Oral vaccine composition for aquatic organisms
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- KYORITSU SEIYAKU
- Filing Date
- 2026-01-30
- Publication Date
- 2026-08-06
Smart Images
Figure JP2026003417_06082026_PF_FP_ABST
Abstract
Description
Oral vaccine composition for aquatic organisms
[0001] The present invention relates to an oral vaccine composition for aquatic organisms that can be applied to solid feed for aquatic organisms. More specifically, it relates to an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated substance.
[0002] Aquaculture is widely practiced for many aquatic organisms because it can increase yields and provide a stable supply at a relatively low cost. Furthermore, in recent years, global aquaculture production has expanded even further due to the rapid increase in global demand for seafood, concerns about the depletion of marine resources, and stricter international fisheries regulations. On the other hand, farmed aquatic organisms are more susceptible to disease than wild organisms because they are raised at higher stocking densities and are more prone to environmental deterioration. Therefore, even if a disease develops in only a few individuals, it can spread to many other individuals in the farm, making them vulnerable to damage. For this reason, vaccination for disease prevention or treatment is essential.
[0003] Methods of administering vaccines to aquatic organisms include injection, immersion, and oral administration.
[0004] The injection method, which involves directly injecting the vaccine into the abdominal cavity or muscle of each individual organism, is a common method of administration and is currently considered to be the most effective. On the other hand, it has several disadvantages, including the need to directly inject the vaccine into each individual organism, which requires a great deal of time and effort, making it impossible to administer the vaccine to many organisms at once, the considerable effort required when administering to large organisms, the stress it causes to aquatic organisms during administration, and the difficulty in applying it to small individuals such as juvenile fish.
[0005] The immersion method involves immersing a large number of individuals in the vaccine solution at once, making administration simple and minimizing stress on aquatic organisms. However, it has drawbacks, such as difficulty in achieving sufficient immunity and often resulting in low vaccine efficacy, and the number of formulations actually marketed using this method is very limited.
[0006] Oral administration involves mixing the vaccine into the food and administering it to aquatic organisms. This method is quick and easy, and causes minimal stress to the organisms during administration. However, it has several drawbacks: it requires a large amount of vaccine solution to soak into the food, the mixing process is complicated, the vaccine is often less effective because the components are digested in the stomach, and multiple doses are necessary to achieve reliable and long-lasting effects. Consequently, relatively few formulations using this method have actually been brought to market.
[0007] Herein, the enteric-coated polymer compounds will be described as a feature of the present invention.
[0008] Enteric-coated polymers are polymers that do not dissolve in environments with a very low pH, such as the stomach, but dissolve in environments with a higher pH. Examples include cellulose phthalate acetate, hydroxypropyl cellulose phthalate, carboxymethylcellulose, methyl acrylate-methacrylic acid copolymer, cellulose succinate acetate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate acetate (hypromellose acetate succinate), polyvinyl phthalate acetate (PVAP), methyl methacrylate-methacrylic acid copolymer, and sodium alginate. Enteric-coated preparations, in which pharmaceutical ingredients are coated with enteric-coated polymers, and enteric-coated capsules filled with pharmaceutical ingredients are used as means to allow pharmaceutical ingredients that would otherwise be broken down by strong acids to reach the intestines without being broken down in the stomach.
[0009] Furthermore, Patent Document 1 discloses a therapeutic vaccine for oral administration against the bacterial kidney disease bacterium (Renibacterium salmoninarum) in fish, comprising sugar beads coated with an antigen and further coated with an enteric coating. Patent Document 2 discloses an orally administered immunostimulant consisting of recombinant cytokines microencapsulated with an enteric protective polymer. In addition, Patent Document 3 describes an oral vaccine formulation for fish streptococcal disease containing antigens derived from the causative agent of fish streptococcal disease and antigens derived from V. anguillarum as active ingredients. (U.S. Patent No. 5,871,751, JP 2012-505193, Japanese Patent No. 6928225)
[0010] Oral vaccines, administered by oral administration, are in high demand in aquaculture and medical settings because they can be administered to a large number of individuals quickly and non-invasively, compared to injectable vaccines. However, oral vaccine administration has several drawbacks, including the need for a large amount of vaccine solution to soak into the feed, the complicated mixing procedure, the often low effectiveness due to digestion of vaccine components in the stomach, and the need for multiple doses to achieve reliable and long-lasting effects.
[0011] Therefore, the present invention aims to provide a means for administering vaccines orally with low effort, high efficiency, and effectiveness.
[0012] The present invention provides an oral vaccine composition for aquatic organisms, which is a composition that can be spread onto solid feed for aquatic organisms and contains an antigen and an enteric-coated polymer compound.
[0013] By applying an oral vaccine composition for aquatic organisms containing an antigen to solid aquatic food and administering it orally, aquatic infections can be effectively prevented and treated through an immune response to the antigen.
[0014] This method involves attaching the antigen to the solid feed rather than soaking it in the antigen solution. This significantly reduces the amount of antigen needed for oral administration, allowing for efficient immunization. Furthermore, it eliminates the need for cumbersome procedures such as mixing the antigen into the feed, greatly simplifying and reducing the labor involved in vaccine administration at aquaculture sites.
[0015] Furthermore, the composition according to the present invention contains an antigen and an enteric-coated polymer compound. In a low pH environment, the antigen is protected by the enteric-coated polymer compound, allowing it to reach the intestines reliably and efficiently without being digested or broken down in the stomach. This enables reliable and effective immunization against aquatic organisms, and reduces the number of immunization cycles required.
[0016] This oral vaccine composition for aquatic organisms preferably has viscosity when applied to solid feed, either due to the addition of an enteric-coated polymer compound or a viscous substance.
[0017] Because the composition is viscous when applied to solid aquatic food, it can be applied to the solid food in a way that coats it, thereby ensuring that the composition is held securely around the solid food without completely sealing in the flavor of the food. Therefore, even when this composition is applied, the solid food can be consumed well without significantly reducing palatability or appetite, and antigens can be administered at the same time.
[0018] Generally, for large fish like tuna, or individuals that have already grown to a certain size through aquaculture, it is necessary to feed them oily feed to promote further growth. However, with oily solid feed, it is difficult to adhere water-soluble antigens to the solid feed.
[0019] In contrast, because this composition is viscous when applied, it is possible to easily and reliably apply water-soluble antigens even to solid aquatic food containing, for example, 7.5% by weight or more of oil. Therefore, antigen administration by oral method can be easily and reliably performed even for individuals that require intake of solid food with a high oil content, such as individuals weighing 300g or more.
[0020] In other words, the present invention has the advantage of being applicable not only to the usual immunization of juvenile fish of small and medium-sized fish species, but also to immunization of individuals that weigh 300g or more even as juveniles, such as tuna, or to supplemental immunization of individuals grown through aquaculture. In particular, considering the considerable effort required to administer vaccines to large individuals by injection, the present invention is highly useful in that it can immunize individuals weighing 300g or more with low effort, high efficiency, and effectiveness.
[0021] Furthermore, the composition according to the present invention generally maintains a viscous or gel-like state even after passing through the stomach and reaching the intestines following oral administration. Therefore, this composition easily adheres to or contacts the intestinal wall and can remain in the intestines for a relatively long period of time, rather than rapidly passing through the intestines in a liquid state. Consequently, the antigen remains largely retained within the composition and continues to contact the intestinal wall, resulting in reliable and highly efficient exposure of the intestinal wall to the antigen, and as a result, an effective immune response is induced. This is presumed to be one of the reasons why the vaccine exhibits remarkably high efficacy despite being administered orally.
[0022] This invention makes it possible to administer vaccines orally with low effort, high efficiency, and effectiveness.
[0023] <About the composition according to the present invention> The present invention broadly encompasses oral vaccine compositions for aquatic organisms that are adhering to solid feed for aquatic organisms and contain an antigen and an enteric-coated polymer compound.
[0024] This composition is an oral vaccine composition that is applied to solid aquatic food and administered orally to aquatic organisms when they ingest the food, and contains an antigen and an enteric-coated polymer compound as active ingredients.
[0025] The antigen broadly includes, and is not particularly limited to, vaccine antigens for aquatic infections, i.e., antigens related to the pathogens of aquatic infections, such as antigens derived from viruses or bacteria. Furthermore, the antigen may be a live vaccine antigen (an uninactivated vaccine antigen) or an inactivated antigen.
[0026] When using live vaccine antigens, they can be obtained, for example, by culturing and growing attenuated strains derived from pathogens of aquatic infectious diseases using known methods. For example, if the pathogen is a virus, an attenuated virus strain may be used to infect and grow cultured cells, and the resulting culture medium may be used as the antigen-containing solution. Alternatively, if the pathogen is bacteria, a culture medium of a bacterial strain that has been attenuated for vaccine use may be used as the antigen-containing solution.
[0027] When using inactivated antigens, they can be obtained, for example, by growing and inactivating a pathogen (a strain isolated for vaccine purposes) of an aquatic infectious disease using a known method. For example, if the pathogen is a virus, the virus strain isolated for vaccine purposes may be used to infect and grow cultured cells, and then the culture medium may be inactivated using a known method to prepare the inactivated antigen. Alternatively, if the pathogen is a bacterium, for example, the culture medium of a bacterial strain isolated for vaccine purposes may be inactivated using a known method to prepare the inactivated bacterial solution (inactivated antigen).
[0028] The method for inactivating the antigen is not particularly limited and can be any known method. For example, the antigen can be inactivated by subjecting the prepared antigen-containing solution to physical treatment (ultraviolet irradiation, X-ray irradiation, heat treatment, ultrasonic treatment, etc.) or chemical treatment (treatment with formalin, treatment with organic solvents such as chloroform or alcohol, acid treatment with weak acids such as acetic acid, treatment with chlorine or mercury, etc.). For example, inactivation with formalin can be performed by adding formalin at a volume concentration of 0.01 to 2.0%, more preferably 0.05 to 1.0%, to the prepared antigen-containing solution and sensitizing the antigen-containing solution at 4 to 30°C for 1 to 10 days. Alternatively, after the inactivation treatment, the inactivating agent such as formalin may be removed by washing with a buffer solution, or neutralization may be performed by adding a neutralizing agent.
[0029] Furthermore, antigens broadly include one or more antigenic proteins derived from pathogens, recombinant proteins thereof, and those artificially prepared using genetic engineering techniques. The preparation of these artificial proteins can employ a wide range of known methods and is not particularly limited.
[0030] As described above, enteric-coated polymer compounds can be any polymer compounds that do not dissolve in environments with a very low pH, such as the stomach, but dissolve in environments with a higher pH, and broadly include known enteric protective polymers. Examples include cellulose phthalate acetate, hydroxypropyl cellulose phthalate, carboxymethylcellulose, methyl acrylate-methacrylic acid copolymer, cellulose succinate acetate, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose succinate acetate (hypromellose acetate succinate), polyvinyl phthalate acetate (PVAP), methyl methacrylate-methacrylic acid copolymer, and sodium alginate. Sodium alginate is viscous in aqueous solution and gels when added to seawater or other environments containing calcium ions. That is, it is viscous when applied to solid bait, gels when mixed with seawater, and maintains its gelled state even when it reaches the intestines of aquatic organisms. For this reason, it is particularly suitable as an enteric-coated polymer compound according to the present invention.
[0031] In addition, this composition may contain an adjuvant.
[0032] A wide range of known adjuvants can be used. For example, oily adjuvants containing animal oils (such as squalene) or their hydrogenated oils, vegetable oils (such as palm oil and castor oil) or their hydrogenated oils, anhydrous mannitol oleate ester, liquid paraffin, polybutene, caprylic acid, oleic acid, higher fatty acid esters, etc., water-soluble adjuvants such as PCPP, saponin, manganese gluconate, calcium gluconate, manganese glycerophosphate, soluble aluminum acetate, aluminum salicylate, acrylic acid copolymer, methacrylic acid copolymer, maleic anhydride copolymer, alkenyl derivative polymers, oil-in-water emulsions, cationic lipids containing quaternary ammonium salts, precipitated adjuvants such as aluminum hydroxide (alum) and sodium hydroxide, microbial toxin components such as cholera toxin and E. coli heat-loopable toxin, and others such as bentonite, muramyl dipeptide derivatives, and interleukins. Mixtures of these may also be used.
[0033] Further, depending on the purpose, application, etc., buffers, isotonic agents, soothing agents, preservatives, antibacterial agents, antioxidants, pH adjusters, dispersants, fragrances, colorants, defoaming agents, etc. may be appropriately added to this composition.
[0034] Suitable examples of buffers include, for example, buffer solutions such as phosphates, acetates, carbonates, citrate, tartrate, tris(hydroxymethyl)aminomethane, and HEPES.
[0035] Suitable examples of isotonic agents include, for example, sodium chloride, glycerin, D-mannitol, etc.
[0036] Suitable examples of soothing agents include, for example, benzyl alcohol, etc.
[0037] Suitable examples of agents for the purpose of preservation include, for example, thimerosal, paraoxybenzoic acid esters, phenoxyethanol, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid, and other various preservatives, antibiotics, synthetic antibacterial agents, etc.
[0038] Suitable examples of antioxidants include, for example, sulfites, ascorbic acid, etc.
[0039] Suitable examples of pH adjusters include, for example, acids such as hydrochloric acid, carbonic acid, acetic acid, citric acid, phosphoric acid, boric acid, sulfuric acid, alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, alkali metal carbonates or bicarbonates such as sodium carbonate, alkali metal acetates such as sodium acetate, alkali metal citrates such as sodium citrate, bases such as trometamol, monoethanolamine, diisopropanolamine, etc.
[0040] Suitable examples of dispersants include, for example, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, polysorbate 80, etc.
[0041] Suitable examples of fragrances include citrus scents such as lemon, orange, and grapefruit, as well as peppermint, spearmint, menthol, pineapple, cherry, fruit, yogurt, and coffee.
[0042] Suitable examples of colorants include, for example, caramel color, gardenia color, anthocyanin color, annatto color, paprika color, safflower color, red yeast rice color, carotene color, carotenoid color, flavonoid color, cochineal color, amaranth (Red No. 2), erythrosine (Red No. 3), Allura Red AC (Red No. 40), New Coccine (Red No. 102), Phloxine (Red No. 104), Rose Bengal (Red No. 105), Acid Red (Red No. 106), Tartrazine (Yellow No. 4), Sunset Yellow FCF (Yellow No. 5), Fast Green FCF (Green No. 3), Brilliant Blue FCF (Blue No. 1), indigo carmine (Blue No. 2), copper chlorophyll, and copper chlorophyllin sodium.
[0043] Suitable examples of defoaming agents include, for example, dimethicone, simethicone, silicone emulsion, sorbitan sesquioleate, and nonionic substances.
[0044] In addition to the above, this composition may also contain auxiliary components, such as light-absorbing dyes (riboflavin, adenine, adenosine, etc.) that act as preservatives and enhance efficacy, chelating and reducing agents (vitamin C, citric acid, etc.) for stabilization, carbohydrates (sorbitol, lactose, mannitol, starch, sucrose, glucose, dextran, etc.), casein digest, and various vitamins.
[0045] As described above, the composition according to the present invention contains an antigen and an enteric-coated polymer compound as a mixture. The method for mixing the antigen and the enteric-coated polymer compound can be any known method and is not particularly limited. For example, the antigen solution and a solution in which the enteric-coated polymer compound is dissolved may be mixed, and the mixture may be dried by spray drying or the like to obtain a powder, or the mixture of the antigen solution and the solution in which the enteric-coated polymer compound is dissolved may be used as is or with the addition of other components.
[0046] The mixing ratio of the antigen and the enteric-coated polymer compound can be set appropriately depending on the purpose, etc., and is not particularly limited. Similarly, regarding the mixing ratio of the solid feed and its composition, in principle it is preferable that the entire amount of the composition adheres to the solid feed, but this too can be set appropriately depending on the purpose, etc., and is not particularly limited.
[0047] The composition according to the present invention is used by spreading it onto solid bait for aquatic organisms. The method of spreading the composition onto the solid bait is not particularly limited and can be broadly employed using known methods. For example, the composition may be spread by gently mixing it with the solid bait to coat it.
[0048] This composition preferably has viscosity when applied to the solid bait for aquatic organisms. The viscosity is preferably, for example, 0.1 to 100 Pa / s, more preferably 0.5 to 60 Pa / s, and most preferably 1 to 30 Pa / s.
[0049] For example, if the mixture of the antigen solution and the enteric-coated polymer compound is viscous, it may be applied as is. Alternatively, if the mixture of the antigen and the enteric-coated polymer compound is in powder form, a viscous substance may be added to adjust the viscosity before application.
[0050] The viscous substance can be a wide range of known substances and is not particularly limited. For example, low-saccharification reduced starch syrup, gluten, monocalcium phosphate, polyethylene glycol, guar gum, sodium polyacrylate, natural gums, etc. may be used.
[0051] The solid feed for aquatic organisms used in this invention can be any solid feed provided to aquatic organisms for growth and development, and can be appropriately selected based on the type, size, and preferences of the aquatic organisms to which it is used, as well as the form and composition of the feed. It can be a wide range of known feeds and is not particularly limited. For example, in addition to dry pellets, granules, crumbles, and extruder pellets, forms such as semi-solid feeds, water-containing feeds, and unformed feeds are also included in the solid feed according to this invention.
[0052] Because the composition according to the present invention is viscous when spread, it can be spread not only on solid feed with low oil content that is fed to juvenile fish of small and medium-sized fish species, but also on solid feed with high oil content that is fed to large fish such as tuna, or to individuals that have already grown to a certain size through aquaculture. For example, it can be spread on solid feed for aquatic organisms with an oil content of 7.5% by weight or more, more preferably 10.0% by weight, and most preferably 12.0% by weight. Therefore, it can be administered to individuals that require the intake of high-oil solid feed, for example, individuals weighing 300g or more. In other words, in addition to the usual immunization given to juvenile fish of small and medium-sized fish species, it can be applied to immunization of individuals that weigh 300g or more even as juveniles, such as tuna, or to additional immunization of individuals that have grown through aquaculture.
[0053] <Regarding the method for preventing and treating aquatic infections according to the present invention> The present invention broadly encompasses a method for preventing and treating aquatic infections, in which an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated polymer compound is applied to solid food for aquatic organisms and administered orally.
[0054] By performing the procedure of spreading the above-described composition onto a solid food for aquatic organisms and then orally administering the solid food to aquatic organisms, an immune response to the antigen is induced, and infectious diseases in aquatic organisms can be effectively prevented and treated.
[0055] The aquatic organisms to which this applies are not limited to any species that are susceptible to infectious diseases for which prevention or treatment is the objective, such as fish or crustaceans.
[0056] In this invention, it is sufficient to feed solid food coated with the composition for a certain period of time, and the dosage and duration of administration of the composition (or solid food coated with it) can be appropriately determined according to the infectious disease or antigen that is the target of prevention or treatment, and are not particularly limited, as they depend on the type of antigen. For example, if the antigen is bacteria, the amount of bacterial cells (or the amount before inactivation in the case of inactivated antigens) is 10 3 ~10 11 Continuous administration may be performed by allowing ad libitum feeding of solid feed coated with an antigen solution in the CFU / mL range for 1 to 20 days.
[0057] In Example 1, an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated polymer compound was prepared, and its efficacy as a vaccine was verified when administered orally after being applied to solid aquatic food. Inactivated Lactococcus garvieae type I cells were used as the antigen, and sodium alginate was used as the enteric-coated polymer compound.
[0058] L. garvieae type I (KS-7M strain) was used as the test antigen, and the amount of antigen was measured as 5.1 × 10⁶ viable cells before inactivation. 10 A solution was prepared by mixing 1 mL of antigen solution, which had been adjusted to CFU / mL and inactivated by adding formalin to a concentration of 0.5 vol%, with a 3 wt% sodium alginate aqueous solution (8-12 Pa / s). Fish solid bait was then placed in this solution and coated with the antigen, spreading almost the entire amount. A sufficient amount of seawater was then added to gel the antigen-containing sodium alginate.
[0059] Twenty-four amberjacks (average weight 50g) were prepared as test fish and divided into three groups of eight. The first group (group given spread-on feed) was orally administered solid feed (crude fat content 18%) with the antigen spread on it for five days. The antigen dose per fish was 2.0 × 10⁶ 9 The dosage was adjusted to CFU / day. For comparison, in the second group (water-soluble oral vaccine administration group), the dosage was 2.0 × 10 per fish. 9 CFU antigen solution was mixed with an equal amount of solid feed without being spread on it, and administered orally for 5 days. In the third group (control group), the antigen was not administered, and the same amount of solid feed was given.
[0060] Subsequently, each group was reared at 25°C for 14 days on a normal solid diet, and then, under anesthesia, 0.1 mL of the antimicrobial solution per fish (antimicrobial strain: KGLG01, antimicrobial titer: 1.73 × 10⁴) was administered. 4 The attack was carried out by injecting CFU (Cellular Fault Untreated / tail) into the abdominal cavity. After the attack, the fish were kept in a tank with a set water temperature of 25°C for 13 days, and the survival rate was compared.
[0061] The results are shown in Figure 1. Figure 1 is a graph showing the survival rate of amberjack after immunization with spreading bait followed by attack. The horizontal axis of the graph (days after attack) represents the number of days elapsed since the attack, and the vertical axis (survival rate, unit: %) represents the survival rate after the attack. In the graph, the line labeled "Spreading Bait" represents the survival rate when immunized with spreading bait, the line labeled "Antigen Solution" represents the survival rate when immunized with a water-soluble oral vaccine (oral administration of antigen solution) as a comparative example, and the line labeled "Control" represents the survival rate when no antigen was administered as a control.
[0062] As shown in Figure 1, the survival rate 13 days after attack was 0% in the control group (no antigen administration) and 12.5% in the group immunized with a water-soluble vaccine (water-soluble oral vaccine (antigen solution) administration group), while it was 50% in the group immunized with a spreading bait (spreading bait administration group), showing a significantly higher survival rate.
[0063] These results demonstrate that an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated polymer compound can be effectively administered orally by being applied to solid aquatic food, thereby effectively inducing an immune response and effectively preventing aquatic infections.
[0064] In Example 2, similar to Example 1, an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated polymer compound was prepared, and its efficacy as a vaccine was verified when administered orally after being applied to solid aquatic food. Inactivated Vibrio anguillarum cells were used as the antigen, and sodium alginate was used as the enteric-coated polymer compound.
[0065] V. anguillarum (KT-5 strain) was used as the test antigen, and the amount of antigen was measured as 5.1 × 10⁶ viable cells before inactivation. 10 A solution was prepared by mixing 1 mL of antigen solution, which had been adjusted to CFU / mL and inactivated by adding formalin to a concentration of 0.5 vol%, with a 3 wt% sodium alginate aqueous solution (8-12 Pa / s). Fish solid bait was then placed in this solution and coated with the antigen, spreading almost the entire amount. A sufficient amount of seawater was then added to gel the antigen-containing sodium alginate.
[0066] As test fish, 30 yellowtails (average weight: 40 g) were prepared and divided into three groups of 10 fish each. In the first group (the spreading feed administration group), solid feed (crude fat content: 18%) with the antigen spread thereon was orally ingested for 5 days. The antigen dosage was adjusted to 2.0×10 9 CFU per fish per day. Also, as a comparative example, in the second group (the water-soluble oral vaccine administration group), an antigen solution of 2.0×10 9 CFU per fish that was not spread on the same amount of solid feed but mixed was orally administered for 5 days. In the third group (the control group), the same amount of solid feed was ingested without administering the antigen.
[0067] After that, in each group, after feeding and raising with normal solid feed at 25°C for 14 days, under anesthesia, 0.1 mL of the challenge bacterial solution per fish (challenge strain: V09K01 strain, challenge titer: 9.6×10 7 CFU / fish) was injected intraperitoneally to conduct the challenge. For 13 days after the challenge, the fish were raised in a water tank with a set water temperature of 25°C, and the survival rates were compared.
[0068] As a result, the survival rate 10 days after the challenge was 0% when no antigen was administered (control group) and also 0% when immunized with the water-soluble vaccine (the water-soluble oral vaccine (antigen solution) administration group), whereas the survival rate was 30% when immunized with the spreading feed (the spreading feed administration group).
[0069] From these results, it was shown that even with an antigen other than Lactococcus garvieae type I, by spreading an oral vaccine composition for aquatic organisms containing the antigen and an enteric polymer compound on an aquatic organism solid feed and orally administering it, an immune response can be effectively induced and an aquatic organism infectious disease can be effectively prevented, similar to the case of Lactococcus garvieae type I.
[0070] In Example 3, an oral vaccine composition for aquatic organisms similar to that in Example 1 and the like was prepared, spread on an aquatic organism solid feed, and the efficacy as a vaccine when orally administered to rainbow trout was verified.
[0071] Similar to Example 2, V. anguillarum (VA1669 strain) was used as the test antigen, and the antigen amount was 5.1×10 in terms of the viable bacteria count before inactivation 10A solution was prepared by mixing 1 mL of antigen solution, which had been adjusted to CFU / mL and inactivated by adding formalin to a concentration of 0.5 vol%, with a 3 wt% sodium alginate aqueous solution (8-12 Pa / s). Fish solid bait was then placed in this solution and coated with the antigen, spreading almost the entire amount. A sufficient amount of seawater was then added to gel the antigen-containing sodium alginate.
[0072] Thirty rainbow trout (Salmonidae, Salmoniformes, average weight 15g) were prepared as test fish and divided into two groups of 15: a group receiving bait with the antigen applied and a control group. The group receiving bait with the antigen applied was orally administered solid bait (crude fat content 8%) with the antigen applied for 5 days. The antigen dose per fish was 2.0 × 10⁶ 9 The CFU / day was adjusted. As a comparative example, the control group was given the same amount of solid feed without being administered the antigen.
[0073] Subsequently, each group was reared at 25°C for 14 days on a normal solid diet, and then, under anesthesia, 0.1 mL of the antimicrobial solution per fish (antimicrobial strain: N-7802 strain, antimicrobial titer: 9.9 × 10⁴) was administered. 6 CFU (Cellular Fault Undead / tail) were subjected to immersion attacks. After the attacks, they were kept in a tank with flowing water (average water temperature approximately 17°C) for 13 days, and the survival rates were compared.
[0074] As a result, the survival rate 13 days after attack was 0% in the control group (no antigen administration), compared to 60% in the group immunized with spreading bait (spreading bait administration group).
[0075] These results demonstrate that, in fish species other than yellowtail and amberjack, as with yellowtail, an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated polymer compound can be effectively administered orally by being applied to solid aquatic food, thereby effectively stimulating an immune response and effectively preventing aquatic infections.
[0076] In Example 4, the efficacy of an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated polymer compound as a booster vaccine was verified when it was applied to solid aquatic food and administered orally. Inactivated Lactococcus formosensis was used as the antigen, and sodium alginate was used as the enteric-coated polymer compound.
[0077] L. formosensis (LG13E strain) was used as the test antigen, and the amount of antigen was measured in terms of viable bacteria before inactivation: 2.0 × 10⁶ 9 A solution was prepared by mixing 1 mL of antigen solution, which had been adjusted to CFU / mL and inactivated by adding formalin to a concentration of 0.5 vol%, with a 3 wt% sodium alginate aqueous solution (8-12 Pa / s). Fish solid feed was then placed in this solution and coated with the antigen, allowing almost the entire amount to adhere.
[0078] Eighty yellowtail were prepared as test fish and divided into four groups of 20 each. The first group (feeding administration group) received additional immunization via feeding, the second group (primary immunization group) received only primary immunization, the third group (positive control group) received additional immunization via transanal administration as a positive control, and the fourth group (control group) received neither primary nor additional immunization.
[0079] First, as an alternative to primary immunization, groups 1 through 3 were intraperitoneally injected with less than the minimum effective dose of the inactivated LG13E strain. Group 4 was not administered the strain and was fed a normal solid diet (crude fat content 14%).
[0080] Next, in the first group (the group administered with the antigen attached), as a booster immunization, solid food with the antigen attached was orally administered for 5 days starting 7 days after administration of less than the minimum effective dose of antigen (2.0 × 10 per fish). 9 (CFU / day x 5 days). In the third group (positive control group), as an additional immunization, instead of spreading the antigen, 2.0 x 10 per fish was administered 7 days after the administration of less than the minimum effective dose of antigen. 9 CFU antigens were administered via transanal injection. Groups two and four did not receive booster immunization and were fed a normal solid diet.
[0081] Subsequently, each group was reared at 25°C for 14 days on a normal solid diet, and then, under anesthesia, 0.1 mL of the antimicrobial solution per individual (antimicrobial strain: LG16K strain, antimicrobial titer: 1.08 × 10⁴) was administered. 5 The attack was carried out by injecting CFU (Cellular Fault Untreated / tail) into the abdominal cavity. After the attack, the fish were kept in a tank with a set water temperature of 25°C for 13 days, and the survival rate was compared.
[0082] The results are shown in Figure 2. Figure 2 is a graph showing the survival rate of yellowtail after being immunized via spreading bait and then attacked. The horizontal axis of the graph (days after attack) represents the number of days since the attack, and the vertical axis (survival rate, unit: %) represents the survival rate after the attack. In the graph, the line labeled "Spreading Bait" represents the survival rate when immunized via spreading bait, the line labeled "Primary Immunization" represents the survival rate when only primary immunization was performed and no additional immunization was given, the line labeled "Positive Control" represents the survival rate when positive controls were immunized via transanal administration, and the line labeled "Control" represents the survival rate when controls were not immunized either primary or additionally.
[0083] As shown in Figure 2, the survival rate 13 days after attack was 20% in the control group (no primary or booster immunization) and 35% in the primary immunization group (primary immunization only), while it was significantly higher at 65% in the group that received booster immunization via feeding (feeding administration group).
[0084] These results demonstrate that, in individuals that have already received primary immunization, administering an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated polymer compound orally, applied to solid aquatic food, can effectively induce an immune response and effectively prevent aquatic infections.
[0085] In Example 5, an oral vaccine composition for aquatic organisms containing an antigen and an enteric polymer compound was applied to a solid aquatic food using a methyl acrylate-methacrylic acid copolymer as the enteric polymer compound, and its efficacy as a booster vaccine when administered orally was verified. Inactivated Lactococcus formosensis cells were used as the antigen.
[0086] (1) 50% methyl acrylate-methacrylic acid copolymer (product name "Polylid PA-30", manufactured by Sanyo Chemical Industries, Ltd.), (2) L. forsemensis (LG13E strain) was used as the test antigen, and the amount of antigen was measured in terms of viable bacteria count before inactivation to 2.0 × 10⁶. 9(3) 48.5% of the antigen solution, which was adjusted to CFU / mL and inactivated by adding formalin to a concentration of 0.5 vol%, was mixed with 1.5% polyethylene glycol (trade name "Macrogol 400", manufactured by Sanyo Chemical Industries, Ltd.). The solution was spray-dried and then collected to obtain antigen powder.
[0087] 2.0g of low-saccharification reduced starch syrup (product name "SE30", manufactured by Bussan Food Science Co., Ltd.) was mixed with 1.5g of antigen powder, and then solid fish feed was added and coated to the mixture, allowing almost the entire amount to adhere.
[0088] Eighty yellowtail were prepared as test fish and divided into four groups of 20 each. Similar to Example 2, the first group (feeding administration group) received additional immunization via feeding, the second group (primary immunization group) received only primary immunization, the third group (positive control group) received additional immunization via transanal administration as a positive control, and the fourth group (control group) received neither primary nor additional immunization.
[0089] First, as a substitute for primary immunization, groups 1 through 3 received 3.4 × 10⁶ of inactivated LG13E strain antigen as a dose less than the minimum effective dose. 4 CFU was administered intraperitoneally. Group 4 was not given the CFU and was fed a normal solid diet (crude fat content 14%).
[0090] Next, in the first group (the group administered with the antigen attached), as a booster immunization, solid food with the antigen attached was orally administered for 5 days starting 7 days after administration of less than the minimum effective dose of antigen (2.0 × 10 per fish). 9 (CFU / day x 5 days). In the third group (positive control group), as an additional immunization, instead of spreading the antigen, 2.0 x 10 per fish was administered 7 days after the administration of less than the minimum effective dose of antigen. 9 CFU antigens were administered via transanal injection. Groups two and four did not receive booster immunization and were fed a normal solid diet.
[0091] Subsequently, each group was reared at 25°C for 14 days on a normal solid diet, and then, under anesthesia, 0.1 mL of the antimicrobial solution per individual (antimicrobial strain: LG16K strain, antimicrobial titer: 1.08 × 10⁴) was administered. 5 The attack was carried out by injecting CFU (Cellular Fault Untreated / tail) into the abdominal cavity. After the attack, the fish were kept in a tank with a set water temperature of 25°C for 13 days, and the survival rate was compared.
[0092] The results are shown in Figure 3. Figure 3 is a graph showing the survival rate when an attack was carried out after additional immunization was performed using a methyl acrylate-methacrylate copolymer as an enteric-coated polymer via a spreading bait. The horizontal axis of the graph (days after attack) represents the number of days elapsed since the attack, and the vertical axis (survival rate, unit: %) represents the survival rate after the attack. In the graph, the line labeled "Spreading Bait" represents the survival rate when additional immunization was performed via a spreading bait, the line labeled "Primary Immunization" represents the survival rate when only primary immunization was performed and no additional immunization was carried out, the line labeled "Positive Control" represents the survival rate when additional immunization was performed via transanal administration as a positive control, and the line labeled "Control" represents the survival rate when neither primary nor additional immunization was performed as a control.
[0093] As shown in Figure 3, the survival rate 13 days after attack was 20% in the control group (no primary or booster immunization) and 35% in the primary immunization group (primary immunization group), while the survival rate was significantly higher at 62% in the group that received booster immunization using a spreading bait containing methyl acrylate-methacrylic acid copolymer (spreading bait administration group).
[0094] These results demonstrate that even when methyl acrylate-methacrylic acid copolymer is used as the enteric-coated polymer, administering an oral vaccine composition for aquatic organisms containing the antigen and the enteric-coated polymer, applied to solid aquatic food, as a booster immunization to individuals that have already received primary immunization, can effectively induce an immune response and effectively prevent aquatic infections.
[0095] The graph in Example 1 shows the survival rate of amberjack after immunization with L. garvieae type I using a spreading bait and then attack. The graph in Example 4 shows the survival rate of yellowtail after additional immunization with a spreading bait and then attack. The graph in Example 5 shows the survival rate of amberjack after additional immunization with a spreading bait using an enteric-coated polymer compound with methyl acrylate-methacrylate copolymer and then attack.
Claims
1. An oral vaccine composition for aquatic organisms, which is applied to solid feed for aquatic organisms and contains an antigen and an enteric-coated polymer compound.
2. The oral vaccine composition for aquatic organisms according to claim 1, which has viscosity when applied to the solid feed for aquatic organisms.
3. The oral vaccine composition for aquatic organisms according to claim 2, wherein the solid feed for aquatic organisms contains 7.5% by weight or more of oil.
4. The oral vaccine composition for aquatic organisms according to claim 1, wherein the solid feed for aquatic organisms is administered to individuals weighing 300 g or more.
5. The oral vaccine composition for aquatic organisms according to claim 1, wherein the enteric-coated polymer compound is methyl acrylate-methacrylate copolymer or sodium alginate.
6. The oral vaccine composition for aquatic organisms according to claim 1, wherein the antigen is derived from bacteria.
7. A method for preventing and treating aquatic infections, comprising applying an oral vaccine composition for aquatic organisms containing an antigen and an enteric-coated polymer compound to solid food for aquatic organisms and administering it orally.