Agent for preventing and treating scuticociliatosis in marine fish

Abstract

To provide an agent that is capable of effectively preventing and treating scuticociliatosis in marine fish.SOLUTION: Provided is an agent for preventing and treating scuticociliatosis in marine fish, containing an ionophore antibiotic as an active ingredient.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a preventive and therapeutic agent for scuticosis caused by ciliate protozoa parasitic on marine fish. [Background technology]

[0002] Scuticosis is a disease caused by facultative parasitism of Scuticida ciliata, classified in the class Oligomenotometa of the phylum Ciliophora. There are multiple species of parasites that cause it, and many of them have not yet been identified. Among them, Miamiensis avidus has been isolated in Japan and Korea, and is the main cause of scuticosis in flounder, causing significant economic damage to the flounder farming industry (Non-Patent Document 1). Since the Scutica ciliate multiplies by feeding on organic matter even in an environment without a host, it is easy to grow in an aquarium with a lot of organic matter such as excrement and leftover food. When it parasitizes a flounder, it invades the fish's brain and internal organs through the fish's surface and gills, feeding on the host's tissue and multiplying, causing anemia, impaired osmotic adjustment, and impaired respiratory and excretory functions, resulting in death (Non-Patent Document 2).

[0003] For example, in flounder farms, infection is common among young fish, with the incidence peaking at around 21°C, and many fish become unsellable due to lesions on the surface of the body. In red sea bream, the disease occurs in both young and adult fish, and adult fish in particular often develop lesions on the surface of the body, making them unsellable. The disease also frequently occurs in tiger puffer farms.

[0004] It has been reported that immersion therapy using formalin or hydrogen peroxide is effective when parasites infest the body surface, but this has not been approved for aquaculture. Vaccines have also been developed, but they are effective only for limited species, making commercialization difficult. When rearing in land-based aquariums, it is said that outbreaks can be suppressed by installing a copper ion generator (copper ion concentration 0.2-0.3 ppm), but the suppression rate is unknown. Furthermore, since control is more difficult when parasites infest the body, there is a need to develop a preventive treatment that is highly effective and can be commercially marketed. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Detection and identification of Miamiensis avidus causing scuticiliatosis by PCR. Fish Pathology, 45(3), 130-132.(2010) [Non-Patent Document 2] Pathological study on the scuticiliatosis affecting farmed Japanese flounder (Paralichthys olivaceus) in Japan. Journal of Veterinary Medical Science,(2010) 1004230231-1004230231. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a drug capable of effectively preventing and treating scuticosis in marine fish. [Means for solving the problem]

[0007] The inventors therefore conducted various studies to find an ingredient that is effective against marine fish scuticosis, and discovered that ionophore antibiotics have an insecticidal effect against scuticosis ciliates, and that at concentrations that do not show toxic effects on marine fish such as flounder, they have a preventive and therapeutic effect against scuticosis when administered orally, thus completing the present invention.

[0008] That is, the present invention provides the following inventions [1] to [9]. [1] A preventive and therapeutic agent for marine fish scutellar disease, the active ingredient of which is an ionophore antibiotic. [2] The agent for preventing or treating marine fish scuticosis according to [1], wherein the ionophore antibiotic is one or more selected from the group consisting of salinomycin, lasalocid, monensin, valinomycin, ionomycin, calcimycin, and salts thereof. [3] The agent for preventing and treating marine fish scutellar disease according to [1] or [2], wherein the administration route is oral administration. [4] The agent for preventing or treating marine fish scuticosis according to any one of [1] to [3], wherein the marine fish is a marine fish selected from the order Pleuronectiformes, Perciformes and Tetraodontiformes. [5] A method for preventing and treating marine fish scuticosis, which comprises administering an ionophore antibiotic to marine fish. [6] The preventive and therapeutic method described in [5], which comprises orally administering an ionophore antibiotic. [7] The preventive and therapeutic method according to [5] or [6], in which an ionophore antibiotic is administered by mixing it with feed. [8] The preventive and therapeutic method according to any one of [5] to [7], wherein the ionophore antibiotic is one or more selected from salinomycin, lasalocid, monensin, valinomycin, ionomycin, calcimycin, and salts thereof. [9] The preventive and therapeutic method according to any one of [5] to [8], wherein the marine fish is a marine fish selected from the order Pleuronectiformes, Perciformes and Tetraodontiformes. Effect of the Invention

[0009] According to the present invention, the present invention has an insecticidal effect and a growth inhibitory effect against the Scutigera ciliata that parasitizes the bodies of marine fish, and can significantly reduce the number of deaths caused by Scutigera in marine fish. [Brief description of the drawings]

[0010] [Figure 1] In vitro insecticidal effect of ionophore antibiotics on Scutica ciliata (96 hours). This figure shows the average of experimental results using three wells per concentration in a 96-well plate. Blank: experimental group without drug; DMSO: dimethyl sulfoxide; A: salinomycin sodium salt; B: lasalocid; C: monensin sodium salt. [Diagram 2] The therapeutic effect of continuous administration of salinomycin sodium salt (A) is shown. This figure shows the survival rate of six flounders in each tank for two weeks. Con: infected and untreated group; Uninfected: uninfected and untreated group; A: salinomycin sodium salt (A)-treated group. [Diagram 3] The therapeutic effect of continuous administration of lasalocid (B) is shown. This figure shows the survival rate of six flounders in each tank over two weeks. Con: infected and untreated; Uninfected: uninfected and untreated; B: lasalocid (B)-treated. [Figure 4] The therapeutic effect of continuous administration of monensin sodium salt (C) is shown. This figure shows the survival rate of six flounders in each tank for two weeks. Con: infected and untreated; Uninfected: uninfected and untreated; C: monensin sodium salt (C)-treated group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] Terms used in this specification are used in the same manner as commonly used in the art unless otherwise specified.

[0012] As used herein, "scuticociliatosis" refers to an infection with Scuticociliatida ciliates, as described in "Selected Fish Disease Names (Revised 2023)" in Fish Pathology, 58(1), 24-36, 2023.3. Fish that are known to be infected with scutellar disease include flounder, red sea bream, and tiger pufferfish, but infections have also been reported in sharks and crustaceans. As mentioned above, the damage caused by this disease is serious in fish farms. Therefore, the target fish is preferably a marine fish selected from the order Pleuronectiformes, Perciformes, and Tetraodontiformes, and more preferably a marine fish selected from the order flounder, red sea bream, and tiger pufferfish. Miamiensis avidus is a well-known ciliate that causes scuticosis.

[0013] As mentioned above, Scutica ciliates multiply by feeding on organic matter even in an environment without a host, so they tend to grow in aquariums with a lot of organic matter such as excrement and leftover food. When parasitizing flounder, they invade the fish's brain and internal organs through the fish's body surface and gills, feeding on the host's tissue and multiplying, causing anemia, impaired osmotic pressure regulation, and reduced respiratory and excretory functions, resulting in death (Non-Patent Document 2). These characteristics of Scutica ciliates make them different from other parasites.

[0014] In the present specification, the term "ionophore antibiotic" is a general term for lipid-soluble molecules capable of increasing the permeability of specific ions in biological membranes, and is an antibiotic mainly produced by bacteria. Examples of ionophore antibiotics include one or more selected from salinomycin, lasalocid, monensin, valinomycin, ionomycin, calcimycin, and salts thereof. Among these, one or more selected from salinomycin, lasalocid, monensin, and salts thereof are preferred from the viewpoint of efficacy and safety, and one or more selected from salinomycin sodium salt, lasalocid, and monensin sodium salt are more preferred.

[0015] As used herein, the term "preventive and therapeutic agent" refers to a drug that has the effect of preventing the onset of schoolworm disease by its insecticidal effect on schoolworm ciliates, inhibiting the growth of schoolworm ciliates in fish, and inhibiting surface injuries and death of fish caused by schoolworm disease.

[0016] One aspect of the present invention is a preventive and therapeutic agent for marine fish scuticosis, which contains an ionophore antibiotic as an active ingredient. Another aspect of the present invention is a method for preventing and treating marine fish scuticosis, which comprises administering an ionophore antibiotic to marine fish.

[0017] The active ingredient of the preventive and therapeutic agent for marine fish scuticosis of the present invention is an ionophore antibiotic. The ionophore antibiotic may be one or more selected from salinomycin, lasalocid, monensin, valinomycin, ionomycin, calcimycin, and salts thereof. Among them, from the viewpoint of efficacy and safety, one or more selected from salinomycin, lasalocid, monensin, and salts thereof are preferred. Here, the salt is preferably an alkali metal salt such as sodium potassium. Furthermore, one or more selected from salinomycin sodium salt, lasalocid, and monensin sodium salt are more preferred.

[0018] As shown in the Examples below, ionophore antibiotics have an insecticidal effect against the ciliate scuticle (e.g., Miamiensis avidus) and have the effect of significantly suppressing death due to scuticosis when orally administered at concentrations that do not show toxic effects on marine fish such as flounder. Therefore, administration of ionophore antibiotics can prevent and treat scuticosis in marine fish.

[0019] The means of administration of the ionophore antibiotic is not particularly limited, but oral administration is preferred because it is simple and safe. For oral administration of the ionophore antibiotic, it is preferred to mix it with a feed (feed). The concentration of the ionophore antibiotic in the feed or feed additive is preferably 0.01% by mass or more, more preferably 0.01 to 100% by mass, and particularly preferably 0.02 to 100% by mass. The dosage of the ionophore antibiotic per kg of body weight of marine fish is preferably 0.1 to 50 mg, more preferably 0.25 to 25 mg per day.

[0020] The preventive and therapeutic agent for scuticosis of the present invention may be administered separately from the feed, without being mixed with the feed, in the form of powder, granules, pellets, etc.

[0021] When the compound is used in the above-mentioned feed, feed additive or other forms, it may be mixed and diluted with, for example, calcium carbonate, calcium phosphate, calcium sulfate, wheat flour, starch, dextrin, feed yeast, or feed raw materials such as cereals, algae, and meal, in addition to the ionophore antibiotic, or may be added to a premix of vitamins, minerals, etc., or to a fish feed containing such a premix. In the case of live feed, additives such as sodium alginate and guar gum may be used at the same time.

[0022] The marine fish that can be targeted by the Scutigeria ciliate of the present invention are not limited to marine fish that can be infested with Scutigeria ciliate, but marine fish that are actually affected by Scutigeria are preferred, such as marine fish selected from the orders Pleuronectiformes, Perciformes and Tetraodontiformes, and marine fish selected from flounder, red sea bream and tiger pufferfish are more preferred.

[0023] The preventive and therapeutic agent for scuticosis of the present invention can be administered before the onset of parasitism by scuticosis ciliate, or at the time when symptoms of scuticosis are discovered. EXAMPLES

[0024] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to these examples.

[0025] Materials and Methods (1) Obtaining and passage of Scutellaria ciliata The ciliate Miamiensis avidus was used in the experiment by subculturing and maintaining the Nakajima strain (Parasitology international, 67(2), 196-202. (2018)) in a culture flask provided by Professor Kitamura of the Faculty of Science, Ehime University. Usually, Scutigera ciliata were subcultured once a month. The medium for Scutigera ciliata was prepared by dissolving 2% proteose peptone and 1% yeast extract in filtered seawater (filtered through a 5 μm filter) diluted 3 times with distilled water, referring to Fish Pathology, 28(3), 131-134. (1993), autoclaving it, and adding 10% inactivated FBS and 1% antibiotic solution (10000 IU / ml penicillin G potassium, 10000 μg / ml streptomycin sulfate, 0.85% NaCl). When subcultured, 100 μL of the suspension of the worms was added to a new culture flask containing 10 ml of the above medium, gently shaking to disperse the worms evenly, and incubating at 23°C.

[0026] (2) Verification of the growth inhibitory effect of ionophore antibiotics The Scutigera ciliata grown according to the above method were used in the experiment. The number of worms in the Scutigera ciliata suspension was counted using a hemocytometer. 100 μL of the Scutigera ciliata suspension and 10% formalin (diluted with filtered seawater) were mixed thoroughly in a 96-well plate and fixed, and the worm suspension was then poured into the sample injection side of the hemocytometer. The Birker-Türk hemocytometer was placed on the microscope stage and counted into nine counting chambers (10 -4 cm 3 The number of worms in each of the samples was counted sequentially using a counter and totaled, and the total number of cells was taken as N to calculate the worm density using the following formula. Note that counting was performed three times, and the average value calculated was used. Insect density=(N / 9)×10 4 pieces / ml Three types of ionophore antibiotics were used: salinomycin sodium salt (A), lasalocid (B), and monensin sodium salt (C). Appropriate amounts of the above drugs were weighed and dissolved in DMSO (dimethyl sulfoxide) to a concentration of 15 ppm. DMSO was then used to serially dilute the drugs 10-fold, so that the final concentrations of the drugs used in the experiment were 15 ppm, 1.5 ppm, 0.15 ppm, and 0.015 ppm. In addition, a negative control using DMSO and a blank test using only the medium without DMSO were also prepared. 990 μL of the medium containing the drug was placed in each well of a 24-well plate, and 10 μL of the suspension of Scutella scutellaris (10 worms) was added. 3 The cells were seeded at 100 cells / well, mixed thoroughly with a pipette, and cultured in an incubator set at 23° C. Three microwell plates were prepared with three wells for each experimental group. After the microplates were incubated in an incubator at 23°C for 24, 48, and 96 hours, the worm density in each well was counted using a Burker-Türk hemocytometer as described above. The recorded number of surviving Scutica ciliates in each experimental group was compared with that in the control group.

[0027] (3) Effects of ionophore antibiotics on flounder (3-1) Acquiring fish and preliminary breeding Approximately 300 flounder fry (average weight 1.7g) were purchased from a fish farmer and raised in an indoor aquarium at 20°C, fed commercially available pellets twice a day, morning and evening. Before the experiment began, 80 fish were randomly taken from the aquarium, anesthetized with 2-phenoxyethanol, and their total length and weight were measured. After waking up from anesthesia, the fish were divided into groups of eight and housed in ten 60 L aquariums at 23°C. Each aquarium was individually aerated using an air stone, and a top filter (Gex) was also installed. The lights in the breeding room were set to be on between 15:00 and 3:00.

[0028] (3-2) Feed preparation To 50 g of commercially available feed (Otohime EP0), 5 ml of 99.5% ethanol in which different amounts of ionophore antibiotics had been thoroughly dissolved was added, and the mixture was quickly shaken and then left to stand in a refrigerator for 4 to 5 hours to allow the ethanol to evaporate. The drugs selected were salinomycin sodium salt (A), lasalocid (B), and monensin sodium salt (C), which showed insecticidal effects against Scutica ciliata in the aforementioned experiment. The amount of drug added was adjusted to 0, 0.2, 0.5, 1.0, and 2.0 g / kg feed weight so that the dose when the feed was fed to the fish at 5% of their body weight per day was 0 (control group), 10, 25, 50, and 100 mg / kgBW / day. After the ethanol had completely evaporated, 0.2% of the feed was mixed with a spreading agent Apifac-W (Kokin Chemical) and 10% water, and the mixture was shaken well and stored in a refrigerator. The fish were taken out of the refrigerator one hour before administration in the experiment and allowed to return to room temperature before being fed.

[0029] (3-3) Toxicity experiment on flounder The experiment began three days after the fish were transferred to the breeding tanks. Water was added to the feed prepared using the procedure described above, and the fish were fed 5.5% of their body weight in each tank every day thereafter. During daily feeding, the water temperature, water quality (NH3, NO2), pH, and salinity of each breeding tank were checked, and residual food and dirt that had accumulated at the bottom of the tank were removed using a cleaning pump, and an appropriate amount of water was added. If any fish were found to have died during breeding, the dead fish were quickly removed, and the date of death and the number of fish in each tank were recorded, and the control group and each treatment group were compared. On the 14th day of the experiment, all surviving flounder fish were removed from each tank, anesthetized with 2-phenoxyethanol, and the total length and weight of each individual fish were measured. In addition, the degree of obesity of the fish at the end of the experiment was calculated as "obesity degree = weight / (total length x total length x total length)" and compared with the control group and each treatment group.

[0030] (3-4) Therapeutic effect of continuous administration of ionophore antibiotics The preparation of the Scutigera ciliata suspension was based on Journal of fish diseases, 41(3), 451-462. (2018). In other words, the suspension in the culture flask was transferred to a 15 ml tube and centrifuged at 980 × g for 25 minutes to remove the supernatant, after which 5 ml of PBS (-) was added and gently shaken to disperse the worms evenly. The number of worms in the Scutigera ciliata suspension was then counted using the method described above using a Birker-Türk hemacytometer and used for the infection experiment. The infection challenge with Scutica ciliata was carried out according to the method described in Aquaculture, 416, 192-195. (2013). In other words, the above Scutica ciliata suspension was added to a 60-L tank containing 50 L of seawater with a salinity of 30‰, and the number of worms in the tank was increased to 5 × 10 7 The tank was set at 100 / 50 L. 42 flounders (average weight: 6.1 g) were placed in this tank and challenged with infection for 3 days at 23°C with aeration and no food. The flounders were then randomly divided into 7 groups of 6 flounders each and housed in 7 tanks. Six uninfected flounders were also housed in a 60 L tank under the same conditions. The breeding tanks were individually aerated using air stones, and an upper filter (Gex) was also installed. The lights in the breeding room were set to be on between 15:00 and 3:00. The day the fish were transferred to the 60L breeding tanks was the first day of the experiment. The above-mentioned sodium salinomycin (A), lasalocid (B), and sodium monensin (C) were added to the feed prepared, and the fish were fed 5.5% of their body weight in each of the six tanks every day. The other two tanks were treated with drug-free feed and uninfected, and were fed with drug-free feed. During daily feeding, the water temperature, water quality (NH3, NO2), pH, and salinity of each tank were checked, and residual food and dirt accumulated at the bottom of the tank were removed using a cleaning pump, and an appropriate amount of water was added. If any fish were found to have died during breeding, the dead fish were quickly removed, and the date of death and the number of fish in each tank were recorded, and the control and each treatment group were compared.

[0031] <Result> (1) Verification of the insecticidal effect of ionophore antibiotics In the blank test and negative control areas, the population of Scutica ciliata increased to 10 3 Approximately 9 × 10 4 The population continued to grow even after 96 hours, with the final population number reaching approximately 14 × 10 4 This resulted in 1 (Figure 1). In the experimental areas containing salinomycin sodium salt (A), lasalocid (B), and monensin sodium salt (C) at a concentration of 15 ppm, the insects were completely killed within 48 hours, and in the experimental areas containing these drugs at a concentration of 1.5 ppm, the insect population was reduced to 2 x 10 3 In the 0.15 ppm experimental group of all drugs, the population of Scutella scutella was 3.5 × 10 4 Individuals ~ 5.5 x 10 4 At a concentration of 0.015 ppm, the insect population increased by 5.4 × 10 after 48 hours. 4 Individuals ~ 8.3 x 10 4 The insects became individuals, and their reproduction was significantly reduced compared to the control area. Taking these results together, salinomycin sodium salt (A), lasalocid (B) and monensin sodium salt (C) showed clear insecticidal effects. Within 24 hours after the start of the experiment, the three drugs significantly inhibited the proliferation of Scutigera ciliata even at low concentrations of 0.015 ppm and 0.15 ppm, but did not completely kill the insects, so the insect density increased significantly over the next 48 hours. This may be due to the gradual weakening of the drug's effectiveness at 23°C, and it was thought that administration at least once every two days would be necessary to treat Scutigera in flounder.

[0032] (2) Effects of ionophore antibiotics on flounder Three to five days after the start of the experiment, all the flounders died in the groups administered 25, 50, and 100 mg / kgBW / day of all three drugs (Table 1). Approximately two weeks after the start of the experiment, all the experimental fish died in the groups administered 100 mg / kgBW / day of salinomycin sodium salt (A) and monensin sodium salt (C). Furthermore, the survival rate of the fish in the groups administered 50 mg / kgBW / day of salinomycin sodium salt (A), lasalocid (B), and monensin sodium salt (C) was 50%, indicating clear toxicity to flounders. In contrast, no deaths were observed in the 10 mg / kg BW / day treatment group, as in the control group. As a result, deaths thought to be drug-related were observed at doses of 25 mg / kg BW / day or more for salinomycin sodium (A), lasalocid (B), and monensin sodium (C). In addition, when the 10 mg / kg BW / day group was compared with the untreated group by Steel's multiple comparison, all drugs showed significantly higher obesity levels (Table 2). Taking these results together, salinomycin sodium (A), lasalocid (B) and monensin sodium (C) were considered to be safe at a dose of 10 mg / kg BW / day.

[0033] [Table 1]

[0034] [Table 2]

[0035] (3) Therapeutic effect of continuous administration of ionophore antibiotics In the non-treated group, flounder died 4 days after the start of the experiment, and all experimental fish had died 9 days later (Figs. 2-4). In contrast, in the lasalocid (B) and monensin sodium (C) treated groups, 4-5 fish survived at the end of the experiment regardless of the dose. In the salinomycin sodium (A) treated group at 10 mg / kg BW / day, 4 fish survived at the end of the experiment (Figs. 2-4). When dead and dying fish were observed under a microscope during the experiment, Scutica ciliata were observed on their body surfaces. Taking these results together, it was found that salinomycin sodium salt (A), lasalocid (B) and monensin sodium salt (C) have therapeutic effects against scuticosis in flounder.

Claims

1. A preventive and therapeutic agent for marine fish scuticosis, containing an ionophore antibiotic as its active ingredient.

2. 2. The agent for preventing and treating marine fish scuticosis according to claim 1, wherein the ionophore antibiotic is one or more selected from the group consisting of salinomycin, lasalocid, monensin, valinomycin, ionomycin, calcimycin and salts thereof.

3. 2. The agent for preventing and treating marine fish scuticosis according to claim 1, which is administered orally.

4. 2. The agent for preventing and treating marine fish scuticosis according to claim 1, wherein the marine fish is a marine fish selected from the order Pleuronectiformes, Perciformes and Tetraodontiformes.

5. A method for preventing and treating marine fish scuticosis, which comprises administering an ionophore antibiotic to marine fish.

6. The preventive and therapeutic method according to claim 5, wherein the ionophore antibiotic is orally administered.

7. The method of claim 6, wherein the ionophore antibiotic is administered by mixing with feed.

8. 6. The method for preventing and treating according to claim 5, wherein the ionophore antibiotic is one or more selected from the group consisting of salinomycin, lasalocid, monensin, valinomycin, ionomycin, calcimycin and salts thereof.

9. 6. The method for preventing and treating according to claim 5, wherein the marine fish is a marine fish selected from the order Pleuronectiformes, Perciformes and Tetraodontiformes.

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