JP2015134734A - Live vaccine formulation and preventive method for edwardsiella disease - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an effective preventive means against Edwardsiella disease in Sparidae or Carangidae fishes.SOLUTION: The live vaccine formulation contains a live virus of a typical strain of Edwardsiella trade (e.g., HET-1 strain) which shows strong pathogenicity to fishes except Sparidae or Carangidae fishes, but has low pathogenicity or non-pathogenicity to Sparidae or Carangidae fishes, such as Anguilla japonica and Paralichthys olivaceus, and has strong immunogenicity against Edwardsiella disease in Sparidae or Carangidae fishes.

Description

  The present invention relates to a vaccine preparation for edwardiadiasis of Thai fish or phlogopaceae, and a method for preventing edovazierasis. More specifically, the present invention relates to a live vaccine preparation containing a viable bacterium of Edwardsiella tarda standard strain and a method for preventing Edwardsiella disease.

  Thai fishes belong to the order Persian and Persian and include red sea bream, chidai and black sea bream. The genus Dendrobidae also belongs to the order of the periwinkle, perch, and includes the yellowtail, yellowtail, amberjack, and yellowtail.

  Many Thai fishes and phlogopid fishes are used for food, and many have been put to practical use. In particular, yellowtail and red sea bream have a high production volume and aquaculture is widespread.

  In general, in fish farming, many fish are bred in a relatively small area, so that infectious diseases are easily generated and prevalent. If an infectious disease prevails during the aquaculture process, it can cause significant economic damage. Therefore, from the viewpoint of preventing the occurrence of infectious diseases, the development of vaccines for each disease has been attempted, and many have been put into practical use.

  Edwardsiella tarda (scientific name, the same applies hereinafter) is a bacterium classified into the genus Edwardiella. E. tarda has a wide host range, and in addition to fish, invertebrates, amphibians, reptiles, birds and mammals have been isolated.

  E. tarda is broadly classified into a typical strain (wild-type) and an atypical strain (biogroup 1). A typical strain is a strain that has motility and does not ferment mannitol, arabinose, or saccharose, and there are separation examples such as eel, flounder, catfish, tilapia, turbot, striped bath, masunosuke, and human. An atypical strain is a strain that does not have motility and ferments mannitol, arabinose, and saccharose, and there are separation examples of tilapia, red sea bream, and red sea bream. It is only tilapia that has a separation example of both standard and atypical stocks.

  The bacterial infection in fish caused by E. tarda is called edwardiellosis. For example, in flounder edovadierosis, typical strains were isolated and presented with symptoms such as poor feeding, darkening of the body, abdominal distension, ileum, ascites, etc. It is isolated and an abscess lesion is formed in the back of the kidney. Symptoms such as an enlarged protrusion of the anus and redness swelling around it occur. On the other hand, in a red sea bream, an atypical strain is isolated and exhibits symptoms such as slow swimming and ulceration in the body surface and in the cavity.

Research on vaccines against fish edwardiellosis has been widely conducted, mainly for flounder. Currently, inactivated vaccines for flounder have been put to practical use, but vaccines for red sea bream have not been put to practical use. . As a live vaccine against edwardiadiosis of fish, for example, Non-Patent Document 1 shows an example of immunizing tilapia using a typical mutant that shows pathogenicity against tilapia, and Non-Patent Document 2 shows a human-derived standard. Examples of immunizing flounder using a strain are disclosed in Non-Patent Document 3, respectively, in which examples of immunizing flounder using a strain isolated from eel are disclosed. In addition, as an inactivated vaccine against red sea bream, Non-Patent Document 4 showed that the vaccine effect was confirmed as a result of intraperitoneal injection immunization with an inactivated bacterial solution of a red sea bream-derived strain followed by intraperitoneal injection attack with a homologous strain. Has been reported. In addition, Patent Document 1 discloses that an inactivated vaccine against edwardiellosis caused by an atypical strain containing an inactivated microbial cell of a typical strain, or an edible type of edeziella disease caused by a typical strain containing an inactivated microbial cell of an atypical strain. Patent Document 2 discloses an inactivated vaccine against fish infection caused by E. tarda, which contains an antigen derived from E. tarda that is not pathogenic to the fish species to be administered. ing.
Japanese Patent No. 4892296 JP 2007-238505 A Arisa Igarashi and Takaji Iida, “A Vaccination Trial Using Live Cells of Edwardsiella tarda in Tilapia”; Fish pathology, 37 (3), 145-148,2002.9 Shuang Cheng, Yong-hua Hu, Min Zhang and Li Sun, “Analysis of the vaccine potential of a natural avirulent Edwardsiella tarda isolate”; Vaccine 28 (2010) 2716-2721 Tomokazu Takano, Tomomasa Matsuyama, Norihisa Oseko, Takamitsu Sakai, Takashi Kamaishi, Chihaya Nakayasu, Motohiko Sano and Takaji Iida, “The efficacy of five avirulent Edwardsiella tarda strains in a live vaccine against Edwardsiellosis in Japanese flounder, Paralichtyhs olivaceus”; Fish & Shellfish Immunology Tomokazu Takano, Tomomasa Matsuyama, Takamitsu Sakai and Chihaya Nakayasu, “Protective Efficacy of a Formalin-Killed Vaccine against Atypical Edwardsiella tarda Infection in Red Sea Bream Pagrus major”; Fish pathology, 46 (4), 120-122,2011.12

  An object of the present invention is to provide an effective preventive measure against edwardiadiasis of Thai fishes or phlogid fishes.

  The inventors of the present invention have established that a viable E. tarda derived from a standard strain of Edwardsiella tarda, i.e. eels, flounder, etc. By inoculating Thai fish or horse mackerel fish without attenuating or inactivating live bacteria of E. tarda standard strains, while demonstrating that they are sexually or non-pathogenic and have strong immunogenicity The present inventors have newly found that it is possible to effectively prevent edwadierasis in Thai fishes or phlogid fishes.

  Therefore, the present invention provides a vaccine preparation for Thai edible fish or phlogopaceae edovadierosis, which contains a live bacterium of Edwardsiella tarda standard strain.

  The live E. tarda strains show strong pathogenicity in fishes other than Thai fish and shark fish such as eel and flounder. On the other hand, it is low-pathogenic or non-pathogenic to Thai fishes or Carpidae fishes, and has strong immunogenicity against Edwardsiellasis of Thai fishes or Carpidae fishes. Therefore, the E. tarda standard strain live bacteria is effective as a live vaccine against edwardiellosis of Thai fishes and phlogid fishes, and can be applied as vaccines without being attenuated or inactivated.

  In the present invention, since the E. tarda standard strain live bacteria can be applied without being attenuated or inactivated, the preparation and preparation of the preparation can be performed easily and at low cost. Moreover, since it can be applied as a live vaccine, the effect as a vaccine is high, and it can immunize with a small amount of inoculation.

  In addition, the present invention can eliminate the concern that the marine area is contaminated with the live bacteria by using it as a preparation to be applied in an area isolated from the marine area in the natural environment, such as a seedling production facility. It is possible to suppress the risk that fish other than the Thai fishes or the horse mackerel fishes present in the plant will be infected with the live bacteria.

  According to the present invention, it is possible to effectively prevent edwadierasis of Thai fishes or phlogid fishes.

<About live vaccine formulation according to the present invention>
The live vaccine preparation according to the present invention includes all vaccine preparations against edwardiaria disease of Thai fishes or phlogid fishes, which contain live E. tarda strains.

  As described above, the E. tarda wild-type strain is a strain that has motility and does not ferment mannitol, arabinose, or saccharose, and includes eel, flounder, catfish, tilapia, turbot, striped bath, trout, and human. It can be isolated from individuals suffering from Edwardsiella disease. The present invention relates to an E. tarda standard strain that is virulent or non-pathogenic to Thai fishes or phlogid fishes, and is an immunogen against edawadiera disease of Thai fishes or phlogid fishes. Widely applicable to those having properties.

  For example, the sequence described in SEQ ID NO: 1 in the upstream region of the ciliary gene group in the genome of the E. tarda strain, or preferably 92% or more, more preferably 95% or more, most preferably with the sequence. A strain containing a sequence having a homology of 97% or more is suitable for viable E. tarda standard strains used in the present invention.

  That is, in the sequence of the upstream region of the cilia gene group in the genome of the E. tarda strain, there is a difference between a typical strain (wild-type) and an atypical strain (biogroup 1). Based on the homology between the partial sequence of the upstream region of the ciliary gene group and the sequence described in SEQ ID NO: 1, appropriately select a strain that is low or non-pathogenic to Thai fish You can choose.

  In addition, when PCR was performed using the genomic DNA extracted from the bacterial cells as a template and the primers of SEQ ID NO: 2 and SEQ ID NO: 3, the sequence in the upstream region of the cilia gene group was amplified, and the primers of SEQ ID NO: 4 and SEQ ID NO: 5 were used. A strain in which the sequence in the upstream region of the cilia gene group is not amplified when PCR is performed is suitable for viable bacteria of the E. tarda routine strain used in the present invention.

  If the sequence in the upstream region of the cilia gene group does not amplify even when PCR is performed using the primers of SEQ ID NO: 2 and SEQ ID NO: 3, it does not fall into either the E. tarda typical strain or the atypical strain. The immunogenicity of edwardierosis in fish or phlogopaceae may be low. On the other hand, when PCR is performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 5, if the sequence in the upstream region of the cilia gene group does not amplify, it may be an atypical strain of E. tarda. Or it may be highly pathogenic to phlogid fish. Therefore, by performing PCR using the primers of SEQ ID NOs: 2 to 5, a strain that has immunogenicity and is less pathogenic or non-pathogenic to Thai fishes or phlogid fishes is appropriately selected. it can.

  In the present invention, it is possible to use attenuated or inactivated cells of the E. tarda standard strain, but those containing live E. tarda standard strain are most preferable. By containing viable bacteria of E. tarda standard strains, it is possible to reduce the labor required for the attenuation or inactivation, such as the establishment of attenuated strains, the manufacture and preparation of pharmaceuticals, and the cost of drugs such as inactivating agents it can. Moreover, the fall of the immunogenicity by attenuation or inactivation can be prevented, the effect as a vaccine is high, or an inoculation amount can be decreased. In addition, it is possible to eliminate the concern that the chemical used in the inactivation treatment remains in the fish body.

  For example, Edwardsiella tarda is a typical E. tarda strain that is hypopathogenic or non-pathogenic to Thai fish or phlogid fish, and is immunogenic for edwardiosis of Thai fish or phlogid fish. HET-1 strain (Accession number NITE BP-01759, Depositary: National Institute for Product Evaluation Technology Patent Microorganisms Deposit Center, Location: 2-5-8 Kazusa Kamashi, Kisarazu, Chiba, Japan, Date of accession: 2013 A strain collected in Japan on November 28) may be used.

  The morphological characteristics of the HET-1 strain are consistent with those of the normal E. tarda standard strain and show the shape of a Gram-negative facultative anaerobic gonococcus. Has periflagellate flagella, motility, no spore formation. As a culture property, a black colony is formed by hydrogen sulfide in SS agar medium, DHL agar medium, or the like. Moreover, since lactose is not decomposed, a colorless colony is formed in the MacConkey medium. The optimum culture temperature is 25-30 ° C.

The biochemical properties of HET-1 strain are shown below.
(1) Gram staining: Gram negative
(2) Catalase: +
(3) Cytochrome oxidase: −
(4) Glucose degradability: fermentation
(5) Sugar fermentability with TSI slope medium: Lactose and white sugar are not decomposed on the slope, and glucose fermentation, gas production, and hydrogen sulfide production are observed in the upper layer.
(6) β-galactosidase activity: −
(7) Arginine dihydrolase:-
(8) Lysine decarboxylase: +
(9) Ornithine decarboxylase: +
(10) Citric acid availability:-
(11) Hydrogen sulfide production: +
(12) Urease:-
(13) Tryptophan deaminase: −
(14) Indole production: +
(15) Acetoin production: −
(16) Gelatinase: −
(17) Use of glucose: +
(18) Use of mannitol:-
(19) Use of isositol 1:
(20) Use of sorbitol:-
(21) Use of rhamnose:-
(22) Use of sucrose:
(23) Use of melibiose:-
(24) Use of amygdalin:-
(25) Use of arabinose: −
(26) Nitrogen dioxide production: +
(27) Reduction to nitrogen gas:-
(28) Growth on MacConkey medium: +

  In the live vaccine preparation according to the present invention, a buffer, an isotonic agent, an antiseptic, an antibacterial agent, an antioxidant and the like may be appropriately added according to the purpose and use.

  As a suitable example of a buffering agent, buffer solutions, such as a phosphate, acetate, carbonate, citrate, etc. can be used, for example.

  As a suitable example of an isotonizing agent, sodium chloride, glycerin, D-mannitol etc. can be used, for example.

  Suitable examples of antiseptic agents include, for example, thimerosal, paraoxybenzoates, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, other antiseptics, antibiotics, and synthetic antibacterials. An agent or the like can be used.

  As a suitable example of an antioxidant, a sulfite, ascorbic acid, etc. can be used, for example.

  In addition, this drug includes auxiliary ingredients such as light-absorbing dyes (riboflavin, adenine, adenosine, etc.) that serve as storage and efficacy aids, and chelating / reducing agents (vitamin C, citric acid, etc.) for stabilization. , Carbohydrates (sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, etc.), casein digests, various vitamins, and the like may be included.

  About the dosage form etc. of a vaccine formulation, a well-known thing can be employ | adopted and it does not specifically limit. For example, it may be used as a liquid formulation, or after treatment such as lyophilization, it is mixed with food and administered orally, or lyophilized is dissolved by adding water at the time of use. You may inoculate by oral method, spray method, transanal method.

<Regarding the method for preventing Edwardsiellosis according to the invention>
The present invention includes all methods for preventing edwardiellosis of Thai fishes or phlogid fishes, including at least a procedure for inoculating Thai fishes or phlogid fishes with the above live vaccine preparation.

  As described above, the live vaccine preparation according to the present invention has low pathogenicity or non-pathogenicity against Thai fish or phlogid fish and has strong immunogenicity. It is effective for the prevention of Edwardsiellosis.

  Applicable objects include all fish susceptible to E. tarda atypical strains (biogroup 1), that is, fish that develops Ewadierosis due to infection with E. tarda atypical strains. For example, Thai fishes such as red sea bream, red sea bream, and black sea bream, and genus fishes such as yellowtail fish (such as yellowtail, amberjack, and kingfish), and horse mackerel fish (such as sea bream) are included in the application target.

  Examples of the method for inoculating the live vaccine preparation according to the present invention include an injection method, a dipping method, an oral method, a spray method, and a transanal method.

In the case of injection method, oral method, spray method or transanal method, for example, 10 ¹ to 10 ⁷ CFU / tail may be administered at a time by injection method, oral method, spray method or transanal method. It is preferred to administer 10 ³ to 10 ⁶ CFU / tail, and most preferred to administer 10 ³ to 10 ⁵ CFU / tail.

In the case of the immersion method, for example, it is preferable to immerse in a vaccine solution of 10 ³ to 10 ⁸ CFU / mL at a time by the immersion method, and to immerse in a vaccine solution of 10 ³ to 10 ⁷ CFU / mL. Is more preferable, and it is most preferable to immerse in a vaccine solution of 10 ³ to 10 ⁶ CFU / mL (concentration at the time of use). About immersion time, for example, it is preferable to immerse for 1 to 120 minutes, it is more preferable to immerse for 1 to 90 minutes, and it is most preferable to immerse for 1 to 60 minutes.

  Of these, intraperitoneal administration by the injection method is most preferred because of its high infection prevention effect and long immunity duration.

  The number of inoculations of the vaccine preparation may be one as long as the action continues, but it may be inoculated several times at intervals of 1 to 60 days depending on the size of the target fish, the degree of vaccine effect, and the like. In addition, the target fish may be inoculated with the vaccine preparation by appropriately combining a plurality of inoculation methods.

  The present invention may be applied in an area isolated from the sea area in the natural environment, such as a seedling production facility. Thereby, the concern that the sea area is contaminated with the live bacteria can be solved, and the risk that fish other than the Thai fish or the horse mackerel fish existing in the natural environment can be suppressed.

  In Example 1, pathogenicity of Japanese flounder-derived E. tarda (typical strain) against red sea bream was examined.

Red sea bream (n = 25 to 27) was administered with 10 ⁴ to 10 ⁵ CFU / tail, intraperitoneal injections of standard E. tarda strains isolated from Japanese flounder (7 strains including HET-1 strain). As a result, in any case of administration of any strain, almost no deaths were observed 13 days after infection.

Also, red sea bream (n = 25-27) was immersed and infected with E. tarda standard strains (total 7 including HET-1 strain) isolated from Japanese flounder at a concentration of 10 ⁵ to 10 ⁶ CFU / mL. It was. As a result, as in the case of intraperitoneal injection administration, almost no deaths were observed 14 days after infection in any strain.

In addition, red sea bream (n = 25-27), E. tarda typical strains (total 7 including HET-1) isolated from Japanese flounder were injected intraperitoneally at a high dose of 10 ⁶ to 10 ⁷ CFU / tail. It was administered by injection. As a result, when any strain was administered, the cumulative mortality rate was 80 to 100% 9 days after infection. Most of the dead individuals died within 2 days after administration.

Thus, almost no deaths were observed in intraperitoneal injection at a dose of 10 ⁴ to 10 ⁵ CFU / tail or immersion infection at a concentration of 10 ⁵ to 10 ⁶ CFU / mL. In addition, most intraperitoneal injections at high doses have died within 2 days after administration, so it is estimated that the death was due to septic shock caused by the administration of a large amount of cells into the body. Therefore, these results suggest that the common strain E. tarda derived from Japanese flounder is less pathogenic to red sea bream.

  In Example 2, it was examined whether E. tarda derived from Japanese flounder, which has low pathogenicity against red sea bream, has an effect as a vaccine against red sea bream edodesia.

Red sea bream (n = 20) was immunized by intramuscular injection of 1.1 × 10 ⁴ CFU / tail of E. tarda HET-1 strain isolated from Japanese flounder. Further, as a comparative example, red sea bream (n = 20) was immunized by injecting 5.5 × 10 ⁸ CFU / tail intramuscularly with an inactivated bacterial solution of E. tarda UT-1 strain isolated from red sea bream. In addition, red sea bream (n = 20) was prepared as a non-administration group.

Fourteen days after immunization, E. tarda ETE1005 strain derived from red sea bream was administered by intraperitoneal injection to each individual at 4.4 × 10 ⁴ CFU / tail and challenged.

  Observation was continued for 30 days from the day of attack with red sea bream E. tarda, and the survival rate was calculated.

  The results are shown in Figure 1. FIG. 1 is a graph showing the survival rate in the case of immunization with E. tarda HET-1 strain isolated from Japanese flounder followed by intraperitoneal injection challenge with red sea bream E. tarda strain. In the figure, the horizontal axis represents the number of days from the day of attack with the red sea bream E. tarda strain, and the vertical axis represents the survival rate (%). In the figure, “HET-1 (live)” is the result of immunization with live E. tarda HET-1 strain isolated from Japanese flounder, and “UT-1 (inactivated)” is isolated from red sea bream. The results of immunization with a formalin-inactivated bacterial solution of E. tarda UT-1 strain, and “control group” represent the results of the non-administered group (non-immunized group), respectively. In addition, in the result of FIG. 1, the statistical significance by the one-sided test (p <0.025) of Fisher direct probability calculation method was recognized between each group.

  As shown in Figure 1, in the non-administered group, the survival rate against attack by red sea bream E. tarda was 0%, whereas in the group immunized with E. tarda HET-1 strain isolated from Japanese flounder, The survival rate was 65.0%, and a high immune effect was recognized. In addition, a markedly high immunity effect was observed in a small amount even when immunized with an inactivated bacterial solution of red sea bream-derived E. tarda strain.

  This result shows that the live E. tarda strain isolated from Japanese flounder is effective for the prevention of red sea bream edovadierosis.

  In Example 3, when immunized with an E. tarda strain isolated from Japanese flounder, the survival rate in the case of immersion attack was examined.

Red sea bream (n = 23) was immunized with 8.8 × 10 ³ CFU / tail, intramuscular injection of E. tarda HET-1 strain isolated from Japanese flounder. In addition, red sea bream (n = 26) in the non-administration group was prepared separately.

14 days after immunization, E. tarda ETE1005 strain derived from red sea bream was prepared at a concentration of 2.6 × 10 ⁵ CFU / mL or 2.6 × 10 ⁶ CFU / mL, and then red sea bream was immersed therein and attacked.

  Observation was continued for 30 days from the day of attack with red sea bream E. tarda, and the survival rate was calculated.

The results are shown in FIGS. 2A and 2B. FIG. 2A is a graph showing the survival rate in the case of immersing and attacking at a concentration of 2.6 × 10 ⁵ CFU / mL after immunization with E. tarda HET-1 strain isolated from Japanese flounder, FIG. 2B is also 2.6 × It is a graph which shows the survival rate at the time of immersion attack at the density | concentration of 10 < ⁶ > CFU / mL. In the figure, the horizontal axis represents the number of days from the day of attack with the red sea bream E. tarda strain, and the vertical axis represents the survival rate (%). In the figure, “HET-1 (live)” is the result of immunization with E. tarda HET-1 strain isolated from Japanese flounder, and “control group” is the result of non-administration group (non-immunized group). Respectively. In the results of FIGS. 2A and 2B, a statistically significant difference was found between the groups by the one-sided test (p <0.025) of the Fisher direct probability calculation method.

  As shown in FIG. 2A and FIG. 2B, even in the case of immersion attack with red sea bream E. tarda strain, the group immunized with E. tarda strain isolated from Japanese flounder remarkably survived compared to the non-treated group. The rate was high.

  In general, the immersion attack can be tested under the same conditions as in the case of infection in a normal aquaculture environment, and good results cannot be obtained unless the effect as a vaccine is high. On the other hand, in this example, even when immersion attack was performed, the survival rate was remarkably high. This result indicates that the E. tarda strain isolated from Japanese flounder has high efficacy as a live vaccine for red sea bream edodezieriosis.

  In Example 4, the base sequence of the upstream region of the cilia gene group of the flounder-derived E. tarda strain was determined.

  The flounder-derived E. tarda strain HET-1 was inoculated on a SCDb agar medium and cultured at 28 ° C. for 20 hours. 1 mL of ultrapure water was put into a 1.5 mL tube, and 1 colony of the test strain was picked up from the agar medium, placed in it, suspended, centrifuged, and the supernatant was removed. Add 200 μL of “InstaGene Matrix (BioRad)”, heat at 56 ° C. for 15 minutes, mix vigorously, heat at 100 ° C. for 8 minutes, mix vigorously, collect by centrifugation, and collect supernatant with DNA It recovered as a liquid.

  Using the DNA-containing solution as a template and the sequences of SEQ ID NO: 2 (forward) and SEQ ID NO: 3 (reverse) as primers, the sequence of the upstream region of the cilia gene group of E. tarda strain was amplified by PCR. The PCR product was purified using “MinElute PCR Purification Kit (Qiagen)”, and TA cloning was performed using “TOPO TA Cloning Kit for Sequencing (Invitrogen)”. Colonies with confirmed PCR product inserts are picked again, inoculated into 5 mL of LB liquid medium, cultured at 37 ° C for 16 hours with shaking, and then cultured with "QIAprep Spin Miniprep Kit (Qiagen)". Plasmid DNA was extracted from the solution. For the extracted plasmid DNA, the base sequence of the amplified portion (upstream region of the cilia gene group of E. tarda strain) was determined by the cycle sequence method.

  SEQ ID NO: 1 shows the base sequence of the upstream region of the cilia gene group of HET-1 strain, which is an E. tarda strain derived from Japanese flounder. The base sequence of the upstream region of the ciliary gene group of the HET-1 strain was 100% identical with the base sequence of the region of the known flounder-derived E. tarda strain. On the other hand, the homology was 91.4% (775/848 bp) as a result of comparison with the nucleotide sequence of the region of the UT-1 strain, which was the E. tarda strain derived from red sea bream.

  In Example 5, it was examined whether amplification was observed when PCR was performed using DNA of flounder-derived E. tarda strain as a template and using red sea bream-derived E. tarda strain-specific primers.

  As DNA containing the base sequence of the ciliary gene group upstream region of the flounder E. tarda strain, the plasmid DNA derived from the HET-1 strain prepared in Example 4 was used as a template, and the sequence of SEQ ID NO: 4 (forward) and sequence as primers An attempt was made to amplify the sequence in the upstream region of the cilia gene group of E. tarda strain by PCR using the sequence of No. 5 (reverse). At the same time, as a positive control, a DNA containing the base sequence of the ciliary gene group upstream region of the UT-1 strain, a red sea bream-derived E. tarda strain, was prepared by the same method as in Example 4, and the DNA was used as a template and the same primer was used. The sequence in the upstream region of the cilia gene group of E. tarda strain was amplified by PCR. Both PCR products were subjected to agarose gel electrophoresis and then stained with ethidium bromide to detect the presence or absence of amplification of the upstream region of the cilia gene group of the E. tarda strain.

  As a result, when PCR was performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 5, amplification of the upstream region of the cilia gene group of the positive control UT-1 strain was detected, whereas that of the HET-1 strain No amplification in the upstream region of the cilia gene group was detected.

  As a result, when the E. tarda strain having low pathogenicity against red sea bream and having high immunogenicity is subjected to PCR using the primers of SEQ ID NO: 2 and SEQ ID NO: 3, the sequence in the upstream region of the cilia gene group is amplified, When PCR is performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 5, the sequence in the upstream region of the cilia gene group is not amplified, that is, when PCR is performed using the primers of SEQ ID NO: 2 and SEQ ID NO: 3, cilia The sequence of the upstream region of the gene group is amplified, and when PCR is performed using the primers of SEQ ID NO: 4 and SEQ ID NO: 5, the sequence of the upstream region of the cilia gene group does not amplify. Suggest that it is applicable.

The graph which shows the survival rate at the time of intraperitoneal injection attack with the red sea bream origin E.tarda strain | stump | stock after immunizing with the live E. tarda strain | stump | stock HET-1 isolate | separated from Japanese flounder in Example 2. FIG. The graph which shows the survival rate at the time of carrying out immersion attack at the density | concentration of 2.6 * 10 < ⁵ > CFU / mL after immunizing with the live microbe of E. tarda HET-1 isolate | separated from the Japanese flounder in Example 3. FIG. The graph which shows the survival rate at the time of carrying out immersion attack at the density | concentration of 2.6 * 10 < ⁶ > CFU / mL after immunizing with the living microbe of E. tarda HET-1 isolate | separated in Example 3.

Claims (6)

A vaccine preparation for edwardiadiosis of Thai fish or Carp family,
A live vaccine preparation containing viable bacteria of Edwardsiella tarda standard strain.   2. The live vaccine preparation according to claim 1, wherein the live bacteria of the Edwardsiella tarda standard strain is low pathogenic or non-pathogenic to Thai fishes or Carpidae fishes.   The live according to claim 1 or 2, wherein the sequence of the upstream region of the ciliary gene group in the genome of the Edwardsiella tarda strain includes the sequence described in SEQ ID NO: 1 or a sequence having 92% or more homology with the sequence. Vaccine formulation.   The live vaccine preparation according to any one of claims 1 to 3, wherein the Edwardsiella tarda fixed strain is Edwardsiella tarda HET-1 strain (Accession No. NITE BP-01759). Administer 10 ¹ to 10 ⁷ CFU / tail at a time by injection, oral, spray, or transanal, or 10 ³ to 10 ⁸ CFU / mL at a time by immersion The live vaccine formulation according to any one of claims 1 to 4, which is immersed in a vaccine solution for 1 to 120 minutes.   A method for preventing edwardiellosis of Thai fishes or phlogid fishes, wherein the live vaccine preparation according to any one of claims 1 to 5 is inoculated into Thai fishes or phlogid fishes.

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