KR101944233B1 - novel streptococcus strain inactivated vaccine against bacterial diseases in starry flounder using the streptococcus strain - Google Patents

Abstract

The present invention relates to a method for producing an alpha-hemolytic Streptococcus parabeuris strain HFTC0045 Streptococcus parauberis (microorganism deposit number KCTC18522P), alpha-hemolytic streptococcus parauberis HFTC0049 Streptococcus parauberis (microorganism deposit number KCTC18523P) And a method for immunizing a fish using the vaccine.

Description

[0001] The present invention relates to a novel streptococcus strain inactivated vaccine against bacterial diseases using starved flounder using the streptococcus strain,

The present invention relates to a novel Streptococcus strain and a mixed inactivated vaccine for preventing bacterial disease of the intact leg using the same. The present invention also relates to a novel Streptococcus strain, an inactivated vaccine for the prevention of bacterial disease of a fish which contains an antigen of a known strain.

Bacterial diseases in fish are caused by a myriad of bacterial infections. In the case of intestinal flora, which is a typical aquaculture, the flora is cultured with flounder, and infection and spread of diseases caused by flounder are very high. In particular, it is always exposed to the occurrence of staphylococcus aureus, which is increasingly damaged in flounder, and the risk of production loss due to it. Streptococci were isolated from internal organs such as sardinops melanosticta and canary (Ammodytes personatus), which are used as flounder-feeding feedstocks (Kitao, 1979) In the case of anthropomorphic infection, the symptoms of typical staphylococcal disease were reproduced in the experimentally infected abdominal cavity, and the causative organisms survived for more than 6 months in the feed fish kept frozen (Minami, 1979). Romalde et al. (1996) found that Atlantic salmon, Salmo salar, and a host of Enterococcus species, a pathogenic enterococcus species causing disease in S. maximus, Reported that streptococcicosis is transmitted horizontally to S. maximus, a turbot, through the fecal-oral route in the transporter and seawater. Lactococcus garvieae, Streptococcus iniae and Streptococcus parauberis are known to be causative agents of Streptococcus pneumoniae (Oh et al., 1998; Lee et al., 2001; Kim et al, 2005; Baeck et al., 2006). Examples of infections caused by L. garvieae and S. iniae, causative organisms of Streptococcus, include Nile tilapia, Plecoglossus altivelis, Oncorhynchus mykiss, Anguilla japonicam, Olive flounder (Paralichthys olivaceus) It has been reported in a variety of fish species such as: Seriola quinqueradiata, Sciaenips ocellatus, Sparus aurata, European sea bass, Dicentrarchus labrax, barramundi and Lates calcarifer 1999, Zlotkin et al., 1998, Bromage et al., 1999 (Kitao et al., 1981; Ugajin, 1981; Kawahara and Kusuda, 1987; Sakai et al., 1989; Eldar and Ghittino, 1999; Eldar et al., 1999;

Recently, S and parauberis have been detected at a high rate among the various streptococci isolated from the aquaculture legs, and the frequency of detection is also increasing. In Spain, a 0.8 to 2.0 kg sized turbot, which is raised in warm water, has a mortality rate of 0.1 to 5.0% due to infection with S. parauberis (Domenech et al., 1996). Infection caused by S. parauberis is known to be detected in high temperature as well as in autumn when water temperature begins to fall. The main symptoms are external darkening of the coloration, fin bleeding, proptosis and congestion, ocular opacification and abdominal distension, , Liver congestion, congestive kidney, and swelling of the spleen. (Baeck et al., 2006; Woo et al., 2006; Jeong et al., 2004). Antibacterial chemotherapy has been widely used as a method for treating such streptococcal disease. However, resistance to antibiotics is increasing. Especially, resistant pathogens, which are the cause of fish diseases, are frequently isolated by R plasmid transfer (Aoki, 1998) (Aoki et al., 1990). In addition, due to the economic loss due to the administration of antibiotics and the growth rate of the flounder, the industry is suffering from double problems. Streptococcal disease is very difficult to treat once it develops, so prevention of disease is very important. However, improvement of breeding environment and breeding management has limitations in suppressing disease occurrence. Therefore, the streptococcal vaccine of Streptococcus pneumoniae is the best way to reduce the amount of antibiotics used, and it will be generalized in the future, and it is proved to be economical in terms of cost effectiveness.

 Therefore, the inventors of the present invention have identified a novel Streptococcus strain while studying a method for preventing bacterial disease which is a serious problem in fish culture, and confirmed that the vaccine treated with the microorganism was effective in preventing bacterial diseases of fish And completed the present invention.

It is therefore an object of the present invention to provide a novel strain of Streptococcus isolated from an intact leg infected with bacterial diseases. It is another object of the present invention to provide an inactivated vaccine for the prevention of bacterial disease of a fish containing an antigen of the above strain.

It is still another object of the present invention to provide a method for producing an inactivated vaccine for preventing bacterial disease in a fish comprising a novel Streptococcus strain.

In order to accomplish the above object, the present invention provides novel α-hemolytic Streptococcus parauberis HFTC 0045 , α-hemolytic Streptococcus parauberis HFTC The strain is provided.

To achieve these and other objects, the present invention provides an inactivated vaccine for the prevention of bacterial disease of fish, which comprises an inactivated strain of Streptococcus parauberis HFTC 0045 , HFTC 0049 . The present invention provides a method for producing a vaccine containing the above bacteria.

The vaccine according to the present invention can be effectively used for aquaculture industry because it can effectively and economically prevent disease caused by streptococci by administering an inactivated vaccine once with a vaccine for intense legs.

Figs. 1 and 2 are photographs of a purely isolated colony of the strain of S. parauberis grown in BHIA (Difco, USA) medium supplemented with 1.5% NaCl and in a rabbit serum medium (BAP).
FIG. 3 shows specific primer information having a size of 718 bp as a 23S rRNA target primer sequence of the strain Streptococcus parauberis.
FIG. 4 is a photograph showing an end product of 718 bp confirmed by a gel-Doc (Bio-Rad) electrophoresis after PCR, and the results are shown in bands.
Figures 5, 6, and 7 show the results of the novel alpha-hemolytic streptococcus parauberis HFTC 0045, HFTC [0049] The photographs showing the external symptoms and autopsy findings of the dead animals after the attack inoculated with the fishes showing the strain on the legs of the fishes. The photographs showing the lesions of the intense legs infected with the above strains showed mainly ocular protrusion, periocular abscesses and bleeding, , External symptoms such as abdominal distension, and internal symptoms such as liver congestion and congestion.
In FIGS. 8, 9 and 10, the test legs of 11 to 14 cm (17 g to 40 g) were used for each test gripper. As a result of immunohistochemical analysis, it was observed that the ellipsoid hypertrophy was not observed even in the general form of MMC. This suggests that the systemic infection level of the pathogen is low or does not reach the threshold value that will induce the inflammatory reaction. The normal tissue findings were observed in all the organs of the vaccine treatments, so that the inoculation vaccine was not toxic.
11, 12, and 13, the test legs of 11 to 14 cm (17 to 40 g) were used for each test organ. In contrast to the vaccination group, macrophage activation was markedly observed in experimental animals that did not receive the immunization of the vaccine but only in the attacked group. In particular, the tendency of the spleen to observe such tendency and the tendency of the ellipsoid to enlarge was observed. It is considered that the pathogen or internal stiffness factor is actively observed.

The present invention provides novel strains of Streptococcus parauberis HFTC 0045, HFTC 0049, isolated from the intact legs.

As a result of the nucleotide sequence analysis of 16S rDNA, the strain showed 99% homology with Streptococcus parauberis KCTC11537 strain and showed higher pathogenicity, that is, mortality rate compared to the conventionally known Streptococcus parauberis .

The new strain according to the present invention is a strain derived from a strain derived from a preliminary field isolate of Yongdeok branch of Gyeongsangbuk-do Fisheries Technical Center through a growth test and the like. The strains are HFTC 0045, HFTC 0049] The origin and main information of the strain are shown in [Table 1] (main information of vaccine strain).

The present invention provides an inactivated vaccine using the strain. There is provided an inactivated vaccine for bacterial disease prevention comprising an externally inactivated Streptococcus parauberis antigen. Such inactivated vaccines may be prepared using methods well known in the art and may be prepared, for example, using inactivating agents known in the art. The inactivated vaccine according to the present invention can prevent bacterial diseases that are most commonly caused in fish by inhibiting streptococcal disease. The vaccine according to the present invention can be used by sealing the vaccine solution in an appropriate volume, for example, about 10 to 500 ml volume. Such a vaccine may be used not only as a liquid but also as a desiccant agent by separately injecting and then freeze-drying. The dry preparations can be used by completely re-dissolving the dry substance with the stimulant added immediately before use. The mixed inactivated vaccine according to the present invention can be used in any age fish at risk of infection. However, from the viewpoint of preserving the fish, it is preferable to use a small fish or a fish. The present invention includes a method for producing the vaccine. Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: New Streptococcus  Isolation and Identification of Strain

The strain of Streptococcus strains according to the present invention was isolated from the intestinal flora of the aquaculture farm in Gyeongbuk province, which was massively dead due to ocular bulge, ocular pericardial eruption and bubble generation, sputum redness, abdominal distension, and ascites symptoms. Blood was collected by aseptically harvesting the internal organs, ascites, heart, brain, etc. of the diseased fish using the medium supplemented with 1.5% NaCl (Difco, USA) And cultured for 24 to 28 hours. The dominant colonies were selected according to the culture condition and cultured separately. Seven Gram-positive strains were isolated from pure colonies and named PH0710, PH0711, PH1012, PH1108, UJ1106, HFTC 0045 and HFTC 0049, respectively. A purely separated colony photograph of each of the strains was shown in Fig.

Among the above strains, HFTC 0045 and HFTC 0049, which have the highest activity, were selected through preliminary experiments. 16S rDNA was cloned into the 23S rRNA target primer of Streptococcus parauberis (Bionics, korea) according to the method of Mata et al. (Appl. Environ. Microbiol., 68: 5177-5180, 2004) (PCR) using Spa2152 (SEQ ID NO: 1) and Spa2870 (SEQ ID NO: 2).

PCR was performed using Accupower PCR premix (Bioneer, Korea). The PCR conditions were predenaturation (94 ℃ for 5 min), denaturation (95 ℃ for 30 sec), annealing (55 ℃ for 30 sec), extension (72 ℃, 30 seconds) was repeated 30 cycles long and final extension (72 ℃, 5 minutes) was performed. The polymerase chain reaction (PCR) product was electrophoresed on 1.0% agarose gel with 0.5 μg / ml EtBr at 100 V for 30 min, and the final product was identified as 718 bp by Gel-Doc (Bio-Rad). The amplified polymerase chain reaction (PCR) products were analyzed for nucleotide sequences using AccuPrep PCR purification kit (Bioneer) and AccuPrep PCR plasmid extension kit (Bioneer).

The 23S rRNA base sequence of HFTC 0045 and the genetic information of Streptococcus bacteria registered in GenBank were subjected to multiple alignment using the Squman program and the homology between the respective species was compared. As a result of analyzing the base sequence for the 23S rRNA gene, it showed 99% homology with Streptococcus parauberius KCTC11537 strain registered in GenBank, HFTC 0045 and HFTC 0049 were identified as Streptococcus parauberi. The HFTC 0045 and HFTC 0049 were designated as Streptococcus parauberis HFTC 0045 and HFTC 0049, respectively. After polymerase chain reaction (PCR), 718 bp of the final product was confirmed by gel electrophoresis (Bio-Rad), and the result is shown in FIG.

Example 2: Streptococcus parauberis HFTC 0045  , HFTC 0049 Characterization of

Streptococcus parauberis HFTC 0045 identified in Example 1, HFTC In order to analyze the properties of 0049, compared to the prior known Streptococcus para Ube-less type strain (KCTC3651) and Example 1, Gram-positive strains obtained from the discrete colony in five kinds (PH0710, PH0711, PH1012, PH1108, UJ1106). The strains used are shown in Table 2 (notation of the above strain).

<2-1> Pathogenic experiment

HFTC 0045 and HFTC 0049 were inoculated in a water tank of 10 to 20 g of each of the legs of 17 to 25 g to be inoculated into the peritoneal cavity at a dose of about 1 × ^{10 8} CFU / And observed clinical signs and mortality for 14 days. Clinical symptoms include head haemorrhage, abdominal distension, mucus hyperplasia, and exophthalmos. Autopsy findings of the lungs show multiple ascites, leprosy, and hepatic bleeding. The pathologic findings of the clinical fish showed leukocyte infiltration of the kidney, liver atrophy, and leukocytosis of the spleen.

Each of the strains to ^{1.0 × 10 8 CFU / ㎖ (} 1 × 10 7 CFU / 0.1㎖) was diluted with 10-fold was adjusted such that the lowest concentration of ^{1.0 ~ 1.5 × 10 2 CFU /} ㎖ (1 × 10 1 CFU / 0.1 ml). LD50 confirmation experiment was carried out, and 0.1 ml of the prepared bacterial solution was intraperitoneally injected into 10 test fishes per test group. In the control group, the same amount of physiological saline was intraperitoneally injected and the cumulative mortality rate for 2 weeks after the infection was examined. The results of the investigation are shown in the following [Table 3].

As shown in the above Table 3, the strains of Streptococcus parauberius HFTC 0045 and HFTC 0049 of the present invention showed mortality from the 4th day of injection and a cumulative mortality rate of 60% or more at the 8th day, , And the anatomical symptoms and external findings of the dead animal after the inoculation were as shown in FIG. 5, FIG. 6, and FIG. 7.

<2-2> Enzyme activity investigation

To investigate the enzymatic activity of Streptococcus parauberius HFTC 0045 and HFTC 0049 , 65 μl of each was dispensed into the cups of each kit using API ZYM (Biomerieux), followed by reaction at 25 ° C. for 4 hours to determine the result Respectively. The results of the enzyme activity test are shown in Table 4 (results of microbial enzyme activity test (API ZYM) of HFTC 0045, HFTC 0049).

ensyme assayed for HFTC 0045 HFTC 0049 One control colorless 2 Alkaline phosphatase + + 3 Esterase + + 4 Esterase Lipase + + 5 Lipase - - 6 Leucine arylamidase + + 7 Valine arylamidase + + 8 Crystine arylamidase + + 9 Trypsin - - 10 alpha-chymotrypsin + + 11 Acid phosphatase + + 12 Naphtol-AS-BI-phosphohydrolase + + 13 alpha -galactosidase - - 14 β-galactosidase - - 15 β-glucuronidase - - 16 α-glucosidase + + 17 β-glucosidase + + 18 N-acetyl-β-glucosaminidase - - 19 alpha-mannosidase - - 20 alpha-fucosidase - -

The fermentation and enzyme activity of the sugar were measured using the API ZYM kit (BioMerieux, France) as a kit for the study of microbial enzyme activity. As shown in the table, there was a slight difference in activity depending on the strains, Valine arylamidase and β-glucosidase all showed negative reaction in all positive reactions, Lipase, α-mannosidase and α-fucosidase. In case of Crystine arylamidase and Trypsin,

<2-3> Hemolytic activity investigation

Hemolysis type was investigated using blood agar medium, and α-hemolysis was observed in both the standard strain and the isolated strain, and there was no difference between the strains.

Example  3: Preparation of strain for vaccine preparation

The following two strains identified in Example 1 were prepared for vaccine production.

Alpha-hemolytic streptococcus parauberis HFTC 0045,

Alpha-hemolytic streptococcus parauberis HFTC 0049

Example  4: Pathogenicity of strains for vaccine production

The LD value of the vaccine strain of Example 3 was determined by a method well known in the art (Hall, JA and Golding, LA: Report of the MFE 80205. NIWA report for standard methods for whole effluent toxicity testing the ministry for environment, Wellington, New Zealand, 1988). The results were as shown in Table 5 (LD value of back-borne strains).

Example  5: Preparation and inoculation of mixed inactivated vaccine

<5-1> Preparation of vaccine

Streptococcus parauberis inactivated bacterial antigen (HFTC 0045 strain, HFTC 0049 strain), which has been confirmed as final identification, was used as an outdoor isolate from the intact leg body. (-), hippurate (+), esculin (-), lactose (-), and lactose resolutions were not observed in the media supplemented with 6.5% NaCl. (+), And biochemical markers such as PCR, sequencing, and genetic testing, and serologically confirmed strains were used as vaccine strains). Strain and preservation of the original strains were carried out. Bacterial strains were plated on Blood agar or THBA (Todd Hewitt Broth agar supplemented with 1.5% NaCl) and incubated overnight at 25 ° C for 18 24 hours. Single colonies were selected and 1.5% NaCl The cells were transplanted into added THB (Todd-Hewitt Broth) and cultured at 25 ° C for 15-18 hours. The cells were lyophilized and stored in cold dark place at -20 ° C or lower.

After lyophilization and dissolution of the strain in a cold dark place at -20 ° C or lower in sterile saline, the cells were plated on THBA supplemented with blood agar or 1.5% NaCl, incubated at 25 ° C for 24-48 hours, Were selected and transplanted into THB supplemented with 1.5% NaCl and cultured at 25 ° C for 15-18 hours with shaking. The primary culture medium of THB supplemented with 1/100% of 1.5% NaCl in the present culture medium is inoculated with 1% of the primary culture medium and shake cultured for 10 to 15 hours.

When the culture solution is prepared, the culture and inactivation are performed. The THB medium supplemented with 1.5% NaCl was completely dissolved in the fermenter, and steam sterilized at 121 ° C for 20 minutes. After completion of the sterilization, the mixture was cooled with cooling water, inoculated with the final prepared seed culture at 25 ° C, - While culturing for 18 hours, measure the OD value at 600 nm at appropriate intervals using UV-spectrophotometer (SHIMADZU Co.) and cultivate to the required concentration. When the OD ₆₀₀ of the final culture reaches 1.1 or higher (about 1x10 ⁹ cfu / ml), take a sample to investigate whether or not the bacteria are mixed and add 0.3% of formalin in the culture medium and inactivate it for 18-24 hours. The inactivated bulk was collected by using a continuous centrifuge (Tomoe-sharples centrifuges), resuspended in sterile phosphate buffered saline (PBS), concentrated, and formalin was added at a concentration of 0.5% After inactivation in a constant temperature room for 72 hours, the mixture is inactivated and stored at 5 ° C. The bulk of the inactivated cells is incubated for 3 days at 25 ° C. No bacterial growth should occur.)

The inactivated Streptococcus parauberis HFTC 0045 antigen and the inactivated alpha-hemolytic Streptococcus parauberis HFTC 0049 antigen were mixed at a ratio of 1: 1, A mixed inactivated vaccine for the prevention of hypertension is prepared.

<5-2> Inoculation of vaccine

Strength legs were used as a test fish. Strength legs were mixed with 1: 1 equivalent of each test vaccine by intraperitoneal injection. Percutaneous injection of 0.1 ml per fish was used. In the control group, sterilized physiological saline was intraperitoneally administered by the same amount.

Example 6: Immunogenicity test of inactivated vaccine

<6-1>: Investigation of Defense

To investigate the effect of the test vaccine on the defense, three weeks after the vaccination, the same strain S. parauberis HFTC 0045, and HFTC 0049 to LD ₆₀ values for 2 weeks after intraperitoneal injection . After 4 weeks from the attack, check the survival of the experimental strength legs and calculate the relative survival rate according to the following formula.

Relative survival rate was calculated as shown in Table 6 (relative survival rate method).

Relative Percent Survival (RPS)
RPS = (1 - X / Y) x100
(X is the cumulative mortality rate of the vaccine group, and Y is the cumulative cumulative mortality rate of the control group)

As a result of investigating the protective effect against each strain after the vaccination for the antigen amount determination test, the antigen amount was determined to be 1 × ^{10 8} cfu / ㎖, and RPS value of 90% or more was obtained as shown in FIGS.

Example  7: Safety investigation and vaccination control against mixed inactivated vaccine

<7-1> In order to investigate the safety of the vaccine treatment according to the present invention, the mixed vaccine peritoneal injection group and the control group of Example 6 were subjected to pathological examination. The liver, kidney and spleen tissues of the test specimens were fixed on 10% neutral formalin on day 1, 1 week, and 4 weeks after vaccination. H & E staining was performed according to the conventional method, and the specimens were examined. As a result, normal tissues were observed in all the organs of the vaccine treatments, and it was confirmed that the vaccine was not toxic through [Figures 8, 9, 10]. The results of histological examination of the dead organisms in the vaccinated group not vaccinated were as shown in Figs. 11, 12 and 13.

[Explanation of table]

Table 1: Origin and main information of the vaccine strain in the strain.

Table 2 is a table listing used strains of five gram-positive strains (PH0710, PH0711, PH1012, PH1108, and UJ1106) for comparison with the strains.

Table 3 is a table showing the log of death after two weeks of intensive inoculation of the intact legs of five gram-positive strains (PH0710, PH0711, PH1012, PH1108, and UJ1106) for comparison with the strains.

[Table 4] Results of microbial enzyme activity test (API ZYM) of HFTC 0045 and HFTC 0049 strains.

[Table 5] HFTC 0045 and HFTC 0049 strains (10 ² to 10 ¹⁰ ) were diluted 10 ² to 10 ¹⁰ , and 0.1 ml of each strain was inoculated into the intestinal peritoneal stomach for each dilution, After the observation, the number of inoculations in the maximum dilution ratio of 60% or more was taken as the attacked dose, and the LD50 value of the final vaccine strain was confirmed.

[Table 6] = Relative survival rate.

Claims (6)

A mixed inactivation vaccine for the prevention of streptococcal syndrome comprising an inactivated mixed strain isolated from an intact strain infected with streptococci, the inactivated mixed strain comprising an inactivated alpha-hemolytic Streptococcus parauberis HFTC The strain (microorganism deposit number KCTC18522P) and the inactivated alpha-hemolytic Streptococcus parauberis strain HFTC 0049 (microorganism deposit number KCTC18523P),
Or a pharmaceutically acceptable salt thereof. The mixed inactivated vaccine according to claim 1, comprising an inactivated mixed strain isolated from an intact leg infected with streptococci, wherein the inactivated mixed strain is an inactivated alpha-hemolytic Streptococcus paraubesi 1: 1 mixture of Streptococcus parauberis strain HFTC 0045 (microorganism deposit number KCTC18522P) and inactivated alpha-hemolytic Streptococcus parauberis strain HFTC 0049 (microorganism deposit number KCTC18523P) Mixed inactivated vaccine for prevention. A mixed inactivation vaccine for the prevention of streptococcal syndrome comprising an inactivated mixed strain isolated from an intact strain infected with streptococci, the inactivated mixed strain comprising an inactivated alpha-hemolytic Streptococcus parauberis HFTC hemolytic Streptococcus para Ube lease (Streptococcus parauberis) HFTC 0049 strain (microorganism deposit number KCTC18523P) mixed streptococci increased prevention mixed inactivated vaccine which comprises - 0045 strain (microorganism deposit number KCTC18522P) and inactivation of the alpha Gt; 4. The mixed inactivated vaccine according to claim 3, wherein the inactivated mixed strain is an inactivated mixed strain isolated from the intact leg infected with the streptococci, wherein the inactivated mixed strain is an inactivated alpha-hemolytic streptococcus para A strain of Streptococcus parauberis HFTC 0045 (microorganism deposit number KCTC18522P) and a strain of Streptococcus parauberis HFTC 0049 (microorganism deposit number KCTC18523P) were mixed at a ratio of 1: 1 A method for producing a mixed inactivated vaccine for the prevention of streptococcal disease. The vaccine of claim 1 or 2; Or a vaccine prepared by the method of claim 3 or 4 to fish. 6. The method according to claim 5, wherein the vaccine is injected into the abdominal cavity of the fish.

Images