US20130011369A1 - Method for Prevention and Treatment of Salmonella Infection - Google Patents

Method for Prevention and Treatment of Salmonella Infection Download PDF

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US20130011369A1
US20130011369A1 US13/176,458 US201113176458A US2013011369A1 US 20130011369 A1 US20130011369 A1 US 20130011369A1 US 201113176458 A US201113176458 A US 201113176458A US 2013011369 A1 US2013011369 A1 US 2013011369A1
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salmonella
bacteriophage
treatment
prevention
composition
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US13/176,458
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Seongjun Yoon
Sooyoun Jun
Hyoungrok Paik
Gimo Jung
Yoonsik Shin
Sanghyeon Kang
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Intron Biotechnology Inc
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Intron Biotechnology Inc
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Priority to US13/176,458 priority Critical patent/US20130011369A1/en
Assigned to INTRON BIOTECHNOLOGY, INC. reassignment INTRON BIOTECHNOLOGY, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JUN, SOO YOUN, JUNG, GIMO, KANG, SANG HYEON, PAIK, HYOUNGROK, SHIN, YOONSIK, YOON, SEONG JUN
Publication of US20130011369A1 publication Critical patent/US20130011369A1/en
Priority to US14/934,920 priority patent/US9540616B2/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/00032—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a composition applicable for prevention or treatment of Salmonella infection comprising bacteriophage that is capable of destroying Salmonella once infected to Salmonella as an active ingredient and a method for prevention and treatment of Salmonella infection by using the said composition. That is, the present invention relates to a composition used for the purpose of prevention or treatment of Salmonella infection and a method for prevention and treatment of Salmonella infection using the same.
  • Salmonella is similar to E. coli in the aspects of morphology or physiology but is categorized in an independent genus for the convenience in clinical use by the proposal of K. Kauffmann et al. Salmonella has been isolated from enteritis and gastroenteritis patients and from animals with diverse diseases since Salmonella choleraesuis was first isolated from a pig died of hog cholera by Salmon and Smith in 1885. Salmonella has also been isolated from health animals such as chicken, cow, pig, goat, dog, and cat and from our environment.
  • Salmonella is a genus of rod-shaped, Gram-negative and non-spore-forming It is a parasite living in a variety of animals.
  • Salmonella infectious disease is developed in different forms. Enteritis is the most general form of Salmonella infection. Once infected with Salmonella , such symptoms as rough skin, anorexia, conjunctivitis, depression, pale feces, spleen enlargement, and even death are observed.
  • Bacteriophage is an extremely small microorganism infecting bacteria, which is generally called phage in short. Bacteriophage is an obligate intracellular parasite that multiplis inside bacteria by making use of some or all of the host biosynthetic machinery. Upon completion of the multiplication, offspring bacteriophages are coming out of the host cell with destroying the host bacteria.
  • the infection of bacteriophage in bacteria is very unique and specific, so only specific bacteria can be infected with a specific bacteriophage. That is, there is a limitation in bacteria that can be infected with bacteriophage. Thus, bacteriophage can only kill specific target bacteria without effecting on any other bacteria.
  • bacteriophage Owing to its capability of killing bacteria, bacteriophage has been in the center of our interest to fight with bacterial infection and studies followed thereon. However, since Flemming found out penicillin, antibiotics have been supplied and the study on bacteriophage has been limited in some east European countries and old Soviet Union. It was not until 2000 that the conventional antibiotics demonstrated their problems in use because of increasing antibiotic-resistant bacteria. So, once again, bacteriophage draws out attention as an alternative anti-bacterial agent that can take the place of the conventional antibiotics.
  • the present inventors tried to develop a composition for prevention or treatment of Salmonella infection by using bacteriophage that can destroy Salmonella selectively and further tried to establish a method for prevention and treatment of Salmonella infection using the same.
  • the inventors isolated a proper bacteriophage from the nature and secured the gene sequence of its genome for the distinguishment from other bacteriophages.
  • the present inventors succeeded in developing a composition comprising the said bacteriophage as an active ingredient and further completed this invention by confirming that the said composition of the invention can be effectively used for prevention and treatment of Salmonella infection.
  • the present invention provides a composition comprising bacteriophage as an active ingredient which is capable of destroying Salmonella by infecting Salmonella , and a method for prevention and treatment of Salmonella infection by using the said composition.
  • Bacteriophage used as the active ingredient in the composition of the present invention is bacteriophage SP-1 having DNA represented by SEQ. ID. NO: 1 as its genome. Bacteriophage SP-1 was isolated by the present inventors and deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on Jul. 30, 2010 (Accession No: KCTC 11737BP).
  • the present invention also provides a disinfectant, a drinking water additive, and a feed additive that can be used for prevention or treatment of Salmonella infection.
  • Bacteriophage SP-1 included in the composition of the present invention is able to kill Salmonella effectively, so that it can have a preventive and/or treating effect on diverse infectious diseases caused by Salmonella . Therefore, the composition of the present invention can be used for the purpose of prevention and treatment of disease caused by Salmonella.
  • the pharmaceutical composition of the present invention can be used for prevention and treatment of salmonellosis, the representative disease caused by Salmonella including enteritis, Bacteremia, Fowl typhoid, Pullorum disease, and cholera.
  • Salmonellosis in this invention generally indicates all the symptoms caused by Salmonella infection including fever, headache, diarrhea, vomiting, etc.
  • the term “treat” or “treatment” in this description indicates (i) to suppress disease caused by Salmonella ; and (ii) to relieve disease symptoms caused by Salmonella.
  • the representative Salmonella targeted by this invention is exemplified by Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Dubulin, and Salmonella Durby, but not always limited thereto.
  • composition of the present invention can include pharmaceutically acceptable carriers such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but not always limited thereto.
  • the composition of the present invention can additionally include lubricants, wetting agents, sweetening agents, flavors, emulsifiers, suspensions and preservatives.
  • the composition of the present invention contains bacteriophage SP-1 as an active ingredient.
  • the bacteriophage SP-1 is included at the concentration of 1 ⁇ 10 1 pfu/mL-1 ⁇ 10 30 pfu/mL or 1 ⁇ 10 1 pfu/g-1 ⁇ 10 30 pfu/g, and more preferably at the concentration of 1 ⁇ 10 4 pfu/mL-1 ⁇ 10 15 pfu/mL or 1 ⁇ 10 4 pfu/g-1 ⁇ 10 15 pfu/g.
  • composition of the present invention can be formulated by the method that can be performed by those in the art by using a pharmaceutically acceptable carrier and/or excipient in the form of unit dose or in multi-dose containers.
  • the formulation can be in the form of solution, suspension, or emulsion in oil or water-soluble medium, extract, powder, granule, tablet or capsule.
  • a dispersing agent or a stabilizer can be additionally included.
  • composition of the present invention can be produced in the form of a disinfectant, a drinking water additive, and a feed additive, but not always limited thereto.
  • composition of the present invention and the method for prevention and treatment of Salmonella infection using the same have an advantage of high specificity against Salmonella , compared with other conventional chemical compositions and methods using thereof. That is, this composition does not have any effect on other useful resident flora and can be used only for the purpose of prevention and treatment of Salmonella infection. Thus, side effects are hardly accompanied. In general, when other chemicals such as the conventional antibiotics are used, general resident bacteria are also targeted and destroyed, resulting in the decrease of immunity in animals and bringing other side effects. In the meantime, the present invention provides an advantage of nature-friendly effect by using the composition containing natural bacteriophage as an active ingredient.
  • FIG. 1 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Enteritidis.
  • FIG. 2 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Gallinarum.
  • FIG. 3 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Pullorum.
  • FIG. 4 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Typhimurium.
  • FIG. 5 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Choleraesuis.
  • FIG. 6 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Dubulin.
  • FIG. 7 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Durby.
  • FIG. 8 is a graph illustrating the result of chick test. “Number” of Y axis indicates the number of dead chicks. “Control” indicates those chicks administered with Salmonella Gallinarum alone. “SP-1” indicates those chicks treated with bacteriophage SP-1 of the present invention and “Gentamycin” indicates those chicks treated with gentamycin.
  • Salmonella Enteritidis Bacteriophage that can destroy Salmonella was isolated from the nature or from animal samples. In the bacteriophage isolation, Salmonella Enteritidis was used as the representative Salmonella . This Salmonella Enteritidis (SE51) was isolated previously by the present inventors and then identified as Salmonella Enteritidis by the inventors.
  • TSB Tryptic Soy Broth
  • TSB Tryptic Soy Broth
  • casein digest 17 g/L; soybean digest, 3 g/L; dextrose, 2.5 g/L; NaCl, 5 g/L; dipotassium phosphate, 2.5 g/L
  • TSB Tryptic Soy Broth
  • Pure bacteriophage was isolated from the filtrate confirmed to contain the bacteriophage capable of killing Salmonella .
  • the isolation of pure bacteriophage was performed by plaque assay. More precisely, one of plaques formed from plaque assay was recovered by using a sterilized tip, which was then added to Salmonella culture solution, followed by culture for 4-5 hours. Upon completion of the culture, centrifugation was performed at 8,000 for 20 minutes to obtain supernatant. Salmonella culture solution was added to the obtained supernatant at the ratio of 1:50, followed by further culture for 4-5 hours. To increase the number of bacteriophage, this procedure was repeated at least 5 times and then centrifugation was performed at 8,000 for 20 minutes to obtain supernatant.
  • Plaque assay was performed with the supernatant. Generally, pure bacteriophage separation cannot be accomplished simply by performing the above procedure once. Thus, the previous steps were repeated again using one of plaques formed from plaque assay. After repeating the procedure at least 5 times, the solution comprising pure bacteriophage was obtained. The repetition of this pure bacteriophage separation processes was not finished until the sizes and shapes of plaques were all similar. Pure bacteriophage separation was confirmed at last by observing under electron microscope. If pure bacteriophage was not confirmed, the above processes were repeated again.
  • the preparation of bacteriophage suspension was performed as follows. Salmonella culture solution was added to the solution comprising pure bacteriophage at the ratio of 1:50, followed by culture for 4-5 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. To obtain enough amount of bacteriophage, the said process was repeated 5 times in total. The final supernatant was filtered with 0.45 ⁇ m filter, followed by precipitation by using polyethylene glycol (PEG). Particularly, PEG and NaCl were added to 100 mL of the filtrate (10% PEG 8000/0.5 M NaCl), which stood at room temperature for 2-3 hours.
  • PEG polyethylene glycol
  • bacteriophage precipitate was suspended in 5 mL of buffer (10 mM Tris-HCl, 10 mM MgSO 4 , 0.1% Gelatin, pH 8.0). This suspension was called bacteriophage suspension or bacteriophage solution.
  • bacteriophage SP-1 Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on Jul. 30, 2010 (Accession No: KCTC 11737BP).
  • Bacteriophage SP-1 genome was extracted as follows using the bacteriophage suspension obtained in Example 1. To eliminate Salmonella DNA and RNA which might be included in the suspension, DNase I and RNase A were added to 10 mL of the bacteriophage suspension (200 U each), which stood at 37° C. for 30 minutes. 30 minutes later, to neutralize DNase I and RNase A activity, 500 ⁇ L of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added, which stood for 10 minutes. The solution stood at 65° C. for another 10 minutes, then 100 ⁇ L of proteinase K (20 mg/mL) was added, followed by reaction at 37° C. for 20 minutes to break the outer wall of the bacteriophage.
  • EDTA ethylenediaminetetraacetic acid
  • Sequencing was performed with the obtained genome as follows. Particularly, gene fragments were obtained by digesting gDNA, the bacteriophage SP-1 genome, with Hpa II according to the conventional method. Linear vector fragment which would be used for the insertion of the gene fragments was also prepared by treating pBluescript II SK(+) phagemid vector (Stratagene) with Cla I. The prepared gene fragments and the vector fragment were ligated by using T4 ligase according to the conventional method. The resultant recombinant vector containing the bacteriophage SP-1 gene fragment was introduced into Top 10F′ (Invitrogen), a kind of E. coli , by eletroporation (electro-transformation).
  • the transformant was selected on agar plate medium containing X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), IPTG (isopropyl ⁇ -D-1-thiogalactopyranoside) and ampicillin by the conventional Blue-White colony selection method.
  • the selected single colony was inoculated on the culture medium supplemented with ampicillin, followed by shaking culture for overnight.
  • Plasmid was extracted from the cultured cells by using plasmid purification kit (iNtRON Biotechnology, Korea). The extracted plasmid proceeded to electrophoresis using 0.8% agarose gel to measure the size. Finally the recombinant plasmid was obtained.
  • the clone containing the obtained plasmid was cultured again and then plasmid was extracted from the cultured cells again. Sequencing with the extracted plasmid was performed by using M13 forward primer and M13 reverse primer which have been generally used. The sequence of each primer is as follows.
  • Partial gene sequences of the bacteriophage SP-1 genome were identified by the above method, based on which total gene sequencing was performed according to the conventional method.
  • the identified bacteriophage SP-1 gene sequence was represented by SEQ. ID. NO: 1.
  • BLAST http://www.ncbi.nlm.nih.gov/BLAST/.
  • nucleotide sequence of bacteriophage SP-1 had high similarity to those of bacteriophage SS3e (GenBank Accession No. AY730274) and Salmonella phage SETP3 (GenBank Accession No. EF177456).
  • nucleotide sequence of the bacteriophage SP-1 genome was closer to the nucleotide sequence of the bacteriophage SS3e.
  • the size of the bacteriophage SP-1 genome was 43285 bp, while the size of the bacteriophage SS3e genome was 40794 bp, suggesting that the bacteriophage SP-1 genome was larger.
  • the additional nucleotides in the bacteriophage SP-1 showed high similarity to some of the Salmonella phage SETP3 genome sequence.
  • two open reading frames (ORF) were identified.
  • ORFs had no similarity to ORFs of the bacteriophage SS3e genome. Instead, they demonstrated similarity to those of the Salmonella phage SETP3 genome. However, while those ORFs were similar to the ORF like sequence in the Salmonella phage SETP3, one of them showed higher similarity to the ORF found in the E. coli phage Klindl genome (GenBank Accession No. GU196279.1).
  • the genome of the bacteriophage SP-1 shows combined characteristics of both bacteriophage SS3e and Salmonella phage SETP3. Moreover, it could be assumed that other additional characteristics could be included except the said combined characteristics. Therefore, it can be concluded that the bacteriophage SP-1 is a novel bacteriophage which is completely different from any of the conventional bacteriophages.
  • Salmonella bacteria were used to investigate killing activity of the selected bacteriophage SP-1. For the investigation, clear zone formation was first observed by spot assay by the same manner as described in Example 1. Salmonella bacteria used for this assay were Salmonella Enteritidis (SE51), Salmonella Gallinarum (SG36), Salmonella Pullorum (SP-11), Salmonella Typhimurium (ST2), Salmonella Choleraesuis (ATCC 9120), Salmonella Dubulin (BA584), and Salmonella Durby. Salmonella Choleraesuis (ATCC 9120) was the standard strain, and Salmonella Dubulin (BA584) and Salmonella Durby were the isolates distributed from National Veterinary Research & Quarantine Service Korea.
  • Salmonella Enteritidis SE51
  • Salmonella Gallinarum SG36
  • Salmonella Pullorum SP-11
  • Salmonella Typhimurium ST2
  • SE51 Salmonella Enteritidis
  • SG36 Salmonella Gallinarum
  • SP-11 Salmonella Pullorum
  • ST2 Salmonella Typhimurium
  • the bacteriophage SP-1 can be used as an active ingredient of the composition formulated for the purpose of prevention and treatment of Salmonella infection.
  • bacteriophage SP-1 suspension (1 ⁇ 10 8 pfu/mL) was loaded to 7 tubes each containing 9 mL of TSB medium.
  • Another 7 tubes containing 9 mL of TSB medium alone were also prepared.
  • One of those 7 tubes containing medium and bacteriophage suspension and one of those 7 tubes containing medium alone are paired for one experimental group.
  • To the prepared 7 experimental groups were added Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Dubulin, and Salmonella Durby culture solution (OD 600 : 0.5), respectively. After Salmonella bacteria were added to those tubes, they were all transferred to 37° C.
  • the bacteriophage SP-1 of the present invention not only suppresses the growth of various Salmonella bacteria but also even destroys them, so that it can be used as an active ingredient for the composition formulated for the purpose of prevention of Salmonella infection.
  • Salmonella Gallinarum which was the pathogen of fowl typhoid
  • 10 broiler chickens at 2 days of age were grouped as one, and three groups were prepared in total. All the groups were orally administered with Salmonella Gallinarum suspension (1 ⁇ 10 7 cfu). 10 hours after the administration, the experimental group chicks were orally administered with bacteriophage SP-1 suspension (1 ⁇ 10 9 pfu). Bacteriophage SP-1 suspension was additionally administered orally twice at 24 hours interval after the first administration.
  • the positive control group was prepared by administering the antibiotics, gentamycin (5 mg/kg), to the animals via muscular injection 10 hours after the Salmonella Gallinarum administration. The gentamycin injection was also repeated twice. The remaining one group was prepared as the negative control which was not treated with anything.
  • the lifespan of the animal treated with bacteriophage SP-1 suspension was significantly extended, compared with that of the negative control. Even if complete cure might not be achieved, significant treatment effect of bacteriophage SP-1, which was shown as significantly extended lifespan, was not in doubt, considering that, unlike the natural infection, excessive amount of bacteria was forcefully administered directly to the animal to cause infectious disease at a severe level.
  • Feed additive containing bacteriophage SP-1 at the concentration of 1 ⁇ 10 9 pfu/g was prepared with bacteriophage SP-1 suspension.
  • the preparation method was as follows. Bacteriophage SP-1 suspension was evenly sprayed on maltodextran at the proper weight ratio, which was then vacuum-dried at room temperature, followed by pulverization into fine powders. Silica was added thereto at the weight ratio of 5% and the mixture was well mixed. For the drying process, either reduced pressure drying, drying at elevated temperature, or freeze drying can be used.
  • bacteriophage free feed additive was also prepared by spraying buffer which was used for the preparation of bacteriophage suspension instead of bacteriophage suspension.
  • the above two feed additives were mixed with feed for pig respectively at the weight ratio of 1:1,000. As a result, two different kinds of feeds for pig were prepared. Another two different feed additives were also prepared by the same manner as described above, which were mixed with feed for poultry farming respectively at the weight ratio of 1:1,000. As a result, two different feeds for poultry farming were prepared.
  • Drinking water additive and disinfectant are prepared by the same method because both are formulated in the same form and have only difference in their use.
  • Drinking water additive (or disinfectant) containing bacteriophage SP-1 at the concentration of 1 ⁇ 10 9 pfu/mL was prepared.
  • the method of preparation of drinking water additive (or disinfectant) is as follows. Bacteriophage SP-1 was added to the buffer which was generally used for the preparation of bacteriophage suspension at the concentration of 1 ⁇ 10 9 pfu/mL and well mixed. For the control, the buffer itself was used as the bacteriophage free drinking water additive (or disinfectant).
  • the prepared two different drinking water additives were diluted with water at the ratio of 1:1,000, resulting in the final drinking water additive or disinfectant.
  • the piglets were supplied with the feeds prepared in Example 7 and the drinking water prepared in Example 8 according to the conventional method as shown in Table 3. Disinfection was performed with the conventional disinfectant and the disinfectant of the present invention by taking turns, three times a week. The day when the disinfectant of the present invention was sprayed on, the conventional disinfectant was not used. The results are shown in Table 4.
  • Salmonella is also an important pathogen of chicken disease. So, the effect of the composition of the present invention on chicken farming was also investigated. Improvement of feeding efficacy on chicken farming was investigated by using the feeds, drinking water and disinfectants for chicken farming prepared in Examples 7 & 8. In particular, this investigation was performed by observing death rate. 60 chicks at 2 days of age were divided into three groups (20 chicks/group) (group A: supplied with bacteriophage by the feeds; group B: supplied with bacteriophage by the drinking water; group C: treated with the disinfectants containing bacteriophage). The investigation was performed for 4 weeks. Each group was divided into two subgroups of 10 chicks.
  • the chicks were supplied with the feeds for chicken farming prepared in Example 7 and the drinking water for chicken farming prepared in Example 8 according to the conventional method as shown in Table 5. Disinfection was performed with the conventional disinfectant and the disinfectant of the present invention by taking turns, three times a week. The day when the disinfectant of the present invention was sprayed on, the conventional disinfectant was not used. The results are shown in Table 6.

Abstract

The present invention relates to a composition comprising bacteriophage SP-1, the bacteriophage capable of destroying Salmonella once being infected in Salmonella, as an active ingredient, and a method for prevention and treatment of Salmonella infection using the same. Bacteriophage SP-1, the active ingredient of the composition of the present invention, characteristically has the killing activity to Salmonella and has the genome represented by SEQ. ID. NO: 1.

Description

    FIELD
  • The present invention relates to a composition applicable for prevention or treatment of Salmonella infection comprising bacteriophage that is capable of destroying Salmonella once infected to Salmonella as an active ingredient and a method for prevention and treatment of Salmonella infection by using the said composition. That is, the present invention relates to a composition used for the purpose of prevention or treatment of Salmonella infection and a method for prevention and treatment of Salmonella infection using the same.
    • BACKGROUND
  • Salmonella is similar to E. coli in the aspects of morphology or physiology but is categorized in an independent genus for the convenience in clinical use by the proposal of K. Kauffmann et al. Salmonella has been isolated from enteritis and gastroenteritis patients and from animals with diverse diseases since Salmonella choleraesuis was first isolated from a pig died of hog cholera by Salmon and Smith in 1885. Salmonella has also been isolated from health animals such as chicken, cow, pig, goat, dog, and cat and from our environment.
  • More than 2,000 serotypes of Salmonella have been reported so far and it can be largely divided into two groups, one of which is the group that has host specificity and the other of which is the group that does not have host specificity. Salmonella is a genus of rod-shaped, Gram-negative and non-spore-forming It is a parasite living in a variety of animals.
  • Salmonella infectious disease is developed in different forms. Enteritis is the most general form of Salmonella infection. Once infected with Salmonella, such symptoms as rough skin, anorexia, conjunctivitis, depression, pale feces, spleen enlargement, and even death are observed.
  • Various drugs have been used for the treatment of Salmonella infection. However, those drugs known so far cannot lead complete cure. So, it is more important to prevent the infection than to treat it.
  • Damage in livestock industry caused by Salmonella infection is rather huge. Therefore, it is an urgent request to develop a method for prevention and effective treatment of Salmonella infection.
  • The utilization of bacteriophage is now highly drawing our attention as an effective way of treating bacterial disease. In particular, our interests in bacteriophage grow with the preference of nature-friendly method. Bacteriophage is an extremely small microorganism infecting bacteria, which is generally called phage in short. Bacteriophage is an obligate intracellular parasite that multiplis inside bacteria by making use of some or all of the host biosynthetic machinery. Upon completion of the multiplication, offspring bacteriophages are coming out of the host cell with destroying the host bacteria. The infection of bacteriophage in bacteria is very unique and specific, so only specific bacteria can be infected with a specific bacteriophage. That is, there is a limitation in bacteria that can be infected with bacteriophage. Thus, bacteriophage can only kill specific target bacteria without effecting on any other bacteria.
  • Bacteriophage was first found in 1915 when English bacteriologist Twort was studying on the phenomenon that micrococcus colony was being melted clearly by some reasons. And also, French bacteriologist d'Herelle noticed that Shigella disentriae was melted by something in filtrate of dysentery patient's feces and afterwards he separated bacteriophage independently by the following study and named it bacteriophage which meant ‘eating bacteria’. Since then, bacteriophages corresponding to different pathogenic bacteria including Shigella, Salmonella and Vibrio cholerae have been continuously reported.
  • Owing to its capability of killing bacteria, bacteriophage has been in the center of our interest to fight with bacterial infection and studies followed thereon. However, since Flemming found out penicillin, antibiotics have been supplied and the study on bacteriophage has been limited in some east European countries and old Soviet Union. It was not until 2000 that the conventional antibiotics demonstrated their problems in use because of increasing antibiotic-resistant bacteria. So, once again, bacteriophage draws out attention as an alternative anti-bacterial agent that can take the place of the conventional antibiotics.
  • Therefore, the present inventors tried to develop a composition for prevention or treatment of Salmonella infection by using bacteriophage that can destroy Salmonella selectively and further tried to establish a method for prevention and treatment of Salmonella infection using the same. At last, the inventors isolated a proper bacteriophage from the nature and secured the gene sequence of its genome for the distinguishment from other bacteriophages. Then, the present inventors succeeded in developing a composition comprising the said bacteriophage as an active ingredient and further completed this invention by confirming that the said composition of the invention can be effectively used for prevention and treatment of Salmonella infection.
    • DISCLOSURE Technical Problem
  • It is an object of the present invention to provide a novel bacteriophage capable of killing Salmonella selectively.
  • It is another object of the present invention to provide a composition usable for prevention of Salmonella infection comprising the said bacteriophage as an active ingredient which is capable of killing Salmonella selectively by infecting Salmonella and to provide a method for prevention of Salmonella infection using the same.
  • It is also an object of the present invention to provide a composition usable for treatment of Salmonella infection comprising the said bacteriophage as an active ingredient which is capable of killing Salmonella selectively by infecting Salmonella and to provide a method for treatment of Salmonella infection using the same.
  • It is further an object of the present invention to provide a disinfectant for treatment of Salmonella using the said composition.
  • It is also an object of the present invention to provide a drinking water additive using the said composition.
  • It is also an object of the present invention to provide a feed additive using the said composition.
    • Technical Solution
  • The present invention provides a composition comprising bacteriophage as an active ingredient which is capable of destroying Salmonella by infecting Salmonella, and a method for prevention and treatment of Salmonella infection by using the said composition.
  • Bacteriophage used as the active ingredient in the composition of the present invention is bacteriophage SP-1 having DNA represented by SEQ. ID. NO: 1 as its genome. Bacteriophage SP-1 was isolated by the present inventors and deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on Jul. 30, 2010 (Accession No: KCTC 11737BP).
  • The present invention also provides a disinfectant, a drinking water additive, and a feed additive that can be used for prevention or treatment of Salmonella infection.
  • Bacteriophage SP-1 included in the composition of the present invention is able to kill Salmonella effectively, so that it can have a preventive and/or treating effect on diverse infectious diseases caused by Salmonella. Therefore, the composition of the present invention can be used for the purpose of prevention and treatment of disease caused by Salmonella.
  • The pharmaceutical composition of the present invention can be used for prevention and treatment of salmonellosis, the representative disease caused by Salmonella including enteritis, Bacteremia, Fowl typhoid, Pullorum disease, and cholera. The term “salmonellosis” in this invention generally indicates all the symptoms caused by Salmonella infection including fever, headache, diarrhea, vomiting, etc. The term “treat” or “treatment” in this description indicates (i) to suppress disease caused by Salmonella; and (ii) to relieve disease symptoms caused by Salmonella.
  • The representative Salmonella targeted by this invention is exemplified by Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Dubulin, and Salmonella Durby, but not always limited thereto.
  • The composition of the present invention can include pharmaceutically acceptable carriers such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but not always limited thereto. The composition of the present invention can additionally include lubricants, wetting agents, sweetening agents, flavors, emulsifiers, suspensions and preservatives.
  • The composition of the present invention contains bacteriophage SP-1 as an active ingredient. At this time, the bacteriophage SP-1 is included at the concentration of 1×101 pfu/mL-1×1030 pfu/mL or 1×101 pfu/g-1×1030 pfu/g, and more preferably at the concentration of 1×104 pfu/mL-1×1015 pfu/mL or 1×104 pfu/g-1×1015 pfu/g.
  • The composition of the present invention can be formulated by the method that can be performed by those in the art by using a pharmaceutically acceptable carrier and/or excipient in the form of unit dose or in multi-dose containers. The formulation can be in the form of solution, suspension, or emulsion in oil or water-soluble medium, extract, powder, granule, tablet or capsule. At this time, a dispersing agent or a stabilizer can be additionally included.
  • The composition of the present invention can be produced in the form of a disinfectant, a drinking water additive, and a feed additive, but not always limited thereto.
    • Advantageous Effect
  • The composition of the present invention and the method for prevention and treatment of Salmonella infection using the same have an advantage of high specificity against Salmonella, compared with other conventional chemical compositions and methods using thereof. That is, this composition does not have any effect on other useful resident flora and can be used only for the purpose of prevention and treatment of Salmonella infection. Thus, side effects are hardly accompanied. In general, when other chemicals such as the conventional antibiotics are used, general resident bacteria are also targeted and destroyed, resulting in the decrease of immunity in animals and bringing other side effects. In the meantime, the present invention provides an advantage of nature-friendly effect by using the composition containing natural bacteriophage as an active ingredient.
    • DESCRIPTION OF DRAWINGS
  • The application of the preferred embodiments of the present invention is best understood with reference to the accompanying drawings, wherein:
  • FIG. 1 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Enteritidis.
  • FIG. 2 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Gallinarum.
  • FIG. 3 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Pullorum.
  • FIG. 4 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Typhimurium.
  • FIG. 5 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Choleraesuis.
  • FIG. 6 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Dubulin.
  • FIG. 7 is a photograph showing the result of killing activity test with bacteriophage SP-1 to Salmonella Durby.
  • FIG. 8 is a graph illustrating the result of chick test. “Number” of Y axis indicates the number of dead chicks. “Control” indicates those chicks administered with Salmonella Gallinarum alone. “SP-1” indicates those chicks treated with bacteriophage SP-1 of the present invention and “Gentamycin” indicates those chicks treated with gentamycin.
    •  MODE FOR INVENTION
  • Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
  • However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
    • Example 1 Isolation of Bacteriophage that can Destroy Salmonella
  • Bacteriophage that can destroy Salmonella was isolated from the nature or from animal samples. In the bacteriophage isolation, Salmonella Enteritidis was used as the representative Salmonella. This Salmonella Enteritidis (SE51) was isolated previously by the present inventors and then identified as Salmonella Enteritidis by the inventors.
  • Collected samples were loaded in TSB (Tryptic Soy Broth) medium (casein digest, 17 g/L; soybean digest, 3 g/L; dextrose, 2.5 g/L; NaCl, 5 g/L; dipotassium phosphate, 2.5 g/L) inoculated with Salmonella Enteritidis (1/1000), followed by shaking culture for 3-4 hours at 37° C. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes and the supernatant was recovered. Salmonella was inoculated in the recovered supernatant (1/1000), followed by shaking culture for 3-4 hours at 37° C. This procedure was repeated 5 times in total in order to increase bacteriophage titer if bacteriophage was included in the sample. After repeating the process 5 times, the culture solution proceeded to centrifugation at 8,000 rpm for 20 minutes. Then, the supernatant was filtered using 0.45 nm filter. The obtained filtrate was investigated by using general spot assay to see whether bacteriophage that could kill Salmonella was included.
  • Spot assay was performed as follows. Salmonella was inoculated in TSB medium (1/1000), followed by shaking culture at 37° C. overnight. Then, 3 mL of the obtained Salmonella culture solution (OD600: 2.0) was spread on TSA (Tryptic Soy Agar) plate medium (casein digest, 15 g/L; soybean digest, 5 g/L; NaCl, 5 g/L; agar, 15 g/L). The plate medium stayed on clean bench for about 30 minutes to let the spread solution is dried. After drying, 10 nl of the prepared filtrate was loaded on the plate medium whereon Salmonella was spread, which was dried as it is for 30 minutes. After drying, the plate medium was standing cultured at 37° C. for a day. It was then investigated whether the clear zone was formed on the spot where the filtrate was loaded. If the clear zone was formed thereon, it suggested that bacteriophage that could kill Salmonella was included therein. According to this procedure, the filtrate containing bacteriophage that could destroy Salmonella could be obtained.
  • Pure bacteriophage was isolated from the filtrate confirmed to contain the bacteriophage capable of killing Salmonella. The isolation of pure bacteriophage was performed by plaque assay. More precisely, one of plaques formed from plaque assay was recovered by using a sterilized tip, which was then added to Salmonella culture solution, followed by culture for 4-5 hours. Upon completion of the culture, centrifugation was performed at 8,000 for 20 minutes to obtain supernatant. Salmonella culture solution was added to the obtained supernatant at the ratio of 1:50, followed by further culture for 4-5 hours. To increase the number of bacteriophage, this procedure was repeated at least 5 times and then centrifugation was performed at 8,000 for 20 minutes to obtain supernatant. Plaque assay was performed with the supernatant. Generally, pure bacteriophage separation cannot be accomplished simply by performing the above procedure once. Thus, the previous steps were repeated again using one of plaques formed from plaque assay. After repeating the procedure at least 5 times, the solution comprising pure bacteriophage was obtained. The repetition of this pure bacteriophage separation processes was not finished until the sizes and shapes of plaques were all similar. Pure bacteriophage separation was confirmed at last by observing under electron microscope. If pure bacteriophage was not confirmed, the above processes were repeated again.
  • The preparation of bacteriophage suspension was performed as follows. Salmonella culture solution was added to the solution comprising pure bacteriophage at the ratio of 1:50, followed by culture for 4-5 hours. Upon completion of the culture, centrifugation was performed at 8,000 rpm for 20 minutes to obtain supernatant. To obtain enough amount of bacteriophage, the said process was repeated 5 times in total. The final supernatant was filtered with 0.45 μm filter, followed by precipitation by using polyethylene glycol (PEG). Particularly, PEG and NaCl were added to 100 mL of the filtrate (10% PEG 8000/0.5 M NaCl), which stood at room temperature for 2-3 hours. Then, centrifugation was performed at 8,000 rpm for 30 minutes to obtain bacteriophage precipitate. The obtained bacteriophage precipitate was suspended in 5 mL of buffer (10 mM Tris-HCl, 10 mM MgSO4, 0.1% Gelatin, pH 8.0). This suspension was called bacteriophage suspension or bacteriophage solution.
  • At last, purified pure bacteriophage was obtained and this bacteriophage was named bacteriophage SP-1, which was then deposited at Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology on Jul. 30, 2010 (Accession No: KCTC 11737BP).
    • Example 2 Extraction of Bacteriophage SP-1 Genome and Sequencing Thereof
  • Bacteriophage SP-1 genome was extracted as follows using the bacteriophage suspension obtained in Example 1. To eliminate Salmonella DNA and RNA which might be included in the suspension, DNase I and RNase A were added to 10 mL of the bacteriophage suspension (200 U each), which stood at 37° C. for 30 minutes. 30 minutes later, to neutralize DNase I and RNase A activity, 500 μL of 0.5 M ethylenediaminetetraacetic acid (EDTA) was added, which stood for 10 minutes. The solution stood at 65° C. for another 10 minutes, then 100 μL of proteinase K (20 mg/mL) was added, followed by reaction at 37° C. for 20 minutes to break the outer wall of the bacteriophage. Then, 500 μL of 10% sodium dodecyl sulfate (SDS) solution was added thereto, followed by reaction at 65° C. for one hour. One hour later, 10 μL of the mixed solution comprising phenol:chloroform:isoamylalcohol at the concentration ratio of 25:24:1 was added thereto and the solution was well mixed. Centrifugation was performed at 13,000 rpm for 15 minutes to separate layers, among which the upper most layer was obtained. Isopropyl alcohol was added to the obtained layer at the volume ratio of 1.5, followed by centrifugation at 13,000 rpm for 10 minutes to precipitate genome. The precipitate was recovered, to which 70% ethanol was added, flowed by centrifugation at 13,000 rpm for 10 minutes. The washed precipitate was collected and vacuum-dried, which was then dissolved in 100 μL of water.
  • Sequencing was performed with the obtained genome as follows. Particularly, gene fragments were obtained by digesting gDNA, the bacteriophage SP-1 genome, with Hpa II according to the conventional method. Linear vector fragment which would be used for the insertion of the gene fragments was also prepared by treating pBluescript II SK(+) phagemid vector (Stratagene) with Cla I. The prepared gene fragments and the vector fragment were ligated by using T4 ligase according to the conventional method. The resultant recombinant vector containing the bacteriophage SP-1 gene fragment was introduced into Top 10F′ (Invitrogen), a kind of E. coli, by eletroporation (electro-transformation). The transformant was selected on agar plate medium containing X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside), IPTG (isopropyl β-D-1-thiogalactopyranoside) and ampicillin by the conventional Blue-White colony selection method. The selected single colony was inoculated on the culture medium supplemented with ampicillin, followed by shaking culture for overnight. Plasmid was extracted from the cultured cells by using plasmid purification kit (iNtRON Biotechnology, Korea). The extracted plasmid proceeded to electrophoresis using 0.8% agarose gel to measure the size. Finally the recombinant plasmid was obtained. The clone containing the obtained plasmid was cultured again and then plasmid was extracted from the cultured cells again. Sequencing with the extracted plasmid was performed by using M13 forward primer and M13 reverse primer which have been generally used. The sequence of each primer is as follows.
  • TABLE 1
    Primer Sequence
    M13 forward primer GTCGTGACTGGGAAAACCCTGGCG
    SEQ. ID. NO: 2
    M13 reverse primer TCCTGTGTGAAATTGTTATCCGCT
    SEQ. ID. NO: 3
  • Partial gene sequences of the bacteriophage SP-1 genome were identified by the above method, based on which total gene sequencing was performed according to the conventional method. The identified bacteriophage SP-1 gene sequence was represented by SEQ. ID. NO: 1.
  • Based on the gene sequence of bacteriophage SP-1, similarity to those sequences of the conventional bacteriophages was investigated by using BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). As a result, the nucleotide sequence of bacteriophage SP-1 had high similarity to those of bacteriophage SS3e (GenBank Accession No. AY730274) and Salmonella phage SETP3 (GenBank Accession No. EF177456). Particularly, the nucleotide sequence of the bacteriophage SP-1 genome was closer to the nucleotide sequence of the bacteriophage SS3e. However, the size of the bacteriophage SP-1 genome was 43285 bp, while the size of the bacteriophage SS3e genome was 40794 bp, suggesting that the bacteriophage SP-1 genome was larger. The additional nucleotides in the bacteriophage SP-1 showed high similarity to some of the Salmonella phage SETP3 genome sequence. Among the additional nucleotides in the bacteriophage SP-1, two open reading frames (ORF) were identified.
  • These ORFs had no similarity to ORFs of the bacteriophage SS3e genome. Instead, they demonstrated similarity to those of the Salmonella phage SETP3 genome. However, while those ORFs were similar to the ORF like sequence in the Salmonella phage SETP3, one of them showed higher similarity to the ORF found in the E. coli phage Klindl genome (GenBank Accession No. GU196279.1).
  • The above results indicate that the genome of the bacteriophage SP-1 shows combined characteristics of both bacteriophage SS3e and Salmonella phage SETP3. Moreover, it could be assumed that other additional characteristics could be included except the said combined characteristics. Therefore, it can be concluded that the bacteriophage SP-1 is a novel bacteriophage which is completely different from any of the conventional bacteriophages.
    • Example 3 Killing Activity of Bacteriophage SP-1 to Various Salmonella Bacteria
  • Various Salmonella bacteria were used to investigate killing activity of the selected bacteriophage SP-1. For the investigation, clear zone formation was first observed by spot assay by the same manner as described in Example 1. Salmonella bacteria used for this assay were Salmonella Enteritidis (SE51), Salmonella Gallinarum (SG36), Salmonella Pullorum (SP-11), Salmonella Typhimurium (ST2), Salmonella Choleraesuis (ATCC 9120), Salmonella Dubulin (BA584), and Salmonella Durby. Salmonella Choleraesuis (ATCC 9120) was the standard strain, and Salmonella Dubulin (BA584) and Salmonella Durby were the isolates distributed from National Veterinary Research & Quarantine Service Korea. Salmonella Enteritidis (SE51), Salmonella Gallinarum (SG36), Salmonella Pullorum (SP-11), and Salmonella Typhimurium (ST2) were the isolates isolated and identified by the inventors. The results of the related experiments are shown in FIG. 1-FIG. 7. As shown in FIG. 1-FIG. 7, the bacteriophage SP-1 was confirmed to have Salmonella killing activity against various Salmonella bacteria. In addition, killing activity of the bacteriophage SP-1 to Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Streptococcus agalactiae, Streptococcus mitis, Streptococcus uberis and Pseudomonas aeruginosa was further investigated. As a result, it was confirmed that the bacteriophage SP-1 did not have killing activity against those bacteria.
  • From the above results, it was confirmed that the bacteriophage SP-1 can be used as an active ingredient of the composition formulated for the purpose of prevention and treatment of Salmonella infection.
    • Example 4 Application Example of Bacteriophage SP-1 for Prevention of Salmonella Infection
  • 100 μL of bacteriophage SP-1 suspension (1×108 pfu/mL) was loaded to 7 tubes each containing 9 mL of TSB medium. Another 7 tubes containing 9 mL of TSB medium alone were also prepared. One of those 7 tubes containing medium and bacteriophage suspension and one of those 7 tubes containing medium alone are paired for one experimental group. To the prepared 7 experimental groups were added Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Dubulin, and Salmonella Durby culture solution (OD600: 0.5), respectively. After Salmonella bacteria were added to those tubes, they were all transferred to 37° C. incubator, followed by shaking culture, during which the growth of Salmonella was observed. As shown in Table 2, the growth of Salmonella was suppressed in the tubes containing bacteriophage SP-1 suspension in every experimental group. In the meantime, the growth of Salmonella was not inhibited in the bacteriophage free tubes.
  • TABLE 2
    Suppression of Salmonella growth
    OD600
    Culture culture culture
    0 min. 15 min. 60 min
    Salmonella Bacteriophage− 0.5 0.7 1.5
    Enteritidis Bacteriophage+ 0.5 0.1 0.05
    Salmonella Bacteriophage− 0.5 0.65 1.4
    Gallinarum Bacteriophage+ 0.5 0.1 0.05
    Salmonella Bacteriophage− 0.5 0.8 1.6
    Pullorum Bacteriophage+ 0.5 0.1 0.05
    Salmonella Bacteriophage− 0.5 0.75 1.5
    Typhimurium Bacteriophage+ 0.5 0.1 0.05
    Salmonella Bacteriophage− 0.5 0.65 1.3
    Choleraesuis Bacteriophage+ 0.5 0.1 0.05
    Salmonella Bacteriophage− 0.5 0.7 1.35
    Dubulin Bacteriophage+ 0.5 0.1 0.05
    Salmonella Bacteriophage− 0.5 0.7 1.5
    Durby Bacteriophage+ 0.5 0.1 0.05
  • The above results indicate that the bacteriophage SP-1 of the present invention not only suppresses the growth of various Salmonella bacteria but also even destroys them, so that it can be used as an active ingredient for the composition formulated for the purpose of prevention of Salmonella infection.
    • Example 5 Treatment Example 1 of Salmonella Infectious Disease Using Bacteriophage SP-1
  • 4 chicks at 2 days of age were administered with 1×107 cfu of Salmonella Enteritidis to induce infection forcefully (animal 1-1, animal 1-2, animal 1-3, animal 1-4). Another 4 chicks at 2 days of age were administered with 1×107 cfu of Salmonella Choleraesuis to induce infection forcefully (animal 2-1, animal 2-2, animal 2-3, animal 2-4). Animal 1-1, animal 1-2, animal 2-1, and animal 2-2 were forced to eat the feed containing bacteriophage SP-1 (mixed at the concentration of 1×109 pfu/g feed), while animal 1-3, animal 1-4, animal 2-3, and animal 2-4 were given with bacteriophage free feed. 2 days later, the numbers of Salmonella bacteria in feces and caecum contents were counted. To avoid contamination by other bacteria, Salmonella selective medium (Rambach agar plate; Merck) was used for the counting. As a result, in those animals fed with bacteriophage SP-1 mixed feed, at least 1000 times less Salmonella was detected in feces, compared with that in the control. In caecum contents, at least 200 times less Salmonella was detected in those animals fed with bacteriophage SP-1 mixed feed.
  • From the above results, it was confirmed that the bacteriophage SP-1 of the present invention was very effective in treating Salmonella infection.
    • Example 6 Treatment Example 2 of Salmonella Infectious Disease Using Bacteriophage SP-1
  • In this example, it was investigated how much bacteriophage SP-1 could reduce the death of chicks infected with Salmonella. Salmonella Gallinarum, which was the pathogen of fowl typhoid, was used for the experiment. 10 broiler chickens at 2 days of age were grouped as one, and three groups were prepared in total. All the groups were orally administered with Salmonella Gallinarum suspension (1×107 cfu). 10 hours after the administration, the experimental group chicks were orally administered with bacteriophage SP-1 suspension (1×109 pfu). Bacteriophage SP-1 suspension was additionally administered orally twice at 24 hours interval after the first administration. The positive control group was prepared by administering the antibiotics, gentamycin (5 mg/kg), to the animals via muscular injection 10 hours after the Salmonella Gallinarum administration. The gentamycin injection was also repeated twice. The remaining one group was prepared as the negative control which was not treated with anything. As shown in FIG. 8, the lifespan of the animal treated with bacteriophage SP-1 suspension was significantly extended, compared with that of the negative control. Even if complete cure might not be achieved, significant treatment effect of bacteriophage SP-1, which was shown as significantly extended lifespan, was not in doubt, considering that, unlike the natural infection, excessive amount of bacteria was forcefully administered directly to the animal to cause infectious disease at a severe level.
  • From the above results, it was confirmed that the bacteriophage SP-1 of the present invention was very effective in treating Salmonella infection.
    • Example 7 Preparation of Feed Additive and Feed
  • Feed additive containing bacteriophage SP-1 at the concentration of 1×109 pfu/g was prepared with bacteriophage SP-1 suspension. The preparation method was as follows. Bacteriophage SP-1 suspension was evenly sprayed on maltodextran at the proper weight ratio, which was then vacuum-dried at room temperature, followed by pulverization into fine powders. Silica was added thereto at the weight ratio of 5% and the mixture was well mixed. For the drying process, either reduced pressure drying, drying at elevated temperature, or freeze drying can be used. For the control, bacteriophage free feed additive was also prepared by spraying buffer which was used for the preparation of bacteriophage suspension instead of bacteriophage suspension.
  • The above two feed additives were mixed with feed for pig respectively at the weight ratio of 1:1,000. As a result, two different kinds of feeds for pig were prepared. Another two different feed additives were also prepared by the same manner as described above, which were mixed with feed for poultry farming respectively at the weight ratio of 1:1,000. As a result, two different feeds for poultry farming were prepared.
    • Example 8 Preparation of Drinking Water Additive and Disinfectant
  • Drinking water additive and disinfectant are prepared by the same method because both are formulated in the same form and have only difference in their use. Drinking water additive (or disinfectant) containing bacteriophage SP-1 at the concentration of 1×109 pfu/mL was prepared. The method of preparation of drinking water additive (or disinfectant) is as follows. Bacteriophage SP-1 was added to the buffer which was generally used for the preparation of bacteriophage suspension at the concentration of 1×109 pfu/mL and well mixed. For the control, the buffer itself was used as the bacteriophage free drinking water additive (or disinfectant).
  • The prepared two different drinking water additives (or disinfectants) were diluted with water at the ratio of 1:1,000, resulting in the final drinking water additive or disinfectant.
    • Example 9 Investigation of Feeding Efficacy on Pig Farming
  • Improvement of feeding efficacy on pig farming was investigated by using the feeds, drinking water and disinfectants prepared in Examples 7 & 8. In particular, this investigation was performed by observing death rate. 30 piglets were divided into three groups (10 piglets/group) (group A: supplied with bacteriophage by the feeds; group B: supplied with bacteriophage by the drinking water; group C: treated with the disinfectants containing bacteriophage). The investigation was performed for 4 weeks. Each group was divided into two subgroups of 5 piglets. Those subgroups were either treated with bacteriophage SP-1 (subgroup (1)) or not treated with bacteriophage SP-1 (subgroup (2)). The test piglets were 20 days old. Each group piglets were raised in an isolated cage separated from each other at regular intervals. Each subgroup was sorted and marked as shown in Table 3.
  • TABLE 3
    Subgroup sorting and marking in feeding efficacy test on pig farming
    Subgroup sorting and marking
    Bacteriophage SP-1+ Bacteriophage SP-1−
    Feed A-(1) A-(2)
    Drinking water B-(1) B-(2)
    Disinfectant C-(1) C-(2)
  • The piglets were supplied with the feeds prepared in Example 7 and the drinking water prepared in Example 8 according to the conventional method as shown in Table 3. Disinfection was performed with the conventional disinfectant and the disinfectant of the present invention by taking turns, three times a week. The day when the disinfectant of the present invention was sprayed on, the conventional disinfectant was not used. The results are shown in Table 4.
  • TABLE 4
    Group Death Rate (%)
    A-(1) 0
    A-(2) 20
    B-(1) 0
    B-(2) 40
    C-(1) 0
    C-(2) 20
  • From the above results, it was confirmed that the feeds, drinking water and disinfectants prepared according to the present invention could help to reduce death rate in pig farming Therefore, it was concluded that the composition of the present invention was effective in the improvement of feeding efficacy on pig farming.
    • Example 10 Investigation of Feeding Efficacy on Chicken Farming
  • Salmonella is also an important pathogen of chicken disease. So, the effect of the composition of the present invention on chicken farming was also investigated. Improvement of feeding efficacy on chicken farming was investigated by using the feeds, drinking water and disinfectants for chicken farming prepared in Examples 7 & 8. In particular, this investigation was performed by observing death rate. 60 chicks at 2 days of age were divided into three groups (20 chicks/group) (group A: supplied with bacteriophage by the feeds; group B: supplied with bacteriophage by the drinking water; group C: treated with the disinfectants containing bacteriophage). The investigation was performed for 4 weeks. Each group was divided into two subgroups of 10 chicks. Those subgroups were either treated with bacteriophage SP-1 (subgroup [1]) or not treated with bacteriophage SP-1 (subgroup [2]). The chicks were isolated group by group and raised. Each subgroup was sorted and marked as shown in Table 5.
  • TABLE 5
    Subgroup sorting and marking in feeding efficacy test on chicken farming
    Subgroup sorting and marking
    Bacteriophage SP-1+ Bacteriophage SP-1−
    Feed A-[1] A-[2]
    Drinking water B-[1] B-[2]
    Disinfectant C-[1] C-[2]
  • The chicks were supplied with the feeds for chicken farming prepared in Example 7 and the drinking water for chicken farming prepared in Example 8 according to the conventional method as shown in Table 5. Disinfection was performed with the conventional disinfectant and the disinfectant of the present invention by taking turns, three times a week. The day when the disinfectant of the present invention was sprayed on, the conventional disinfectant was not used. The results are shown in Table 6.
  • TABLE 6
    Group Death Rate (%)
    A-[1] 0
    A-[2] 20
    B-[1] 0
    B-[2] 30
    C-[1] 0
    C-[2] 30
  • From the above results, it was confirmed that the feeds, drinking water and disinfectants prepared according to the present invention could help to reduce death rate in chicken farming Therefore, it was concluded that the composition of the present invention was effective in the improvement of feeding efficacy on chicken farming.
  • Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims (8)

1. A composition for prevention and treatment of Salmonella infection, comprising:
bacteriophage SP-1 harboring the genome represented by SEQ. ID. NO: 1 as an active ingredient.
2. The composition for prevention and treatment of Salmonella infection according to claim 1, wherein the bacteriophage SP-1 has Accession No. KCTC 11737BP.
3. The composition for prevention and treatment of Salmonella infection according to claim 1, wherein the composition is a feed additive, drinking water additive, or disinfectant.
4. The composition for prevention and treatment of Salmonella infection according to claim 1, wherein the Salmonella is Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Dublin, or Salmonella Durby.
5. A method for prevention or treatment of Salmonella infection, comprising the step of administering the composition of claim 1 to a subject animal.
6. The method for prevention or treatment of Salmonella infection according to claim 5, wherein the bacteriophage SP-1 has Accession No. KCTC 11737BP.
7. The method for prevention or treatment of Salmonella infection according to claim 5, wherein the composition is administered to the subject animal in the form of a feed additive, drinking water additive, or disinfectant.
8. The method for prevention or treatment of Salmonella infection according to claim 5, wherein the Salmonella is Salmonella Enteritidis, Salmonella Gallinarum, Salmonella Pullorum, Salmonella Typhimurium, Salmonella Choleraesuis, Salmonella Dublin, or Salmonella Durby.
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KR101649851B1 (en) * 2014-12-30 2016-08-30 주식회사 인트론바이오테크놀로지 Novel Shigatoxin-producing Escherichia coli type F18 bacteriophage Esc-COP-1 and its use for preventing proliferation of Shigatoxin-producing Escherichia coli type F18
KR101761581B1 (en) 2014-12-30 2017-07-26 주식회사 인트론바이오테크놀로지 Novel enteroinvasive Escherichia coli bacteriophage Esc-COP-4 and its use for preventing proliferation of enteroinvasive Escherichia coli

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090030385A (en) * 2007-09-20 2009-03-25 주식회사 인트론바이오테크놀로지 Novel bacteriophage having killing activity specific to salmonella selected from infectious bacteriophages of salmonella enteritidis

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090030385A (en) * 2007-09-20 2009-03-25 주식회사 인트론바이오테크놀로지 Novel bacteriophage having killing activity specific to salmonella selected from infectious bacteriophages of salmonella enteritidis

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Machine translation of KR 1020090030385, dated 03-2009. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140363401A1 (en) * 2011-09-09 2014-12-11 Intron Biotechnology, Inc Method for preventing and treating salmonella typhimurium infection
US9402873B2 (en) * 2011-09-09 2016-08-02 Intron Biotechnology, Inc. Method for preventing and treating Salmonella Typhimurium infection
WO2017180549A1 (en) * 2016-04-12 2017-10-19 Dots Technology Corp. Allergen detection using magnetics

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