WO2002092114A1 - Preventives and/or remedies for fish infections - Google Patents

Abstract

Preventives and/or remedies for fish infections characterized by containing algae belonging to the genera Kjellmaniella and Ecklonia, giant kelp and algae belonging to the genus Ascophyllum or extracts thereof. Using these preventives and/or remedies, infections of fishes with viruses or bacteria, in particular, PRDV-infection can be effectively prevented and treated. These preventives and/or remedies can be also added to feeds in fish cultivation.

Description

 Description Preventive and / or therapeutic agent for infection of fish and shellfish

 The present invention relates to a drug for preventing and / or treating infection of fish and shellfish with a virus or a pathogenic bacterium, a feed containing the same, and a method of cultivating fish and shellfish by administering the same. Background art

 In fish farms such as Kuruma shrimp, red sea bream, and flounder, diseases often occur because fish and shellfish are bred at a high density. The main cause of the disease is infection by pathogenic bacteria, viruses, parasites, etc. Various measures have been taken against this.

 Among the infectious diseases, a virus that kills shrimp, which began to spread rapidly in Taiwan, China, and Southeast Asia several years ago, has been introduced to Japan by importers, as well as PRDV (Penae id Rod-Shaped DNA Virus). Spread and cause serious damage. In addition, Bili-like virus infection and streptococcal disease are also seriously affected as frequently occurring infectious diseases.

Countermeasures such as administration of antibiotics and immunologically active substances have been taken for these infectious diseases. However, when antibiotics were administered, they remained in fish farms, etc., and their effects were not always satisfactory. On the other hand, recently, the effects of polysaccharides on fish infection have become known. For example, Japanese Patent Application Laid-Open No. H02-218865 discloses that water-soluble glucans obtained from mushrooms and the like contain fish. It has been disclosed that it has the effect of preventing and treating infectious diseases. On the other hand, in WO98 / 42204, sulfated polysaccharides prevent and cure viral infection. It is disclosed that it has a therapeutic effect.

 However, although there have been many attempts to prevent and treat fish and shellfish infestation in fish farms, the incidence of damage has not yet decreased, and new, more effective preventive and therapeutic agents have been developed. Requested.

 In addition, mozuku containing a large amount of sulfated polysaccharides such as antibiotics and fucoidan was often expensive and difficult to produce for aquaculture feed.

 Therefore, an object of the present invention is to provide an inexpensive and highly safe agent for preventing and / or treating fish and shellfish, which has excellent preventive and therapeutic effects on viral infection and bacterial infection. Disclosure of the invention

 As a result of intensive research in view of such circumstances, the present inventors have found that

(Kj el lmani el la crass ifol ia), Kajime (Eck Ioni a maxima, Nyaian kelp (Laminar ia), Ascophyllum (Ascophyl lum nodosum), etc.) Prevent infection of fish and shellfish by virus, pathogenic bacteria and Z or The present inventors have found that the present invention is effective for treatment and completed the present invention.

 That is, the present invention relates to a fish and shellfish characterized by containing one or more components selected from the group consisting of algae of the genus Trolocombus, brown algae of the genus Bombyx, giant kelp, algae of the genus Ascophyllum, and extracts thereof. It provides a prophylactic and / or therapeutic agent for infection.

 The present invention also provides a feed for culturing fish and shellfish, which contains the agent for preventing and / or treating infection.

 Further, the present invention provides a method for cultivating fish and shellfish, which comprises administering the preventive and / or therapeutic agent or the feed or the feed.

Further, the present invention relates to an algae of the genus Trolocombus, And a method for preventing and / or treating fish and shellfish infectious diseases characterized by administering an effective amount of one or more components selected from the group consisting of brown algae of the genus Ascophyllum and extracts thereof. . BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows the effect of various seaweed extracts on PRDV infection.

 FIG. 2 is a diagram showing the effects of various seaweed extracts on PRDV infection.

 FIG. 3 is a graph showing the effect of using various seaweed extracts and fucoidan in combination.

 FIG. 4 is a graph showing the effect of using various seaweed extracts in combination with fucoidan and Bacillus pamilus. BEST MODE FOR CARRYING OUT THE INVENTION

 Trolocomb used in the present invention is a brown algae belonging to the genus Trolocomb that frequently breeds in the eastern part of Hokkaido, such as Kushiro and Nemuro, and has a thick, slender strip of about l to 5 m in length and about 10 cm in width. It has leaf-like leaves, and its surface has complex cloud-like irregularities. Algae are known to contain nutrients such as potassium and sodium, vitamin A, chloride and alginic acid.

 As the alga of the genus Trolocombus used in the present invention, for example, Trolocum (Kjellmaniella crassifolia), Gagome (Kjel lmaniel la gyrata) and the like can be mentioned, and any of them can be suitably used.

 Kazime is a brown algae of the genus Ceratophyta that grows on the rocky shores of the Pacific coast south of Kanto.It has a plate-shaped or band-shaped central leaf at the top of its elongated stem, and its lateral lobes have wings spread to the left and right. Is recognized. Algae are rich in nutrients such as mannitol, eel, and alginic acid, and are also used as raw materials for their production.

The brown algae of the genus Ecklonia used in the present invention include, for example, Ecklonia maxima, Ecklonia kurome, and Ecklonia stoloni fera Etc., and any of them can be suitably used.

 The giant kelp used in the present invention is a brown alga of the order Laminaria that grows in a rocky reef along the coast of Power Reformia in North America, and is known to grow to several tens of meters with a long and thin stem attached to many side leaves. I have. Algae contain calcium and vitamins

It is known to contain nutrients such as _BP and alginic acid.

 Ascophyllum algae is a brown alga of the genus Hibamata that frequently breeds along the northern European coast, and contains nutrients such as veins, auxins, cytokinins, abscisic acid, polysaccharides, oligosaccharides, minerals, vitamins, and amino acids. It is known as a seaweed with a high alginic acid content.

 Ascophyllum algae used in the present invention include, for example, Ascophyllum nodosum.

 The above-mentioned seaweeds may be used as they are, or those obtained by pulverization or the like, or those extracted with water, a hydrophilic solvent, or a mixture thereof. The extraction temperature and time may be selected appropriately for each seaweed. For example, the extraction may be carried out at a low or room temperature for several days, or may be performed by heating and extracting for several minutes to several hours. The extract thus obtained can be used as it is, or may be subjected to purification treatment or the like within a range that does not impair the effect.

 It is more preferable that the agent for preventing and / or treating Z or the infection of the present invention further contain fucoidan in addition to the seaweed or its extract. Fucoidan is a sulfated polysaccharide consisting mainly of fucose and having a molecular weight of around hundreds of thousands, and is abundantly contained in the brown alga Mozuku, Mizuki, Macombu, Wakame, Kombu, Amijigusa, etc. and is harmless to the human body belongs to.

The fucoidan used in the present invention may be a product obtained by pulverizing brown algae containing fucoidan, or may be a product obtained by extraction and purification using an appropriate method. Is not particularly limited, but from the viewpoint of cost and the like, Mozuku or Okinawa Mozuku is preferable. The extraction method is usually used The method is not particularly limited as long as it is a method, and examples thereof include acid extraction and hot water extraction. The polymer extracted from seaweed may be used after being reduced in molecular weight or oligosaccharide by hydrolysis, for example, by treatment with a weak acid.

 More preferably, the agent for preventing or treating infection of the present invention further comprises a bacterium belonging to Bacillus pamilus or Bacillus lentus. Bacteria belonging to Bacillus pamilus or Bacillus' lentus used in the present invention include, in particular, Bacillus pamilus A-1 strain (FERM BP-6280) and Bacillus lentus A-2 strain disclosed in W98Z045402. (FERM BP-6278), Bacillus lentus A-3 (FERM BP-6279), and Bacillus pamilus A-4 (FERM BP-6281). Is preferred.

 The agent for preventing and / or treating Z or the infection of the present invention can be administered as it is, or may be used by mixing or blending it with feed materials usually used for culturing fish and shellfish. The amount to be mixed or added to the feed may be determined as appropriate according to the fish and shellfish and the culture method, but for example, about 10 mg / kg to 500 nig / kg, more preferably about 50 mg / kg to 30 Omg / kg, particularly It is preferable to mix 100 mg / kg to 20 Omg / kg. The feed amount of feed containing the preventive and / or Z- or therapeutic agent for infection of the present invention is preferably about 3% of the body weight of fish and shellfish a day.

 On the other hand, the amount of fucoidan may be appropriately determined according to the type of fish and shellfish, etc., and is usually about 50 mg / kg to 100 mg / kg, particularly preferably 60 mg / kg to 80 mg / kg. .

'' As the administration form of bacteria belonging to Bacillus pamilus or Bacillus lentus, the cells may be administered as they are grown in a normal growth medium, for example, dried broth, or the like. The recovered cells may be administered by centrifugation, standing, and removing the supernatant. The cells may be freeze-dried together with excipients such as starch and skim milk. Furthermore, the cells after culturing were supported on a carrier. May be administered. The carrier used here is not particularly limited, but zeolite, maltstone, activated carbon, igneous rock, coral stone, and the like are preferable in terms of the ability to retain cells, and zeolite is particularly preferable in terms of cost.

The seaweed, fucoidan, and Bacillus bacterium can be administered separately. That is, the seaweed as it is or the extract may be fed directly, and fucoidan may be separately administered as it is, or a seaweed or an extract dried product containing the same may be administered. On the other hand, the cells recovered from the Bacillus bacterium bacterium may be separately administered as it is or by being supported on a carrier by a separation means such as centrifugation. These may be mixed or blended with the breeding raw materials usually used for cultivation of fish and shellfish. The amount of the seaweed mixed or mixed in the feed varies depending on the fish and shellfish and the cultivation method, but the amount of the above seaweed is generally 10 mg to 50 mg / kg of seafood per day for prevention of infection. The dose may be set so as to be administered in an amount of about 50 mg to 30 Omg, particularly 100 mg to 20 Omg. The amount of fucoidan may be usually set to be about 50 mg to 100 mg, especially 60 mg to 80 mg per kg of seafood per day. On the other hand, the amount of the bacterium belonging to the genus Bacillus may be appropriately determined according to the type of fish and shellfish, and is usually 1 × 10 ^{5 to} 1 × 10 ⁹ cfu / g—feed, especially 1 × 10 ⁶ to 1 × 10 6 cfu / g. When set to X 10 ⁸ cfu / g_ feed, the yield of the fish, cost, or the point of workability et preferred. The feed amount of feed containing these is preferably about 3% of the body weight of fish and shellfish a day.

The fish and shellfishes to which the agent for preventing or treating infection of the present invention can be applied are not particularly limited, and include freshwater fish, eel, koi, rainbow trout, ayu, tilapia, crucian carp, etc., and saltwater fish, Puri, Thailand, coho salmon. Invertebrates include kuruma shrimp, ゥ shevi (black taiga), white shrimp, korejabi, kazami and the like. Examples of applicable infectious diseases include viral infectious diseases such as iridovirus, birnavirus, and PRDV, and bacterial infectious diseases such as vibrio, pseudomonas, streptococci, and nodular disease. During ~ However, the effect on PRDV is high and preferable. Example

 Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

Example 1 Method for preparing seaweed

 Salted seaweed (trocombe, kajime, ascophyllum nodsam, giant kelp) was used. Each seaweed was homogenized, suspended in the same amount of water as its wet weight, and desalted. Further, the suspension was suspended in water at a rate of 1 liter per 1 kg of algal cells, heated at 100 ° C. for 2 hours, and then allowed to stand at room temperature for extraction. The alga bodies were filtered off with a gauze, and the filtrate was centrifuged (9,000 rpm, 60 minutes). The supernatant was further dried to obtain each seaweed extract as a powder.

Example 2 Preparation method of fucoidan

 The raw material used was salted Okinawa mozuku. Okinawa mozuku was suspended in water in the same amount as its wet weight to remove salts, and then adjusted to pH 3 by adding an aqueous hydrochloric acid solution. This suspension was heated at 100 ° C. for 10 minutes to extract fucoidan. In addition, insoluble components were removed at 7,000 G using a centrifugal separator, and batch hydrofiltration was repeated at pH 3 using an ultrafiltration membrane with a molecular weight cut-off of 6,000. The low molecular weight fraction having a molecular weight of 6,000 or less was removed. Then, sodium hydroxide was added to adjust the pH to a range of 5.5 to 6.0. The concentrated solution containing fucoidan as a main component was dried by spraying to obtain a powder of a fucoidan fraction of 5 g per 1 kg of wet algal cells. This powder is hereinafter referred to as “Fucoidan (F15)”.

Example 3

The effects of the powdered extracts (made by Marine Science Co., Ltd.) of the trolley kelp, cajime, ascophyllum and giant kelp obtained in Example 1 were examined. These pulverized extracts are called "Toro-kombu (X-240)" and " (X-241) "," Ascophilum (X-3) "and" Giant Kelp (X-242) J.

 (Test kuruma shrimp) Using a sample with an average body weight of 8 g obtained from Mitsui Norin, preliminarily reared for one week until the start of the test, and then used for the test.

 (Breeding method) A bottom filtration device was attached to a water tank of 0.6mX 0.4mX 0.3m, and the animals were bred with sufficient aeration.

 (Number of Test Tail) Twenty prawns were provided to each of the control group and the sample administration group. Feeding was carried out at regular intervals in the evening, and the feeding rate was 2% per body weight of shrimp.

 (Feed) For the control, normal feed (manufactured by Evian Kyowa) was used. In test group 1 (trolocomb-administered group), 6 Omg (XF-240) of trolocomb was added per day to 1 kg of prawns in a normal feed, and the mixture was molded. In test group 2 (Kajime administration group), in addition to the normal feed, the mixture was added and mixed at a dose of 6 Omg (XF-241) per day for 1 kg of prawns to form and used. In test group 3 (giant kelp-administered group), in addition to the normal feed, the mixture was added, mixed and molded so that giant kelp 6 Omg (XF-242) was administered per lkg of prawns per day. In test group 4 (ascofilm administration group), in addition to the normal feed, 6% Oscofilm (XF-243) was administered per 1 kg of prawns per day, mixed and molded.

(Test method) Each feed was fed continuously for 17 days. The virus (PRDV) was infected on the 8th day after the start of feed feeding, and the survival rate on the 10th day after the challenge was determined. Infection was carried out by homogenizing the head and thorax of prawns infected with the virus in sterile seawater and centrifuging them at high speed, and suspending the obtained supernatant in seawater so as to be diluted about 8000-fold in seawater. Kuruma shrimp were placed in this solution, and immersed for 2 hours with aeration for infection. The water temperature during the breeding period was 23. In addition, the prawns that died during the experimental period were tested for virus using the PCR method, and it was confirmed that they died due to the virus. Figure 1 shows the survival rate after infection.

 As shown in Fig. 1, the administration of seaweed was able to effectively prevent the death of Pseudomonas prawns by PRDV, and the mortality of trolocomb and ascophyllum was particularly high.

Example 4

 We examined the effect of using trolokin, kajime, ascophyllum or giant kelp together with fucoidan.

 The test prawns, breeding methods, and test methods were the same as in Example 3.

 (Number of Test Tail) Twenty prawns were provided to each of the control group and the sample administration group. Feeding was performed on a regular basis in the evening, and the feeding rate was 2% per body weight of shrimp.

 (Feed) For the control, normal feed (manufactured by Evian Kyowa) was used. Test plots 1 to 4 used the same feed as in Example 3. In test group 5 (combination administration of trolocomb and fucoidan), the usual diet should be such that 15 mg of fucoidan (F15) and 10 Omg of trolocomb (XF-240) per kg of shrimp per day were administered. The mixture was added, molded and used. In test zone 6 (combination administration of kajime and fucoidan), normal feed should be such that 15 mg of fucoidan (F15) and 10 omg of kajime (XF-241) per kg of shrimp per day were administered. , Mixed and molded. Test plot 7

(Diet with giant kelp and fucoidan) was fed with 15 mg of fucoidan (F15) and 10 mg of giant kelp per kg of carme shrimp per day.

(XF-242) was added and mixed so as to be administered, molded and used. In test area 8 (ascofilam and fucoidan combined administration area), 15 mg (F15) of fucoidan and 10 Omg (XF-243) of fucoidan / kg of shrimp per day were administered to normal feed. , And then molded and used.

As shown in Figs. 2 and 3, administration of trolokinite, swordfish, ascophyllum, or giant kelp effectively prevented PRDV death of kuruma prawns. In addition, Fuco, a daily dose that is normally ineffective It was confirmed that the combined use of idadan 15 mg / kg with trolocomb, kajime, ascophyllum, or giant kelp enhances the death-prevention effect of PRDV.

Example 5

 The effect of the combination of trolokin, kajime, ascofilam or giant kelp with fucoidan and Bacillus pamilus was examined.

 The test prawns, breeding methods, and test methods were the same as in Example 3.

 (Number of Test Tail) Twenty prawns were provided to each of the control group and the sample administration group. Sample feeding was performed on a regular basis in the evening, and the feeding rate was 2% per body weight of shrimp.

(Feed) For the control, normal feed (manufactured by Evian Kyowa) was used. In addition to the normal diet to test group 1 (Ba Chirusu group administered), feed 1 g per Bacillus' Parumisu A- 10 ⁶ cells per share (Bacillus) added were mixed and used in molding. Test group 2 (Tororo kelp, Fukoidan, Bacillus administered in combination District) to the normal feed in, the bus Chirusu-Pamirusu A- 1 share per feed lg 10 ⁶ cells (hereinafter to as "Bacillus Pamirusu (B)", and Test mixture 3 (Kajime, Fucoidan and Bacillus combined) was added and mixed to give 15 mg of fucoidan (F15) and 1 mg of trolocomb (XF-240) per kg of prawns per day. The administration group) consisted of 10 ⁶ Bacillus pamilus per g feed (B), 15 mg fucoidan per lkg of prawns per day (F15), and 1 K Omg (KF) per 1 g of feed. In test group 4 (giant gelp and fucoidan bacillus combined administration group), 10 ⁶ Bacillus' palmis per gram of feed (B), and Kle per day 15 mg of fucoidan (F15) and 1 giant kelp were added and mixed to give 1 O Omg (XF-242) per 1 kg of shrimp, and the mixture was used for shaping Test group 5 (Ascofiram combined with fucoidan bacillus) ) Contains 10 ⁶ Bacillus' palmis per gram of feed (B) and 15 mg of fucoidan per kg of shrimp per day (F15) and ascophyllum per gram of feed. The mixture was added and mixed to give a dose of 10 Omg (XF-243), molded and used.

 As shown in Figure 4, PRDV mortality resulted from the combined use of 15 mg / kg fucoidan, a daily dose that would normally be ineffective, with Bacillus' palmis and trolocomb, cajime, ascofilam or giant kelp. It was confirmed that the inhibitory effect was more significantly enhanced.

Example 6

 The effect of combined use of ascophyllum nodosam and fucoidan was confirmed using white shulimps.

 (Test shrimp) White shrimp shrimp shrimp (15 days old postra) were tested.

Using (husbandry) 10, unlined E bi ponds three sides of 000m ^2.

 (Number of test specimens) The control group, the fucoidan-administered group, and the sample-administered group each received 80,000 white shrimp. In each plot, the amount of feed was increased as white shrimp grew, and the total feed rate was 2-20% per shrimp body weight. However, the feeding rate of fucoidan-added feed and fucoidan / ascophyllum-added feed (sample) was set at 2% per body weight of shrimp, and normal feed was given if the total feeding rate was insufficient.

 (Feed) For the control group, normal feed (manufactured by NASA) was used. In test group 1 (fucoidan-administered group), normal feed was mixed with fucoidan at a dose of 60 mg / kg (F15) per 1 kg of white shrimp per day, and the mixture was molded and used to give a 2% feed. . Insufficient portions were fed with normal feed. In test plot 2 (feeding plot), normal feed was fucoidan 15 mg / kg / kg of white shrimp per day.

(F15) and ascophyllum 10 Omg (XF-243) were added and blended, molded and used to give 2% feed. Insufficient portions were fed with normal feed.

(Test method) Each feed was fed continuously for 89 days. Depletion due to natural infection of PRDV was observed. White shrimp that died during the test period used the PCR method And attempted to detect the virus, and confirmed that the virus was the leading cause of death.

 Table 1 shows the survival rates at the end of the breeding test.

表 1に示したように、 通常では効果を示さない 1日あたりの投与量であるフコ ィダン 15mgZkgとァスコフィラムを併用することで、 1日にフコィダン 6 Omg Zkg投与したのと同等の PRDVによる死亡阻止効果が得られた。 産業上の利用可能性 Table 1 Survival rate

As shown in Table 1, combined use of fucoidan 15 mg Zkg, which is usually not effective, and ascofilam prevents PRDV death equivalent to that of fucoidan 6 Omg Zkg daily. The effect was obtained. Industrial applicability

 By using the preventive and / or therapeutic agent of the present invention, infection with fish and shellfish viruses and bacteria, particularly PRDV infection, can be effectively prevented and / or treated. In addition, the prophylactic and / or therapeutic agent or Z may be used in a feed for the cultivation of fish and shellfish.

Claims

The scope of the claims 1. Prevention of infection of fish and shellfish, characterized by containing one or more components selected from the group consisting of algae of the genus Trolocombus, brown algae of the genus Bombyx, giant gelp, algae of the genus Ascophyllum, and extracts thereof. Or therapeutic agents.  2. The preventive and / or therapeutic agent for Z or infection of fish and shellfish according to claim 1, further comprising fucoidan.  3. The agent for preventing and / or treating fish and shellfish infection according to claim 1 or 2, further comprising a bacterium belonging to Bacillus pamilus or Bacillus lentus.  4. A feed for cultivating fish and shellfish, comprising the agent for preventing and / or treating fish and shellfish according to any one of claims 1 to 3.  5. A method for cultivating fish and shellfish, which comprises administering the agent for preventing and / or treating fish and shellfish according to any one of claims 1 to 3.  6. A method for cultivating fish and shellfish, comprising providing the feed according to claim 4.  7. Fish and shellfish infectious disease characterized by administering an effective amount of one or more components selected from the group consisting of algae of the genus Trolocombus, the brown algae of the genus Laceae, the giant kelp, the algae of the genus Ascophyllum, and extracts thereof. Prevention and / or treatment method.  8. The method of claim 7, further comprising administering an effective amount of fucoidan. 9. The method according to claim 7 or 8, further comprising administering an effective amount of a bacterium belonging to Bacillus pamilus or Bacillus' lentus.