KR102543648B1 - Vaccin composition containing inactivated Salmonella Gallinarum cells by the culture medium containing plantaricin of Lactobacillus plantarum for preventing or treating Fowl Typhoid - Google Patents

Abstract

The present invention relates to a vaccine composition for preventing or treating poultry typhus, comprising improved Salmonella gallina room inactivated killed cells by a culture medium containing plantarisin of Lactobacillus plantarum.

Description

Vaccine composition containing inactivated Salmonella Gallinarum cells by the culture medium containing plantaricin of Lactobacillus plantarum for preventing or treating Fowl Typhoid}

The present invention relates to a vaccine composition for preventing or treating poultry typhus, comprising inactivated killed cells of Salmonella gallina room reacted with a culture solution containing plantaricin (antibacterial peptide) of Lactobacillus plantarum.

Poultry typhus is caused by Salmonella enterica Serovar Gallinarum ( S. Gallinarum, poultry typhus), and among chickens, it causes sepsis mainly in brown leghorns, occurs acutely and chronically, and causes enormous economic damage to poultry farms due to high mortality. It is a major intracellular parasitic bacterial disease. Also, poultry typhus occurs mainly in medium chicks and mature chickens rather than young chicks.

The causative agent of poultry typhus, S. Gallinarum is an intracellular parasitic bacterium, and conventionally prepared dead cell vaccines such as formalin are characterized by very low defense efficiency. In other words, the cellular immune response protects against poultry typhus, and for this, live attenuated vaccine is considered the most ideal vaccine. For example, Korean Patent Publication No. 10-2012-0128583 discloses a non-pathogenic Salmonella gallina room mutant strain and a pharmaceutical composition using the same, and Korean Patent Publication No. 10-2011-0016369 includes a Salmonella mutant strain. A vaccine composition for preventing poultry typhus is disclosed. S. Gallinarum live attenuated vaccine was developed in the 1950s and has been used as a live vaccine to prevent poultry typhus in poultry farms worldwide since the 1960s. However, in the case of the live vaccine disclosed above, it is a GMO in which the gene is artificially mutated based on genetic manipulation technology, and due to the trend of avoiding GMO products domestically and internationally, it remains at the research level and has not reached industrialization, and its safety is low. It has not been answered yet. In addition, in the case of probiotic vaccines, there is an inherent concern about returning to the virulence strain by recovering pathogenicity, and in light of the domestic breeding environment, attenuated probiotic vaccines are often required to be vaccinated and treated with antibiotics. There can be many situations in which this cannot fully function. Therefore, it is necessary to develop a vaccine that is safe like a dead vaccine and has excellent efficacy by inducing a cellular immune response like a live vaccine. In particular, it is urgent to develop a vaccine that can be safely treated with antibiotics after vaccination in between seasons like in Korea.

Lactic acid bacteria are bacilli that produce lactic acid by fermenting sugar. They produce useful substances that help our body to be healthy, and produce immunity enhancing, antioxidant, antibacterial, anticholesterol, antiobesity, carcinogenic and mutagenic substances. It is known to have a function to prevent Among them, Lactobacillus plantarum strain, a type of lactic acid bacteria contained in kimchi, is a lactic acid bacillus that undergoes homozygous or heterozygous fermentation and is commonly seen in the intestinal tract of animals, including humans, and in the fermentation process of dairy products and vegetables. Microorganisms of the genus Lactobacillus are known to produce various proteinaceous antibacterial substances, maintain intestinal pH as acidic, suppress the propagation of harmful bacteria such as E. coli and Clostridium, and relieve diarrhea and constipation. It is known to play a role in vitamin synthesis, anti-cancer activity, and lowering serum cholesterol.

Under this background, five plantarisin peptides, which are novel antibacterial peptides isolated from Lactobacillus plantarum strains derived from kimchi, and previously well-known plantarisin N, F/E were expressed, as well as lysozyme, There are excellent antibacterial activities by more than 9 types of AMPs (antimicrobial peptides; bacteriocins), including MazF and holin, and among them, the main antibacterial activity is induced by 7 plantaricins, which are It is proposed to act on cell membranes to form holes. Using this property, Salmonella gallina room, the causative agent of poultry typhus, was induced into inactivated dead cells. Inactivated dead cells derived from Salmonella gallina room with the Lactobacillus plantarum strain induce a cellular immune response in brown leghorn species, and virulence S. The present invention was completed by confirming that Gallinarum strain had a perfect protective effect after challenge infection strain.

The present invention has been made to solve the above problems, the present inventors reacted with a culture solution containing plantarisin of Lactobacillus plantarum to induce inactivation of Salmonella Gallinarum strain, thereby improving Salmonella Galina room inactivated killed cells were prepared, and it was confirmed that the vaccine containing the improved Salmonella gallina room inactivated killed cells induces a cellular immune response like a live vaccine in poultry typhus and has an excellent protective effect, the present invention has been completed.

An object of the present invention is to provide a Salmonella Gallinarum inactivated dead cell strain prepared by reacting Salmonella Gallinarum with a culture medium containing plantaricin of Lactobacillus plantarum .

Another object of the present invention is to provide a method for producing an inactivated killed cell of Salmonella enterica Serovar Gallinarum, comprising the following steps:

(a) inoculating Salmonella enterica Serovar Gallinarum into a culture medium and then culturing;

(B) reacting by mixing a culture solution containing plantarisin of Lactobacillus plantarum in the cultured Salmonella gallina room;

(c) inoculating and culturing the reacted mixture in LB broth containing fetal bovine serum (FBS); and

(d) checking the culture for colony formation.

Another object of the present invention is to provide a vaccine composition for preventing or treating poultry typhus comprising at least one selected from the group consisting of a culture of the above-described Salmonella Gallinarum inactivated dead cell strain and a mixture thereof will be.

Another object of the present invention is the above-described Salmonella Gallina room (Salmonella Gallinarum) inactivated dead cell strain; To provide a feed additive for preventing or improving poultry typhus comprising at least one selected from the group consisting of a culture thereof and a mixture thereof.

Another object of the present invention is the above-described Salmonella Gallina room (Salmonella Gallinarum) inactivated dead cell strain; To provide a method for preventing poultry typhus, comprising feeding or inoculating poultry with at least one selected from the group consisting of a culture thereof and a mixture thereof.

In order to achieve the object of the present invention as described above, the present invention is a Salmonella Gallinarum inactivated yarn prepared by reacting Salmonella Gallinarum with a culture medium containing plantarisin of Lactobacillus plantarum. Bacterial strains can be provided.

The present invention can provide a method for producing an inactivated dead cell of Salmonella enterica Serovar Gallinarum, which is improved, including the following steps.

(a) inoculating Salmonella enterica Serovar Gallinarum into a culture medium and then culturing;

(B) reacting by mixing a culture solution containing plantarisin of Lactobacillus plantarum in the cultured Salmonella gallina room;

(c) inoculating and culturing the reacted mixture in LB broth containing fetal bovine serum (FBS); and

(d) checking the culture for colony formation.

The present invention can provide a vaccine composition for preventing or treating poultry typhus comprising at least one selected from the group consisting of a culture of the above-described Salmonella Gallinarum inactivated dead cell strain and a mixture thereof.

The vaccine composition may further include any one or more selected from the group consisting of a carrier, a diluent and an adjuvant.

The adjuvant is selected from the group consisting of aluminum hydroxide, ISA70 (VG), ISA71 (VG), ISA71R (VG), ISA206 (VG), ISA201 (VG), ISA763 (A VG), IMS1313 (VG N) and GEL01 It can be any one.

The vaccine composition may be administered by one or more routes selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, intradermal, subcutaneous and nasal routes.

The poultry may be any one selected from the group consisting of chicks, chickens, ducks, turkeys, quails, water birds, pigeons and canaries.

The present invention relates to the above-described Salmonella Gallinarum inactivated dead cell strain; It is possible to provide a feed additive for preventing or improving poultry typhus containing at least one selected from the group consisting of a culture thereof and a mixture thereof.

The present invention relates to the above-described Salmonella Gallinarum inactivated dead cell strain; It is possible to provide a method for preventing poultry typhus, comprising feeding or inoculating poultry with at least one selected from the group consisting of a culture thereof and a mixture thereof.

Improvement by the plantaricin-containing culture medium of Lactobacillus plantarum of the present invention After inoculating the inactivated killed cells of Salmonella enterica Serovar Gallinarum into the muscles of chickens, challenge infection was performed with Salmonella gallinarum, an outdoor disease. On the 11th day of infection, all chickens died, and the group inoculated with 3 × 10 ⁹ CFU / 200 μl of formalin-inactivated killed cells had a survival rate of 30%, but the improved Salmonella gallina room inactivated cells of the present invention A survival rate of 100% was observed in the group inoculated with cells at 3×10 ⁹ CFU/200 μl, the group inoculated with commercially available live cell vaccine SG9R, and the group not challenged, indicating that the Lactobacillus plantarum of the present invention Manufactured by reacting with a culture medium containing plantarisin When the Salmonella gallina room inactivated dead cell vaccine is inoculated intramuscularly, it has an excellent protective effect like commercially available live attenuated vaccines, and is effective in preventing or treating poultry typhus. In addition, in the case of commercially available live vaccines, a subcutaneous inoculation method is used, which is difficult to inoculate, requires inoculation by an expert, and is not well inoculated, whereas plantarisin of Lactobacillus plantarum of the present invention Improved by containing culture medium Since Salmonella gallinarum inactivated killed cell vaccine is available for intramuscular and oral inoculation, anyone can easily inoculate it, and the inoculation effect is good.

Figure 1 is an electron microscope of the Salmonella Gallinarum of the present invention (A) Lactobacillus plantarum of the Plantalysin-containing culture medium untreated group and (B) Lactobacillus plantarum of the Plantarycin-containing culture medium treatment group photo result.
Figure 2 shows each antigen [control (PBS); live vaccine (SG9R); formalin-inactivated dead cells 3×10 ⁹ ; Improved Lactobacillus plantarum prepared with plantaricin-containing culture medium This is a result of serum antibody titer against Salmonella Gallinarum inactivated dead cells 3×10 ⁹ ].
Figure 3 shows each antigen [control (PBS); live vaccine (SG9R); formalin-inactivated dead cells 3×10 ⁹ ; Improved Lactobacillus plantarum prepared with plantaricin-containing culture medium It is the result of IFN-r concentration in plenocytes for Salmonella Gallinarum inactivated dead cells 3×10 ⁹ ].
Figure 4 shows each vaccination group [control group (PBS); live vaccine (SG9R); formalin-inactivated dead cells 3×10 ⁹ ; Improved Lactobacillus plantarum prepared with plantaricin-containing culture medium Salmonella Gallinarum ( Salmonella Gallinarum) Inactivated dead cells 3×10 ⁹ ] The result of the survival rate after challenge infection.

Hereinafter, the present invention will be described in more detail.

Poultry typhus caused by Salmonella enterica Serovar Gallinarum ( S. Gallinarum, poultry typhus) causes sepsis in chickens, mainly brown leghorns, and occurs acutely and chronically. It is one of the major intracellular parasitic bacterial diseases. A lot of research is in progress to prevent or treat the poultry typhus. To prevent this most effectively, live attenuated vaccine is considered to be the most ideal vaccine. Currently live S. Gallinaru vaccine strain 9R has been used to prevent poultry typhus for about 60 years (Cheng et al., 2016; Wigley 2017). However, it has been reported that this live vaccine strain still remains toxic, and has disadvantages such as slow growth rate and insufficient defense efficiency (Bouzoubaa et al., 1989; Kwon and Cho, 2011). In addition, the domestic poultry breeding environment has four distinct seasons, and laying hens are stressed due to interseason, hot summer, and cold winter, resulting in frequent diarrhea and loss of appetite. Antibiotics are frequently used to prevent bacterial diseases, so attenuated probiotic vaccine It has a breeding environment that is not suitable for the defense efficacy of . Therefore, the development of an inactivated killed cell vaccine that is safer and more effective than SG9R, which is currently on the market, is actively underway. The present inventors reacted Salmonella enterica Serovar Gallinarum (SG) isolated from a dead brown leghorn species with a culture medium containing Lactobacillus plantarum plantarisin to induce improved Salmonella enterica Serovar Gallinarum (SG) inactivation and killing, As a result of muscle inoculation of chickens with the improved Salmonella enterica Serovar Gallinarum (SG) inactivated killed cells prepared from the plantaricin-containing culture medium of the Lactobacillus plantarum, it was confirmed that it effectively prevented poultry typhus, and the present invention was completed. .

The present invention can provide improved Salmonella Gallinarum inactivated dead cells prepared by reacting Salmonella Gallinarum with a culture medium containing plantaricin of Lactobacillus plantarum.

The researchers of the present invention cultured Salmonella enterica Serovar Gallinarum isolated from laying hens showing poultry typhus symptoms and then induced inactivation with a culture medium containing Lactobacillus plantarum plantarisin, improving Salmonella Gallinarum inactivation Killed mycelium was prepared. As a result of confirming the inactivated dead cells of Salmonella Gallinarum using a transmission electron microscope, an empty cytoplasmic space was observed along with a cell membrane with holes in the inactivated cells of Salmonella Gallinarum. As a result, it was confirmed that Salmonella Gallinarum was inactivated and became inactivated dead cells (FIG. 1B).

In order to obtain a culture medium containing plantarisin of the Lactobacillus plantarum, one Lactobacillus plantarum colony was inoculated into 10 ml of MRS medium and cultured overnight, and then added to a new MRS medium to be 1%, cultured for 24 hours, and 3,000 xg The supernatant was collected by centrifugation for 20 minutes, filtered through a 0.2um filter, and used as a material for inactivating and killing Salmonella gallina room, but is not limited thereto.

The Salmonella gallina room ( Salmonella enterica Serovar Gallinarum) may be deposited under the microorganism accession number KCTC14384BP (microbial name: Salmonella Gallinarum (Fowl typhoid).

The Lactobacillus plantium may be Lactobacillus plantarum NIBR97, and may be deposited under the microorganism accession number KCTC18645P (Lactobacillus plantarum NIBR97).

The Lactobacillus plantarum is one of the lactic acid bacteria and may contain various antibacterial substances, one of which is plantarisin, which has a wide antibacterial spectrum and has bactericidal power against Gram-negative or Gram-positive bacteria, so that the culture medium containing them Salonella gallina room can be used as an improved inactivated dead cell inducer.

The present invention can provide a method for producing inactivated dead cells of Salmonella enterica Serovar Gallinarum comprising the following steps:

(a) inoculating Salmonella enterica Serovar Gallinarum into a culture medium and then culturing;

(B) reacting by mixing a culture solution containing plantarisin of Lactobacillus plantarum in the cultured Salmonella gallina room;

(c) inoculating and culturing the reacted mixture in LB broth containing fetal bovine serum (FBS); and

(d) checking the culture for colony formation;

A method for producing inactivated dead cells of Salmonella Gallinarum, comprising a.

The present invention is the above-described improved Salmonella gallina room inactivated dead cells; It provides a vaccine composition for preventing or treating salmonellosis caused by Salmonella galina room comprising at least one selected from the group consisting of a culture thereof and a mixture thereof.

The present inventors inoculate the improved Salmonella enterica Serovar Gallinarum inactivated cells prepared by reacting Salmonella gallina room with a culture solution containing plantarisin of Lactobacillus plantarum to brown leghorn muscle, resulting in improvement Antibody titers of serum IgG of all brown leghorn species in the inactivated killed cell inoculated group, the formalin-inactivated killed cell inoculated group, and the commercial live cell inoculated group were significantly increased compared to the control group (FIG. 2), and the cellular immune response was A significant increase was observed in the representative IFN-r compared to the control group in the brown leghorn of the improved inactivated cell inoculation group and the commercially available live cell vaccine inoculated group, whereas the formalin-inactivated killed cell inoculation group was significantly higher than the control group. No significant increase was observed (FIG. 3). In addition, as a result of challenge infection with Salmonella Salmonella gallina room (SG), an outdoor disease strain, the untreated control group began to die on the 5th day, and all chickens died on the 11th day of challenge infection. The group inoculated with the killed cells at 3×10 ⁹ CFU/200 μl showed a survival rate of 30%, but the group inoculated with the improved Salmonella gallina room inactivated killed cells of the present invention at 3×10 ⁹ CFU/200 μl, A survival rate of 100% was observed in the group inoculated with the commercially available live cell vaccine, SG9R, and in the group not challenged (FIG. 4). When the improved inactivated killed cell vaccine of the present invention is inoculated intramuscularly, there is an excellent protective effect, and there is an effect of preventing or treating poultry typhus caused by Salmonella gallina room.

In the present invention, the term "vaccine" refers to a biological agent containing an antigen that gives immunity to a living body, and refers to an immunogen or antigenic material that gives immunity to a living body by injecting or orally administering to a human or animal to prevent infection. In vivo immunity is largely divided into autoimmunity, in which in vivo immunity is automatically obtained after infection with pathogens, and passive immunity, which is obtained by externally injected vaccines. Autoimmunity has a long period of antibody production related to immunity and is characterized by continuous immunity, whereas passive immunity by vaccine acts immediately in the treatment of infections, but has a disadvantage of poor durability.

The vaccine composition may further include any one or more selected from the group consisting of a carrier, a diluent and an adjuvant.

In addition, the vaccine composition may include a veterinarily acceptable carrier. As used herein, the term "veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, adjuvants, stabilizers, diluents, preservatives, antibacterial and antifungal agents, isotonic agents, adsorption delaying agents, and the like. Carriers, excipients, and diluents that may be included in the vaccine composition include glucose, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, glycerin, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; and the like.

In addition, the vaccine composition is formulated according to conventional methods, oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, nasal formulations such as drips or sprays, and sterile injection solutions. It can be formulated and used in the form of. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain at least one excipient, for example, starch, calcium carbonate, glucose, It can be prepared by mixing sucrose or lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium styrate and talc may also be used. Liquid preparations for oral administration may include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents. can be included Formulations for parenteral administration include sterilized aqueous solutions, water-insoluble agents, suspensions, emulsions, and lyophilized preparations. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous preparations and suspensions, but are not limited thereto. Penetrants suitable for formulation for intranasal administration are generally known to those skilled in the art. Such suitable formulations are preferably formulated to be sterile, isotonic and buffered for stability and compliance. Formulations for intranasal administration are also formulated to stimulate mucus secretion in several ways to maintain normal ciliary function, as described in Remington's Pharmaceutical Science, 18th Ed., Mack Publishing Co., Easton, PA (1990). As noted, suitable formulations are preferably isotonic, slightly buffered formulations maintaining a pH of 5.5 to 6.5, and most preferably contain antimicrobial preservatives and suitable drug stabilizers.

The vaccine composition is a stabilizer, emulsifier, aluminum hydroxide, aluminum phosphate, pH adjuster, surfactant, liposome, iscom adjuvant, synthetic glycopeptide, bulking agent, carboxypolymethylene, subviral particle adjuvant, cholera toxin , N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide, Marcol-Aracel, Chlorhexidine and mixtures thereof. Any one or more second adjuvants selected from the group consisting of may be further contained. Preferably any one selected from the group consisting of MONTANIDE ISA70 (VG), ISA71 (VG), ISA71R (VG), ISA206 (VG), ISA201 (VG), ISA763 (A VG), IMS1313 (VG N) and GEL01 may be used, and most preferably, MONTANIDE ISA70 (VG) may be used as an adjuvant.

The composition of the present invention can be administered orally or parenterally (for example, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally or topically) depending on the desired method, preferably attenuated SG live cell vaccine. It may be administered orally or intramuscularly, but is not limited thereto. In addition, the dosage of the composition varies depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease of the chicken. The dose of the improved Salmonella gallina room inactivated killed cell vaccine composition treated with the culture medium containing plantarisin of the Lactobacillus plantarum is about 3×10 ⁷ to 3×10 ¹¹ cells per chicken, preferably about 3× It is preferable to administer once or twice with 10 ⁸ to 3×10 ¹⁰ cells, more preferably 3×10 ⁹ cells.

The poultry may be any one selected from the group consisting of chicks, chickens, ducks, turkeys, quails, waterfowl, pigeons and canaries, and the vaccine composition may be inoculated into growing poultry.

The present invention in order to achieve another object, the above-described improved Salmonella gallina room (SG) inactivated dead cells; It provides a feed additive for preventing or improving poultry typhus containing at least one selected from the group consisting of a culture thereof and a mixture thereof.

The feed additive of the present invention uses the above-described improved Salmonella gallina room (SG) inactivated dead cells or a mixture thereof as it is or additionally known carriers such as grains and their by-products acceptable for livestock, stabilizers, etc. Can be added and, if necessary, organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acidic pyrophosphate, polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, Natural antioxidants such as rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid, antibiotics, antibacterial agents, and other additives may be added. It may be in a state, and when supplying the feed additive, it may be supplied alone or mixed with feed for livestock.

The above-described rock-improved Salmonella gallina room (SG) inactivated killed cells; It is possible to provide a method for preventing poultry typhus, comprising feeding or inoculating poultry with at least one selected from the group consisting of a culture thereof and a mixture thereof.

Hereinafter, the present invention will be described in more detail through examples. The following examples are only preferred specific examples of the present invention, and the scope of the present invention is not limited to the scope of the following examples.

Example 1. Bacterial strains, plasmids and growth conditions

The poultry typhus bacteria used in this study was Salmonella enterica Serovar Gallinarum (SG) isolated from brown leghorn species killed by poultry typhus in chickens, and the Salmonella enterica Serovar Gallinarum (SG) was Lactobacillus plantarum In addition to the preparation of the inactivated dead cell vaccine of Salmonella galinarum (SG), which was improved by treatment with the sour-containing culture medium, it was also used as all brown leghorn challenge infection strains after vaccination. In addition, it was also used as a strain for extracting outer membrane proteins (OMPs) to be used as a coating antigen to measure antibody titers after vaccination. All strains were cultured in LB broth, LB agar, and BGA agar supplemented with 5% fetal bovine serum.

Example 2. Preparation of improved Salmonella gallina room (SG) inactivated killed cell vaccine treated with plantaricin-containing culture medium of Lactobacillus plantarum

Lactobacillus plantarum was inoculated with 1% of the culture medium pre-cultured in MRS medium and cultured for 24 hours at 37 ° C. at 150 rpm. The cultured liquid was centrifuged at 3,000 × g for 20 minutes to collect the supernatant, and then filtered through a 0.2 um filter to eliminate bacteria. Salmonella gallina room (SG) was cultured for 24 hours at 150 rpm at 37°C. The cultured liquid was centrifuged at 3,000 × g for 20 minutes to remove the supernatant, and Salmonella gallina room (SG) was collected. The collected Salmonella gallina room (SG) was treated with Lactobacillus plantarum culture solution in the same amount as the culture medium at 37 ° C. for 1 hour to 8 hours to obtain dead cells of Salmonella gallina room (SG). The thus obtained Salmonella gallina room (SG) killed cells were concentrated by centrifugation at 2,000 × g for 30 minutes, washed three times with sterile PBS, and finally 1 × 10 ⁹ CFUs / ml, 1 × 10 ¹⁰ using ISA70 It was diluted to CFUs/ml and 1×10 ¹¹ CFUs/ml, respectively, and used in experiments. After the reaction, in order to confirm the improved inactivation, 100 μl of each reaction solution was inoculated into LB agar medium and broth supplemented with 5% fetal bovine serum, and cultured at 37 ° C for 7 days to determine whether or not colonies were formed. Inactivation of the reaction solution was confirmed. The identified inactivation reaction solution was concentrated by centrifugation at 4000 × g for 30 minutes, washed three times with sterile PBS, and finally diluted to 3 × 10 ⁹ cells / 200 μl using ISA70, respectively, and used in the experiment. .

Example 3. Formalin-treated Salmonella gallina room ( Salmonella enterica Serovar Gallinarum; SG) Manufacture of Inactivated Killed Cell Vaccine

One colony of the SG strain was selected and inoculated into 200 mL of LB liquid medium and cultured at 37°C until the absorbance reached 0.6. Formalin was added to the cultured medium to be 0.3%, followed by reaction at 37°C for 48 hours. After the reaction, 100 μl of each reaction solution was inoculated into BGA Agar medium and broth supplemented with 5% fetal bovine serum to confirm whether or not the cells were killed after the reaction, and incubated at 37 ° C for 5 days to confirm the presence or absence of colony formation. . Formalin-inactivated Salmonella galinarum The dead cells were concentrated by centrifugation at 4,000 rpm for 30 minutes, washed three times with sterile PBS, and finally mixed with ISA70 and diluted to 3 × 10 ⁹ cells / 200 μl to form a formalin-inactivated Salmonella Galina room dead cell vaccine. used

Example 4. Scanning electron microscopy (SEM) of inactivated killed cells of improved Salmonella gallina room (SG) treated with a culture medium containing plantaricin of Lactobacillus plantarum

The improved Salmonella gallina room (SG) inactivated killed cells prepared by the method of Example 2 were pre-fixed with 2.5% glutaraldehyde overnight before mixing with ISA70 adjuvant, and then the bacterial pellet was washed three times with PBS and 2 They were post-fixed with 1% osmium tetroxide in PBS for a period of time. The fixed bacterial cells were washed three times with PBS, dehydrated in various series of ethanol (50%, 70%, 90%, 100%) for 15 min, coated with platinum, and imaged by scanning electron microscopy. As shown in Figure 1A, Salmonella galinarum (SG), which was not treated with the plantaricin-containing culture medium of Lactobacillus plantarum, was observed with intact cell membranes and cell contents filling the cells (FIG. 1A). On the other hand, Salmonella gallina room (SG) inactivated by the plantaricin-containing culture medium of Lactobacillus plantarum was observed with empty cytoplasmic space along with a cell membrane with one or more holes, as shown in FIG. 1B.

Example 5. ELISA for vaccination and sample collection and immune response evaluation

Seventy-five five-week-old brown leghorns were purchased and used in this study, and all brown leghorns were transferred to a barn where breeding was possible by group. In addition, they were fed regular chicken feed without the addition of antibiotics or growth promoters. A total of 75 brown leghorns were divided into 5 groups (15 in each group). Brown leghorns in all groups were vaccinated intramuscularly (once at 6 weeks of age and twice at 9 weeks of age). Brown leghorns of groups A and B were inoculated twice intramuscularly with sterile PBS as negative and positive controls, respectively, and group C was treated with a culture solution containing plantarisin of Lactobacillus plantarum of Example 2, improved Salmonella galina room. (SG) Inactivated dead cell 3X10 ⁹ cells/chicken and ISA70 adjuvant were inoculated twice intramuscularly with a mixture of 3:7 each, and group D brown leghorn species prepared in Example 3 Formalin-Salmonella Galina room inactivated A vaccine containing a mixture of 3X10 ⁹ cells/chickens and ISA70 adjuvant at a ratio of 3:7 was injected intramuscularly twice. For group E brown leghorn species, a commercially available attenuated SG9R live attenuated vaccine was administered subcutaneously according to the manufacturer's recommended dose. Inoculated once. Blood samples were collected at 0 weeks post prime immunization (WPPI) (before the 1st vaccination), 3 WPPI (before the 2nd vaccination), and 6 WPPI (before challenge infection). All specimens were stored at -70 ° C until use and used in the experiment. The animal experiments reported in this experiment were conducted with the approval of the Animal Ethics Committee of Chonbuk National University, which was authorized by the Korean Council on Animal Care. To examine SG OMPs-specific antibody titer in serum To examine SG OMPs-specific antibody titer in serum, a poultry IgG (Bethyl Lab Inc. Montgomery, TX, USA) ELISA quantitation kit was used according to the manufacturer's instructions. For serum titer, serum was diluted 1:200 and used. The antibody titer of serum IgG of each chicken against SG OMPs in vaccinated brown leghorn is shown in FIG. 2 . A significant increase in antibody titer was observed in brown leghorn chickens of all vaccination groups compared to the control group 3 weeks after vaccination (FIG. 2).

Example 6. Cytokine measurement of splenocytes

On day 10 after the second inoculation, 5 chickens from each group were sacrificed to collect the spleens, and the spleens were harvested aseptically. Splenocytes were prepared according to the method described in a previous study by Hur et al. (Hur et al., 2016). That is, spleen were aseptically collected from 5 chickens in each group on the 10th day after the last vaccination and collected in RPMI 1640. Then, red blood cells were lysed using 0.8% ammonium chloride (w/v), centrifuged at 380×4° C. for 10 minutes, and the precipitate was washed three times with sterile PBS. After the last centrifugation, it was resuspended in complete medium (RPMI 1640 containing 100 IU/ml penicillin, 100 ug/ml streptomycine and 10% FCS). After calculating the number of cells for culture and reacting with SG OMPs for 48 hours, centrifugation at 380 × 4 ° C for 10 minutes, and the supernatant was stored at -80 ° C and the cytokine concentration was measured by ELISA. As shown in Figure 3, the group inoculated with the improved Salmonella gallina room (SG) inactivated dead cell vaccine treated with the plantaricin-containing culture medium of Lactobacillus plantarum and the group inoculated with the commercially available SG9R live cell vaccine compared to the control group. A significant increase in IFN-r was observed. However, in the group inoculated with formalin-inactivated dead cells, although there was an increase compared to the control group, no significant increase was observed (FIG. 3).

Example 7. Measurement of survival rate after challenge infection

For the challenge experiment, Salmonella gallina room (SG) HJL456, a virulence strain outdoors, was prepared according to the method described by Hur et al. (Hur et al., 2011). All chickens were challenged by oral inoculation with 2×10 ⁹ CFU at 6 WPPI orally. Thereafter, all chickens were observed for mortality twice daily for 14 days after challenge. As shown in FIG. 4, the control group began to die 5 days after the challenge infection, ^and 100% of the brown leghorn species died on the 11th day. Of the chickens in the group inoculated by mixing at a ratio of :7, only 40% of the brown leghorns survived on the 14th day, the end of the experiment, after one bird died on the 8th day of challenge infection. However, improved Salmonella gallina room (SG) inactivated killed cells 3X10 ⁹ cells/chicken treated with plantaricin-containing culture medium of Lactobacillus plantarum and ISA70 adjuvant were mixed at a ratio of 3:7, respectively, and injected intramuscularly twice In one group and the group inoculated with commercially available SG9R live cells, 100% survived up to 14 days after challenge infection (FIG. 4).

[Accession number]

Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14384BP

Entrusted date: 20201123

Claims (9)

Salmonella Gallinarum prepared by reacting with a culture solution containing plantarisin of Lactobacillus plantarum deposited under accession number KCTC18645P, salmonella gallinarum deposited under accession number KCTC14384BP is inactivated bacterial strains.
(a) inoculating Salmonella enterica Serovar Gallinarum into a culture medium and then culturing;
(b) mixing and reacting a culture solution containing plantarisin of Lactobacillus plantarum deposited with accession number KCTC18645P in the cultured Salmonella gallina room;
(c) inoculating and culturing the reacted mixture in LB broth containing fetal bovine serum (FBS); and
(d) checking the culture for colony formation;
A method for preparing dead cells in which Salmonella Gallinarum is inactivated, deposited with accession number KCTC14384BP, including a.
Salmonella gallina room inactivated dead cells deposited under the improved accession number KCTC14384BP of claim 1; A vaccine composition for preventing or treating poultry typhus, comprising at least one selected from the group consisting of a culture thereof and a mixture thereof.
The vaccine composition according to claim 3, further comprising at least one selected from the group consisting of a carrier, a diluent and an adjuvant.
5. The method of claim 4, wherein the adjuvant is aluminum hydroxide, ISA70 (VG), ISA71 (VG), ISA71R (VG), ISA206 (VG), ISA201 (VG), ISA763 (A VG), IMS1313 (VG N) and GEL01 Any one selected from the group consisting of, vaccine composition.
The vaccine composition according to claim 3, wherein the vaccine composition is administered by one or more routes selected from the group consisting of oral, transdermal, intramuscular, intraperitoneal, intradermal, subcutaneous and nasal routes.
The vaccine composition for preventing or treating poultry typhus according to claim 3, wherein the poultry is any one selected from the group consisting of chicks, chickens, ducks, turkeys, quails, waterfowl pigeons and canaries.
Salmonella gallina room inactivated dead cells deposited under the improved accession number KCTC14384BP of claim 1; A feed additive for preventing or improving poultry typhus, comprising at least one selected from the group consisting of a culture thereof and a mixture thereof.
Salmonella gallina room inactivated dead cells deposited under the improved accession number KCTC14384BP of claim 1; A method for preventing poultry typhus comprising feeding or inoculating poultry with at least one selected from the group consisting of a culture thereof and a mixture thereof.

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