JP2020182455A - Novel serum type vaccine for prevention of fish chain coccus disease - Google Patents

Abstract

To provide novel Streptococcus bacterial strain and, a vaccine for prevention of a chain coccus disease using the novel Streptococcus bacterial strain.SOLUTION: There are provided an inactivation vaccine for prevention of a chain coccus disease of fish including a novel Streptococcus bacterial strain, inactivated Streptococcus parauberis bacterial strain, a production method thereof and an administration method thereof.EFFECT: As an effect, it is possible to effectively prevent a chain coccus disease of fishes, especially cultivated flatfishes for significantly preventing death of fishes due to a chain coccus disease. Therefore, it is possible to increase productivity of fishes, and save cost required for use of an antibiotic. In addition, by a vaccine for preventing bacterial diseases of fishes, it is possible to produce healthy fishes for which no antibiotic has to be used, for contributing to development of a fish breeding industry, for improving significantly fish consumption.SELECTED DRAWING: Figure 9

Description

The present invention relates to a novel streptococcal strain and a vaccine for preventing streptococcus using the same, and more specifically, inactivating a novel streptococcal strain and an inactivated streptococcal disease preventive vaccine in fish including an inactivated streptococcal strain. Regarding vaccine, its production method and administration method.

Streptococcus disease is a disease that induces sepsis in natural and farmed freshwater and marine fish. It is infected with various fish species in the world including yellowtail (Seriolae spp.) And eel (Eel; Angulla japonica), which are marine fish, and induces diseases. Streptococcus parauberis, Streptococcus iniae, Streptococcus agaractiae, Streptococcus agalactiae, Streptococcus agalactiae, Streptococcus agalactiae, Streptococcus agalactiae, Streptococcus agalactiae, Streptococcus agalactiae Giving.

The world production of marine products in 2014 was 195.72 million tons. Of this, the catch production is 94.63 million tons, showing similar catch production for the past few years, but the aquaculture production is 101.09 million tons, increasing annually to 102 million tons in 2025. , Is expected to increase by 39% compared to 2013-2015 (FAO). In 2017, Korean fish farming production was 86,387 tons, and the production by fish type was 41,207 tons (47.7% of the total production), and the production value of the same year was 584.5 billion won. (57.9%) was recorded, indicating that flounder is a representative fish farming variety in Korea (Fish Production Statistics, Statistics Agency, 2018). Flounder is designated as a teenage export strategic item in the fisheries field promoted by the Ministry of Oceans and Fisheries of Korea, and is a high-value-added variety currently being promoted in the aquaculture product strategic item development research and development project.

The damage caused by the disease of cultured flatfish is estimated to be about 12,000 tons per year, which is 30% of the annual production, and scoutica disease, which is a parasitic disease, is the disease that most induced the death of farmed flatfish in 2018. (44.5%), sickness (12%), viral hemorrhagic septicemia (16.6%), and streptococcal disease (9.7%), which is a bacterial disease, have been confirmed (aquaculture mortality trends). Survey and effect verification research of disinfecting equipment for transporting live fish, National Fisheries Science Institute research role, 2018). Recently, the most common diseases of flatfish in Korea are parasitic diseases such as scoutica disease (61%), viral hemorrhagic septicemia (10.6%), and S. cerevisiae. Streptococcal disease (9.8%) caused by parauberis has been reported (Aquaculture Farm Death Status and Dead Body Treatment Plan Research, 2014-2016, National Academy of Fisheries Science). Streptococci, which are problematic in flatfish farming, are S. cerevisiae. iniae, S.M. Known as parauberis, the identification of streptococci isolated from cultured flounder in the Jeju region between 2003 and 2005 revealed that S. cerevisiae. paraubilis 54%, S.A. Iniae was separated at a ratio of 46%. However, in the sick flounder from the latter half of the 2000s to the present, S. The ratio of iniae has decreased, and recently it has hardly been separated, and usually S.I. It is the fact that parauberis is separated.

The pathogen detection rate of farmed fish is increasing every year, and despite the spread of the streptococcal (S. paraubilis) vaccine currently commercialized for flatfish, the disease is still occurring and the current vaccine antigen is improved. Is the actual situation that is necessary. Since the therapeutic efficacy is decreasing due to the emergence of drug-resistant bacteria and multidrug-resistant bacteria due to misuse and abuse of antibiotics and antibacterial agents, it is expected that it will become more difficult to treat with chemotherapeutic agents after the onset of the disease. It is necessary to develop a vaccine that can be prevented in advance. In fact, in South Korea, S. The paraubelis vaccine has been commercialized and spread since 2009, but even after the vaccine spread, S. Parauberis is still occurring (Aquatic Disease Characteristics Study, 2012-2017). In general, scoutica disease, which induces the highest mortality in flatfish, occurs in the fry stage, but streptococcal disease occurs in intermediate breeding fish, adult fish, etc., causing greater economic damage. According to an analysis of the causes of mortality due to diseases that recently occurred in cultured flounder, mortality due to bacterial diseases was the highest at 13%, of which 67.1% was mortality due to streptococcal disease.

In Korea, S. Various studies such as parauberis inactivated vaccines, recombinant protein vaccines, and attenuated vaccines are steadily progressing, but at present, inactivated vaccines produced in the form of combination vaccines with other bacterial pathogens are commercialized and sold. It is only (9 products from 6 companies). S.A. for Talbots in Spain Hipra's ICTHIOVAC (R) -STR inactivated injectable vaccine using paraubelis as an antigen has been developed and sold, but in South Korea, S.A. No single vaccine against paraubelis is sold. Due to the characteristics of the fish farming industry, the development of low-cost fish vaccines is indispensable, and a combination vaccine that can prevent various bacterial pathogens at once has been commercialized, but a survey on the vaccine effect after using the combination vaccine is actually conducted. Is a slight fact. In flatfish, S. Streptococcal disease caused by paraubilis has occurred even after the spread of the vaccine, and it is necessary to verify the efficacy of the vaccine currently on the market.

Multivalent vaccines that are made by mixing many types of the same type, such as influenza vaccines and polio vaccines for humans, that is, A1, A2, and B types for influenza and I, II, and III types for polio. It is called (poliovalent vaccine). Since these pathogens are divided into several antigen types, it is useless if the disease type occurs in a completely different type from the vaccine when vaccinated with a vaccine (unit price vaccine) contained separately, and it is multivalent. We are using vaccines. S. is a streptococcus that induces pneumonia in humans. There are various serotypes of pneumoniae, but a 23-valent polyvalent-capped polysaccharide vaccine that can prevent all of them is used (Song Jun-young, John Hee-jin, 2014, Streptococcus pneumoniae vaccine, J. Korean Med Assoc 57 ( 9), 780-788).

Therefore, the present inventors have introduced S. cerevisiae to induce diseases in flatfish. Molecular mechanics and serotyping were carried out on paraubelis using the MLST (Multilocus sqquecne typing) method, and the flounder streptococcus S. We secured basic data for selecting a strain suitable for the parauberis vaccine, produced a unit price vaccine for each serotype, and evaluated the efficacy of the cross-vaccine. In addition, based on the cross-vaccine efficacy evaluation result, S. For the development of paraubelis multivalent vaccines, S. serotypes of different serotypes. The paraubelis strain was mixed and the vaccine efficacy was evaluated to complete the present invention.

The above information described in this background art portion is merely for the purpose of improving the understanding of the background of the present invention and forms the prior art already known to those having ordinary knowledge in the technical field to which the present invention belongs. It is not necessary to include the information to be used.

An object of the present invention is to provide a strain of Streptococcus paraubelis isolated from a streptococcal-infected scallop.

Another object of the present invention is to provide an inactivated vaccine for the prevention of streptococcal disease, which comprises an inactivated Streptococcus paraubellis strain as an antigen.

Yet another object of the present invention is streptococcal disease comprising the step of inactivating a Streptococcus paraubellis strain isolated from a streptococcal-infected flatfish with formalin to obtain a vaccine. The purpose is to provide a method for producing a preventive inactivated vaccine.
Yet another object of the present invention is to provide a method of immunizing a fish, which comprises administering the vaccine to the fish.

In order to achieve the above object, the present invention provides Streptococcus paraubellis KTCC 13800BP strain, CKTC 13801BP strain, CKTC 13802BP strain and KCTC 13802BP strain isolated from streptococcal-infected flatfish.

The present invention also provides an inactivated streptococcal disease-preventing vaccine comprising an inactivated Streptococcus paraubellis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain or KCTC 13803BP strain as an antigen.

The present invention also obtains Streptococcus paraubellis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain, or KCTC 13802BP BP strain or KCTC 13803BP inactivated vaccine isolated from streptococcal-infected flatfish. Provided is a method for producing an inactivated vaccine for preventing streptococcal disease, which comprises.
The present invention also provides a method of immunizing a fish, comprising administering the vaccine to the fish.

INDUSTRIAL APPLICABILITY The present invention has an effect of effectively preventing streptococcal disease of fish, particularly cultured flounder, and remarkably reducing the mortality of fish due to streptococcal disease. Therefore, not only the productivity of fish can be increased, but also the cost of using antibiotics can be reduced. The present invention also contributes to the development of aquaculture by enabling the production of healthy fish that do not use antibiotics by using a vaccine that can prevent bacterial diseases in fish, and greatly improves fish consumption. Can be done.

S. Five genes of 64 strains of parauberis [DNA gyrase subunit B (gyrB), Surface M-protein (simA), autolysin, capsular polysaccharide biosynthesis protein (about 120system) sequence for palaubilis (gyrB), surface (simA), capsular polysaccharide biosystem It is a figure which shows the phylogenetic tree analysis result of. S. The serotype analysis of parauberis is shown, FIG. 2 (A) is a diagram showing a slide agglutination reaction (I: positive reaction, II: negative reaction), and FIG. 2 (B) is a diagram showing a microtiter agglutination reaction. Korea 2002-2017 S.A. It is a graph which shows the serotype distribution of parauberis bacterium. Korea 2002-2017 S.A. It is a figure which shows the isolation ratio by region and isolation year of parauberis bacterium. S. sold in South Korea. It is a figure which shows the serotype PCR (serotype-PCR) result of the inactivated vaccine containing parauberis antigen. S. It is a graph which shows the cumulative mortality amount of flatfish after artificial infection of 7 strains of parauberis. S. It is a graph which shows the cumulative mortality amount of flatfish by the concentration of 4 strains of parauberis. Flounder S. It is a diagram which schematized the parauberis vaccination and artificial infection experiment schedule. S. It is a graph which shows the antibody measurement result 2 weeks after parauberis multivalent vaccination. S. One week after the parauberis multivalent vaccination, it is the result of histopathological examination of internal organs. S. Two weeks after the parauberis multivalent vaccination, the results of histopathological examination of internal organs. S. 3 weeks after parauberis multivalent vaccination, the results of histopathological examination of internal organs.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by skilled professionals in the art of the invention. In general, the nomenclature used herein is well known and common in the art.

In one example of the present invention, Streptococcus vaccine 19FBSPa0003 (PR5) strain, Streptococcus vaccine FP2331 strain, Streptococcus vaccine serotype serotype serotype serotype serotype serotype strain FP2331 strain, and serotyped serotype strain FP2331 strain As a result of inoculating fish with a vaccine containing the above-mentioned antigen, it was confirmed that all the vaccinated plots showed a low mortality rate and that there was also a cross-vaccination effect by serotype. It was also confirmed that the multivalent vaccine containing the different serotype antigens had a higher vaccine efficacy than the single serotype vaccine.

Therefore, the present invention relates, in one aspect, to the Streptococcus paraubellis KCTC 13800BP strain isolated from streptococcal-infected scallops.

In another aspect, the present invention relates to the Streptococcus paraubellis KCTC 13801BP strain isolated from streptococcal-infected scallops.

In yet another aspect, the present invention relates to the Streptococcus paraubellis KCTC 13802BP strain isolated from streptococcal-infected scallops.

In yet another aspect, the present invention relates to the Streptococcus paraubellis KCTC 13803BP strain isolated from streptococcal-infected scallops.

In the present invention, the KCTC 13800BP strain is a Streptococcus parauberis 19FBSPa0003 strain deposited on January 30, 2019 at the Korean Collection for Type Culture, and the same strain as 19FBSPa0003 is used in the present specification. means.

In the present invention, the KCTC 13801BP strain is the Streptococcus parauberis FP2331 strain deposited at the Korea Biological Resources Center on January 30, 2019, and the KCTC 13802BP strain is the KCTC 13802BP strain deposited at the Korea Bioresource Center on January 30, 2019. The deposited Streptococcus parauberis FPa4365 strain, and the KCTC 13803BP strain is the Streptococcus parauberis FPa4870 strain deposited at the Korea Biological Resources Center on January 30, 2019.

The present invention is further selected from the group composed of inactivated Streptococcus vaccine CKTC 13800BP strain, CKTC 13801BP strain, CKTC 13802BP strain and one or more KCTC 13803BP strains from still another viewpoint. The present invention relates to an inactivated vaccine for preventing streptococcal disease containing a strain of Streptococcus as an antigen.

In the present invention, the vaccine may be characterized by, but is not limited to, a mixture of the KCTC 13802BP strain and the KCTC 13803BP strain in a ratio of 1: 1.

In the present invention, the vaccine may be characterized by, but is not limited to, a mixture of the CKTC 13800BP strain, the CKTC 13802BP strain and the CKTC 13803BP strain in a ratio of 1: 1: 1.

In the present invention, the antigen concentration of the vaccine may be 0.5 to 30 mg / fish. Preferably, it may be characterized by being 1-3 mg / fish, but is not limited thereto.

In one example of the present invention, after vaccination containing Streptococcus parauberis 19FBSPa0003 (PR5) strain, FP2331 strain, FPa4365 strain or FPa4870 strain, the antibody titer of serum was measured by antigen type (serotypes Ia to II). Both formed significantly higher antibody titers than the control group (PBS). Each vaccine group showed the highest antibody titer when measuring antibody titers with antigens of the same serotype.

In addition, S. Artificial infection with the parauberis serotype Ia + Ic vaccine group and the Ia + Ib + Ic vaccine group showed a higher survival rate than the control group. It also showed a higher relative survival rate than the parauberis single serotype vaccine group.

In the present invention, the term "vaccine" refers to an antigen used for immunization to prevent an infectious disease, which is manufactured by an attenuated microorganism or virus and is artificially immunogenic to a specific infectious disease. It means to administer a weakened or killed pathogenic microorganism into an individual in order to obtain the virus. When stimulated by a vaccine, the immune system within the individual works to produce antibodies and maintain their susceptibility, but reinfection overcomes the disease by effectively producing antibodies within a short period of time. be able to.

The inactivated vaccine can be produced by a method known in the art, for example, using an inactivating agent known in the art. Preferably, it may be characterized by being inactivated by formalin treatment, but is not limited thereto.

The mixed inactivated vaccine according to the present invention can further contain an adjuvant. Any of the auxiliary agents known in the art can be used, preferably Montandide ISA35 or Gel01.

Further, as the stabilizer or additive, sugars and amino acids, mineral oil, vegetable oil, sardine, aluminum phosphate, bentonite, silica, muramyl dipeptide derivative, thymosin, interleukin and the like may be used. ..

In the present invention, the vaccine can be used by dispensing the vaccine solution into a vial having an appropriate volume, for example, about 10 to 500 ml, and then sealing the vaccine. In addition to the liquid vaccine, such a vaccine may be injected separately and then freeze-dried to be used as a dry preparation. The dried preparation may be used by completely redissolving the dried substance with a sterilizing solution added immediately before use.

In the present invention, the mixed inactivated vaccine can be used for fish of any age at risk of infection. Although not limited to this, it can be used for intermediate breeding fish including fry and adult fish.

From still another point of view, the present invention is composed of Streptococcus paraubellis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain and KCTC 13803BP strain selected from streptococcal disease-infected flatfish. The present invention relates to a method for producing an inactivated vaccine for preventing streptococcal disease, which comprises a step of inactivating any one or more of the strains with formalin to obtain a vaccine.

In the present invention, the vaccine is produced by culturing and inactivating the Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain or KCTC 13803BP strain, and then adjusting the concentration. In one embodiment of the present invention, cryopreserved S. cerevisiae. Four strains of parauberis were cultured, 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution so as to be 0.5% (v / v) of each culture solution, and the mixture was stirred. It was inactivated in an incubator at 150 rpm and 20 ° C. for 24 hours. After centrifuging the inactivated bacterial solution, washing with sterile physiological saline, collecting the bacterial cells, and finally measuring the weight of the wet bacterial cells and varying the concentration depending on the experimental group. Produced a vaccine.

The present invention relates to a method for immunizing fish, which comprises administering the inactivated vaccine for preventing streptococcal disease to fish from still another viewpoint.

The present invention may feature, but is not limited to, injecting the vaccine into the abdominal cavity of fish.

In the present invention, the fish include, but are not limited to, fish such as flatfish at risk of infection.

In one embodiment of the present invention, four types of S.A. After treatment with paraubelis vaccine, artificial infection tests were conducted for each serotype, and as a result, it was found that the mortality rate was lower in all vaccinated groups than in the non-vaccinated control group, and that there was also cross-vaccination efficacy by serotype. confirmed. In addition, S. It was confirmed that the multivalent vaccination group containing parauberis serotypes Ia to Ic showed higher vaccine efficacy than the single serotype vaccination group.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those of ordinary skill in the art that these examples are merely exemplary of the invention and are not to be construed as limiting the scope of the invention by those examples.

Example 1: Streptococcus S. Separation and identification of paraubilis

2002-2017 S.A. isolated from sick cultured flounder in South Korea, which was preserved at the National Graduate School of Fisheries Sciences. From 199 parauberis strains, 64 representative strains were selected by year of isolation and region and used for analysis.

Strains were coated on tryptic soy agar (TSA, Becton Dickinson, USA) or brain heart infusion agar (BHIA, Becton Dick) (BHIA, Becton Dick) supplemented with 1% NaCl. After that, the cells were cultured at 25 ° C. for 24 hours to confirm the properties of the bacteria, which were subcultured to perform phenotype, serum type, genetic analysis and the like.
Table 1 shows the isolated areas and years of the analyzed strains.

Example 2: S.A. Phenotypic analysis of paraubilis

To analyze the biochemical properties, S. The parauberis strain was cultured in BHIA medium supplemented with 1% NaCl at 25 ° C. for 24 hours. The API 20 strep kit (BIOMERIEUX, France) and API ZYM kit (BIOMERIEUX, France) experimental methods were tested by inoculating and culturing purely cultured bacterial colonies on strips according to the manufacturer's method.

S. As a result of analysis of 64 API 20strep and API zym kits of paraubelis strains, it was confirmed that all the strains examined were non-motile gram-positive cocci, which were negative for catalase and oxidase (oxidase). Table 2).

PA: alkaline phosphatase, AD: arginine dihydrorase, RI: ribose acidification, MA: mannitol acidification, SO: sorbitol acidification, LA: lactose acidification, TR: trehalose acidification, IN: inulin acidification, AM: starch acidification, 2: alkaline phosphatase, 3: esterase (C4), 4: esterase lipase (C8), 5: lipase (C14), 7: valine arylamidase, 8: crystine arylamidase, 10: α-chymotrypsin, 11: acid phosphatase, 14: β-galactosidase, 16 : α-glucosidase.

As a result of analyzing the biochemical properties of API 20strep, all the strains were found to be VP (pyruvate acetatein production), HIP (hippurate hydrorysis), ESC (β-glucosidase), PYRA (pyruvic acid arabinose), PYRA (pyruvic acidyla) α-galactosidase), βGUR (β-galactosidase), ARA (L-arabinose hydrolysis), RAF (Raffinose hydrolysis), and GLYG (Glycogen activation) negative reactions were shown (Table 3).

As a result of analyzing the biochemical properties of the API zym kit, all strains were found to be leucine acetylamide, naphthol-AS-BI-phosphohydrose positive, typsin, β-galactosidase, β-glucuronidase, β-glucuronidase, β-glucuronidase, β-glucuronidase. Negative reactions of α-mannosidase and α-fucosidase were shown (Table 4).

Example 3: S.A. Genotype analysis of paraubilis
Example 3-1: Primer preparation and gene base sequence analysis For genetic characteristic analysis, each isolated strain was cultured in BHIB medium supplemented with 1% NaCl for 24 hours, and then centrifuged at 13,000 rpm for 2 minutes. , The supernatant was removed to prepare bacterial pellets. Bacterial pellets were DNA extracted using the Promega Genomic DNA Purification Kit (Promega, USA). Housekeeping gene groEL, gyrB used the previously reported primers and used S. cerevisiae. From the genomes of parauberis CKTC 11537 (Nho et al., 2011) and CKTC 11980 (Park et al., 2013), 6 genes presumed to be related to antigenicity / pathogenicity (capsule polysaccharide 4 species, autolysis 1 species, M) -Like protein 1 type) was selected to prepare primers that can be specifically amplified and analyzed (Table 5).

In Table 5 above, the base sequence is represented by the IUPAC nucleotide code (Nucleotide ambiguity code), R means A or G, i (denoted as S in the sequence list) means Inosine, and H means A, C or T. N means any base, Y means C or T, and W means A or T.

PCR amplification was performed with the prepared primers, and the PCR product was electrophoresed on a 1.5% agarose gel (Bioneer, Korea) containing EtBr, and then a UV transluminator (Alpha Innotech). , USA) was used to confirm the band, and the nucleotide sequence was analyzed (Tables 6 to 14).

For gene sequence analysis, use the MEGA6 program and GENETYX Ver. Using 8.0 (SDC Software Development, Japan), each nucleotide sequence determined is provided by NCBI (National Center for Biotechnology Information). .. The gene of the paraubelis strain and the gene of the strain analyzed in this example were comparatively analyzed. Based on the nucleotide sequence information of the Blast search, Bioedit Ver. Perform multiple alignment using 7.2.1 and neighbor-joining analysis (NJ; neighbor-) using the MEGA6 program (http://www.megasoftware.net; Tamura et al., 2013). A genetic distance between each base sequence and a phylogenetic tree were created by joining analysis (1,000 rounds of boostrap) (Fig. 1).

Example 3-2: Results of genotype analysis As a result of 16S rRNA analysis of 64 strains isolated from flatfish, S. It was confirmed to be paraubilis, and it was confirmed to have the same base sequence. S. MLST (multilocus sequence typing) analysis was performed on 64 paraubelis strains. The Soa (Sugar O-acrytranstranslation sialic acid) and PB (Polysaccharide bioscience protein) genes were not amplified in some strains. In the 64 strains analyzed, the 16S rRNA and groEL genes all had the same nucleotide sequence, and MLST analysis was performed only on gyrB and 4 antigenic / pathogenic related genes having different nucleotide sequences for each strain. S. separated from flatfish. The 64 parauberis strains were roughly classified into two MLST genotypes, and were confirmed to be 36 strains in the MLST-1 group and 28 strains in the MLST-2 group (Fig. 1). The results of genotype analysis by strain are shown in Table 15 below.

A'-' in the CPB and SOA items indicates that PCR was not amplified.

Example 4: S. Serotype analysis of paraubilis
Example 4-1: Serotype analysis method Although the serotype was analyzed by a slide agglutination reaction, rabbit antisera against the reference strains (S. paraubelis serotype I FPa4164 and S. paraubelis serotype O2 S53) were prepared and used for the analysis. .. On a slide glass, S. 70 μL of each of the two rabbit antisera against paraubelis was dropped, and the flounder-derived S. 64 parauberis strains were collected from McFarland no. In 6 standard, 10 μL of each bacterial solution suspended in sterile physiological saline was dropped and mixed well, and the presence or absence of aggregate formation was confirmed within 30 seconds (FIG. 2 (A)). Aggregation was observed from the side of the slides on a dark background and the control group used sterile saline that did not suspend the fungus.

S. S. by microtiter agglutination to distinguish subserotypes of serotype I of paraubilis. An attempt was made to analyze the serotypes of 64 paraubelis strains (Fig. 2 (B)). S. Rabbit antisera against four strains DK14, DW1, FPa4870, and FP4743, which differ in genotype and phenotypic characteristics from parauberis, were prepared and described by Kanai et al. Subserotyping using the microtiter agglutination reaction was performed by the method of (2015). Rabbit antisera were diluted 2-fold with PBS in a 96-well plate in sequence, and then 4 types of S. The perauberis FKC suspension (2 mg / mL PBS) was added in the same volume of 25 μl each, mixed, O / N incubated at 4 ° C., and then the agglutinated antibody titer was measured. For a cross-absorbed test of antisera, absorbed antisera were prepared (Kanai at al., (2015) Fish pathology 50 (2), 75-80) and the absorbed antisera had agglutinated antibody titers. It was prepared as less than 4. Tu et al. According to the method of (2015), S.I. Serotyping PCR was performed on paraubelis. The base sequence of the PCR primer is as shown in Table 2 above.

Example 4-2: Serotype analysis result Slide agglutination reaction result, S. The paraubelis strains were confirmed to be 58 strains of serotype I, 4 strains of serotype II, and 2 strains of serotype (untypeable serology). The results of serotyping PCR of 64 strains are shown in Table 15 above.

As a result of preparing rabbit antisera against 4 strains DK14, DW1, FPa4870, and FP4743 of Spalaberis and subserotyping by microtiter agglutination reaction, 58 strains of serum type I were anti-DK14, anti-DW1, It shows an agglutination reaction with anti-FPa4870 antiserum, but the agglutination value differs depending on the antiserum type, suggesting that a subserotype is present in serotype I. Depending on the agglutination value, S. As a result of subserotyping of serotype I of parauberis, it was confirmed that Ia 25 strain, Ib 25 strain, Ic 8 strain, and Non-typeable 2 strain (Table 16).

S. Anit-DK14, anti-DW1, and anti-FPa4870 antisera were prepared by a cross-aggregate absorption method for strain-specific absorption antisera (absorbed-antiserum) for classification of subserotypes with serotype I of paraubelis, and aggregated again. The valence was measured. As a result of analysis by the cross-aggregate absorption method, three subtypes (subtypes) were present in serotype I, and in the case of subserotype Ic type, the aggregation value of Ib type was higher than that of Ia type (Table 17). ).

All strains of serotype Ib were included in the MLST-2 group, serotypes II and Ia were included in the MLST-1 group, and serotype Ic was not correlated with genotype (Fig. 1).

S. As a result of analyzing the serotype (genotype) distribution by the parauberis isolated area, no specific serotype was confirmed by the isolated area, and various serotypes were confirmed regardless of the isolated area (Table 18, FIG. 3 and FIG. 4). ..

S. As a result of analyzing the serotype distribution by the parauberis separation year, serotype Ib was dominant before 2010, but serotype Ia (MLST-1 group) was continuously isolated after 2010. Recently, it was confirmed that the serotype Ic separation rate increased (Table 19).

In particular, in the case of serotypes Ia and Ic, S.I. Since the commercialization of the paraubelis flounder vaccine, it has been confirmed that the isolation ratio has increased. This is due to the use of the vaccine, rather than the ineffectiveness of the commercialized vaccine. It is presumed to be due to changes in the serotype of paraubelis (FIGS. 3 and 4).

Example 5: Serotyping of commercial commercial vaccines Flounder S. cerevisiae sold in South Korea in 2018. Seven inactivated vaccines containing paraubelis antigen were purchased. After centrifugation and extraction of DNA with the Promega Generic DNA Purification Kit (Promega, USA), the serotype was analyzed by serotype PCR (Tu et al., 2015). PCR amplification was performed and the PCR product was electrophoresed on a 1.5% agarose gel (Bioneer, Korea) containing EtBr, and then banded using a UV transluminator (Alpha Innotech, USA). confirmed.
As a result, both were confirmed to be serotype Ib and serotype Ib + II (FIGS. 5 and 20).

Example 6: S. Pathogenicity analysis of parauberis Seven representative strains (FPa4870, FP4743, FP2331, FPa4365, PR5 (19FBSPa0003), DK14, DW) with different serotypes were selected and 10 ⁶ cfu / in flatfish (16.5 ± 0.7 cm). An artificial infection experiment (water temperature 23 ± 1 ° C., subcutaneous injection method, 10 animals per test group) was carried out at a fish concentration. Artificial infection experiments using subcutaneous injection are described in Mori et al. (2010) Fish Pathology, 45 (1), 37-42.

A secondary infection experiment was conducted with PR5, FPa4365, FPa4870, and FP2331 strains that showed a relatively high mortality rate by serotype in the primary infection experiment (Fig. 6). After artificially infecting 4 strains of flounder (11.6 ± 0.5 cm) with 10 ⁵ to ¹⁰ cfu / fish (water temperature 25 ± 0.5 ° C, subcutaneous injection), the cumulative mortality rate for 2 weeks was measured. did. As a result, S. FPa4365 (Ia), PR5 (Ib), and FPa4870 (Ic) contained in serotype I of parauberis all showed high pathogenicity to flatfish (Fig. 7). The serotype II FP2331 (II) strain was subcutaneously injected into the flounder at 10 ¹⁰ cfu / fish, but the flounder did not die even at high concentrations, so the FP2331 strain was judged to be non-pathogenic or low in the flounder. Will be done. Finally, serotypes Ia, Ib, and Ic strains were set at 10 ⁸ cfu / fish and serotype II at 10 ¹⁰ cfu / fish as artificial infection concentrations for vaccine efficacy verification.

Example 7: Interserotype S. Efficacy analysis of paraubelis vaccine
Example 7-1: Vaccine preparation and efficacy analysis method S. Parauberis serotypes Ia, Ib, Ic, II strains (FPa4870, FP2331, FPa4365, PR5 (19FBSPa0003)) were treated with formalin to prepare an inactivated vaccine (FKC), which was intraperitoneally inoculated (1 mg / fish) into flatfish and controlled. The plot injected PBS intraperitoneally.

Cryopreserved S. Four parauberis strains were cultured in BHIA medium supplemented with 1.5% (v / v) NaCl at a temperature of 30 ° C. for 24 hours. The cultured plate medium colonies were aseptically inoculated into BHIB supplemented with 1.5% NaCl, and then cultured in a stirring incubator (JS Research Inc.) at 150 rpm at a temperature of 30 ° C. for 48 hours. 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution so as to be 0.5% (v / v) of each culture solution, and 24 at 150 rpm and 20 ° C. in a stirring incubator. Time was inactivated. The inactivation of the cultured bacteria was finally confirmed by BHIA (Difco, USA). After centrifuging the bacterial solution confirmed to be inactivated at 12,000 RCF, the cells were washed 3 times with sterile physiological saline to collect the cells, and finally the wet cell weight was measured and the concentration was adjusted by the experimental group. It was suspended differently. Blood was collected from the tail vein 2 weeks after vaccination, and the antibody titer was measured by the ELISA method. By the antibody titer investigation method using the ELISA method, S.I. Paraubelis FKC was adjusted to a concentration of 10 μg / well and dispensed, and the antigen was attached to the bottom of the plate at 4 ° C. overnight. S. Parauberis Serotype-specific ELISA values of four different serotypes of S. FKC of paraubelis was prepared and the antibody titer against each serum was examined. Block with 3% skim milk at room temperature for 1 hour, dilute flatfish serum and intestinal mucus 10-fold with a primary antibody, dispense and react for 1 hour. Then, after washing 3 times with PBST, the secondary antibody is diluted 1: 1000 with Anti-Japanese flounder IgM Mab and dispensed, and then reacted at 37 ° C. for 1 hour. After washing, AP-conjugated anti-mouse IgG (AP-conjuged anti-mouse IgG) (where molecule) was diluted 1: 1000 and dispensed, reacted at 37 ° C. for 1 hour, washed, and then the substrate (PNPP substrate solution). ) Was added, and the absorbance was confirmed at 405 nm 20 minutes later. Four weeks after vaccination, artificial infection experiments were performed on each vaccinated group with serotypes Ia, Ib, Ic, and II strains, and the cumulative mortality rate was measured for 3 weeks (Fig. 8). The relative survival rate was calculated as follows.
Relative survival rate (%) = {1- (cumulative mortality rate in test plot / cumulative mortality rate in control plot)} x 100

Example 7-2: Efficacy and cross -vacuum efficacy analysis of serotyped vaccine Blood was collected 2 weeks after serotyped vaccination and the antibody titer of the serum was measured by antigen type (serotypes Ia to II). All of them formed significantly higher antibody titers, and each vaccine group showed the highest antibody titer when the antibody titers were measured with antigens of the same serotype (Table 21).

Four different serotypes of S. flounder for flatfish. As a result of performing an artificial infection test for each serotype after treatment with the paraubelis vaccine, a lower mortality rate was shown in all vaccinated plots as compared with the non-vaccinated control plot (Table 22).

As a result of comparing the relative survival rate of the cross-serotype cross-vaccination efficacy with that of the control group, the highest relative survival rate was shown when a strain of the same serotype was artificially infected after vaccination with the same serotype in all test groups. (Table 23). It was confirmed that serotypes Ib and Ic had a high cross-vaccine efficacy response between each other (relative survival rate: 73.9 to 82.5%), but Ia, Ib, and Ic had relatively low cross-vaccine efficacy (relative survival rate: 73.9 to 82.5%). Relative survival rate: 0 to 30.8%) (Table 23).

Example 8: S. Efficacy analysis of parauberis multivalent vaccine
Example 8-1: Multivalent vaccine preparation and efficacy analysis method S.A. Since paraubelis serotype II is not pathogenic to flounder and has not recently been isolated in flounder farms, it was excluded from the vaccine target strains for the development of multivalent vaccines. S. An inactivated vaccine (FKC) was prepared by formalinizing three serotypes of paraubelis (serotype Ia (FPa4365), serotype Ib (PR5), and serotype Ic (FPa4870)) and conducted experiments.

Cryopreserved S. The parauberis strain was cultured in BHIA medium supplemented with 1.5% (v / v) NaCl at 30 ° C. for 24 hours. The colonies of the cultured plate medium were aseptically inoculated into BHIB supplemented with 1.5% NaCl, and then cultured in a stirring incubator (JS Research Inc.) at 150 rpm, 30 ° C., and 48 hours. 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution so as to be 0.5% (v / v) of each culture solution, and 24 at 150 rpm and 20 ° C. in a stirring incubator. Time was inactivated. The inactivation of the cultured bacteria was finally confirmed by BHIA (Difco, USA). After centrifuging the inactivated bacterial solution at 12,000 RCF, the cells were washed 3 times with sterile physiological saline, and then the cells were collected. Finally, the wet cell weight was measured and the concentration was determined by the experimental group. It was suspended differently. Each S. Parauberis Serotype-specific inactivated single vaccine research results confirmed that serotypes Ib and Ic have cross-vaccine efficacy, and the antigen combinations and concentrations in Table 24 show hirame (average total length: 15.05 ± 0.9 cm, average body weight). : 26.7 ± 4.74 g) was vaccinated by the peritoneal injection method. A total of 80 flounders per experimental plot were used in the experiment.

Two weeks after vaccination, blood was collected from the tail vein (n = 10), and S.I. The antibody titer against paraubelis was measured. By the antibody titer investigation method using the ELISA method, S.I. Parauberis FKC was adjusted to a concentration of 10 μg / well and dispensed, and the antigen was attached to the bottom of the plate at 4 ° C. overnight. S. To measure the serotype-specific ELISA value of parauberis, three different serotypes of S. FKC of paraubelis was prepared and the antibody titer against each serum was examined. Blocking was performed at room temperature for 1 hour using 3% skim milk, and the flounder serum and intestinal mucus were diluted 10-fold with a primary antibody, dispensed, and then reacted for 1 hour. Then, after washing three times with PBST, the secondary antibody was diluted 1: 1000 with Anti-Japanese flounder IgM Mab and dispensed, and then reacted at 37 ° C. for 1 hour. After washing, AP-conjugated anti-mouse IgG (AP-conjuged anti-mouse IgG) (where molecule) was diluted 1: 1000 and dispensed, reacted at 37 ° C. for 1 hour, washed, and then the substrate (PNPP substrate solution). ) Was added, and the absorbance was confirmed at 405 nm 20 minutes later. As a result of measuring the antibody titer 2 weeks after vaccination, the antibody titer was not confirmed in the control group, and the antibody titer was significantly higher in the Ia + Ic group and the Ia + Ib + Ic group than in the control group (Table 25, FIG. 9).

Example 8-2: Efficacy analysis of multivalent vaccine S. cerevisiae 3 weeks after vaccination. Parauberis Ia FPa4365 (5.2 × 10 ⁸ cfu / fish), Ib PR5 (7 × 10 ⁸ cfu / fish), Ic FPa4870 (1.4 × 10 ⁸ cfu / fish), water temperature 26-27 ° C. An artificial infection experiment was conducted while maintaining the above, and the mortality rate was confirmed for 3 weeks (Table 26).

S. The parauberis serotypes Ia + Ic vaccine group and the Ia + Ib + Ic vaccine group have three different serotypes of S. cerevisiae. When artificially infected with parauberis, the survival rate was higher than that of the control group, and S.I. Parauberis showed higher relative survival rates compared to single serotypes Ia and Ib vaccine groups (Table 27).

For example, after Ia single serotype vaccination, S. cerevisiae. Artificial infection with the parauberis Ia serotype FPa4365 strain showed a relative survival rate of 55.6% compared to the control group, but artificial infection with the Ia + Ic vaccine group and Ia + Ib + Ic group with the FPa4365 strain compared with the control group. The relative survival rates were 93.4% and 66.7%. In addition, after Ic single serotype vaccination, S. Artificial infection with the parauberis Ic serotype FPa4870 strain showed a relative survival rate of 75.1% compared to the control group, but artificial infection with the Ia + Ic vaccine group and Ia + Ib + Ic group with the FPa4365 strain compared with the control group. It showed a relative survival rate of 50% and 30%, and showed a lower efficacy than the Ic single serotype vaccine. In a comparison of vaccine efficacy between multiserotyped multivalent vaccines, the Ia + Ic vaccine group showed higher relative survival in all three serotype strains than the Ia + Ib + Ic vaccine group. As for the specific antibody titer against parauberis, the Ia + Ib + Ic vaccine group showed a higher antibody titer than the Ia + Ic vaccine group.

Example 9: S. Stability analysis of parauberis multivalent vaccine
Example 9-1: Multivalent vaccine stability analysis method For multivalent vaccine safety investigation, vaccine efficacy against flatfish (average total length: 15.83 ± 1.04 cm, average weight: 27.2 ± 4.75 g) The safety was evaluated by inoculating the abdominal cavity at twice, five, and ten times the antigen concentration used at the time of the survey (Table 28). The mortality rate was confirmed up to 3 weeks after peritoneal inoculation, but the safety was confirmed by blood biochemical properties and histopathological examination every week by collecting blood from the tail vein of the flatfish (n = 5). Blood properties are measured by measuring the concentrations of glucose, ALT, AST and total protein using an automatic blood analyzer, and histopathological investigation is performed by a conventional method after fixing the kidney, spleen, liver, etc. to neutral formalin. did.

Example 9-2: Stability analysis result of multivalent vaccine S. No mortality occurred in all the experimental plots for 3 weeks after injecting the vaccine at a high farming rate of 2 times, 5 times, and 10 times the antigen levels of the parauberis Ia + Ic vaccine group and the Ia + Ib + Ic vaccine group. The blood biochemical properties (AST, ALT, GLU, TP) were not significantly different from those of the control group at 1, 2, and 3 weeks after vaccination in all vaccinated groups, and the administration concentration was safe for flatfish. It was confirmed that the vaccine was (P> 0.05, Table 29).

As a result of histopathological examination after multivalent vaccination, there was no difference in histopathological examination of liver, kidney and spleen between the vaccine group and the control group, confirming that the multivalent vaccine is safe for flatheads. (FIGS. 10 to 12).

As described above, when a specific part of the content of the present invention is described in detail, such a specific description is merely a preferable embodiment for a person having ordinary knowledge in the art, thereby expanding the scope of the present invention. It will be clear that there are no restrictions. Therefore, the substantial scope of the invention will be defined by the appended claims and their equivalents.

Claims (10)

Streptococcus paraubellis KCTC 13800BP strain isolated from streptococcal-infected scallops. Streptococcus paraubellis KCTC 13801BP strain isolated from streptococcal-infected scallops. Streptococcus paraubellis KCTC 13802BP strain isolated from streptococcal-infected scallops. Streptococcus paraubellis KCTC 13803BP strain isolated from streptococcal-infected scallops. A chain containing any one or more inactivated strains selected from the group consisting of inactivated Streptococcus paraubellis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain and KCTC 13803BP strain as an antigen. Activated vaccine. The inactivated vaccine for the prevention of streptococcal disease according to claim 5, wherein the vaccine is a mixture of the KCTC 13802BP strain and the KCTC 13803BP strain in a ratio of 1: 1. The inactivated vaccine for the prevention of streptococcal disease according to claim 5, wherein the vaccine is a mixture of the KCTC 13800BP strain, the KCTC 13802BP strain and the KCTC 13803BP strain in a ratio of 1: 1: 1. The inactivated vaccine for preventing streptococcal disease according to claim 5, wherein the antigen concentration of the vaccine is 0.5 to 30 mg / fish. Streptococcus vaccine isolated from streptococcal disease-infected strains KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain or KCTC 13803BP strain selected from one of the marine strains A method for producing an inactivated vaccine for preventing streptococcal disease, which comprises a step of activating to obtain a vaccine. A method for immunizing a fish, which comprises administering the vaccine of claim 5 to the fish.