Art
[1]
The present invention represents a novel bacteriophage-specific apoptotic function in avian pathogenic E. coli, using the compositions and the novel bacteriophage or the composition containing the same to a method for preventing or treating an infectious disease of birds.
BACKGROUND
[2]
E. coli ( Escherichia coli , or less, ' E.coli ' hereinafter) are intestinal bacteria, and (Enterobacteriaceae) in Escherichia Gram-negative short bacillus belonging to the genus Escherichia (Escherichia), normally present in a variety of animals, including mammals Minister one of the microflora. E. coli may lead to opportunistic infections, most non-pathogenic, some highly pathogenic strains are known to induce various bowel diseases such as sepsis, and in animals, including man.
[3]
These particular birds of the E. coli E. coli (Avian Pathogenic E. coli, APEC) is a bird, such as chicken, duck, turkey, etc. of the respiratory tract caused by an infection of the respiratory mucous membrane of E. coli as to penetrate through the body is known to have . The problem with avian pathogenic E. coli is causing enormous economic damage to the poultry industry, including with regard to the incidence of respiratory disease in birds, mainly to poultry.
[4]
On the other hand, bacteriophage (bacteriophage) refers to specific bacterial virus to inhibit the growth of bacteria infected by infecting bacteria, and specific inhibition. Bacteriophage is growing interest in the use of bacteriophages in accordance with, as well as the strong host specificity, etc. In recent years, the issue of meat residues of antibiotics and the problem of the emergence of resistant bacteria to become antibiotic use severely than the antibiotics.
[5]
This world has become many countries research on bacteriophages actively conducted in a patent application for a bacteriophage as well as Food and Drug Administration with respect to the compositions take advantage of this (Food and Drug Administration, FDA) increase the movement to obtain approval, etc. a trend that is.
[6]
However, bacteriophage-related technology is situation is still insufficient, therefore, the required development of such bacteriophage and related techniques for the prevention and / or treatment of infectious diseases caused by avian pathogenic E. coli has been a major problem in the breeding of birds including poultry being.
Detailed Description of the Invention
SUMMARY
[7]
The present inventors have solved the like the residual problem of appearance and antibiotic resistant bacteria by antibiotics meat and result of extensive research in order to effectively prevent and treat the infectious disease of birds, avian pathogenic E. coli that cause respiratory diseases in poultry for separating reached a new bacteriophage ΦCJ25 (KCCM11463P) in nature has a specific stake killing ability.
[8]
Also, by checking the excellent morphological, biochemical, genetic bacteriophage identify the property and the acid resistance, heat resistance and naegeonseong the like of the new bacteriophage, was developed utilizing this, antibiotics, disinfectants, food additives, other compositions such as, further developed a composition and a method of preventing or treating disease using the same for the prevention or treatment of infectious disease that is endemic in birds.
[9]
The object of the present invention is to provide a novel bacteriophage ΦCJ25 (KCCM11463P) having a specific ability to kill avian pathogenic E. coli.
[10]
Another object of the present invention is to provide a preventive and / or therapeutic composition for infectious disease caused by avian pathogenic E. coli containing the bacteriophage ΦCJ25 (KCCM11463P) as an active ingredient.
[11]
The invention has for its object to provide an antibiotic, feed additives, drinking water additives, feed, drinking water, cleaning agent or disinfectant containing the bacteriophage ΦCJ25 (KCCM11463P) as an active ingredient.
[12]
In addition, it is an object of the invention to provide a method for using a composition including as the bacteriophage ΦCJ25 (KCCM11463P) or active ingredients which, for preventing and / or treating infectious diseases caused by avian pathogenic E. coli.
Technical Solution
[13]
One aspect of the invention, E. coli birds (Avian Pathogenic Escherichia provides novel bacteriophage ΦCJ25 (KCCM11463P) having a specific ability to kill coli).
[14]
According to another aspect of the invention provides a composition for the prevention or treatment of the bacteriophage comprising the ΦCJ25 (KCCM11463P) as an active ingredient, an infectious disease caused by avian pathogenic E. coli.
[15]
According to another aspect of the present invention, there is provided the antibiotic bacteriophage containing ΦCJ25 (KCCM11463P) as an active ingredient, feed additives, drinking water additives, feed, drinking water, disinfectant or cleanser.
[16]
According to another aspect of the present invention, the bacteriophage provides a method for preventing or treating ΦCJ25 (KCCM11463P), or infectious diseases caused by avian pathogenic E. coli comprising the step of administering a composition comprising the same as an active ingredient in the bird.
Effects of the Invention
[17]
Bacteriophage ΦCJ25 (KCCM11463P) of the present invention has the effect of killing the algae E. coli specifically.
[18]
Further, the present invention bacteriophage ΦCJ25 (KCCM11463P) not only can be used as acid resistance, heat resistance and naegeonseong is superior to different temperatures, pH, and preventing or substances for the treatment of infectious diseases caused by avian pathogenic E. coli in a dry condition range, the bacteriophage containing ΦCJ25 (KCCM11463P) as an active ingredient and has an effect which can be utilized as antibiotics, feed additives, drinking water additives, feed, drinking water, disinfectant or detergent.
[19]
In addition, the invention can provide an antibiotic comprising a said bacteriophage ΦCJ25 (KCCM11463P) or the active ingredient, conventional compared with the antibiotic specificity for avian pathogenic E. coli is very high ikgyun is without killing killing only a specific pathogen, drug resistance do not induce has an effect extending the life of the product in comparison with the conventional antibiotics.
[20]
In addition, the present invention has the effect that by administering a composition comprising as the bacteriophage ΦCJ25 (KCCM11463P) or the active ingredient in this bird, can prevent or treat infectious diseases caused by avian pathogenic E. coli.
Brief Description of the Drawings
[21]
1 is an electron micrograph of a novel bacteriophage ΦCJ25 (KCCM11463P) (hereinafter, 'ΦCJ25').
[22]
Figure 2 illustrates the result of new bacteriophage ΦCJ25 PFGE.
[23]
Figure 3 shows the SDS-PAGE results of the new bacteriophage ΦCJ25.
[24]
4 is a graph showing the experimental results of the acid resistance of new bacteriophage ΦCJ25.
[25]
5 is a graph showing experimental results of the heat resistance of new bacteriophage ΦCJ25.
[26]
Figure 6 is a graph showing the experimental results of the new bacteriophage naegeonseong ΦCJ25.
Mode for the Invention
[27]
It will be described below in more detail with respect to the present invention. If the information is not described in the present specification is the art or similar to those skilled in the art because it can be sufficiently recognized and inferred the description thereof is omitted.
[28]
[29]
One aspect of the invention, E. coli birds (Avian Pathogenic Escherichia provides having a specific function in the apoptotic coli, APEC), New bacteriophage ΦCJ25 (KCCM11463P) (hereinafter, 'ΦCJ25').
[30]
Avian pathogenic E. coli in chickens, ducks, birds of infectious diseases as E. coli infection through the respiratory system of birds, such as turkeys, specifically, can cause algae daejanggyunjeung. Specifically, the bird E. coli is E. coli that cause various diseases such as sepsis, granulomas, envelope salts, salts oviduct, arthritis to penetrate into the body of the bird through the respiratory mucosa. The bird E. coli is a Gram-negative bacilli of generic E. coli as the main maternal flagellum is aerobic or facultative anaerobic bacteria to decompose movement and lactose (Lactose), fructose (Fructose) to produce gas in the mountains.
[31]
E. coli is the bird grows well in normal growth medium available from about 7 ℃ to 48 ℃ of temperature and optimal growth temperature may be about 35 ℃ to 37 ℃. In particular, the expression of virulence factors made effective in about 42 ℃ temperature and close to the birds. Also, birds pathogenic E. coli can be developed in the range of pH 4.5 to pH 9.0.
[32]
Bacteriophages (bacteriophage) a particular bacterial infection to relevant bacterial growth inhibition and inhibition of bacteria-specific viruses, single or double chains of DNA (Deoxyribonucleic acid) or RNA (Ribonucleic acid) genetic material to contain the virus meaning do.
[33]
Specifically, the invention of one aspect of the bacteriophage ΦCJ25 Bird pathogenic E. coli to selectively infect that species specificity of having bacteriophage as, regular icosahedron head (isometric capsid) to have shrinking is that the tail (contractile tail) to the configured structure ( with FIG. 1), a bacteriophage belonging to the morphologically ohbiri de Mai (Myoviridae). After a nucleotide sequence of bacteriophage ΦCJ25 and comparing the homology of the base sequence of the other bacteriophage decryption is as shown in [Table 1]. In the acid resistance of the bacteriophage ΦCJ25 without losing the activity to pH 3.5 to pH 11.0 stable (Fig. 4), in the heat resistance more than 50 ℃: even when exposed for 2 hours (example 53 ℃) did not lose significant activity (Fig. 5) . In naegeonseong bacteriophage it was reduced to approximately 2 logs after drying of the tie ΦCJ25 emitter (Fig. 6). DNA sequencing of the bacteriophage ΦCJ25 are as SEQ ID NO: 1 on the sequence list.
[34]
ΦCJ25 the bacteriophage was deposited by the present inventors as a novel bacteriophage isolation, Korea Culture Center of Microorganisms of 25 October 2013 on the deposit (Korean Culture Center of Microorganisms, Seodaemun-gu, Seoul 361-221 East Hongje 1) Number KCCM11463P.
[35]
[36]
According to another aspect of the invention provides a composition for the prevention or treatment of the bacteriophage comprising the ΦCJ25 as an active ingredient, an infectious disease caused by avian pathogenic E. coli.
[37]
ΦCJ25 the bacteriophage may be used for the purpose of prevention of diseases caused by infection by avian pathogenic E. coli or treatment because it indicates that the antimicrobial activity can be an avian pathogenic E. coli apoptosis specifically. It includes birds daejanggyunjeung (Avian colibacillosis) Examples of the avian infectious disease pathogenic E. coli, but are not limited thereto.
[38]
The bird daejanggyunjeung Iran, a disease that occurs in birds respiratory, etc. are infected with E. coli, the envelope salt thereof symptoms (airsacculitis), ganpo pericarditis (perihepatitis), peritonitis (peritonitis), the heart peri (pericarditis), oviduct salt (salpingitis ), umbilical salt (omphalitis), osteomyelitis (represented by a variety of lesions, such as osteomyelitis) or sepsis (septicemia), a disease that causes decreased growth and mortality of infected birds.
[39]
As used herein, the term "prevention" refers to the bacteriophage ΦCJ25 and / or the bacteriophage by providing a composition comprising as an effective ingredient to a subject ΦCJ25 refers to all acts to inhibit the disease or delaying the onset.
[40]
As used herein, the term "treatment" refers to the bacteriophage used in the ΦCJ25 and / or the bacteriophage provides a composition comprising a ΦCJ25 as an effective ingredient to a subject by means all actions that have already improved or improving the symptoms of infection the disease.
[41]
The composition for prevention or treatment of this aspect of the infectious disease caused by the avian pathogenic E. coli is the bacteriophage ΦCJ25 5 × 10 2 to 5 × 10 12 may contain as pfu / ㎖, specifically, 1 × 10 the bacteriophage the ΦCJ25 6 to 1 × 10 10 it may further contain pfu / ㎖.
[42]
Composition for preventing or treating infectious diseases caused by the avian pathogenic E. coli of the present embodiment is provided with, and can further comprise a pharmaceutically acceptable carrier, it is formulated with the support food, medicine, feed additive or drinking water additive It can be.
[43]
It means a carrier or diluent that does not stimulate the term "pharmaceutically acceptable carrier" as used in the invention is an organism which does not affect the biological activity and properties of the administered compound.
[44]
Type of the carrier is available in the present aspect is used in a conventional in particular shall not limit the art it may be used if the carrier is pharmacologically acceptable to either. May include the non-limiting examples of the carriers include, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or a mixture of two or more.
[45]
In addition, as an antioxidant, a buffer and / or bacteriostatic agents, etc. it may be used by the addition of other conventional additives, diluents, dispersants, surfactants, binders and / or lubricants, such as an addition solution was added, suspension, emulsion, if necessary and formulated in such injectable formulations, pills, capsules, granules or tablets, etc., it can be used.
[46]
The mode of administration of the composition for preventing or treating infectious diseases caused by the avian pathogenic E. coli of the present embodiment are merely exemplary and are not particularly restricted, and may be in accordance with a method that conventionally used in the art. The administration of non-limiting example of the method, may be administered as a composition administered orally or parenteral modes of administration.
[47]
Non-limiting examples of formulations for the oral administration, troches (troches), lozenges (lozenge), tablets, aqueous suspensions, oily suspensions, preparing powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs (elixirs ) and the like can be given.
[48]
In order to formulate the composition of the present aspect to the formulation, such as tablets or capsules, lactose, saccharose (Saccharose), sorbitol (Sorbitol), mannitol (Mannitol), starch, amylopectin (Amylopectin), cellulose (Cellulose) or gelatin (Gelatin) as such as a binder; Excipients such as dicalcium phosphate (dicalcium phosphate); The collapse, such as corn starch or sweet potato starch; Magnesium stearate (magnesium stearate), calcium stearate (calcium stearate), stearyl fumarate, sodium (sodium stearyl fumarate) or polyethylene glycol wax (polyethylene glycol wax) such as lubricants for inclusion to be, and the capsule dosage form in the case the above-mentioned in addition to the material it may further contain a liquid carrier such as fatty oil.
[49]
A method for parenteral administration of a composition of the present aspect, for example, intravenous administration, intraperitoneal administration, intramuscular administration, can be used for subcutaneous or local administration, such as, a method for applying or spraying the said composition to the disease site also It is used, but not limited to these.
[50]
The parenteral dosage form as is for administration, such as injectable forms, such as subcutaneous injection, intravenous injection or intramuscular injection; Suppositories, injection; Or it may be formulated for aerosol spray or the like which enables the inhalation through the respiratory tract, but not limited thereto. The injectable composition can be manufactured according to the present aspect to a formulation formulated as solutions or suspensions in a mixture of water and formulated with a stabilizer or buffer that for the unit dosage of the ampoule (ampoule) or vial (vial). When formulated for the spray, such as aerosols, it may be dispersed such that dispersed concentrate or a wet powder is to be formulated with a propellant and the like additives.
[51]
Suitable coating, spraying or dosage of a composition for preventing or treating infectious diseases caused by the avian pathogenic E. coli of the present aspect is formulated method of the composition, mode of administration, time of administration and / or the route of administration, of course, that the administration subject the age of the animal, the degree of weight, sex, disease symptoms, intake of food, and can be vary by factors such as the excretion rate, generally experienced veterinarian will readily determine and prescribe an effective dosage for the desired treatment can.
[52]
[53]
According to another aspect of the present invention, the bacteriophage comprises a ΦCJ25 provide antibiotic as an effective ingredient.
[54]
And it is provided to a subject, including humans, by the term "antibiotic" means a dosage form used in the present invention means a preparation having a potency capable of killing bacteria, and corresponds to the generic concept of a preservative, a fungicide and antimicrobial agent.
[55]
Containing the bacteriophage ΦCJ25 or a composition containing the same of the present embodiment as an active ingredient, antibiotic conventional specificity for comparison with antibiotics in birds E. coli very high ikgyun can be killed only certain pathogens without killing, not induce drug resistance It does can exhibit the effect that the product life is extended as compared to the conventional antibiotics.
[56]
[57]
According to another aspect of the present invention, the bacteriophage provide bird feed additive or drinking water additive for ΦCJ25 containing as an active ingredient.
[58]
"Bird" target birds application of the feed additive or drinking water additives is not particularly limited, the birds in the present embodiment may be particularly poultry.
[59]
As used herein the term "poultry" refers to cattle of a concept ranging collectively animals belonging to the birds. The poultry is not a not particularly limited, and may include one or more selected from the group consisting of chickens, ducks and turkeys and the like.
[60]
The birds feed additive or drinking water additive of the present aspect is used or in a manner to separately manufacture a composition forms a feed additive or drinking water additive containing the bacteriophage ΦCJ25 or them to mix in the feed or drinking water, the bacteriophage ΦCJ25 or compositions containing them when the feed or drinking water produced can be used in a way that directly added.
[61]
ΦCJ25 the bacteriophage and / or compositions containing the bacteriophage as the active ingredient a ΦCJ25 used in the feed additive or drinking water additive of the present embodiment may be a liquid or dry state, it may be in dry powder form.
[62]
For example, bacteriophage ΦCJ25 of the present invention can be mixed in a composition ratio of 0.05 to 10% by weight of the feed additive by weight in the form of a powder, preferably from 0.1 to 2% by weight.
[63]
Drying method for preparing a dry powder form for the feed additive or drinking water additive of the present embodiment are merely exemplary and are not particularly limited and may be a method for commonly used in the art. There may be mentioned non-limiting examples of the drying method is blow-dried, air-dried, spray-drying, freeze-drying and the like. These may be used alone or carried out in a manner with the use of two or more methods.
[64]
The feed additive or drinking water additive of the present embodiment has other pathogenic microorganisms can be further added. Bacillus subtilis, such as the non-limiting examples of microorganisms that can be added are proteases, Bacillus subtilis which can produce a lipolytic enzyme and / or a sugar-converting enzyme (Bacillus subtilis); Lactobacillus strain having the physiological activity and the organic material in anaerobic conditions, such as resolution of cattle above ( Lactobacillus lactic acid bacteria, such as sp.) And the like; To increase the weight of the animal's increasing Aspergillus having the effect of enhancing the digestive absorption of the milk production of milk feed ducks party ( Aspergillus oryzae ), such as fungi; And a saccharide in MRS serenity busy ( Saccharomyces cerevisiae may be selected from the group consisting of yeast, such as). These may be used alone or a mixture of two or more.
[65]
Feed additive or drinking water additive containing the bacteriophage of the present ΦCJ25 aspect as an active ingredient may further comprise other additives, if necessary. The used binder is added in order to prevent the non-limiting examples of the additive, the feed or the drinking water of the quality degradation, emulsifiers, preservatives and the like; Adding an amino acid agents, vitamins, enzymes, probiotics, flavoring agents, non-protein nitrogen compounds, silicate agent, buffering agents, coloring agents, extracts and the like agent or oligosaccharide, feed mixtures, etc. In addition to that added in order to increase the utility of the feed or drinking water it can include. These may be used alone or added with two or more.
[66]
With respect to the feed additive is 100 parts by weight of the feed of the present aspect, and may be contained from 0.05 to 10 parts by weight, may be particularly containing from 0.1 to 2 parts by weight. The drinking water additive of the present embodiment is with respect to 100 parts by weight of the drinking water, may be contained O.0001 to 0.01 parts by weight, it may be contained in concrete, from 0.001 to 0.005 parts by weight. The activity of the bacteriophage ΦCJ25 for avian pathogenic E. coli within this range there is an advantage to be sufficiently exhibited.
[67]
[68]
According to yet another aspect, provides a feed additive or drinking water additive of the feed or the drinking water prepared by adding directly to the feed or addition to drinking water or the bacteriophage feed the ΦCJ25 or drinking water containing the bacteriophage ΦCJ25 as an active ingredient of the present invention do.
[69]
Feed to be used in the present invention it shall not be particularly limited and may be used to feed commonly used in the art. The non-limiting examples of feed, cereals, roots and acids, food processing logistics Busan, birds, fiber flow, logistics constraints Busan, fats, starches, vegetable or grain feed residues such as Busan logistics; It may be an animal feed, such as proteins and inorganic logistics, oils, mineral acids, oils, single cell proteins, zooplankton flow or food. These may be used alone or a mixture of two or more.
[70]
Shall not be particularly limited drinking water is to be used in the present invention, the drinking water may be conventionally used in the art.
[71]
[72]
According to another aspect of the invention provides a bacteriophage comprising the active ingredient in the ΦCJ25, disinfectants or cleansers. The formulation of the disinfectant or cleaning agents are merely exemplary and are not particularly restricted, and may be used is prepared in the dosage form known in the art.
[73]
The birds can be sprayed disinfectants to remove pathogenic E. coli it can be sprayed activity areas, slaughterhouses, birds of our area, cooking area or cooking equipment, etc., but not limited thereto.
[74]
The cleaning agent may be used for the purpose of cleaning the surface of the skin, such as body or the parts of the birds, which can be exposed or exposed to avian pathogenic E. coli, but not limited thereto.
[75]
[76]
According to another aspect of the present invention, by using a composition containing the bacteriophage as the active ingredient or ΦCJ25 this end, provides a method for preventing or treating infectious diseases caused by avian pathogenic E. coli.
[77]
The prevention or treatment method of the present embodiment is particularly, the in birds which are at risk to be infected or infected by avian pathogenic E. coli bacteriophage ΦCJ25 or the bacteriophage step of administering a composition comprising an ΦCJ25 as an active ingredient a pharmaceutically effective amount of It includes. Suitable total daily usage of the compositions containing the bacteriophage ΦCJ25 or it may be determined by the physician in the correct medical judgment range, which is made apparent to one of ordinary skill in the art.
[78]
Specific pharmaceutically effective amount of the composition containing the bacteriophage ΦCJ25 or the bacteriophage ΦCJ25 as an active ingredient for a particular algae, the type of reaction to be achieved and the degree, age, body weight, general health, sex, or expression as well as for the object the bacteriophage ΦCJ25 or may be determined in consideration of the time of administration, route of administration, and the ratio of the composition of the composition, treatment period and so on containing them, various factors and medicine, including the components such as drugs and other compositions to be used together at the same time or Ishihara according to similar factors well known in the field it may be varied.
[79]
The bacteriophage ΦCJ25 or composition containing the bacteriophage as the active ingredient of the present embodiment is either intranasal aerosol minutes in birds in the form of pharmaceutical preparations, are added directly to feed or drinking water of the birds may be administered in a way that feeding them, in the form of a feed additive or drinking water additive can be administered mixed in the feed or drinking water.
[80]
Administration route and manner of administration of the composition containing the bacteriophage ΦCJ25 or the bacteriophage as the active ingredient of the present embodiment is administered any that can reach the composition containing the bacteriophage ΦCJ25 or them to the organization that shall not be particularly limited, the objective It may depend on the route and mode of administration. That is, within the bacteriophage composition comprising a ΦCJ25 or the bacteriophage as the active ingredient may be administered via the oral or parenteral sphere different paths, non-limiting examples of the route of administration, oral, rectal, topical, intravenous, intraperitoneal , intramuscular, it may be mentioned to be administered through intraarterial, transdermal, or by inhalation such as non cheuknae.
[81]
[82]
It will now be described in more detail to the present invention the following examples. However, these examples are not intended the scope of the invention serves to explain the present invention by way of example to be limited to these Examples.
[83]
[84]
Example 1 - Isolation of bacteriophage to infect birds in E. coli
[85]
[86]

[87]
Screening and separating the single bacteriophage bacteriophage
[88]
Chungcheong Province Hongseong after the mineral region Samhwa progenitor 10 minutes centrifugation of the sample 50 ㎖ obtained from chicken manure on the farm at 4,000 rpm separated by filtration and the supernatant with 0.45 ㎛ filter to prepare a sample solution, by using this soft agar overlay ( soft agar overlay) method was performed. The soft agar overlay method is the top - by the host cells growing in agar (to put it on a solid medium using a top-agar, 0.7% agar) refers to a method for the lysis observed that bacteriophage.
[89]
Specifically, a bird E. coli obtained from National University College of Veterinary Medicine (E09-19) shaking culture (OD 600 = 2) and 10 × 150 ㎕ LB medium (tryptophane (tryptone) 10 g / ℓ; yeast 5 g / ℓ extract; a mixture of 18 ㎖ the sample filtrate to NaCl 10 g / ℓ) 2 ㎖ and incubated them for 18 hours at 30 ℃ and then, the culture solution by a separation at 4,000 rpm 10 bungan centrifuged and filtered and the supernatant with 0.45 ㎛ filter. Then, 0.7% (w / v) agar and bird 3 ㎖ pathogenic E. coli (E09-19) shaking culture (OD on the LB plate medium 600 and then hardened by pouring = 2), a mixed solution of 150 ㎕, that the sample solution over 10 ㎕ added dropwise and it was confirmed gyunban are formed by incubation for 18 hours at 30 ℃.
[90]
Since known to the one type of bacteriophage has a gyunban for the present, it was to separate the single bacteriophage from gyunban for said formed. Specifically, the for gyunban the 400 ㎕ of SM solution (NaCl 5.8 g / ℓ; MgSO 4 7H 2 O 2 g / ℓ; 1M Tris-Cl (pH 7.5) 50 ㎖) was added at room temperature 4 hours dongan left by bacteriophage to give a solution.
[91]
Then, the bacteriophage solution 100 ㎕ a 0.7% (w / v) agar 12 ㎖ and avian pathogenic E. coli (E09-19) shaking culture (OD 600 = 2) 500 ㎕ and mixed, 150 mm diameter of the LB plate medium for was performed using the soft agar overlay method, it was incubated until completely lysed. After the culture was completed, the solution was added to the SM 15 ㎖ on the LB plate medium and allowed to stand at room temperature for 4 hours to give the bacteriophage solution.
[92]
Chloroform was added to the solution, and recovering, 1% (v / v) and then mixed for 10 minutes and the supernatant was obtained by centrifugal separation bungan 10 to 4,000 rpm to give a final sample by filtering the supernatant with 0.45 ㎛ filter.
[93]
[94]

[95]
Mass culture and purification of the bacteriophage
[96]
A bacteriophage culture obtained in the above Example 1-1, a large amount using Escherichia coli birds (E09-19), and the bacteriophage were purified therefrom.
[97]
Specifically, by shaking culture avian pathogenic E. coli (E09-19) 1.5 × 10 10 after frequency division by separating from the 4,000 rpm centrifugation bungan 10 such that the cfu, were resuspended in this solution 4 ㎖ SM. The bacteriophage 1.5 × 10 here six inoculated with pfu and MOI (multiplicity of infection) = 0.0001 appropriate then allowed to stand at room temperature to 20 bungan.
[98]
[99]
Then, it was inoculated in 150 ㎖ LB medium and incubated for 6 hours at 30 ℃. After the culture is finished, to a final 1% of the volume (v / v) was added to chloroform and was stirred for 20 minutes, restriction enzymes, the addition of DNase I and RNase A to a final concentration of 1 ㎍ / ㎖, respectively, and at 30 ℃ allowed to stand for 30 minutes. Then, the final concentration was added to each of 1M and 10% (w / v) is sodium chloride and polyethylene glycol (polyethylene glycol, PEG) that was added value brought for 3 hours at 4 ℃ 20 minutes at from 4 ℃ 12,000 rpm centrifugation to give a precipitate.
[100]
Allowed to stand in the obtained precipitate was suspended in a 5 ㎖ SM 20 minutes at room temperature to the solution and then added to 4 ㎖ chloroform and stirred with 4,000 rpm and centrifuged for 20 minutes at 4 ℃ to give a supernatant. The supernatant was filtered through a 0.45 filter and then ㎛, glycerol density gradient method by performing a second centrifugation (35,000 rpm, 1 sigan, 4 ℃) using (density 40%, 5% glycerol) was purified bacteriophage.
[101]
[102]
The present inventors have farms of fecal samples collected from the sample to the birds pathogenic E. coli (Avian Pathogenic E. coli, APEC) specifically for the killing ability of the bacteriophage with the "Bacteriophage ΦCJ25" naming and October 25, 2013 Korea Culture Center of microorganisms (Korean Culture Center of microorganisms, Seodaemun-gu, Seoul 361-221 East Hongje 1) accession No. KCCM11463P was deposited on the arc.
[103]
[104]

[105]
Types of observed ΦCJ25
[106]
Diluting the purified bacteriophage ΦCJ25 in Example 1 in 0.01% gelatin solution, which was fixed with 2.5% glutaraldehyde (glutaraldehyde) solution. This carbon-coated mica plate (carbon-coated mica plate) (ca. 2.5 mm × 2.5 mm) was added dropwise in 10 min to adapt was then washed with sterile distilled water it was.
[107]
Then, the carbon film (carbon film) copper grid (copper grid) in the fitted 4% uranyl acetate (uranyl acetate) at 60 chogan dyed and dried, a transmission electron microscope (JEM-1011, 80 kV, magnification × 200,000) was speculum (FIG. 1).
[108]
FIG. 1 is a bacteriophage ΦCJ25 of transmission electron micrographs and shown to be about 83 nm size of the regular icosahedron head and contractility is in the tail (contractile tail) to the configured form type (morphotype A1), My ohbiri to (myobiridae) in the fall.
[109]
[110]

[111]
[112]
Size analysis of the genome DNA of ΦCJ25
[113]
Genomic DNA was extracted from the bacteriophage ΦCJ25 purified in Example 1.
[114]
Specifically, the purified bacteriophage ΦCJ25 culture medium in 20 mM EDTA (Ethylenediaminetetraacetic acid), 50 ㎍ / ㎖ proteolytic enzyme (proteinase) K and 0.5% (w / v) SDS (sodium dodecyl sulfate) was added and 50 ℃ 1 hour dongan value was stirring added to phenol (pH 8.0) of equal volume, and then at room temperature at 12,000 rpm centrifugal separation to obtain a supernatant liquid 10 bungan.
[115]
PC equal volume of the supernatant (phenol: chloroform = 1: 1) and mixed at room temperature, remove 10 bungan centrifuged at 12,000 rpm to obtain a supernatant. The supernatant was mixed with an equal volume of chloroform and centrifuged to separate 10 bungan at room temperature at 12,000 rpm to obtain a supernatant. Was added a mixture of 3M sodium acetate (sodium acetate) in the supernatant so that 10% (v / v) of the total volume, and the mixture was added to cold 95% ethanol of two times volume, then allowed to stand for 1 hour at -20 ℃ .
[116]
Then, the precipitate obtained by removing the supernatant after centrifugation at 12,000 rpm for 10 minutes and then 0 ℃ was dissolved was added to this 50 ㎕ TE buffer (Tris-EDTA, pH 8.0). Diluted 10-fold in the extracted DNA by the OD 260 was measured for the concentration by measuring the absorbance at.
[117]
Then, 1 ㎍ of DNA a 1% PFGE (pulse-field gel electrophoresis) agarose gel in the loading and BioRad (BIORAD) PFGE systems 7 program (size range 25 kb to about 100 kb; switch-time lamp (switch time ramp) 0.4 seconds to 2.0 seconds, a linear (linear shape); forward voltage (forward voltage), 180 V; reverse voltage (reverse voltage), 120 V) by using the ambient temperature at 20 was sigan during deployment (FIG. 2).
[118]
2 is an electrophoresis photo of genomic DNA of bacteriophage ΦCJ25, the genomic DNA of bacteriophage ΦCJ25 was confirmed to represent a size of about 39 kbp.
[119]
[120]

[121]
Analysis of protein patterns ΦCJ25
[122]
10 11 mix the purified bacteriophage ΦCJ25 15 ㎕ solution and 5X SDS sample solution of 3 ㎕ pfu / ㎖ titer (titer) and boiled for 5 minutes. The mixture was subjected to a 12% SDS-PAGE (Fig. 3).
[123]
Figure 3 is an electrophoresis image showing the SDS-PAGE results of a bacteriophage to target ΦCJ25, it confirmed the major protein of about 43 kDa, 49.3 kDa, 60.4 kDa and 94.9 kDa in size.
[124]
[125]

[126]
Analysis of genetic characteristics of ΦCJ25
[127]
In order to examine the genetic properties of a bacteriophage ΦCJ25 purified in Example 1 were analyzed for DNA of the bacteriophage ΦCJ25 using genetic analysis device FLX Titanium sequencer (sequencer titanium) (Roche). (Note) was a combination of genes using Macrogen GS and de novo assembly software (de novo assembler software) (Roche) in (Macrogen Inc.). Transcription analysis frames (open reading frame) was performed by using GeneMArk.hmm, Glimmer and FGENESB v3.02 software. The name of the analysis framework was named Champion (annotation) using the BLASTP and inter-professional scan (InterProScan) program.
[128]
Nucleotide sequence of the bacteriophage is bacteriophage was found not to match the 100%, or any fragment showed a different nucleotide sequence and the similarity of the bacteriophage (Enterobacteria phage EcoDS1) reported. Thus the bacteriophage was confirmed that the newly separated bacteriophage.
[129]
[130]
Table 1 shows a comparison of the homology of the base sequence of the bacteriophage ΦCJ25 and decode the base sequence of the other bacteriophage.
[131]
[132]
Table 1 [Table 1]
Query Subject 　 Identities
Name Length Start End Description E-Value Match/Total Pct.(%)
SEQ ID NO: 1 39618 1 9030 Enterobacteria phage EcoDS1, complete genome 0 8653/9041 95

[133]
Above for the preparation of a DNA bacteriophage ΦCJ25 analyzed using gene analysis apparatus entire base sequence result is shown in SEQ ID NO: 1.
[134]
[135]

[136]
Stability investigation of the pH according to the ΦCJ25
[137]
To various pH ranges to determine the bacteriophage ΦCJ25 ability to retain the stability at low pH as shown in the condition in the stomach (pH2.5, 3.0, 3.5, 4.0, 5.5, 6.5, 7.0, 8.0, 9.0, 10.0 and 11.0) stability tests were carried out at research.
[138]
Experiments to various pH solutions (sodium acetate buffer solution (pH 4.0, pH 4.5, pH 5.0 and pH 5.5), sodium citrate buffer solution (pH 2.5, pH 3.0 and pH 3.5), sodium phosphate buffer solution (pH 6.5 and pH 7.0 ), Tris-HCl solution (pH 8.0, pH 9.0, pH 10.0 and pH 11.0) were prepared in 0.2 M, respectively.
[139]
The pH of each solution with 1.0 × 180 ㎕ 10 10 Mix 20 ㎕ PFU / ㎖ of titer bacteriophage solution is then presented a 1M concentration of each pH solution, it was allowed to stand at room temperature for 2 hours. The control group, 1.0 × 10 in the same way 10 was allowed to stand at room temperature gave a mixture PFU / ㎖ bacteriophage solution 20㎕ the SM 180 ㎕ solution, for 2 hours. Then, these diluted phase, and by using a soft agar overlay method and cultured for 18 hours at 30 ℃ after dropping a diluted solution in each step by 10 ㎕ measuring the titer over whether lysis (FIG. 4).
[140]
Figure 4 shows the experimental results of the acid resistance ΦCJ25 bacteriophage. As shown in FIG 4, compared to the control ΦCJ25 bacteriophage was confirmed that stable without losing the activity to pH 11.0 from pH 3.5.
[141]
[142]

[143]
Stability investigation of the temperature according to the ΦCJ25
[144]
When used as a feed additive of the bacteriophage product formulation of the dosage form, it may cause heat from the process of the bacteriophage, the experiment was performed to determine the stability of a column.
[145]
More specifically, 1.25 × 10 10 10 min PFU / ㎖ 37 ℃ the solution 100 ㎕ of bacteriophage ΦCJ25 of concentration, 45 ℃, 53 ℃ and 60 ℃, 30 minutes, 60 minutes, and then allowed to stand for 120 minutes, the experiment culture medium the step was diluted with a soft agar overlay method by culturing for 18 hours at 30 ℃ back dropped by 10 ㎕ dilutions of each step measured titer over whether lysis (FIG. 5).
[146]
Figure 5 shows the experimental results of the heat resistance ΦCJ25 bacteriophage. As shown in Fig. 5, is the bacteriophage ΦCJ25 ℃ 53 did not decrease the activity up to 120 minutes in 60 ℃ was found to be about 1 log (log) decreases to less than 120 minutes.
[147]
[148]

[149]
Stability investigation of dried
[150]
When using a bacteriophage feed additive formulation of the product of the drying process so mounted in the formulation process of the bacteriophage, the experiment was performed in order to verify the stability of the ΦCJ25 on drying conditions.
[151]
Based on the results derived from the heat-resistant confirmation experiment, and concentrated using a vacuum centrifuge (SpeedVac concentrator) was performed on the dry experiment. 1.2 X 10 10 to PFU / ml titer completely before re-suspension for a day for two hours a solution of 200 ㎕ ΦCJ25 of titer and dried in vacuum at 60 ℃ the pellet (pellet) into the SM 200㎕ solution at 4 ℃ was measured (Fig. 6).
[152]
Figure 6 shows the experimental results of bacteriophage naegeonseong ΦCJ25. As shown in FIG. 6, after drying when compared to the original titer and stability relative activity when dried for 2 hours at 60 ℃, was found to be reduced to less than about 2 log (log).
[153]
[154]

[155]
It isolates infected wild birds ΦCJ25 range of research on pathogenic E. coli
[156]
Bacteriophage ΦCJ25 the bird pathogenic E. coli (E09-19) In addition to separate wild birds 46 weeks pathogenic E. coli, the E. coli pathogenic avian pathogenic E. coli separated from the birds of 10 weeks, Chonbuk National University College of Veterinary Quarantine Service in separate separated from Konkuk University College of Veterinary Medicine, used in experiments 7 weeks, about 26 weeks in neuropathic feelings pathogenic E. coli isolated from birds komipam farm was to determine whether the lytic activity.
[157]
More specifically, each strain of shaking culture (OD 600 = 2) 150 ㎕ the mix soft agar overlay method proceeds to 10 titer of bacteriophages ΦCJ25 of the solution 10 ㎕ by dropped after 30 ℃ in 18 hours culture gyunban formation was observed for the presence or absence (Table 2).
[158]
[159]
To the results shown in Table 2 below.
[160]
Table 2 [Table 2]
No. Konkuk strains serotyping ØCJ25 No. Quarantine Service strains serotyping ØCJ25
1 E09-1 O 48 06Q-035 The 78- O
2 E09-2 O 49 06D-044 The 78-
3 E09-3 O 50 06Q-140 The 78- O
4 E09-4 O 51 07D-001 The 78- O
5 E09-5 O 52 07D-022 The 78- O
6 E09-6 The 78- O 53 07Q-039 The 78- O
7 E09-7 O 54 KWU-02 The 78- O
8 E09-8 The 78- O 55 KWU-32 The 78- O
9 E09-9 The 78- O 56 KWU-33 The 78-
10 E09-10 O 57 KWU-43 The 78- O
11 E09-11 The 78- O No. Chonbuk strains serotyping ØCJ25
12 E09-12 The 125- O
13 E09-13 O 58 A12-MRA-076-① O
14 E09-14 O 59 A10-LSf-005
15 E09-15 O 60 A11-LSF-043
16 E09-16 O 61 A12-MRA-076-②
17 E09-17 O 62 D12-JW-058 O
18 E09-18 63 A12-LSF-083 The 78-
19 E09-19 O 64 A12-MRA-076-③ O
20 E09-20 O No. Komipam strains serotyping ØCJ25
21 E09-21 O
22 E09-22 O 65 12-001-3 O
23 E09-23 O 66 12-053 O
24 E09-24 O 67 12-055 O
25 E09-25 O 68 12-086-1 The 78- O
26 E09-26 O 69 12-086-2 The 78- O
27 E09-27 O 70 12-096-3 O
28 E09-28 O 71 12-175 O
29 E09-29 O 72 12-187 The 78- O
30 E09-30 O 73 12-211-5 O
31 E09-31 O 74 12-220-4 O
32 E09-32 O 75 12-220-6 O
33 E09-33 O 76 12-248 O
34 E09-34 O 77 12-261-1 O
35 E09-35(297) The 78- 78 12-266 O
36 E09-36(343) O 79 12-274-1 O
37 E09-37(343) O 80 12-275-2 The 78- O
38 E09-38(343) O 81 12-286-2 O
39 E09-39(353) O 82 12-300 The 78-
40 E09-40(353) O 83 12-303-2 The 78- O
41 E09-41(376) O 84 12-304-3 O
42 E09-42(376) O 85 12-299-1 The 78- O
43 E102 O-1 O 86 12-299-2 The 78- O
44 E103 The 78- O 87 12-299-3 The 78- O
45 E104 The 78- O 88 12-324 The 78- O
46 E105 The 78- 89 12-338-1 The 78-
47 E106 90 12-338-4 The 78-

[161]
As shown in Table 2, the most frequent of bacteriophage ΦCJ25 is the causative agent of avian pathogenic E. coli daejanggyunjeung birds from the poultry farms in general is shown with O-serotypes 78 serotypes (O-1, 0-125) in addition to that it was confirmed that seem to infect a wide effective performance.
[162]
[163]
Attached to the sequence listing submitted by submission
[164]

Claims
[Claim 1]
Avian pathogenic E. coli (Avian pathogenic Escherichia coli having a specific function in apoptosis), new bacteriophage ΦCJ25 (KCCM11463P).
[Claim 2]
Of claim 1 wherein the bacteriophage ΦCJ25 composition for the prevention or treatment of infectious diseases caused by bird E. coli containing (KCCM11463P) as an active ingredient.
[Claim 3]
According to claim 2, wherein the avian infectious disease is daejanggyunjeung the prophylaxis or treatment composition for infectious diseases caused by avian pathogenic E. coli.
[[4]
Of claim 1 wherein the bacteriophage comprising the ΦCJ25 (KCCM11463P) as an active ingredient, an antibiotic.
[Claim 5]
Paragraph 1 of the bacteriophage containing the ΦCJ25 (KCCM11463P) as an active ingredient, feed additives for birds.
[6.]
Including the paragraph 5 Feed additives for birds, Fodder for birds.
[7.]
Of claim 1 wherein the bacteriophage comprising the ΦCJ25 (KCCM11463P) as an active ingredient, an additive for drinking water algae.
[8.]
Article 7 paragraph that contains the birds drinking water additives, drinking water for the birds.
[9.]
Of claim 1 wherein the bacteriophage comprising the ΦCJ25 (KCCM11463P) as an active ingredient, a disinfectant.
[10.]
Of claim 1 wherein the bacteriophage comprising the ΦCJ25 (KCCM11463P) as an active ingredient, detergent.
[11.]
Article of claim 1 comprising the step of administering the bacteriophage ΦCJ25 (KCCM11463P) or the composition of claim 2 in birds, the method for preventing or treating infectious diseases caused by avian pathogenic E. coli.
[12.]
According to claim 11, wherein the avian infectious disease is daejanggyunjeung, the method for preventing or treating infectious diseases caused by avian pathogenic E. coli.
drawing
[Figure 1]

[Figure 2]

[Figure 3]

[Figure 4]

[5]

[6]