JPWO2020050403A1 - How to prevent salmonid muscle melting - Google Patents

Abstract

It is a method for preventing muscle melting that occurs in the muscles of salmonid fish, which comprises administering a microsporidia extermination agent containing a benzimidazole compound as an active ingredient. It is an agent for exterminating microsporidia parasitizing the muscles of salmonid fish, which contains a benzimidazole compound as an active ingredient. The benzimidazole compound is one or more compounds selected from albendazole, fevantel, fenbendazole, oxfendazole, mebendazole, flubendazole, oxybendazole, triclabendazole, ricobendazole and thiabendazole. ..

Description

The present invention relates to a method of preventing muscle melting that occurs in salmonid fish. Furthermore, the present invention relates to a drug and a method for exterminating microsporidia parasitizing the muscles of salmonid fish by oral administration.

In many prefectures in Japan, sea surface aquaculture of salmonids called local salmon has become popular in recent years. Many salmonids cultivated in Japan are also offered as sashimi.

Benzimidazole compounds are known as antiparasitic drugs. In Japan, mebendazole is a therapeutic drug for worms, albendazole is a therapeutic drug for psoriasis, and flubendazole is for animals for roundworms and roundworms. As pharmaceuticals, fevantel and fenbendazole are approved as veterinary drugs against nematodes and streaks. For fisheries, Febantel is licensed for blowfish.

There is a report testing the effect of albendazole on Loma salmonae, a microsporidia that parasitizes the gills of rainbow trout (Non-Patent Document 1). There is a report testing the effects of albendazole, mebendazole, and fenbendazole on Glugea anomala, a microsporidia that parasitizes the three-spined stickleback (Gasterosteiformes) (Non-Patent Document 2). There are reports that benzimidazole drugs are effective against microsporidia parasitizing fish and shellfish of the order Perciformes or Flatfish (Patent Documents 1 and 2). There is also a report on the use of albendazole, fenvantel, or the like for the treatment of bedding and myxosporea (Patent Document 3).

JP-A-2017-186306 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 the muscle of the fish used for sashimi melts, that part cannot be used as sashimi, and its commercial value is greatly impaired. In addition, the depression of muscles due to melting makes the appearance of grilled fish poor and reduces its commercial value. It is an object of the present invention to provide a method for identifying and solving the cause of melting occurring in the muscles of salmonid fish. Further, it is also an object of the present invention to provide a method for preventing melting occurring in the muscles of salmonids and improving the appearance of the muscles of salmonids.

We have noticed that the muscles under the salmon skin have melting symptoms. The dents due to the melting symptoms that can be visually found are about 2 to 3 mm in diameter when they are small, and about 15 mm or more when they are large, and become dents with bleeding. It cannot be found on the skin without careful observation. Small ones are difficult to find on the skin, but when they are large, they appear to appear as spots even when viewed from the top of the skin. In order to identify the cause of muscle melting found in the salmon muscles, examination revealed innumerable microsporidia spores (Fig. 4) from the melting site. Cysts were also observed (FIGS. 1 and 3), and microsporidia spores were observed in the cysts. From these results, it was considered that this disease was caused by microsporidia parasitizing muscles. Muscle melting by microsporidia in sea-cultured salmon has not been reported so far and is a new discovery.

In search of an orally-administered drug effective for exterminating microsporidia of the type that parasitizes the muscles of salmon fish, we searched for various existing antiparasitic drugs for animals and substances derived from natural products. As a result, they have found that albendazole, which is sold as an antiparasitic drug for animals, can be orally administered without reducing the appetite of salmon and has an anthelmintic effect, and completed the present invention.

The present invention is a method for preventing muscle melting due to microsporidia occurring in the following salmonid fishes (A1) to (A11), and microsporidia parasitizing the muscles of salmonid fishes (B1) to (B12) below. Includes repellents.
(A1) A method for preventing muscle melting by microsporidia occurring in salmonid fish, which comprises administering a microsporidia exterminating agent containing a benzimidazole compound as an active ingredient.
(A2) The method according to (A1), wherein a administration period is provided in which a microsporidia exterminating agent is continuously administered to a fish in an environment infected with microsporidia for 5 days or more.
(A3) For salmonids, which are farmed fish, a period of administration of a microsporidia extermination agent and a drug holiday are provided in a cycle of 20 to 60 days (A1) or (A2). The method described.
(A4) For salmonids, which are farmed fish, a period of administration of a microsporidia repellent and a period of withdrawal are provided in a cycle of 260 to 780 ° C. at an integrated water temperature (A1) or (A1). The method described in A2).
(A5) One or more benzimidazole compounds selected from albendazole, fevantel, fenbendazole, oxfendazole, mebendazole, flubendazole, oxybendazole, triclabendazole, ricobendazole and thiabendazole. The method according to any one of (A1) to (A4), which is a compound.
(A6) The method according to any one of (A1) to (A5), wherein the salmonid fish is a salmonid fish or a smelt family fish.
(A7) Salmonids are fish belonging to any of the genus Salmon, Salmo, Iwana and Taimen, and fish of the family Smelt are fish belonging to the genus Smelt and Ayu (A6). The method described in.
(A8) The method according to any one of (A1) to (A7), wherein the benzimidazole compound is orally administered.
(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 dose of the pesticide 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 exterminating microsporidia parasitizing the muscles of salmonid fish, which contains a benzimidazole compound as an active ingredient.
(B2) One or more of the benzimidazole compounds selected from albendazole, fevantel, fenbendazole, oxfendazole, mebendazole, flubendazole, oxybendazole, triclabendazole, ricobendazole and thiabendazole. The repellent according to compound (B1).
(B3) The pesticide according to (B1) or (B2), wherein the salmonid fish is a salmonid fish or a smelt family fish.
(B4) Salmonids are fish belonging to any of the genus Salmon, Salmo, Iwana and Taimen, and fish of the family Smelt are fish belonging to the genus Smelt and Ayu (B3). The repellent described.
(B5) The method according to any one of (B1) to (B4) for use by oral administration.
(B6) The repellent according to any one of (B1) to (B5) for use in administration of a benzimidazole compound at 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 more.
(B8) The pesticide according to any one of (B1) to (B7), wherein a administration period and a drug holiday are provided in a cycle of 20 to 60 days for the salmonid fish which are farmed fish. ..
(B9) For salmonids, which are farmed fish, a period of administration of a microsporidia extermination agent and a drug holiday are provided in a cycle of a cumulative water temperature of 260 to 780 ° C. (B1) to (B1). The pesticide according to any one of B7).
(B10) The pesticide according to any one of (B8) to (B9), wherein the total dose of the pesticide per cycle is 5 to 50 mg / kg.
(B11) The pesticide according to any one of (B1) to (B6), which is administered once.
(B12) A method for exterminating microsporidia, which comprises a step of administering the exterminating agent according to (B1) to (B11) to salmonid fish.

Further, the invention of the present application includes the inventions described in the following (C1) to (C11).
(C1) A method for preventing muscle melting due to microsporidia generated in the muscles of salmonid fish, which comprises administering a microsporidia extermination agent containing a benzimidazole compound as an active ingredient.
(C2) The method according to (C1), wherein a administration period is provided in which a microsporidia exterminating agent is continuously administered to fish in an environment infected with microsporidia for 5 days or more.
(C3) For fish in an environment infected with microsporidia, a period of administration of a microsporidia exterminating agent and a drug holiday are provided in a cycle of 20 to 60 days (C1) or (C1). Method of C2).
(C4) An agent for exterminating microsporidia parasitizing the muscles of salmonid fish, which contains a benzimidazole compound as an active ingredient.
(C5) One or more of the benzimidazole compounds selected from albendazole, fevantel, fenbendazole, oxfendazole, mebendazole, flubendazole, oxybendazole, triclabendazole, ricobendazole and thiabendazole. A repellent for compound (C4).
(C6) An extermination agent of (C4) or (C5) in which the salmonids are salmonids or smelts.
(C7) Salmonids are fish belonging to any of the genus Salmon, Salmo, Iwana and Taimen, and fish of the family Smelt are fish belonging to the genus Smelt and Ayu (C6). Disinfectant.
(C8) The pesticide according to any one of (C4) to (C7), wherein the benzimidazole compound is administered at a dose 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 more.
(C10) Described in any of (C4) to (C9), wherein a administration period and a drug holiday are provided in a cycle of 20 to 60 days for fish in an environment infected with microsporidia. Pesticide.
(C11) The pesticide according to (C10), wherein the total dose of the pesticide per cycle is 5 to 50 mg / kg.

According to the present invention, microsporidia parasitizing the muscles of salmonid fish can be exterminated by oral administration. As a result, the melting of muscles found in salmonids can also be suppressed. Furthermore, the present invention can provide a method for preventing melting that occurs in the muscles of salmonids and improving the appearance of the muscles of salmonids.

It is a photograph showing the whole of the coho salmon fillet. In the enlarged photograph of the part (1) in FIG. 1, the arrow indicates the melting of the muscle. In the enlarged photograph of the part (2) in FIG. 1, the arrow indicates a cyst. It is a photograph of microsporidia spores commonly observed from (1) and (2) of FIG.

The present invention has elucidated that the cause of the melting symptoms observed in the muscles of salmonid fish is microsporidia, and has found a drug that is effective by oral administration for exterminating the microsporidia. be.

The fish that is the subject of the present invention is a fish that is classified as a salmonid fish in terms of biological taxonomy. Unless otherwise specified in the present specification, the term "salmonid fish" means fish including fish that can be classified as salmonid suborder (such as Smelt family) in some cases. When "salmonids" is understood in a narrow sense, the fishes of the present invention are "salmonids" or "salmonids". For example, the target fish of the present invention is a fish belonging to the family Salmonidae or Osmeriformes.

Fish species belonging to the family Salmon include Karafutomas (Oncorhynchus gorbuscha), White salmon (Oncorhynchus keta), Benizake (Oncorhynchus nerka), Sakuramasu (Yamame) (Oncorhynchus masou), Satsukimas (Oncorhynchus Oncorhynchus Oncorhynchus Oncorhynchus masou) masou rhodurus), coho salmon (Oncorhynchus kisutch), masnosuke (Oncorhynchus tshawytscha), steelhead, oncorhynchus mykiss, brown trout belonging to the genus Atlantic salmon (Salmo trutta), belonging to the genus Atlantic salmon (Salmo salar) Examples include Iwana (Salvelinus Richardson) and Ito (Parahucho perryi) belonging to the genus Ito.

Examples of fish species belonging to the family Smelt include Chika (Hypomesus japonicus) belonging to the genus Smelt, Hypomesus nipponensis (Hypomesus nipponensis), and Ayu (Plecoglossus altivelis) belonging to the genus Ayu.
In a preferred embodiment, the parasite repellent of the present invention is used for farmed fish of these fish.

The active ingredient of the parasite repellent of the present invention is an agent classified as a benzimidazole compound. The benzimidazole compound is a drug having benzimidazole as a basic skeleton, and is known as a parasite repellent or a fungicide. Albendazole (methyl N- (5-propylsulfanyl-1H-benzimidazol-2-yl) carbamate), Febantel (methyl (NE) -N-[[2-[(2-methoxyacetyl) amino] -4- phenylsulfanylanilino]-(methoxycarbonylamino) methylidene] carbamate), fenbendazole (Fenbendazole; methyl N- (5-phenylsulfanyl-1H-benzimidazol-2-yl) carbamate), oxfendazole (Oxfendazole; methyl N- [5- (benzenesulfinyl) ) -1H-benzimidazol-2-yl] carbamate), mebendazole (methyl [5- (Benzoyl) benzimidazol-2-yl] carbamate), flubendazole (Flubendazole; methyl N- [5- (4-fluorobenzoyl)- 1H-benzimidazol-2-yl] carbamate), oxybendazole, triclabendazole, ricobendazole, thiabenzazole and the like. Fevantel is known to be a prodrug, the active ingredients of which are fenbendazole and oxfendazole.

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

The microsporidia targeted by the pesticide of the present invention are microsporidia that parasitize the muscles of salmonids and cause muscle melting. FIG. 1 is a photograph of a fillet of coho salmon (fillet), and muscle melting occurs at the portion shown in (1). FIG. 2 is an enlarged photograph of the melting portion of the muscle shown in (1). Microsporidia cysts have been confirmed at the location shown in FIG. 1 (2). FIG. 3 is an enlarged photograph thereof, and a cyst is present at the position of the arrow. Microsporidia spores were commonly detected in (1) and (2), and FIG. 4 is an enlarged photograph thereof. In the photograph of FIG. 4, polar vesicles, polar tubes, and posterior vacuoles can be confirmed. Parasitizing the muscles of salmonids and causing muscle melting is understood to be a distinctly different feature from previously known microsporidia. In one embodiment of the invention, the causative parasite is a microsporidia that parasitizes the muscles of salmonids and causes muscle melting.

In one embodiment, the causative parasite can be identified by analyzing the base sequence of the DNA, the base sequence of the ribosomal DNA region, or the base sequence of the SSU ribosomal DNA region. Microsporidia, which is the causative parasite, is, for example, 92% or more, 93% or more, 94% or more, 95% or more, when comparing the base sequences corresponding to any one of the base sequences of SEQ ID NOs: 1 to 8. It has 96% or more, 97% or more, 98% or more, or 99% or more similarity (homology, identity). The comparison can be made, for example, with respect to a base sequence 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. A known tool (for example, Clustal W (https://clustalw.ddbj.nig.ac.jp/)) can be used for the comparison of the base sequences.

The parasite repellent of the present invention can exert its effect by oral administration. In addition, administration by a drug bath in which fish is immersed in a solution in which a drug is dissolved or administration by injection is also possible.
The dosage of the anthelmintic agent is not limited to the present invention. As shown in the examples, the feeding activity decreases when the dose is large, but the single administration or short-term administration does not affect the survival. Depending on the state of parasite infection, it is desirable to control the parasite by administering a large amount in a single dose or for a short period of time. After that, there is a method of continuing the low dose for a certain period of time and withdrawing the drug while observing the infection state. In addition, if the infection is mild, the low dose can be continued for a certain period from the beginning and the drug can be withdrawn. It is preferable to grasp the infection state of the parasite and administer the parasite while changing the dosage as appropriate at the discretion of the veterinarian. In this case, the high dose is preferably 5 to 25 mg / kg / day from the viewpoint of effects and side effects, and the low dose is preferably 1 to 5 mg / kg / day.

On the other hand, there is always a risk of microsporidia infection when breeding in a living cage, and it is difficult to detect the symptoms of microsporidia infection in the early stages. However, when infection is observed, a method of alternately repeating a fixed dose administration period and a drug holiday is practical. In this case, one administration period and drug holiday are defined as 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 drug holiday can be 15 to 40 days. For example, one cycle is set to 4 weeks, and the drug is administered for 1 week and withdrawn for 3 weeks. Alternatively, one cycle can be set to 6 weeks, and the drug can be set to be administered for 2 weeks and withdrawn for 4 weeks. These are repeated until the fish is transferred to the cage and then shipped. The same cycle may be repeated, or the administration period and drug holiday may be changed for each cycle. The preferred dose is 1-5 mg / kg / day. For example, 1 mg / kg / day can be administered for 2 weeks and then withdrawn for 4 weeks. Parasites tend to grow and grow easily at low water temperatures, and tend to grow and grow easily at high water temperatures. It is also effective to adjust the dosage according to the water temperature. In either case, the total dose per cycle can be 5-50 mg / kg.

One cycle including one administration period and drug holiday in the practice of the present invention can also be specified by the integrated water temperature. The integrated water temperature is the value obtained by adding the average daily water temperature every day. One cycle can be, for example, 260-780 ° C, 390-780 ° C, or 390-585 ° C. The administration period in one cycle can be 65 to 260 ° C., and the drug holiday can be 195 to 520 ° C. For example, one cycle is defined as 364 ° C., with 91 ° C. dosing and 273 ° C. withdrawal. Alternatively, one cycle can be set to 546 ° C, with 182 ° C dosing and 364 ° C withdrawal. These are repeated until the fish is transferred to the cage and then shipped. The same cycle may be repeated, or the administration period and drug holiday may be changed for each cycle. In one example, the preferred dose per 13 ° C. integrated temperature is 1-5 mg / kg. For example, it can be designed such that 1 mg / kg / day is administered during a period of an integrated temperature of 182 ° C., and then the drug is suspended for a period of an integrated temperature of 364 ° C. Parasites tend to grow and grow easily at low water temperatures, and tend to grow and grow easily at high water temperatures. It is also effective to adjust the dosage according to the water temperature. In either case, the total dose per cycle can be 5-50 mg / kg.

In aquaculture of salmon and sweetfish, in many cases, the period of breeding in cages at risk of infection is about 3 months to 6 months. If the breeding period is short, it may be completed with one cycle of administration. During the half-year aquaculture period, administration will be performed in 3 to 6 cycles.

As described above, the administration period of the parasite repellent of the present invention is not particularly limited. For example, the administration period can be appropriately selected from 1 to 20 days, 3 to 20 days, and 5 to 14 days.

The administration of the parasite repellent of the present invention may be provided with a drug holiday, which is a period during which the parasite repellent is not administered. As shown in Example 2, microsporidia was significantly reduced even by the method of first administering the fish infected with microsporidia on the 21st day after bringing the fish infected with microsporidia by breeding in a cage for 42 days. I was able to suppress it. It is not necessary to administer the drug for the entire period even if it is kept in a cage at risk of infection. As shown in Example 2, a single dose of 1 day is first administered, and after a 17-day washout period, 14 consecutive days of continuous administration are performed. The administration period can be set.

Further, the administration period of the parasite repellent of the present invention can be appropriately determined based on the integrated 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 dose of the anthelmintic agent of the present invention is, for example, 1 mg to 25 mg of the benzimidazole compound per 1 kg of fish body weight per day in any fish (hereinafter, "1 to 25 mg / kg / day"). It is described, preferably 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 only once in the above daily dose.

Although the detailed mechanism of action of the parasite repellent of the present invention has not been clarified, the effect is sustained for a certain period of time. The dose of the anthelmintic agent can also be adjusted by the total dose administered per cycle rather than the daily dose. The total amount during the entire administration period can be, for example, 5 to 50 mg / kg and 10 to 25 mg / kg. The frequency of administration in one cycle is not particularly limited as long as the dose per cycle is secured, for example, once a day, twice a day, three times, or more, or two days. It may be once, once every three days, once every four days, or once every five days or more. The dose may be secured as one cycle, and the frequency of feeding may be changed during one cycle.

The therapeutic agent or parasite repellent of the present invention uses the compound as an active ingredient alone, and if necessary, supplements other substances such as carriers, stabilizers, solvents, excipients, diluents and the like. It can be used in combination with the ingredients. In addition, the form may be any of the forms usually used for these compounds, such as powders, granules, tablets, and capsules. In the case of fish that are sensitive to the taste and odor of the compound, a method such as coating can prevent a decrease in the palatability of the feed and prevent the compound from leaking.

In the case of fish, orally administered drugs are usually added to feed. When the therapeutic agent or parasite repellent of the present invention is added to the feed, it is preferable to use a feed in which the nutritional components and physical characteristics required for each fish species are taken into consideration. The feed is usually pellets made by mixing fish meal, bran, starch, minerals, vitamins, fish oil, etc., or powdered feed (mash) made by adding vitamins to frozen fish such as sardines and fish meal. Is used as a mixture of pellets. Since the daily feed amount is almost determined by the type and size of the fish, the therapeutic agent or the anthelmintic agent of the present invention in an amount converted to the above-mentioned dosage is added to the feed. The therapeutic agent or parasite repellent of the present invention may be administered in a single daily dose or in several divided doses. Since the therapeutic agent of the present invention is used by being added to a fish feed, it is preferable to make a preparation suitable for adding an appropriate concentration to the feed that the fish ingests per day. Specifically, it is preferable to formulate and use the benzimidazole compound as an active ingredient in the preparation so as to contain 1 to 50% by weight, preferably 5 to 30% by weight, and more preferably 10 to 20% by weight.

In one embodiment of the invention, a feed for salmon fish used in the control of muscle thawing by microspores, 0.0001 to 5% by weight, preferably 0.0005 to 2.5% by weight of a benzimidazole compound by dry weight. The feed is provided which contains by weight, more preferably 0.001 to 0.5% by weight.

The "environment in which microsporidia are infected" is not particularly limited as long as the fish can be infected with microsporidia, and the fish are exposed to the outside world in which microsporidia may exist, for example, using a cage. Includes situations where fish are cultivated using sea surface cages. In addition, even in a closed system environment, if there is a situation in which microsporidia can be infected in the fish breeding environment, the environment is also included in the "environment in which microsporidia is infected".

As shown in the examples, coho salmon were transferred from freshwater to a sea surface cage and kept in the cage for 42 days to infect microsporidia. Twenty-one days after returning the infected coho salmon to land, administration of the drug was started, and a high anthelmintic effect could be obtained. In the actual aquaculture site, the first drug is administered 20 to 60 days or 30 to 60 days after the sea surface, or after a period of 260 to 780 ° C or 390 to 780 ° C at the cumulative water temperature. Since microsporidia infection continues in the sea surface cage, the second drug is administered 20 to 60 days or 30 to 60 days after the end of drug administration, or after a period of 260 to 780 ° C or 390 to 780 ° C at the cumulative water temperature. Is preferable. Therefore, the extermination agent of the present invention is used every 20 to 60 days or 30 to 60 days when the fish is placed in an environment infected with microsporidia, or every period of 260 to 780 ° C or 390 to 780 ° C at the integrated water temperature. By setting an administration period and repeating it, microsporidia can be exterminated and the onset can be prevented.

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

Examples of the present invention will be described below, but the present invention is not limited thereto.

<Effect of albendazole administration on fish feeding habits>
Marine trout (marine rainbow trout), yellowtail, and tiger puffer were housed in the aquarium. The size of the aquarium was 500 liters for marine trout and 200 liters for yellowtail and tiger puffer. Sand filtration and UV sterilization seawater was injected into each tank under the condition of 2.4 liters / minute. Fish weight was measured the day before albendazole administration. After acclimatization, the test feed was fed to each fish species and each plot for 5 consecutive days. Albendazole was administered once daily. To prepare the feed containing albendazole, a predetermined amount of commercially available feed (trade name: Otohime, manufacturer: Marubeni Nisshin Feed) and albendazole (manufactured by Tokyo Chemical Industry Co., Ltd.) were placed in a polyethylene bag and diluted 2-fold. A spreading agent (low-saccharified reduced water candy, trade name: SE30, manufacturer: Bussan Food Science Co., Ltd.) was added in an amount of 4% of the feed weight and stirred. The feed for the control group was prepared by adding only diluted SE30 in an amount of 4% of the feed weight and stirring. The amount of feed was set at 1% by weight for marine trout, 2% by weight for yellowtail, and 1.5% by weight for tiger puffer fish. The effect of albendazole on food intake was evaluated by observing the food status. Table 1 shows the water temperature, the body weight of the fish to be tested at the start of the test, the number of fish to be tested, the dose of albendazole, and the results.

In yellowtail and tiger puffer, daily administration of albendazole 40 mg / kg fish body weight for 5 days did not affect feeding habits. In yellowtail, administration of 200 mg / kg fish body weight per day for 5 days did not affect the feeding ability, but in tiger puffer, administration of 80 mg / kg fish body weight per day for 5 days affected the feeding ability. .. On the other hand, marine trout was affected by feeding habits at a very small dose of 10 mg / kg fish body weight per day for 5 days. From these results, it was found that the effect of albendazole administration on the diet differs depending on the fish species. In particular, marine trout was affected by feeding even at a small dose of albendazole 10 mg / kg fish body weight per day compared to other fish species. It was confirmed that salmonids are less tolerated by benzimidazole compounds.

<Effect of albendazole on coho salmon feeding and anthelmintic effect on microspore disease>
Fry of coho salmon was introduced into the cage of the fishing ground and bred for 42 days. By breeding this sea surface cage, juvenile coho salmon were naturally infected with microsporidia. The coho salmon brought into the onshore facility were divided into three groups, and each group was housed in a separate 2t aquarium. The average fish weight at this time was 466 g. Table 2 shows the test plots and the number of test tails. Water was injected into the water tank under the same conditions as in Example 1. The breeding 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 necropsied to observe the number of cysts on the side muscles and the presence or absence of muscle melting.

At the end of the breeding test, all fish were picked up from all test plots, cysts on the side muscles were counted by autopsy, and the fish in which cysts were observed were regarded as the onset fish. The observed cysts were measured in length and width to calculate the area. The evaluation was performed by comparing the incidence rate (number of affected fish / number of test fish x 100), the number of cysts of the affected fish, and the cyst area between the control group and the albendazole oral administration group.

Results and discussion In the albendazole-administered group, the feeding became inactive the day after the single administration, and the feeding was half of the prescribed amount. As with marine trout, administration of 10 mg / kg or more of fish body weight to coho salmon adversely affected feeding habits. From these results, it was found that salmonids are more susceptible to feeding adverse effects than other fish species even at a small dose of albendazole 10 mg / kg fish body weight. However, the growth and survival during the breeding period were superior to those of the control group (Table 3). This was presumed to be due to the microsporidia anthelmintic effect described later.

Table 4 shows the anthelmintic effect of albendazole on microsporidia. The incidence of albendazole 20 mg / kg fish body weight single-dose group was clearly lower than that of the control group, demonstrating that albendazole exerts an anthelmintic effect on this causative insect. Furthermore, the insecticidal rate in the 10 mg / kg fish body weight single dose + 1 mg / kg fish body weight 14-day continuous administration group was clearly lower than that in the 20 mg / kg fish body weight single dose group, and the 1 mg / kg fish body weight was less. It was found that the worm can be eradicated by long-term administration even in a large amount. In addition, no decrease in food intake was observed during administration of 1 mg / kg fish body weight for 14 days.

Salmon fish can be adversely affected by feeding at a low dose of 10 mg / kg fish body weight, and administration of 1 mg / kg fish body weight for 14 days can be administered without reducing the feeding and can exterminate microsporidia. It became clear. In addition, no death was observed due to this disease. This causes the infection to be difficult to notice until the fish infected with this insect is shipped.

Here, the number of onsets in Table 4 is the ratio of fish having cysts or melting symptoms in the muscle, and the number and size of cysts are the number and size of cysts or melting. That is, it was shown that administration of albendazole suppressed the occurrence of cyst and muscle melting.

<Anthelmintic effect of albendazole and fevantel on coho salmon microspore disease-1>
Fry of coho salmon was introduced into the cage of the fishing ground and bred for 35 days. By breeding this sea surface cage, juvenile coho salmon were naturally infected with microsporidia. The coho salmon brought into the onshore facility were divided into four groups, and each group was housed in a separate 500L aquarium. The average fish weight at this time was 252 g. Table 5 shows the test plots and the number of test tails. Water was injected into the water tank under the same conditions as in Example 1. The breeding period after delivery was 89 days. The water temperature during the test period was 11.6-15.3 ° 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 necropsied to observe the number of cysts on the side muscles and the presence or absence of muscle melting.

At the end of the breeding test, all fish were picked up from all test plots, cysts on the side muscles were counted by autopsy, and the fish in which cysts were observed were regarded as the onset fish. The evaluation was performed by comparing the incidence rate (number of affected fish / number of test fish x 100) and the number of cysts of the affected fish in the control group and the drug oral administration group. During the test period, some test plots had low survival due to the occurrence of dead fish due to caudal fin defects due to territorial behavior. Therefore, the anthelmintic effect was evaluated by adding the autopsy results of dead fish 65 days after breeding in the onshore aquarium to the results of surviving fish.

Results and discussion When the muscles of 10 fish were examined when they were brought into the land tank, no cysts were observed. Table 6 shows the fish weight, fork length, and mortality rate of each group at the end of the test. The growth and survival of albendazole and fevantel were superior to those of the control group, suggesting that administration of these drugs did not adversely affect coho salmon. In addition, when the dead fish were observed, the caudal fin defect was remarkable, and the fish became anemic. Therefore, the death in this study was due to a caudal fin defect due to territorial behavior.

The anthelmintic effects of albendazole and fevantel on microsporidia are shown in Table 7. The incidence was 10% in the control group, compared with 0% in the albendazole and fevantel-treated groups. It was found that this insect can be exterminated by long-term administration even at a low dose of 1 mg / kg fish body weight per day. In addition, it was revealed that Fevantel, like albendazole, has an anthelmintic effect on microsporidia that thaw newly developed muscles in salmonids.

<Anthelmintic effect of albendazole and fevantel on coho salmon microspore disease-2>
Fry of coho salmon was introduced into the cage of the fishing ground and bred for 28 days. By breeding this sea surface cage, juvenile coho salmon were naturally infected with microsporidia. The coho salmon brought into the onshore facility were divided into four groups, and each group was housed in a separate 500L aquarium. The average fish weight at this time was 261 g. Table 8 shows the test plots and the number of test tails. Water was injected into the water tank under the same conditions as in Example 1. The breeding period after delivery was 77 days. The water temperature during the test period was 10.5-14.6 ° C, with an average of 13.0 ° C. In addition, in order to understand the onset of microsporidia at the start of the test, 10 fish were necropsied to observe the number of cysts on the side muscles and the presence or absence of muscle melting.

At the end of the breeding test, all fish were picked up from all test plots, cysts on the side muscles were counted by autopsy, and the fish in which cysts were observed were regarded as the onset fish. The evaluation was performed by comparing the incidence rate (number of affected fish / number of test fish x 100) and the number of cysts of the affected fish in the control group and the drug oral administration group. During the test period, some test plots had low survival due to the occurrence of dead fish due to caudal fin defects due to territorial behavior. Therefore, the anthelmintic effect was evaluated by adding the autopsy results of dead fish 38 days after breeding in the onshore aquarium to the results of surviving fish.

Results and discussion When the muscles of 10 fish were examined when they were brought into the land tank, no cysts were observed. Table 9 shows the fish weight, fork length, and mortality rate of each group at the end of the test. The growth and survival of albendazole and fevantel were superior to those of the control group, suggesting that the administration of these drugs did not adversely affect coho salmon. In addition, when the dead fish were observed, the caudal fin defect was remarkable, and the fish became anemic. Therefore, the death in this study was due to a caudal fin defect due to territorial behavior.

Table 10 shows the anthelmintic effects of albendazole and fevantel on microsporidia. The incidence was 11.8% in the control group, compared with 0% in the albendazole and fevantel-treated groups. The results of Example 3 were reproduced. 1 mg / kg per day Even a small dose of fish can be administered for a long period of time to exterminate this insect, and like albendazole, Fevantel is also an anthelmintic against microsporidia that melts newly developed muscles in salmonids. It was shown again to have an effect.

<Base sequence analysis of microsporidia that causes muscle melting>
Tissues of sea surface-cultured coho salmon lesions showing this symptom were collected, and DNA was extracted after confirming the presence of causative microsporidia spores under a microscope. Then, according to previously reported literature (Bell, AS, et al., J. Eukaryot. Microbiol. 2001, 48, 258-265.), 530f (5'-GTGCATCCAGCCGCGG-3') (SEQ ID NO: 9), and 580r ( The nucleotide sequence of the causative microsporidia of the ribosomal DNA region of about 1500 bp amplified by the primer pair of 5'-GGTCCGTGTTTCAAGACGG-3') (SEQ ID NO: 10) was determined. The obtained nucleotide sequence was subjected to a BLAST search (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm whether it was known. Furthermore, in the obtained nucleotide sequence, the SSU ribosomal DNA region of about 940 bp was compared with the sequences of five known microsporidia species that have been reported to infest salmonids. Clustal W (https://clustalw.ddbj.nig.ac.jp/) was used for the comparison.

DNA having 8 patterns of nucleotide sequences (SEQ ID NOs: 1 to 8) including mutations was collected from the causative microsporidia. The following studies were conducted using type 1 (SEQ ID NO: 1) of them. In general, the rDNA region has a repeat structure, and it is known that even within the same individual, the sequences may not match in each repeat. The similarity of each sequence was 99% or more. The obtained base was about 1.5 kbp. Homology was compared with other species using about 900 pb on the 5-terminal side of the acquired base, which corresponds to the nucleotide sequence registered for other microsporidia.

The resulting sequence did not match any of the species registered in the database. In addition, the similarity to the previously reported microsporidia base sequence in salmonids remained at about 68-84%. Moreover, the symptoms observed in the affected fish were also different from any of these species. Therefore, it was clarified that this species is an unknown species that has not been reported to occur so far.

Symptoms of microsporidia species in salmonids have been reported in the following literature: Dis Aquat Org 101: 43-49, 2012; Dis Aquat Org 44: 223-230, 2001; Infectious Diseases / Parasitic Diseases ”, supervised by Shuzo Egusa, published by Hoshisha Koseikaku. Pages 309-312 and 318-320 (2004).

Oral administration of microsporidia parasitizing the muscles of salmonid fish can provide an effective repellent. Since no deaths due to this disease are observed, the commercial value will be lost if the fish raised to the shipping size at a high cost are infected. If the melting of muscles can be prevented by the present invention, it is possible to prevent a decrease in commercial value and have industrial value.

Claims (14)

A method for preventing muscle melting by microsporidia occurring in salmonid fish, which comprises administering a microsporidia exterminating agent containing a benzimidazole compound as an active ingredient. The method according to claim 1, wherein a administration period is provided in which a microsporidia extermination agent is continuously administered to a farmed fish, Salmonids, for 5 days or more. The method according to claim 1 or 2, wherein a period of administration of a microsporidia repellent and a period of withdrawal are provided for a farmed fish of the order Salmonids in a cycle of 20 to 60 days. The first or second claim, wherein the salmonids, which are farmed fish, are provided with a administration period and a drug holiday of the microsporidia extermination agent in a cycle of 260 to 780 ° C. at an integrated water temperature. Method. An agent for exterminating microsporidia parasitizing the muscles of salmonids, which contains a benzimidazole compound as an active ingredient. The benzimidazole compound is one or more compounds selected from albendazole, fevantel, fenbendazole, oxfendazole, mebendazole, flubendazole, oxybendazole, triclabendazole, ricobendazole and thiabendazole. The repellent according to claim 5. The exterminating agent according to claim 4 or 5, wherein the salmonid fish is a salmonid fish or a smelt family fish. The extermination according to claim 7, wherein the salmonid fish belongs to any of the genus Salmon, the genus Salmo, the genus Iwana, and the genus Taimen, and the fish of the family Smelt belongs to the genus Smelt and the genus Ayu. Agent. The pesticide according to any one of claims 5 to 8, wherein the benzimidazole compound is administered at a dose of 1 to 25 mg / kg fish body weight / day. The pesticide according to any one of claims 5 to 9, wherein the administration period is 5 days or more. The pesticide according to any one of claims 5 to 10, wherein a administration period and a drug holiday are provided in a cycle of 20 to 60 days for the salmonid fish which is a farmed fish. Any of claims 5 to 10, wherein a period of administration of a microsporidia extermination agent and a period of withdrawal are provided for salmonids, which are farmed fish, in a cycle of a cumulative water temperature of 260 to 780 ° C. The method described in. The pesticide according to claim 11 or 12, wherein the total dose of the pesticide per cycle is 5 to 50 mg / kg fish body weight. The pesticide according to any one of claims 5 to 9, which is administered in a single dose.

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