TWI403580B - The new Edwardsi strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33), its vaccine, and methods of using the vaccine to protect fish - Google Patents

Description

Novel Edwards strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33), vaccine thereof, and method for protecting fish using the same

The present invention relates to a novel Escherichia coli strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33), a vaccine derived therefrom, particularly a soaking type vaccine and an oral dosage form vaccine; and a method of using the same to protect fish .

Edwardsiella is a small Gram-negative bacterium with a wide range of hosts, including fish such as catfish, catfish, tilapia and other warm water fish. Edwardsiella ictaluri is a member of Edwardsiella , which causes Edwardsiella Speticemia (ESC) in the intestines of salmon . Acute Edwards sepsis (ESC) induces typical bacterial sepsis with a high mortality rate; whereas chronic Edwards sepsis (ESC) slowly progresses and induces infected fish to develop "head cavity disease" lesions (Hole -in-the-head syndrome) may cause local residual signs of fish or develop sepsis or even death. Because fish may turn into acute ESC after 3 ^to 4 weeks of infection with chronic Edwards septicaemia (ESC), resulting in reduced fish production, causing huge losses to the aquaculture industry.

Edward's sepsis occurs in salmon farming around the world, especially in Vietnam and the United States, where infection with Edwards has led to high mortality and severe economic losses. In the squid farms in Vietnam, the infection of microorganisms is mainly caused by E. ictaluri . The infection was first discovered in the Mekong River Delta in 1999 (Ferguson HW, et al. J Fish Dis 2001; 24: 509-13), and also in the American river otter (channel catfish, Ictalurus). The punctatus), and the Thai pond carp or the walking catfish (Kasornchandra J, et al. J Fish Dis 1987; 10: 137-8) found that the fish were infected with the bacterium. Today, all species of squid in Vietnam are susceptible to E. ictaluri , a disease that has caused significant economic damage to the Basa Tra fish and other farming areas in the Mekong Delta. Breeders have tried a variety of methods to protect fish from E. ictaluri , but so far no satisfactory results have been obtained.

In order to control Edward's sepsis, antibiotics and various vaccination methods are widely used in aquaculture fisheries, but vaccination is not effective in preventing Edward's sepsis specific to known carp. In some cases, although antibiotics are effective against bacterial infections, farms that do not use antibiotics are effective against Edward's infection. In addition, large-scale and uncontrolled single or mixed use of multiple antibiotics has led to the general development of resistance to Edwards. It is also caused by the fact that Edwards can survive in the soil of the pond for up to 3 months, which worsens the infection of Edwards. Furthermore, the use of antibiotics and chemical drugs not only has the problem of drug residues, but also the long-term use of antibiotics and the inability to continuously control fish diseases. In addition, one of the earliest clinical symptoms of Edward's sepsis is that the fish develop symptoms of anorexia. Therefore, oral antibiotics can only effectively limit the outbreak of the disease, and can not achieve effective prevention and early treatment.

Vaccination has long been used as a preventive measure to control Edward's infection of various salmon. However, examples of so far no single vaccination or mixed vaccination have been reported to be successful in effectively preventing the infection of the disease. Although attenuated vaccines can help with disease control, so far this product has not yet made significant progress.

In addition, oral delivery of vaccine antigens to fish is preferred for some reasons, but the limitation is in the lack of immune efficacy (Gudding R, et al. Vet Immunol Immunopathol 1999; 72: 203-12; Hastein T, et Al., Dev Biol Stand 2005; 121: 55-74; Thune RL, ey al. . J Appl Aquacult 1994; 3(1-2): 11-24.).

It can be seen that there are still many shortcomings in the above-mentioned conventional methods, which is not a good designer, but needs to be improved.

In view of the shortcomings derived from the above-mentioned conventional methods, the inventor of the present invention has improved and innovated, and after years of painstaking research, he finally succeeded in research and development of a novel E. sinensis strain E-ict-VL33 ( Edwardsiella ictaluri E-ict) - VL33), its vaccine, and methods of using the vaccine to protect fish.

The technical features of this case were previously published in the Journal of Fish & Shellfish Immunology on September 10, 2009 (Thinh NH, Fish Shellfish Immunol. 2009 Dec; 27(6): 773-6. Epub 2009 Sep 10.).

All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be common to those of ordinary skill in the art.

It is an object of the present invention to provide a novel E. sinensis strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33) which has been identified as a novel E. faecalis strain.

A second object of the present invention is to provide a novel Edwardia vaccine which is prepared by the inactivated treatment of the above E. sinensis strain E-ict-VL33, and comprises an infusion formulation and an oral dosage form.

Another object of the present invention is to provide a method for using these vaccines to enhance fish immunity against Edwards, thereby preventing and protecting fish from infection with Edwards.

In order to achieve the above object, the present invention provides a novel E. serovar E-ict-VL33 strain isolated from an infected squid ( Pangasius hypophthalmus , tra catfish), which is cultured and identified. Afterwards, it was identified as a novel Edwardian strain named Edwards E-ict-VL33 strain, and deposited in the Bioresource Conservation and Research Center of Hsinchu Food Industry Development Research Institute on January 26, 1999. For BCRC 910460.

In the second part of the invention, an immunological composition comprising the novel E. faecalis strain is provided. In a preferred embodiment, the immunological composition is a vaccine. The vaccine of the present invention is prepared by injecting an effective amount of the inactivated Edwardian strain E-ict-VL33, and a pharmaceutically acceptable vehicle into a dosage form suitable for use in the vaccine of the present invention. The method is briefly described as follows: after the above-mentioned E. facilis E-ict-VL33 strain is cultured, it is inactivated, and then the type of the preparation preparation is prepared, and the appropriate pharmaceutically acceptable carrier can be used. Inactivated Edwardella vaccines of various dosage forms are prepared, wherein the dosage forms include, but are not limited to, oral dosage forms, infusion dosage forms, injectable dosage forms, and other dosage forms similar or suitable for use in the present invention.

When preparing a soaking dosage form, the Edwards strain E-ict-VL33 can be inactivated by a deactivating agent, and then the inactivated Edwards strain E-ict-VL33 is further suspended in a buffer solution, and A soaking vaccine can be prepared by adding an appropriate carrier.

When an oral dosage form is to be prepared, the Escherichia E-ict-VL33 may be inactivated by a deactivating agent, and then the inactivated E. faecalis E-ict-VL33 is further suspended in a buffer solution, and The oral dosage form vaccine is prepared by mixing with an adjuvant and homogenizing the mixture by a homogenizer. The oral dosage form vaccine can be sprayed on the outer layer of the feed as needed, and further coated with edible oils such as vegetable oil or animal oil, especially fish oil.

The inactivated treatment of the present invention includes, but is not limited to, a deactivated treatment, a heat treatment, and the like, which are conventionally applicable. The deactivating agent includes, but is not limited to, formalin treatment, binary ethyleneimine (BEI) and the like.

Wherein the pharmaceutically acceptable carrier may comprise one or more agents selected from the group consisting of: solvents, emulsifiers, suspending agents, decomposers, binding agents ), excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative, lubricant, interface Surfactants, adjuvants, and other carriers similar or suitable for use in the present invention.

Wherein the adjuvant may include, but is not limited to, an oil adjuvant (eg, mineral oil, vegetable oil, animal oil, Freund's complete adjuvant, Freund's incomplete adjuvant, etc.), a water adjuvant (eg, aluminum hydroxide), A two-phase oily adjuvant (eg, a water-in-oil-in-water formulation, w/o/w), a biologic adjuvant (eg, a CpG oligonucleotide, a bacterial toxoid). Wherein the dual phase oily adjuvant comprises a surfactant and an oil phase material; the surfactant comprises one or more of the following selected groups: sorbitol fatty acid ester; sorbitol fatty acid Ester and ethylene oxide or propylene oxide concentrate; mannitol fatty acid ester; mannitol fatty acid ester and ethylene oxide or propylene oxide concentrate; mannitol fat Acid esters with the following selected hydrophilic groups: carboxylic acid, amine, amide, alcohol, polyester, ether, oxygen Oxide conjugate; anhydrous mannitol fatty acid ester; anhydrous mannitol fatty acid ester with the following selected hydrophilic groups: carboxylic acid, amine, decylamine, alcohol, polyester polyol, ether a conjugate of an oxy group; a saccharose fatty acid ester; a sucrose fatty acid ester with an ethylene oxide or propylene oxide concentrate; a glycerin fatty acid ester; a glycerin fatty acid ester with ethylene oxide or propylene oxide Concentrate; fatty acid and ethylene oxide or propylene oxide concentrate; fatty alcohol and epoxy Dioxane or propylene oxide concentrate; and glycerophospholipids (glycerophospholipid). The oil phase material comprises one or more of the following selected groups: mineral oil, vegetable oil, and animal oil. In a preferred embodiment, the oil phase material is animal oil, particularly fish oil.

In addition, the present invention also provides a composition containing the novel Edwardia, which can be applied to various applicable products, such as: a challenge composition (the bacteria is not subjected to non-activation treatment), and a vaccine composition. The bacteria are subjected to non-activation treatment) and the like.

Another part of the present invention provides a method for using the vaccines to enhance fish immunity against Edwardella, thereby preventing and protecting fish from infection with Edwards, including using the above-mentioned two types of infusion and oral dosage forms to enhance fish. Immunity against Edwards. The method comprises first soaking the inoculated fish with the above-mentioned soaking type vaccine (administered by primary immune reaction treatment), and then administering the oral dosage form vaccine to the oral administration (excitation of the immune response) after the appropriate period. The results according to the examples described below show that soaking (primary immune response) combined with oral vaccination (stimulating an immune response) can elicit a strong and sustained immune effect. The fish referred to herein refers to a susceptible by E. ictaluri infection, especially a squid.

In addition, the present invention also provides a method or use of the aforementioned E. sinensis E-ict-VL33 for preparing a vaccine for protecting fish from infection with Edwards.

The novel E. sinensis isolate E-ict-VL33 of the present invention should also contain its subcultured progeny, or mutant strain, but still have the characteristics, genomic, and genomics of the present invention. Or the same pathogenicity.

The term " effective amount " means that the amount of the non-activated strain is effective to enhance and improve the immunity of the fish against Edwardella, thereby preventing and protecting the fish from infection with Edwards and related diseases.

The term " prevention, protection " means that the vaccine of the present invention can effectively enhance the immunity of fish against Edwardella, increase its survival rate, prevent and protect, compared to those who do not use the vaccine of the present invention. Fish are free of infection with Edwards and its associated diseases.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Example 1 Isolation and Identification of Edwards E-ict-VL33

An E. sinensis isolate isolated from an infected carp ( P. hypophthalmus , tra catfish) cultured in Brain Heart Infusion (BHI broth) at 25-30 ° C 18 After the culturing, the bacteria were cultured in BHI medium (components shown in Table 1) at 30 ° C for 48 hours, and then analyzed for bacteriological characteristics and strain identification.

Bacteriological characteristics

Referring to Fig. 1, the isolate E-ict-VL33 is a bacillus; Gram staining results show that the isolate E-ict-VL33 is a Gram-negative bacterium. In addition, please refer to Table 2 to Table 4 for the growth of the isolate E-ict-VL33 under different temperature, salinity and pH conditions. Wherein + represents growth but the concentration is not high; ++ means there is a little precipitation; +++ means growth and precipitation is large; - represents no growth. In addition, the strain can be cultured in tryptic soy borth (TSB) or brain heart infusion (BHI). See also Table 5 for analysis of available carbon sources for the isolate E-ict-VL33, where + represents the available carbon source.

Table 5 Analysis of available carbon sources of isolate E-ict-VL33

Species identification

The isolate E-ict-VL33 isolated in the present invention was analyzed by 16S rDNA, and its sequence is shown in SEQ ID No: 1. Based on the 16S rDNA analysis and the National Center for Biotechnology Information (NCBI) database, it was confirmed that the isolate E-ict-VL33 was similar to E. ictaluri with a similarity of over 99%. Therefore, the results showed that the isolate E-ict-VL33 was a member of E. ictaluri .

Since some Edwards strains contain the plastid pEI1, the inventors designed a PCR primer according to the sequence of the E. sinensis pEI1 ORF (open reading frame) of GenBank number AF244083.1 on the NCBI to isolate the Edwards strain of the present invention. E-ict-VL33 was subjected to plastid pEI1 sequencing, and the resulting partial sequence of the Escherichia coli E-ict-VL33 plastid pEI1 was as shown in SEQ ID No: 2. The sequence of SEQ ID No: 2 was aligned with the NCBI library, and the results showed that the 1st ^to 1443 bp fragment of the SEQ ID No: 2 sequence (full length 1876 bp) and the Edwardian plastid pEI1 sequence fragment of GenBank No. AF244083.1 were 99%. The similarity, but the 1444 ^~ 1876 bp fragment of the SEQ ID No: 2 sequence is different from the AF244083.1 plastid pEI1 sequence, indicating that although both are Edwards, they are different strains. This result confirmed that the E. sinensis strain E-ict-VL33 of the present invention is indeed a novel E. faecalis isolate.

The above results confirmed that the isolate E-ict-VL33 provided by the present invention is a novel Edwardsella ictaluri , named Edwards strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33), It was deposited at the Bioresource Conservation and Research Center of Hsinchu Food Industry Development Research Institute on January 26, 1999. The registration number is BCRC 910460.

Example 2 Preparation of Edwards strain E-ict-VL33 vaccine Step 1 Bacterial culture

The Edwards strain E-ict-VL33 was inoculated into BHI liquid medium, and the bacterial liquid was harvested after culturing at 28 ° C for 48 hours.

Step 2 Inactivated process

37% formaldehyde solution (formaldehyde) was added to the Escherichia coli strain E-ict-VL33 collected in step 1, and the formaldehyde content was adjusted to a final concentration of 0.5% (w/v) and continued at 70 rpm at 25 ° C. Shake for at least 24 hours, preferably for 48 hours, for non-activation treatment; after confirming that the Edwards has not been activated, the Edwards solution containing formaldehyde is centrifuged at 9,000 × g , and the supernatant is removed. The liquid is used to remove formaldehyde, and the Edwards is suspended in a buffer solution (such as distilled water or phosphate buffered saline, PBS) to prepare an antigen-containing vaccine stock solution, which is stored at 4 ° C for use.

Example 3 Preparation of Escherichia E-ict-VL33 non-activated immersion vaccine

Adding a buffer solution (such as distilled water or PBS) to the non-activated vaccine stock solution prepared in the second embodiment, adjusting the Edwardian content to 5.5×10 ⁹ cfu/ml, and preparing the Epsteinella vaccine of the infusion type; formaldehyde content The analysis showed that the formaldehyde content of the Soybean vaccine of the infusion type was less than 0.2% (v/v).

Example 4 Preparation of Edwards E-ict-VL33 inactivated oral vaccine

To prepare a 400-liter oral dosage form vaccine, mix 300 liters of vaccine stock (containing at least 1.65 × 10 ¹⁵ cfu of Edwards) with 4 liters of surfactant (polysorbate 80. polysorbate 80) and 96 liters of fish oil. The mixture is then homogenized by a high shear homogenizer at 10,000 rpm, and the Edwardian antigen is embedded in the water-in-oil (w/o/w) form in the dual phase oil adjuvant. The oral dosage form vaccine is obtained. Among them, fish oil accounts for 24% (v/v) of 400 liters of oral dosage form vaccine; surfactants account for 1% (v/v) of 400 liters of oral dosage form vaccine; and about 3.85 × 10 ⁸ cases of oral dosage form vaccine per milliliter Cfu does not activate Edwardella; the formaldehyde content analysis results show that the oral dose of the Edwards vaccine has a formaldehyde content of less than 0.2% (v/v).

Instructions

The present invention further provides a method of using the oral vaccine and a composition thereof, which does not limit the scope of application of the oral vaccine of the present invention. The prepared oral dosage form vaccine is sprayed on the outer layer of the granular feed at a ratio of 2% (vol/vol), and the cod liver oil is coated with the sprayed vaccine at a ratio of 0.1% (vol/vol). . The pelleted feed is an AQUAXCEL 7434 pelleted feed or other commercially available feed.

Example 5 Efficacy of Edwards strain E-ict-VL33 inactivated vaccine 5-1. Test method

Healthy, uninfected squid ( P. hypophthalmus , also known as Basa Tra fish) was used as a test material, and vaccinated by soaking, oral or a combination thereof, the different vaccination methods, their combination, and their challenge (challenge The test was divided into two groups of tests (Test 1 and Test 2), and the tests were repeated at least three times. among them:

Trial 1: Establish a poisoning mode

This attack mode will determine the test 2 series of immersion or injection infection for the challenge test, a total of 1500 squid (the number can be changed according to the test needs), the two methods of attack test are briefly described as follows: Bath cha1lenge : soak the squid in water at a final concentration of 5.5 × 10 ³ , 5.5 × 10 ⁴ , 5.5 × 10 ⁵ , 5.5 × 10 ⁶ cfu of E. ictaluri E-ict-VL33 per ml, at least Soak for 30 minutes for soaking and attacking. During the soaking and attacking, the test tank needs to be fully aerated. The control group was immersed in fully aerated purified water. After 14 days of challenge, the efficacy (mortality) of the soaking challenge test was evaluated.

Injection challenge: After anesthetizing the squid with 0.2% MS222 aqueous solution, 0.1 ml of the bacterial dilution solution (final concentration: 5.5 × 10 ³ , 5.5 × 10 ⁴ , 5.5 × 10 ⁵ , 5.5 per ml respectively) ×10 ⁶ cfu) The challenge test was performed by intraperitoneal injection, and the control group was injected with physiological saline. After 14 days of challenge, the efficacy (mortality) of the soaking challenge test was evaluated.

In the soaking and injecting challenge tests, the dying or just-dead fish were isolated from the liver, spleen, and kidney, and the surviving fish were tested for bacterial infection at the end of the observation period.

Test 2: Efficacy test in combination with oral dosage form vaccine and infusion type vaccine

For the test grouping, please refer to Table 6, where:

Group A (immersion-prime only): Primary immersion vaccination was performed on the first day of the experiment; and immersion challenge was performed on days 48 and 121, respectively.

Group C (soak-oral boost-1, imm-oral boost-1): first oral boost was administered from day 8 to day 21 after the first soak inoculation on the first day of the test; Soaking and attacking were carried out on the 48th day and the 121st day respectively.

Group F (immersion/oral boost-2, imm-oral boost-2): After primary inoculation on the first day of the test, the first oral inoculation was given from the 8th day to the 21st day, and then on the 101st day. The second oral boost was administered to the 107th day, and finally the immersion challenge was performed on the 121st day.

Group E (oral-prime only): Primary oral vaccination was administered only from day 8 to day 21, and immersion challenge was performed on days 48 and 121, respectively.

Control group (Ct): No oral vaccine or immersion vaccine of the present invention was inoculated, and immersion challenge was performed on the 48th day and the 121st day, respectively.

Where Ct represents the control group (Control); "-" represents that the test was not performed.

The aforementioned soaking inoculation, oral inoculation, and post-inoculation challenge test procedures are detailed as follows: Soaking inoculation: The infusion vaccine is a suspension consisting of 5 x ¹⁰⁹ cfu of inactivated bacteria per ml of sterile water. Use 2 liters of immersion vaccine to add 18 liters of water to dilute (final concentration of about 5.56 × 10 ⁸ cfu) and strong aeration for 1 minute to soak 1,200 fish.

Oral vaccination: The oral dosage form vaccine prepared in Example 4 (in water-in-oil-in-water form, w/o/w, the concentration of inactivated bacteria per ml is 3.85×10 ⁸ cfu) is about 2% (v/m). The amount was sprayed on the outer layer of the feed pellets, and then sprayed with squid oil in an amount of about 0.1% (v/m) for coating. These vaccine-coated feeds are prepared daily and used within 1-2 days of preparation. The feed may be two granular (J3, J4 feed) catfish feeds commercially available from WOOSUNGVINA Co. or other commercially available feeds, and may be fed after being embedded in the above manner.

Attack test: immersion and poisoning in a 96-liter culture tank, which contains 70 liters of water. The first challenge was carried out on the 48th day. 40 or 50 fish (the number of fish depends on the experiment) were transferred from the culture tank to another bucket containing 10 liters of purified water, and the bacteria cultured in BHI medium were added to it. The bacterial concentration per ml of purified water was 7.6 × 10 ⁶ cfu (Test 2). The test fish were exposed to the aforementioned dose for at least 1 hour. The second challenge was carried out on day 121, changing the bacterial concentration per ml of purified water to 8.1 × 10 ⁶ cfu, and soaking the test fish in the same manner. Fish that were not challenged (control group) were immersed in aerated water. All fish must be observed for 14 days after being challenged.

Statistical Analysis

Fisher's exact test was used to analyze the differences between the groups of experiments. A P-value of less than 0.05 is considered to be a significant difference between the test groups.

5-2. Results Test 1

Test 1 test, compare the results of soaking challenge and injection attack. In immersion challenge only way challenged fish, mortality its end (end-point mortality) of between ^{5.5 × 10 3 cfu / 1.3%} (± 1.15 SD) at time of ml to 5.5 × 10 ⁶ cfu / ml of 66% (±8.5 SD). Fish that were challenged by injection challenge had a mortality rate of 93% (±1.5 SD) to 99.3% (±0.6 SD) within the dose range tested. Based on these results, immersion doses above 10 ⁶ cfu will be effective in responding to effective mortality, ie greater than 60% control mortality; therefore, subsequent trials (Test 2 and Trial 3) will be soaked The mode of attack is used to evaluate the efficacy of the vaccine of the present invention.

Test 2

Referring to Figures 2 and 7, the soaking with oral inoculation (test 2) showed that by the 48th day, there was a cumulative mortality rate of 87% in the unvaccinated control group. In group A (only immersion), the mean cumulative mortality was 65% ± 3.1 ( p < 0.02), while in group E (only oral), the mean cumulative mortality was 74% ± 3.5 ( p > 0.1). And in group C (soaked/oral reinforced-1), the mean cumulative mortality was 42% ± 4.0 ( p <0.001); its relative survival rate [RPS, Relative Percent Survival = (1-(% mortality in vaccinated fish) /% mortality in control x 100))] values are 25, 15, and 52, respectively. This result clearly indicates that the prevention effect of squid vaccination combined with oral vaccine reinforcement (group C) is better than that of other single immunization groups (group A or group E).

Please refer to Figure 3 and Table 8. On the 121st day of the experiment, the second attack was carried out. The attacking dose of the carp was 8.1×10 ⁶ cfu per ml of water, and the squid of each vaccinated group and the control group. The cumulative mortality rates are shown in Table 10. On the 121st day of the experiment, the cumulative mortality rate in the control group was 90%. In group A (soaking), the cumulative mortality rate was 80% (RPS = 11, p = 0.26), while in group E (oral), the mortality was 82% (RPS = 9, p = 0.388). In group C (soaked/oral reinforced-1), the mortality rate was 64% (RPS = 29, p = 0.0037). In group F (soaked/orally reinforced-2), the cumulative mortality rate was 48% (RPS = 47, p = 0.0001). From the statistical value, it was found that only the immersion/oral vaccine vaccination had significant protective effects, and the effect also exceeded the control group and the single immunization group.

This result shows that in long-term immunization, immunization with oral immunization is more effective than single immunization or oral immunization. The second immunization is preferably started 80 days after the first immunization and completed 21 days before the challenge. This immunization method will increase the protection and protection time of the fish, and its relative survival rate (RPS). Up to 47 (the mortality rate of the control group reached 90%).

In summary, the use of immersion vaccines combined with oral vaccines as the primary means of immunization can induce strong and long-lasting immunity in fish. The immunized fish can produce better immunity by the second boost immunization, and the immunity is compared with the immunity of the unvaccinated control fish and the fish that are vaccinated only once. , has statistical significance.

Accordingly, the present inventors further include the following technical features such as novelty, advancement, and industrial applicability and their advantages:

1. The present invention provides a novel novel E. sinensis strain E-ict-VL33 (Easonsiella ictaluri E-ict-VL33).

2. The present invention provides a novel Edwardian vaccine prepared by the novel Edwardian strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33) to prepare a soaking dosage form and an oral dosage form, and other suitable dosage forms. .

3. The present invention provides a method for enhancing the immunity of a fish against Edwards by using the vaccines, so as to protect the fish from infection with Edwards, including first applying a soaking dosage form, and then administering an oral dosage form vaccine to enhance fish confrontation. The immunity of Edwards; this method is better than the conventional method of oral or soaking.

The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

In summary, the technical features disclosed in this case have fully complied with the statutory invention patent requirements of novelty and progressiveness. If you apply in accordance with the law, you are requested to approve the application for this invention patent to encourage invention.

<110> Fuda Biotech Co., Ltd.

<120> Novel Escherichia coli strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33), vaccine thereof, and method for protecting fish using the same

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<223> 16S rDNA of Edwards E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33)

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<213> Edwards E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33)

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<223> Partial sequence of ORF1 of Escherichia E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33) plastid pEI1

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Figure 1 shows the morphology of E. ictaluri E-ict-VL33 under the microscope.

Figure 2 shows the cumulative mortality measured by the immersion challenge test on the 48th day after the vaccination of groups A, C, and E.

Figure 3 shows the cumulative mortality measured by immersion challenge on the 121st day after vaccination with groups A, C, E, and F.

Claims (15)

An Escherichia coli strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33), which is deposited at the Bioresource Conservation and Research Center of the Hsinchu Food Industry Development Research Institute, and its registration number is BCRC 910460. An immunological composition comprising the Edwards strain E-ict-VL33 ( Edwardsiella ictaluri E-ict-VL33) according to claim 1 of the patent application, which is an Edwardian strain as described in claim 1 E-ict-VL33 was prepared after inactivation with a deactivating agent. The immunological composition of claim 2, wherein the deactivating agent is formaldehyde or 2-bromoethylamine. The immunological composition of claim 2, wherein the immunological composition further comprises a pharmaceutically acceptable carrier. The immunological composition of claim 4, wherein the carrier comprises one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposers, binders, excipients, stabilizers, Chelating agents, diluents, gelling agents, preservatives, lubricants, surfactants, and adjuvants. The immunological composition of claim 5, wherein the adjuvant is selected from the group consisting of an oil adjuvant, a water adjuvant, a biphasic oil adjuvant, and a biologic adjuvant. The immunological composition of claim 6, wherein the oleaginous adjuvant is selected from the group consisting of mineral oil, vegetable oil, animal oil, Freund's complete adjuvant, and Freund's incomplete adjuvant. The immunological composition of claim 6, wherein the aqueous adjuvant is selected from aluminum hydroxide. The immunological composition of claim 6, wherein the dual phase oily adjuvant comprises a surfactant and an oil phase material. The immunological composition of claim 9, wherein the surfactant comprises one or more selected groups of the following: sorbitol fatty acid ester; sorbitol fatty acid ester and epoxy Ethylene oxide or propylene oxide concentrate; mannitol fatty acid ester; mannitol fatty acid ester and ethylene oxide or propylene oxide concentrate; mannitol fatty acid ester and the following Selected hydrophilic groups: carboxylic acid, amine, amide, alcohol, polyester, ether, oxide A conjugate; anhydromannitol fatty acid ester; an anhydrous mannitol fatty acid ester and a hydrophilic group selected below: a carboxylic acid, an amine group, a guanamine, an alcohol, a polyester polyol, an ether, an oxy group Conjugate; saccharose fatty acid ester; sucrose fatty acid ester and ethylene oxide or propylene oxide concentrate; glycerol fatty acid ester; glycerol fatty acid ester and ethylene oxide or propylene oxide concentrate; Ethylene oxide or propylene oxide concentrate; fatty alcohol with ethylene oxide or propylene oxide An alkane concentrate; and a glycerophospholipid. The immunological composition of claim 9, wherein the oil phase material comprises one or more of the following selected groups: mineral oil, vegetable oil, and animal oil. The immunological composition of claim 6, wherein the biologic adjuvant is selected from the group consisting of an oligonucleotide and a bacterial toxoid. A method of detoxifying Edwards in the preparation of an immunological composition for enhancing the immunity of a fish against Edwards, comprising using a deactivated love as described in claim 2 The immune composition of Deuterobacter E-ict-VL33 is first vaccinated with a primary vaccination method, and the immune composition containing Edwardella is administered orally to the fish to enhance the fish against Edwards. The immunity of the bacteria. The method of claim 13, wherein the immune composition comprising Edwardella is sprayed on the outer layer of the feed prior to oral administration to the fish, and then coated with the oil phase substance. To prepare a feed containing an oral dosage form of the immunological composition. The method of claim 14, wherein the oil phase material comprises one or more of the following selected groups: mineral oil, vegetable oil, and animal oil.