JP2008239541A - Infection prophylactic of fish and shellfish - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To improve infectious diseases of fishes and shellfishes in farms by providing an infection prophylactic having high prophylactic effects to infectious diseases of fishes and shellfishes without orally administering to the fishes and the shellfishes, and an infection prophylactic method of fishes and shellfishes, comprising administration of the prophylactic. <P>SOLUTION: An infection prophylactic of fishes and shellfishes, which comprises a sulfation-treated disaccharide as an active ingredient, the infection prophylactic of fishes and shellfishes, whose monosaccharides composing the disaccharide are substituted their all H of hydroxy groups with SO<SB>3</SB>group, the infection prophylactic of fishes and shellfishes, whose disaccharide is maltose and/or sucrose, and a prophylactic method of infectious diseases of fishes and shellfishes, which comprises administering an effective amount of the infection prophylactic of fishes and shellfishes, are provided. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a drug for preventing infection of fish and shellfish by viruses or pathogenic bacteria, and a method for preventing infection of fish and shellfish by administering the drug.

  Crustaceans such as prawns (Penaeus japonicus) and cattle shrimp (Penaeus monodon), and fish farms such as yellowtail, red sea bream, and flounder often cause diseases due to the high density of seafood. The cause of the disease is mainly infection by pathogenic bacteria, viruses, parasites, etc., and various countermeasures have been taken conventionally.

  Among the infectious diseases, in recent years, prawne acute viremia (PAV), which is a viral infection that kills shrimp, has caused serious damage. This infectious disease was first confirmed in Japan in 1993, but is mediated by a pathogenic virus called Penaeid rod-shaped DNA virus (PRDV). . As a similar pathogenic virus, yellowhead virus causing yellowhead disease is known. In addition, infects with high incidences such as viral infections and streptococcal diseases are known in fishes to be cultivated, all of which cause serious damage.

  For these infectious diseases, countermeasures such as administration of antibiotics and immunologically active substances have been taken. However, in the case of administering antibiotics, there is concern about remaining in the fish and shellfish that are cultivated, and the effect is not always satisfactory.

On the other hand, in recent years, attempts have been made to prevent and treat seafood infections at farms using seaweed-derived fucoidan, but the incidence of damage has not been reduced, and new infections that are more effective. (See Patent Documents 1 to 8).
JP 2004-30736 A JP 2000-336035 A Japanese Patent Laid-Open No. 11-80003 International Publication No. 2002/092114 International Publication No. 98/42204 JP-A-6-234651 Japanese Patent Laid-Open No. 11-180813 Japanese Patent Laid-Open No. 6-234652

In aquaculture ponds, there is a problem of how quickly the spread of infection can be controlled when an infection, especially a fatal infection occurs, but with conventional preventive and therapeutic agents such as fucoidan, However, there is a problem that the effect cannot be fully exerted unless eating. In addition, there is a method of treating fish and shellfish in the aquaculture pond with a conventional preventive / therapeutic agent, but in this case too, it cannot be said that sufficient effects are achieved.
Therefore, an object of the present invention is to provide an infection preventive agent having a high preventive effect on fish and shellfish infections without oral administration of fish and shellfish, and a method for preventing infection of fish and shellfish by administering this preventive agent. To provide and improve seafood infections in the farm.

  Therefore, as a result of intensive studies, the present inventors have found that disaccharides that have undergone sulphation treatment are less susceptible to viruses and the like even if fish and shellfish are not taken into the body, compared to oral preventive and therapeutic agents such as conventional fucoidan. It was found that the effect of preventing further spread of fish and shellfish infectious diseases was significantly high, and the present invention was completed.

That is, the gist of the present invention is as follows.
[1] Infection preventive agent for fish and shellfish containing a sulfated disaccharide as an active ingredient,
[2] The anti-infection agent for fish and shellfish according to [1] above, wherein in the monosaccharide constituting the disaccharide, hydrogens of all hydroxyl groups are substituted with SO ₃ groups,
[3] The fish and shellfish infection preventive agent according to [1] or [2], wherein the disaccharide is maltose and / or sucrose,
[4] The present invention relates to a method for preventing fish and shellfish infections, which comprises administering an effective amount of the fish and shellfish infection preventive agent according to any one of [1] to [3] to a fishery pond.

  By using the infection preventive agent of the present invention, it is possible to remarkably prevent infectious diseases of fish and shellfish in the culture pond. This can prevent the death of fish and shellfish caused by infection and the reduction of commercial value, and can minimize the economic loss of fish farmers.

  The fish and shellfish infection preventive agent of the present invention (hereinafter referred to as infection preventive agent) comprises a disaccharide subjected to a sulfation treatment as an active ingredient.

  The disaccharide used in the present invention has a structure in which two monosaccharides form a glycosidic bond between the anomeric carbon of one monosaccharide and the hydroxyl group of the other monosaccharide. Examples of the disaccharide include maltose, sucrose, cellobiose, lactose, and trehalose. Among them, maltose and sucrose are preferable, and sucrose is more preferable from the viewpoint of excellent effects of preventing fish and shellfish infections. Two or more of these disaccharides may be mixed and used.

In the present invention, the disaccharide obtained by sulfation treatment is used.
The sulfation treatment refers to a treatment in which all hydroxyl group hydrogens are replaced with SO ₃ groups in monosaccharides constituting disaccharides.

  In the present invention, the sulfation treatment is performed, for example, by mixing the disaccharide with an amine sulfide compound.

  Examples of the amine sulfide compound include a pyridine complex containing a sulfate group, a trimethylamine complex having a sulfate group, and sulfoxide. These sulfurized amine compounds may be used alone or in admixture of two or more.

  The mixing ratio of the disaccharide and the sulfurized amine compound is not particularly limited. For example, the disaccharide: sulfurized amine compound (weight ratio) may be adjusted to be 1: 2 to 1:15. preferable.

  Further, if necessary, the amine sulfide compound may be used in combination with other components such as amine. There are no particular limitations on the amount of amine or the like.

The sulfation treatment is preferably performed by mixing the disaccharide and the sulfurized amine compound with an organic solvent. The organic solvent is used for dissolving the sulfurized amine compound and the disaccharide. By mixing with the sulfurized amine compound in such a state, the disaccharide can be efficiently sulfated.
Examples of the organic solvent include methanol, chloroform, dioxane, dimethylformamide, toluene, ethyl ether and the like. These organic solvents can be used individually or in mixture of 2 or more types. Of these, dimethylformamide is preferable from the viewpoint of efficient reaction.

  The amount of the organic solvent is preferably adjusted so that the organic solvent is 10 to 30 mL with respect to 1 g of the disaccharide.

  As a sulfation treatment condition performed by mixing the disaccharide and the amine sulfide compound with the organic solvent, for example, when mixing at a temperature exceeding room temperature, it is preferable to leave it for about 1 to 2 days. From the viewpoint of efficiently carrying out the treatment, it is more preferable to leave it at around 60 ° C. for about 20 to 30 hours. Moreover, you may stir etc. at the time of mixing.

  After the sulfation treatment, the mixture is centrifuged to obtain a precipitate, and the precipitate is washed with an organic solvent that does not dissolve the precipitate such as acetonitrile having a concentration of 3 to 8 times. Disaccharide (hereinafter referred to as sulfated disaccharide) can be obtained. After washing, the precipitate may be mixed with trifluoroacetic acid and left at room temperature for about 0.5 to 2 hours to decompose the remaining amine sulfate compound.

  In addition, the sulfated disaccharide can be made solid by degassing and removing the organic solvent from the washed product as described above and then drying.

  The sulfated disaccharide obtained as described above has a higher surface tension than the disaccharide before sulfation treatment. For example, the surface tension of the sulfated disaccharide at 25 ° C. and a humidity of 40% is preferably 66.2 mN / m or more at a 5% concentration, and more preferably 70.0 mN / m or more.

  The infection-preventing agent of the present invention contains the sulfated disaccharide as an active ingredient, and one or more of the sulfated disaccharides may be used as they are, and depending on the form, other ingredients may be contained. Also good.

  As said other component, you may contain the monosaccharide which carried out the sulfation process from a viewpoint which the prevention effect improves more, for example. Examples of monosaccharides include glucose, mannose, fructose, galactose and the like. The sulfation treatment may be performed in the same manner as the method for obtaining the sulfated disaccharide. Moreover, you may mix | blend another component according to the form of an infection prevention agent.

  The form of the agent for preventing infection according to the present invention is not particularly limited, and for example, liquids such as syrups, emulsions, suspensions, injections, tablets, fine granules, granules, powders, pills, capsules, troches It can be a solid preparation such as an agent. In preparing these preparations, conventional carrier components can be used depending on the kind of preparation.

  For the preparation of solid preparations, for example, excipients, binders, lubricants, disintegrants, disintegration aids, sugars, humectants, surfactants and the like can be used. In addition, for example, a solvent, a solubilizing agent, a buffering agent, a suspending agent, a tonicity agent, a surfactant, a soothing agent, glucose, an amino acid, and the like can be used for preparing the liquid agent. In addition, preservatives, antioxidants, solubilizers, emulsifiers, thickeners, plasticizers, adsorbents, fragrances, colorants, dispersants, sweeteners, preservatives, and the like can be used for solid preparations and liquids.

  The infection-preventing agent of the present invention can be produced by a common method such as mixing, kneading, granulation, tableting, coating, sterilization, emulsification, etc., depending on the form.

  Moreover, in this invention, the infectious disease of fishery products can be prevented by administering the effective amount of the infection prevention agent manufactured as mentioned above to a culture pond.

  The administration method may be carried out according to conventional means, and the infection preventive agent of the present invention may be used as it is or on a carrier component, or an aqueous solution of these agents may be administered to the culture pond. In addition, an effective amount means the amount with which a preventive effect is show | played.

  The culture pond is preferably a closed type from the viewpoint of high preventive effect. Examples of closed aquaculture ponds include rice fields, ponds, and aquaculture tanks on land. In addition, in a large water environment such as a river or the sea, a farm where the degree of water circulation to the outside is reduced by using a fine mesh or the like is also included in the culture pond of the present invention.

  The dose of the infection preventive agent of the present invention varies depending on the volume of water in the culture pond, the kind of seafood, body weight, symptoms, etc., and is appropriately set in consideration of these. As the amount of the sulfated disaccharide as a component, the capacity of the water concentration in the culture pond is preferably 1 g / L or more. In the case of seafood other than shrimps, the dosage may be adjusted according to the above-mentioned shrimps.

  In addition, the infection preventive agent of the present invention is, for example, a treatment liquid containing shrimp in the aquaculture farm containing the infection preventive agent of the present invention when infection with a pathogenic virus such as PRDV is confirmed in the shrimp farm. Or by immersing shrimp in the treatment solution for a predetermined time. The treatment liquid can be prepared, for example, by blending an effective amount of the preventive agent of the present invention in seawater, fresh water, physiological saline, or the like near the farm.

  The infection preventive agent of the present invention includes, for example, tiger shrimp, cattle shrimp (black tiger), tiger shrimp, bear shrimp, lobster, Penaeus vannamei, crustacean and other crustaceans, carp, crucian carp, eel, rainbow trout, ayu, goldfish, etc. It can be administered to seawater fish such as freshwater fish, puffer fish, puffer fish, tie, flounder, flatfish, horse mackerel and yellowtail.

  Infectious diseases that can be applied include viral infections such as PRDV, yellow head virus, iridovirus and birnavirus, and bacterial infections such as Vibrio, Pseudomonas and Streptococcus. Among them, it is preferable because it is highly effective against PRDV and yellow head virus having a high lethality after infection.

Although the mechanism by which the infection preventive agent used in the present invention has a remarkable effect in preventing fish and shellfish infections is not yet clear, the sulfate group in the monosaccharide constituting the sulfated disaccharide and one of the monosaccharides It is conceivable that the oxygen bonded to the glycoside between the anomeric carbon and the other monosaccharide acts on viruses and pathogens to prevent infection. For example, in sulfated disaccharides, the presence of many sulfate groups in the space adjacent to the oxygen of glycosidic bonds in the three-dimensional structure facilitates the binding of sulfated disaccharides to viruses and pathogens. Because the molecular weight of saccharides is small, the rate of binding is high, the infectivity of viruses and pathogenic bacteria cells to which sulfated disaccharides are bound can be significantly reduced, and the binding strength is high, so it is taken into the body of seafood. However, it is inferred that pathogenicity is difficult to develop.
Moreover, since the therapeutic agent for infection according to the present invention does not exhibit a significant immune activation effect such as fucoidan, it is presumed that it acts by a mechanism different from that of conventional oral preventive and therapeutic agents.

(Example 1) Preparation method of sulfated disaccharide (sucrose) 200 mg of sucrose and 400 mg of trimethyl octoside were placed in 6 mL of dimethylformamide and stirred and mixed at 60 ° C. and 130 rpm for 24 hours for sulfation treatment. Next, centrifugation (6000 g, 10 minutes) was performed, and the precipitate was collected, washed twice with acetonitrile, and then dried at 40 ° C. to obtain a white powdery solid (yield: sulfuric acid). Sucrose (93%).

In order to confirm the position of the sulfate group (SO ₃ group) in the obtained sulfated sucrose, structural analysis was performed as follows.

[Structural analysis of synthetic products]
The synthesized product was dissolved in 80% acetonitrile to prepare a sample, which was subjected to HPLC under the following conditions. The total peak area with an elution time of 15 to 16 minutes was calculated and quantified. Each peak was subjected to structural analysis by LC-MS / MS.
Column: “Amide80” (5μm, 4.6mm x 250mm)
Column temperature: 65 ° C
Flow rate: 1.0mL / min
Eluent: 80% acetonitrile
Injection volume: 10μL
Detector: PDA

As a result of separation by HPLC, there are two peaks, a peak (8%) that appeared in the vicinity of 14 minutes and a peak (91.5%) that appeared in the vicinity of 15 minutes. As shown in Fig. 1, the MW413.4 fragment peak was strongly confirmed in the substance present at 91.5% in the vicinity of 15 minutes. Therefore, sulfated sucrose is contained in glucose and fructose constituting sucrose (MW: 342.3). It can be seen that the H of a certain OH group is all converted to SO ₃ . The peak near 14 minutes was a substance in which seven of the OH groups of sucrose were bound together (M.W .: 395.4).

(Example 2) Preparation method of sulfated disaccharide (maltose) 200 mg of maltose and 400 mg of trimethyl octoside were placed in 6 mL of dimethylformamide, and stirred and mixed at 60 ° C. and 130 rpm for 24 hours for sulfation treatment. Next, centrifugation (6000 g, 10 minutes) was performed, and the precipitate was collected, washed twice with acetonitrile, and then dried at 40 ° C. to obtain a white powdery solid (yield: sulfuric acid). Maltose (91%).

In order to confirm the position of the sulfate group (SO ₃ group) in the obtained sulfated maltose, structural analysis was performed in the same manner as in Example 1. It was found that the hydroxyl groups of one glucose constituting sulfated maltose were all substituted with SO ₃ groups, and the other hydroxyl groups of glucose were all substituted with SO ₃ groups.

(Experimental example 1) [Functional effect 1 as a preventive agent]
(Preparation of prawns)
Shrimp (purchased from Matsumoto Suisan Co., Ltd., about 12 g per fish) was rested in a tank of seawater (60 L, aeration at 23 ° C, 43 mL / min) for 3 days, and then tested for preventive effects Provided.

(Preparation of PRDV solution)
The heart of two prawns infected with PRDV was taken out, pulverized in physiological saline, and diluted 10 times to obtain a PRDV solution.

(Preparation of infection prevention solution)
The sulfated disaccharides obtained in Examples 1 and 2 were mixed in physiological saline so as to be 1 mg / mL to prepare an infection prevention solution. As a control, a physiological saline alone was used.

(Experimental procedure)
An equal amount of PRDV solution was added to the infection prevention solution and allowed to stand at room temperature for 30 minutes, and then passed through a 0.45 μm aperture filter to obtain three types of treatment solutions for injection.
Using an injection needle of model No. 28G, each prawn (15 fish per test section) was injected into the first joint so that each injection treatment solution was 200 μL / tail, returned to the water tank, water temperature 23 ° C., aeration (43 mL / Min).

  The body weight and survival rate of the prawn 1-8 days after returning to the water tank were measured. These results are shown in FIG.

As shown in FIG. 2, it can be seen that sulfated sucrose and sulfated maltose both have a significantly lower mortality rate than the control. In particular, sulfated sucrose maintains a survival rate of nearly 70% even on the 8th day, so that it can be seen that even when a large amount of lethal PRDV is present in the culture pond, it has a significant preventive effect. .
In addition, from the results shown in FIG. 2, sulfated sucrose and sulfated maltose, particularly sulfated sucrose, have excellent binding properties to PRDV, and even when PRDV is taken into the shrimp body, its pathogenicity is expressed. It can be seen that it can be significantly suppressed.

(Experimental example 2) [Operation effect 2 as a preventive agent]
An immersion experiment was conducted on prawns as follows, and the change in mortality over time was confirmed, and the effectiveness of the anti-PRDV drug was confirmed. The result is shown in FIG.

〔Method〕
Test sample: Shrimp (about 16 g) from Matsumoto Suisan, purchased, rested in a water tank for 2 days, and used for immersion experiments.
PRDV solution: Remove all 4 shrimp heads infected with PRDV, crush them in physiological saline and dilute 10-fold to make a PRDV solution, and infect in a 2 L water bath for 2 hours.
Anti-PRDV dipping solution: A solution prepared by preparing 1 mg / ml each of the anti-PRDV3 solution (A group, B group, C group) is used as an anti-PRDV attack solution, and is applied to each solution for 10 minutes.
Test group: Sulfated sucrose (A group), Fucoidan (B group), iodine 2ppm (C group)
Number of experimental animals: 15 water tanks per test area: Water temperature 23 ° C, Aeration (43 ml / min)
Death confirmation time: 1, 3, 5, 7, 9 days after returning prawns to water tank Measurement item: Number of deaths, time

As shown in the results of FIG. 2, among the A to C zones, it is recommended for fucoidan (B zone) used in the current aquaculture site and shrimp treatment in aquaculture ponds where PRDV infection was seen It can be seen that sulfated sucrose shows a significantly higher survival rate even in shrimp already infected with PRDV, compared to the 2 ppm iodine solution (section C).
In addition, from the results of FIG. 3, sulfated sucrose excellent in binding to PRDV rapidly binds to PRDV that is already attached to the body of shrimp, thereby significantly manifesting its pathogenicity. It can be suppressed.

(Experiment 3) (Safety test)
2L of 100 mg / ml concentration of sulfated sucrose aqueous solution was made, and prawns were dipped there for about 2 hours, returned to the 60L water tank, and mortality was confirmed. No death was confirmed even after 10 days. . Therefore, it can be seen that sulfated sucrose is excellent in safety. Moreover, the safety | security which was similarly excellent in the sulfated maltose was shown.

(Experimental example 4)
The surface tension at 25 ° C. and 40% humidity in the 5% solution of raw material sucrose and the 5% solution of sulfated sucrose obtained in Example 1 was measured to find that raw material sucrose was 66.2 mN / m, sulfated sucrose 70.2 mN / m. m.

1 is a graph showing the results of structural analysis of sulfated sucrose obtained in Example 1. FIG. FIG. 2 is a graph showing the results of the preventive effect in Experimental Example 1. FIG. 3 is a graph showing the results of the preventive effect in Experimental Example 2.

Claims (4)

  An infection preventive agent for fish and shellfish containing a sulfated disaccharide as an active ingredient. The agent for preventing infection of fish and shellfish according to claim 1, wherein in the monosaccharide constituting the disaccharide, hydrogen atoms of all hydroxyl groups are substituted with SO ₃ groups.   The agent for preventing infection of fish and shellfish according to claim 1 or 2, wherein the disaccharide is maltose and / or sucrose.   A method for preventing fish and shellfish infections, comprising administering an effective amount of the fish and shellfish infection preventive agent according to any one of claims 1 to 3 to a fish and fish culture pond.

Images