KR20220075555A - 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 typhus in poultry, comprising an improved Salmonella gallinarum inactivated dead cell by a plantaricin-containing culture medium of Lactobacillus plantarum.

Description

Vaccin composition for preventing or treating poultry typhus comprising inactivated Salmonella gallinarum dead cells reacted with a culture solution containing plantaricin of Lactobacillus plantarum {Vaccin 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 the prevention or treatment of poultry typhus comprising inactivated dead cells of Salmonella gallinarum 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 it mainly causes sepsis in brown leghorn breeds among chickens, and it occurs acutely and chronically. It is a major intracellular parasitic bacterial disease. Also, poultry typhus occurs mainly in middle-aged and mature chickens rather than young chicks.

The causative agent of poultry typhus, S. Gallinarum is an intracellular parasitic bacterium, and a dead cell vaccine made with formalin, etc., which is traditionally manufactured, is characterized by very low protective efficiency. That is, the cellular immune response protects against poultry typhus, and for this purpose, live attenuated vaccine is considered the most ideal vaccine. For example, Korean Patent Publication No. 10-2012-0128583 discloses a non-pathogenic Salmonella gallinarum mutant 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 for the prevention of poultry typhus in poultry farms worldwide since the 1960s. However, in the case of the live vaccine disclosed above, it is a GMO whose 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 the level of industrialization, and its safety is low. It has not been answered. In addition, in the case of live vaccines, there are concerns about a return to a sickly strain by recovering the hospital history. There can be many situations where this completely does not work. Therefore, it is necessary to develop a vaccine that is as safe as a dead cell vaccine and has excellent efficacy by inducing a cellular immune response like a live vaccine.

Lactic acid bacteria are bacteria that ferment sugar and produce lactic acid. They produce useful substances that help our body stay healthy, and produce immune-enhancing, antioxidant, antibacterial, anti-cholesterol, anti-obesity, carcinogenic and mutant substances. It is known to have a function to prevent Among them, the Lactobacillus plantarum strain, a type of lactic acid bacteria contained in kimchi, is a lactic acid bacillus that performs homozygous or heterozygous fermentation, and is commonly found in the intestinal tract of animals, including humans, and in the fermentation process of dairy products or vegetables. The microorganisms of the genus Lactobacillus are known to produce several proteinaceous antibacterial substances, and by maintaining the intestinal pH at an acidic level, it suppresses the reproduction of harmful bacteria such as E. coli and Clostridium, and relieves diarrhea and constipation. It is known to play a role in vitamin synthesis, anticancer action, and lowering serum cholesterol as well as improving.

Under this background, five types of plantaricin peptides, which are novel antibacterial peptides isolated from the Lactobacillus plantarum strain derived from kimchi, and the well-known plantaricin N, F/E are expressed as well as lysozyme, It has excellent antibacterial activity by more than 9 types of AMPs (antimicrobial peptides; bacteriocins) such as MazF and holin, and among them, the main antibacterial activity is induced by 7 plantaricins, which are It is suggested to act on the cell membrane to form a hole. Using this property, Salmonella gallinarum, the causative bacteria of poultry typhus, was induced as inactivated dead cells. Salmonella gallinarum-induced inactivated dead cells 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 the Gallinarum strain had a perfect protective effect after the challenge-infected strain.

The present invention has been devised to solve the above problems, the present inventors reacted with a culture solution containing plantaricin of Lactobacillus plantarum to induce inactivation of the Salmonella Gallinarum strain, thereby improving Salmonella Galinarum inactivated dead cells were prepared, and the vaccine containing the improved Salmonella gallinarum inactivated cells induces a cellular immune response like a live vaccine in poultry typhus By confirming that it has an excellent protective effect, the present invention was completed.

It is an object of the present invention to provide a Salmonella Gallinarum (Salmonella Gallinarum ) inactivated dead cell strain prepared by reacting with a culture solution containing plantaricin of Lactobacillus plantarum in Salmonella Gallinarum.

Another object of the present invention is to provide a method for preparing an improved Salmonella enterica Serovar Gallinarum inactivated dead cells comprising the following steps:

(A) Salmonella gallinarum ( Salmonella enterica Serovar Gallinarum) inoculated in the medium and then culturing;

(b) reacting the cultured Salmonella galinarum by mixing a culture solution containing plantaricin of Lactobacillus plantarum;

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

(d) confirming the presence or absence of colony formation in the culture.

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-mentioned Salmonella Gallinarum inactivated dead cell strain and a mixture thereof. will be.

Another object of the present invention is the above-mentioned Salmonella Gallinarum (Salmonella Gallinarum) inactivated dead cell strain; It is to provide a feed additive for preventing or improving poultry typhus comprising any one or more selected from the group consisting of a culture thereof and a mixture thereof.

Another object of the present invention is the above-mentioned Salmonella Gallinarum (Salmonella Gallinarum) inactivated dead cell strain; It is to provide a method for preventing poultry typhus comprising; feeding or inoculating poultry with any one or more 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 Salmonella Gallinarum ( Salmonella Gallinarum ) Lactobacillus plantarum in Lactobacillus plantarum Plantaricin prepared by reacting with a culture medium containing a lysin Salmonella gallinarum (Salmonella Gallinarum) inactivation A bacterial strain can be provided.

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

(A) Salmonella gallinarum ( Salmonella enterica Serovar Gallinarum) inoculated in the medium and then culturing;

(b) reacting the cultured Salmonella galinarum by mixing a culture solution containing plantaricin of Lactobacillus plantarum;

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

(d) confirming the presence or absence of colony formation in the culture.

The present invention can provide a vaccine composition for preventing or treating poultry typhus comprising any one or more selected from the group consisting of a culture of the above-mentioned 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 carriers, diluents and adjuvants.

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 any 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, waterfowl pigeons and canaries.

The present invention is the above-mentioned Salmonella Gallinarum (Salmonella Gallinarum) inactivated dead cell strain; It is possible to provide a feed additive for preventing or improving poultry typhus comprising any one or more selected from the group consisting of a culture thereof and a mixture thereof.

The present invention is the above-mentioned Salmonella Gallinarum (Salmonella Gallinarum) inactivated dead cell strain; It can provide a method for preventing poultry typhus, including; feeding or inoculating poultry with any one or more selected from the group consisting of a culture thereof and a mixture thereof.

Improved by the plantaricin-containing culture medium of Lactobacillus plantarum of the present invention After inoculating the inactivated dead cells of Salmonella enterica Serovar Gallinarum into chicken muscles, challenge infection was performed with Salmonella gallinarum, an outdoor poisoning strain. As a result, the untreated control group started to die from the 5th day On the 11th day of infection, all chickens died, and a 30% survival rate was observed in the group inoculated with 3×10 ⁹ CFU/200 μl of formalin-inactivated dead cells, but the improved Salmonella gallinarum inactivated death of the present invention A survival rate of 100% was observed in the group inoculated with 3×10 ⁹ CFU/200 μl of the cells, the group inoculated with SG9R, a commercially available live cell vaccine, and the group that did not perform the challenge infection. Prepared by reacting with a culture solution containing plantaricin When the Salmonella gallinarum inactivated dead cell vaccine is intramuscularly inoculated, it has an excellent protective effect like a commercially available live attenuated vaccine, thereby preventing or treating typhus in poultry. In addition, in the case of a commercially available live vaccine, a subcutaneous inoculation method is used, which is difficult to inoculate, requires inoculation by a professional, and is not well inoculated, whereas the Lactobacillus plantarum plantaricin of the present invention is difficult to inoculate. improved by the containing culture medium Salmonella gallinarum inactivated dead cells vaccine can be administered intramuscularly or orally, so anyone can easily inoculate it, and it has a good inoculation effect.

1 is an electron microscope of (A) Lactobacillus plantarum plantaricin-containing culture untreated group and (B) Lactobacillus plantarum plantaricin-containing culture solution treated group in Salmonella Gallinarum of the present invention; This is the photo result.
Figure 2 shows each antigen [control (PBS); live vaccine (SG9R); formalin-inactivated dead cells 3×10 ⁹ ; An improved solution prepared with a plantaricin-containing culture solution of Lactobacillus plantarum This is the result of the 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 ; An improved solution prepared with a plantaricin-containing culture solution of Lactobacillus plantarum IFN-r concentration results in plenocytes against Salmonella Gallinarum inactivated dead cells 3×10 ⁹ ].
4 shows each vaccination group [control (PBS); live vaccine (SG9R); formalin-inactivated dead cells 3×10 ⁹ ; An improved solution prepared with a plantaricin-containing culture solution of Lactobacillus plantarum Salmonella Gallinarum ( Salmonella Gallinarum ) Inactivated dead cells 3×10 ⁹ ] is a survival rate result 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, especially brown leghorns, and occurs acutely and chronically, causing enormous economic damage to poultry farmers due to high mortality. It is one of the major intracellular parasitic bacterial diseases. Many studies are being conducted to prevent or treat the poultry typhoid bacteria. In order to most effectively prevent it, the 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 disadvantages such as slow growth rate and insufficient protective efficiency still exist (Bouzoubaa et al., 1989; Kwon and Cho, 2011). In addition, the domestic poultry breeding environment is characterized by the frequent use of antibiotics to prevent bacterial diseases due to frequent diarrhea and decreased appetite due to stress on laying hens due to interseasonal seasons, hot summers, and cold winters due to the four distinct seasons. has an unsuitable breeding environment for its protective efficacy. Therefore, the development of an inactivated dead cell vaccine that is safer and more effective than the currently marketed SG9R is being actively developed. The present inventors reacted with a plantaricin-containing culture solution of Lactobacillus plantarum to Salmonella enterica Serovar Gallinarum (SG) isolated from a dead brown leghorn species to induce improved Salmonella enterica Serovar Gallinarum (SG) inactivation and death, As a result of intramuscular inoculation of the improved Salmonella enterica Serovar Gallinarum (SG) inactivated dead cells prepared with 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 an improved Salmonella Gallinarum (Salmonella Gallinarum ) inactivated dead cells prepared by reacting Salmonella Gallinarum with a culture solution containing plantaricin of Lactobacillus plantarum.

Researchers of the present invention, after culturing Salmonella enterica Serovar Gallinarum isolated from laying hens showing poultry typhoid symptoms, induce inactivation with a plantaricin-containing culture medium of Lactobacillus plantarum, improved Salmonella Gallinarum inactivation A dead cell was prepared. As a result of confirming the Salmonella Gallinarum inactivated dead cells using a projection electron microscope, an empty cytoplasmic space was observed along with a perforated cell membrane in the Salmonella Gallinarum inactivated dead cells. Accordingly, it was confirmed that inactivation of Salmonella Gallinarum was induced and became inactivated dead cells (FIG. 1B).

In order to obtain a plantaricin-containing culture solution of the Lactobacillus plantarum, one Lactobacillus plantarum colony was inoculated into 10ml of MRS medium and cultured overnight, then added to a new MRS medium so as to be 1%, and cultured for 24 hours to 3,000 x g was centrifuged for 20 minutes, the supernatant was collected, filtered through a 0.2um filter, and used as a Salmonella gallinarum inactivation deadening inducing material, but is not limited thereto.

The Salmonella gallinarum ( Salmonella enterica Serovar Gallinarum) may be deposited as microorganism accession number KCTC1438BP (microorganism name: Salmonella Gallinarum (Fowl typhoid)).

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

The Lactobacillus plantarum may contain various antibacterial substances as one of the lactic acid bacteria, and one of them is plantaricin, which has a broad antibacterial spectrum and has sterilizing power against gram-negative or gram-positive bacteria. Salonella gallinarum can be used as an improved inactivated dead cell inducer.

The present invention may provide a method for preparing an inactivated dead cell of Salmonella enterica Serovar Gallinarum comprising the steps of:

(A) Salmonella gallinarum ( Salmonella enterica Serovar Gallinarum) inoculated in the medium and then culturing;

(b) reacting the cultured Salmonella galinarum by mixing a culture solution containing plantaricin of Lactobacillus plantarum;

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

(d) confirming the presence or absence of colony formation in the culture;

Containing, Salmonella Gallinarum ( Salmonella Gallinarum) method for producing inactivated dead cells.

The present invention is the above-mentioned improved Salmonella gallinarum inactivated dead cells; It provides a vaccine composition for preventing or treating salmonellosis caused by Salmonella gallinarum, comprising any one or more selected from the group consisting of a culture thereof and a mixture thereof.

The present inventors inoculated the improved Salmonella enterica Serovar Gallinarum inactivated dead cells prepared by reacting Salmonella gallinarum with a culture solution containing plantaricin of Lactobacillus plantarum into brown leg hybrid muscle, improvement The antibody titer of serum IgG of all brown leghorn species in the inactivated dead cell inoculation group, formalin-inactivated dead cell inoculation group, and commercial live cell inoculation group was significantly increased compared to the control group (Fig. 2), and the cellular immune response was In the representative IFN-r, a significant increase was observed compared to the control group in the brown Leghorn species of the improved inactivated dead cell inoculation group and the commercially available live vaccine, whereas the formalin-inactivated dead cell inoculation group compared to the control group. No significant increase was observed ( FIG. 3 ). In addition, as a result of challenge infection with Salmonella Salmonella gallinarum (SG), an outdoor poisonous strain, the untreated control group started to die on the 5th day, and all chickens died on the 11th day of the challenge infection, and formalin-inactivated inactivation The group inoculated with 3×10 ⁹ CFU/200 μl of dead cells showed a survival rate of 30%, but the group inoculated with 3×10 ⁹ CFU/200 μl of the improved Salmonella gallinarum inactivated dead cells of the present invention, A survival rate of 100% was observed in the group inoculated with SG9R, a commercially available live cell vaccine, and in the group not subjected to challenge infection (FIG. 4). When the improved inactivated dead cell vaccine of the present invention is intramuscularly inoculated, there is an excellent protective effect, thereby preventing or treating poultry typhus caused by Salmonella gallinarum.

As used herein, 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 substance that induces immunity in a living body by injecting or oral administration to a person or animal for the prevention of infection. In vivo immunity is largely divided into automatic immunity, which is obtained automatically after infection with a pathogen, and passive immunity, which is obtained by an externally injected vaccine. Autoimmune has a long period of generation of immunity-related antibodies and has a characteristic of continuous immunity, whereas passive immunity by vaccine acts immediately on the treatment of infection, but has a disadvantage in that the durability is lowered.

The vaccine composition may further include any one or more selected from the group consisting of carriers, diluents and adjuvants.

In addition, the vaccine composition may include a veterinary acceptable carrier. As used herein, the term "veterinary 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.

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

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 It may further contain any one or more second adjuvants selected from the group consisting of. 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 can be used, and most preferably, MONTANIDE ISA70 (VG) can be used as an adjuvant.

The composition of the present invention may be administered orally or parenterally (eg, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally or topically) according to a desired method, preferably live attenuated SG vaccine may be administered orally or intramuscularly, but is not limited thereto. In addition, the dosage of the composition varies according to the weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease of the chicken of the chicken. The dose of the Lactobacillus plantarum plantaricin-containing culture medium treatment improved Salmonella gallinarum dead cell vaccine composition is about 3×10 ⁷ to 3×10 ¹¹ cells per chicken, preferably about 3× 10 ⁸ to 3×10 ¹⁰ cells, more preferably 3×10 ⁹ cells, is preferably administered once or twice.

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 to growing poultry.

The present invention to achieve another object, the improved Salmonella gallinarum (SG) inactivated dead cells described above; It provides a feed additive for preventing or improving poultry typhus comprising any one or more selected from the group consisting of a culture thereof and a mixture thereof.

The feed additive of the present invention uses the above-mentioned improved Salmonella gallinarum (SG) inactivated dead cells or a mixture thereof as it is, or additionally known carriers such as grains and by-products allowed for livestock, stabilizers, etc. can be added. If necessary, organic acids such as citric acid, humic acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid 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. In the case of supplying the feed additive, it may be supplied alone or mixed with feed for livestock and the like.

The above-mentioned lock-improved Salmonella gallinarum (SG) inactivated dead cells; It can provide a method for preventing poultry typhus, including; feeding or inoculating poultry with any one or more 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 embodiments 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 typhoid bacteria used in this study was Salmonella enterica Serovar Gallinarum (SG) isolated from a brown leghorn species that died from poultry typhus in chickens, and the Salmonella enterica Serovar Gallinarum (SG) was Lactobacillus plantarum plantari. In addition to the preparation of the improved Salmonella gallinarum (SG) inactivated dead cell vaccine, it was also used as all brown leg hybrid challenge-infected strains after vaccination. In addition, it was also used as a strain for extracting outer membrane proteins (OMPs) for use as a coating antigen for measuring antibody titer after vaccination. All strains were cultured in LB broth supplemented with 5% fetal bovine serum, LB agar, and BGA agar.

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

Lactobacillus plantarum was inoculated with 1% of the pre-cultured medium in MRS medium, and cultured at 37° C. at 150 rpm for 24 hours. The cultured solution 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 gallinarum (SG) was cultured at 37°C at 150 rpm for 24 hours. The cultured solution was centrifuged at 3,000 × g for 20 minutes to remove the supernatant, and then Salmonella gallinarum (SG) was collected. The collected Salmonella gallinarum (SG) was treated with the Lactobacillus plantarum culture treatment solution in the same amount as the culture solution at 37° C. for 1 hour to 8 hours to obtain a dead cell body of Salmonella gallinarum (SG). The Salmonella gallinarum (SG) dead cells thus obtained 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 CFUs/ml and 1×10 ¹¹ CFUs/ml were respectively diluted and used in the experiment. In order to check whether the inactivation was improved after the reaction, 100 μl of each reaction solution was inoculated into LB agar medium and broth supplemented with 5% fetal bovine serum, respectively, and cultured at 37° C. for 7 days to determine whether colonies were formed or not. It was checked whether the reaction solution was inactivated. The confirmed 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 and used in the experiment. .

Example 3. Formalin-treated Salmonella galinarum ( Salmonella enterica Serovar Gallinarum; SG) Preparation of inactivated dead cell vaccine

One colony of the SG strain was selected and inoculated into 200 mL of LB liquid medium, and then cultured at 37°C until absorbance reached 0.6. Formalin was added to the cultured medium at a concentration of 0.3% and reacted at 37°C for 48 hours. After the reaction, in order to check whether the reaction solution is dead, 100ul of each reaction solution was inoculated into BGA Agar medium and Broth supplemented with 5% fetal bovine serum, respectively, and cultured at 37°C for 5 days to check whether the reaction solution was killed by 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, diluted to 3×10 ⁹ cells/200 μl, and used as a formalin-inactivated Salmonella gallinarum dead cell vaccine. was used.

Example 4. Scanning electron microscope (SEM) of improved Salmonella gallinarum (SG) inactivated dead cells treated with plantaricin-containing culture medium of Lactobacillus plantarum

After pre-fixing the improved Salmonella gallinarum (SG) inactivated dead cells prepared by the method of Example 2 with 2.5% glutaraldehyde overnight before mixing with ISA70 adjuvant, the bacterial pellet was washed 3 times with PBS and 2 After fixing with 1% osmium tetroxide in PBS for hours. The fixed bacterial cells were washed three times with PBS, dehydrated in various series of ethanol (50%, 70%, 90%, 100%) for 15 minutes, and then coated with platinum and photographed by scanning electron microscopy. As shown in FIG. 1A , in Salmonella gallinarum (SG), which was not treated with the plantaricin-containing culture medium of Lactobacillus plantarum, intact cell membranes and cell contents filled inside the cells were observed ( FIG. 1A ). On the other hand, Salmonella galinarum (SG) inactivated by the plantaricin-containing culture medium of Lactobacillus plantarum was observed to have an empty cytoplasmic space with one or more perforated cell membranes as shown in FIG. 1B .

Example 5. ELISA for vaccination and specimen 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 that can be reared in groups. In addition, general chicken feed without antibiotics or growth promoters was fed. A total of 75 brown leghorns were divided into 5 groups (15 in each group). All groups of brown leghornoma were vaccinated intramuscularly (1 dose at 6 weeks of age, 2 doses at 9 weeks of age). Group A and B brown leghorn species were inoculated intramuscularly twice with sterile PBS as negative and positive controls, respectively, and group C was improved Salmonella galinarum treated with the plantaricin-containing culture solution of Lactobacillus plantarum of Example 2 (SG) Inactivated dead cells 3X10 ⁹ cells/chicken and ISA70 adjuvant were inoculated twice intramuscularly with a mixed vaccine at a ratio of 3:7, respectively, and group D brown leg hybrids were formalin-Salmonella galinarum inactivated prepared in Example 3 3X10 ⁹ cells/chickens and ISA70 adjuvant 3X10 9 cells/chickens were inoculated intramuscularly twice, respectively. For group E brown leghorn, the commercially available attenuated SG9R live attenuated vaccine was administered subcutaneously according to the manufacturer's recommended dose. It was inoculated once. Blood samples were collected at 0 weeks post prime immunization (WPPI) (before the 1st immunization), 3 WPPI (before the 2nd immunization), and 6 WPPI (before the challenge infection). All specimens were used in the experiment while being stored at −70° C. until use. The animal experiments reported in this experiment were carried out with the approval of the Chonbuk National University Animal Ethics Committee, which was approved by the Korean Council on Animal Care. To investigate SG OMPs-specific antibody titers in serum To investigate SG OMPs-specific antibody titers 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. Antibody titers of serum IgG of each chicken against SG OMPs in vaccinated brown leghorn breeds are shown in FIG. 2 . Brown Leghorn chickens of all vaccination groups showed a significant increase in antibody titers compared to the control group from 3 weeks after inoculation ( FIG. 2 ).

Example 6. Measurement of cytokines in splenocytes

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

Example 7. Measurement of survival rate after challenge infection

For the challenge experiment, Salmonella gallinarum (SG) HJL456, an outdoor toxic strain, was prepared according to the method described by Hur et al. (Hur et al., 2011). All chickens were challenged orally by oral inoculation with 2×10 ⁹ CFU at 6 WPPI days. After that, all chickens were observed for mortality twice a day for 14 days after challenge infection. As shown in FIG. 4 , the control group started to die 5 days after the challenge infection, and 100% of the brown leg hybrids died on the 11th day, and formalin-inactivated dead cells 3×10 ⁸ cells/mouse were used as adjuvants ISA70 and 3 After one chicken died on the 8th day of challenge in the group inoculated with a mixing ratio of :7, only 40% of the brown leghorn species survived on the 14th day, the end of the experiment. However, the improved Salmonella gallinarum (SG) inactivated dead cell 3X10 ⁹ cells/chicken treated with the plantaricin-containing culture medium of Lactobacillus plantarum was mixed with 3X10 9 cells/chicken and ISA70 adjuvant at 3:7 intramuscular inoculation twice. In one group and the group inoculated with commercially available live SG9R cells, 100% survived until 14 days after challenge infection (FIG. 4).

[ Deposit No.]

Name of depositary institution: Korea Research Institute of Bioscience and Biotechnology

Accession number: KCTC14384

Deposit date: 20201123

Claims (9)

Salmonella gallinarum ( Salmonella Gallinarum ) Lactobacillus plantarum in Lactobacillus plantarum was prepared by reacting with a culture solution containing plantaricin, Salmonella gallinarum (Salmonella Gallinarum) inactivated dead cell strain.
(A) Salmonella gallinarum ( Salmonella enterica Serovar Gallinarum) inoculated in the medium and then culturing;
(b) reacting the cultured Salmonella galinarum by mixing a culture solution containing plantaricin of Lactobacillus plantarum;
(c) inoculating the reacted mixture in LB broth containing fetal bovine serum (FBS) and culturing; and
(d) confirming the presence or absence of colony formation in the culture;
Containing, Salmonella Gallinarum ( Salmonella Gallinarum) method for producing inactivated dead cells.
The improved Salmonella gallinarum inactivated dead cells of claim 1; A vaccine composition for preventing or treating typhus in poultry, comprising any one or more selected from the group consisting of cultures and mixtures thereof.
The vaccine composition of claim 3, wherein the vaccine composition further comprises any one or more 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, a vaccine composition.
The vaccine composition of claim 3, wherein the vaccine composition is administered by any 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 typhus in poultry 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.
The improved Salmonella gallinarum inactivated dead cells of claim 1; A feed additive for preventing or improving poultry typhus, comprising any one or more selected from the group consisting of cultures and mixtures thereof. The improved Salmonella gallinarum inactivated dead cells of claim 1; A method for preventing poultry typhus, including; feeding or inoculating poultry with any one or more selected from the group consisting of a culture thereof and a mixture thereof.

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