JP2021004195A - Agent for preventing and treating fish disease - Google Patents

Abstract

To provide an agent for preventing and treating fish disease that has low concentration and has small environmental load, and also has excellent safety in operation.SOLUTION: The agent is used for preventing and treating the infection of fish disease in a fish, and contains salt of monoalkyl sulfate or benzalkonium chloride.SELECTED DRAWING: Figure 1

Description

The present invention relates to a preventive and therapeutic agent for fish diseases that are effective in preventing or treating fish from diseases such as bacterial infections.

In aquaculture farms and aquariums, the breeding density is high, and fish are easily infected with fish diseases, so prevention and treatment are important.
Currently, vaccines for disease prevention are used, and various antibiotics and the like are used for treatment, but operations such as administration are complicated, and the development of resistant bacteria is also a problem.

Patent Document 1 discloses an infection preventive / therapeutic agent for fish containing an algae extract and lactic acid cocci or polyphenols.
However, the prophylactic / therapeutic agents disclosed in the same gazette have a relatively high concentration and use the oral method and the drug bath method, which makes the administration operation difficult.

On the other hand, the present inventors have investigated measures against fish diseases using the biological defense mechanism originally possessed by fish.
For example, as disclosed in Non-Patent Document 1 published by the present inventors as a research result, L-amino acid oxidase (LAO) was identified as an antibacterial protein from the body surface mucus of fish, and L-amino acid oxidase was also identified in serum. Has been identified.
It has been suggested that LAO in Hong Kong grouper serum is about 450 kDa and is a hexamer of about 70 kDa subunits.
Since this LAO is presumed to exhibit antibacterial activity via hydrogen peroxide, it was examined that if a substance capable of stimulating and enhancing this LAO activity could be obtained, it would be effective as a countermeasure against fish diseases. It came to.

Japanese Unexamined Patent Publication No. 2004-307346

Yoichiro Kitani, Yuto Kosaka, "L-amino-acid oxidase contained in serum of Hong Kong grouper Epinephelus akaara" 2018 Spring Meeting of the Japanese Society of Fisheries Science, Tokyokaiyo University, Tokyo (2018.3.26-30)

An object of the present invention is to provide a preventive and therapeutic agent for fish diseases having a low concentration, a low environmental load, and excellent operational safety.

The fish disease prophylaxis and therapeutic agent according to the present invention contains a salt of monoalkyl sulfate or benzalkonium chloride for use in the prevention or treatment of fish disease infection in fish.
Here, the salt of the monoalkyl sulfate ester is preferably sodium lauryl sulfate or ammonium lauryl sulfate.

The salt of monoalkyl sulfate ester is a surfactant belonging to anionic properties.
Sodium lauryl sulfate, also called sodium dodecyl sulfate (SDS), has relatively excellent storage stability and is highly safe for humans.
Benzalkonium chloride is a type of cationic surfactant.

The action of the preventive and therapeutic agent for fish disease according to the present invention will be described below.
It has L-amino acid oxidase (LAO) as a biological defense substance in the epidermal cells and serum of fish, and this LAO acts on the substrate L-amino acid to produce hydrogen peroxide as shown in FIG. 8, and this peroxidation It is presumed that hydrogen has an antibacterial effect.
In FIG. 1, "FAD" is an abbreviation for flavin adenine dinucleotide and is a cofactor for redox reaction.
It is presumed that the preventive and therapeutic agent for fish diseases according to the present invention stimulates the activity of LAO and enhances the antibacterial activity.
Although the details will be described later, the drug according to the present invention has an antibacterial activity effect at an extremely thin concentration of about 0.5 to 5 mM.

The agent for preventing and treating a bacterial infection in fish according to the present invention has a low environmental load because the concentration used is low.
Further, since these are also anionic or cationic surfactants, they can be stored for a long period of time, and there is no problem in safety to humans during handling.
Further, when used in a water tank, a cleaning action in the water tank can be expected.

The results of antibacterial activity tests against the fish disease bacteria Vibrio anguillarum and Aeromonas salmonicida are shown. The test result of LAO activity with respect to SDS concentration is shown. The test result of LAO activity with respect to the NaCl concentration is shown. The test result 2 of the LAO activity with respect to the SDS concentration is shown. The test result 3 of the LAO activity with respect to the SDS concentration is shown. The test result of LAO activity with respect to the concentration of ammonium lauryl sulfate is shown. The test result of LAO activity with respect to the concentration of benzalkonium chloride is shown. It is explanatory drawing of the reaction system which LAO shows antibacterial activity.

The antibacterial activity of the preventive and therapeutic agent for fish disease according to the present invention has been tested and evaluated, and will be described below.
Hong Kong grouper serum contains L-amino acid oxidase (hereinafter, simply referred to as LAO if necessary), and this LAO acts on the L-amino acid substrate to exhibit antibacterial activity via hydrogen peroxide.
Therefore, the following test was conducted.
Mueller Hinton the medium (NaCl-free) to prepare a 2-fold serial dilution series of Kijihata serum were added 1 × 10 ⁵ fish diseases bacteria were adjusted to Vibrio anguillarum or Aeromonas salmonicida in the same medium.
Based on this, a group in which sodium dodecyl sulfate (SDS) was added to a final concentration of 0.04% in the medium, a group in which catalase was added to a final concentration of 20 units / mL, and a catalase having the same concentration. The group to which the catalase was added was cultured at the same time.
Catalase was added for the purpose of exhausting hydrogen peroxide produced by L-amino acid oxidase contained in Hong Kong grouper serum.
After culturing these at 20 ° C. for 16 hours, if the growth of bacteria was suppressed, the medium did not become turbid. Therefore, the maximum dilution ratio at which turbidity of the medium was not visually observed was evaluated as the value of antibacterial activity.
As a result, as shown in FIG. 1, the antibacterial activity in the SDS-added group was 4096 times or more against Vibrio anguillarum, but the antibacterial activity in the other groups was not detected.
In addition, the antibacterial activity of the SDS-added group was 2048 times higher than that of Aeromonas salmonicida, and no antibacterial activity was observed in the group to which SDS and catalase were added at the same time.
The reagent-free group and the catalase-only group showed 32-fold antibacterial activity, which was expected to be due to other antibacterial substances inactivated by SDS.
From the above results, it was confirmed that the antibacterial activity of Hong Kong grouper serum was enhanced by SDS and that the activity was decreased by the addition of catalase.
That is, it was confirmed that the serum alone or SDS alone does not show antibacterial activity, but the presence of SDS in the serum shows a strong antibacterial action via hydrogen peroxide.

Next, the LAO activity was evaluated using various reagents, which will be described below.

Since the addition of NaCl has been observed to enhance LAO activity by the present inventors, they were compared and evaluated while changing the concentrations of sodium dodecyl sulfate (SDS) and NaCl.
<Measurement method of LAO activity>
To 25 microliters of serum diluted with 1 M Tris-HCl buffer (pH 7.5), 25 microliters of an aqueous sodium chloride solution or an aqueous sodium dodecyl sulfate solution was added to a final concentration of 0-2M or 0-1.4 mM.
Next, 50 microliters of 1M Tris-HCl buffer (pH 7.5) containing 0.25 mg / mL of ortho-phenylenediamine and 0.01 unit / mL of horseradish peroxidase were added and reacted at 37 ° C. for 150 minutes.
After the reaction, 100 microliters of 1M sulfuric acid was added to stop the reaction, and the absorbance at 492 nm was measured with a spectrophotometer.
To evaluate the activity, hydrogen peroxide serially diluted to 0-0.5 mM was measured as a sample, and the activity (unit) was estimated from the amount of hydrogen peroxide produced and the reaction time obtained from the calibration curve.

FIG. 2 shows the LAO activity for changes in SDS concentration, and FIG. 3 shows the LAO activity for changes in NaCl concentration.
Comparing FIGS. 2 and 3, the LAO activity does not become high unless the concentration of NaCl is 0.5 M or higher, whereas the SDS has a low concentration of 0.5 mM to 1.4 mM, that is, the concentration of NaCl. LAO showed high activity at a low concentration of about 1/1000 or less.

4 and 5 show the LAO activity when the SDS concentration is high.
FIG. 4 is a graph showing changes in LAO activity at a low concentration of SDS of 1.4 mM or less, and FIG. 5 is a graph showing changes to the high concentration side up to 140 mM.
As a result, the SDS concentration should be in a thin concentration range of about 0.5 mM to 5 mM, and when it exceeds 10 mM, the LAO activity is conversely decreased.

FIG. 6 shows the measurement results of the change in LAO activity with respect to the change in the concentration of ammonium lauryl sulfate.
From this result, ammonium larryl sulfate also showed the same LAO activity as SDS.
FIG. 7 shows the measurement results of the change in LAO activity with respect to the change in the concentration of benzalkonium chloride.
Benzalkonium chloride, which is a type of cationic surfactant, showed LAO activity at a low concentration of 0.01 to 0.04%.

Next, LAO activity was evaluated using various reagents.
As a result, LAO activity was not observed with the following reagents.
(1) Since SDS and ammonium lauryl sulfate are also used as anionic surfactants, the trade name Triton X-100 {29- [4- (1,1)) belongs to the nonionic surfactants. , 3,3-Tetramethylbutyl) Phenoxy] -3,6,9,12,15,18,21,24,27-Nonaoxanonanacosan-1-ol}, and trade name Tween20 (polyoxyethylene sorbitanmono) Laurat) was evaluated in the concentration range of 0 to 2%, but the LAO activating effect was weak.
(2) Cetylpyridinium chloride, which belongs to a cationic surfactant, was evaluated, but it did not show LAO activity.
(3) In addition, urea and tetramethylammonium chloride were evaluated, but they did not have a LAO activating effect.

From the above, it was clarified that it is effective to use a salt of monoalkyl sulfate or benzalkonium chloride to stimulate LAO activity and enhance antibacterial activity.
The above monoalkyl has 8 or more carbon atoms, preferably 12 or more carbon atoms.

Claims (4)

A preventive and therapeutic agent for fish diseases, which comprises a salt of monoalkyl sulfate or benzalkonium chloride for use in the prevention or treatment of fish disease infection in fish. The preventive and therapeutic agent for fish diseases according to claim 1, wherein the salt of the monoalkyl sulfate ester is sodium lauryl sulfate or ammonium lauryl sulfate. The preventive and therapeutic agent for fish diseases according to claim 1 or 2, wherein antibacterial activity can be obtained by activating L-amino acid oxidase possessed by fish. A method for preventing and treating fish disease, which comprises adding the preventive and therapeutic agent for fish disease according to any one of claims 1 to 3 to the breeding water of fish so as to have a concentration of 0.5 to 5 mM. ..