KR102616625B1 - Vaccine composition for preventing or treating of caseous lymphadenitis and uses thereof - Google Patents

Abstract

The present invention relates to a vaccine composition for the prevention or treatment of caseous lymphadenitis and its use, and specifically includes Corynebacterium pseudotuberculosis , its culture filtrate, and an adjuvant as active ingredients. It relates to a vaccine composition for the prevention or treatment of caseous lymphadenitis and a method of preventing caseous lymphadenitis by administering the vaccine composition to small ruminants.

Description

Vaccine composition for preventing or treating caseous lymphadenitis and uses thereof}

The present invention relates to a vaccine composition for preventing or treating caseous lymphadenitis and its use.

Caseous lymphadenitis (CLA) is a bacterial disease caused by Corynebacterium pseudotuberculosis and causes chronic debility in small ruminants such as goats and sheep. This disease has been reported worldwide, including in Europe, Oceania, America, Asia, and Africa. When infected with the disease, abscesses like lumps of cheese are formed in the lymph nodes on the surface of the skin, or abscesses are formed in internal organs, deteriorating the quality of wool or meat. Mainly due to competition for rank between animals or structures in the breeding environment, the abscess mass on the skin surface ruptures, bacteria leak out, and are spread through the animal's wounds. Infected animals form abscesses with a thick coating and are difficult to cure with antibiotics, so it is necessary to cull infected animals and improve the breeding environment. This disease has a low mortality rate, but causes economic losses to breeding farms due to contamination of lesions and abscesses, weight loss and reduced milk flow due to chronic weakness.

Goat caseous lymphadenitis is a disease that is prevalent in domestic goat farms and is infectious to humans. It is an important disease not only for the economic damage to farms but also for public health. However, when caseous lymphadenitis currently occurs, passive measures such as culling or isolating the affected individuals and disinfecting the breeding farm are the best, and active management is insufficient due to the absence of a vaccine to prevent the disease.

Due to the absence of a caseous lymphadenitis vaccine, there is no special vaccination program for caseous lymphadenitis other than management of the breeding environment, early detection, and prompt treatment. In a situation where antibody induction by vaccine is not possible, the antibody positivity rate of black goats for caseous lymph node abscess is over 50% nationwide, confirming that infection is widespread. In addition, as a result of investigating the occurrence of abscesses in goats shipped in 2020, internal and external abscesses were confirmed in 15.85 to 22.99%.

Not only bacteria but also various bacterial proteins are involved in the pathogenesis of caseous lymphadenitis. The most widely known substances are PLD (Phospholipase D), which is involved in cytotoxic and tissue necrotic activity and acts on the spread of bacteria in the early stage of infection, and secreted protein, a type of serine protease that induces humoral immunity during early stage of infection. CP40 and others are beginning to be known as major antigens in the development of vaccines for caseous lymphadenitis, and many exotoxins are being identified.

Meanwhile, Korean Patent Publication No. 2021-0133986 discloses ‘Bacterial Membrane Preparation’, a pharmaceutical composition containing an isolated bacterial membrane preparation, and WO2012-149622 discloses ‘Skin test for diagnosing subclinical caseous lymphadenitis in goats and sheep. ' is disclosed, but the 'vaccine composition for preventing or treating caseous lymphadenitis and its use' containing the Corynebacterium pseudotuberculosis ( C. pseudotuberculosis ) cells and their culture filtrate of the present invention is not described. does not exist.

The present invention was developed in response to the above-mentioned needs, and the present inventors sought to develop an effective vaccine against caseous lymphadenitis. For this purpose, a test vaccine was prepared by varying the content of Corynebacterium pseudotuberculosis ( C. pseudotuberculosis ) and its culture filtrate, and then antibody titer evaluation and challenge vaccination tests were performed on 3-4 month old goats. As a result, 100% positive antibody titers were confirmed from the 2nd week of vaccination in individuals inoculated with the test vaccine containing more than 10 ⁹ CFU/ml and 50% culture filtrate, and there was no death or abscess formation for 4 weeks after challenge vaccination. As this was not observed, the present invention was completed by confirming the excellent defense ability against pathogens.

In order to solve the above problems, the present invention includes Corynebacterium pseudotuberculosis , its culture filtrate, and an adjuvant as active ingredients, for the prevention or treatment of caseous lymphadenitis. A therapeutic vaccine composition is provided.

Additionally, the present invention provides a method of preventing caseous lymphadenitis by administering the vaccine composition to small ruminants.

Since the vaccine composition of the present invention is non-toxic and has excellent immunogenicity, it can be usefully used for the prevention of caseous lymphadenitis in goats or sheep.

Figure 1 shows the Western blot results of the culture filtrate of Corynebacterium pseudotuberculosis strain 51-12A, where A is the result of reaction using Corynebacterium pseudotuberculosis negative mouse serum as the primary antibody, B is the result of reaction using mouse serum inoculated only with Corynebacterium pseudotuberculosis cells as the primary antibody, and C is the result of reaction using mouse serum inoculated with Corynebacterium pseudotuberculosis cells and culture filtrate as the primary antibody. This is the result of a reaction using antibodies.

In order to achieve the object of the present invention, the present invention provides a treatment for caseous lymphadenitis, comprising Corynebacterium pseudotuberculosis , its culture filtrate, and an adjuvant as active ingredients. A vaccine composition for prophylaxis or treatment is provided.

In the present invention, the term "vaccine" refers to a biological agent containing an antigen that provides immunity to a living body, and refers to an immunogen or antigenic substance that creates immunity in a living body by injecting or orally administering it to humans or animals to prevent infectious diseases. 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. Automatic immunity is characterized by a long production period of antibodies related to immunity and sustained immunity, while passive immunity through vaccines acts immediately in treating infectious diseases, but has the disadvantage of being less durable.

In the present invention, the term “prevention” refers to all actions that inhibit or delay the occurrence, spread, and recurrence of caseous lymphadenitis by administering the vaccine composition according to the present invention.

In the present invention, the term “treatment” refers to any action in which the symptoms of caseous lymphadenitis are improved or beneficial due to administration of the vaccine composition according to the present invention.

In addition, the vaccine composition for preventing or treating caseous lymphadenitis according to the present invention is preferably an inactivated vaccine, and the Corynebacterium pseudotuberculosis and its culture filtrate may be inactivated. , the inactivation method may be any method known in the art without limitation.

Additionally, in the vaccine composition according to the present invention, the Corynebacterium pseudotuberculosis may preferably be included in the vaccine composition at 10 ⁹ CFU/ml or more, and more preferably in the vaccine composition at 10 ⁹ ~ It may be included at 10 ¹⁰ CFU/ml, but is not limited thereto.

In addition, in the vaccine composition according to the present invention, the culture filtrate may be included at 40 to 60% (v/v) based on the vaccine composition, and more preferably at 45 to 55% (v/v) based on the vaccine composition. It may be included, more preferably, it may be included at 48 to 52% (v/v) based on the vaccine composition, and most preferably it may be included at 50% (v/v) based on the vaccine composition. However, it is not limited to this.

In the vaccine composition according to one embodiment of the present invention, the culture filtrate is a Corynebacterium pseudotuberculosis strain in BHI (brain heart infusion) medium containing Tween 80 at a temperature of 35 to 39 ° C. It may be cultured for 48 hours and then filtered and concentrated through a 0.2 ㎛ membrane, but is not limited thereto.

In addition, the culture filtrate may contain an exotoxin secreted by Corynebacterium pseudotuberculosis, and the exotoxin is preferably PLD (phospholipase D), CP40 (corynebacterial protease 40 kDa), and PknG. (protein kinase G), NanH (neuramidase H), SpaC (putative adhesive pili tip protein), and SodC (superoxide dismutase C) proteins, but are not limited thereto.

In the present invention, the term "exotoxin" is also called exotoxin and refers to a toxin secreted outside the bacterial cell as it proliferates. Exotoxins are unstable to heat, have protein properties, and are detoxified with formaldehyde, but maintain antigenicity.

In addition, in the vaccine composition according to the present invention, the adjuvant may be included at 15 to 25% (v/v) based on the vaccine composition, and more preferably at 18 to 22% (v/v) based on the vaccine composition. It may be included, and more preferably, it may be included at 20% (v/v) based on the vaccine composition, but is not limited thereto.

In the present invention, the term “immune adjuvant” refers to a pharmaceutical or immunological agent administered for the purpose of improving the immune response to a vaccine. The above immune enhancers are not limited to this, but include aluminum hydroxide (Al(OH) ₃ ), aluminum phosphate (AlPO ₄ ), alum (potassium aluminum sulfate), MF59, virosome, AS04 [aluminum hydroxide] mixture of roxide and monophosphoryl lipid A (MPL)], AS03 (a mixture of DL-α-tocopherol, squalene and the emulsifier polysorbate 80), CpG, Flagellin, Poly I:C, AS01, AS02, ISCOMs or ISCOMMATRIX. can be used

In addition, the vaccine composition of the present invention may further include a pharmaceutically acceptable carrier and/or excipient and/or diluent.

As used herein, the term "pharmaceutically acceptable" refers to a substance that is physiologically acceptable and does not typically cause gastrointestinal upset, allergic reactions such as dizziness, or similar reactions when administered to a mammal. Examples of the carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Examples include polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may be additionally included, but are not limited thereto.

The vaccine composition of the present invention can be formulated using methods known in the art to allow rapid, sustained or delayed release of the active ingredient when administered to a mammal. Dosage forms include powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, and sterile powder forms.

The vaccine composition according to the present invention can be administered by various routes, for example, orally, parenterally, such as suppository, transdermal, intravenous, intraperitoneal, intramuscular, intralesional, nasal, intraspinal administration. It can also be administered using an implantable device for sustained or continuous or repetitive release. The frequency of administration can be administered once a day or in several divided doses within the desired range, and the administration period is not particularly limited.

The present invention also provides a method of preventing caseous lymphadenitis by administering the vaccine composition of the present invention to small ruminants.

In the method for preventing caseous lymphadenitis according to the present invention, the small ruminant animal may preferably be a goat or a sheep, but is not limited thereto.

In the present invention, the term "administration" means introducing a predetermined vaccine composition into a small ruminant individual by an appropriate method, and the route of administration of the vaccine composition of the present invention is through any general route as long as it can reach the target tissue. It may be administered, preferably intramuscularly, but is not limited thereto. Additionally, the vaccine composition can be administered by any device capable of transporting the active agent to target cells.

Additionally, the vaccine composition may be administered alone or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art.

Hereinafter, the present invention will be described in detail by examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

Materials and Methods

1. Corynebacterium pseudotuberculosis ( Corynebacterium pseudotuberculosis ) Culture medium preparation

The present invention used the Corynebacterium pseudotuberculosis 51-12A strain acquired from the Agriculture, Forestry and Livestock Quarantine Headquarters on January 4, 2022. As a result of BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis of the 16S rRNA (SEQ ID NO: 1) nucleotide sequence of the 51-12A strain, it was confirmed that it was C. pseudotuberculosis .

The received seed was inoculated onto a blood agar plate, a single colony was selected, and inoculated into BHI-T medium (Table 1) supplemented with 0.05% Tween80. The bacterial solution was cultured with shaking at 37°C for 12 to 48 hours, lyophilized, and transformed into the original strain. It was used as. The culture medium inoculated into BHI-T medium and freeze-dried was mixed with sterilized 4X TPGG protectant, aliquoted into 10 ml glass vials (3 ml each), and placed in a freeze dryer to freeze-dry. In addition, the strain freeze-dried using the same passage method as the original strain was inoculated into BHI medium, and the bacterial solution cultured with shaking at 37°C was freeze-dried and used as a seed strain for production.

BHI-T medium composition ingredient content Beef heart (infusion from 250g) 5g Calf brains (infusion from 200g) 12.5g NaCl 5g _{Na2HPO4 _} 2.5g D(+)-glucose 2g Peptone 10 g Tween80 0.5 ml Distilled water up to 1,000 ㎖

2. Production of antigens for vaccines

The production seed was inoculated into BHI-T medium and cultured with shaking at 37°C. The culture medium was inoculated within 10% of the main culture medium and cultured in an incubator for 12 to 48 hours. A portion of the culture medium incubated at 37°C for 12 to 48 hours was collected for testing, and the remaining culture medium was concentrated with a 0.2 ㎛ membrane, and the pellet and culture filtrate were taken respectively and dissolved in formalin (formaldehyde gas 37% in pure water). After dissolving, add 10-15% of methanol to prevent polymer formation to a final concentration of 0.2%, inactivate by reacting at 37℃ for more than 96 hours, and store in a cool, dark place at 2-8℃. did.

Before the inactivation, the culture was decimal diluted with PBS-T, inoculated into blood agar plates step by step, cultured at 37°C for 24 to 48 hours, and then the number of bacteria was measured to confirm that the number of bacteria in 1 ml was more than 4x10 ⁹ CFU. . In addition, the inactivated bacterial solution was cultured on a blood agar plate at 37°C for more than 72 hours, and no colonies were observed, confirming that the bacterial cells were inactivated.

3. Verification of exotoxin content in culture filtrate

PLD and CP40 proteins, known as exotoxins of Corynebacterium pseudotuberculosis, are proteins secreted outside the cell during the culture process without separate cell disruption, and are used in the culture medium of the 51-12A strain according to the present invention. Protein expression was confirmed by performing SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). BHI-T medium before and after bacterial culture were loaded and developed on a polyacrylamide gel, and the gels were stained with Coomassie blue for comparison. As a result, no protein components were identified in the pre-culture and PBS-T samples. After culturing the 51-12A strain, a band of a specific size was confirmed in the culture medium. Among the proteins of each size, the most prominent bands were PLD of 32 kDa and CP40 of 40 kDa, and protein bands were identified between 50 and 70 kDa. .

In addition, Western blot was performed on proteins identified in the culture medium of strain 51-12A. The bacterial culture supernatant was loaded, and mouse serum inoculated with Corynebacterium pseudotuberculosis cells alone or Corynebacterium pseudotuberculosis cells and culture filtrate was used as the primary antibody. After reacting with an anti-mouse Ab to which AP (alkaline phosphatase) was attached as a secondary antibody, color development was induced by treating the substrate. As a result, clear bands could be identified for proteins that were not identified due to low concentration in the SDS-PAGE gel, including PknG of 83 kDa, NanH of 72 kDa, CP40 of 40 kDa, PLD of 32 kDa, SpaC of 86 kDa, and It was confirmed to be an 18 kDa SodC protein (Figure 1).

The results showed that the culture filtrate of Corynebacterium pseudotuberculosis used in the present invention contained various types of exotoxins.

4. Serum titer test

One dose (1.0 ml) of the vaccine was intramuscularly inoculated into the target animal (goat/sheep), and 4 weeks later, the second dose was administered. Three weeks after the second vaccination, blood was collected and serum was separated. In the separated serum, the antibody titer for caseous lymphadenitis against Corynebacterium pseudotuberculosis was confirmed by ELISA (ELITEST CLA, manufacturer HYPHEN BioMed, Cat.no CK105A).

Example 1. Production and characterization of test vaccine

A test vaccine for the prevention of caseous lymphadenitis was prepared with the composition shown in Table 2 below.

Composition of test vaccine for preventing caseous lymphadenitis Contents (per 1 dose) C. pseudotuberculosis 51-12A strain 10 ⁹ CFU or more C. pseudotuberculosis culture filtrate 50% Aluminum hydroxide gel (AI(OH) ₃ ) 20%

The test vaccine was tested for color, turbidity, uniformity, foreign matter or off-flavor according to the general test method 1-10-20-01 of the National Testing Standard for Animal Drugs and Biological Products. As a result of the analysis, the test vaccine according to the present invention was a yellow-white suspension. When left to stand, a slight sediment layer was formed, but when shaken, it became a uniform liquid. There were no foreign substances or off-flavors, and the contents were uniform in appearance.

In addition, as a result of measuring the hydrogen ion concentration of the test vaccine according to the general test method 1-10-20-03 of the National Testing Standard for Animal Drugs and Biological Products, it was confirmed that the hydrogen ion concentration was within pH 6.0 to 8.0.

In addition, the test vaccine was inoculated into tryptic soy agar (TSA), tryptic soy borth (TSB), and fluid thioglycollate medium (Thio) in accordance with the National Standards for Veterinary Drug Biological Products 1-10-20-06 and incubated at 22°C and 32°C. As a result of observing the incorporation of bacteria while culturing at ℃ for 14 days, no bacterial growth was observed.

In addition, as a result of measuring the formalin content of the test vaccine according to the general test method 1-10-20-09 of the National Testing Standard for Animal Drugs and Biological Products, the formalin content was confirmed to be 0.13 to 0.15%, which is less than 0.2%.

Example 2. Toxicity (safety) evaluation of test vaccine

To confirm the safety of the test vaccine, safety tests were conducted on laboratory animals (mice, guinea pigs) and target animals (goats). Mice were vaccinated with 1/5 doses, guinea pigs with 1 dose, and goats were vaccinated with 2 doses and a single-dose repeated vaccination test. Specifically, 8 mice weighing 15-20 g were prepared and inoculated with 0.2 ml intraperitoneally or subcutaneously and observed for 7 days. Four guinea pigs weighing 300-350 g were prepared and inoculated with 1 ml subcutaneously. They were observed for 7 days. Using two healthy 3-month-old goats, they were inoculated intramuscularly with 2 doses (2.0 ml) of the vaccine once and observed for 7 days.

As a result of the experiment, all healthy individuals were observed with no deaths during the test period after vaccination with the test vaccine, showing that the caseous lymphadenitis test vaccine according to the present invention was not toxic.

Example 3. Setting up the challenge model and determining antigen content

To confirm the efficacy of the caseous lymphadenitis vaccine, a challenge vaccination model setting test and an antigen content determination test were conducted in target animals.

3-1. Attack inoculation model setting test

Using 24 healthy 3-4 month old goats that were negative for caseous lymphadenitis ELISA antibodies, the attack strain C.pseudotuberculosis was diluted by concentration and inoculated intramuscularly into the axillary area at 1.0 ml each. After 4 weeks of observation, death and survival were observed. An autopsy was performed on the subject, and the presence or absence of internal organ abscess formation was confirmed, referring to Jesse et al. (Microbial pathogenesis 139 (2020): 103852).

After observing clinical symptoms for 4 weeks after the challenge vaccination, autopsies were performed on dead and surviving animals to confirm the presence or absence of internal organ abscess formation. 1x10 ^8.0 CFU and 1x10 ^7.0 CFU The challenge vaccination group suffered acute death within 1 to 2 days of vaccination, and in the case of individuals that died acutely within 1 week of challenge vaccination, no abscesses were observed in internal organs.

Analysis of death and abscess formation of experimental animals after challenge with various numbers of bacteria Group Titer
(CFU/ml) No. of
goats No. of total
death Death CLA abscesses (No. of goats) 1w 2w 3w 4w Lung lymph nodes Spleen Kidney Liver One 1x10 ⁸ 4 4 4 - - - 0 0 0 0 0 2 1x10 ⁷ 4 4 3 One - - One 0 0 0 0 3 1x10 ⁶ 4 3 0 2 One 0 2 3 0 0 0 4 1x10 ⁵ 4 One 0 One 0 0 2 One 0 0 0 5 1x10 ⁴ 4 One 0 0 0 One One 3 0 0 0 6 Control 4 0 0 0 0 0 0 0 0 0 0

An autopsy was performed on the number of deaths and surviving individuals in each group, and individuals with no abscesses formed in internal organs were set as the final surviving individuals, and 1x10 ^6.0 CFU/ml, in which more than half of the mice died, was set as the challenge inoculation concentration. As a result of an autopsy of all animals, it was confirmed that abscesses were mainly formed in the goat's lungs, jaw, and parotid lymph nodes, and the presence of abscesses in the lungs and lymph nodes was selected as a key indicator for future pathogenicity evaluation.

3-2. Antigen content determination test

To determine the antigen content for the caseous lymphadenitis vaccine, a test vaccine was produced by varying the content of Corynebacterium pseudotuberculosis ( C. pseudotuberculosis ) cells and their culture filtrate as follows.

Antigen content of caseous lymphadenitis test vaccine No. Contents (per 1 dose) One C. pseudotuberculosis 51-12A strain 10 ⁸ CFU or more
Aluminum hydroxide gel (AI(OH) ₃ ) 20% 2 C. pseudotuberculosis 51-12A strain 10 ⁸ CFU or more
C. pseudotuberculosis culture filtrate 50%
Aluminum hydroxide gel (AI(OH) ₃ ) 20% 3 C. pseudotuberculosis 51-12A strain 10 ⁹ CFU or more
Aluminum hydroxide gel (AI(OH) ₃ ) 20% 4 C. pseudotuberculosis 51-12A strain 10 ⁹ CFU or more
C. pseudotuberculosis culture filtrate 50%
Aluminum hydroxide gel (AI(OH) ₃ ) 20% 5 C. pseudotuberculosis 51-12A strain 10 ¹⁰ CFU or more
Aluminum hydroxide gel (AI(OH) ₃ ) 20% 6 C. pseudotuberculosis 51-12A strain 10 ¹⁰ CFU or more
C. pseudotuberculosis culture filtrate 50%
Aluminum hydroxide gel (AI(OH) ₃ ) 20%

Using 32 healthy goats aged 3 to 4 months and negative for caseous lymphadenitis ELISA antibodies, 4 goats each were intramuscularly inoculated with 1.0 ml of each test vaccine. The vaccination interval was 4 weeks after the first vaccination, and the second vaccination was administered, and 3 weeks later, challenge vaccination was performed in the same manner with 1.0 ㎖ of the attack strain (1x10 ⁶ CFU/㎖). During the vaccination period, weekly ELISA antibody titers were checked, clinical symptoms and mortality were observed for 4 weeks after challenge vaccination, and autopsies were performed on surviving individuals to observe the formation of abscesses in internal organs.

Antigen content determination experiment plan Group Composition of the vaccines No. goats Vaccinaiton Challenge Bacterins (CFU) Culture filtrate (v/v) Primary Booster One 1x10 ⁸ - 4 0 week 4 weeks 7 weeks 2 1x10 ⁸ 50% 4 3 1x10 ⁹ - 4 4 1x10 ⁹ 50% 4 5 1x10 ¹⁰ - 4 6 1x10 ¹⁰ 50% 4 7 Challenge control 4 none none with other groups 8 Negative Control 4 none none none

A positive caseous lymphadenitis antibody titer (ELITEST CLA, manufacturer HYPHEN BioMed, Cat.no CK105A) was confirmed through blood collection at the week of vaccination. Positive/negative judgments were calculated using the formula provided by the manufacturer, and positive values are indicated in bold in Table 6.

The antibody titer of the vaccinated group was 1x10 ⁹ + culture filtrate, 1x10 ¹⁰ + culture filtrate. In the group vaccinated, the antibody titer was confirmed to be 100% positive from the second week of vaccination. In addition, both the attack control and negative control groups were negative. In the case of group 1, one individual showed a positive seroconversion after the second vaccination, but the level was low and the serum titer did not seem to be maintained. Group 2 also confirmed partial positive conversion after the second vaccination, and from Group 3, positive antibody titers were confirmed after the first vaccination. In addition, in the case of groups 4 and 6 containing culture filtrate, it was confirmed that all inoculated groups had positive antibodies.

As a result of comparing vaccine groups 1, 3, and 5 consisting of only bacterial cells, antibody titer formation was found in proportion to the bacterial concentration. In the group with more than 1x10 ⁹ CFU of bacteria containing culture filtrate, it was confirmed that all individuals were 100% positive.

Antibody titer results according to antigen content Group No. goats weeks post-vaccination 0 One 2 3 4 5 6 7 One One 0.240 0.283 0.280 0.305 0.293 0.427 0.515 0.417 2 0.172 0.146 0.130 0.118 0.095 0.130 0.146 0.126 3 0.288 0.265 0.260 0.285 0.284 0.377 0.368 0.237 4 0.290 0.239 0.231 0.224 0.203 0.277 0.406 0.319 2 5 0.232 0.189 0.360 0.385 0.373 0.507 0.595 0.497 6 0.261 0.313 0.310 0.335 0.323 0.457 0.545 0.447 7 0.226 0.176 0.160 0.148 0.125 0.160 0.176 0.156 8 0.268 0.395 0.390 0.465 0.564 0.557 0.548 0.682 3 9 0.232 0.324 0.267 0.251 0.210 0.274 0.311 0.249 10 0.261 0.179 0.194 0.177 0.165 0.226 0.200 0.285 11 0.226 0.219 0.283 0.389 0.374 0.492 0.499 0.408 12 0.268 0.215 0.450 0.769 0.946 0.856 0.787 0.585 4 13 0.132 0.088 0.614 0.644 0.861 0.765 0.902 1.125 14 0.328 0.203 0.688 0.653 0.985 0.899 1.102 1.115 15 0.198 0.350 1.787 2.056 1.730 1.775 1.539 1.348 16 0.171 0.168 0.928 1.259 0.847 0.903 0.737 0.699 5 17 0.186 0.353 0.434 0.401 0.325 0.511 0.549 0.570 18 0.114 0.419 0.797 0.948 0.880 0.959 1.158 1.584 19 0.134 0.366 0.538 0.767 0.845 0.815 0.800 0.909 20 0.292 0.277 0.465 0.590 0.253 0.367 0.295 0.230 6 21 0.120 0.337 0.958 1.115 1.259 1.150 1.267 1.657 22 0.105 0.424 0.968 1.014 0.955 1.318 1.244 1.428 23 0.125 0.288 0.753 0.983 1.061 1.031 1.015 1.124 24 0.185 0.359 0.766 0.898 0.901 0.868 1.116 1.242 7 25 0.256 0.201 0.231 0.192 0.185 0.311 0.254 0.231 26 0.385 0.264 0.265 0.205 0.216 0.249 0.381 0.289 27 0.285 0.127 0.258 0.224 0.279 0.274 0.249 0.203 28 0.100 0.317 0.139 0.117 0.212 0.252 0.224 0.188 8 29 0.274 0.176 0.203 0.186 0.198 0.224 0.310 0.275 30 0.213 0.156 0.193 0.148 0.147 0.179 0.176 0.159 31 0.264 0.262 0.217 0.243 0.176 0.210 0.271 0.228 32 0.240 0.227 0.195 0.169 0.182 0.217 0.227 0.202

Example 4. Efficacy analysis of caseous lymphadenitis vaccine - challenge vaccination

As a result of the challenge, death occurred in the challenge control and vaccine groups 1, 2, and 3, and a lymph node abscess was confirmed in group 5. No deaths or abscesses were observed in vaccinated groups Group 4 and Group 6.

Challenge inoculation test results Group No. of
goats No. of total
death Death CLA abscesses (No. of goats) 1w 2w 3w 4w Lung lymph nodes Spleen Kidney Liver One 4 2 0 2 0 0 2 2 0 0 0 2 4 2 0 One One 0 One One 0 0 0 3 4 2 0 One One 0 One 2 0 0 0 4 4 0 0 0 0 0 0 0 0 0 0 5 4 0 0 0 0 0 0 One 0 0 0 6 4 0 0 0 0 0 0 0 0 0 0 7 4 3 0 2 One 0 2 3 0 0 0 8 4 0 0 0 0 0 0 0 0 0 0

Based on the results of serum titer following vaccination and the confirmation of death and abscess formation according to challenge test, the bacterial content was ^1x10 , which showed sufficient protective ability against Corynebacterium pseudotuberculosis ( C. pseudotuberculosis ) infection. The condition of more than CFU + 50% culture filtrate was determined as the final composition of the vaccine for preventing caseous lymphadenitis.

Claims (9)

A vaccine composition for the prevention or treatment of caseous lymphadenitis, comprising Corynebacterium pseudotuberculosis , its culture filtrate, and an immune enhancer as active ingredients,
The Corynebacterium pseudotuberculosis and culture filtrate are inactivated,
The Corynebacterium pseudotuberculosis is included in the vaccine composition at more than 10 ⁹ CFU/ml,
The culture filtrate contains 40 to 60% (v/v) of the vaccine composition,
The culture filtrate contains PLD (phospholipase D), CP40 (corynebacterial protease 40 kDa), PknG (protein kinase G), NanH (neuramidase H), SpaC (putative adhesive pili tip protein), and SodC (superoxide dismutase C) proteins. ,
A vaccine composition, characterized in that the immune adjuvant is contained in 15 to 25% (v/v) based on the vaccine composition. delete delete delete delete delete The vaccine composition according to claim 1, wherein the adjuvant is aluminum hydroxide (Al(OH) ₃ ). A method of preventing caseous lymphadenitis by administering the vaccine composition of claim 1 or 7 to goats or sheep. delete