BACTERIOPHAGE AND ANTIBACTERIAL COMPOSITION COMPRISING THE SAME

Abstract

The present invention relates to a novel bacteriophage having a specific bactericidal activity against salmonella, a composition for the prevention or treatment of infectious diseases comprising the bacteriophage as an active ingredient, an antibiotic comprising the bacteriophage as an active ingredient, an animal feed or drinking water comprising the bacteriophage as an active ingredient, and a sanitizer or cleaner comprising the bacteriophage as an active ingredient. The novel bacteriophage of the present invention has a specific bactericidal activity against Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis or Salmonella newport with no influences on beneficial bacteria, as well as excellent acid- and heat-resistance and desiccation tolerance. Therefore, the novel bacteriophage can be used for the prevention or treatment of salmonellosis or salmonella food poisoning, which is an infectious disease caused by Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis or Salmonella newport, and also widely used in animal feeds, drinking water for livestock, sanitizers, and cleaners.

Claims

1. A composition for the retarding or treating infectious diseases caused by Salmonella bacteria selected from the group consisting of Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby Salmonella infantis, Salmonella newport, and combinations thereof, comprising a bacteriophage as an active ingredient and a pharmaceutically acceptable carrier, wherein the bacteriophage has nucleic acid sequences of SEQ ID NOs: 1 to 4, and wherein the composition comprises a concentration of the bacteriophage in an amount of 110.sup.2 to 110.sup.12 PFU/ml.

2. The composition according to claim 1, wherein the composition comprises a concentration of the bacteriophage in an amount of 110.sup.6 to 110.sup.10 PFU/ml.

3. The composition according to claim 1, wherein the bacteriophage is identified by accession number KCCM11208P.

4. An antibiotic, comprising a bacteriophage as an active ingredient and a pharmaceutically acceptable carrier, wherein the bacteriophage has nucleic acid sequences of SEQ ID NOs: 1 to 4, and wherein the composition comprises a concentration of the bacteriophage in an amount of 110.sup.2 to 110.sup.12 PFU/ml.

5. The antibiotic according to claim 4, wherein the bacteriophage is identified by accession number KCCM11208P.

6. An animal feed or drinking water, comprising the bacteriophage as an active ingredient and additives for long term preservation, wherein the bacteriophage has nucleic acid sequences of SEQ ID NOs: 1 to 4, wherein the bacteriophage is in a liquid or in a solid form, wherein the animal feed or drinking water comprises 0.05% to 10% of the solid form by weight or a concentration of the bacteriophage in an amount of 110.sup.6 to 110.sup.10 PFU/ml of the liquid form.

7. The animal feed or drinking water according to claim 6, wherein the bacteriophage is identified by accession number KCCM11208P.

8. A sanitizer or cleaner, comprising the bacteriophage having nucleic acid sequences of SEQ ID NOs: 1 to 4 and identified by accession number KCCM11208P as an active ingredient, wherein the sanitizer or cleaner comprises a concentration of the bacteriophage in an amount of 110.sup.2 to 110.sup.12 PFU/ml.

9. A method for feeding animals to retard or treat infectious diseases caused by Salmonella bacteria selected from the group consisting of Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby Salmonella infantis, Salmonella newport, and combinations thereof, comprising administering a bacteriophage with feed to animals in need thereof, wherein the bacteriophage has nucleic acid sequences of SEQ ID NOs: 1 to 4.

10. The method feeding animals according to claim 9, wherein the bacteriophage is identified by accession number KCCM11208P.

11. The method feeding animals according to claim 9, the feed further comprises other non-pathogenic microorganisms.

12. The method feeding animals according to claim 11, the other non-pathogenic microorganisms are selected from the group consisting of Bacillus subtilis, Lactobacillus sp, strain, filamentous fungi, and yeast including Saccharomyce scerevisiae.

Description

DESCRIPTION OF DRAWINGS

[0017] FIG. 1 is an electron microscopy photograph of CJ11, which belongs to the family Siphoviridae of morphotype B1, characterized by an isometric capsid and a long non-contractile tail;

[0018] FIG. 2 is a photograph showing the formation of CJ11 plaques in a lawn of salmonella bacteria, in which (A,B; SC, C; SG, D; ST, E; SI, F,G; SD, H; SN), plaque formation was observed in lawns of SC, ST, SI, SD and SN, but not in lawns of SG;

[0019] FIG. 3 is the result of SDS-PAGE of the isolated bacteriophage CJ11, in which the major proteins were detected at 33 kDa, 55 kDa and 69.5 kDa, and Precision plus protein standard (BIO-RAD) was used as a marker;

[0020] FIG. 4 is the result of PFGE of the isolated bacteriophage CJ11, in which a total genome size of CJ11 was approximately 140 kbp, and the CHEF DNA Size Standard Lambda Ladder (Bio-Rad) was used as a DNA size marker;

[0021] FIG. 5 is the result of PCR, performed using each primer set for the CJ11 genomic DNA, in which A; a primer set of SEQ ID NOs. 5 and 6, B; a primer set of SEQ ID NOs. 7 and 8, C; a primer set of SEQ ID NOs. 9 and 10, and D; a primer set of SEQ ID NOs. 11 and 12, and all of A, B, C and D lanes had PCR products of approximately 1 kbp or more to 2 kbp or less;

[0022] FIG. 6 is the result of acid-resistance assay on the bacteriophage CJ11, showing the number of surviving bacteriophage at pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 6.9, 7.4, 8.0, 9.0, 9.8 and 11.0, in which the bacteriophage CJ11 did not lose its activity until pH 5.5, but the bacteriophage CJ11 showed reduced activity at pH 4 and pH 3.5, and completely lost its activity at pH 3.0 or lower, as compared to a control;

[0023] FIG. 7 is the result of heat-resistance assay on the bacteriophage CJ11, showing the number of surviving bacteriophage at 37, 45, 53, 60, and 70 C. for 0, 10, 30, 60 and 120 minutes, in which the bacteriophage CJ11 maintained its activity even though exposed to 60 C. for up to 2 hours;

[0024] FIG. 8 is the result of desiccation tolerance assay on the bacteriophage CJ11 dried with the aid of a SpeedVac concentrator, in which when titer changes under the dry condition were measured in comparison with pre-drying titers, the activity was maintained at 60 C. for up to 1 hour; and

[0025] FIG. 9 is the results of body weight changes due to toxicity after single oral administration of Sprague-Dawley rats with CJ11, in which observation of body weight changes before and 1, 3, 7, 10 and 14 days after administration with CJ11 showed no significant changes in comparison with the control group (.square-solid.; male control, ; CJ11 male, ; female control, ; CJ11 female).

BEST MODE

[0026] In one aspect to achieve the above objects, the present invention provides a novel bacteriophage having a specific bactericidal activity against Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis or Salmonella newport.

[0027] The present inventors collected fecal and sewage samples from swinery, and isolated therefrom bacteriophages that can lyse the host cell SC. They were also found that these bacteriophages can lyse ST, SD, SI and SN (FIG. 2 and Table 1). A morphological examination under an electron microscope confirmed that the bacteriophage (CJ11) belongs to the family Siphoviridae of morphotype B1 (FIG. 1). Further, the bacteriophage CJ11 was found to have major structural proteins of approximately 69.5 kDa, 55 kDa and 33 kDa, as measured by a protein pattern analysis (FIG. 3), and a genome analysis showed that CJ11 has a total genome size of approximately 97-145.5 kbp (FIG. 4). Furthermore, the results of analyzing its genetic features showed that the bacteriophage includes nucleic acid molecules represented by SEQ ID NOs. 1 to 4 within the total genome (Example 6). Based on SEQ ID NOs. 1 to 4, genetic similarity with other species was compared. It was found that the bacteriophage showed very low genetic similarity with the known bacteriophages, indicating that the bacteriophage is a novel bacteriophage (Table 2). For more detail analysis of genetic features, the CJ11-specific primer sets, namely, SEQ ID NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11 and 12 were used to perform PCR. Each PCR product was found to have a size of 1.4 kbp, 1.2 kbp, 1.25 kbp and 1.5 kbp (FIG. 5).

[0028] Meanwhile, when SC, ST, SD, SI and SN were infected with CJ11, the phage plaques (clear zone on soft agar created by host cell lysis of one bacteriophage) were observed (FIG. 2). The stability of CJ11 was examined under various temperature and pH conditions, resulting in that CJ11 stably maintains in a wide range of pH environments from pH 3.5 to pH 11.0 (FIG. 6) and in high temperature environments from 37 C. to 70 C. (FIG. 7), and even after desiccation (FIG. 8). Also, the wild-type strains SC, ST, SD, SI and SN were also found to fall within the host cell range of CJ11 (Table 3).

[0029] Finally, the results of dermal and ocular irritation tests on CJ11 in specific-pathogen-free (SPF) New Zealand White rabbits showed that the primary irritation index (PII) was 0.33, indicating no irritant, and the index of acute ocular irritation (IAOI) was 0 in washing and non-washing groups during the whole experimental periods, indicating no irritant. The results of oral administration of CJ11 showed no changes in weight gain (FIG. 9). As well, mortality, general symptoms (Table 4) and organ abnormality (Table 5) were not observed, indicating no toxicity.

[0030] Accordingly, the present inventors designated the bacteriophage as Bacteriophage CJ11, in which the bacteriophage was isolated from fecal and sewage samples from swinery and has a specific bactericidal activity against SC, ST, SD, SI and SN and the above characteristics, and deposited at the Korean Culture Center of Microorganisms (361-221, Honje 1, Seodaemun, Seoul) on Sep. 9, 2011 under accession number KCCM11208P.

[0031] In another aspect to achieve the above objects, the present invention provides a composition for the prevention or treatment of infectious disease caused by one or more Salmonella bacteria selected from the group consisting of Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis and Salmonella newport, comprising the bacteriophage as an active ingredient.

[0032] Having specific bactericidal activity against Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis and Salmonella newport, the bacteriophage of the present invention may be used for the purpose of preventing or treating the diseases caused by these bacteria. Preferably, examples of the infectious diseases include porcine salmonellosis and Salmonella food poisoning caused by Salmonella choleraesuis or Salmonella typhimurium, and acute or chronic porcine enteritis caused by Salmonella derby, Salmonella infantis, Salmonella newport, but are not limited thereto.

[0033] As used herein, the term salmonellosis refers to symptoms following salmonella infection, such as fever, headache, diarrhea, and vomiting. That is, salmonellosis is an infection with bacteria of the genus Salmonella, which is defined with two clinical forms: an acute septicemic form that resembles typhoid fever and an acute gastroenteritis, including enteritis, food poisoning, and acute septicemia.

[0034] As used herein, the term prevention means all of the actions in which disease progress is restrained or retarded by the administration of the composition.

[0035] As used herein, the term treatment means all of the actions in which the condition has taken a turn for the better or been restrained or modified favorably by the administration of the composition.

[0036] The composition of the present invention includes CJ11 in an amount of 110.sup.2 to 110.sup.12 PFU/mL, and preferably in an amount of 110.sup.6 to 110.sup.10 PFU/mL.

[0037] On the other hand, the composition of the present invention may further include a pharmaceutically acceptable carrier.

[0038] As used herein, the term pharmaceutically acceptable carrier refers to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. For formulation of the composition into a liquid preparation, a pharmaceutically acceptable carrier which is sterile and biocompatible may be used such as saline, sterile water, Ringer's solution, buffered physiological saline, albumin infusion solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more thereof. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. Further, diluents, dispersants, surfactants, binders and lubricants may be additionally added to the composition to prepare injectable formulations such as aqueous solutions, suspensions, and emulsions, or oral formulations such as pills, capsules, granules, or tablets.

[0039] The prophylactic or therapeutic compositions of the present invention may be applied or sprayed to the afflicted area, or administered by oral or parenteral routes. The parenteral administration may include intravenous, intraperitoneal, intramuscular, subcutaneous or topical administration.

[0040] The dosage suitable for applying, spraying, or administrating the composition of the present invention will depend upon a variety of factors including formulation method, the mode of administration, the age, weight, sex, condition, and diet of the patient or animal being treated, the time of administration, the route of administration, the rate of excretion, and reaction sensitivity. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the composition required.

[0041] Examples of the oral dosage forms including the composition of the present invention as an active ingredient include tablets, troches, lozenges, aqueous or emulsive suspensions, powder or granules, emulsions, hard or soft capsules, syrups, or elixirs. For formulation such as tablets and capsules, the following are useful: a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; and a lubricant such as magnesium stearate, calcium stearate, sodium stearylfumarate, or polyethylene glycol wax. For capsules, a liquid carrier such as lipid may be further used in addition to the above-mentioned compounds.

[0042] The parenteral dosage forms including the composition of the present invention as an active ingredient may be formulated into injections via subcutaneous, intravenous, or intramuscular routes, suppositories, or sprays inhalable via the respiratory tract, such as aerosols. Injection forms may be prepared by dissolving or suspending the composition of the present invention, together with a stabilizer or a buffer, in water and loading the solution or suspension onto ampules or vial unit forms. For sprays, such as aerosols, a propellant for spraying a water-dispersed concentrate or wetting powder may be used in combination with an additive.

[0043] In still another aspect to achieve the above objects, the present invention provides an antibiotic comprising the bacteriophage as an active ingredient.

[0044] As used herein, the term antibiotic means any drug that is applied to animals to kill pathogens, and used herein as a general term for antiseptics, bactericidal agents and antibacterial agents. The animals are mammals including human. The bacteriophage of the present invention, unlike the conventional antibiotics, has a high specificity to Salmonella so as to kill the specific pathogens without affecting beneficial bacteria, and does not induce drug resistance so that it can be provided as a novel antibiotic with a comparatively long life cycle.

[0045] In still another aspect to achieve the above objects, the present invention provides an animal feed or drinking water comprising the bacteriophage as an active ingredient.

[0046] In-feed antibiotics used in the livestock and fishery industries are intended to prevent infections. However, most of the currently available in-feed antibiotics are problematic in that they are apt to induce the occurrence of resistant strains and may be transferred to humans, due to remaining in livestock products. The uptake of such residual antibiotics may make human pathogens resistant to antibiotics, resulting in the spread of diseases. In addition, since there are a variety of in-feed antibiotics, the increasing global emergence of multidrug-resistant strain is a serious concern. Therefore, the bacteriophage of the present invention can be used as an in-feed antibiotic that is more eco-friendly and able to solve the above problems.

[0047] The animal feed of the present invention may be prepared by adding the bacteriophage directly or in separate feed additive form to an animal feed. The bacteriophage of the present invention may be contained in the animal feed as a liquid or in a solid form, preferably in a dried powder. The drying process may be performed by air drying, natural drying, spray drying, and freeze-drying, but is not limited thereto. The bacteriophage of the present invention may be added as a powder form in an amount of 0.05 to 10% by weight, preferably 0.1 to 2% by weight, based on the weight of animal feed. The animal feed may also include other conventional additives for long-term preservation, in addition to the bacteriophage of the present invention.

[0048] The feed additive of the present invention may additionally include other non-pathogenic microorganisms. The available additional microorganism may be selected from the group consisting of Bacillus subtilis that can produce protease, lipase and invertase, Lactobacillus sp. strain that can exert physiological activity and a function of decomposing under anaerobic conditions, such as in the stomach of cattle, filamentous fungi including Aspergillus oryzae (J Animal Sci 43:910-926, 1976) that increases the weight of domestic animals, enhances milk production and helps the digestion and absorptiveness of feeds, and yeast including Saccharomyce scerevisiae (J Anim Sci 56:735-739, 1983).

[0049] The feed including CJ11 of the present invention may include plant-based feeds, such as grain, nut, food byproduct, seaweed, fiber, drug byproduct, oil, starch, meal, and grain byproduct, and animal-based feeds such as protein, inorganic matter, fat, mineral, fat, single cell protein, zooplankton, and food waste, but is not limited thereto.

[0050] The feed additive including CJ11 of the present invention may include binders, emulsifiers, and preservatives for the prevention of quality deterioration, amino acids, vitamins, enzymes, probiotics, flavorings, non-protein nitrogen, silicates, buffering agents, coloring agents, extracts, and oligosaccharides for efficiency improvement, and other feed premixtures, but is not limited thereto.

[0051] Further, the supply of drinking water mixed with the bacteriophage of the present invention can reduce the number of Salmonella bacteria in the intestine of livestock, thereby obtaining Salmonella-free livestock.

[0052] In still another aspect to achieve the above objects, the present invention provides a sanitizer or cleaner comprising the bacteriophage as an active ingredient.

[0053] In still another aspect to achieve the above objects, the present invention provides a method for treating infectious diseases caused by Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis or Salmonella newport using the bacteriophage or the composition.

[0054] In detail, the therapeutic method of the present invention comprises the step of administering a pharmaceutically effective amount of the bacteriophage or the composition to an individual having infectious diseases caused by Salmonella choleraesuis, Salmonella typhimurium, Salmonella derby, Salmonella infantis or Salmonella newport.

[0055] The bacteriophage or the composition of the present invention may be administered in the form of a pharmaceutical formulation into animals or may be ingested as a mixture with animal feed or drinking water by animals and preferably as a mixture with animal feed.

[0056] As long as it reaches target tissues, any route, whether oral or parenteral, may be taken for administering the bacteriophage or the composition of the present invention. In detail, the composition of the present invention may be administered in a typical manner via any route such as oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, and inhalation routes.

[0057] It will be obvious to those skilled in the art that the total daily dose of the bacteriophage or the composition of the present invention to be administered by the therapeutic method should be determined through appropriate medical judgment by a physician. Preferably, the therapeutically effective amount for given patients may vary depending on various factors well known in the medical art, including the kind and degree of the response to be achieved, the patient's condition such as age, body weight, state of health, sex, and diet, time and route of administration, the secretion rate of the composition, the time period of therapy, concrete compositions according to whether other agents are used therewith or not, etc.

MODE FOR INVENTION

[0058] Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.

Example 1: Salmonella Bacteriophage Isolation

Example 1-1: Bacteriophage Screening and Single

Bacteriophage Isolation

[0059] 50 mL of sample from swinery and sewage effluent was transferred to a centrifuge tube, and centrifuged at 4000 rpm for 10 minutes. Then, the supernatant was filtered using a 0.45 m filter. 18 mL of sample filtrate was mixed with 150 l of Salmonella choleraesuis (SC) shaking culture medium (OD.sub.600=2) and 2 mL of 10 Luria-Bertani medium (tryptone 10 g/L, yeast extract 5 g/L and NaCl 10 g/L: LB medium). The mixture was cultured at 37 C. for 18 hours, and the culture medium was centrifuged at 4000 rpm for 10 minutes. The supernatant was filtered using a 0.2 m filter. 3 mL of 0.7% agar (w/v) and 150 l of SC shaking culture medium (OD.sub.600=2) were mixed, and plated onto LB plate (top-agar), and allowed to solidify. 10 l of the culture filtrate was spread thereon, and cultured for 18 hours at 37 C., and the titration of phage lysate was performed on the top-agar, called soft agar overlay method.

[0060] The sample culture medium containing the phage lysate was properly diluted, and mixed with 150 l of SC shaking culture medium (OD.sub.600=2), followed by soft agar overlay method, so that single plaques were obtained. A single plaque represents one bacteriophage and thus, for isolation of single bacteriophages, one phage plaque was added to 400 l of SM solution (NaCl, 5.8 g/L, MgSO.sub.47H.sub.2O, 2 g/L, 1 M Tris-Cl (pH 7.5) 50 ml/L), and left for 4 hours at room temperature to isolate single bacteriophages. To purify the bacteriophage in large quantities, 100 l of supernatant was taken from the single bacteriophage solution, and mixed with 12 mL of 0.7% agar and 500 l of SC shaking culture medium, followed by soft agar overlay method on LB plate having a diameter of 150 mm. When lysis was completed, 15 mL of SM solution was added to the plate. The plate was gently shaken for 4 hours at room temperature to elute the bacteriophages from the top-agar. The SM solution containing the eluted bacteriophages was recovered, chloroform was added to a final volume of 1%, and mixed well for 10 minutes. The solution was centrifuged at 4000 rpm for 10 minutes. The obtained supernatant was filtered using a 0.45 m filter, and stored in the refrigerator.

Example 1-2: Large-Scale Batches of Bacteriophage

[0061] The selected bacteriophages were cultured in large quantities using SC. SC was shaking-cultured, and an aliquot of 1.510.sup.10 cfu (colony forming units) was centrifuged at 4000 rpm for 10 minutes, and the pellet was resuspended in 4 ml of SM solution. The bacteriophage of 9.010.sup.8 PFU (plaque forming unit) was inoculated thereto (MOI: multiplicity of infection=0.001), and left at 37 C. for 20 minutes. The solution was inoculated into 150 ml of LB media, and cultured at 37 C. for 5 hours. Chloroform was added to a final volume of 1%, and the culture solution was shaken for 20 minutes. DNase I and RNase A were added to a final concentration of 1 g/ml, respectively. The solution was left at 37 C. for 30 minutes. NaCl and PEG (polyethylene glycol) were added to a final concentration of 1 M and 10% (w/v), respectively and left at 4 C. for an additional 3 hours. The solution was centrifuged at 4 C. and 12,000 rpm for 20 minutes to discard the supernatant. The pellet was resuspended in 5 mL of SM solution, and left at room temperature for 20 minutes. 4 mL of chloroform was added thereto and mixed well, followed by centrifugation at 4 C. and 4000 rpm for 20 minutes. The supernatant was filtered using a 0.2 m filter, and the bacteriophage was purified by glycerol density gradient ultracentrifugation (density: 40%, 5% glycerol at 35,000 rpm and 4 C. for 1 hour). The purified bacteriophage was designated as Bacteriophage CJ11, and resuspended in 300 l of SM solution, followed by titration. The bacteriophage CJ11 was deposited at the Korean Culture Center of Microorganisms (361-221, Honje 1, Seodaemun, Seoul) on Sep. 9, 2011 under accession number KCCM11208P.

Example 2: Examination on CJ11 Infection of Salmonella

[0062] To analyze the selected bacteriophage for lytic activity on Salmonella species other than SC, attempts were made of cross infection with other Salmonella species. As a result, CJ11 infected SC (Salmonella choleraesuis), ST (Salmonella typhimurium), SD (Salmonella derby), SN (Salmonella newport), SI (Salmonella infantis), SA (Salmonella arizonae) and SB (Salmonella bongori), but did not infect SE (Salmonella enteritidis), SG (Salmonella gallinarum), and SP (Salmonella pullorum) (Table 1 and FIG. 2).

TABLE-US-00001 TABLE 1 CJ11 Infection of Salmonella Phage plaque Serotype Strain name formation SC ATCC 10708 SN SL 317 SD ATCC 2468 SE SGSC 2282 X SG SGSC 2293 X SA ATCC 12398 SB ATCC 12397 ST 13 SI SARB 26 SP SGSC 2295 X * ATCC: American Type Culture Collection * SGSC: Salmonella Genetic Stock Center

[0063] Moreover, FIG. 2 is a photograph showing the formation of CJ11 plaques in a lawn of salmonella bacteria. As shown in FIG. 2 (A,B; SC, C; SG, D; ST, E; SI, F,G; SD, H; SN), plaque formation was observed in lawns of SC, ST, SI, SD and SN, but not in lawns of SG.

Example 3: Morphology of CJ11

[0064] The purified CJ11 was diluted in the SM buffer solution, and then mounted on a copper grid, stained with 2% uranyl acetate for 3 to 5 seconds, and dried. Examination under a transmission electron microscope (LIBRA 120, Carl Zeiss transmission electron Microscope, 80 kV, magnification of 120,000200,000) was performed (FIG. 1). FIG. 1 is an electron microscopy photograph of CJ11. As shown in FIG. 1, it was found that the purified CJ11 belongs to the family Siphoviridae of morphotype B1, characterized by an isometric capsid and a long non-contractile tail.

Example 4: Protein Pattern Analysis of CJ11

[0065] 15 L of a CJ11 solution purified at a titer of 10.sup.11 PFU/mL was mixed with 3 L of a 5SDS sample solution, and heated for 5 minutes. 12% SDS-PAGE was performed, and then the gel was stained with Coomassie blue for 1 hour at room temperature (FIG. 3). FIG. 3 is the result of SDS-PAGE of the isolated bacteriophage CJ11, in which Precision plus protein standard (BIO-RAD) was used as a marker. As shown in FIG. 3, the major proteins were detected at 33 kDa, 55 kDa and 69.5 kDa.

Example 5: Analysis of Total Genomic DNA Size of CJ11

[0066] Genomic DNA of the purified CJ11 was isolated using ultracentrifugation. In detail, to the purified CJ11 culture medium were added EDTA (ethylenediaminetetraacetic acid (pH 8.0)), proteinase K, and SDS (sodium dodecyl sulfate) at a final concentration of 20 mM, 50 ug/mL, and 0.5% (w/v), respectively, followed by incubation at 50 C. for 1 hour. An equal volume of phenol (pH 8.0) was added and mixed well. After centrifugation at room temperature and 12,000 rpm for 10 minutes, the supernatant was mixed well with an equal volume of PC (phenol:chloroform=1:1). Another centrifugation was performed at room temperature and 12,000 rpm for 10 minutes. Then, a supernatant was obtained, and mixed with an equal volume of chloroform, followed by centrifugation at room temperature and 12,000 rpm for 10 minutes. The obtained supernatant was mixed with 1/10 volume of 3 M sodium acetate and two volumes of cold 95% ethanol, and left at 20 C. for 1 hour. After centrifugation at 0 C. and 12,000 rpm for 10 minutes, the supernatant was completely removed, and the DNA pellet was dissolved in 50 L of TE (Tris-EDTA (pH 8.0)). The extracted DNA was diluted 10-fold, and measured for absorbance at OD.sub.260 to determine its concentration. 1 g of the total genomic DNA was loaded onto 1 PFGE (pulse-field gel electrophoresis) agarose gel, and electrophoresed at 14 C. for 22 hours in a BIORAD CHEF DR II PFGE system under the conditions of switch time ramp for 50-90 seconds, 6 V/cm (200V). The CHEF DNA Size Standard Lambda Ladder (Bio-Rad) was used as a DNA size marker (FIG. 4). FIG. 4 is the result of PFGE of the isolated bacteriophage CJ11. As shown in FIG. 4, DNA of approximately 140 kbp present between 48.5 to 1,000 kbp was observed.

Example 6: Genetic Analysis of CJ11

[0067] For the genetic analysis of the purified CJ11, 5 g of the genomic DNA of CJ11 was double digested with the restriction enzymes PstI, XbaI and BamHI, EcoRI and SalI. The vector pCL1920 (Promega) was digested with the restriction enzymes PstI, XbaI and BamHI, EcoRI and SalI, and then treated with CIP (calf intestinal alkaline phosphatase). The digested genomic DNA was mixed at a ratio of 3:1 with the vector, and ligated at 16 C. for 2 hours. The resulting recombinant vector was transformed into E. coli DH5a which was then plated on an LB plate containing specinomycin and X-gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) for selection of blue/white colonies. The selected colonies were cultured for 16 hours in a culture medium containing the antibiotic with shaking. Then, plasmids were extracted using a Plasmid purification kit (Promega).

[0068] The cloning of the plasmids was confirmed by PCR using primer sets of FTR135 and FTR136 (SEQ ID NOs. 13 and 14) and selection was made only of insert fragments having a size of 1 kb or longer. Their nucleotide sequences were analyzed using the primer sets. The nucleotide sequences thus obtained were given in SEQ ID NOs. 1 to 4, respectively, each having a size of 1 to 2 kbp or less, and analyzed for sequence similarity with the aid of NCBI blastx and blastn program, and the results are summarized in Table 2, below.

TABLE-US-00002 TABLE 2 Comparison of Sequence Similarity between CJ11 and Other Bacteriophages Blastx Organism Protein Query Subject Identity e-value 1 Enterobacteria hypothetical protein 436-627 1-64 63/64 7e32 phage T5 (98%) Enterobacteria hypothetical protein 10-423 1-139 127/139 3e31 phage SPC35 (91%) Enterobacteria hypothetical protein 3-863 30-315 264/291 2e116 phage SPC35 (91%) Enterobacteria hypothetical protein 3-863 30-315 255/291 1e113 phage T5 (88%) Enterobacteria hypothetical protein 3-761 29-281 235/253 7e107 phage EPS7 (93%) Klebsiella phage hypothetical protein 9-761 22-276 217/255 4e100 KP15 (85%) Enterobacteria putative SPFH domain- 9-839 18-257 223/281 7e99 phage RB43 containing protein (79%) Enterobacteria hypothetical protein 9-761 18-272 219/255 2e98 phage RB16 (86%) 2 Enterobacteria hypothetical protein 412-771 1-120 109/120 7e57 phage EPS7 (91%) Enterobacteria hypothetical protein 490-771 1-94 94/94 3e46 phage T5 (100%) Enterobacteria D11 protein 532-2 1-177 175/177 5e96 phage T5 (99%) Enterobacteria D11 protein 532-2 1-177 174/177 2e95 phage SPC35 (98%) Enterobacteria D11 protein 532-8 1-175 163/175 4e90 phage EPS7 (93%) Enterobacteria hypothetical protein 872-528 5-120 96/116 2e46 phage EPS7 (83%) 3 Enterobacteria tail protein Pb4 777-4 1-258 221/258 4e128 phage SPC35 (86%) Enterobacteria tail protein Pb4 777-4 1-258 197/258 6e115 phage T5 (76%) Enterobacteria tail protein Pb3 1322-780 769-949 178/181 2e96 phage SPC35 (98%) Enterobacteria tail protein Pb3 1322-780 769-949 160/181 9e88 phage EPS7 (88%) Enterobacteria structural tail 1322-780 769-949 156/181 8e84 phage T5 protein Pb3 (86%) Enterobacteria tail protein Pb4 423-4 1-140 112/140 2e62 phage EPS7 (80%) 4 Enterobacteria flap endonuclease 980-564 153-291 138/139 1e73 phage SPC35 (99%) Enterobacteria flap endonuclease 980-564 153-291 138/139 1e73 phage T5 (99%) Enterobacteria flap endonuclease 980-564 153-291 135/139 4e72 phage EPS7 (97%) Enterobacteria putative deoxyUTP 564-157 1-136 130/136 3e70 phage SPC35 pyrophosphatase (96%) Enterobacteria putative deoxyUTP 564-160 1-135 130/135 2e69 phage T5 pyrophosphatase (96%) Enterobacteria putative deoxyUTP 564-157 1-136 128/136 2e67 phage EPS7 pyrophosphatase (94%)

Example 7: Design of CJ11-Specific Primer Sequences

[0069] In order to identify CJ11, CJ11-specific primers were designed on the basis of SEQ ID NOS. 1 to 4. PCR was performed using each primer set of SEQ ID NOS. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11 and 12. 0.1 g of the genomic DNA of bacteriophage and 0.5 pmol of each primer were added to a pre-mix (Bioneer), and the final volume was adjusted to 20 L. PCR was performed with 30 cycles of denaturation; 94 C. 30 seconds, annealing; 55 C. 30 seconds, and polymerization; 72 C., 1.5 minutes (FIG. 5). FIG. 5 is the result of PCR, performed using each primer set for the CJ11 genomic DNA. A; a primer set of SEQ ID NOs. 5 and 6, B; a primer set of SEQ ID NOs. 7 and 8, C; a primer set of SEQ ID NOs. 9 and 10, and D; a primer set of SEQ ID NOs. 11 and 12. All of A, B, C and D lanes had PCR products of approximately 1 to 2 kbp. As shown in FIG. 5, the PCR products thus obtained had a size of approximately 1 kbp or more to 2 kbp or less, with the primer sets of SEQ ID NOs. 5 and 6, SEQ ID NOs. 7 and 8, SEQ ID NOs. 9 and 10, and SEQ ID NOs. 11 and 12.

Example 8: pH Stability of Bacteriophage

[0070] In order to determine whether CJ11 survives with stability under the low pH environment in the stomach of pig, CJ11 was assayed for stability in a wide range of pH (pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 6.9, 7.4, 8.2, 9.0, 9.8 and 11.0). Various pH solutions (sodium acetate buffer (pH 2.1, pH 4.0, pH 5.5, and pH 6.4), sodium citrate buffer (pH 2.5, pH 3.0, and pH 3.5), sodium phosphate buffer (pH 6.9 and pH 7.4) and Tris-HCl (pH 8.2, pH 9.0, pH 9.8 and pH 11.0)) were prepared to have a concentration of 0.2 M. 180 L of each pH solution was mixed with 20 L of a bacteriophage solution (1.010.sup.11 PFU/mL) to give each pH solution a concentration of 1 M, followed by incubation at room temperature for 2 hours. The reaction solution was serially diluted, and 10 L of each dilution was cultured at 37 C. for 18 hours by a soft agar overlay method to determine the titers of the phage lysates (FIG. 6). FIG. 6 is the result of acid-resistance assay on the bacteriophage CJ11, showing the number of surviving bacteriophage at pH 2.1, 2.5, 3.0, 3.5, 4.0, 5.5, 6.4, 6.9, 7.4, 8.0, 9.0, 9.8 and 11.0. The bacteriophage CJ11 did not lose its activity until pH 5.5. However, the bacteriophage CJ11 showed reduced activity at pH 4 and pH 3.5, and completely lost its activity at pH 3.0 or lower, as compared to a control. As shown in FIG. 6, the bacteriophage did not lose its activity and remained stable down to pH 5.5 whereas it lost its activity at pH 3.0 or lower.

Example 9: Heat Stability of Bacteriophage

[0071] For use as a feed additive, the bacteriophage was assayed for stability to the heat generated during a formulation process. In this regard, 200 L of a CJ11 solution with a titer of 1.010.sup.11 PFU/mL was incubated at 37 C., 45 C., 53 C., 60 C., and 70 C. for 0 minute, 10 minutes, 30 minutes, 60 minutes and 120 minutes. The solution was serially diluted, and 10 L of each dilution was cultured at 37 C. for 18 hours by a soft agar overlay method to determine the titers of phage lysates (FIG. 7). FIG. 7 is the result of heat-resistance assay on the bacteriophage CJ11, showing the number of surviving bacteriophage at 37 C., 45 C., 53 C., 60 C., and 70 C. for 0, 10, 30, 60 and 120 minutes. As shown in FIG. 7, the bacteriophage CJ11 maintained its activity even though exposed at 60 C. up to 2 hours.

Example 10: Desiccation Tolerance of Bacteriophage

[0072] For use as a feed additive, the bacteriophage CJ11 was assayed for tolerance to the dry condition set for a formulation process. On the basis of the results obtained from the heat stability assay, a desiccation assay was performed using a SpeedVac concentrator. 200 L of a CJ11 solution having a titer of 1.010.sup.11 PFU/mL was dried under vacuum at 60 C. for 2 hours, and the pellet thus obtained was completely re-suspended in 200 L of the SM solution at 4 C. for one day, and measured for titer values (FIG. 8). FIG. 8 is the result of desiccation tolerance assay on the bacteriophage CJ11 dried with the aid of a SpeedVac concentrator. As shown in FIG. 8, when titer changes under the dry condition were measured in comparison with pre-drying titers, the activity was maintained at 60 C. up to 1 hour.

Example 11: Infection Spectrum of Bacteriophage

[0073] CJ11 was assayed for lytic activity against the wild-type (2 strains), Salmonella choleraesuis (5 strains), Salmonella typhimurium (17 strains), Salmonella infantis (4 strains), Salmonella newport (6 strains), Salmonella derby (2 strains) and Salmonella dublin (3 strains), obtained from Laboratory of Avian Diseases, College of Veterinary Medicine, Seoul National University, in addition to SC (ATCC SC10708) used in the experiment. 150 L of each strain shaking culture medium (OD.sub.600=2) was mixed, and 10 L of CJ11 solution having a titer of 10PFU/mL was cultured at 37 C. for 18 hours using a soft agar overlay method to monitor the formation of plaques (Table 3). Formation of phage plaque was observed in 8 strains of SC.

TABLE-US-00003 TABLE 3 Wild-type strains SC, ST, SD, SI, SN infected by CJ11 Phage plaque Phage plaque Serotype Strain name formation serotype Strain name formation SC S. choleraesuis ATCC SI S. infantis SARB 26 2929 S. choleraesuis ATCC S. infantis SARB 27 2930 S. choleraesuis ATCC S. infantis S1326/28 2932 S. choleraesuis ATCC S. infantis B09-106 2933 S. choleraesuis ATCC SN S. newport SARB 36 2425 S. choleraesuis ATCC S. newport SARB 37 10708 S. choleraesuis SNU#1 S. newport SARB 38 S. choleraesuis SNU#2 S. newport 7257 ST S. typhimurium SNU S. newport SL 317 ST1 S. typhimurium SNU S. newport SL 254 ST2 S. typhimurium SNU SD S. derby ATCC ST4 2466 S. typhimurium SNU S. derby ATCC ST7 2468 S. typhimurium SNU SD S. dublin SA 4405 ST8 S. typhimurium SNU S. dublin RKS 4699 ST11 S. typhimurium SNU S. dublin 88/6 ST12 S. typhimurium SNU SA S. arizonae ATCC ST13 12398 S. typhimurium SNU SB S. bongori ATCC ST14 12397 S. typhimurium SNU SH S. heidelberg SARA 33 ST17 S. typhimurium SNU S. heidelberg SARA 23 ST18 S. typhimurium SNU SM S. maimi SARB 28 ST19 S. typhimurium SNU S. maimi SARB 29 ST20 S. typhimurium SNU SP S. panama SARB 39 ST26 S. typhimurium SNU S. panama SARB 40 ST38 S. typhimurium SNU S. panama SARB 41 ST41 S. typhimurium SNU S. panama 7261 ST42 SGSC: Salmonella genetic stock center ATCC: American Type Culture Collection SNU: Laboratory of Avian Diseases, College of Veterinary Medicine, Seoul National University

Example 12: Toxicity Assay of Bacteriophage

[0074] Dermal and ocular irritation tests were performed in specific-pathogen-free (SPF) New Zealand white rabbits, which are commonly used in the toxicity test of bacteriophage CJ11 for the prevention of salmonellosis and salmonella food poisoning, and of which experimental data were accumulated to allow easy analysis of experiment results. The normal abdominal skin (non-injured skin) and injured abdominal skin of rabbits were covered and contacted with 2.5 cm2.5 cm of gauze applied with the test substance, and each 0.5 mL/site was applied. No changes in general symptoms were observed, and a slight weight loss was observed 1 day after application of the test substance, which can likely to be attributed to stress due to occlusive application of the test substance. In the dermal irritation test, the primary irritation index (PII) was 0.33, indicating no irritant. For the ocular irritation test, the left eye of a rabbit was applied with the test substance, and then compared to the right eye, which was not applied with the test substance. During the experimental period, general symptoms and abnormal changes in body weight related to application of the test substance were not observed. After application of the test substance, the eye examination showed that the index of acute ocular irritation (IAOI) was 0, indicating no irritant. Therefore, these results indicate that the novel bacteriophage CJ11 has no toxicity.

[0075] Further, toxicity assay was performed by single oral administration of Sprague-Dawley rats with CJ11. A test substance-administered group treated with 110.sup.11 PFU/kg of CJ11 and an excipient control group treated with a vehicle [20 mM Tris-HCl (pH 7.0)+2 mM MgCl.sub.2] as an excipient were prepared, and 10 rats of each group (5 each of female and male sexes) were orally administered with a single dosage. Mortality, general symptoms, changes in body weight, and autopsy findings were monitored for 2 weeks and compared to each other. Monitoring was conducted every 6 hours, starting from 30 minutes to 1 hour after administration on the day of administration. Then, general symptoms were monitored once a day for 14 days, and recorded thereof (Tables 4 and 5).

TABLE-US-00004 TABLE 4 Mortality and general symptoms after oral administration of CJ11 Done Day after treatment Sex (pfu) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Mortality Male Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10.sup.11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Female Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10.sup.11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

TABLE-US-00005 TABLE 5 Autopsy findings after oral administration of CJ11 Done Sex (PFU) gross finding Frequency.sup.A Male Control No gross 5/5 finding 10.sup.11 No gross 5/5 finding Female Control No gross 5/5 finding 10.sup.11 No gross 5/5 finding .sup.ANumber of animal with the sign/number of animals examined.

[0076] As shown in Tables 4 and 5, none of them died, and neither toxic symptoms nor noticeable clinical symptoms were generated by CJ11. The results are summarized in Tables 4 and 5. Body weights were recorded before administration and 1, 3, 7, 10 and 14 days after administration. No significant changes were observed in body weight compared to the control group.

[0077] Meanwhile, the results of body weight changes indicate that CJ11 does not cause a toxic reaction sufficient to reduce appetite or to change body weight. These results are shown in FIG. 9. FIG. 9 is the results of body weight changes due to toxicity after single oral administration of Sprague-Dawley rats with CJ11. As shown in FIG. 9, observation of body weight changes before administration and 1, 3, 7, 10 and 14 days after administration with CJ11 showed that no significant changes in body weight were found in comparison with the control group (.square-solid.; male control, ; CJ11 male, ; female control, ; CJ11 female).

[0078] Therefore, it was found that ADL of the novel bacteriophage CJ11 exceeds 110.sup.11 PFU/kg in both female and male rats, and thus it is non-toxic.