JP2000325026A - Immunomodulator for fish - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an immunomodulator for fish, capable of controlling death rate without polluting the environments, and useful as a therapeutic agent, or the like, for viral diseases, by including, as the active ingredient, a polypeptide which is produced by synthesis or the like to contain basic amino acids. SOLUTION: This immunomodulator is produced by synthesis and/or microorganismic fermentation, and contains a polypeptide as the active ingredient, which contains at least one or more than two basic amino acids selected from the group consisting of lysine, arginine and histidine. The polypeptide has a molecular weight of 200 to 12,500, preferably 700 to 5,000, is contained in the modulator at 0.01 μg to 100 mg, preferably 1 μg to 10 mg as the effective quantity for gram of fish weight, and is preferably incorporated with a digestion inhibitor (e.g. trypsin inhibitor), when the modulator is to be orally administered.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a fish immunomodulator containing a polypeptide containing a basic amino acid as an active ingredient.

[0002]

2. Description of the Related Art It has been known that polylysine, which is a kind of basic polypeptide, can be used as a carrier for drugs and high molecular compounds, and has the ability to induce interferon production. It is also known that conjugates with oligomers exhibit antiviral activity against specific viruses. Regarding the prevention and treatment methods for fish disease, protection methods with vaccines and treatment methods with antibiotics are known. Also, β-1,3 glucan and the like are known as immunostimulants (Japanese Patent Laid-Open No. 218615/1990), but it has been known that the polypeptide used in the present invention is used in such a fishery technical field. Not been.

[0003]

In the recent fisheries industry,
The shift from catching to making fisheries is advancing, and the cultivation of many fish species is becoming widespread. Under these circumstances, a major problem is the occurrence of diseases at farms caused by bacteria, viruses, parasites, etc., and the industrial loss due to them is considered to be enormous. ing. As one of the solutions to these fish disease problems, many drugs including antibiotics have been developed and used. However, the usage of antibiotics is limited due to resistance bacteria and safety issues. In addition, there are drugs that are concerned about environmental problems.
Furthermore, practical vaccines have been developed,
Development of new drugs is desired due to limited applicable fish species and period. Accordingly, an object of the present invention is to solve the above-mentioned problems and to provide a drug, a feed additive, or a composition thereof that is effective for disease prevention.

[0004]

Means for Solving the Problems In order to achieve the above-mentioned object, the present inventors have continued research on drugs for improving the disease resistance of fish, which are different from antibiotics, or feed compositions and feed additives. Was. As a result, a basic polypeptide was found to have an effective and effective prophylactic or therapeutic effect on various fish diseases. In addition, it has been found that a basic polypeptide has a similar effect. The present invention provides a fish immunomodulator containing a polypeptide containing a basic amino acid as an active ingredient. The present invention also provides a prophylactic / therapeutic agent for bacterial infections or viral infections in fish containing a basic polypeptide as an active ingredient. The present invention solves the above problems by these.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. The first invention of the present application is a fish immunomodulator containing the polypeptide as an active ingredient. That is,
As described above, the polypeptide exerts a nonspecific therapeutic and preventive effect on bacterial and viral diseases by activating the immune system of fish,
As a result, it is possible to reduce mortality due to fish diseases. Polylysine, a kind of basic polypeptide, is known for use as an antibacterial preparation (Japanese Patent Application Laid-Open No. 7-17 / 1995).
957). However, in the present invention, as shown in the examples, it has been found that after a certain period of time after administration, the effect is enhanced, or that it is also effective against viral diseases. The effect is not an antimicrobial effect due to the polypeptide contacting the pathogen. In addition, the immunomodulator as used herein includes a vaccine itself that is contained in a vaccine as an adjuvant or includes the vaccine itself. In the present invention, the immunomodulatory activity of the polypeptide enhances the protective effect of the vaccine against infectious diseases.

In the present invention, the term "basic amino acid" refers to all amino acids having a basic side chain.
Arginine and histidine are preferred. Further, a mixture of these may be used. Generally, those having 10 or less amino acid residues may be referred to as oligopeptides, but the polypeptide in the present invention refers to those having 2 or more amino acid residues. The immunomodulator in the present invention is intended to prevent or promote the infection and onset of infectious diseases temporarily or for a long period of time or to promote healing by stimulating or improving cell-mediated immunity and humoral immunity, Also included are vaccine adjuvants, such as vaccine adjuvants, which can improve the effect when used in combination with other drugs. The effective amount of the polypeptide in the immunomodulator is fish weight 1
The effective amount is 0.01 μg to 100 mg per g, and particularly preferably 1 μg to 10 mg per 1 g of fish body weight. If the molecular weight of the polypeptide is 200 or less, it becomes amino acid alone, and 125
If it is more than 00, it may act as an immunogen and may produce immunized antibodies. Therefore, the effect may not be exhibited in the administration after the first administration. Most preferably, the molecular weight is from 700 to 5,000. In the present invention, the method of administering the polypeptide to fish is not particularly limited. That is, administration to the fish body by injection method, such as abdominal cavity, muscle, etc., administration by immersing the fish body in an aqueous solution containing the polypeptide, oral administration by tablets or microcapsules, etc. Or oral administration by adding it as a feed composition. Further, in the case of oral administration, the effect is enhanced by mixing a digestive inhibitor such as a trypsin inhibitor.

The fish disease to which the present invention is applied includes, for example, yellowtail, flounder, tiger pufferfish, sweetfish, carp, salmonids, butterflyfish, wakin and the like, marine aquaculture, ornamental fish, or freshwater aquaculture, vibrio in ornamental fish. Disease (Vibrio anguillarum), tuberculosis (Pastourella piscicida), streptococcal disease (Enterococcus cerioricida), edwajellosis (Edwojera talda), rosacea (Eromonas salmonicida), cold water disease (Cytoferga cyclo) Various bacterial infectious diseases such as Fira) and bacterial kidney disease (Renibacterium salmoninum). Viruses such as iridovirosis, viral epidermal hyperplasia, rhabdovirosis, ascites, lymphocystis, viral neuronecrosis, infectious hematopoietic necrosis, infectious pancreatic necrosis, herpesvirosis, and erythrocyte inclusion body syndrome Sexually transmitted diseases and the like.

[0008]

Example 1 Example 1 is an example in which the effect of rainbow trout on vibriopathy was examined. Rainbow trout (average fish weight 37 g) reared at 18 ° C. were divided into 20 fish, and polylysine (average molecular weight: about 3000) dissolved in physiological saline was singly intraperitoneally adjusted to 0.025 mg / g (BW). Dose was administered once. The administration volume was 5 μl / g (BW), and saline was administered to the control. After breeding this at 18 ° C. for one week and three weeks, Vibrio anguillarum was administered to a saline-administered fish and a polylysine-administered fish at 2.6 × 10 ³ per g of fish body weight.
CFU was inoculated and infected. Thereafter, observation was performed for one week, and the survival rate was examined. The results obtained when rainbow trout were infected with Vibrio anguillarum are shown in the following table.

[0009]

[Table 1]

[0010]

Example 2 Example 2 is an example in which the effect of red sea bream on vibriopathy was examined. 25 red sea bream (average fish weight 2.49 g) bred at 24 ° C. were divided into three sections, and the polylysine (average molecular weight: about 3000) -administered group was treated with 0.025 mg / mg polylysine dissolved in physiological saline. A single dose was given intraperitoneally to give g (BW). In the glucan administration group, β-1,3 glucan, which is a conventional immunostimulating substance, was added in an amount of 0.025.
The dose was administered so as to be mg / g (BW). The administration liquid volume was 5 μl / g (BW) in all the groups, and physiological saline was administered to the control group. After breeding this at 24 ° C for 3 weeks, these fish were fed with Vibrio anguillarum at 1.0 × 10 ⁶ CFU / g of fish weight.
Inoculated and infected. Thereafter, observation was performed for one week, and the survival rate was examined. The results obtained when the red sea bream was infected with Vibrio anguillarum are shown in the following table.

[0011]

[Table 2]

[0012]

Example 3 Example 3 shows infectious hematopoietic necrosis of rainbow trout.
This is an example of examining the effect on IHN. At a water temperature of 14 ° C
75 bred rainbow trout (average fish weight 3.81g)
Virus 10 ^ThreeInoculate at a concentration of TCID50 / 5μl / g (B.W.), 14
They were bred at ℃. 48 hours after virus inoculation
Thereafter, the rainbow trout were divided into three sections each having 25 fish. In one ward
Is polylysine dissolved in saline (average molecular weight: about 300
0) to 0.025mg / g (B.W.)
Polyarginine dissolved in salt water (average molecular weight: about 400
0) was intraperitoneally administered to give 0.025 mg / g (B.W.).
Was. The dose volume was 5 μl / g (B.W.), and the remaining sections were
And saline was administered. Then reared at 14 ° C for 2 weeks
Then, the survival rate was examined. When rainbow trout was inoculated with IHNV
Table 3 shows the cumulative survival rates.

[0013]

[Table 3]

[0014]

Example 4 Example 4 is an example of examining the adjuvant effect of sweetfish on the vibriovirus vaccine. Ayu (average fish weight: 1.1 g) reared at 18 ° C. was divided into 5 groups, and 3 groups were divided into 3 groups. Polylysine (average molecular weight: about 3000) dissolved in physiological saline and polyarginine (average molecular weight: About 4000) and 0.025 mg / g (BW) of polyhistidine (average molecular weight: about 3,500).
Was administered intraperitoneally so that The same amount of physiological saline was administered to the other two groups. The next day, one of the groups to which physiological saline was administered and the other three groups to which the polypeptide was administered were immunized with a formalin-inactivated vaccine of Vibrio anguillarum by an immersion method. The vaccine is Vibrio
Anguillarum (type A) field isolate was cultured, and a cell obtained by inactivating all the cells with 0.3% formalin was prepared. The immersion concentration was 10 ⁸ CFU / ml and the immersion time was 2 minutes. The other group performed the same operation using physiological saline as a control.
These fish were bred at 18 ° C., and 21 days after the vaccine administration, all fish were inoculated intraperitoneally with Vibrio anguillarum at an inoculation amount of 10 ³ CFU / 5 μl / g, and an attack test was performed. Thereafter, observation was performed at 18 ° C. for 2 weeks, and the survival rate was examined. Table 4 shows the cumulative survival rate when inoculating Vibrio anguillarum to ayu after the vaccine treatment.

[0015]

[Table 4]

[0016]

As is clear from Examples 1 and 2, the group to which physiological saline was administered (control group) was infected with pathogenic bacteria under conditions showing a high mortality, but the group to which polylysine was administered (3). In all cases, the mortality rate was suppressed to 0%. This is considered to be due to the immunostimulatory effect of polylysine, but it was shown that the immunostimulatory effect was clearly higher than that of a conventional immunostimulant. Also,
These effects were confirmed in freshwater rainbow trout and marine red sea bream, and were not applied only to specific fish species. Also, as is clear from Example 3,
When administered to fish after infection, the mortality rate decreases, so it is thought that a therapeutic effect on viral diseases can be expected, indicating the possibility of a therapeutic agent for fish viral diseases that has never been seen before. there were. Furthermore, as is clear from Example 4, the effect of the vaccine can be expected as an adjuvant because the effect can be enhanced by using the vaccine together.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/00 C12P 21/00 C12P 21/00 A61K 37/02 F term (Reference) 2B005 GA01 GA02 MB07 2B150 AA08 AB10 DA47 DA48 DC23 4B064 CA01 DA04 4C084 AA02 BA04 BA08 BA18 CA62 NA14 ZB072 ZC652 4H045 AA10 AA30 BA10 CA11 CA15 EA22 FA20 FA73

Claims (3)

[Claims] An immunomodulator of fish containing a polypeptide containing a basic amino acid as an active ingredient, which is produced by synthesis and / or fermentation of a microorganism. 2. The fish immunomodulator according to claim 1, wherein the basic amino acid is at least one or more of lysine, arginine and histidine. (3) the polypeptide has a molecular weight of 200 to 125;
The immunomodulator for fish according to claim 1, wherein the number is 00.