KR20220127524A - A vaccine composition for preventing Fowl Typhoid comprising live attenuated and killed Salmonella gallinarum - Google Patents

Abstract

It relates to a vaccine composition for preventing poultry typhus comprising live and dead cells of the attenuated Salmonella gallinarum. The vaccine composition according to one aspect provides a live and dead toxin vaccine in which live and dead cells of the live and dead cells of the attenuated Salmonella vaccine are provided. do.

Description

A vaccine composition for preventing poultry typhoid comprising live and dead cells of attenuated Salmonella gallinarum {A vaccine composition for preventing Fowl Typhoid comprising live attenuated and killed Salmonella gallinarum}

It relates to a vaccine composition for preventing poultry typhus comprising live and dead cells of the attenuated Salmonella gallinarum.

Fowl typhoid is an important bacterial disease caused by Salmonella enterica subsp. enterica serovar Gallinarum biovar Gallinarum, S. Gallinarum, a morphologically Gram-negative bacillus. It is an acute and chronic infectious disease. The disease can appear at any age, and high mortality is caused by sepsis. Common symptoms of poultry typhus infection in chickens include acute death, enlarged liver, spleen, and kidneys, white necrotic spots and bronze liver, watery or mucinous yellow diarrhea, decreased egg production, etc. Sporadic mortality over long periods of time can occur at the time of onset.

Salmonella gallinarum, the causative agent of poultry typhus, has serologically D-type O-antigens, among them O1, O9 and O12. It is the same as Salmonella pullorum, so it is difficult to distinguish it by serological examination. In addition, non-specific reactions may occur due to infection of other Salmonella or other bacterial diseases in the test through plate aggregation reaction. Therefore, recently, serological tests through ELISA are common, but even if a rough strain in which a part of the O-antigen LPS (lipopolysaccharide) is lost is inoculated, some of the test results are detected as positive, which may cause confusion in monitoring. .

In Salmonella, H-antigen, which is generally a flagellate protein, stimulates the initial immune response of the host, but Salmonella gallinarum lacks H-antigen, so motor ability is lost and the immune response is delayed. As a result, the probability of systemic infection is high, and pathogenicity is stronger than that of general Salmonella. Salmonella can cause high fever, shock, and organ failure due to endotoxin that Gram-negative bacteria generally possess. SPI-2 (Salmonella Pathogenicity Island-2), a collection of pathogenic genes, is Secretion system (T3SS) is activated to help infiltration into endothelial cells. The spv locus present in the pathogenic plasmid of Salmonella not only exerts a toxic effect on host cells by inhibiting actin polymerization, but also plays a key role in pathogenicity by inhibiting autophage.

Typhoid poultry can be transmitted to other flocks through drinking water or feed through contact with infected birds and bacteria excreted in the feces of infected birds. Vertical transmission from an infected maternal line is also possible. Although poultry typhus occurs worldwide, in developed poultry countries such as Canada, the United States, and some European countries, no outbreak of poultry has been reported since the 1980s. However, the incidence of poultry typhus in Korea has gradually increased from the first report in 1992 until 2001, and the number of cases in 2017 was 121,495, 112,009 in 2018, and 101,204 in 2019, and continues to occur recently. In addition, the actual occurrence frequency is expected to be higher, and the economic damage is enormous, considering that the symptoms of poultry typhus in the stellar or white hen do not show severe symptoms and antibiotic treatment is performed in the field.

Several vaccines have been developed for the prevention of poultry typhus, and live attenuated vaccines such as SG9R and SR2-N6 are preferred. However, recently, in outdoor farms, the case of additional vaccination is increasing due to the incomplete protection of the live attenuated vaccine. to be.

Accordingly, the present inventors inoculated a vaccine composition in which live and dead cells of the attenuated poultry strain were inoculated. As a result, not only cellular immunity related to the defense of poultry typhus but also humoral immunity were strengthened, thereby effectively preventing and alleviating poultry typhus. By confirming that there is, the present invention was completed. As such, there has been no study on the protective efficacy of a vaccine in which live and dead cells of the poultry typhus attenuated vaccine are mixed.

One aspect is to provide a vaccine composition for preventing poultry typhus comprising live and dead cells of the attenuated Salmonella Gallinarum.

Another aspect is to provide a method for preventing poultry typhus comprising inoculating poultry with the vaccine composition.

One aspect provides a vaccine composition for preventing poultry typhus comprising live and dead cells of the attenuated Salmonella Gallinarum.

As used herein, the term "prevention" refers to any action that suppresses or delays poultry typhus infection by administration of the vaccine composition according to an aspect.

As used herein, the term "vaccine" refers to an immunogen or antigenic substance that generates immunity in a living body by injecting or oral administration to a person or animal for the prevention of infection as a biological preparation containing an antigen that gives immunity to the living body.

There are two types of vaccines: an attenuated vaccine in which pathogenicity is attenuated and an inactivated vaccine or killed vaccine in which a pathogen is completely killed. The attenuated vaccine is also referred to as an attenuated vaccine or a live vaccine, and the inactivated vaccine is also referred to as a dead cell or dead vaccine.

The vaccine composition according to one aspect may be characterized in that it contains both live and dead cells of the attenuated Salmonella gallinarum.

In the present specification, the term "live cell" may be used to include live vaccines or live vaccines, and the live cells are viruses that weaken the pathogenicity of bacteria or viruses and show no or reduced clinical symptoms of disease when administered to animals. it means The attenuated Salmonella gallinarum can be isolated by methods known in the art, for example, virus subculture.

In the present specification, the term "dead cells" may be used to include both dead cell vaccines or dead poison vaccines, and the dead cells are pathogenic inactive without heating bacteria or viruses or losing immunogenicity by chemicals such as formalin or phenol. it means done The inactivation may be performed without limitation as long as it is a method known in the art, and preferably, it may be inactivated by heating live attenuated Salmonella gallinarum.

In one embodiment, the dead cells of the attenuated Salmonella gallinarum may be inactivated by heating the live attenuated Salmonella gallinarum cells at 55 to 70 ℃, 30 minutes to 3 hours, preferably 60 to 70 ℃, It may be inactivated by heating for 1 hour 30 minutes to 2 hours 30 minutes, and most preferably may be inactivated by heating at 65° C. for 1 hour.

In one embodiment, the live and dead cells of the attenuated Salmonella galinarum may be included in the vaccine composition at 1 x 10 ⁶ cfu (colony forming unit) / to 1 x 10 ¹⁰ cfu / each, preferably 1 x 10 ⁷ cfu (colony forming unit) / may be included in 1 x 10 ⁹ cfu / horse. More preferably, live and dead cells of the attenuated Salmonella gallinarum may be included in the vaccine composition in a bacterial ratio of 1:100, for example, when the vaccine composition is inoculated to poultry, the attenuated Salmonella gallinarum Live cells may be inoculated at 1 x 10 ⁷ cfu/animal, and dead cells may be inoculated at 1 x 10 ⁹ cfu/animal, but the present invention is not limited thereto.

The dosage of the vaccine composition may vary depending on the weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease of the chicken, and may be administered once to several times.

In one embodiment, the live and dead cells of the attenuated Salmonella gallinarum may be mixed in a volume ratio of 1:1, but is not limited thereto.

In one embodiment, 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, preferably administered by intramuscular route may be, but is not limited thereto.

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

The carrier may be a veterinarily 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 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, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil.

In addition, the vaccine composition can be prepared according to a conventional method in oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, and nasal dosage forms such as drips or sprays, and sterile injection solutions. It can be formulated and used in the form. 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, sucrose Alternatively, it may be prepared by mixing lactose, gelatin, or the like.

In addition to simple excipients, lubricants such as magnesium stearate talc can also be used. As liquid formulations for oral administration, suspensions, internal solutions, emulsions, syrups, etc. can be used. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, and freeze-dried preparations. Examples of the non-aqueous preparation and the suspending agent 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 above, 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, MONTANIDE ISA70 (VG), ISA71 (VG), ISA71R (VG), ISA206 (VG), ISA201 (VG), ISA763 (A VG), IMS1313 (VG N) or GEL01 may be used, most preferably may use MONTANIDE ISA70 (VG) as an adjuvant.

Another aspect provides a feed additive comprising live and dead cells of Salmonella Gallinarum.

The feed additive contains live and dead cells of Salmonella gallinarum at the same time, as well as a protective effect against poultry typhus, as well as effectively induces both cellular and humoral immune responses of poultry. It can contribute to prevention and immunity enhancement.

As the feed additive, live and dead cells of Salmonella gallinarum may be used as they are, or known carriers, stabilizers, etc., such as grains and by-products allowed for poultry breeding, may be added in addition, and citric acid, humic acid, adipic acid may be added as necessary. , organic acids such as lactic acid and malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, polyphosphate (polyphosphate), polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, Natural antioxidants such as chitosan, tannic acid, and phytic acid, antibiotics, antibacterial agents and other additives may be added, and the shape may be in an appropriate state such as powder, granule, pellet, suspension, etc., and when the feed additive is supplied can be supplied alone or mixed with feed for poultry, etc.

Another aspect provides a method for preventing poultry typhus comprising inoculating poultry with the vaccine composition according to the aspect.

In one embodiment, the poultry may be any one selected from the group consisting of chickens, ducks, turkeys, geese, quails, pheasants and geese, but is not limited thereto.

Preferably, the poultry may be a chicken, and more preferably a chick under 3 weeks of age, but is not limited thereto.

A method for preventing poultry typhus according to an aspect is by inoculating poultry with the vaccine composition (eg, by intramuscular route), wherein the vaccine composition includes both live and dead cells of the attenuated Salmonella gallinarum Salmonella It is possible to effectively prevent poultry typhus by inducing a humoral immunity response as well as cell-mediated immunity to Galinarum.

The vaccine composition according to one aspect provides a live and dead toxin vaccine in which live and dead cells of the live and dead cells of the attenuated Salmonella vaccine are provided. do.

1 is a diagram showing the evaluation results of the humoral immune response measured using ELISA under various conditions. A-D are ELISA results using OmpA-N-L3(A), OmpA-N-L4(B), OMP-12(C) and Omp-ELISA(D), respectively, and SG9R is dead cell/oil emulsion adjuvant (OE). ), live and dead cells mixed (MX), or dead cells/PBS suspension (PS) conditions, and the results were compared with the negative control (CONT). * indicates a significant difference compared to the negative control.

Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Example 1. Preparation of live and dead poison mixed vaccine

1-1. live vaccine

Vaccine strains were isolated from liver and feces of domestic poultry inoculated with SG9R, a live attenuated poultry vaccine. Specifically, the Salmonella gallinarum strain isolated from the practical world that was requested for disease evaluation in this laboratory was named SNU5161, and as a result of bacterial identification, SNU5161 was genetically identical to SG9R. The live attenuated vaccine strain was incubated in a liquid medium (LB: Difco, USA) at 150ml, 37°C for 16 hours, and then the CFU was measured and diluted with PBS to 1 x 10 ⁸ cfu/ml.

1-2. Production of heat-treated dead-venom vaccine and live-dead poison mixed vaccine

As in Example 1-1, the attenuated Salmonella gallinarum vaccine strain SNU5161 isolated from domestic poultry was cultured in a liquid medium at 150ml, 37°C for 16 hours. The cultured bacteria were centrifuged at 3000 rpm for 20 minutes, the supernatant was discarded, and the bacteria were concentrated in 5 ml of PBS. The colony forming unit (CFU) of the concentrated bacteria was measured, and inactivation was carried out by bathing so that the entire tube was immersed in a water bath at 65° C. for 2 hours. After 2 hours, the power of the water bath was turned off and cooled slowly with a hot water bath to prevent changes in protein structure due to sudden temperature change. When the temperature reached room temperature, 100 μl of the MHA medium was inoculated to ensure complete inactivation, and then the inactivated strain was diluted with PBS to 1 x 10 ¹⁰ cfu/ml.

A total of 200 μl was prepared by mixing the diluted live attenuated cells and dead cells prepared above in a 1:1 ratio. That is, when inoculated to chickens, live and dead cells were inoculated at 1 x 10 ⁷ cfu / horse and 1 x 10 ⁹ cfu / animal, respectively. In addition, in order to observe the effects of the live and dead poison mixed vaccine, as a comparative control, a dead poison vaccine (OE_killed) using an oil emulsion as an adjuvant and a dead poison vaccine only diluted in PBS (PS_killed) were prepared. The dead poison vaccine (PS_killed) diluted in PBS was prepared at 1 x 10 ⁹ cfu/animal, and the dead poison vaccine (OE_killed) using an oil emulsion as an adjuvant was diluted to 1 x 10 ¹⁰ cfu/ml and then ISA70 and 3:7 was mixed to obtain a final titer of about 3 x 10 ⁸ cfu/animal.

Example 2. Vaccination and Blood Collection

Salmonella gallinarum-negative 3-week-old brown chickens (chicks) were purchased, and 10 or 5 numbers were distributed to each group to constitute a total of 5 experimental and control groups. Dead poison vaccine (OE_killed), live attenuated vaccine (Live), mixed vaccine (Mix), dead poison vaccine diluted in PBS (PS_killed) and control (CONT) using the oil emulsion prepared in Example 1 as an adjuvant To evaluate the preventive effect of poultry typhus, the vaccine was intramuscularly inoculated, and serological tests were performed weekly for 3 weeks [1 week post injection (wpi) ~ 3wpi]. The dose and the number of chickens inoculated in each group are shown in Table 1 below.

Vaccine type menstruum Dose (cfu/animal) number of chickens inoculation route OE_killed PBS adjuvanted with ISA 70 3x10 ⁸ 10 muscle Live PBS 1x10 ⁷ 10 MIX PBS 1x10 ⁷ (Live)+1x10 ⁹ (PS_killed) 10 PS_killed PBS 1x10 ⁹ 5 CONT PBS - 5

Example 3. Vaccine antibody titer evaluation and comparison

In order to compare and evaluate the increase in antibody titer due to vaccination, ELISA (Enzyme-Linked Immunosorbent Assay) using an outer membrane protein as an antigen was performed. Two peptides (L3, L4) were prepared by synthesizing the amino acid sequence of a loop structure exposed on the surface of the fungus from OmpA, a protein known to have high immunogenicity in Salmonella, and Salmonella galinarum outer membrane protein (outer membrane protein: OMP) whole was extracted using sodium lauryl sarcosine. The amino acid sequences of the prepared synthetic peptides are shown in Table 2 below.

protein Peptide (N'-C') OmpA_N-terminal TKSNV PGGPS (OmpA_L3) TNNIG DANTI GTR (OmpA_L4)

The synthetic peptides of Table 2 were diluted to 1 μg/ml and the total OMP was 105 ng/ml, and 100 mM bicarbonate buffer (bicarbonate buffer, pH 9.6) was used at 4° C. overnight and coated on immunoplate Maxi (SPL, Korea). The ELISA plate coated with each antigen was washed twice with PBS (PBST) containing 0.5% Tween-20 and then blocked with 1% bovine serum albumin (BSA) at room temperature for 2 hours. The plate was washed in the same manner as above, and the primary antibody, ie, serum or bile, was diluted 1:300 in PBST containing 1% BSA, added to the plate, incubated for 30 minutes, and washed 4 times with PBST. After that, HRP-conjugated goat-anti chicken IgG or IgA antibody, which is an antibody that specifically binds to each antigen-binding antibody, was diluted 1:10,000 in PBST containing 1% BSA and added for 30 minutes, The plate was washed. After inducing color development by adding TMB substrate for 10 minutes, the color reaction was stopped with a stop solution, and absorbance was measured at 450 nm. The results are shown in FIG. 1 .

As shown in FIG. 1 , in ELISA using OmpA, it was confirmed that the antibody titer significantly increased in all of the OE_killed, Live, and Mix groups compared to the control group. The antibody titer fell as time passed after inoculation, but in the case of the Mix group, the antibody titer significantly increased compared to the control group in all sections after inoculation, and it was confirmed that the OD value was higher than that of the live attenuated cell inoculation alone (Live). (FIG. 1A, B). In the ELISA using OmpA_L4, it was confirmed that the Mix group only showed a significantly higher antibody titer than the control group at the 3rd week. In ELISA using the entire OMP, the OE_killed, Live, and PS_killed groups all showed an increase in antibody titer over time, and a significant difference was observed compared to the control group, but the Mix group did not show a significant difference compared to the other groups.

Through the above results, the vaccine composition according to an aspect contains both live and dead cells of the attenuated Salmonella strain, thereby maintaining a stronger and more persistent antibody titer than when inoculating live or dead cells alone, thereby attenuating existing poultry typhus with enhanced humoral immunity. It was confirmed that it was able to induce a much stronger immune response than the live bacterial vaccine.

Claims (8)

Attenuated Salmonella Gallinarum ( Salmonella Gallinarum ) Vaccine composition for preventing poultry typhus comprising live and dead cells. The vaccine composition according to claim 1, wherein live and dead cells of the attenuated Salmonella galinarum are each included in an amount of 1 x 10 ⁷ cfu / to 1 x 10 ⁹ cfu / animal. The vaccine composition according to claim 1, wherein live and dead cells of the attenuated Salmonella gallinarum are mixed in a volume ratio of 1:1. The vaccine composition of claim 1, 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 of claim 1, wherein the vaccine composition further comprises any one or more selected from the group consisting of carriers, diluents and adjuvants. A method for preventing poultry typhus comprising inoculating poultry with the vaccine composition of any one of claims 1 to 5. The method according to claim 6, wherein the poultry is any one selected from the group consisting of chickens, ducks, turkeys, geese, quails, pheasants and geese. The method of claim 7 , wherein the poultry is a chick under 3 weeks of age.

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