JP7654122B2 - Methods for preventing muscle melting in salmonid fish - Google Patents

Description

The present invention relates to a method for preventing muscle melting that occurs in salmonid fish. It also relates to an agent and a method for exterminating microsporidia that parasitize the muscles of salmonid fish by oral administration.

In recent years, marine aquaculture of salmonid fish, known as local salmon, has become popular in many prefectures in Japan. Many of the salmonid fish farmed in Japan are also served as sashimi.

Benzimidazole compounds are known as antiparasitic drugs, and in Japan, mebendazole is approved as a treatment for pinworm infection, albendazole as a treatment for hydatid disease, flubendazole as a veterinary drug for strongyle and roundworm nematodes, and febantel and fenbendazole as veterinary drugs for nematodes and cestodes. For aquatic use, febantel is approved for use on pufferfish.

There is a report that tested the effectiveness of albendazole against Loma salmonae, a microsporidia that parasitizes the gills of rainbow trout (Non-Patent Document 1). There is a report that tested the effectiveness of albendazole, mebendazole, and fenbendazole against Glugea anomala, a microsporidia that parasitizes three-spined sticklebacks (Order Sticklebacks, Family Sticklebacks) (Non-Patent Document 2). There are reports that benzimidazole drugs are effective against microsporidia that parasitize fish and shellfish of the order Perciformes or Pleuronectiformes (Patent Documents 1 and 2). There is also a report on the use of albendazole or fenbantel to treat beko disease and myxosporidiasis (Patent Document 3).

JP 2017-186306 A WO 2018/062246 A1 WO 2018/062246 A1

D.J. Speare, et al., J.Comp. Path. 121, 241-248, 1999. “A Preliminary Investigation of Alternatives to Fumagillin for the Treatment of Loma salmonae Infection in Rainbow Trout”. Schmahl G., Benini J., Parasitol Res., 84(1), 41-49, 1998. “Treatment of fish parasites. 11. Effects of different benzimidazole derivatives (albendazole, mebendazole, fenbendazole) on Glugea anomala, Monies, 1887 (Microsporidia): ltrastructural aspects and efficacy studies.”

When melting occurs in the muscle of a fish used for sashimi, that part cannot be used as sashimi, and its commercial value is greatly diminished. Furthermore, collapse of the muscle due to melting leads to a poor appearance even when the fish is grilled, lowering its commercial value. An object of the present invention is to identify the cause of melting that occurs in the muscle of salmoniformes and to provide a method for solving the problem. Another object of the present invention is to provide a method for preventing melting that occurs in the muscle of salmoniformes and improving the appearance of the muscle of salmoniformes.

The inventors noticed that the muscles under the skin of salmon were melting. The smallest pits caused by the melting symptoms can be seen with the naked eye, with a diameter of about 2 to 3 mm, and the larger ones can reach a diameter of about 15 mm or more, and are accompanied by bleeding. They cannot be seen from above the skin unless you look carefully. Small ones are difficult to see from above the skin, but the larger ones can be seen as spots even when seen from above the skin. In order to identify the cause of the melting of the muscles of these salmon, they were examined, and numerous microsporidian spores (Figure 4) were observed in the melted areas. Cysts were also observed (Figures 1 and 3), and microsporidian spores were observed within the cysts. From these results, it was thought that the disease was caused by microsporidian parasites in the muscles. Muscle melting caused by microsporidian parasites in marine farmed salmon has not been reported before, so this is a new discovery.

In search of an orally administered drug that is effective in eliminating microsporidia that parasitize the muscles of salmonid fish, we searched through various existing animal antiparasitic drugs and substances derived from natural products. As a result, we found that albendazole, which is sold as an animal antiparasitic drug, can be orally administered without reducing the appetite of salmon and has an anthelmintic effect, leading to the completion of this invention.

The present invention includes the following methods (A1) to (A11) for preventing muscle lysis caused by microsporidia that occur in salmoniform fish, and (B1) to (B12) for controlling microsporidia that parasitize the muscles of salmoniform fish.
(A1) A method for preventing muscle lysis caused by microsporidia that occur in salmonid fish, which comprises administering an insecticide for microsporidia containing a benzimidazole compound as an active ingredient.
(A2) The method according to (A1), characterized in that an administration period is set up in which a microsporidia-killing agent is administered to fish in an environment where microsporidia are infected for 5 or more consecutive days.
(A3) The method according to (A1) or (A2), characterized in that a period of administration of a microsporidia pesticide and a period of withdrawal are set for farmed salmoniform fish in a cycle of 20 to 60 days.
(A4) The method according to (A1) or (A2), characterized in that for farmed salmoniform fish, a cycle of 260 to 780°C in accumulated water temperature is performed, with administration and withdrawal of a microsporidia pesticide.
(A5) The method according to any one of (A1) to (A4), wherein the benzimidazole compound is one or more compounds selected from albendazole, febantel, fenbendazole, oxfendazole, mebendazole, flubendazole, oxibendazole, triclabendazole, ricobendazole and thiabendazole.
(A6) The method according to any one of (A1) to (A5), wherein the salmoniformes are salmonids or smelts.
(A7) The method according to (A6), wherein the salmonid fish is a fish belonging to any one of the genera Salmon, Salmonella, Salvelinus, and Siberian char, and the Osmeridae fish is a fish belonging to the genus Smelt or Ayu.
(A8) The method according to any one of (A1) to (A7), wherein the benzimidazole compound is administered orally.
(A9) The method according to any one of (A1) to (A8), wherein the benzimidazole compound is administered at a dose of 1 to 25 mg/kg/day.
(A10) The method according to any one of (A3) to (A9), wherein the total amount of the pesticide administered per cycle is 5 to 50 mg/kg.
(A11) The method according to (A1), wherein the pesticide is administered in a single dose.

(B1) An agent for controlling microsporidia that parasitize the muscles of salmonid fish, comprising a benzimidazole compound as an active ingredient.
(B2) The pesticide according to (B1), wherein the benzimidazole compound is one or more compounds selected from albendazole, febantel, fenbendazole, oxfendazole, mebendazole, flubendazole, oxibendazole, triclabendazole, ricobendazole and thiabendazole.
(B3) The pesticide according to (B1) or (B2), wherein the salmoniform fish is a salmonidae fish or a fish of the Osmeridae family.
(B4) The pesticide according to (B3), wherein the salmonid fish is a fish belonging to any one of the genus Salmon, Salmonella, Salvelinus or Sakhalinus, and the Osmeridae fish is a fish belonging to the genus Smelt or Ayu.
(B5) The method according to any one of (B1) to (B4), for use by oral administration.
(B6) The pesticide according to any one of (B1) to (B5), for use in administering the benzimidazole compound in an amount of 1 to 25 mg/kg/day.
(B7) The pesticide according to any one of (B1) to (B6), wherein the administration period is 5 days or longer.
(B8) The pesticide according to any one of (B1) to (B7), which is administered to farmed salmoniform fish in a cycle of 20 to 60 days with an administration period and a withdrawal period.
(B9) The pesticide according to any one of (B1) to (B7), characterized in that a period of administration of the microsporidia pesticide and a period of withdrawal are set for farmed salmoniform fish in a cycle of 260 to 780°C in accumulated water temperature.
(B10) The pesticide according to any one of (B8) to (B9), wherein the total amount of the pesticide administered per cycle is 5 to 50 mg/kg.
(B11) The pesticide according to any one of (B1) to (B6), which is administered in a single dose.
(B12) A method for exterminating microsporidia, comprising a step of administering the extermination agent according to (B1) to (B11) to salmoniform fish.

The present invention further includes the inventions described in (C1) to (C11) below.
(C1) A method for preventing muscle lysis caused by microsporidia that occur in the muscles of salmonid fish, comprising administering an insecticide for microsporidia that contains a benzimidazole compound as an active ingredient.
(C2) The method according to (C1), characterized in that a microsporidia extermination agent is administered to fish in an environment where microsporidia are infected for a period of 5 or more consecutive days.
(C3) The method according to (C1) or (C2), characterized in that a period of administration of a microsporidia-killing agent and a period of withdrawal are set for fish in an environment where microsporidia are infected, in a cycle of 20 to 60 days.
(C4) An agent for controlling microsporidia that parasitize the muscles of salmonid fish, comprising a benzimidazole compound as an active ingredient.
(C5) A pesticide according to (C4), wherein the benzimidazole compound is one or more compounds selected from albendazole, febantel, fenbendazole, oxfendazole, mebendazole, flubendazole, oxibendazole, triclabendazole, ricobendazole and thiabendazole.
(C6) The pesticide according to (C4) or (C5), wherein the salmoniform fish is a fish of the family Salmonidae or a fish of the family Osmeridae.
(C7) The pesticide according to (C6), wherein the salmonid fish is a fish belonging to any one of the genus Salmon, Salmonella, Salvelinus or Sakhalinus, and the Osmeridae fish is a fish belonging to the genus Smelt or Ayu.
(C8) The pesticide according to any one of (C4) to (C7), wherein the benzimidazole compound is administered at a dosage of 1 to 25 mg/kg/day.
(C9) The pesticide according to any one of (C4) to (C8), wherein the administration period is 5 days or longer.
(C10) The pesticide according to any one of (C4) to (C9), characterized in that an administration period and a withdrawal period are set for fish in an environment infected with microsporidia in a cycle of 20 to 60 days.
(C11) The pesticide according to (C10), wherein the total amount of the pesticide administered per cycle is 5 to 50 mg/kg.

The present invention makes it possible to eradicate microsporidia that parasitize the muscles of salmoniform fish by oral administration. This also makes it possible to inhibit muscle melting observed in salmoniform fish. Furthermore, the present invention provides a method for preventing muscle melting in salmoniform fish and improving the appearance of the muscles of salmoniform fish.

1 is a photograph showing the entire fillet of a coho salmon. In the enlarged photograph of part (1) in Figure 1, the arrow indicates muscle melting. In this enlarged photograph of part (2) in Figure 1, the arrow indicates a cyst. This is a photograph of microsporidia spores observed in common in Figures 1 (1) and (2).

The present invention clarifies that the cause of the lysis symptoms observed in the muscles of salmonid fish is microsporidia, and discovers a drug that is effective when administered orally to eradicate the microsporidia.

The fishes that are the subject of the present invention are those classified as Salmoniformes in biological taxonomy. Unless otherwise specified in this specification, the term "Salmoniformes" refers to fishes that may be classified as Salmoniformes (such as Osmeridae). When "Salmoniformes" is interpreted in a narrower sense, the fishes that are the subject of the present invention are "Salmoniformes" or "Salmoniformes". For example, the fishes that are the subject of the present invention are fishes that belong to the Salmonidae family or the Osmeridae family.

Examples of fish species belonging to the Salmonidae family include pink salmon (Oncorhynchus gorbuscha), chum salmon (Oncorhynchus keta), sockeye salmon (Oncorhynchus nerka), cherry salmon (Oncorhynchus masou), satsukimasu (Oncorhynchus masou ishikawae), Biwa salmon (Oncorhynchus masou rhodurus), coho salmon (Oncorhynchus kisutch), chinook salmon (Oncorhynchus tshawytscha), steelhead, rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), Atlantic salmon (Salmo salar), char (Salvelinus Richardson), and huchen (Parahucho perryi).

Examples of fish species belonging to the family Osmeridae include Hypomesus japonicus and Hypomesus nipponensis, which belong to the genus Hypomesus, and Plecoglossus altivelis, which belongs to the genus Ayu.
In a preferred embodiment, the parasiticide of the present invention is used on farmed fish of these fish species.

The active ingredient of the parasiticide of the present invention is a drug classified as a benzimidazole compound. Benzimidazole compounds are drugs that have benzimidazole as the basic structure and are known as parasiticides and fungicides. Albendazole (methyl N-(5-propylsulfanyl-1H-benzimidazol-2-yl)carbamate), Febantel (methyl (NE)-N-[[2-[(2-methoxyacetyl)amino]-4-phenylsulfanylanilino]-(methoxycarbonylamino)methylidene]carbamate), Fenbendazole (methyl N-(5-phenylsulfanyl-1H-benzimidazol-2-yl)carbamate), Oxfendazole (methyl N-[5-(benzenesulfinyl)-1H-benzimidazol-2-yl]carbamate), Mebendazole (methyl [5-(Benzoyl)benzimidazol-2-yl]carbamate), Flubendazole (methyl N-[5-(4-fluorobenzoyl)-1H-benzimidazol-2-yl]carbamate), oxibendazole, triclabendazole, ricobendazole, or thiabendazole. Febantel is known to be a prodrug, and its active ingredients are fenbendazole and oxfendazole.

In a preferred embodiment, the parasiticide of the present invention contains albendazole as an active ingredient.

The microsporidian parasites targeted by the pesticide of the present invention are microsporidian parasites that parasitize the muscles of salmoniform fishes and cause muscle melting. Figure 1 is a photograph of a fillet of coho salmon, and muscle melting has occurred at the location indicated by (1). Figure 2 is an enlarged photograph of the melted muscle location indicated by (1). Microsporidian cysts have been confirmed at the location indicated by (2) in Figure 1. Figure 3 is an enlarged photograph, and cysts are present at the location indicated by the arrow. Microsporidian spores have been detected in both (1) and (2), and Figure 4 is an enlarged photograph of the same. In the photograph of Figure 4, polar vacuoles, polar tubes, and posterior vacuoles can be confirmed. The fact that the microsporidian parasites parasitize the muscles of salmoniform fishes and cause muscle melting is considered to be a characteristic that is clearly different from previously known microsporidian parasites. In one embodiment of the present invention, the causative parasite is a microsporidian parasite that parasitizes the muscles of salmoniform fishes and causes muscle melting.

In one embodiment, the causative parasite can be identified by analyzing the DNA base sequence, the base sequence of the ribosomal DNA region, or the base sequence of the SSU ribosomal DNA region. When comparing the base sequence of the causative parasite, the microsporidia have, for example, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more similarity (homology, identity) with the base sequence corresponding to any one of the base sequences of SEQ ID NOs: 1 to 8. The comparison can be performed, for example, with respect to base sequences of 200 bp or more, 300 bp or more, 400 bp or more, 500 bp or more, 600 bp or more, 700 bp or more, 800 bp or more, or 900 bp or more. For comparing base sequences, known tools (e.g., Clustal W (https://clustalw.ddbj.nig.ac.jp/), etc.) can be used.

The parasiticide of the present invention can be effective when administered orally. It can also be administered by immersing fish in a solution containing the drug in a medicinal bath or by injection.
The dosage of the parasiticide is not limited in any way in the present invention. As shown in the examples, a large dosage reduces feeding activity, but a single dose or short-term administration does not affect survival. Depending on the infection state of the parasite, it is desirable to suppress the parasite by administering a large amount once or for a short period of time. Thereafter, a low dose is continued for a certain period while observing the infection state, and then the drug is suspended. Also, if the infection state is mild, a low dose can be continued for a certain period from the beginning and then the drug is suspended. It is preferable to grasp the infection state of the parasite and administer the drug while changing the dosage as appropriate at the discretion of a veterinarian. In this case, the high dose is preferably 5 to 25 mg/kg/day from the viewpoint of efficacy and side effects, and the low dose is preferably 1 to 5 mg/kg/day.

On the other hand, because there is always a risk of infection with microsporidia when rearing in a fish cage, and because symptoms of microsporidia infection are difficult to detect in the early stages, it is practical to alternate between a certain dosage period and a withdrawal period when even a slight infection is observed, in hopes of achieving both a therapeutic and preventive effect. In this case, one administration period and withdrawal period are considered to be one cycle. One cycle can be 20 to 60 days, 30 to 60 days, or 30 to 45 days. The administration period in one cycle can be 5 to 20 days, and the withdrawal period can be 15 to 40 days. For example, one cycle can be set to 4 weeks, with one week of administration and three weeks of withdrawal. Alternatively, one cycle can be set to 6 weeks, with two weeks of administration and four weeks of withdrawal. These are repeated until the fish are transferred to the cage and shipped. The same cycle can be repeated, or the administration period and withdrawal period can be changed for each cycle. The preferred dosage is 1 to 5 mg/kg/day. For example, it can be designed to administer 1 mg/kg/day for two weeks followed by a four-week break. Parasites tend to develop and grow less easily in low water temperatures and more easily in high water temperatures. It is also effective to adjust the dosage according to the water temperature. In either case, the total dosage for one cycle can be 5-50 mg/kg.

In the practice of the present invention, one cycle including one administration period and withdrawal period can also be specified by the accumulated water temperature. The accumulated water temperature is the value obtained by adding up the average water temperature for one day. One cycle can be, for example, 260 to 780°C, 390 to 780°C, or 390 to 585°C. The administration period in one cycle can be 65 to 260°C, and the withdrawal period can be 195 to 520°C. For example, one cycle can be set to 364°C, with administration at 91°C and withdrawal at 273°C. Alternatively, one cycle can be set to 546°C, with administration at 182°C and withdrawal at 364°C. These are repeated until the fish are moved to the fish pond and shipped. The same cycle can be repeated, or the administration period and withdrawal period can be changed for each cycle. In one example, the preferred dosage is 1 to 5 mg/kg per 13°C of accumulated temperature. For example, it can be designed so that 1 mg/kg/day is administered for a period of accumulated temperature 182°C, followed by a period of rest for an accumulated temperature of 364°C. Parasites tend to develop and grow less easily in low water temperatures, but more easily in high water temperatures. It is also effective to adjust the dosage and administration method according to the water temperature. In either case, the total dosage for one cycle can be 5-50 mg/kg.

In the case of farming salmon, sweetfish, etc., the period during which they are kept in fish pens etc. where there is a risk of infection is usually around three to six months. If the rearing period is short, one cycle of administration may be sufficient. In a six-month farming period, three to six cycles of administration will be required.

As mentioned above, there is no particular limit to the administration period of the parasiticide of the present invention. For example, the administration period can be appropriately selected from 1 to 20 days, 3 to 20 days, or 5 to 14 days.

The administration of the parasiticide of the present invention may include a rest period during which the parasiticide is not administered. As shown in Example 2, microsporidiosis was significantly suppressed by a method in which fish were infected with microsporidia by rearing them in a fish cage for 42 days, and then the first administration was administered on the 21st day after they were brought to a land facility. Even when rearing in a fish cage where there is a risk of infection, it is not necessary to administer the drug for the entire period. As shown in Example 2, the administration period can be set appropriately depending on the health condition of the fish and the occurrence of parasites, for example, a single administration for one day at first, a 17-day rest period, and then 14 consecutive days of administration.

The administration period of the parasiticide of the present invention can be appropriately determined based on the accumulated water temperature and is not particularly limited. For example, the administration period can be appropriately selected from a period of 13 to 260°C, a period of 39 to 260°C, and a period of 65 to 182°C.

The dosage of the parasiticide of the present invention is, for example, for any fish, 1 mg to 25 mg of the benzimidazole compound per kg of fish body weight per day (hereinafter referred to as "1 to 25 mg/kg/day"), and is preferably orally administered in the range of 1 to 10 mg/kg/day, 1 to 8 mg/kg/day, or 1 to 5 mg/kg/day. In one embodiment of the present invention, the benzimidazole compound can be administered in the above daily amount only once.

Although the detailed mechanism of action of the parasiticide of the present invention is not clear, the effect is sustained for a certain period of time. The dosage of the parasiticide can be adjusted not by the dosage per day, but by the total amount administered in one cycle. The total amount for the entire administration period can be, for example, 5 to 50 mg/kg, or 10 to 25 mg/kg. The frequency of administration in one cycle is not particularly limited as long as the dosage per cycle is ensured, and may be, for example, once a day, twice, three times or more a day, once every two days, once every three days, once every four days, or once every five days or more. As long as the dosage for one cycle is ensured, the frequency of feeding may be changed during one cycle.

The therapeutic agent or parasiticide of the present invention may be used alone as the active ingredient, or may be used in combination with other substances, such as auxiliary ingredients such as carriers, stabilizers, solvents, excipients, and diluents, as necessary. The form may be any of the forms normally used for these compounds, such as powder, granules, tablets, and capsules. In the case of fish that are sensitive to the taste or smell of the compound, a coating method or the like can be used to prevent a decrease in the palatability of the feed and to make it difficult for the compound to leak out.

In the case of fish, drugs for oral administration are usually added to feed. When adding the therapeutic agent or parasiticide of the present invention to feed, it is preferable to use feed that takes into consideration the nutritional components and physical properties required for each fish species. As feed, a pellet-shaped mixture of fish meal, bran, starch, minerals, vitamins, fish oil, etc., or a pellet-shaped mixture of frozen fish such as sardines and powdered feed (mash) in which vitamins and other ingredients have been added to fish meal, etc., is usually used. Since the daily feed intake is almost determined depending on the type and size of the fish, the therapeutic agent or parasiticide of the present invention is added to the feed in an amount converted to the above-mentioned dosage. The therapeutic agent or parasiticide of the present invention may be administered in a single dose or in several doses. Since the therapeutic agent of the present invention is used by adding it to fish feed, it is preferable to prepare a formulation suitable for adding an appropriate concentration to the feed consumed by the fish per day. Specifically, it is preferable to formulate the drug so that it contains 1 to 50% by weight, preferably 5 to 30% by weight, and more preferably 10 to 20% by weight, of a benzimidazole compound as an active ingredient.

In one embodiment of the present invention, there is provided a feed for salmonid fish used in preventing muscle lysis caused by microsporidia, the feed containing 0.0001 to 5% by dry weight, preferably 0.0005 to 2.5% by dry weight, and more preferably 0.001 to 0.5% by dry weight of a benzimidazole compound.

The "environment where microsporidia can infect" is not particularly limited as long as it is an environment where fish can be infected with microsporidia, and includes, for example, a situation where fish are exposed to the outside world where microsporidia can exist by using fish cages, or an environment where fish are cultivated using marine fish cages. In addition, even if it is a closed environment, if there is a situation in which microsporidia can infect in the fish breeding environment, the environment is also included in the "environment where microsporidia can infect".

As shown in the examples, Coho salmon were transferred from freshwater to a marine surface cage and were infected with microsporidia by rearing in the cage for 42 days. Administration of the drug was started 21 days after the infected Coho salmon was returned to land, and a high deworming effect was obtained. In actual aquaculture sites, the first drug is administered 20 to 60 days or 30 to 60 days after the salmon is released onto the sea surface, or after a period of time when the accumulated water temperature is 260 to 780°C or 390 to 780°C. Since microsporidia infection continues in marine surface cages, it is preferable to administer the second drug 20 to 60 days or 30 to 60 days after the end of drug administration, or after a period of time when the accumulated water temperature is 260 to 780°C or 390 to 780°C. Therefore, when fish are placed in an environment where they are infected with microsporidia, the pesticide of the present invention can be administered every 20 to 60 days or every 30 to 60 days, or every period of accumulated water temperature of 260 to 780°C or 390 to 780°C, and by repeating this administration period, microsporidia can be eradicated and the onset of the disease can be prevented.

Microsporidia infection does not cause death or affect growth, leading to delayed detection. If skin melting is found at the shipping stage, the commercial value of the fish will inevitably decrease. Muscle melting caused by microsporidia is frequently observed just below the epidermis, and the color of the epidermis appears whiter than the surrounding area. In one embodiment of the present invention, the condition of the fish is observed, and if a fish exhibiting such an appearance is observed, a drug is immediately administered.

The following are examples of the present invention, but the present invention is not limited to these in any way.

<Effect of albendazole on feeding in fish>
Marine trout (rainbow trout), yellowtail, and tiger pufferfish were housed in tanks. The tanks were 500 liters for marine trout and 200 liters for yellowtail and tiger pufferfish. Sand-filtered and UV-sterilized seawater was poured into each tank at 2.4 liters/min. Fish weights were measured the day before administration of albendazole. After acclimation, each fish species and each group was fed the test feed for 5 consecutive days. Albendazole was administered once a day. The albendazole-added feed was prepared by placing a specified amount of commercial feed (product name: Otohime, manufacturer: Nisshin Marubeni Feed) and albendazole (manufactured by Tokyo Kasei Kogyo Co., Ltd.) in a polyethylene bag, adding 2-fold diluted spreading agent (low-sugar reduced starch syrup, product name: SE 30, manufacturer: Bussan Food Science Co., Ltd.) at 4% of the feed weight and stirring. The control group feed was prepared by adding only diluted SE 30 at 4% of the feed weight and stirring. The feeding amount was set at 1% by weight of the fish body weight for marine trout, 2% by weight for yellowtail, and 1.5% by weight for tiger pufferfish. The effect of albendazole on feeding was evaluated by observing the feeding behavior. The water temperature, test fish body weight at the start of the test, number of test fish, albendazole dosage, and results are shown in Table 1.

In yellowtail and tiger pufferfish, feeding was not affected by administration of 40 mg albendazole/kg of fish body weight per day for five days. Feeding was not affected in yellowtail even when administered at 200 mg/kg of fish body weight per day for five days, but feeding was affected in tiger pufferfish by administration of 80 mg/kg of fish body weight per day for five days. On the other hand, feeding was affected in marine trout by an extremely low dose of 10 mg/kg of fish body weight per day for five days. These results show that the effect of albendazole administration on feeding differs depending on the fish species. In particular, feeding was affected in marine trout by a low dose of 10 mg albendazole/kg of fish body weight per day compared to other fish species. It was confirmed that salmonid fish have low tolerance to benzimidazole compounds.

<Effect of albendazole on feeding of coho salmon and its anthelmintic effect against microsporidia>
Coho salmon fry were introduced into a fish pen at the fishing ground and reared for 42 days. The fry were naturally infected with microsporidia by rearing in the marine fish pen. The coho salmon brought to the land facility were divided into three groups, and each group was housed in a separate 2-ton tank. The average fish weight at this time was 466 g. The test area and the number of test fish are shown in Table 2. Water was poured into the tank under the same conditions as in Example 1. The rearing period after delivery was 99 days. The water temperature during the test period was 10.9 to 14.9°C, with an average of 13.2°C. In addition, in order to understand the onset of microsporidia at the start of the test, 10 fish were dissected to observe the number of cysts in the lateral muscles and the presence or absence of muscle lysis.

At the end of the rearing experiment, all fish from all test groups were removed and the number of cysts in the lateral muscles was counted by necropsy, and fish in which cysts were observed were designated as diseased fish. The length and width of the observed cysts were measured and their area calculated. Evaluation was performed by comparing the incidence rate (number of diseased fish/number of test fish x 100) and the number and cyst area of diseased fish in the control group and the albendazole oral administration group.

Results and Discussion: The day after the single dose, feeding became inactive in the albendazole-treated group, and feeding was reduced to half the prescribed amount. As with marine trout, administration of 10 mg/kg or more of albendazole per kg of body weight had a detrimental effect on feeding in Coho salmon. These results demonstrate that compared to other fish species, salmonids are more susceptible to adverse effects on feeding even at doses as low as 10 mg/kg of albendazole per kg of body weight. However, growth and survival during the rearing period were superior to those in the control group (Table 3). This is presumably due to the anthelmintic effect of microsporidia, which will be described later.

The anthelmintic effect of albendazole against microsporidia is shown in Table 4. The incidence rate in the group administered a single dose of 20 mg/kg of fish weight of albendazole was clearly lower than in the control group, demonstrating that albendazole has an anthelmintic effect against this causative worm. Furthermore, the anthelmintic rate in the group administered a single dose of 10 mg/kg of fish weight plus 1 mg/kg of fish weight for 14 consecutive days was clearly lower than that in the group administered a single dose of 20 mg/kg of fish weight, demonstrating that this worm can be anthelminticated by long-term administration even at a low dose of 1 mg/kg of fish weight. Furthermore, no decrease in food intake was observed during administration of 1 mg/kg of fish weight for 14 days.

It was found that feeding is adversely affected in salmonid fish at a small dose of 10 mg/kg of fish body weight, and that administration of 1 mg/kg of fish body weight for 14 days can be done without reducing feeding and can eradicate the microsporidia. Furthermore, no deaths due to the disease were observed. This makes it difficult to detect the infection until the infected fish are shipped.

In Table 4, the incidence rate refers to the percentage of fish that had cysts or lysis in the muscles, and the number and size of cysts refers to the number and size of cysts or lysis. In other words, it was shown that administration of albendazole suppressed the occurrence of cysts and lysis in the muscles.

<Anthelmintic effect of albendazole and febantel against microsporiosis in coho salmon - 1>
Coho salmon fry were introduced into a fish pen at the fishing ground and reared for 35 days. The fry were naturally infected with microsporidia by rearing in the marine fish pen. The coho salmon brought to the land facility were divided into four groups, and each group was housed in a separate 500 L tank. The average fish weight at this time was 252 g. The test area and the number of test fish are shown in Table 5. Water was poured into the tank under the same conditions as in Example 1. The rearing period after the introduction was 89 days. The water temperature during the test period was 11.6 to 15.3 °C, with an average of 13.2 °C. In addition, in order to grasp the onset status of microsporidia at the start of the test, 10 fish were dissected to observe the number of cysts in the lateral muscles and the presence or absence of muscle melting.

At the end of the rearing test, all fish were removed from all test areas and the number of cysts in the lateral muscles was counted by necropsy. Fish in which cysts were observed were designated as diseased fish. Evaluation was performed by comparing the incidence rate (number of diseased fish/number of test fish x 100) and the number of cysts in diseased fish in the control and orally administered drug areas. During the test period, some fish died due to caudal fin defects caused by territorial behavior, and there were test areas where survival was low. Therefore, the results of the necropsy of fish that died 65 days or later after rearing in the land tank were added to the results of the surviving fish to evaluate the anthelmintic effect.

Results and Discussion When the muscles of 10 fish were examined upon transport to the land tank, no cysts were found. The body weight, fork length, and mortality rate of fish in each group at the end of the experiment are shown in Table 6. Growth and survival rates of albendazole and febantel were superior to those of the control group, suggesting that administration of these drugs had no adverse effect on the Coho salmon. Furthermore, when the dead fish were observed, they had noticeable caudal fin loss and were anemic. Therefore, the deaths in this experiment were caused by caudal fin loss due to territorial behavior.

Table 7 shows the anthelmintic effect of albendazole and febantel against microsporidia. The incidence rate in the control group was 10%, while it was 0% in the albendazole and febantel-treated groups. It was found that this worm can be anthelminticized by long-term administration even at a low dose of 1 mg/kg fish body weight per day. It was also found that febantel, like albendazole, has an anthelmintic effect against newly developed muscle-dissolving microsporidia in salmonid fish.

<Anthelmintic effect of albendazole and febantel against microsporidia in coho salmon - 2>
Coho salmon fry were introduced into a fish pen at the fishing ground and reared for 28 days. The fry were naturally infected with microsporidia by rearing in the marine fish pen. The coho salmon brought to the land facility were divided into four groups, and each group was housed in a separate 500 L tank. The average fish weight at this time was 261 g. The test area and the number of test fish are shown in Table 8. Water was poured into the tank under the same conditions as in Example 1. The rearing period after the introduction was 77 days. The water temperature during the test period was 10.5 to 14.6 °C, with an average of 13.0 °C. In addition, in order to grasp the onset status of microsporidia at the start of the test, 10 fish were dissected to observe the number of cysts in the lateral muscles and the presence or absence of muscle melting.

At the end of the rearing test, all fish from all test groups were removed and the number of cysts in the lateral muscles was counted by necropsy, and fish in which cysts were observed were designated as diseased fish. Evaluation was performed by comparing the incidence rate (number of diseased fish/number of test fish x 100) and the number of cysts in diseased fish in the control group and the group orally administered drug. During the test period, some fish died due to caudal fin defects caused by territorial behavior, and there were test groups where survival was low. Therefore, the results of the necropsy of fish that died 38 days or later after rearing in the land tank were added to the results of the surviving fish to evaluate the anthelmintic effect.

Results and Discussion When the muscles of the 10 fish were examined upon transport to the land tank, no cysts were found. The weight of the fish, fork length, and mortality rate for each group at the end of the experiment are shown in Table 9. The growth and survival rates of the fish treated with albendazole and febantel were superior to those of the control group, suggesting that the administration of these drugs had no adverse effects on the Coho salmon. Furthermore, when the dead fish were observed, they had noticeable caudal fin loss and were anemic. Therefore, the deaths in this experiment were caused by caudal fin loss due to territorial behavior.

The anthelmintic effects of albendazole and febantel against microsporidia are shown in Table 10. The incidence rate in the control group was 11.8%, while it was 0% in the albendazole and febantel-treated group. The results of Example 3 were reproduced. It was demonstrated once again that this worm can be anthelminticated by long-term administration even at a low dose of 1 mg/kg fish body weight per day, and that febantel, like albendazole, has an anthelmintic effect against newly developed muscle-dissolving microsporidia in salmonid fish.

<Base sequence analysis of muscle-dissolving microsporidia>
Tissues from the lesions of the marine cultured coho salmon showing this symptom were collected, and the presence of spores of the causative microsporidia was confirmed under a microscope, after which DNA was extracted. Then, according to a previously published literature report (Bell, AS, et al., J. Eukaryot. Microbiol. 2001, 48, 258-265.), the base sequence of the causative microsporidia in the ribosomal DNA region of approximately 1500 bp amplified with the primer pair 530f (5'-GTGCATCCAGCCGCGG-3') (SEQ ID NO: 9) and 580r (5'-GGTCCGTGTTTCAAGACGG-3') (SEQ ID NO: 10) was determined. The obtained base sequence was subjected to a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm whether it was a known sequence. In addition, we compared the sequence of the SSU ribosomal DNA region (approximately 940 bp) of the obtained sequence with sequences of five known microsporidia that have been reported to parasitize salmonid fish using Clustal W (https://clustalw.ddbj.nig.ac.jp/).

DNA with eight patterns of base sequences (sequence numbers 1 to 8), including mutations, was collected from the causative microsporidia. Of these, type 1 (sequence number 1) was used for the following study. In general, the rDNA region has a repeat structure, and it is known that even within the same individual, the sequences may not match between the individual repeats. The similarity of each sequence was 99% or higher. The obtained base was approximately 1.5 kbp. The homology with other species was compared using approximately 900 pb on the 5' end side of the obtained base, which corresponds to base sequences registered for other microsporidia.

Results: The obtained sequence did not match any of the species registered in the database. In addition, the similarity to previously reported microsporidia sequences in salmonid fish was only about 68-84%. Furthermore, the symptoms observed in the diseased fish were different from those of any of the previously reported species. Therefore, it was revealed that this is an unknown species that has not been reported to occur before.

Symptoms of microsporidia in salmonid fish have been reported in the following publications: Dis Aquat Org 101: 43-49, 2012; Dis Aquat Org 44: 223-230, 2001; and "Infectious and Parasitic Diseases of Fish and Shellfish," edited by Egusawa Shuzo, published by Koseisha Kouseikaku, pp. 309-312 and 318-320 (2004).

It is possible to provide an effective extermination agent for microsporidia that parasitize the muscles of salmonid fish by oral administration. Since no deaths due to this disease have been observed, fish that have been raised to shipping size at great expense lose their commercial value if they are infected. If the melting of muscles can be prevented by this invention, the decline in commercial value can be prevented and it will be of industrial value.

Claims (12)

A method for preventing muscle lysis caused by microsporidian parasites in salmonid fish, comprising administering an agent for controlling microsporidian parasites containing a benzimidazole compound as an active ingredient, wherein the benzimidazole compound is one or more compounds selected from mebendazole, flubendazole, and oxibendazole. The method according to claim 1, characterized in that the microsporidia extermination agent is administered to farmed salmonid fish for a period of 5 or more consecutive days. The method according to claim 1 or 2, characterized in that the microsporidia extermination agent is administered to farmed salmonid fish in a cycle of 20 to 60 days, followed by a rest period. The method according to claim 1 or 2, characterized in that for farmed salmonid fish, a cycle of 260 to 780°C in accumulated water temperature is used, with administration and withdrawal periods of a microsporidia pesticide. An anthelmintic agent for controlling microsporidia that parasitize the muscles of salmonid fish, containing a benzimidazole compound as an active ingredient, for use in preventing muscle melting, wherein the benzimidazole compound is one or more compounds selected from mebendazole, flubendazole, and oxibendazole. The pesticide according to claim 5, wherein the salmonid fish is a fish belonging to any one of the genera Salmon, Salmonella, Salvelinus, and Sakhalinus. The pesticide according to claim 5 or 6, wherein the benzimidazole compound is administered at a dosage of 1 to 25 mg/kg of fish body weight/day. The pesticide according to any one of claims 5 to 7, wherein the administration period is 5 days or more. The pesticide according to any one of claims 5 to 8, characterized in that it is administered to farmed salmonid fish in a 20 to 60 day cycle with a drug-withdrawn period. The pesticide according to any one of claims 5 to 8, characterized in that the microsporidia pesticide is administered and withdrawn during a cycle of 260 to 780°C accumulated water temperature for farmed salmonid fish. The pesticide according to claim 9 or 10, wherein the total amount of the pesticide administered per cycle is 5 to 50 mg/kg of fish body weight. The pesticide according to any one of claims 5 to 7, which is administered in a single dose.