KR102106168B1 - Combination Vaccines Against Streptococcal Disease in Fish - Google Patents

Abstract

The present invention relates to a novel strain of Streptococcus, and a vaccine for the prevention of streptococcosis using the same, and more specifically, to a novel strain of Streptococcus; an inactivated vaccine for the prevention of streptococcosis in fish, comprising an inactivated strain of Streptococcus parauberis; a method of preparing the same; and an administration method of the same. The present invention can effectively prevent streptococcosis in fish, particularly in farmed halibut, and thus can significantly reduce the death of fish due to streptococcosis. Moreover, the present invention can increase the productivity of fish and can reduce the costs associated with the use of antibiotics. Further, by using a vaccine capable of preventing bacterial diseases in fish, the present invention makes it possible to produce healthy fish without using antibiotics, thus contributing to the development of fish farming, and remarkably increasing fish consumption.

Description

Combination Vaccines Against Streptococcal Disease in Fish}

The present invention relates to a vaccine for preventing streptococcal disease using a Streptococcus strain, and more specifically, a multivalent vaccine for preventing streptococcal disease in fish containing the inactivated Streptococcus parauberis strain, a method of manufacturing and administration thereof It is about.

Streptococcosis is a disease that causes sepsis in fresh and marine freshwater and marine fish. It is infected with various fish species worldwide, such as yellowtail ( Seriolae spp.) And eel (Eel; Anguilla japonica ), causing disease. Streptococcus parauberis , Streptococcus iniae , Streptococcus agalactia e, and Streptococcus dysgalactiae species occur severely in fish anywhere in the world.

In 2014, the world production of marine products was 193.57 million tons, of which the production of fish was 94.3 million tons, similar to the level in recent years, while the production of aquaculture increased to 109.11 million tons annually. Therefore, it is expected to increase by 39% from 2013 to 2015 level (FAO). In 2017, domestic fish farming production amounted to 86,387 tons, and the production by fish type was 41,207 tons (47.7% of total production) in 2017, and in the same year, the production amount of halibut was 584 billion won (57.9%). It can be seen that it is a representative Korean fish farming variety (Fish Production Statistics, Statistics Korea, 2018). Olive flounder has been designated as one of the top 10 export strategic items in the fisheries sector promoted by the Ministry of Oceans and Fisheries, and is currently a high value-added variety promoted as a research and development project for aquaculture products.

The damage caused by diseases of farmed halibut is estimated to be about 120,000 tons per year, which accounts for 30% of the annual production, and the most common cause of mortality in farmed halibut in 2018 is the parasite-related disease, Scutica disease (44.5%), nymphosis ( 12%), viral hemorrhagic septicemia (16.6%), and bacterial disease, streptococcal disease (9.7%) (investigation of mortality of aquatic organisms and research on the effectiveness of disinfection devices for live fish transportation, National Fisheries Research Institute, 2018) ). Recently, the most common diseases in Korean flounder have been reported, parasitic diseases such as Scutica disease (61%), viral hemorrhagic sepsis (10.6%), and streptococcal disease caused by S. parauberis (9.8%). Research on the status of mortality and treatment of dead bodies, 2014-2016, National Institute of Fisheries Science). Streptococcus that is problematic in flounder farming is known as S. iniae and S. parauberis, and as a result of identifying streptococcus isolated from cultured flounder in Jeju from 2003 to 2005, S. parauberis 54%, S. iniae 46% Was separated at the rate of. But in the diseased flounder from the late 2000s to the present low proportion of S. iniae are not separated in the last few, most of the situation with S. parauberis separated.

The detection rate of pathogens in aquaculture fish is constantly increasing every year, and despite the spread of the S. parauberis vaccine, which is currently commercialized for halibut, the disease continues to occur, and thus it is necessary to improve the current vaccine antigen. With the emergence of drug-resistant bacteria and multi-drug-resistant bacteria caused by misuse of antibiotics and antibacterial agents, treatment efficacy with chemotherapeutic agents after the disease is gradually becoming difficult, so it is necessary to develop a vaccine that can prevent disease in advance. Indeed, in Korea, the vaccine of S. parauberis from halibut has been commercialized and distributed since 2009, but S. parauberis has been continuously occurring even after the vaccine was supplied (Aquatic Products Disease Research, 2012-2017). In general, Scutica disease, which causes the highest mortality in halibut, occurs in sting, but streptococcal disease occurs in medium-growth fish, adult fish, and the like, causing greater economic damage. As a result of the analysis of the causes of mortality caused by diseases in aquaculture halibut, mortality caused by bacterial disease was 13%, and the death caused by streptococcal disease was the highest (67.1%).

In Korea, various studies such as S. parauberis inactivated vaccine, recombinant protein vaccine, and attenuated vaccine have been conducted steadily, but currently only inactivated vaccines produced in the form of mixed vaccines with other bacterial pathogens are commercially available and commercially available (6 companies) 9 products). In Spain, Hipra's ICTHIOVAC (R) -STR inactivating vaccine, which targets S. parauberis as an antigen for turbots, has been developed and marketed, but in Korea, a single vaccine against S. parauberis has not been sold. Due to the nature of the fish farming industry, the development of low-cost fish vaccines is essential, so mixed vaccines that can prevent various bacterial pathogens are commercially available. However, the investigation of vaccine effects after using the mixed vaccines is insignificant. Streptococcus disease caused by S. parauberis in halibut continues to occur even after the vaccine is supplied, and it is necessary to verify the efficacy of the currently commercialized vaccine.

A vaccine made by mixing many types of the same type of human influenza vaccine or polio vaccine, that is, A1, A2, B type for influenza, and I, I, III type for polio, is called a polyvalent vaccine. , These pathogens are divided into several types of antigens, so if they are vaccinated with a vaccine (unit price vaccine) that is included separately, the multivalent vaccine is used because it does not help if the disease type occurs in a completely different type from the vaccine. In the case of S. pneumoniae , a streptococcus that causes pneumonia in humans, there are various serotypes, and 23 multivalent polysaccharide vaccines are used to prevent them (Jun-Young Song, Hee-Jin Jeong 2014, pneumococcal vaccine, J. Korean Med Assoc 57 (9 ), 780-788).

Accordingly, the present inventors conducted molecular epidemiology and serotype investigation through the Multilocus sqquecne typing (MLST) method for S. parauberis causing disease in halibut, selecting a suitable strain for the halibut streptococcus S. parauberis vaccine in Korea. Basic data for the following were secured, and cross-vaccination efficacy was evaluated by producing a unit price vaccine for each serotype. Based on the results of the cross vaccine efficacy evaluation, the vaccine efficacy was evaluated by mixing S. parauberis strains with different serotypes for the development of the S. parauberis multivalent vaccine, and the present invention was completed.

The above information described in this background section is only for improving the understanding of the background of the present invention, and thus does not include information that forms prior art already known to those of ordinary skill in the art. It may not.

An object of the present invention is to provide an inactivated vaccine for preventing streptococcal disease comprising an inactivated Streptococcus parauberis strain as an antigen.

Another object of the present invention comprises the step of obtaining a vaccine by inactivating Streptococcus parauberis strain isolated from halibut infected with Streptococcus disease with formalin to obtain a vaccine. It is to provide a method for producing a vaccine.

Another object of the present invention is to provide a method for immunizing fish comprising administering the vaccine to the fish.

In order to achieve the above object, the present invention is an inactivated Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain or KCTC 13803BP strain as an antigen, an inactivated vaccine for preventing streptococcal disease Gives

The present invention also provides a step of obtaining a vaccine by inactivating Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain, or KCTC 13803BP strain isolated from halibut infected with streptococcal disease. It provides a method for producing an inactivated vaccine for preventing streptococcal disease characterized in that it comprises.

The present invention also provides a method of immunizing fish comprising administering the vaccine to the fish.

The present invention has the effect of significantly preventing the death of fish caused by streptococcal disease by effectively preventing streptococcal disease of fish, especially cultured halibut. Therefore, it is possible to reduce the cost of using antibiotics as well as increasing the productivity of fish. In addition, the present invention, by using a vaccine that can prevent the bacterial disease of fish, by enabling the production of healthy fish without antibiotics, can contribute to the development of aquaculture and significantly improve fish consumption.

Figure 1 shows the nucleotide sequence of 5 genes of S. parauberis 64 strains [DNA gyrase subunit B ( gyrB ), Surface M-protein (simA), autolysin, capsular polysaccharide biosynthesis protein, tyrosine-protein kinase (wze)] This is a diagram showing the results of analysis of the main strain of about 4,120 bp).
Figure 2 shows the S. parauberis serotype analysis, Figure 2 (A) is a slide aggregation reaction (I: positive reaction, II: negative reaction), Figure 2 (B) is a diagram showing the microtiter aggregation reaction.
Figure 3 is a graph showing the serotype distribution of S. parauberis bacteria in Korea 2002-2017.
Figure 4 is a diagram showing the separation ratio by region and year of separation of S. parauberis in Korea between 2002 and 2017.
5 is a diagram showing the serotype-PCR results of an inactivated vaccine containing the S. parauberis antigen commercially available in Korea.
6 is a graph showing the cumulative mortality of flounder after artificial infection of 7 strains of S. parauberis .
7 is a graph showing the cumulative mortality of flounder by concentration of four strains of S. parauberis .
8 is a diagram schematically showing the schedule of S. parauberis vaccination and artificial infection experiments of halibut.
9 is a graph showing the results of antibody titer 2 weeks after S. parauberis multivalent vaccine inoculation.
10 is a result of the pathological histologic examination of the internal organs 1 week after inoculation of the S. parauberis polyvalent vaccine.
Figure 11 shows the pathological histologic results of internal organs 2 weeks after S. parauberis multivalent vaccine inoculation.
Figure 12 is the pathological histologic results of the internal organs 3 weeks after inoculation of the S. parauberis polyvalent vaccine.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein is well known in the art and commonly used.

In one embodiment of the present invention, after inactivating Streptococcus parauberis 19FBSPa0003 (PR5) strain, Streptococcus parauberis FP2331 strain, Streptococcus parauberis FPa4365 strain and Streptococcus parauberis FPa4870 strain isolated from halibut infected with streptococcal disease, comprising this as an antigen As a result of inoculating the vaccine with fish, all vaccinations showed low mortality, and it was confirmed that there was also a cross vaccine efficacy by serotype. In addition, it was confirmed that in the case of a polyvalent vaccine containing antigens according to the different serotypes, the vaccine efficacy was further increased compared to a single serotype vaccine.

Therefore, in one aspect, the present invention includes, as an antigen, any one or more strains selected from the group consisting of inactivated Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain, and KCTC 13803BP strain. It relates to an inactivated vaccine for preventing streptococcal disease.

In the present invention, the KCTC 13800BP strain is a Streptococcus parauberis 19FBSPa0003 strain deposited as of January 30, 2019 at the Korean Collection for Type Culture, where 19FBSPa0003 means the same strain as PR5.

In the present invention, the KCTC 13801BP strain is a Streptococcus parauberis FP2331 strain deposited in January 30, dated 2019 in Korea, Biological Resource Center, the KCTC 13802BP strains of Streptococcus deposited in January 30, dated 2019 in Korea, Biological Resource Center The parauberis FPa4365 strain, and the KCTC 13803BP strain is a Streptococcus parauberis FPa4870 strain deposited with the Korea Biological Resource Center on January 30, 2019.

In the present invention, the vaccine may be characterized in that the KCTC 13802BP strain and the KCTC 13803BP strain are mixed in a ratio of 1: 1, but is not limited thereto.

In the present invention, the vaccine may be characterized in that the KCTC 13800BP strain, KCTC 13802BP strain and KCTC 13803BP strain are mixed in a ratio of 1: 1: 1, but is not limited thereto.

In the present invention, the antigen concentration of the vaccine may be characterized in that 1 to 30mg / fish.

In one embodiment of the present invention, as a result of measuring the antibody titer of serum by the type of antigen (serum type Ia ~ II) after vaccination including Streptococcus parauberis 19FBSPa0003 (PR5) strain, FP2331 strain, FPa4365 strain or FPa4870 strain, control Compared to (PBS), all antibody titers formed significantly higher, and each vaccine cell showed the highest antibody titer when measuring the antibody titer with the same serotype antigen.

In addition, artificial infection with S. parauberis serotype Ia + Ic vaccine and Ia + Ib + Ic vaccine showed high survival rate compared to control, and high relative survival rate compared to S. parauberis single serotype vaccine.

In the present invention, the term "vaccine" is an antigen used for immunity to prevent an infectious disease, and is made of attenuated microorganisms or viruses, and is a hospital that is attenuated or killed to artificially obtain immunogenicity against a specific infectious disease It means that the microorganism is administered in the individual. When stimulated by a vaccine, the immune system in the individual acts to produce antibodies, and if it maintains sensitivity and re-infection occurs, it is possible to overcome the disease by effectively generating antibodies in a short time.

The inactivated vaccine may be prepared using methods well known in the art, for example, using an inactivating agent known in the art. Preferably, it can be characterized in that it is inactivated by formalin treatment, but is not limited thereto.

The mixed inactivated vaccine according to the present invention may further include an adjuvant. The adjuvant can be used without limitation as long as it is known in the art, but preferably, Montanide ISA35 or Gel01 can be used.

In addition, as stabilizers or additives, sugars or amino acids, mineral oil, vegetable oil, alum, aluminum phosphate, bentonite, silica, muramyl dipeptide derivatives, cymosin, interleukin, and the like may be used.

In the present invention, the vaccine may be used by dispensing the vaccine solution into a vial having an appropriate volume, for example, about 10 to 500 ml, and sealing the vaccine solution. These vaccines can be used as dry preparations, as well as in liquid form, by injecting and then lyophilizing. The dry formulation may be used by completely re-dissolving the dry substance with the antiseptic 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. It is not limited to this, but can be used for medium-grown fish, including pompoms, and adult words.

According to another aspect of the present invention, Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain, and KCTC 13803BP strain isolated from flounder infected with streptococcal disease It relates to a method for producing an inactivated vaccine for preventing streptococcal disease, characterized in that it comprises the step of obtaining a vaccine by inactivating with formalin.

In the present invention, the vaccine is prepared by incubating 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, 4 strains of cryopreserved S. parauberis were cultured, and 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution, and 0.5% (v / v) of each culture solution was obtained. The mixture was inactivated at 150 rpm and 20 ° C. in a stirred incubator for 24 hours. After centrifugation of the incubation solution, the inactivation was confirmed, washed with sterile physiological saline, the cells were collected, and finally, the weight of the cells was measured, and the vaccine was prepared by varying the concentration according to the experimental group.

In another aspect, the present invention relates to a method for immunizing fish comprising administering to the fish an inactivated vaccine for preventing streptococcal disease.

In the present invention, the vaccine may be characterized in that it is injected into the abdominal cavity of fish, but is not limited thereto.

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

In one embodiment of the present invention, after treatment with four S. parauberis vaccines, as a result of performing an artificial infection test for each serotype, it showed a low mortality rate in all vaccinated cells compared to the control group not vaccinated, It was confirmed that there was also a cross vaccine efficacy by serotype. In addition, it was confirmed that the multivalent vaccine comprising S. parauberis serotypes Ia to Ic showed higher vaccine efficacy compared to the single serotype vaccine.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Streptococcus S. parauberis Separation and identification

Among the 199 S. parauberis strains isolated from domestic diseased flounder from 2002 to 2017, which were preserved by the National Institute of Fisheries Science and Pathology, 64 representative strains were selected and used for analysis.

After spreading the strain on a tryptic soy agar (TSA, Becton Dickinson, USA) or brain heart infusion agar (BHIA, Becton Dickinson, USA) medium with 1% NaCl, the cultures were incubated at 25 ° C for 24 hours to confirm the properties of the bacteria. Then, it was subcultured to perform phenotype, serotype, and genetic analysis.

Table 1 shows the separation regions and years of the analyzed strains.

S. parauberis strain isolation region information year Strain (number) Jeju Wando Pohang Ulsan Geoje Tongyeong sum 2002 One One 2003 One One 2004 One One 2 2005 One 2 3 2006 4 One 5 2007 One One 2 2008 5 One 6 2010 2 2 2012 2 3 4 9 2013 One One 2014 8 4 12 2015 9 9 2016 2 One 2 5 2017 One One 2 2 6 sum 25 9 17 6 5 2 64

Example 2:  S. parauberis Phenotypic analysis of

In order to analyze the biochemical properties, S. parauberis strains were incubated for 24 hours at 25 ° C. in BHIA medium with 1% NaCl added. The API 20strep kit (BIOMERIEUX, France) and API ZYM kit (BIOMERIEUX, France) test methods were tested by inoculating and culturing the pure cultured colonies on a strip according to the manufacturer's method.

As a result of analysis of API 20strep and API zym kit of 64 strains of S. parauberis , all the investigated strains were identified as catalase and oxidase negative as motility gram-positive cocci (Table 2).

Analysis of biochemical properties of 64 S. parauberis strains from halibut API20stretp (Number of positive-reacting strains) APIzym (number of positive reaction strains) PA AD RI MA SO LA TR IN AM 2 3 4 5 7 8 10 11 14 16 3 5 7 13 12 17 51 7 One 43 One 43 2 21 11 25 61 5 23

PA: alkaline phospatase, 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 phospatase, 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 API 20strep biochemical characterization, all strains are pyruvate acetoin production (VP), hippurate hydrolysis (HIP), β-glucosidase (ESC), pyrrolidonyl arylamidase (PYRA), leucine arylamidase (LAP) positive, α-galactosidase (αGAL) , β-galactosidase (βGUR), L-arabinose acidification (ARA), Raffinose acidification (RAF), and Glycogen acidification (GLYG) showed negative reactions (Table 3).

Results of API 20strep biochemical characterization of 64 S. parauberis strains No. FP No. VP HIP ESC PYRA αGAL βGUR βGAL PAL LAP ADH RIB ARA MAN SOR LAC TRE INU RAF AMD GLYG βHEM One FPa2041 ^Ia + + + + - - - - + - - - + + + + - - - - 2 FPa3132 + + + + - - - - + - - - - - - + - - - - 3 FP4076 + + + + - - - - + - + - + + - + + - - - 4 FP4114 + + + + - - - - + - - - - - - + - - - - 5 FP2199 + + + + - - - - + - - - - - - + - - - - 6 FP5042 + + + + - - - + + - + - + + - + - - - - 7 FP3197 + + + + - - - - + - + - - - - + - - - - 8 FP2333 + + + + - - - - + - - - + - - + - - - - 9 FP3335 + + + + - - - - + - - - - - - + - - - - 10 FP3380 + + + + - - - - + - - - - - - + - - - - 11 FP3441 + + + + - - - - + - - - - - - + - - - - 12 FP3446 + + + + - - - - + - - - - - - + - - - - 13 FP3826 ^Ia + + + + - - - - + - + - + + + + + - - - 14 FPa4365 ^Ia + + + + - - - - + - - - - - + + - - - - 15 FPa4369 ^Ia + + + + - - - - + - - - - - + + - - - - 16 FPa4403 ^Ia + + + + - - - - + - - - + + + + - - - - 17 FPa4404 ^Ia + + + + - - - - + - - - + + + + - - - - 18 Fpa4290 ^Ia + + + + - - - - + - - - - - - - - - - - 19 Fpa4293 ^Ia + + + + - - - - + - - - - - - - - - - - 20 Fpa4325 + + + + - - - - + - - - + - - + + - + - 21 Fpa4406 ^Ia + + + + - - - - + - - - - - - - - - - - 22 Fpa4407 + + + + - - - - + - - - - - - + - - - - 23 Fpa4599 ^Ia + + + + - - - - + - - - - - - - - - - - 24 Fpa4616 ^Ia + + + + - - - - + - - - - - - - - - - - 25 Fpa4624 ^Ia + + + + - - - - + - - - - - - - - - - - 26 Fpa4658 + + + + - - - - + - - - - - - + - - - - 27 Fpa4660 ^Ia + + + + - - - - + - - - - - - + - - - - 28 Fpa4661 + + + + - - - - + - - - - - - + - - - - 29 Fpa4683 ^Ia + + + + - - - - + - - - - - - + - - - - 30 Fpa4710 ^Ia + + + + - - - - + + + - + + + + + - - - 31 Fpa4743 ^II + + + + - - - - + - - - - - - + - - - - 32 Fpa4804 ^Ia + + + + - - - - + + - - + + - + - - - - 33 Fpa4805 ^Ia + + + + - - - - + - - - - - - + - - - - 34 Fpa4806 ^Ia + + + + - - - - + - - - - - - - - - - - 35 Fpa4807 ^Ia + + + + - - - - + - - - - - - - - - - - 36 Fpa4850 + + + + - - - - + - - - - - - + - - - - 37 Fpa4860 ^Ia + + + + - - - - + - - - - - - + - - - - 38 Fpa4870 + + + + - - - - + - - - - - - + - - - - 39 Fpa4894 + + + + - - - - + - - - - - - + - - - - 40 BS1K (Pohang-Buseong 1K) ^Ia + + + + - - - - + - - - - - + + + - - - 41 DK14 (Daeguan Fisheries 14) ^Ia + + + + - - - - + - - - - - + + - - - - 42 DW C7-3 (September Daewon C7-3) + + + + - - - + + - - - - - - + - - - - 43 DW1 (Daewon Hospital 1 in September) + + + + - - - + + - - - - - - + - - - - 44 YL3 (Jeju Younglim 3) + + + + - - - - + - - - - - - + - - - - 45 PR 5KJK (Blue Blue 5KJK) + + + + - - - - + - - - - - - + - - - - 46 FP3825 ^Ia + + + + - - - - + - - - - - + + - - - - 47 FPa4577 ^Ia + + + + - - - - + - - - - - - - - - - - 48 FPa4578 ^Ia + + + + - - - - + - - - - - - + - - - - 49 FPa4586 ^Ia + + + + - - - - + + - - - + + + - - - - 50 FPa4587 + + + + - - - - + - - - + - - + - - - - 51 SPOF3K ^Ia + + + + - - - - + - - - - - + + - - - - 52 KSP28 + + + + - - - - + - - - - - - + - - - - 53 PH0710 ^Ia + + + + - - - - + - - - - - + + - - - - 54 FP2331 ^II + + + + - - - - + - - - - - - - - - - - 55 FP2332 ^II + + + + - - - - + - - - - - - - - - - - 56 YJ3 (Geoje Breeding Center 3) ^Ia + + + + - - - - + + + - + + + + + - - - - 57 YJ7 (Geoje Breeding Center 7) ^Ia + + + + - - - - + + + - + + + + + - - - - 58 DB1 ^Ia + + + + - - - - + - - - - - + + - - - - 59 FP2313 + + + + - - - - + - - - - - - + - - - - 60 FP3439 + + + + - - - - + - - - - - - + - - - - 61 FP3440 + + + + - - - - + - - - - - - + - - - - 62 DK-M3 + + + + - - - - + - - - - - - + - - - - 63 DK-L2 ^Ia + + + + - - - - + - - - - - - - - - - - 64 best7-1 ^Ia + + + + - - - - + - - - - + + - - - - -

^Ia is serotype Ia, and ^II is serotype II.

As a result of API zym kit biochemical analysis, all strains are leucine acrylamidase, Naphtol-AS-BI-phosphohydrolase positive, typsin, β-galactosidase, β-glucuronidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase , α-fucosidase showed a negative reaction (Table 4).

Results of API zym kit biochemical characterization of 64 S. parauberis strains No. FP No. One 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 One FPa2041 ^Ia C + - + - ++ - - - - + + - - - - - - - - 2 FPa3132 C + - + - ++ - - - + + + - - - - - - - - 3 FP4076 C + - + - ++ - - - - + + - - - - - - - - 4 FP4114 C + - + - ++ - - - + ++ + - - - - - - - - 5 FP2199 C + - + - ++ - - - + + + - - - - - - - - 6 FP5042 C + + + - ++ + + - - ++ + - - - + - - - - 7 FP3197 C + - + - ++ - - - + + + - - - - - - - - 8 FP2333 C + - + - ++ + - - + ++ + - - - - - - - - 9 FP3335 C + - + - ++ - - - + ++ + - - - + - - - - 10 FP3380 C + - + - ++ - - - - + + - - - - - - - - 11 FP3441 C + - + - ++ + - - + ++ + - - - + - - - - 12 FP3446 C + - + - ++ - - - - + + - - - - - - - - 13 FP3826 ^Ia C + - + - ++ - - - - + + - - - - - - - - 14 FPa4365 ^Ia C + - + - ++ - - - + + + - - - - - - - - 15 FPa4369 ^Ia C + - + - ++ - - - - + + - - - - - - - - 16 FPa4403 ^Ia C + - + - ++ - - - - + + - - - - - - - - 17 FPa4404 ^Ia C + - + - ++ - - - - + + - - - - - - - - 18 Fpa4290 ^Ia C + - + - ++ + + - + ++ + - - - - - - - - 19 Fpa4293 ^Ia C + - - - ++ + - - + + + - - - - - - - - 20 Fpa4325 C - - - - ++ - - - + + + - - - - - - - - 21 Fpa4406 ^Ia C + - + - ++ + - - + ++ + - - - - - - - - 22 Fpa4407 C - - - - ++ - - - + + + - - - - - - - - 23 Fpa4599 ^Ia C - - - - ++ - - - - - + - - - - - - - - 24 Fpa4616 ^Ia C - - - - ++ - - - - - + - - - - - - - - 25 Fpa4624 ^Ia C - - - - ++ - - - - - + - - - - - - - - 26 Fpa4658 C + - + - ++ - - - - ++ + - - - - - - - - 27 Fpa4660 ^Ia C + - + - ++ - - - - + + - - - - - - - - 28 Fpa4661 C + - + - ++ - - - - ++ + - - - + - - - - 29 Fpa4683 ^Ia C - - + - ++ - - - - + + - - - - - - - - 30 Fpa4710 ^Ia C - - + - ++ - - - - + + - - - - - - - - 31 Fpa4743 ^II C - - - - ++ - - - - + + - - - - - - - - 32 Fpa4804 ^Ia C - - + - ++ - - - - + + - - - - - - - - 33 Fpa4805 ^Ia C + - + - ++ - - - - + + - - - - - - - - 34 Fpa4806 ^Ia C - - + - ++ - - - - + + - - - - - - - - 35 Fpa4807 ^Ia C - - + - ++ - - - - + + - - - - - - - - 36 Fpa4850 C - - - - ++ - - - - + + - - - - - - - - 37 Fpa4860 ^Ia C - - + - ++ - - - - + + - - - + - - - - 38 Fpa4870 C - - - - ++ - - - - + + - - - - - - - - 39 Fpa4894 C - - - - ++ - - - + + + - - - - - - - - 40 BS1K (Pohang-Buseong 1K) ^Ia C + - + - ++ - - - - + + - - - + - - - - 41 DK14 (Daeguan Fisheries 14) ^Ia C - - + - ++ - - - + + + - - - - - - - - 42 DW C7-3 (September Daewon C7-3) C - - - - ++ - - - + + + - - - - - - - - 43 DW1 (Daewon Hospital 1 in September) C - - - - ++ - - - + + + - - - - - - - - 44 YL3 (Jeju Younglim 3) C + - + - ++ + - - + + + - - - - - - - - 45 PR 5KJK (Blue Blue 5KJK) C + - + - ++ - - - - + + - + - + - - - - 46 FP3825 ^Ia C + - - - ++ + - - - + + - - - + - - - - 47 FPa4577 ^Ia C + - + - ++ + + - + + + - - - + - - - - 48 FPa4578 ^Ia C + - + + ++ + + - + ++ + - - - + - - - - 49 FPa4586 ^Ia C - - - - ++ - - - - + + - - - + - - - - 50 FPa4587 C - - - - ++ + + - + ++ + - + - + - - - - 51 SPOF3K ^Ia C + - + - ++ + + - + ++ + - + - + - - - - 52 KSP28 C + - - - ++ + - - + + + - - - + - - - - 53 PH0710 ^Ia C + - + + ++ + + - + ++ + - - - + - - - - 54 FP2331 ^II C + - - - ++ + - - - ++ + - - - + - - - - 55 FP2332 ^II C + - - - ++ + - - - ++ + - - - + - - - - 56 YJ3 (Geoje Breeding Center 3) ^Ia C + - + - ++ + + - - + + - - - - - - - - 57 YJ7 (Geoje Breeding Center 7) ^Ia C + - + - ++ + + - - + + - - - - - - - - 58 DB1 ^Ia C + - + - ++ + + - - + + - - - + - - - - 59 FP2313 C + - - - ++ - - - - + + - + - + - - - - 60 FP3439 C - - - - ++ - - - - + + - - - + - - - - 61 FP3440 C + - + - ++ - - - - + + - - - + - - - - 62 DK-M3 C + - + - ++ + + - + + + - + - + - - - - 63 DK-L2 ^Ia C + - - - ++ - - - - + + - - - + - - - - 64 best7-1 ^Ia C + - + - ++ + - - - ++ + - - - - - - - -

^Ia is serotype Ia, and ^II is serotype II.

Example 3:  S. parauberis Genotyping

Example 3-1: Primer preparation and gene sequence analysis

To analyze the genetic properties, each isolate was cultured in BHIB medium with 1% NaCl added for 24 hours, followed by centrifugation at 13,000 rpm for 2 minutes and removal of the supernatant to prepare a bacterial pellet. For bacterial pellets, DNA was extracted using a Promega Genomic DNA purification kit (Promega, USA). Housekeeping gene groEL, gyrB was used as a Primer reporting period, S. parauberis KCTC 11537 (Nho et al., 2011) and KCTC 11980 (Park et al., 2013) are estimated to be antigenic, pathogenic genomes from related Six genes (4 capsule polysaccharides, 1 autolysin, and 1 M-like protein) were selected and primers capable of specifically amplifying were prepared and analyzed (Table 5).

PCR primer information for genes related to housekeeping, antigen, and pathogenicity of S. parauberis gene Targets Primer Sequence
(5´to 3´) PCR product size
(bp) References
(Genbank accession no./protein ID) 16S rRNA 16S rRNA 63F 5'-CAGGCCTAACACATGCAAGTC-3 '
(SEQ ID NO: 1) 1,343 Han et al. (2015) Fisheries and aquatic sciences 18 (3): 333-339 1406R 5'-ACGGGCGGTGTGTRC-3 '
(SEQ ID NO: 2) groEL Molecular chaperon GroEL StreptogroELd 5'-GAHGTNGTiGAAGGiATGCA-3 '
(SEQ ID NO: 3) 796 Glazunova et al. (2009) Int J Syst Evol Microbiol 59, 2317-2322 StreptogroELr 5'-ATTTGRCGiAYWGGYTCTTC-3 '
(SEQ ID NO: 4) gyrB DNA gyrase subunit B StreptogyrBd 5'-GGTTCiTCNGTiGTTAATGC-3 '
(SEQ ID NO: 5) 498 Glazunova et al. (2009) Int J Syst Evol Microbiol 59, 2317-2322 StreptogyrBr 5'-GTTCRTGNGTTCCiCCTTCAT-3 '
(SEQ ID NO: 6) simA Surface M-protein simAF 5'-GCTAAGGTCATGACCCATTC-3 '
(SEQ ID NO: 7) 800 This study
(CP002471.1 / AEF25898.1) simAR 5'-AGGCGTGCAATTTCAGCTTG-3 '
(SEQ ID NO: 8) atlI Autolysin
(type I) autoF 5'-CTCAAACTGAGGCTAGTAAT-3 '
(SEQ ID NO: 9) 1,360 This study
(CP002471.1 / AEF25898.1) autoR 5'-GTCCCGGCTAATCCAACTGT-3 '
(SEQ ID NO: 10) cpb Capsular polysaccharide biosynthesis protein CPB-F 5'-CTTGGTGGATCCCAAGCAC-3 '
(SEQ ID NO: 11) 428 This study
(CP002471.1 / AEF25344.1) CPB-R 5'-CCAAACAACCACTCTCCAGC-3 '
(SEQ ID NO: 12) Soa Sugar O-acyltransferase, sialic acid SOA-F 5'-GCAAGCGGTCACGGAAAAGT-3 '
(SEQ ID NO: 13) 559 This study
(CP002471.1 / AEF25347.1) SOA-R 5'-CACCTACATAGGTTCCACTA-3 '
(SEQ ID NO: 14) cpb Polysaccharide biosynthesis protein PB-F 5'-GCAAATCTGTCGGCTTTTCG-3 '
(SEQ ID NO: 15) 1,290 This study
(CP002471.1 / AEF25350.1) PB-R 5'-ACGACTACCAAGGACGTTAC-3 '
(SEQ ID NO: 16) Wze Tyrosine-protein kinase TPK-F 5'-CCGAAGAGTACTATAACTCC-3 '
(SEQ ID NO: 17) 641 This study
(CP002471.1 / AEF25351.1) TPK-R 5'-TGGCTTACCATAATTCCCGT-3 '
(SEQ ID NO: 18) Wzy Polysaccharide polymerase gene For-Ia ATTGTTAGTCATTCAGTTGT
(SEQ ID NO: 19) 213 Tu et al., (2015) Fish Pathology. 50 (4), 213-215 Rev-Ia AATTATAGTCAACAGTCCAG
(SEQ ID NO: 20) For-Ib / c ATTTCTACCAGGTTACTTTG
(SEQ ID NO: 21) 303 Rev-Ib / c ATTTCTACCAGGTTACTTTG
(SEQ ID NO: 21) For-II GAACTACTTAGGTTTAGCAT
(SEQ ID NO: 22) 413 Rev-II AACTTGTAAATAGGATTGCT
(SEQ ID NO: 23)

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

PCR was amplified with the prepared primer, and the PCR product was electrophoresed using a 1.5% agarose gel (Bioneer, Korea) containing EtBr, and then the band was confirmed using a UV transilluminator (Alpha Innotech, USA), and the sequence was analyzed. (Tables 6 to 14).

16S rRNA gene sequence (1,287bp) of 64 Streptococcus parauberis strains isolated from flounder Gene Name (Type) Base sequence (5 '→ 3') 16S rRNA GCCTAGTCAGATGAGTTGCGAACGGGTGAGTAACGCGTAGGTAACCTACCTCATAGCGGGGGATAACTATTGGAAACGATAGCTAATACCGCATGACAATTAAGTACTCATGTACTAAATTTAAAAGGAGCAATTGCTTCACTATGAGATGGACCTGCGTTGTATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATACATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGGGGCAACCCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGAGAAGAACGGTAATGGGAGTGGAAAATCCATTACGTGACGGTAACTAACCAGAAAGGGACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTCTCGAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTATTTAAGTCTGAAGTTAAAGGCCGTGGCTCAACCATGGTTCGCTTTGGAAACTGGATAACTTGAGTGCAGAAGGGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCGGTGGCGAAAGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAGGTGTTAGGCCCTTTCCGGGGCTTAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCCTCTGACCGTCCTAGAGATAGGACTTTCCTTCGGGACAGAGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGT GAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTGGTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCAATCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGC (SEQ ID NO: 24)

GyrB gene sequence of 4 strains of Streptococcus parauberis isolated from flounder (465 bp) Gene typing Base sequence (5 '→ 3') gyrB-A TCATCTCAGTTAGATGTTCGTGTCTTCAAAAATGGTAGCATTCATTATCAAGAGTTTAAACGTGGAATCGTTTATGATGATTTGAAAATTATTGGAGAAACGGATTTAACAGGAACAACAGTTCACTTTACTCCGGACCCAGAAATTTTTACAGAAACAGTTGAATTTGATTTTGAAAAGCTTGCTAAGCGTGTCCAAGAATTAGCATTTTTAAACCGTGGATTACGAATCTCAATTGCCGATAAACGTAATGGAATTGAACAAGTCAAAGATTACCATTATGAAGGTGGTATCGCTAGCTATGTTGAATTTTTAAATGAGAAAAAAGATGTTATTATCGAACATCCTATCTTTACTGATGGAGAGATGGATGGCATTGCTGTCGAAGTTGCGATGCAATATACAACAGGCTATCATGAAAATGTAATGAGTTTTGCCAACAATATTCATACACATGAAGGCGGA (SEQ ID NO: 25) gyrB-B TCATCTCAGTTAGATGTTCGTGTCTTCAAAAATGGTAGCATTCATTATCAAGAGTTTAAACGTGGAATCGTTTATGATGATTTGAAAATTATTGGAGAAACGGATTTAACAGGCACAACAGTTCACTTTACTCCGGACCCAGAAATTTTTACAGAAACAGTTGAATTTGATTTTGAAAAGCTTGCTAAGCGTGTCCAAGAATTAGCATTTTTAAACCGTGGATTACGAATCTCAATTGCCGATAAACGTAATGGAATTGAACAAGTCAAAGATTACCATTATGAAGGTGGTATCGCTAGCTATGTTGAATTTTTAAATGAGAAAAAAGATGTTATTATCGAACATCCTATCTTTACTGATGGAGAGATGGATGGCATTGCTGTCGAAGTTGCGATGCAATATACAACAGGCTATCATGAAAATGTAATGAGTTTTGCCAACAATATTCATACACATGAAGGCGGA (SEQ ID NO: 26)

The sequence of groE gene of 64 strains of Streptococcus parauberis isolated from flounder (769 bp) Gene typing Base sequence (5 '→ 3') groE-A GGATGCAGTTTGACCGTGGTTACCTTTCTCAATACATGGTAACAGATAACGAAAAAATGGTTGCTGACTTAGAAAATCCATTTATTTTGATCACTGATAAAAAAGTATCAAACATTCAAGAAATTCTTCCACTCTTAGAAGAAGTCTTAAAAACAAACCGCCCATTATTGATTATTGCTGATGATGTTGATGGTGAAGCCTTACCAACATTAGTTTTAAATAAAATTCGCGGAACATTTAATGTTGTTGCCGTTAAAGCACCAGGATTTGGCGACCGTCGTAAGGCTATGTTAGAGGATATTGCCATTCTAACAGGTGGAACTGTGATTACTGAAGATCTTGGTTTAGAGCTTAAGGATGCGACAATAGCAGCGCTAGGCCAAGCAGCTAAAATCTCTGTGGATAAAGATTCAACAGTTATTGTTGAAGGTTCTGGCGGTGCGGATGCTATTGCCAATCGTGTTGGCCTAATTAAATCCCAATTAGAAACAACAACGTCTGAGTTCGACCGTGAGAAATTGCAAGAACGTCTAGCTAAGTTAGCGGGTGGTGTGGCAGTTATCAAGGTTGGTGCTGCGACAGAAACTGAGTTGAAAGAAATGAAGCTCCGCATTGAAGATGCTTTGAATGCTACTCGTGCTGCTGTTGAAGAAGGGATCGTAGCTGGTGGAGGAACTGCCTTTATTTCAGTGATTGAGAAAGTGCAAGCTCTAGAACTTGAAGGTGATAATGCGACTGGTCGTAACATTGTCCTTCGTGCCCTTGAAGA (SEQ ID NO: 27)

SimA gene sequence (714bp) of 64 Streptococcus parauberis isolates from flounder Gene typing Base sequence (5 '→ 3') simA-A GCTAAAGAGGCTGAAGTTGCTAACAATCGTGTTATTGTTTTAAGTAATGCTTTAAATCAAGCTAAAGAAGAATTTGATTTGAAAGCTGCTGAAGCACAAAAACTGAATAATAACGTCACTGTATTATCAAATGCCCTACAACAAGCTAAAGAAGAATTTGACAAAAAAGCCCAGGACTATCAAGCAGCTCTTGCAGAAAAAGATGCTATTGACGGACAATTAGAAACTGCTTTGACTAAAGTTTCAGACCTAGAAAGTCAATTGGCAAGTTTAAAAGAAGCTAATGAAGCTGCTAATCAAAAGATTACTCAATTAGAACAAGAAGTTGCTGATGGTGGGGTTGCCTTTAAAGACCTTAAAGGGCAAAAAGAAAAATCAGACGCACAAGTTAAAGCTCAAGCAGATAAGATTAAAGAACTGGAATCTGACTTAGAAGCGACTCAATCAGAATTAAACCAGGTGAAAGAGCAACGTGCTAATTTAGAACAAGAAGTTGCTGAGCTTGGAACTAAGGTAAAAGATTTAGAAAAAGAAAAAGCTGATTTAGAGAAAAAAGCGATAGACTTGGAAGCTGACAAAGCGGAATTTGAAGCAGAAATGGATCGTTTAAAAGCCTTACTAGCTGATAAAGACTTAGCAAATGATAAATTACTTAAAGAAATGGAAGATCTACAAAAAGACTTTGATACTAAGAAAAATCATAGTGACAGAGAA (SEQ ID NO: 28) simA-B GCTAAAGAGGCTGAAGTTGCTAACAATCGTGTTATTGTTTTAAGTAATGCTTTAAATCAAGCTAAAGAAGAATTTGATTTGAAAGCTGCTGAAGCACAAAAACTGAATAATAACGTCACTGTATTATCAAATGCCCTACAACAAGCTAAAGAAGAATTTGACAAAAAAGCCCAGGACTATCAAGCAGCTCTTGCAGAAAAAGATGCTATTGACGGACAATTAGAAACTGCTTTGACTAAAGTTTCAGACCTAGAAAGTCAATTGGCAAGTTTAAAAGAAGCTAATGAAGCTGCTAATCAAAAGATTACTCAATTAGAACAAGAAGTTGCTGATGGTGGGGTTGCCTTTAAAGACCTTAAAGGGCAAAAAGAAAAATCAGACGCACAAGTTAAAGCTCAAGCAGATAAGATTAAAGAACTGGAATCTGACTTAGAAGCGACTCAATCAGAATTAAACCAGGTGAAAGAGCAACGTGCTAATTTAGAACAAGAAGTTGCTGAGCTTGGAACCAAGGTAAAAGATTTAGAAAAAGAAAAAGCTGATTTAGAGAAAAAAGCGATAGACTTGGAAGCTGACAAAGCGGAACTTGAAGCAGAAATGGATCGTTTAAAAGCCTTACTAGCTGATAAAGACTTAGCAAATGATAAATTACTTAAAGAAATGGAAGATCTACAAAAAGACTTTGATACTAAGAAAAATCATAGTGACAGAGAA (SEQ ID NO: 29)

Autolysin gene sequence of 12 strains of Streptococcus parauberis isolated from flounder (1237bp) Gene typing Base sequence (5 '→ 3') autolysin-A GTGAAGCAATTGCTATTGATGCATCTACTCAAAAGTCAGAACTAAATAGTAATACAGAAAGTTCAACTCCCGAAATAGAATCAGTTCCAAGTGCTCCAACTGAAGTCACAAATCAGGTTCAAACTCAAACTGCTATCCAGTCAAAAAGTGTCGCACCAGTCGAAACAGTCGAAACAGTCTCAGCTGATGTGAGTGGTCATGTTTTAAAAATTGTCTATAATGGGCAACTAGAACCAACTAAGAAGATAAAGTATGCAGTTTGGACAGATAATGCTGGCCAAGATGATTTAGTTTGGTATACTGCTGATCAAGTTGGTGCGGCATATATTGATTTATCTAAAAAACATAGAGCATATGGGCTATATAATATTCATACCTATTCGCAAGATGTAACAGGTAAGATGTCTGGCTTAAATGCGAGACAATTTACTATTCTTAAACCAACAGTAAGTACATCGTTTAACGTACAAACTAATGGTCTTGTTGATATTGTCGTTTCAAATGTGAGAGGTGATATTAGCTCAATTAAAGTTCCTGTTTGGTCAGATTTAGGTGGCCAAAACGATATTAAATGGTATCAAGCTACTCAATCAACTGATGGAACATACAAAGTTTCAGTAAAGATTTCAGATCATTCAAATGATACTGGCCATTTTGCTGTTCATGTTTATGGGAATAGTACAATCACGAATAGTCAAATCGGATTAGGGACAACAGAAGGTTTTACAATTGCTACCCCAGACCCCCAAAATGTAGTGTCAGTCGTAGTAGCTAATGATGGTTTCCACCTAGCATTAAATTCGAATGTTGTTAAAGAATTTACCAAAGTGAAATTTGCTGTTTGGTCGGATCAAGCTGGTCAAGATGACCTTCATTGGTACACAGCCAATGCTCAGGGTCAAGTCATTGTTCCTTATGTTAATCATAGTAATTATGGTCTATACAACATCCATACCTATAGTTTTGAGTCGGGAAGTGCTAAAGGTCTAAATACAAGAAC AATTACAGTACCAAATCCAACTGCTAGCGCAGCCATTACACAGAAATCAGATCTTGAATTTTTAGTATCAGTTACGAATGTCCCAGCTTATATCACAAAAGTTATGTTACCAAGTTGGAGTGAAATTAATGGCCAAGATGATATCAAGTGGGTGACGGCAAGTAAGCTAGCTGATAATATAATATAGATATAGATA autolysin-A1 GTGAAGCAATTGCTATTGATGCATCTACTCAAAAGTCAGAACTAAATAGTAATACAGAAAGTTCAACTCCCGAAATAGAATCCCTTCCAAGTGCTCCAACTGAAGTCACAAATCAGGTTCAAACTCAAACTGCTATCCAGTCAAAAAGTGTCGCACCAGTCGAAACAGTCGAAACAGTCTCAGCTGATGTGAGTGGTCATGTTTTAAAAATTGTCTATAATGGGCAACTAGAACCAACTAAGAAGATAAAGTATGCAGTTTGGACAGATAATGCTGGCCAAGATGATTTAGTTTGGTATACTGCTGATCAAGTTGGTGCGGCATATATTGATTTATCTAAAAAACATAGAGCATATGGGCTATATAATATTCATACCTATTCGCAAGATGTAACAGGTAAGATGTCTGGCTTAAATGCGAGACAATTTACTATTCTTAAACCAACAGTAAGTACATCGTTTAACGTACAAACTAATGGTCTTGTTGATATTGTCGTTTCAAATGTGAGAGGTGATATTAGCTCAATTAAAGTTCCTGTTTGGTCAGATTTAGGTGGCCAAAACGATATTAAATGGTATCAAGCTACTCAATCAACTGATGGAACATACAAAGTTTCAGTAAAGATTTCAGATCATTCAAATGATACTGGCCATTTTGCTGTTCATGTTTATGGGAATAGTACAATCACGAATAGTCAAATCGGATTAGGGACAACAGAAGGTTTTACAATTGCTACCCCAGACCCCCAAAATGTAGTGTCAGTCGTAGTAGCTAATGATGGTTTCCACCTAGCATTAAATTCGAATGTTGTTAAAGAATTTACCAAAGTGAAATTTGCTGTTTGGTCGGATCAAGCTGGTCAAGATGACCTTCATTGGTACACAGCCAATGCTCAGGGTCAAGTCATTGTTCCTTATGTTAATCATAGTAATTATGGTCTATACAACATCCATACCTATAGTTTTGAGTCGGGAAGTGCTAAAGGTCTAAATACAAGAAC AATTACAGTACCAAATCCAACTGCTAGCGCAGCCATTACACAGAAATCAGATCTTGAATTTTTAGTATCAGTTACGAATGTCCCAGCTTATATCACAAAAGTTATGTTACCAAGTTGGAGTGAAATTAATGGCCAAGATGATATCAAGTGGGTGACGGCAAGTAAGCTAGCTGATAATATAATAGATATATAGATATAGATATAGATAT autolysin-B GTGAAGCAATTGCTATTGATGCATCTACTCAAAAGTCAGAACTAAATAGTAATACAGAAAGTTCAACTCCCGAAATAGAATCAGTTCCAAGTGCTCCAACTGAAGTCACAAATCAGGTTCAAACTCAAACTGCTATCCAGTCAAAAAGTGTCGCACCAGTCGAAACAGTCTCAGCTGATGTGAGTGGTCATGTTTTAAAAATTGTCTATAATGGGCAACTAGAACCAACTAAGAAGATAAAGTATGCAGTTTGGACAGATAATGGTGGCCAAGATGATTTAGTTTGGTATACTGCTGATCAAGTTGGTGCGGCATATATTGATTTATCTAAAAAACATAGAGCATATGGGCTATATAATATTCATACCTATTCGCAAGATGTAACAGGTAAGATGTCTGGCTTAAATGCGAGACAATTTACTATTCTTAAACCAACAGTAAGTACATCGTTTAACGTACAAACTAATGGTATTGTTGATATTGTCGTTTCAAATGTGAGAGGTGATATTAGCTCAATTAAAGTTCCTGTTTGGTCAGATTTAGGTGGCCAAAACGATATTAAATGGTATCAAGCTACTCAATCAACTGATGGAACATACAAAGTTTCAGTAAAGATTTCAGATCATTCAAATGATACTGGCCATTTTGCTGTTCATGTTTATGGGAATAGTACAATCACGAATAGTCAAATCGGATTAGGGACAACAGAAGGTTTTACAATTGCTACCCCAGACCCCAAAAATGTAGTGTCAGCCGTAGTAGCTAATGATGGTTTCCACCTAGCATTAAATTCGAATGTTGTTAAAGAATTTACCAAAGTGAAATTTGCTGTTTGGTCGGATCAAGCTGGTCAAGATGACCTTCATTGGTACACAGCCAATGCTCAGGGTCAAGTCATTGTTCCTTATGTTAATCATAGTAATTATGGTCTATACAACATCCATACCTATAGTTTTGAGTCGGGAAGTGCTAAAGGTCTAAATACAAGAACAATTACAGT ACCAAATCCAACTGCTAGCGCAGCCATTACACAGAAATCAGATCTTGAATTTTTAGTATCAGTTACGAATGTCCCAGCTTATATCACAAAAGTTATGTTACCAAGTTGGAGTGAAATTAATGGCCAAGATGATATCAAGTGGGTGACGGCAAGTAAGCTAGCTGATAATAGTTACCAAGTATAATTAGATTATATTA autolysin-B1 GTGAAGCAATTGCTATTGATGCATCTACTCAAAAGTCAGAACTAAATAGTAATACAGAAAGTTCAACTCCCGAAATAGAATCCGTTCCAAGTGCTCCAACTGAAGTCACAAATCAGGTTCAAACTCAAACTGCTATCCAGTCAAAAAGTGTCGCACCAGTCGAAACAGTCTCAGCTGATGTGAGTGGTCATGTTTTAAAAATTGTCTATAATGGGCAACTAGAACCAACTAAGAAGATAAAGTATGCAGTTTGGACAGATAATGGTGGCCAAGATGATTTAGTTTGGTATACTGCTGATCAAGTTGGTGCGGCATATATTGATTTATCTAAAAAACATAGAGCATATGGGCTATATAATATTCATACCTATTCGCAAGATGTAACAGGTAAGATGTCTGGCTTAAATGCGAGACAATTTACTATTCTTAAACCAACAGTAAGTACATCGTTTAACGTACAAACTAATGGTATTGTTGATATTGTCGTTTCAAATGTGAGAGGTGATATTAGCTCAATTAAAGTTCCTGTTTGGTCAGATTTAGGTGGCCAAAACGATATTAAATGGTATCAAGCTACTCAATCAACTGATGGAACATACAAAGTTTCAGTAAAGATTTCAGATCATTCAAATGATACTGGCCATTTTGCTGTTCATGTTTATGGGAATAGTACAATCACGAATAGTCAAATCGGATTAGGGACAACAGAAGGTTTTACAATTGCTACCCCAGACCCCAAAAATGTAGTGTCAGCCGTAGTAGCTAATGATGGTTTCCACCTAGCATTAAATTCGAATGTTGTTAAAGAATTTACCAAAGTGAAATTTGCTGTTTGGTCGGATCAAGCTGGTCAAGATGACCTTCATTGGTACACAGCCAATGCTCAGGGTCAAGTCATTGTTCCTTATGTTAATCATAGTAATTATGGTCTATACAACATCCATACCTATAGTTTTGAGTCGGGAAGTGCTAAAGGTCTAAATACAAGAACAATTACAGT ACCAAATCCAACTGCTAGCGCAGCCATTACACAGAAATCAGATCTTGAATTTTTAGTATCAGTTACGAATGTCCCAGCTTATATCACAAAAGTTATGTTACCAAGTTGGAGTGAAATTAATGGCCAAGATGATATCAAGTGGGTGACGGCAAGTAAGCTAGCTGATAATAGTTACCAAGAGATATTATATTAT

Capsular polysaccharide biosynthesis protien gene sequence of 4 strains of Streptococcus parauberis isolated from flounder (465bp) Gene typing Base sequence (5 '→ 3') CPB-A CCAAGCACTAATAATGAATATTTACAGAAATATTGATCGTACACAAATTCAATTTGATTTTATAATCGATCATCCAAGTATGAACTACTATCAGGATGAAATAGAAAGACTAGGTGGACGAGTATATTCTTTTCCAACATTTACAGGAAGAAATGTTCGTAATGTGCGTAATGAATGGGATAAGTTTTTTAAAGAACATGTAGAGTACTCGATTATTCATTTTCATGTTAGAAGTTATATTTCTTTATTAATTCCTATTGCAAAAAGATACGGTTTAATAACAATATCACATAGTCATAGTATTTCTAATGGACTAGGACTAAAATCAAAGATTAAGAATCTTTTACAAATTCCGATTAGATATCAAGCTGATTATCTTTTTGCTTGCTCTAAAGAGGCTGGAGA (SEQ ID NO: 34)

Sugar-O-acyltransferase sialic acid gene sequence (533bp) of 64 Streptococcus parauberis isolates from flounder Gene typing Base sequence (5 '→ 3') SOA-A GAAAAGTTGTTGCAGAAATTGCTAAATTATCTGGTTATAACGACATTATTTTTCTTGATGATTATTCTAACGAAAAACTTTGTTCTGGTTATCCAGTTGTCGGAAAAGTTTCTGAAATTGTTAATTTCAAAAATGAAGATGTTTTTATAGCTATTGGATCAAGTGCTGTTAGAGAGAAAATTGCTAAACATTTGAAGGACCACAAAATAGTATCCTTAATACATCCGGCCGCAGTTGTAAGTGAAAAAGCTAAAATTGGCAAAGGGAGTGTTATTATGGCTGGTGCTGTAGTAAACCCTGATACTGAAATTGGTGAATTTTGTATAGTCAATACTTGTTCTTCTGTAGATCATGATTGTATTATAGGTGATTTTTCACACGTCTCAGTTGGCAGTCACGTTGCAGGAACAGTAACAGTTGGTTCTCATGTATGGATAGGTGCTGGGGCGACAATAATTAATAACATCGAAACACACAATAATATTTGTATTGGAGCAGGAGCAACGGTTATAAATAATTTAGTAGATAGTGGA (SEQ ID NO: 35)

Polysaccharide biosynthesis protein gene sequence (1119bp) of 64 Streptococcus parauberis isolates from flounder Gene typing Base sequence (5 '→ 3') PB-A AATAAGAGCTCGAATCTTTTCCATTATCAATCGCTTTACCGATTATAAAGTCATCTTTATCCTAATAGCTAACATGTTCTTCGCATCCCTATTAAGTTATTTGGTGGACGTTCTTTTCCTAGATACATTCAGTCGTCGTTTTCTTTTCTTATCATTCCTTTTTGGAACATTTTTAATTATCCTTCCTCGGATGATTTGGCGGATGTGGCATGAACAAAATTTGTTTGTCAAACATAATAAAAAAGACCAAAAGACAAAAATGTTGGTTGTTGGTGCTGGTGAAGGTGGTAGTGCCTTTATTCAAACAATTCTGAATAAGAGTAAAGATATTGACATTGTCGGTATTGTTGATGCTGATATCAATAAATTAGGCACCTACTTACACGGGATTAAAGTACTGGGAAATAAAAATTCCATTCCAAGATTACTAGCAGAATATGAAGTTAAACAGGTTACGATTGCCATCCCAAGTTTATCTGGGGAAGAACGAGAATCAATCTTAGATATCTGCCGTAACGCAAACGTTCACGTAAACAATATGCCTAGTATTGAGAATATCGTTCTAGGTAATGTGTCACTTAATAAATTTAAAGAAATTGAGATTGCTGACTTACTTGGACGAAAGGAAGTAGTATTAGATCAAACGTCTTTAAATTCATTCTTTAACGGGAAAACAGTTCTTGTAACTGGTGCAGGAGGATCAATTGGTTCAGAAATCTGTCGTCAAGTTTCTAAATTTAATCCAGCACGCATTTTACTTTTAGGGCATGGTGAAAATTCCATTTATCTGATTCATCGTGAATTATCAGCACTGTTAAAGGGACGAATTGACATTGTCCCAATTATCGCGGACATTCAAGATCGAGACTTGATTTTTGAAATTATGGCGAATTATCGACCTGATATAGTCTATCATGCTGCAGCACATAAACATGTGCCATTGATGGAATACAATCCAAAAGAAGCTGTTAAAAATAATATCTTTGGGACAAAAAATGTG GCGGAAGCAGCCAAGGCTGCTGGGATTCCTAAATTTATCATGGTCTCAACTGACAAAGCTGTTAACCCACCTAATGTGATGGGTGCAACTAAGCGCTTCGCTGAAATGATCGTCACTGG (SEQ ID NO: 36) PB-A1 AATAAGAGCTCGAATCTTTTCCATTATCAATCGCTTTACCGATTATAAAGTCATCTTTATCCTAATAGCTAACATGTTCTTCGCATCCCTATTAAGTTATTTGGTGGACGTTCTTTTCCTAGATACATTCAGTCGTCGTTTTCTTTTCTTATCATTCCTTTTTGGAACATTTTTAATTATCCTTCCTCGGATGATTTGGCGGATGTGGCATGAACAAAATTTGTTTGTCAAACATAATAAAAAAGACCAAAAGACAAAAATGTTGGTGCTGGTGAAGGTGGTAGTGCCTTTATTCAAACAATTCTGAATAAGAGTAAAGATATTGACATTGTCGGTATTGTTGATGCTGATATCAATAAATTAGGCACCTACTTACACGGGATTAAAGTACTGGGAAATAAAAATTCCATTCCAAGATTACTAGCAGAATATGAAGTTAAACAGGTTACGATTGCCATCCCAAGTTTATCTGGGGAAGAACGAGAATCAATCTTAGATATCTGCCGTAACGCAAACGTTCACGTAAACAATATGCCTAGTATTGAGAATATCGTTCTAGGTAATGTGTCACTTAATAAATTTAAAGAAATTGAGATTGCTGACTTACTTGGACGAAAGGAAGTAGTATTAGATCAAACGTCTTTAAATTCATTCTTTAACGGGAAAACAGTTCTTGTAACTGGTGCAGGAGGATCAATTGGTTCAGAAATCTGTCGTCAAGTTTCTAAATTTAATCCAGCACGCATTTTACTTTTAGGGCATGGTGAAAATTCCATTTATCTGATTCATCGTGAATTATCAGCACTGTTAAAGGGACGAATTGACATTGTCCCAATTATCGCGGACATTCAAGATCGAGACTTGATTTTTGAAATTATGGCGAATTATCGACCTGATATAGTCTATCATGCTGCAGCACATAAACATGTGCCATTGATGGAATACAATCCAAAAGAAGCTGTTAAAAATAATATCTTTGGGACAAAAAATGTGGCGGAAG CAGCCAAGGCTGCTGGGATTCCTAAATTTATCATGGTCTCAACTGACAAAGCTGTTAACCCACCTAATGTGATGGGTGCAACTAAGCGCTTCGCTGAAATGATCGTCACTGG (SEQ ID NO: 37) PB-B AATAAGAGCTCGAATCTTTTCCATTATCAATCGCTTTACCGATTATAAAGTCATCTTTATCCTAATAGCTAACATGTTCTTCGCATCCTTATTAAGTTATTTGGTCGACGTTCTTTTCCTAGATACATTCAGTCGTCGTTTTCTTTTCTTATCATTCCTTTTTGGAACATTTTTAATTATCCTTCCTCGGATGATTTGGCGGATGTGGCATGAACAAAATTTGTTTGTCAAACATAATAAAAAAGACCAAAAGACAAAAATGTTGGTTGTTGGTGCCGGTGAAGGTGGTAGTGCCTTTATTCAAACAATTCTGAATAAGAGTAAAGATATTGACATTGTCGGTATTGTTGATGCTGATATCAATAAATTAGGCACCTACTTACATGGGATTAAAGTACTGGGAAATAAAAATTCCATTCCAAGATTAGTAGCAGAATATGAAGTTAAACAAGTTACGATTGCCATCCCAAGTTTATCTGGGGAAGAACGAGAATCAATCTTAGATATTTGCCGTAACGCAAATGTTCACGTAAACAATATGCCTAGTATTGAGAATATCGTTCTAGGTAATGTGTCACTTAATAAATTTAAAGAAATTGAGATTGCTGACTTACTTGGACGAAAGGAAGTAGTATTAGATCAAACGTCTTTAAATTCATTCTTTAACGGGAAAACAGTTCTTGTAACTGGTGCAGGAGGATCAATTGGTTCAGAAATCTGTCGTCAAGTTTCTAAATTTAATCCAGCACGCATTTTACTTTTAGGGCATGGTGAAAATTCCATTTATCTGATTCATCGTGAATTATCAGCACTGTTAAAGGGACGAATTGACATTGTCCCAATTATCGCGGACATTCAAGATCGAGACTTGATTTTTGAAATTATGGCGAATTATCGACCTGATATAGTCTATCATGCTGCAGCACATAAACATGTGCCATTGATGGAATACAATCCAAAAGAAGCTGTTAAAAATAATATCTTTGGGACAAAAAATGTG GCGGAAGCAGCCAAGGCTGCTGGGATTCCTAAATTTATCATGGTCTCTACTGACAAAGCAGTTAATCCGCCTAATGTGATGGGTGCAACTAAGCGCTTCGCTGAAATGATCGTCACTGG (SEQ ID NO: 38) PB-B1 AATAAGAGCTCGAATCTTTTCCATTATCAATCGCTTTACCGATTATAAAGTCATCTTTATCCTAATAGCTAACATGTTCTTCGCATCCTTATTAAGTTATTTGGTCGACGTTCTTTTCCTAGATACATTCAGACGTCGTTTTCTTTTCTTATCATTCCTTTTTGGAACATTTTTAATTATCCTTCCTCGGATGATTTGGCGGATGTGGCATGAACAAAATTTGTTTGTCAAACATAATAAAAAAGACCAAAAGACAAAAATGTTGGTTGTTGGTGCCGGTGAAGGTGGTAGTGCCTTTATTCAAACAATTCTGAATAAGAGTAAAGATATTGACATTGTCGGTATTGTTGATGCTGATATCAATAAATTAGGCACCTACTTACATGGGATTAAAGTACTGGGAAATAAAAATTCCATTCCAAGATTAGTAGCAGAATATGAAGTTAAACAAGTTACGATTGCCATCCCAAGTTTATCTGGGGAAGAACGAGAATCAATCTTAGATATTTGCCGTAACGCAAATGTTCACGTAAACAATATGCCTAGTATTGAGAATATCGTTCTAGGTAATGTGTCACTTAATAAATTTAAAGAAATTGAGATTGCTGACTTACTTGGACGAAAGGAAGTAGTATTAGATCAAACGTCTTTAAATTCATTCTTTAACGGGAAAACAGTTCTTGTAACTGGTGCAGGAGGATCAATTGGTTCAGAAATCTGTCGTCAAGTTTCTAAATTTAATCCAGCACGCATTTTACTTTTAGGGCATGGTGAAAATTCCATTTATCTGATTCATCGTGAATTATCAGCACTGTTAAAGGGACGAATTGACATTGTCCCAATTATCGCGGACATTCAAGATCGAGACTTGATTTTTGAAATTATGGCGAATTATCGACCTGATATAGTCTATCATGCTGCAGCACATAAACATGTGCCATTGATGGAATACAATCCAAAAGAAGCTGTTAAAAATAATATCTTTGGGACAAAAAATGTG GCGGAAGCAGCCAAGGCTGCTGGGATTCCTAAATTTATCATGGTCTCTACTGACAAAGCAGTTAATCCGCCTAATGTGATGGGTGCAACTAAGCGCTTCGCTGAAATGATCGTCACTGG (SEQ ID NO: 39) PB-B2 AATAAGAGCTCGAATCTTTTCCATTATCAATCGCTTTACCGATTATAAAGTCATCTTTATCCTAATAGCTAACATGTTCTTCGCATCCTTATTAAGTTATTTGGTCGACGTTCTTTTCCTAGATACATTCAGTCGTCGTTTTCTTTTCTTATCATTCCTTTTTGGAACATTTTTAATTATCCTTCCTCGGATGATTTGGCGGATGTGGCATGAACAAAATTTGTTTGTCAAACATAATAAAAAAGACCAAAAGACAAAAATGTTGGTTGTTGGTGCCGGTGAAGGTGGTAGTGCCTTTATTCAAACAATTCTGAATAAGAGTAAAGATATTGACATTGTCGGTGTTGTTGATGCTGATATCAATAAATTAGGCACCTACTTACATGGGATTAAAGTACTGGGAAATAAAAATTCCATTCCAAGATTAGTAGCAGAATATGAAGTTAAACAAGTTACGATTGCCATCCCAAGTTTATCTGGGGAAGAACGAGAATCAATCTTAGATATTTGCCGTAACGCAAATGTTCACGTAAACAATATGCCTAGTATTGAGAATATCGTTCTAGGTAATGTGTCACTTAATAAATTTAAAGAAATTGAGATTGCTGACTTACTTGGACGAAAGGAAGTAGTATTAGATCAAACGTCTTTAAATTCATTCTTTAACGGGAAAACAGTTCTTGTAACTGGTGCAGGAGGATCAATTGGTTCAGAAATCTGTCGTCAAGTTTCTAAATTTAATCCAGCACGCATTTTACTTTTAGGGCATGGTGAAAATTCCATTTATCTGATTCATCGTGAATTATCAGCACTGTTAAAGGGACGAATTGACATTGTCCCAATTATCGCGGACATTCAAGATCGAGACTTGATTTTTGAAATTATGGCGAATTATCGACCTGATATAGTCTATCATGCTGCAGCACATAAACATGTGCCATTGATGGAATACAATCCAAAAGAAGCTGTTAAAAATAATATCTTTGGGACAAAAAATGTG GCGGAAGCAGCCAAGGCTGCTGGGATTCCTAAATTTATCATGGTCTCTACTGACAAAGCAGTTAATCCGCCTAATGTGATGGGTGCAACTAAGCGCTTCGCTGAAATGATCGTCACTGG (SEQ ID NO: 40) PB-B3 AATAAGAGCTCGAATCTTTTCCATTATCAATCGCTTTACCGATTATAAAGTCATCTTTATCCTAATAGCTAACATGTTCTTCGCATCCTTATTAAGTTATTTGGTCGACGTTCTTTTCCTAGATACATTCAGTCGTCGTTTTCTTTTCTTATCATTCCTTTTTGGAACATTTTTAATTATCCTTCCTCGGATGATTTGGCGGATGTGGCATGAACAAAATTTGTTTGTCAAACATAATAAAAAAGACCAAAAGACAAAAATGTTGGTTGTTGGTGCCGGTGAAGGTGGTAGTGCCTTTATTCAAACAATTCTGAATAAGAGTAAAGATATTGACATTGTCGGTATTGTTGATGCTGATATCAATAAATTAGGCACCTACTTACATGGGATTAAAGTACTGGGAAATAAAAATTCCATTCCAAGATTAGTAGCAGAATATGAAGTTAAACAAGTTACGATTGCCATCCCAAGTTTATCTGGGGAAGAACGAGAATCAATCTTAGATATTTGCCGTAACGCAAATGTTCACGTAAACAATATGCCTAGTATTGAGAATATCGTTCTAGGTAATGTGTCACTTAATAAATTTAAAGAAATTGAGATTGCTGACTTACTTGGACGAAAGGAAGTAGTATTAGATCAAACGTCTTTAAATTCATTCTTTAACGGGAAAACAGTTCTTGTAACTGGTGCAGGAGGATCAATTGGTTCAGAAATCTGTCGTCAAGTTTCTAAATTTAATCCAGCACGCATTTTACTTTTAGGGCATGGTGAAAATTCCATTTATCTGATTCATCGTGAATTATCAGCACTGTTAAAGGGACGAATTGACATTGTCCCAATTATCGCGGACATTCAAGATCGAGACTTGATTTTTGAAATTATGGCGAATTATCGACCTGATATAGTCTATCATGCTGCAGCACATAAACATGTGCCATTGATGGAATACAATCCAAAAGAAGCTGTTAAAAATAATATCTTTGGGACAAAAAATGTG GCGGAAGCAGCCAAGGCTGCTGGGATTCCTAAATTTATCATGGTCTCTACTGACAAAGCAGTTAATCCGCCTAATGTGATGGGTGCAACTAAGCGCTTCGCTGAAATGATCGTCACTGG (SEQ ID NO: 41)

The tyrosin -protein kinase gene sequence (594bp) of 64 Streptococcus parauberis isolates from flounder Gene typing Base sequence (5 '→ 3') Wze-A CTCCATTCGGACTAACATCCAATTTAGTGGTCGAGATTTAAAAGTTATCACCTTAACATCAGTACAACCTGGTGAAGGGAAATCGACAACATCCGCAAATATTGCTATCTCATTTGCTAAAGCAGGTCTAAAAACCCTATTAATCGATGCAGACATCCGTAATTCAGTTATGTCTGGTACATTTAAAGCTGATGAAAAGTATGAAGGTCTATCAAGTTACCTGTCAGGTAATGCAGAATTATCAGCAGTTATCTCTCATACAAATATTGAAAACTTAATGTTGATTCCAGCAGGACATGTTCCTCCTAATCCAACAACTTTACTCCAAAATAGCAATTTTAATTTCATGATTGATACTGTAAAAGAGTTATTTGATTATGTGATTATCGATACCCCACCTATTGGCCTTGTTATCGACTCAGCGATTATTTCACAAAAAGCTGACGCAAACATCTTAGTAACAGAAGCTGGGGCTATTAAACGACGCTTTATCCAAAAAGCAAAAGAACAAATGGAACAAAGTGGTGCTTTGTTCTTGGGTGTTATTTTAAATAAAGTAGAAGAAACACTTGATTCATATGGTGGTTATGGTAG (SEQ ID NO: 42) Wze-A1 CTCCATTCGGACTAACATCCAATTTAGTGGTCGAGATTTAAAAGTTATCACCTTAACATCAGTACAACCTGGTGAAGGGAAATCGACAACATCCGCAAATATTGCTATCTCATTTGCTAAAGCAGGTCTAAAAACCCTATTAATCGATGCAGACATCCGTAATTCAGTTATGTCTGGTACATTTAAAGCTGATGAAAAGTATGAAGGTCTATCAAGTTACCTGTCAGGTAATGCAGAATTATCAGCAGTTATCTCTCATACAAATATTGAAAACTTAATGTTGATTCCAGCAGGACATGTTCCTCCTAATCCAACAACTTTACTCCAAAATAGCAATTTTAATTTCATGATTGATACTGTAAAAGAGTTATTTGATTATGTGATTATCGATACCCCACCTATTGGCCTTGTTATCGACTCAGCGATTATTTCACAAAAAGCTGACGCAAACATCTTAGTAACAGAAGCTGGGGCTATTAAACGACGCTTTATCCAAAAAGCAAAAGAACAAATGGAACAAAGTGGTGCTTTGTTCTTGGGTGTTATTTTAAATAAAGTAGAAGAATCACTTGATTCATATGGTGGTTATGGTAG (SEQ ID NO: 43) Wze-B CTCCATTCGGACTAACATCCAATTTAGTGGTCGAGATTTAAAAGTTATCACCTTAACATCAGTACAACCTGGTGAAGGGAAATCGACATTATCCGCAAATATTGCTATCTCATTTGCTAAAGCAGGTCTTAAAACCCTATTAATCGATGCAGACATCCGTAATTCAGTTATGTCTGGTACATTTAAAGCTGATGAAAAGTATGAAGGTCTATCAAGTTACCTATCAGGTAATGCAGAATTATCAGCAGTTATTTCTCATACAAATATTGAAAACTTAATGTTGATTCCAGCAGGACATGTTCCTCCTAATCCAACAACTTTACTCCAAAATAGCAATTTTAATTTCATGATTGATACTGTAAAAGAGTTATTTGATTATGTGATTATCGATACCCCACCTATTGGCCTTGTTATAGACTCAGCGATTATTTCACAAAAAGCTGACGCAAACATTTTAGTAACAGAAGCAGGGGCTATTAAACGACGCTTTATCCAAAAAGCAAAAGAACAAATGGAACAAAGTGGTGCCTTGTTCTTGGGTGTTATTTTAAATAAAGTAGAAGAAACACTTGATTCATATGGTGGTTATGGTAG (SEQ ID NO: 44) Wze-B1 CTCCATTCGGACTAACATCCAATTTAGTGGTCGAGATTTAAAAGTTATCACCTTAACATCAGTACAACCTGGTGAAGGGAAATCGACATTATCCGCAAATATTGCTATCTCATTTGCTAAAGCAGGTCTTAAAACCCTATTAATCGATGCAGACATCCGTAATTCAGTTATGTCTGGTACATTTAAAGCTGATGAAAAGTATGAAGGTCTATCAAGTTACCTATCAGGTAATGCAGAATTATCAGCAGTTATTTCTCATACAAATATTGAAAACTTAATGTTGATTCCAGCAGGACATGTTCCTCCTAATCCAACAACTTTACTCCAAAATAGCAATTTTAATTTCATGATTGATACTGTAAAAGAGTTATTTGATTATGTGATTATCGATACCCCACCTATTGGCCTTGTTATAGACTCAGCGATTATTTCACAAAAAGCTGACGCAAACATTTTAGTAACAGAAGCAGGGGCTATTAAACGACGCTTTATCCAAAAAGCAAAAGAACAAATGGAACAAAGTGGTACCTTGTTCTTGGGTGTTATTTTAAATAAAGTAGAAGAAACACTTGATTCATATGGTGGTTATGGTAG (SEQ ID NO: 45)

For gene sequencing, MEGA6 program and GENETYX Ver.8.0 (SDC Software Development, Japan) were used, and each determined sequencing was performed using the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Institute (NCBI). Genes of the previously reported S. parauberis strain and genes of the strain analyzed in this example were compared and analyzed. Based on the base sequence information of the Blast search, multiple alignments were performed using Bioedit Ver.7.2.1, and neighborhood binding analysis using the MEGA6 program (http://www.megasoftware.net; Tamura et al., 2013) ( The genetic distance and phylogenetic tree between each sequence were prepared through NJ; neighbor-joining analysis, 1,000 rounds of boostrap (FIG. 1).

Example 3-2: Results of genotyping

As a result of 16S rRNA analysis of 64 strains isolated from halibut, it was identified as S. parauberis , and was identified with the same nucleotide sequence. Multilocus sequence typing (MLST) analysis was performed for 64 S. parauberis strains. Sugar O-acryltransferase sialic acid (Soa) and polysaccharide biosynthesis protein (PB) genes were not amplified in some strains. In the 64 strains analyzed, 16S rRNA and groEL genes all had the same base sequence, and MLST analysis was performed only on gyrB and antigen-pathogenicity-related genes with different base sequences for each strain. The 64 strains of S. parauberis isolated from halibut were largely divided into two MLST genotypes, and were identified as 36 strains of MLST-1 group and 28 strains of MLST-2 group (FIG. 1). The results of genotyping for each strain are shown in Table 15 below.

Genotyping analysis by 64 strains of S. parauberis serotype PCR Serotype Slide coagulation reaction No. FP No. Resource gathering area Gathering 16S rRNA groEL gyrB simA auto CPB SOA PB TPK Ia Ib / c II I II One FPa2041 Jeju Island 2002-07-03 A A B B B - - B B ++ Ia + 2 FPa3132 Pohang, Gyeongbuk 2003-08- A A A A A A A A A ++ Ib + 3 FP4076 Wando, Jeonnam 2004-08-06 A A A A A A A A A ++ Ib + 4 FP4114 Jeju (North) 2004-08-31 A A A A A A A A A ++ Ib + 5 FP2199 Wando, Jeonnam 2005-05-31 A A A A A A A A A ++ Ib + 6 FP5042 Jeju Island 2005-06-15 A A A A A A A A A ++ Ib + 7 FP3197 Ulsan, Gyeongnam 2006-10-01 A A A A A A A A A ++ Ib + 8 FP2333 Yeonwoo Fisheries 2006-12-01 A A A A A A A A A ++ Ib + 9 FP3335 Pohang, Gyeongbuk 2007-08-01 A A A A A A A A A1 ++ Ib + 10 FP3380 Ulsan, Gyeongnam 2007-10-01 A A A A A A A A1 A ++ Ic X X 11 FP3441 Wando, Jeonnam 2008-05-06 A A A A A A A A A ++ Ib + 12 FP3446 Pohang, Gyeongbuk 2008-07-01 A A A A A A A A A ++ Ib + 13 FP3826 Aquaculture Environment Research Institute (Tongyeong) 2010-01-04 A A B B B - - B B + Ia + 14 FPa4365 Pohang, Gyeongbuk 2012-07-14 A A B B B - - B3 B + Ia + 15 FPa4369 Pohang, Gyeongbuk 2012-07-14 A A B B B - - B3 B + Ia + 16 FPa4403 Gyeongnam Geoje Breeding Center 2012-09-05 A A B B B - - B B + Ia + 17 FPa4404 Gyeongnam Geoje Breeding Center 2012-09-05 A A B B B - - B B + Ia + 18 Fpa4290 Jeju Island 2012-04-18 A A B B B - - B B + Ia + 19 Fpa4293 Jeju Island 2012-04-18 A A B B B - - B B + Ia + 20 Fpa4325 Pohang, Gyeongbuk 2012-05-08 A A A A A A A A A ++ Ib + 21 Fpa4406 Gyeongnam Geoje 2012-09-10 A A B B B - - B B + Ia + 22 Fpa4407 Gyeongnam Geoje 2012-09-10 A A A A A A A A A ++ Ib + 23 Fpa4599 Jeju Island 2014-04-14 A A B B B - - B B + Ia + 24 Fpa4616 Pohang, Gyeongbuk 2014-05-26 A A B B B - - B B + Ia + 25 Fpa4624 Pohang, Gyeongbuk 2014-05-14 A A B B B - - B B + Ia + 26 Fpa4658 Jeju Island 2014-07-25 A A A A A A A A A ++ Ib + 27 Fpa4660 Jeju Island 2014-07-25 A A B B B - - B B + Ia + 28 Fpa4661 Jeju Island 2014-07-25 A A A A A A A A A ++ Ib + 29 Fpa4683 Pohang, Gyeongbuk 2014-09-26 A A B B B - - B B + Ia + 30 Fpa4710 Pohang, Gyeongbuk 2014-09-15 A A B B B - - B B + Ia + 31 Fpa4743 Jeju Island 2015-03-05 A A A A B - - B2 B ++ II + 32 Fpa4804 Jeju Island 2015-07-15 A A B B B - - B B + Ia + 33 Fpa4805 Jeju Island 2015-07-15 A A B B B - - B B + Ia + 34 Fpa4806 Jeju Island 2015-07-15 A A B B B - - B B + Ia + 35 Fpa4807 Jeju Island 2015-07-15 A A B B B - - B B + Ia + 36 Fpa4850 Jeju Island 2015-09-16 A A A A A A A A A ++ Ib + 37 Fpa4860 Jeju Island 2015-10-14 A A B B B - - B B + Ia + 38 Fpa4870 Jeju Island 2015-10-14 A A A A A A A A A ++ Ic X X 39 Fpa4894 Jeju Island 2015-11-18 A A A A A A A A A ++ Ib + 40 BS1K (Pohang-Buseong 1K) Pohang, Gyeongbuk 2016-06-21 A A B B B - - B B + Ia + 41 DK14 (Daeguan Fisheries 14) Pohang, Gyeongbuk 2016-08-21 A A B B B - - B B + Ia + 42 DW C7-3 (September Daewon C7-3) Ulsan, Gyeongnam 2016-09-08 A A A A A A A A A ++ Ib + 43 DW1 (Daewon Hospital 1 in September) Ulsan, Gyeongnam 2016-09-08 A A A A A A A A A ++ Ib + 44 YL3 (Jeju Younglim 3) Jeju Island 2016-11-00 A A A A A A A A A ++ Ib + 45 PR 5KJK (Blue Blue 5KJK) Jeju Island 2016-12-23 A A A A A A A A A ++ Ib + 46 FP3825 Aquaculture Environment Research Institute (Tongyeong) 2010-01-04 A A B B B - - B B + Ia + 47 FPa4577 Jeju Island 2014-03-20 A A B B B - - B B + Ia + 48 FPa4578 Jeju Island 2014-03-20 A A B B B - - B B + Ia + 49 FPa4586 Jeju Island 2014-03-20 A A B B B - - B B + Ia + 50 FPa4587 Jeju Island 2014-03-20 A A A A A A A A A + Ic X X 51 SPOF3K Geoje Island In 2013 A A B B B - - B B + Ia + 52 KSP28 Wando, Jeonnam 2005's A A A A A A A A A + Ib + 53 PH0710 A A B B B - - B B1 + Ia + 54 FP2331 2006-11-01 A A A A B - - B2 B + II + 55 FP2332 2006-12-01 A A A A B - - B2 B + II + 56 YJ3 (Geoje Breeding Center 3) 2017-05-17 A A B B B - - B B + Ia + 57 YJ7 (Geoje Breeding Center 7) 2017-05-17 A A B B B - - B B + Ia + 58 DB1 Jeju Island 2017-07-18 A A B B B - - B  B + Ia + + 59 FP2313 Pohang, Gyeongbuk 2006 A A A A B - - B2 B II 60 FP3439 Wando, Jeonnam 2008 A A A A A A A A A + Ib + 61 FP3440 Wando, Jeonnam 2008 A A A A A A A A A + Ib + 62 DK-M3 Busan millet 2017-08-17 A A A A A A A A A + Ib + 63 DK-L2 Wando, Jeonnam 2017-09-07 A A B B B - - B B + Ia + 64 best7-1 Busan millet 2017-08-17 A A B B B - - B B + Ia +

In the CPB and SOA categories, the ones marked with-are not PCR amplified.

Example 4:  S. parauberis Serotype analysis

Example 4-1: Serotype analysis method

The serotype was analyzed by the slide agglutination reaction, and rabbit antiserum against the reference strains ( S. parauberis serotype I FPa4164 and S. parauberis serotype O2 S53) was prepared and used for analysis. Two kinds of rabbit antisera against S. parauberis were added to the slide glasses by 70 μL each, and 64 strains of S. parauberis from halibut were added to McFarland no. With 6 standard, each 10 μL of the suspension suspended in sterile physiological saline was mixed well to confirm the presence or absence of aggregation within 30 seconds (FIG. 2 (A)). Aggregation was observed from the side of the slide on a dark background, and a sterile physiological saline solution without suspension was used as a control.

To distinguish subserotypes in serotype I of S. parauberis , a serotype analysis of 64 strains of S. parauberis was attempted through microtiter aggregation (FIG. 2 (B)). Rabbit antisera was prepared for four strains DK14, DW1, FPa4870, and FP4743 with different S. parauberis genotypes and phenotypic characteristics, and sub-serotyping through microtiter aggregation reaction was performed by the method of Kanai et al. (2015). After serially diluting rabbit antisera twice with PBS in 96 well plates, add 4 S. parauberis FKC suspensions (2mg / mL PBS) at the same volume of 25 µl and mix and then incubate at 4 ° C. Was measured. For the cross-absorbed test of antisera, absorbed-antisera was prepared (Kanai at al., (2015) Fish pathology 50 (2), 75-80), and absorbed-antisera produced less than 4 aggregated antibodies. Serotyping PCR for S. parauberis was carried out according to the method of Tu et al. (2015). The base sequence of the PCR primer is shown in Table 2 above.

Example 4-2: Serotype analysis results

As a result of the slide aggregation reaction, S. parauberis strains were identified as 58 strains of serotype I, 4 strains of serotype II, and 2 strains of untypable serotype. Serotyping-PCR results of 64 strains are shown in Table 15 above.

S parauberis 4 strains DK14, DW1, FPa4870, and FP4743 were prepared with rabbit antiserum, and sub-serotyping through microtiter aggregation reaction resulted in 58 strains of Serotype I anti-DK14, anti-DW1, and anti-FPa4870 antisera It shows hyperagglutination reaction, but the agglutination value differs depending on the type of antiserum, suggesting the presence of subserotype in serotype I. As a result of subserotyping of S. parauberis serotype I according to the cohesive value, it was identified as 25 strains of Ia, 25 strains of Ib, 8 strains of Ic, and 2 strains of non-typeable (Table 16).

Classification of S. parauberis serotypes according to aggregation to rabbit antisera Antiserum
(Antiserum) Agglutination titers Subserotype
Ia (n = 25) Subserotype
Ib (n = 25) Subserotype
Ic (n = 8) serotype II (n = 4) Non-typeable
(n = 2) Anti-DK14 (Ia) 64 16-32 ≤64 <2 32-2048 Anti-DW1 (Ib) ≤32 64 ≤64 <2 32-2048 Anti-FPa4870 (Ic) ≤32 8-64 ≥128 <2 64-1028 Anti-FP4743 (II) <2 <2 <2 128 32-2048

In order to classify subserotypes in S. parauberis serotype I, anit-DK14, anti-DW1, and anti-FPa4870 antiserum were prepared by cross-agglomerate absorption method, and absorbed-antiserum for each strain was prepared to measure the aggregation value again. As a result of analyzing through the cross-aggregation absorption method, there are three subtypes in serotype I, and in the case of subserotype Ic type, it shows a higher aggregation value in Ib type than Ia type (Table 17).

Agglomerated results comparison for Absorbed and unabsorbed rabbit antisera Antiserum
(Antiserum) Absorbed FKC Agglutination titers for FKC for each strain DK14 (Ia) DW1 (Ib) FPa4870 (Ic) Anti-DK14
(Ia) Unabsorbed 64 32 32 DK14 <2 <2 <2 DW1 8 <2 <2 4870 16 16 <2 Anti-DW1
(Ib) Unabsorbed 32 64 64 DK14 <2 8 8 DW1 <2 <2 <2 4870 16 16 <2 Anti-FPa4870
(Ic) Unabsorbed 64 64 128 DK14 <2 8 32 DW1 <2 <2 64 4870 <2 <2 <2

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

As a result of analyzing the distribution of serotypes (genotypes) according to the S. parauberis isolates, specific serotypes could not be identified according to the isolates, and various serotypes were identified regardless of the isolates (Table 18, FIGS. 3 and 3). 4).

2002-2017, S. parauberis Serotype distribution by segregation area area Jeju Jeonnam Gyeongbuk Gyeongnam Wando Pohang Busan and Ulsan Geoje Tongyeong Number of strains 25 9 17 6 5 2 Serotype Ia = 9, Ib = 9, Ic = 5, II = 1, NT = 1 Ia = 1, Ib = 8 Ia = 9, Ib = 5, II = 3 Ia = 1, Ib = 2, Ic = 1, NT = 1 Ia = 1, Ib = 4 Ia = 1, Ic = 1

As a result of analyzing the serotype distribution according to the S. parauberis separation year, serotype Ib was dominant before 2010, serotype Ia (MLST-1 group) was continuously isolated after 2010, and recently the serotype Ic separation rate was It was confirmed that the increase (Table 19).

Serotype distribution by 2002.2017 S. parauberis isolates year 2002 2003 2004 2005 2006 2007 2008 2010 2012 2013 2014 2015 2016 2017 Number of strains One One 2 3 5 2 6 2 10 One 12 9 4 6 Serotype Ia = 1 Ib = 1 Ib = 2 Ib = 3 Ib = 2, II = 3 Ib = 1, NT = 1 Ib = 6 Ia = 1, Ic = 1 Ia = 5 Ib = 5 Ia = 1 Ia = 10,
Ib = 2 Ic = 5, Ib = 3, II = 1 Ia = 1,
Ib = 3 Ia = 4, Ic = 1, NT = 1

In particular, in the case of serotype Ia and Ic, the separation ratio has been increased since the flounder vaccine of S. parauberis was commercialized in 2009. This is presumed to be a change in the serotype of S. parauberis due to the use of the vaccine rather than the effect of the commercialized vaccine (FIGS. 3 and 4).

Example 5: Serotype investigation of commercial commercial vaccines

In 2018, 7 types of inactivated vaccines containing the halibut S. parauberis antigen sold in Korea were purchased. After centrifugation and DNA extraction using Promega Genomic DNA purification kit (Promega, USA), serotype was analyzed by serotype-PCR (Tu et al., 2015). The PCR was amplified and the PCR product was electrophoresed using a 1.5% agarose gel (Bioneer, Korea) containing EtBr, and then the band was confirmed using a UV transilluminator (Alpha Innotech, USA).

As a result, both were identified as serotype Ib and serotype Ib + II (FIG. 5 and Table 20).

Serotype -PCR results of inactivated vaccines containing Streptococcus parauberis antigens on the market in Korea product name Company Name Serotype PCR results Ⅰa Ⅰb / c Ⅱ One Poseidon 4 Green Cross Pharmaceutical - + + 2 Pro Bag Penta Comey Farm - + - 3 Pro Bag-3ST Comey Farm - + - 4 Ezilbang ES3 Plus Koryo B & P - + - 5 Willo Marine M Bag SIP3 Yuhan Corporation - + + 6 Strep-Keeper 3 Woosung Yang - + + 7 Daesung Chain, Ed Fishback Daesung Microbiological Research Institute - + -

Example 6: S. parauberis Pathogenicity analysis

7 strains (FPa4870, FP4743, FP2331, FPa4365, PR5 (19FBSPa0003), DK14, DW) of 7 different strains of serotypes were selected, and artificial infection experiments at 10 ⁶ cfu / fish concentration in flounder (16.5 ± 0.7cm) (water temperature) 23 ± 1 ° C., subcutaneous injection, 10 animals per test sphere). Experiments on artificial infections by subcutaneous injection were conducted by Mori et al. (2010) Fish Pathology, 45 (1), 37-42.

In the first infection experiment, a second infection experiment was performed with PR5, FPa4365, FPa4870, and FP2331 strains with relatively high mortality rates for each serotype (FIG. 6). After 4 strains of halibut (11.6 ± 0.5cm) were artificially infected with 10 ⁵ ~ 10 ¹⁰ cfu / fish (water temperature 25 ± 0.5 ℃, subcutaneous injection method), the cumulative mortality for 2 weeks was measured. As a result, all of FPa4365 (Ia), PR5 (Ib), and FPa4870 (Ic) included in serotype I of S. parauberis showed high pathogenicity to halibut (FIG. 7). The FP2331 (II) strain of Serotype II was injected subcutaneously to the halibut with 10 ¹⁰ cfu / fish, but mortality did not occur in the halibut even at high concentrations, so the FP2331 strain is judged to have no or low pathogenicity to the halibut. Finally, serotypes Ia, Ib, Ic strain is 10 ⁸ cfu / fish, serotype II was set to the artificial infection levels for vaccine efficacy validated the concentration of ¹⁰ 10 cfu / fish.

Example 7: Serotype liver S. parauberis  Vaccine efficacy analysis

Example 7-1: vaccine production and efficacy analysis method

S. parauberis serotypes Ia, Ib, Ic, and II strains (FPa4870, FP2331, FPa4365, PR5 (19FBSPa0003)) were prepared by inactivation vaccine (FKC) treated with formalin, and intraperitoneally inoculated (1 mg / fish) to the flounder, control PBS was injected intraperitoneally.

Four strains of cryopreserved S. parauberis were cultured in BHIA medium to which 1.5% (v / v) NaCl was added at a temperature of 30 ° C. for 24 hours. Colonies of the cultured plate medium were inoculated into BHIB to which 1.5% NaCl was added aseptically, and then incubated for 48 hours at 150 rpm and 30 ° C. in a stirred incubator (JS Research Inc.). 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution to be 0.5% (v / v) of each culture, and inactivated at 150 rpm and 20 ° C. in a stirred incubator for 24 hours. The inactivation of the culture was finally confirmed in BHIA (Difco, USA). The inactivated bacteria were centrifuged at 12,000 RCF, washed three times with sterile physiological saline, the cells were collected, and finally, the weight of the cells was measured and suspended at different concentrations according to the experimental group. After 2 weeks of vaccination, blood was collected from the arrhythmia and the antibody titer was measured by ELISA. Antibodies using the ELISA method were used to control S. parauberis FKC to a concentration of 10 μg / well in 96-well plates and dispensed, and antigen was attached to the bottom of the plate at 4 ° C over night. In order to measure the ELISA value of each S. parauberis serotype, FKCs of four different serotypes of S. parauberis were prepared and antibody titers for each serum were examined. Blocking is performed at room temperature for 1 hour using 3% skim milk, and after diluting and dissolving halibut serum and intestinal mucus 10 times with primary antibody, react for 1 hour. Then, after washing three times with PBST, the secondary antibody was diluted and diluted with 1: 1000 Anti-Japanese flounder IgM Mab and reacted at 37 ° C. for 1 hour. After washing, the AP-conjugated anti-mouse IgG (whole molecule) was diluted 1: 1000, dispensed, reacted at 37 ° C. for 1 hour, washed, and then added to the substrate (PNPP substrate solution) at 405 nm after 20 minutes. Absorbance was confirmed. After 4 weeks of vaccination, each vaccinated sphere was artificially infected with serotypes Ia, Ib, Ic, and II strains, and cumulative mortality was measured for 3 weeks (FIG. 8). The relative survival rate was calculated as follows.

Relative survival rate (%) = {1- (cumulative mortality rate of the test zone / control mortality rate)} × 100

Example 7-2: Efficacy of cross-type vaccine and analysis of cross vaccine efficacy

After 2 weeks of vaccination by serotype, blood was collected and the antibody titer of the serum was measured by antigen type (serum type Ia ~ II). When the antibody titer was measured with the antigen of, it showed the highest antibody titer (Table 21).

Antibody titer results after 2 weeks of vaccination by serotype Antigen (serotype) Absorbance (OD) of ELISA FPa4365 (Ia)
Vaccine PR5 (Ib)
Vaccine FPa4870 (Ic)
Vaccine FP2331 (II)
Vaccine Control
(PBS) FPa4365 (Ia) 0.469 ± 0.219 0.376 ± 0.134 0.866 ± 0.227 0.409 ± 0.199 0.144 ± 0.063 PR5 (Ib) 0.249 ± 0.12 0.473 ± 0.180 0.837 ± 0.278 0.421 ± 0.212 0.161 ± 0.072 FPa4870 (Ic) 0.223 ± 0.14 0.386 ± 0.106 1.078 ± 0.251 0.332 ± 0.197 0.156 ± 0.067 FP2331 (II) 0.2 ± 0.133 0.392 ± 0.094 0.823 ± 0.261 0.446 ± 0.159 0.159 ± 0.074

After treatment with S. parauberis vaccine with 4 different serotypes for halibut, an artificial infection test for each serotype resulted in a lower mortality rate in all vaccinated cells compared to the unvaccinated control group (Table). 22).

Cumulative mortality according to artificial infection test after vaccination of 4 serotypes of S. parauberis Experiment Mortality rate according to artificial infection experiment (%) FPa4365 infection (Ia) PR5 infection (Ib) FPa4870 infection (Ic) FP2331 infection (II) FPa4365 vaccine (Ia) 33.3 61 33.3 0 PR5 vaccine (Ib) 66.6 15.4 16.6 0 FPa4870 vaccine (Ic) 75 23 16.6 0 FP2331 vaccine (II) 66.6 26.5 16.6 0 Control (PBS) 75 88.2 66.6 0

As a result of comparing the relative survival rate of the cross vaccine efficacy between the serotypes and the control group, the highest relative survival rate was shown when all strains were artificially infected with the same serotype strain after vaccination of the same serotype (Table 23). Serotypes Ib and Ic were found to have a high cross vaccine efficacy response (relative survival rate: 73.9 to 82.5%), but relatively low cross vaccine efficacy (relative survival rate: 0 to 30.8%) to Ia, Ib, and Ic. (Table 23).

R. Survival Rate of Artificial Infection after Vaccination of S. parauberis 4 Serotypes Experiment Relative survival rate (%) FPa4365 infection (Ia) PR5 infection (Ib) FPa4870 infection (Ic) FPa4365 vaccine (Ia) 55.6 30.8 49.5 PR5 vaccine (Ib) 11.2 82.5 75.1 FPa4870 vaccine (Ic) 0 73.9 75.1 FP2331 vaccine (II) 11.2 70.0 75.1

Example 8: S. parauberis  Analysis of the efficacy of polyvalent vaccines

Example 8-1: Multivalent vaccine production and efficacy analysis method

Since S. parauberis serotype II is not pathogenic to halibut and is not isolated from halibut farms recently, it was excluded from the vaccine strain for multivalent vaccine development. Three types of S. parauberis (serum type Ia (FPa4365), serotype Ib (PR5), and serotype Ic (FPa4870)) strains were prepared by formalin-treated inactivation vaccine (FKC).

The cryopreserved S. parauberis strain was cultured in BHIA medium to which 1.5% (v / v) NaCl was added at 30 ° C. for 24 hours. Colonies of the cultured plate medium were aseptically inoculated into BHIB to which 1.5% NaCl was added, and then incubated at 150 rpm, 30 ° C., 48 hours in a stirred incubator (JS Research Inc.). 37% (v / v) formalin (Merck, Germany) was added to the cultured bacterial solution to be 0.5% (v / v) of each culture solution, and inactivated at 150 rpm and 20 ° C. for 24 hours in a stirred incubator. The inactivation of the culture was finally confirmed in BHIA (Difco, USA). The inactivated bacteria were centrifuged at 12,000 RCF, washed three times with sterile physiological saline, the cells were collected, and finally, the weight of the cells was measured and suspended at different concentrations according to the experimental group. Based on the results of the inactivated single vaccine study for each S. parauberis serotype, serotypes Ib and Ic were found to have mutual cross-vaccination efficacy and flounder (average length: 15.05 ± 0.9cm, with the antigen combination and concentration in Table 24). The average body weight: 26.7 ± 4.74g) was vaccinated by an intraperitoneal injection method. A total of 80 halibut per experiment were used for the experiment.

Types and concentrations of S. parauberis FKC for intraperitoneal injection division Mixed serotype (concentration) / fish Final inoculation injection / fish Ia + Ic vaccine sphere Ia-FKC 1.5mg + Ic-FKC 1.5mg 0.1ml Ia + Ib + Ic vaccine sphere Ia-FKC 1mg + 1b-FKC 1mg + Ic-FKC 1mg 0.1ml Control Sterile physiological saline 0.1ml

After 2 weeks of vaccination, blood was collected from the arrhythmia ( n = 10), and the antibody titer to S. parauberis was measured by ELISA. Antibodies using the ELISA method were used to control S. parauberis FKC to a concentration of 10 μg / well in 96-well plates and dispensed, and antigen was attached to the bottom of the plate at 4 ° C over night. In order to measure ELISA values by S. parauberis serotype, FKCs of S. parauberis of three different serotypes were prepared and antibody titers for each serum were examined. Blocking is performed at room temperature for 1 hour using 3% skim milk, and after diluting and dissolving halibut serum and intestinal mucus 10 times with primary antibody, react for 1 hour. Then, after washing three times with PBST, the secondary antibody was diluted and diluted with 1: 1000 Anti-Japanese flounder IgM Mab and reacted at 37 ° C. for 1 hour. After washing, the AP-conjugated anti-mouse IgG (whole molecule) was diluted 1: 1000, dispensed, reacted at 37 ° C. for 1 hour, washed, and then added to the substrate (PNPP substrate solution) at 405 nm after 20 minutes. Absorbance was confirmed. As a result of measuring the antibody titer 2 weeks after vaccination, no antibody titer was found in the control group, and significantly higher antibody titers were formed in the Ia + Ic and Ia + Ib + Ic groups compared to the control group (Table 25, Fig. 9).

Antibody titer measurement result (Mean ± SD) 2 weeks after S. parauberis multivalent vaccine inoculation Antigen (serotype) Absorbance value (OD) of ELISA Control Ia + Ic vaccine sphere Ia + Ib + Ic vaccine sphere FPa4365 (Ia) 0.0028 ± 0.0016 0.4178 ± 0.2556 0.1699 ± 0.1699 PR5 (Ib) 0.0053 ± 0.0023 0.2307 ± 0.1909 0.0491 ± 0.0349 FPa4870 (Ic) 0.0045 ± 0.0026 0.3340 ± 0.2800 0.2600 ± 0.0400

Example 8-2: Analysis of the efficacy of the polyvalent vaccine

Three weeks after vaccination, the water temperature is 26 ~ with three strains of S. parauberis Ia FPa4365 (5.2 × 10 ⁸ cfu / fish), Ib PR5 (7 × 10 ⁸ cfu / fish), and Ic FPa4870 (1.4 × 10 ⁸ cfu / fish). An artificial infection experiment was performed while maintaining 27 ° C. and mortality was confirmed for 3 weeks (Table 26).

Cumulative mortality according to artificial infection test after S. parauberis multivalent vaccine Experiment Mortality rate according to artificial infection experiment (%) FPa4365 infection (Ia) PR5 infection (Ib) FPa4870 infection (Ic) Ia + Ic vaccine sphere 5 0 25 Ia + Ib + Ic vaccine sphere 25 5 35 Control (PBS) 75 55 50

The relative survival rate compared to the control was calculated as follows.

Relative survival rate (%) = {1- (cumulative mortality rate of the test zone / control mortality rate)} × 100

When S. parauberis serotype Ia + Ic and Ia + Ib + Ic vaccines were artificially infected with three different serotypes of S. parauberis , the survival rate was higher than that of the control group, and S. parauberis single serotype of Table 23. It showed a high relative survival rate compared to the Ia and Ib vaccine groups (Table 27).

Relative survival rate according to artificial infection test after S. parauberis multivalent vaccine Experiment Relative survival rate (%) FPa4365 infection (Ia) PR5 infection (Ib) FPa4870 infection (Ic) Ia + Ic vaccine sphere 93.4 100 50 Ia + Ib + Ic vaccine sphere 66.7 90.1 30

For example, when artificially infected with the FPa4365 strain of the S. parauberis Ia serotype after vaccination of a single Ia serotype, the relative survival rate of 55.6% was compared to that of the control group, but FPa4365 in the Ia + Ic vaccine and Ia + Ib + Ic spheres. When artificially infected with the strain, the relative survival rates of 93.4% and 66.7% were shown compared to the control. In addition, when artificially infected with FPa4870 strain of S. parauberis Ic serotype after inoculation of Ic single serotype vaccination, the relative survival rate of 75.1% was compared with that of the control group, but the Ia + Ic vaccine and Ia + Ib + Ic spheres were used as FPa4365 strains. In the case of artificial infection, the relative survival rates of 50% and 30% were compared to those of the control group, and the efficacy was lower than that of the Ic single serotype vaccine. In comparison of vaccine efficacy between multivalent vaccines mixed with serotypes, the Ia + Ic vaccines showed higher relative survival rates compared to all three serotype strains compared to the Ia + Ib + Ic vaccines, and specific antibodies to S. parauberis Thin Ia + Ib + Ic vaccines showed higher antibody titers than Ia + Ic vaccines.

Example 9: S. parauberis  Stability analysis of polyvalent vaccine

Example 9-1: Multivalent vaccine stability analysis method

To investigate the safety of the multivalent vaccine, safety was assessed by intraperitoneal injection of flounder (average length: 15.83 ± 1.04 cm, average weight: 27.2 ± 4.75 g) at a concentration of 2, 5, and 10 times the antigen concentration used for vaccine efficacy investigation. Table 28. Mortality was confirmed up to 3 weeks after intraperitoneal vaccination, and blood was collected from the arrhythmia of the flounder weekly ( n = 5) to confirm safety through blood biochemical properties and histopathological examination. Blood properties were measured using an automatic blood analyzer, and the concentrations of glucose, ALT, AST, and total protein were measured. Histopathological investigation was performed by fixing kidney, spleen, liver, etc. in neutral formalin and following the conventional method.

Types and concentrations of S. parauberis FKC for intraperitoneal injection division Mixed serotype (concentration) / fish Final inoculation injection / fish A Ia + Ic 2x Ia-FKC 3mg + Ic-FKC 3mg 0.1ml B Ia + Ic 5 times Ia-FKC 7.5mg + Ic-FKC 7.5mg 0.1ml C Ia + Ic 10 times Ia-FKC 15mg + Ic-FKC 15mg 0.1ml D Ia + Ib + Ic 2x Ia-FKC 2mg + 1b-FKC 2mg + Ic-FKC 2mg 0.1ml E Ia + Ib + Ic 5 times Ia-FKC 5mg + 1b-FKC 5mg + Ic-FKC 5mg 0.1ml F Ia + Ib + Ic 10 times Ia-FKC 10mg + 1b-FKC 10mg + Ic-FKC 10mg 0.1ml G Control Sterile physiological saline 0.1ml

Example 9-2: Results of stability analysis of polyvalent vaccine

No death occurred in all experimental groups for 3 weeks after the vaccine was injected at high concentrations of 2, 5, and 10 times the antigen levels of the S. parauberis Ia + Ic vaccine and Ia + Ib + Ic vaccine. Blood biochemical properties (AST, ALT, GLU, TP) did not show a significant difference from the control group at 1, 2, or 3 weeks after vaccination in all vaccination groups, and the concentration was confirmed to be safe for flounder (P> 0.05, Table 29).

Changes in hematologic properties of each group after vaccination (Mean ± SD) exam
Item After inoculation (Week) A
(Ia + Ic 2x) B
(Ia + Ic 5 times) C
(Ia + Ic 10 times) D
(2 times Ia + Ib + Ic) E
(Ia + Ib + Ic 5 times) F
(Ia + Ib + Ic 10 times) G
(Control) AST
(U / L) One 21 ± 4.12 16.4 ± 10.08 17.4 ± 1.52 22.2 ± 8.75 15.6 ± 3.91 12.2 ± 1.30 17.8 ± 4.76 2 16.2 ± 5.45 16.6 ± 7.33 14.6 ± 4.04 20 ± 19.59 18.8 ± 3.42 21.2 ± 8.1 15.8 ± 4.76 3 17 ± 8.41 16 ± 6.40 10 ± 1.3 10 ± 2.3 12 ± 2.88 10 ± 1.48 25 ± 5.61 ALT
(U / L) One 4.8 ± 0.83 4.8 ± 0.84 4.4 ± 2.19 4 ± 2.24 3.2 ± 0.45 4.2 ± 0.83 3.4 ± 1.14 2 4.8 ± 0.44 1.8 ± 1.09 2.6 ± 0.89 1.2 ± 0.45 1.8 ± 1.30 2.2 ± 2.17 3.6 ± 0.89 3 4 ± 1.14 2.4 ± 1.14 5 ± 0.54 2 ± 0.84 2 ± 0.87 3 ± 0.45 5 ± 1.14 GLU
(mg / dl) One 10 ± 2.45 15.2 ± 6.72 48.2 ± 16.85 22.2 ± 3.27 15.8 ± 5.93 14.6 ± 7.27 15.8 ± 5.54 2 40 ± 8.15 45 ± 20.55 36.2 ± 11.21 48 ± 23.09 43 ± 12.34 36.6 ± 17.13 30.6 ± 5.32 3 21 ± 10.87 19 ± 8.29 29 ± 16.38 25 ± 8.08 18 ± 4.22 20 ± 2.77 30 ± 12.70 TP
(g / dl) One 3.76 ± 0.45 3.6 ± 0.14 3.54 ± 0.36 3.1 ± 1.69 3.18 ± 0.30 3.34 ± 0.54 3.6 ± 0.54 2 3.88 ± 0.42 3.6 ± 0.55 3.6 ± 0.46 3.24 ± 0.53 3.1 ± 0.25 3.46 ± 0.53 3.88 ± 0.35 3 4 ± 0.19 4 ± 0.27 4 ± 0.45 3 ± 0.27 3 ± 0.29 3 ± 0.22 4 ± 0.11

As a result of the histopathological examination after inoculation of the polyvalent vaccine, no differences were observed in the liver, kidney and spleen pathological histologic examinations of the vaccine and control groups, and the polyvalent vaccine was confirmed to be safe for flounder (FIGS. 10 to 12).

Since specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that this specific technique is only a preferred embodiment, and the scope of the present invention is not limited thereby. will be. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Korea Biological Resource Center KCTC13800BP 20190130 Korea Biological Resource Center KCTC13801BP 20190130 Korea Biological Resource Center KCTC13802BP 20190130 Korea Biological Resource Center KCTC13803BP 20190130

<110> Republic of Korea (National Institute of Fisheries Science) <120> Combination Vaccines Against Streptococcal Disease in Fish <130> P19-B252 <160> 45 <170> KoPatentIn 3.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 caggcctaac acatgcaagt c 21 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 acgggcggtg tgtrc 15 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gahgtngtsg aaggsatgca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 atttgrcgsa ywggytcttc 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ggttcstcng tsgttaatgc 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gttcrtgngt tccsccttca t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gctaaggtca tgacccattc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 aggcgtgcaa tttcagcttg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 ctcaaactga ggctagtaat 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gtcccggcta atccaactgt 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 cttggtggat cccaagcac 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ccaaacaacc actctccagc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gcaagcggtc acggaaaagt 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cacctacata ggttccacta 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 gcaaatctgt cggcttttcg 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 acgactacca aggacgttac 20 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ccgaagagta ctataactcc 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 tggcttacca taattcccgt 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 attgttagtc attcagttgt 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 aattatagtc aacagtccag 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 atttctacca ggttactttg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 gaactactta ggtttagcat 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 aacttgtaaa taggattgct 20 <210> 24 <211> 1287 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA <400> 24 gcctagtcag atgagttgcg aacgggtgag taacgcgtag gtaacctacc tcatagcggg 60 ggataactat tggaaacgat agctaatacc gcatgacaat taagtactca tgtactaaat 120 ttaaaaggag caattgcttc actatgagat ggacctgcgt tgtattagct agttggtgag 180 gtaacggctc accaaggcca cgatacatag ccgacctgag agggtgatcg gccacactgg 240 gactgagaca cggcccagac tcctacggga ggcagcagta gggaatcttc ggcaatgggg 300 gcaaccctga ccgagcaacg ccgcgtgagt gaagaaggtt ttcggatcgt aaagctctgt 360 tgttagagaa gaacggtaat gggagtggaa aatccattac gtgacggtaa ctaaccagaa 420 agggacggct aactacgtgc cagcagccgc ggtaatacgt aggtctcgag cgttgtccgg 480 atttattggg cgtaaagcga gcgcaggcgg ttatttaagt ctgaagttaa aggccgtggc 540 tcaaccatgg ttcgctttgg aaactggata acttgagtgc agaaggggag agtggaattc 600 catgtgtagc ggtgaaatgc gtagatatat ggaggaacac cggtggcgaa agcggctctc 660 tggtctgtaa ctgacgctga ggctcgaaag cgtggggagc aaacaggatt agataccctg 720 gtagtccacg ccgtaaacga tgagtgctag gtgttaggcc ctttccgggg cttagtgccg 780 cagctaacgc attaagcact ccgcctgggg agtacgaccg caaggttgaa actcaaagga 840 attgacgggg gcccgcacaa gcggtggagc atgtggttta attcgaagca acgcgaagaa 900 ccttaccagg tcttgacatc cctctgaccg tcctagagat aggactttcc ttcgggacag 960 aggtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt gagatgttgg gttaagtccc 1020 gcaacgagcg caacccctat tgttagttgc catcattaag ttgggcactc tagcgagact 1080 gccggtaata aaccggagga aggtggggat gacgtcaaat catcatgccc cttatgacct 1140 gggctacaca cgtgctacaa tggttggtac aacgagtcgc aagccggtga cggcaagcta 1200 atctcttaaa gccaatctca gttcggattg taggctgcaa ctcgcctaca tgaagtcgga 1260 atcgctagta atcgcggatc agcacgc 1287 <210> 25 <211> 465 <212> DNA <213> Artificial Sequence <220> <223> gyrB-A <400> 25 tcatctcagt tagatgttcg tgtcttcaaa aatggtagca ttcattatca agagtttaaa 60 cgtggaatcg tttatgatga tttgaaaatt attggagaaa cggatttaac aggaacaaca 120 gttcacttta ctccggaccc agaaattttt acagaaacag ttgaatttga ttttgaaaag 180 cttgctaagc gtgtccaaga attagcattt ttaaaccgtg gattacgaat ctcaattgcc 240 gataaacgta atggaattga acaagtcaaa gattaccatt atgaaggtgg tatcgctagc 300 tatgttgaat ttttaaatga gaaaaaagat gttattatcg aacatcctat ctttactgat 360 ggagagatgg atggcattgc tgtcgaagtt gcgatgcaat atacaacagg ctatcatgaa 420 aatgtaatga gttttgccaa caatattcat acacatgaag gcgga 465 <210> 26 <211> 465 <212> DNA <213> Artificial Sequence <220> <223> gyrB-B <400> 26 tcatctcagt tagatgttcg tgtcttcaaa aatggtagca ttcattatca agagtttaaa 60 cgtggaatcg tttatgatga tttgaaaatt attggagaaa cggatttaac aggcacaaca 120 gttcacttta ctccggaccc agaaattttt acagaaacag ttgaatttga ttttgaaaag 180 cttgctaagc gtgtccaaga attagcattt ttaaaccgtg gattacgaat ctcaattgcc 240 gataaacgta atggaattga acaagtcaaa gattaccatt atgaaggtgg tatcgctagc 300 tatgttgaat ttttaaatga gaaaaaagat gttattatcg aacatcctat ctttactgat 360 ggagagatgg atggcattgc tgtcgaagtt gcgatgcaat atacaacagg ctatcatgaa 420 aatgtaatga gttttgccaa caatattcat acacatgaag gcgga 465 <210> 27 <211> 769 <212> DNA <213> Artificial Sequence <220> <223> groE-A <400> 27 ggatgcagtt tgaccgtggt tacctttctc aatacatggt aacagataac gaaaaaatgg 60 ttgctgactt agaaaatcca tttattttga tcactgataa aaaagtatca aacattcaag 120 aaattcttcc actcttagaa gaagtcttaa aaacaaaccg cccattattg attattgctg 180 atgatgttga tggtgaagcc ttaccaacat tagttttaaa taaaattcgc ggaacattta 240 atgttgttgc cgttaaagca ccaggatttg gcgaccgtcg taaggctatg ttagaggata 300 ttgccattct aacaggtgga actgtgatta ctgaagatct tggtttagag cttaaggatg 360 cgacaatagc agcgctaggc caagcagcta aaatctctgt ggataaagat tcaacagtta 420 ttgttgaagg ttctggcggt gcggatgcta ttgccaatcg tgttggccta attaaatccc 480 aattagaaac aacaacgtct gagttcgacc gtgagaaatt gcaagaacgt ctagctaagt 540 tagcgggtgg tgtggcagtt atcaaggttg gtgctgcgac agaaactgag ttgaaagaaa 600 tgaagctccg cattgaagat gctttgaatg ctactcgtgc tgctgttgaa gaagggatcg 660 tagctggtgg aggaactgcc tttatttcag tgattgagaa agtgcaagct ctagaacttg 720 aaggtgataa tgcgactggt cgtaacattg tccttcgtgc ccttgaaga 769 <210> 28 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> simA-A <400> 28 gctaaagagg ctgaagttgc taacaatcgt gttattgttt taagtaatgc tttaaatcaa 60 gctaaagaag aatttgattt gaaagctgct gaagcacaaa aactgaataa taacgtcact 120 gtattatcaa atgccctaca acaagctaaa gaagaatttg acaaaaaagc ccaggactat 180 caagcagctc ttgcagaaaa agatgctatt gacggacaat tagaaactgc tttgactaaa 240 gtttcagacc tagaaagtca attggcaagt ttaaaagaag ctaatgaagc tgctaatcaa 300 aagattactc aattagaaca agaagttgct gatggtgggg ttgcctttaa agaccttaaa 360 gggcaaaaag aaaaatcaga cgcacaagtt aaagctcaag cagataagat taaagaactg 420 gaatctgact tagaagcgac tcaatcagaa ttaaaccagg tgaaagagca acgtgctaat 480 ttagaacaag aagttgctga gcttggaact aaggtaaaag atttagaaaa agaaaaagct 540 gatttagaga aaaaagcgat agacttggaa gctgacaaag cggaatttga agcagaaatg 600 gatcgtttaa aagccttact agctgataaa gacttagcaa atgataaatt acttaaagaa 660 atggaagatc tacaaaaaga ctttgatact aagaaaaatc atagtgacag agaa 714 <210> 29 <211> 714 <212> DNA <213> Artificial Sequence <220> <223> simA-B <400> 29 gctaaagagg ctgaagttgc taacaatcgt gttattgttt taagtaatgc tttaaatcaa 60 gctaaagaag aatttgattt gaaagctgct gaagcacaaa aactgaataa taacgtcact 120 gtattatcaa atgccctaca acaagctaaa gaagaatttg acaaaaaagc ccaggactat 180 caagcagctc ttgcagaaaa agatgctatt gacggacaat tagaaactgc tttgactaaa 240 gtttcagacc tagaaagtca attggcaagt ttaaaagaag ctaatgaagc tgctaatcaa 300 aagattactc aattagaaca agaagttgct gatggtgggg ttgcctttaa agaccttaaa 360 gggcaaaaag aaaaatcaga cgcacaagtt aaagctcaag cagataagat taaagaactg 420 gaatctgact tagaagcgac tcaatcagaa ttaaaccagg tgaaagagca acgtgctaat 480 ttagaacaag aagttgctga gcttggaacc aaggtaaaag atttagaaaa agaaaaagct 540 gatttagaga aaaaagcgat agacttggaa gctgacaaag cggaacttga agcagaaatg 600 gatcgtttaa aagccttact agctgataaa gacttagcaa atgataaatt acttaaagaa 660 atggaagatc tacaaaaaga ctttgatact aagaaaaatc atagtgacag agaa 714 <210> 30 <211> 1237 <212> DNA <213> Artificial Sequence <220> <223> autolysin-A <400> 30 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcagttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc gaaacagtct 180 cagctgatgt gagtggtcat gttttaaaaa ttgtctataa tgggcaacta gaaccaacta 240 agaagataaa gtatgcagtt tggacagata atgctggcca agatgattta gtttggtata 300 ctgctgatca agttggtgcg gcatatattg atttatctaa aaaacataga gcatatgggc 360 tatataatat tcatacctat tcgcaagatg taacaggtaa gatgtctggc ttaaatgcga 420 gacaatttac tattcttaaa ccaacagtaa gtacatcgtt taacgtacaa actaatggtc 480 ttgttgatat tgtcgtttca aatgtgagag gtgatattag ctcaattaaa gttcctgttt 540 ggtcagattt aggtggccaa aacgatatta aatggtatca agctactcaa tcaactgatg 600 gaacatacaa agtttcagta aagatttcag atcattcaaa tgatactggc cattttgctg 660 ttcatgttta tgggaatagt acaatcacga atagtcaaat cggattaggg acaacagaag 720 gttttacaat tgctacccca gacccccaaa atgtagtgtc agtcgtagta gctaatgatg 780 gtttccacct agcattaaat tcgaatgttg ttaaagaatt taccaaagtg aaatttgctg 840 tttggtcgga tcaagctggt caagatgacc ttcattggta cacagccaat gctcagggtc 900 aagtcattgt tccttatgtt aatcatagta attatggtct atacaacatc catacctata 960 gttttgagtc gggaagtgct aaaggtctaa atacaagaac aattacagta ccaaatccaa 1020 ctgctagcgc agccattaca cagaaatcag atcttgaatt tttagtatca gttacgaatg 1080 tcccagctta tatcacaaaa gttatgttac caagttggag tgaaattaat ggccaagatg 1140 atatcaagtg ggtgacggca agtaagctag ctgataatag ttaccaagcc attattaata 1200 ttggagatca taagtataat ttaggtcatt atttagt 1237 <210> 31 <211> 1237 <212> DNA <213> Artificial Sequence <220> <223> autolysin-A1 <400> 31 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcccttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc gaaacagtct 180 cagctgatgt gagtggtcat gttttaaaaa ttgtctataa tgggcaacta gaaccaacta 240 agaagataaa gtatgcagtt tggacagata atgctggcca agatgattta gtttggtata 300 ctgctgatca agttggtgcg gcatatattg atttatctaa aaaacataga gcatatgggc 360 tatataatat tcatacctat tcgcaagatg taacaggtaa gatgtctggc ttaaatgcga 420 gacaatttac tattcttaaa ccaacagtaa gtacatcgtt taacgtacaa actaatggtc 480 ttgttgatat tgtcgtttca aatgtgagag gtgatattag ctcaattaaa gttcctgttt 540 ggtcagattt aggtggccaa aacgatatta aatggtatca agctactcaa tcaactgatg 600 gaacatacaa agtttcagta aagatttcag atcattcaaa tgatactggc cattttgctg 660 ttcatgttta tgggaatagt acaatcacga atagtcaaat cggattaggg acaacagaag 720 gttttacaat tgctacccca gacccccaaa atgtagtgtc agtcgtagta gctaatgatg 780 gtttccacct agcattaaat tcgaatgttg ttaaagaatt taccaaagtg aaatttgctg 840 tttggtcgga tcaagctggt caagatgacc ttcattggta cacagccaat gctcagggtc 900 aagtcattgt tccttatgtt aatcatagta attatggtct atacaacatc catacctata 960 gttttgagtc gggaagtgct aaaggtctaa atacaagaac aattacagta ccaaatccaa 1020 ctgctagcgc agccattaca cagaaatcag atcttgaatt tttagtatca gttacgaatg 1080 tcccagctta tatcacaaaa gttatgttac caagttggag tgaaattaat ggccaagatg 1140 atatcaagtg ggtgacggca agtaagctag ctgataatag ttaccaagcc attattaata 1200 ttggagatca taagtataat ttaggtcatt atttagt 1237 <210> 32 <211> 1228 <212> DNA <213> Artificial Sequence <220> <223> autolysin-B <400> 32 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tcagttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc tcagctgatg 180 tgagtggtca tgttttaaaa attgtctata atgggcaact agaaccaact aagaagataa 240 agtatgcagt ttggacagat aatggtggcc aagatgattt agtttggtat actgctgatc 300 aagttggtgc ggcatatatt gatttatcta aaaaacatag agcatatggg ctatataata 360 ttcataccta ttcgcaagat gtaacaggta agatgtctgg cttaaatgcg agacaattta 420 ctattcttaa accaacagta agtacatcgt ttaacgtaca aactaatggt attgttgata 480 ttgtcgtttc aaatgtgaga ggtgatatta gctcaattaa agttcctgtt tggtcagatt 540 taggtggcca aaacgatatt aaatggtatc aagctactca atcaactgat ggaacataca 600 aagtttcagt aaagatttca gatcattcaa atgatactgg ccattttgct gttcatgttt 660 atgggaatag tacaatcacg aatagtcaaa tcggattagg gacaacagaa ggttttacaa 720 ttgctacccc agaccccaaa aatgtagtgt cagccgtagt agctaatgat ggtttccacc 780 tagcattaaa ttcgaatgtt gttaaagaat ttaccaaagt gaaatttgct gtttggtcgg 840 atcaagctgg tcaagatgac cttcattggt acacagccaa tgctcagggt caagtcattg 900 ttccttatgt taatcatagt aattatggtc tatacaacat ccatacctat agttttgagt 960 cgggaagtgc taaaggtcta aatacaagaa caattacagt accaaatcca actgctagcg 1020 cagccattac acagaaatca gatcttgaat ttttagtatc agttacgaat gtcccagctt 1080 atatcacaaa agttatgtta ccaagttgga gtgaaattaa tggccaagat gatatcaagt 1140 gggtgacggc aagtaagcta gctgataata gttaccaagc cattattaat attggagatc 1200 ataagtataa tttaggtcat tatttagt 1228 <210> 33 <211> 1228 <212> DNA <213> Artificial Sequence <220> <223> autolysin-B1 <400> 33 gtgaagcaat tgctattgat gcatctactc aaaagtcaga actaaatagt aatacagaaa 60 gttcaactcc cgaaatagaa tccgttccaa gtgctccaac tgaagtcaca aatcaggttc 120 aaactcaaac tgctatccag tcaaaaagtg tcgcaccagt cgaaacagtc tcagctgatg 180 tgagtggtca tgttttaaaa attgtctata atgggcaact agaaccaact aagaagataa 240 agtatgcagt ttggacagat aatggtggcc aagatgattt agtttggtat actgctgatc 300 aagttggtgc ggcatatatt gatttatcta aaaaacatag agcatatggg ctatataata 360 ttcataccta ttcgcaagat gtaacaggta agatgtctgg cttaaatgcg agacaattta 420 ctattcttaa accaacagta agtacatcgt ttaacgtaca aactaatggt attgttgata 480 ttgtcgtttc aaatgtgaga ggtgatatta gctcaattaa agttcctgtt tggtcagatt 540 taggtggcca aaacgatatt aaatggtatc aagctactca atcaactgat ggaacataca 600 aagtttcagt aaagatttca gatcattcaa atgatactgg ccattttgct gttcatgttt 660 atgggaatag tacaatcacg aatagtcaaa tcggattagg gacaacagaa ggttttacaa 720 ttgctacccc agaccccaaa aatgtagtgt cagccgtagt agctaatgat ggtttccacc 780 tagcattaaa ttcgaatgtt gttaaagaat ttaccaaagt gaaatttgct gtttggtcgg 840 atcaagctgg tcaagatgac cttcattggt acacagccaa tgctcagggt caagtcattg 900 ttccttatgt taatcatagt aattatggtc tatacaacat ccatacctat agttttgagt 960 cgggaagtgc taaaggtcta aatacaagaa caattacagt accaaatcca actgctagcg 1020 cagccattac acagaaatca gatcttgaat ttttagtatc agttacgaat gtcccagctt 1080 atatcacaaa agttatgtta ccaagttgga gtgaaattaa tggccaagat gatatcaagt 1140 gggtgacggc aagtaagcta gctgataata gttaccaagc cattattaat attggagatc 1200 ataagtataa tttaggtcat tatttagt 1228 <210> 34 <211> 405 <212> DNA <213> Artificial Sequence <220> <223> CPB-A <400> 34 ccaagcacta ataatgaata tttacagaaa tattgatcgt acacaaattc aatttgattt 60 tataatcgat catccaagta tgaactacta tcaggatgaa atagaaagac taggtggacg 120 agtatattct tttccaacat ttacaggaag aaatgttcgt aatgtgcgta atgaatggga 180 taagtttttt aaagaacatg tagagtactc gattattcat tttcatgtta gaagttatat 240 ttctttatta attcctattg caaaaagata cggtttaata acaatatcac atagtcatag 300 tatttctaat ggactaggac taaaatcaaa gattaagaat cttttacaaa ttccgattag 360 atatcaagct gattatcttt ttgcttgctc taaagaggct ggaga 405 <210> 35 <211> 533 <212> DNA <213> Artificial Sequence <220> <223> SOA-A <400> 35 gaaaagttgt tgcagaaatt gctaaattat ctggttataa cgacattatt tttcttgatg 60 attattctaa cgaaaaactt tgttctggtt atccagttgt cggaaaagtt tctgaaattg 120 ttaatttcaa aaatgaagat gtttttatag ctattggatc aagtgctgtt agagagaaaa 180 ttgctaaaca tttgaaggac cacaaaatag tatccttaat acatccggcc gcagttgtaa 240 gtgaaaaagc taaaattggc aaagggagtg ttattatggc tggtgctgta gtaaaccctg 300 atactgaaat tggtgaattt tgtatagtca atacttgttc ttctgtagat catgattgta 360 ttataggtga tttttcacac gtctcagttg gcagtcacgt tgcaggaaca gtaacagttg 420 gttctcatgt atggataggt gctggggcga caataattaa taacatcgaa acacacaata 480 atatttgtat tggagcagga gcaacggtta taaataattt agtagatagt gga 533 <210> 36 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-A <400> 36 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatccct attaagttat ttggtggacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgctggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacacgggat taaagtactg ggaaataaaa attccattcc 420 aagattacta gcagaatatg aagttaaaca ggttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatctg ccgtaacgca aacgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctcaac tgacaaagct gttaacccac ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 37 <211> 1112 <212> DNA <213> Artificial Sequence <220> <223> PB-A1 <400> 37 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatccct attaagttat ttggtggacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggtgct ggtgaaggtg gtagtgcctt tattcaaaca 300 attctgaata agagtaaaga tattgacatt gtcggtattg ttgatgctga tatcaataaa 360 ttaggcacct acttacacgg gattaaagta ctgggaaata aaaattccat tccaagatta 420 ctagcagaat atgaagttaa acaggttacg attgccatcc caagtttatc tggggaagaa 480 cgagaatcaa tcttagatat ctgccgtaac gcaaacgttc acgtaaacaa tatgcctagt 540 attgagaata tcgttctagg taatgtgtca cttaataaat ttaaagaaat tgagattgct 600 gacttacttg gacgaaagga agtagtatta gatcaaacgt ctttaaattc attctttaac 660 gggaaaacag ttcttgtaac tggtgcagga ggatcaattg gttcagaaat ctgtcgtcaa 720 gtttctaaat ttaatccagc acgcatttta cttttagggc atggtgaaaa ttccatttat 780 ctgattcatc gtgaattatc agcactgtta aagggacgaa ttgacattgt cccaattatc 840 gcggacattc aagatcgaga cttgattttt gaaattatgg cgaattatcg acctgatata 900 gtctatcatg ctgcagcaca taaacatgtg ccattgatgg aatacaatcc aaaagaagct 960 gttaaaaata atatctttgg gacaaaaaat gtggcggaag cagccaaggc tgctgggatt 1020 cctaaattta tcatggtctc aactgacaaa gctgttaacc cacctaatgt gatgggtgca 1080 actaagcgct tcgctgaaat gatcgtcact gg 1112 <210> 38 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B <400> 38 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 39 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B1 <400> 39 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agacgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 40 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B2 <400> 40 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtgttgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 41 <211> 1119 <212> DNA <213> Artificial Sequence <220> <223> PB-B3 <400> 41 aataagagct cgaatctttt ccattatcaa tcgctttacc gattataaag tcatctttat 60 cctaatagct aacatgttct tcgcatcctt attaagttat ttggtcgacg ttcttttcct 120 agatacattc agtcgtcgtt ttcttttctt atcattcctt tttggaacat ttttaattat 180 ccttcctcgg atgatttggc ggatgtggca tgaacaaaat ttgtttgtca aacataataa 240 aaaagaccaa aagacaaaaa tgttggttgt tggtgccggt gaaggtggta gtgcctttat 300 tcaaacaatt ctgaataaga gtaaagatat tgacattgtc ggtattgttg atgctgatat 360 caataaatta ggcacctact tacatgggat taaagtactg ggaaataaaa attccattcc 420 aagattagta gcagaatatg aagttaaaca agttacgatt gccatcccaa gtttatctgg 480 ggaagaacga gaatcaatct tagatatttg ccgtaacgca aatgttcacg taaacaatat 540 gcctagtatt gagaatatcg ttctaggtaa tgtgtcactt aataaattta aagaaattga 600 gattgctgac ttacttggac gaaaggaagt agtattagat caaacgtctt taaattcatt 660 ctttaacggg aaaacagttc ttgtaactgg tgcaggagga tcaattggtt cagaaatctg 720 tcgtcaagtt tctaaattta atccagcacg cattttactt ttagggcatg gtgaaaattc 780 catttatctg attcatcgtg aattatcagc actgttaaag ggacgaattg acattgtccc 840 aattatcgcg gacattcaag atcgagactt gatttttgaa attatggcga attatcgacc 900 tgatatagtc tatcatgctg cagcacataa acatgtgcca ttgatggaat acaatccaaa 960 agaagctgtt aaaaataata tctttgggac aaaaaatgtg gcggaagcag ccaaggctgc 1020 tgggattcct aaatttatca tggtctctac tgacaaagca gttaatccgc ctaatgtgat 1080 gggtgcaact aagcgcttcg ctgaaatgat cgtcactgg 1119 <210> 42 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-A <400> 42 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacaac atccgcaaat attgctatct catttgctaa 120 agcaggtcta aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctgtcaggta atgcagaatt 240 atcagcagtt atctctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatcgactc 420 agcgattatt tcacaaaaag ctgacgcaaa catcttagta acagaagctg gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgctt tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594 <210> 43 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-A1 <400> 43 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacaac atccgcaaat attgctatct catttgctaa 120 agcaggtcta aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctgtcaggta atgcagaatt 240 atcagcagtt atctctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatcgactc 420 agcgattatt tcacaaaaag ctgacgcaaa catcttagta acagaagctg gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgctt tgttcttggg 540 tgttatttta aataaagtag aagaatcact tgattcatat ggtggttatg gtag 594 <210> 44 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-B <400> 44 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacatt atccgcaaat attgctatct catttgctaa 120 agcaggtctt aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctatcaggta atgcagaatt 240 atcagcagtt atttctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatagactc 420 agcgattatt tcacaaaaag ctgacgcaaa cattttagta acagaagcag gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtgcct tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594 <210> 45 <211> 594 <212> DNA <213> Artificial Sequence <220> <223> Wze-B1 <400> 45 ctccattcgg actaacatcc aatttagtgg tcgagattta aaagttatca ccttaacatc 60 agtacaacct ggtgaaggga aatcgacatt atccgcaaat attgctatct catttgctaa 120 agcaggtctt aaaaccctat taatcgatgc agacatccgt aattcagtta tgtctggtac 180 atttaaagct gatgaaaagt atgaaggtct atcaagttac ctatcaggta atgcagaatt 240 atcagcagtt atttctcata caaatattga aaacttaatg ttgattccag caggacatgt 300 tcctcctaat ccaacaactt tactccaaaa tagcaatttt aatttcatga ttgatactgt 360 aaaagagtta tttgattatg tgattatcga taccccacct attggccttg ttatagactc 420 agcgattatt tcacaaaaag ctgacgcaaa cattttagta acagaagcag gggctattaa 480 acgacgcttt atccaaaaag caaaagaaca aatggaacaa agtggtacct tgttcttggg 540 tgttatttta aataaagtag aagaaacact tgattcatat ggtggttatg gtag 594

Claims (7)

Inactivated Streptococcus parauberis KCTC 13800BP, KCTC 13801BP, KCTC 13802BP and KCTC 13803BP As described above, the KCTC 13800BP strain, the KCTC 13801BP strain, the KCTC 13802BP strain, and the KCTC 13803BP strain are inactivated for the prevention of streptococcal disease, which are strains of serotype Ib, serotype II, serotype Ia and serotype Ic, respectively. vaccine.
According to claim 1, wherein the vaccine is an inactivated vaccine for preventing streptococcal disease, characterized in that the KCTC 13802BP strain and KCTC 13803BP strain are mixed in a ratio of 1: 1.
According to claim 1, wherein the vaccine is an inactivated vaccine for preventing streptococcal disease, characterized in that the KCTC 13800BP strain, KCTC 13802BP strain and KCTC 13803BP strain are mixed in a ratio of 1: 1: 1.
The method of claim 1, wherein the antigen concentration of the vaccine is 1 to 30mg / fish inactivated vaccine for preventing streptococcal disease.
Vaccines by inactivating any two or more strains selected from the group consisting of Streptococcus parauberis KCTC 13800BP strain, KCTC 13801BP strain, KCTC 13802BP strain and KCTC 13803BP strain isolated from halibut infected with streptococcal disease As a method for preparing an inactivated vaccine for preventing streptococcal disease, comprising the steps of obtaining, the KCTC 13800BP strain, the KCTC 13801BP strain, the KCTC 13802BP strain and the KCTC 13803BP strain are serotype Ib and serotype II, respectively. , Serotype Ia and serotype Ic strains, characterized in that the method of producing an inactivated vaccine for preventing streptococcal disease.
A method of immunizing fish comprising administering the vaccine of claim 1 to fish.
7. The method of claim 6, wherein the vaccine is injected into the abdominal cavity of the fish.

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