JP2011241192A - Disease controlling agent for crustacean, and feed containing the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a disease controlling agent having effects for enhancing biophylaxis ability naturally included in a crustacean, and reducing damage caused by various diseases including crustacean acute viremia.SOLUTION: The disease controlling agent contains lectin originated in a plant, and has the effect for reducing damage caused by various diseases including crustacean acute viremia.

Description

  The present invention provides a crustacean that has an effect of reducing the damage caused by various diseases including crustacean acute viremia, which is a serious threat to the industry by enhancing the biological defense ability inherent to the crustacean. The present invention relates to a composition for controlling diseases and a method for controlling crustacean diseases using the composition.

  In recent years, shrimp farming such as prawns and bovine shrimp has increased tremendously, especially in Southeast Asia and Latin America. However, due to overcrowded farming and the environmental degradation of farms, Diseases such as illness and bacterial disease are widespread, causing remarkable drowning.

  Conventionally, such crustacean diseases have been treated with drugs such as antibiotics and antiviral agents. However, the use of these drugs can cause drug-resistant bacteria and drug-resistant viruses, and there is a risk of drug residues in the organism, and its use is currently limited.

  In recent years, a control agent that obtains an antiviral effect without using a drug has been developed (Patent Documents 1-2). However, these control agents are effective only for specific pathogenic virus species, and cannot be expected to have an effect on other pathogenic virus species, and due to their pharmacological action, there is concern about the emergence of resistant viruses. Furthermore, crustacean infection prevention and treatment agents containing oligosaccharides derived from animals and plants and feeds containing the same have been developed (Patent Documents 3-4). However, since these therapeutic agents use various purified products derived from animals and plants, production costs are required.

  Therefore, at present, crustacean farms or seedling production facilities are eager to develop a drowning reduction method that can be expected to have a safe, inexpensive, and reliable effect different from conventional methods.

  Many plants are traditionally used in Chinese medicine and the like, and are considered to contain various health benefits such as medicinal and immunostimulatory functions. For example, lectins that are abundantly contained in plant seeds bind to sugar chains of cell membrane complex carbohydrates (e.g., glycoproteins, glycolipids), thereby causing cell aggregation, glycoconjugate precipitation, mitogenesis, functional activation, It is a protein that has effects such as cytotoxicity, and has been shown to have a mitogenic function (cell division activation function of T cells and B cells) and an effect of aggregating diseased microorganisms (non-patented) Literature 1-3). However, to date, there has been no report that plant-derived lectins are effective in controlling diseases of crustaceans and have the effect of enhancing the crustacean biological defense function.

Japanese Patent Laid-Open No. 9-206067 JP 2008-169131 A Japanese Patent Laid-Open No. 10-45600 Japanese Patent Laid-Open No. 10-45605

Vania M.M.Melo et al., `` Plant Science '', 2005, 169, p.629-639 Ana Paula de A. Boleti et al., "Journal of Agricultural and Food Chemistry", 2007, 55, p.2653-2658 Li Huanga et al., "Immunology letters", 2008, Vol. 121, p.148-156

  The present invention is a crustacean which has an effect of reducing the damage caused by various diseases including crustacean acute viremia, which is a serious threat to the industry by enhancing the biological defense ability inherent to the crustacean. And a method for controlling crustacean diseases using the composition are provided.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a composition containing a lectin derived from a plant, particularly bitter gourd, enhances the body defense ability inherent in crustaceans, and crustaceans. The inventors have found that the present invention is effective in controlling various diseases including acute viremia, and have completed the present invention.

That is, the present invention is as follows.
[1] A composition for controlling crustacean diseases, comprising plant powder, crude plant lectin and / or purified plant lectin.
[2] The composition of [1], wherein the plant is bitter gourd.
[3] The composition of [1] or [2], which can enhance the crustacean biological defense function.
[4] The composition according to any one of [1] to [3], wherein the crustacean disease is crustacean acute viremia.
[5] The composition according to any one of [1] to [4], which is in the form of a shellfish feed or an injection.
[6] A method for controlling crustacean diseases, comprising administering the composition according to any one of [1] to [5] to a crustacean.
[7] The method according to [6], wherein the composition of any one of [1] to [5] is administered to the crustacean to enhance the crustacean biological defense function.
[8] The method of [6] or [7], wherein the crustacean disease is crustacean acute viremia.
[9] The composition according to any one of [1] to [5], or the method according to any one of [6] to [8], wherein the crustacean is a prawn family.

  According to the present invention, a crustacean that has an effect of reducing the damage caused by various diseases including crustacean acute viremia, which is a serious threat to the industry, is strengthened by the natural defense ability of the crustacean. A composition for controlling a disease and a method for controlling a crustacean disease using the composition can be provided, and as a result, productivity in a farm or a seed production facility can be improved.

FIG. 1 is a characteristic diagram showing the crustacean acute viremia control effect by the administration (injection) of crude purified bitter gourd lectin. FIG. 2 is a characteristic diagram showing the crustacean acute viremia control effect by administration (injection) of purified bitter gourd lectin. FIG. 3 (A) shows a schematic scheme for the production of crustacean feed to which bitter gourd lectin has been added. FIG. 3 (B) is a photograph showing an embodiment of a crustacean feed to which the produced bitter lectin is added. FIG. 4 is a characteristic diagram showing the protective effect of crustacean acute viremia by the administration (oral) of a feed supplemented with bitter gourd seed powder. (A) 1% concentration of virus inoculation; (B) 2% concentration of virus inoculation FIG. 5 is a characteristic diagram showing the protective effect of crustacean acute viremia by oral administration of purified goya lectin-added feed. FIG. 6 is a graph showing changes in blood count (× 10 ⁶ cells / ml) of prawn prawns by oral administration of purified goya lectin-added feed (p <0.05, 0.01). The alphabets attached to the bar graphs indicate that there is no significant difference when compared with the treatment sections with the same alphabets in the same treatment days. FIG. 7 is a graph showing changes over time in the blood cell composition of prawn prawns by oral administration of purified goya lectin-added feed (p <0.05, 0.01). (A): A region; (B): B region. The alphabets attached to the bar graphs indicate that there is no significant difference when compared with the treatment sections with the same alphabets in the same treatment days. FIG. 8 is a graph showing changes over time in the phenol oxidase activity of prawn prawns by oral administration of purified goya lectin-added feed (p <0.05, 0.01). The alphabets attached to the bar graphs indicate that there is no significant difference when compared with the treatment sections with the same alphabets in the same treatment days.

  The composition of the present invention contains plant powder, crude purified plant lectin and / or purified plant lectin.

  In the present invention, the term “plant” refers to red beans, peanuts, red-bellied beans, dolicos beans, soybeans, banderia beans, lentils, Miyakogusa, Inuenju, green beans, lima beans, peas, winged bean, Enju, harineishida, faba beans, purple beans, mayumi, sea lion , Elderberry, yellow-crowned cucumber, castor beetle, squirrel moth, snowy hannah, amaryllis, daffodil, wheat, damselfly, tomato, potato, nettle, pokeweed, amaranth, jackfruit, American cricket, fuji, hairy vetch, banana, garlic, garlic It refers to Kikuimo, bitter gourd, etc. (not limited to these). Preferably, the “plant” is bitter gourd. The kind of bitter gourd used in the present invention is not particularly limited.

  In the present invention, the term “plant” refers to a plant body leaf, stem, root, fruit, bulb, seed, or one or more combinations thereof. Lectins are present in the leaves, stems, roots, fruits, bulbs, seeds, etc. of plant bodies, and one or more of these can be used in combination for extraction. Generally, however, lectins are contained in large amounts in seeds. Since it is contained, it is preferable to use seeds. The seeds may be mature or immature, but preferably mature seeds are used. Moreover, it is preferable to use what dried the plant.

  “Plant powder”, “crudely purified plant lectin” and “purified plant lectin” used in the present invention are based on known methods, for example, the methods described in Japanese Patent No. 3849945, Japanese Patent Application Laid-Open No. 2006-126025, etc. It can prepare by the method of.

  “Plant powder” is pulverized to a desired size by a known method such as coffee mill, ultrasonic treatment, French press, stone mortar, mortar, crushing with homogenizer, crushing with glass beads, coffee mill, etc. Can be obtained.

  “Roughly purified plant lectin” is an aqueous solution for extraction generally used for protein extraction (eg, distilled water, Tris buffer solution, phosphate buffer solution, Tricine buffer solution, Hepes buffer solution, MOPS buffer solution). , Carbonate buffer, citrate buffer, borate buffer, MES buffer, PIPES buffer, etc.), and the resulting extract is subjected to ethanol precipitation. Can be obtained by drying, adding to physiological saline and centrifuging, and collecting the supernatant. The extract may be subjected to one or a plurality of filtration and / or centrifugation steps as required before being subjected to ethanol precipitation. The obtained crude purified plant lectin contains contaminating proteins, sugars and the like together with the plant lectin, and the required plant lectin activity is also sufficient for the crude purified product.

  "Purified plant lectin" is a known method (Patent No. 3849945, JP 2006-126025 A, J. Toyama et al., "Asian Journal of Plant Sciences", 2008, 7 ( 7), p. 647-653), and can be prepared by the following method.

  That is, the crudely purified plant lectin is applied to a sugar-binding affinity column to adsorb the lectin, and after washing impurities, the lectin is eluted using a high-concentration sugar solution or the like. The sugar to be bound to the affinity column is selected according to the sugar specificity of the lectin to be purified. For example, in the case of bitter gourd, a lectin can be efficiently purified by an affinity column to which galactose is bound. Further, if necessary, the degree of purification can be further improved by adding a technique such as gel filtration.

  In preparing crude plant lectin and purified plant lectin, instead of or in addition to the above methods, known methods used for protein purification, for example, ammonium sulfate salting out, precipitation with organic solvents (ethanol, methanol, acetone, etc.) Separation, ion exchange chromatography, isoelectric focusing chromatography, gel filtration chromatography, hydrophobic chromatography, adsorption column chromatography, affinity chromatography using substrates or antibodies, chromatography such as reverse phase column chromatography, precision It is possible to purify by using one or a combination of filtration, ultrafiltration, filtration treatment such as reverse osmosis filtration and the like.

  Plant lectins contained in plant powders, crude purified plant lectins and purified plant lectins are proteins having an affinity for sugar, and the molecular weight varies depending on the plant species, and is generally in the range of 8,000 to 170,000. Many plant lectins can aggregate blood cells. Goya lectin has affinity for human H antigen sugar chains, has a molecular weight in the range of 100,000 to 170,000, and can aggregate blood cells.

  The plant powder, crude plant lectin and purified plant lectin obtained in the present invention may be dissolved or suspended in an appropriate solvent according to the use, or may be a general drying method (for example, natural drying, heat Drying, freeze-drying, etc.) to form a dry powder.

  In the composition of the present invention, in addition to the above-mentioned plant powder, crude purified plant lectin and / or purified plant lectin, an appropriate carrier, diluent, emulsion, excipient, extender, binder, infiltrant, disintegrant , Surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives, colorants, flavoring agents, stabilizers, water-soluble solvents (water, lower alcohols, polyols, etc.), hydrophobic Additives such as solvents (such as higher fatty acid esters and lipophilic alcohol) may be included.

  The content of the plant powder, the roughly purified plant lectin and / or the purified plant lectin in the composition of the present invention is appropriately determined according to the administration target, the type and shape of the composition, the type and severity of the disease, and the like. Each can contain an amount appropriately selected from the range of 0.0001 mg to 100 mg per mL of the composition.

  The composition of the present invention can be prepared in an appropriate form according to the administration method. For example, as an oral administration agent, powder, granule, sheet, paste, liquid (solution, suspension, Mixed liquid, etc.), tablets, capsules, granules, powders, syrups and the like, and parenteral agents include injections, inhalants, ointments, patches, absorbents, etc. Not. Preferably, the composition of the present invention is in the form of injection or oral administration.

  An injection can be prepared using a known technique, and the above-mentioned plant powder, crude purified plant lectin and / or purified plant lectin is mixed with a common solvent for injection (for example, distilled water for injection, physiological saline). , Glucose aqueous solution, vegetable oil for injection, sesame oil, peanut oil, soybean oil, sorghum oil, propylene glycol, polyethylene glycol and the like, but not limited thereto). If necessary, an isotonic agent, a solubilizer, a stabilizer, a preservative, a suspending agent, an emulsifier, or the like may be added. Moreover, an injection may be prepared by re-preparing a liquid preparation from a plant powder, a crudely purified plant lectin and / or a lyophilized product of a purified plant lectin immediately before use using the solvent for injection.

  The oral administration agent can be prepared using a known technique, and a mixture comprising the above-mentioned plant powder, the roughly purified plant lectin and / or the purified plant lectin and, if necessary, the above additive is dried, It can be produced by molding by a known method such as granulation, compression, molding, etc., and applying a treatment such as coating as necessary. In particular, the oral preparation is preferably prepared as a crustacean feed.

  The crustacean feed containing the composition of the present invention can be produced using a general feed production method. That is, plant powder, crude purified plant lectin and / or purified plant lectin is added to and mixed with the basic feed material and then molded and dried to obtain a dry pet, or plant powder, crude purified plant lectin and / or After adding and mixing the purified plant lectin, an appropriate amount of water may be added to form a moist pellet. The basic feed materials are mainly fish meal, squid meal, krill meal, cereals, and cholesterol, lecithin, vegetable oil meal, various binders (CMC, sodium alginate, guar gum, etc.), vitamin mixtures, mineral mixtures, etc. A well-known feed material for crustaceans, such as a combination with raw food, can be used, and is not particularly limited. The addition of plant powder, crude plant lectin and / or purified plant lectin to the basic feed material can be done before or after cooking the basic feed material, but in order to avoid inactivation of the plant lectin It is preferable to add after cooking. If necessary, the feed may be mixed with commonly used excipients such as yeast powder, wheat flour, defatted rice bran, soybean meal, calcium carbonate, anhydrous silicic acid and the like. In addition, vitamins such as vitamin E, various minerals such as calcium, phosphorus, zinc and selenium, amino acids such as methionine, lysine and tryptophan, enzymes such as protease and lipase, squid extract and krill extract that have an effect of improving palatability Fish solubil, yeast extract and the like may be further added. A person skilled in the art would be able to appropriately prepare and shape the feed in order to maintain sufficient shape retention in water and ensure that the active ingredient is predated by the crustaceans.

  The content of plant powder, crude purified plant lectin and / or purified plant lectin in the above injections and crustacean feed is treated with erythrocytes of various animals such as rabbits, cows, humans, etc., or these are treated with enzymes such as sialidase and trypsin. It can be expressed by the erythrocyte agglutination titer of lectin. Plant-derived lectins bind to agglutinogens on the surface of the red blood cell membrane and cause red blood cells to aggregate. The type and titer of red blood cells that can be aggregated by plant lectins vary depending on the type of plant lectin. For example, bitter gourd-derived lectin specifically binds to H antigen among agglutinogens on the erythrocyte membrane surface consisting of A, B and H antigens of human erythrocytes, and causes erythrocytes to aggregate. The hemagglutination titer of lectin can be determined based on a known method (for example, Japanese Patent No. 3849945, Japanese Patent Application Laid-Open No. 2006-126025).

  The content of the plant powder, the roughly purified plant lectin and / or the purified plant lectin in the injection and the crustacean feed varies depending on the type of plant used. For example, when the plant is bitter gourd, if it is an injection, the total titer of the above hemagglutination titer is set to 4 to 1024, preferably 8 to 512, more preferably 64, and purified goya lectin. Each can be included in the range of 3.2 to 640, preferably 6.4 to 640, and more preferably 6.4. Here, the total titer of the injection can be determined by multiplying the erythrocyte aggregation titer (value per mL) by the amount of solution to be injected. The injection may contain bitter gourd seed powder having a total titer in the above-mentioned range. Even when other plants are used, the injection can contain at least an amount of plant powder, crude purified plant lectin and / or purified plant lectin corresponding to the total titer in the above range.

  In addition, for example, when the plant is bitter gourd, if it is a feed for crustaceans, the total titer of the above-mentioned hemagglutination titer per kg of feed, 51200-512000 bitter gourd powder, 2130-213000 roughly purified bitter gourd lectin, Purified bitter gourd lectins can each be included in the range 2130-213000. Here, the total titer of the crustacean feed can be determined by multiplying the erythrocyte aggregation titer (value per mL) by the daily feed intake of the crustacean. The daily feed intake of the crustacean can be appropriately selected from the range of 50 to 500 mg. Even when other plants are used, the feed for crustaceans can contain at least an amount of plant powder, crude purified plant lectin and / or purified plant lectin corresponding to the total titer in the above range.

  The administration conditions for the composition of the present invention can be appropriately determined according to the administration subject, the type and shape of the composition, the type and severity of the disease, and the like. For example, administration of the composition of the present invention can be performed by injection, immersion, spraying or oral administration. Preferably, the composition of the present invention is administered by intramuscular, intraperitoneal, etc., preferably by injection into the second abdominal node, or orally via the crustacean feed. The dosage of the composition of the present invention can be appropriately determined in the range of 0.01 to 1.5 mL in the case of the injection, and can be appropriately determined in the range of 50 to 500 mg in the case of the crustacean feed. . The amount of plant powder, crude plant lectin and / or purified plant lectin to be administered per animal per day depends on the type of plant used. For example, when the plant is bitter gourd, the total titer of the above hemagglutination titer is 3.2 to 16, preferably 6.4 equivalent amount of bitter gourd seed powder, crude purified bitter gourd lectin and / or purified bitter gourd per animal per day. What is necessary is that lectins can be administered. Even when other plants are used, it is only necessary to administer an amount of plant powder, crude plant lectin and / or purified plant lectin in an amount corresponding to at least the total titer in the above range per animal per day. Administration of the composition of the present invention may be performed only once a day, but may be repeated two or more times at intervals, or intermittently throughout the day by ingestion by an animal. Also good.

  The composition of the present invention exhibits a high effect in controlling crustacean diseases by being administered to crustaceans.

  The crustacea to which the composition is administered in the present invention may be any crustacea, and examples include shrimp, crab, crayfish, crayfish, krill, daphnia and the like. Particularly suitable examples of these crustaceans include, but are not limited to, decapod crustaceans, such as the Shrimp Family, the Shrimp Family, the Shrimp Family, the Shrimp Family, the Red Shrimp Family, the Shrimp Family, the Shrimp Family, the American Crayfish Family And other organisms belonging to the genus, such as the Decapoda crabs, spider crabs, crabs, crabs, crabs, and crabs. More specific examples of crustaceans suitable for administration include, but are not limited to, organisms in the family Penaeidae, such as Farfantepenaeus, Fenneropenaeus, Litopenaeus, Marsupenaeus, Melicertus, Metapenaeopsis, Metapenaeus, Penacheus, Examples include shrimp belonging to the genus Xiphopenaeus. Among the shrimps that are suitable for administration, for example, edible shrimp include shrimp (Marsupenaeus japonicus), southern shrimp (Melicertus canaliculatus), shrimp (black tiger) (Penaeus monodon), red shrimp (Penaeus chinensis), bear shrimp (Penaeus semi) ), Yellow shrimp (Penaeus latisulcatus), Indian shrimp (Fenneropenaeus indicus), reed shrimp (Metapenaeus ensis), horse shrimp (Metapenaeus intermedius) and the like, but are not limited thereto.

  “Crustacean diseases” include infections caused by pathogenic bacteria and viruses, such as Vibrio disease, shell disease, epiphytic fungal disease, ragenidiosis, halocrustosis, haliftrossis, fusarium disease, aphanomyces Disease, acute viremia (PAV), midgut necrosis (BMN), Taura syndrome (TSV), infectious subcutaneous hematopoietic necrosis (IHHNV), yellowhead disease (YHV), etc. It is not limited. Particularly preferred is acute viremia (PAV). “Acute viremia (PAV)” means a crustacean infection caused by white spot syndrome virus (WSSV) (also called white spot syndrome, vitiligo disease, etc.). This disease is typically characterized by white spots appearing on the body surface and death at an extremely high rate.

  In the present invention, “control of crustacean disease” refers to the elimination of pathogenic bacteria or viruses that have invaded the living body and / or the suppression of their growth, thereby preventing the development of crustacean diseases or It means to reduce the incidence significantly. In particular, the composition of the present invention controls crustacean diseases by enhancing the body defense function of crustaceans. “Bioprotective function” refers to the immune system that crustaceans naturally have. The crustacean immune system, like vertebrates, is divided into cellular and humoral factors. Cellular factors include phagocytosis / envelopment and nodule formation by blood cells, and humoral factors include phenol oxidase precursor (proPO) activation, lysozyme, lectin and complement-related factors. Crustaceans are thought to exert biological defense functions by the mutual cooperation of these cellular factors and humoral factors upon infection with pathogenic bacteria or viruses.

  The control effect of crustacean disease obtained by administration of the composition of the present invention is typically shown by an increase in survival rate (decrease in mortality rate) in the presence of pathogenic bacteria or viruses. Survival can be obtained by dividing the number of crustacean individuals alive in a given number of days after virus exposure by the total number of individuals before virus exposure. For example, the survival rate of crustaceans administered the composition of the present invention in the presence of pathogenic bacteria or viruses is approximately 1.5 times or 2 times that of crustaceans not administered the composition of the present invention. Can be 3x, 4x, 5x, 6x, 7x or more.

  Moreover, the control effect of the crustacean disease obtained by the administration of the composition of the present invention is shown by the activation of the biological defense function of the crustacean administered with the composition of the present invention. “Activation of the biological defense function” means an increase in the total blood cell count and a change in blood cell composition (for example, blood cells with small cells and few cytoplasmic granules (eg, transparent cells) or blood cells with large cells and many cytoplasmic granules). (E.g. increased granulocytes, hemi-granulocytes, etc.)), increased phagocytic activity of blood cells, and increased phenol oxidase activity. Regarding the phagocytic activity of blood cells, aseptically remove blood from crustaceans, mix with latex beads in a test tube, and then smear on slide glass to phagocytose the number of cells and phagocytic beads. Calculate phagocytic activity. For phenol oxidase activity, homogenize the collected crustacean blood, add it to a buffer containing L-DOPA (3,4-dihydroxy-L-phenylalanine), leave it still, and measure the absorbance at a wavelength of 640 nm. The degree of melanin production is measured (T. Itami et al., “Aquaculture”, 1998, 164, 277-288). For example, the total blood count of crustaceans administered the composition of the present invention is increased by approximately 1.2 times, 1.5 times or more compared to that of crustaceans not administered the composition of the present invention. obtain. In addition, the predetermined blood cell count in the crustacean administered with the composition of the present invention is approximately 1.2 times, 1.5 times, 2 times or about that of the crustacean not administered with the composition of the present invention. It can increase more than that. In addition, the phagocytic activity of blood cells in crustaceans administered with the composition of the present invention is increased by about 1.3 times, 1.5 times or more compared to that of crustaceans not administered with the composition of the present invention. Can do. Furthermore, the phenol oxidase activity in crustaceans administered with the composition of the present invention is approximately 2-fold, 3-fold, 4-fold, 5-fold or greater compared to that of crustaceans not administered with the composition of the present invention. It can rise above.

  Since the control effect of the crustacean disease obtained by the administration of the composition of the present invention depends partly or completely on the biological defense function inherent in the crustacean, the effect is against specific pathogenic bacteria or viruses. It is not limited to an effect and may be effective against various pathogenic bacteria or viruses. In addition, such characteristics of the composition of the present invention make it possible to avoid the development of drug-resistant bacteria or viruses. Furthermore, since the lectin contained in the composition of the present invention as an active ingredient is derived from a plant, it is possible to avoid the problem of drug residue.

  The following examples illustrate the present invention in more detail. However, the present invention is not limited to these examples.

Example 1. Control effect of acute viremia (hereinafter referred to as “PAV”) by administration (injection) of crude purified bitter gourd lectin A crude purified bitter gourd lectin solution was prepared by a known technique (Japanese Patent No. 3849945, JP 2006-126025 A). ) To adjust the hemagglutination titer (hereinafter referred to as “titer”). The crude purified bitter gourd lectin was added to physiological shrimp saline for the following composition.

Immersed attack with white spot syndrome virus (hereinafter referred to as “WSSV”) after injecting 0.1 mL of the crude purified bitter gourd lectin solution (titer 8, 64, 512) to the shrimp prawn (1 fish, 15 fish) Went. That is, WSSV was adjusted so that the amount of virus was 10 ¹⁴ copies / μL in the breeding seawater, and prawns were immersed in this virus suspension for 4 hours and then returned to the breeding tank. As the control group, a group immersed in breeding seawater containing no virus for 4 hours was used. After that, the survival rate of the shrimp for 2 weeks was investigated.

  The results are shown in FIG. Eleven days after immersion attack with WSSV, the survival rate of the prawns was 50% in the control group, while the roughly inoculated group with the purified goya lectin solution showed a high survival rate of 67 to 93%. In particular, the prawns inoculated with the roughly purified bitter gourd lectin solution having a titer of 64 showed a very high survival rate of 93%.

  This result shows that injection administration of a crude purified bitter lectin solution has a high PAV control effect.

  Next, purified goya lectin solution was prepared by a known method (Patent No. 3849945, Japanese Patent Application Laid-Open No. 2006-126025, J. Toyama et al., “Asian Journal of Plant Sciences”, 2008, Vol. 7 (7), p. 647-653) and the titer was adjusted.

  After injecting 0.1 mL of purified goya lectin solution (titer 6.4, 64, 640) into tiger prawns (15 fish in 1 ward), immersion attack with WSSV was performed, and then the survival rate for 2 weeks was investigated.

  The results are shown in FIG. Survival rate of tiger prawns was 27% in the control group, whereas high viability was shown in the purified indian lectin solution with titers of 6.4 and 640. In particular, prawn shrimp inoculated with a roughly purified bitter gourd lectin solution having a titer of 6.4 showed a high survival rate of 50%.

  This result shows that injection administration of purified bitter gourd lectin solution has a high PAV control effect.

Example 2. Production of crustacean feed containing bitter gourd lectin A crustacean feed containing bitter gourd lectin was prepared. FIG. 3A schematically shows the manufacturing process. In order to prevent inactivation of bitter gourd lectin, usually bitter gourd seed powder, crude purified bitter gourd lectin, or purified bitter gourd lectin was added to the basic feed material after cooking. However, a feed prepared by adding steamed bitter gourd seed powder to the basic feed material and then cooking it was also prepared (referred to as “5-fold cooking”). The feed after molding is shown in FIG. The basic added amount of bitter gourd seed powder, crude purified bitter gourd lectin, or purified bitter gourd lectin is the total titer of 6.4 (= titer 64 × injected dose 0.1 mL) equivalent to the daily feed intake (about 300 mg) (ie total titer 21000 / kg feed).

  The bitter gourd lectin activity in the prepared feed is shown in Table 1 below.

The prepared feed generally retained the expected bitter gourd lectin activity. In addition, the feed prepared by cooking the basic feed material containing bitter gourd seed powder (shown as “5-fold cooking” in the table) still had some activity. Each feed was good in sedimentation in water, shape retention and shrimp palatability.
These feeds were used for the following oral administration experiments.

Example 3 PAV protection effect by administration (oral) of feed containing bitter gourd seed powder Oral administration experiment of feed added with bitter gourd seed powder was conducted. In the experiment, a predetermined diet was administered to prawns for 7 days at the beginning, and the virus was inoculated with 1% or 2% concentration on the 8th day. Thereafter, the same feed was continued and the survival rate for 10 days after virus inoculation was observed. The experiment was carried out in the lectin-added group to which each feed shown in Table 1 in the 1-fold, 5-fold and 5-fold (steamed after addition) groups of the bitter gourd seed powder and the control group without lectin was added. The control group replaced bitter gourd seed powder with casein.

  The results are shown in FIG. When the virus was inoculated at a concentration of 1%, there was no clear difference in the survival rate of prawns in the 1-fold, 5-fold and 5-fold (steamed after addition) and control plots (FIG. 4 (A)). On the other hand, in the case of 2% virus inoculation, the control prawns were completely annihilated 6 days after the inoculation, whereas the survival rate of the prawns in the 1-fold, 5-fold and 5-fold (steamed after addition) was 60 respectively. %, 44%, and 30% (FIG. 4B). From this result, the effect of controlling PAV by oral administration of the feed containing bitter gourd seed powder was confirmed.

Example 4 PAV protective effect by administration (oral) of purified goya lectin-added feed An oral administration experiment of feed to which purified goya lectin was added was conducted. In the experiment, a predetermined diet was first administered to prawns for 7 days, and the immersion attack by WSSV was performed by the above method on the 8th day. Thereafter, the same feed was continuously administered, and the survival rate for 10 days after the virus challenge was observed. The prawns were 12-13 fish per test group, and each test group was repeated twice. The experiment was carried out in the lectin-added group to which each feed shown in Table 1 in the purified goya lectin 0.1-fold, 1-fold, and 10-fold groups was administered, and in the control group to which no lectin was added.

  The results are shown in FIG. On the 10th day after the virus attack, the prawn survival rate was 4% in the control group, compared to 28% in the purified goya lectin-added group, 28%, and 48% in the 0.1 and 1-fold groups. it was high. From these results, the effect of controlling PAV by oral administration of purified goya lectin-added feed was confirmed.

Example 5 FIG. Dynamic observation of biological defense-related factors by administration (injection) of purified bitter gourd lectin As shown in the above Examples, bitter gourd lectin was found to have a PAV control effect by administration to prawns. Therefore, for the purpose of clarifying the mechanism of action, purified goya lectin solution was administered to prawns (0.1 mL injection), and the total blood cell count, blood cell flow site on days 1, 3, 5, 7, and 10 after administration Gram, phenol oxidase (PO) activity and phagocytic activity were measured. The purified bitter gourd lectin solution used in this example has a titer of 6.4, which has the highest protective effect against WSSV in the experiment of Example 1 above. The control group was inoculated only with saline for prawns.
The results are shown in Table 2-5 below.

  As shown in Table 2 below, on the first and third days after injection of the purified bitter gourd lectin solution, an increase in the total blood cell count was observed in the group administered with the purified bitter gourd lectin compared to the control group.

  In addition, as shown in Table 3 below, from the flow cytogram on days 1 and 3 after injection of the purified goya lectin solution, blood cells (referred to as “A region”) with small cells and few cytoplasmic granules and large cells with cytoplasmic An increase in blood cells rich in granules (referred to as “B region”) was observed.

  Furthermore, as shown in Table 4 and Table 5 below, phenol oxidase activity (described as “PO activity”) (Table 4) 3 and 5 days after injection of purified goya lectin solution and blood cells 1 and 10 days after injection It was observed that the phagocytic activity (Table 5) was significantly higher than that of the control group.

  From the above results, it was shown that the injection administration of purified bitter gourd lectin solution changes the total blood cell count and blood cell composition of prawns and can improve the natural defense ability of the prawns by raising the phagocytic activity and PO activity. .

Example 6 Dynamic observation of biological defense-related factors by administration of purified bitter gourd lectin (oral) The blood cell count, flow cytogram and PO activity in prawns after administration of purified goya lectin supplemented feed were measured over time. In other words, purified goya lectin-added feed was orally administered to prawns, and 5 fish were collected on days 1, 3, 5, 7, 10 and 15 after administration, and the blood count, flow cytogram and PO activity were measured. did. The experiment was carried out in the lectin-added group to which each feed shown in Table 1 in the purified goya lectin 0.1-fold, 1-fold, and 10-fold groups was administered, and in the control group to which no lectin was added.
The results are shown in FIGS. 6-8.

  The total blood count in the prawns after administration of the feed containing purified goya lectin increased compared to the control group (FIG. 6).

  The flow cytograms of blood cells in the prawns after administration of the feed containing purified goya lectin were observed to be higher in the A region and the B region than in the control group (FIG. 7).

  The PO activity in the prawns after administration of the feed containing purified goya lectin was higher than that in the control group throughout the test period (FIG. 8).

  Based on the above results, oral administration of purified goya lectin-added feed can change the total blood cell count and blood cell composition of prawns as well as injection administration, and increase the PO activity, thereby improving the natural defense ability inherent to the prawns It has been shown. This result shows that the administration of the purified goya lectin-added feed is effective in improving the body defense ability of prawns.

  The control agent containing the plant-derived component of the present invention can be used in the aquaculture industry as an effective treatment and / or prevention means for crustacean diseases that cause enormous damage, particularly crustacean viremia. Moreover, the control agent containing the plant origin component of this invention can improve the biological defense ability of a crustacean. From these characteristics, the present invention is expected as a new preventive and therapeutic agent for crustacean infections.

Claims (9)

  A composition for controlling crustacean diseases, comprising plant powder, crude purified plant lectin and / or purified plant lectin.   The composition of claim 1, wherein the plant is bitter gourd.   The composition according to claim 1 or 2, which is capable of enhancing a crustacean biological defense function.   The composition according to any one of claims 1 to 3, wherein the crustacean disease is crustacean acute viremia.   The composition according to any one of claims 1 to 4, which is in the form of a shellfish feed or an injection.   A method for controlling a crustacean disease, comprising administering the composition according to any one of claims 1 to 5 to a crustacean.   The method according to claim 6, wherein the biological defense function of the crustacean is enhanced by administering the composition according to any one of claims 1 to 5 to the crustacean.   The method according to claim 6 or 7, wherein the crustacean disease is crustacean acute viremia.   The composition according to any one of claims 1 to 5, or the method according to any one of claims 6 to 8, wherein the crustacean is a lobster family.

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