feed additives thus prepared was mixed with 250 times the fish feed for fish in a weight ratio to prepare the final two feeds.

Example  7: Bath  Produce

The bacteriophage Edw-TAP-1 solution was used to prepare a bath so that 1 × 10 ⁸ pfu of bacteriophage Edw-TAP-1 per ml of the bath detergent was included. The method of preparing the bath detergent is prepared by adding the bacteriophage Edw-TAP-1 solution so that 1 × 10 ⁸ pfu of bacteriophage Edw-TAP-1 is included per 1 ml of the buffer used to prepare the bacteriophage solution. It was. For the control experiment, as a bath agent that does not contain bacteriophage, the buffer itself used in the preparation of the bacteriophage solution was used as it is.

The two types of baths thus prepared were diluted with 1,000 times water by volume and used as the final bath.

Example  8: Confirmation of specification effect in flounder breeding

Using the feed prepared in Example 6 and Example 7 and the bath was investigated whether the improvement of the specifications results when flounder breeding. In particular, the survey was conducted in terms of mortality. A total of 500 halibuts were divided into two groups (group-A fed and group-B treated with a bath) for four weeks. Each group was divided into 125 subgroups, and each subgroup was divided into a small group (small group-①) to which the bacteriophage Edw-TAP-1 was applied and a small group (small group-②) to which the bacteriophage was not applied. The flounder was a four-month-old flounder fry, and flounders from each subgroup were raised in separate tanks at regular intervals. Each subgroup is divided and referred to as Table 3 below.

Classification and marking of small groups in flounder specification test apply Small Group Separation and Display Applying bacteriophage Edw-TAP-1 Bacteriophage is not applicable A group fed at a feed A-① A-② Group treated with a bath B-① B-②

In the case of feed feeding, the feed prepared according to the feed preparation method described in Example 6 was fed according to the conventional feed feeding method according to the classification of Table 3, and in the case of the treatment of the bathing agent, the bathing bath described in Example 7 The bath preparation prepared according to the preparation method was treated according to the conventional bath treatment method according to the classification of Table 3. The test results are shown in Table 4.

Mortality in flounder specification test group Mortality (%) A-① 4.0 A-② 22.4 B-① 5.6 B-② 33.6

As a result, it was confirmed that the feed of the feed prepared according to the present invention and the bath treatment according to the present invention are effective in reducing mortality in flounder breeding. From this, it can be concluded that the application of the composition of the present invention is effective for improving the specifications results of the flounder.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Depositary: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC12740BP

Trust Date: 20150108

[Revision 25.01.2016 under Rule 26]

Claims (5)

Myobiridae bacteriophage Edw-TAP-1 (Accession No. KCTC 12740BP) characterized by having a genome represented by SEQ ID NO: 1 having the ability to specifically kill the Edward sielar tarda bacteria isolated from nature. . The bacteriophage Edw-TAP-1 of claim 1, wherein the composition for preventing and treating the infection of Ed Ward Siela tarda. The method of claim 2, wherein the composition is a composition for preventing and treating eddsiella tarda bacteria, characterized in that it is used for the preparation of a bath or feed additives. A method for preventing or treating an infection caused by Eddsiella tarda, comprising administering to a non-human animal a composition comprising the bacteriophage Edw-TAP-1 according to claim 2 or 3 as an active ingredient. Way. The method according to claim 4, wherein the composition is administered to animals other than humans for use as a bath or feed additive.

Images