JP2000060437A - Prophylactic or inhibitive agent for virus disease of aquatic life - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a prophylactic or inhibitive agent for virus disease of aquatic life capable of prophylactic or inhibition of a virus disease of aquatic life, specifically an iridovirus infections disease of cultured fish or shell fish in an effective safe manner by oral administration, which is easy for administration by including a eucalyptus extract. SOLUTION: This agent is composed of a eucalyptus extract. Eucalyptus globulus and Eucalyptus sitriodora are preferably used as the eucalyptus. The extraction of eucalyptus is preferably carried out by using ethanol at 20-90 deg.C. The obtained extract is preferably fractionated with an alcohol or fractionally purified by chromatography. The eucalyptus extract may be formulated during the manufacturing process of feed, or it may be formulated in a commercially available feed product. The formulation amount of the eucalyptus extract is preferably 0.1-10% based on the weight of feed in terms of the dry weight of the extract. The cultured fish and shell fish to which the diet feed is applied include especially high class cultured fishes such as red sea beam, yellowtail, prawn and eel.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a preventive or suppressive agent for a viral infectious disease in aquatic animals, particularly an infectious disease caused by one of the iridviruses, and is useful in the fishing industry.

[0002]

2. Description of the Related Art In recent years, with the active cultivation of fish, the damage caused by fish diseases has increased, which has become a major problem for the industry. Among them, the iridvirus infection found in the red sea bream farms in Shikoku in 1990 caused great damage to the farms in various parts of Japan. It has been found that iridovirus infections subsequently occur not only in red sea bream, but also in high-grade farmed fish such as yellowtail, amberjack, striped horse mackerel, sea bass, stone bream, oyster oysters, and flounder, and the number of these cases has recently been increasing. In the example of the red sea bream survey, the maximum annual mortality rate reaches 20%, and the serious problem is that the damage occurs in 20 to 100g of juveniles. This disease is a novel disease, and there is currently no effective prevention or control method.
As a countermeasure at the farm, there are preventive measures to prevent further spread of the disease, such as replacement of aquaculture nets, movement of aquaculture areas, reduction of aquaculture density, interruption of feeding, disposal of infected dead fish. Is not effective, and the labor and cost required for the work are large.

[0003] As another drug treatment, there is an antiviral agent for fish having glutathione, glycyrrhizin, etc. (Japanese Patent Laid-Open No. 8-38065), and the iridovirus of the flounder fry by oral administration and medicated bath is used. Reduced mortality from infectious diseases has been reported. However, the medicinal bath has not been put into practical use because it is time-consuming and costly, and has not been shown to be effective against fishes of the order Perciformes, which are important damaging fish such as red sea bream. On the other hand, among chemotherapeutic agents for fish viral diseases (Japanese Patent Publication No. 52-39897 and Japanese Patent Publication No. 56-52883), no effective ones for iridovirus have been reported.
Further, chemotherapeutic agents have many problems in terms of appearance of resistant bacteria, persistence in fish and safety, and it is strongly desired to develop safe and effective preventive or suppressive agents for iridvirus infections instead of antibiotics. ing.

In addition to the above methods, a method using a vaccine can be considered, but it has not been put into practical use at present. further,
There is an example in which the eucalyptus extract is used for the prevention and treatment of bacterial fish disease (Japanese Patent Laid-Open No. 360839/1992), but its effectiveness against viral diseases has not been shown.

[0005]

The object of the present invention is effective by oral administration, which is easy to administer, against viral infectious diseases of aquatic animals, specifically, iridovirus infectious diseases of cultured fish and crustaceans. And to provide a safe preventive or suppressive agent.

[0006]

[Means for Solving the Problems] As a result of intensive studies aimed at achieving the above-mentioned object, the present inventors focused their attention on eucalyptus, which is a natural product and is safe, and this extract suppresses the iridvirus infection of cultured fish cells. The present invention was completed based on such findings that the degeneration of cells is reduced, and that the mortality rate of fish due to an iridvirus infection is reduced by orally administering it to a feed of fish. Is.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below. Eucalyptus has been used as a medicinal ingredient for a long time, and its essential oil has been described as Eucalyptus oil in the Japanese Pharmacopoeia Law, the official compendial of food additives, etc., and its effectiveness and safety have been confirmed. However, the findings on the iridvirus infection of fish obtained this time are those newly discovered by the inventors as shown in the examples below, and in the present situation where there is no effective countermeasure, an extremely excellent effect. Can be said.
Here, the eucalyptus used in the present invention has a scientific name of Eucaly.
ptus globulus Labill., Japanese name, Eucalyptus typified by Eucalyptus and other closely related plants, such as E. polybractea RT Baker, E. dives
Schauer, E. smithii RT Baker, E. australiana B
aker and Smith, E. elaeophora F. von Mueller, E. si
Any eucalyptus such as deroxylon Woolls, E. leucoxylon F. von Mueller, etc. may be used, but preferably Eu
Use calyptus globulus and Eucalyptus sitriodora.

The method for producing the eucalyptus extract is not particularly limited, but the extract can be obtained as follows, for example.
The portion of eucalyptus used includes any of eucalyptus leaves, branches and bark, or mixtures thereof. The eucalyptus part is extracted as it is in a raw state or after being dried, using water, an organic solvent or a mixture thereof at various temperatures. The organic solvent used for extraction and fractionation, for example, methanol, ethanol, propanol, isopropanol, butanol, acetone, ethyl acetate,
Examples include chloroform, petroleum ether, hexane, benzene and the like. Preferably ethanol is used. The extraction is carried out at various temperatures generally used in the extraction, for example, 0 to 100 ° C, preferably 20 to 90 ° C from the viewpoint of extraction efficiency.

Then, the active ingredient is fractionated with various organic solvents, preferably alcohol, or fractionated and purified by chromatography or the like to obtain a eucalyptus extract. If necessary, these may be optionally concentrated and dried. The extract may be in any form such as liquid and solid. The method of adding the eucalyptus extract to the fish feed is not particularly limited, and the eucalyptus extract can be added by a conventional method. That is, the eucalyptus extract is mixed and stirred at the time of producing a feed such as a solid pellet or a paste.
Alternatively, a eucalyptus extract may be impregnated and added to a commercially available feed produced in advance. The content of eucalyptus extract is 0.01% to dry weight of extract per feed weight.
It is 20%, preferably 0.1% to 10%. If it is less than 0.01%, the effect becomes weak, and if it is more than 20%, the biting property as feed is deteriorated, which is not preferable.

The eucalyptus extract thus obtained is mixed with feed and orally administered, or by direct injection,
Or you may apply by bathing. Applicable aquatic animals include farmed fish and crustaceans, especially red sea bream, yellowtail, amberjack,
Striped horse mackerel, sea bass, rockfish, rockfish, flounder,
There are high-grade farmed fish such as eels, sweetfish, and prawns. Hereinafter, the details will be described with reference to Examples and Test Examples of the present invention, but the scope of the present invention is not limited thereby. In addition, it is added that there is no clinical knowledge that the disease caused by the iridovirus has been prevented or suppressed by chemotherapy or the like, and that the present invention is remarkably large in the productivity improving effect of the fishery industry.

[0011]

EXAMPLES Example 1 Production of Eucalyptus Extract 20 kg of fresh leaves of Eucalyptus (Eucalyptus globulus Labill.)
Was dried at 90 ° C for 1 hour to obtain 9.6 kg of dried leaves. To this, add 60 liters of ethanol and stir at room temperature (20
It was left to stand at (° C.) and extracted for 3 days. After repeating this 3 times, the extract was filtered through a filter paper, and the filtrate was concentrated under reduced pressure to dryness to obtain 1.04 kg of a dry extract of a eucalyptus extract.

Example 2 Preparation of 1% by Weight Eucalyptus-Containing Pellet Feed for Fish 10 g of the dry extract of the eucalyptus extract obtained in Example 1 was dissolved in 20 ml of ethanol. This was impregnated with 1 kg of a pelleted feed for red sea bream (Madai EP2.5, manufactured by ITOCHU Feed), mixed with air, and then air-dried to prepare a 1% by weight eucalyptus extract-containing pellet feed for fish.

Example 3 Production of 0.5% by Weight Eucalyptus Blended Pellet Feed for Yellowtail 5 g of the dry extract of the eucalyptus extract obtained in Example 1 was added to 1 kg of moist pellets consisting of 60% by weight of frozen sardines and 40% by weight of blended feed. By kneading, a 0.5 wt% eucalyptus-containing pellet feed for yellowtail was prepared.

Test Example 1 Antivirus against iridovirus infection of cultured fish cells
Effect test GF cultured in 24-well microplate in BME-4 medium
Cells (3.4 × 10 ⁵ cells / ml) were dispensed at 500 μl / well and cultured in a monolayer. On the other hand, undiluted iridovirus solution (10
^-2.7 TCID ₅₀ / ml) was diluted with BME-4 medium to give 1
Prepare 0, 10 ² , 10 ³ , and 10 ^4- fold diluted solutions, and add 500
Dispense by μl each. Here, TCID ₅₀ is a measurement index of the viral cytopathic effect. Specifically, a series of serial dilutions of virus were inoculated into many cell cultures cultured under the same conditions, the number of culture bottles showing cytopathic effect was counted, and 50% of the culture bottles showed cell change effect. The virus dilution factor is TCID ₅₀ . Then add 10 to each virus dilution.
Add 0ppm eucalyptus extract, stir, and mix at 25 ℃ for 1
Time processed. Then, the medium was extracted from the 24-well myloplate in which GF cells were monolayer-cultured, and 300 μl × 2 wells of each eucalyptus-treated virus dilution were inoculated. This was stirred with a plate mixer to adsorb the virus to the cells. After 1 hour, the stirred liquid was removed and the BME-4 medium was added.
500 μl / well was dispensed and the cytopathic effect (CPE) was observed while culturing at 25 ° C. (Eucalyptus section).

As a comparative example, the same test was conducted by setting a group (control group) to which the eucalyptus extract was not added. BME-
4 media is Feta on Basal Medium Eagle (Gibco BRL)
It was prepared by adding 4% of Bovine Serum (manufactured by Gibco BRL). The antiviral effect of the Eucalyptus extract in this test example

FIG. 1 shows. Comparing the number of virus-denatured cells (CPE) per 400x field of view (per 0.0625 mm ² ) 3 days after inoculation with iridovirus, eucalyptus plots have much less CPE than control plots, and eucalyptus extracts have antiviral effects. Was shown.

Test Example 2 Antivirus against Iridovirus infection of juvenile red sea bream
Effect test (1) Experiment period: September 23, 1997-October 10, 1997 (2) Experiment place: Oita Prefectural Marine Fisheries Research Center (3) Setting of test group: 10 red sea bream juveniles weighing about 20 g each Divided into 2 groups, 1 group as a control group
P2.5) and 2 groups were used as test groups for 10 days using the 1% by weight eucalyptus-containing pellet feed for fish prepared in Example 2 of the present invention. The daily feed amount of the pellet feed containing 1% by weight of eucalyptus was 2.5% of the fish body weight, and 5 g / day was given to each group. The dose of Eucalyptus extract was 250 mg / kg per day. (4) Infection method and aquarium breeding: For red sea breams bred for 10 days by the feeding method as in (3),
^-2.5 TCID ₅₀ / ml) by intraperitoneal injection of 0.1 ml
Each group was divided into two 500-liter aquariums and reared at 25 ° C for 7 days, and the number of survivors was investigated. (5) Experimental results and discussion: The number of surviving red sea bream larvae is shown in [Table 1] with the virus solution inoculation day set to 0 day. In the test group administered with the eucalyptus extract, all 10 fish survived even 7 days after the virus inoculation, and a significant antiviral effect was confirmed. The cause of death of the dead fish in the control group was confirmed by the pathological anatomical findings and histological examination by histological examination. In the test group, no pathological condition due to iridovirus was observed.

[0017]

[Table 1] Table 1 Survival number of juvenile red sea breams after virus inoculation Date Elapsed days [days] Control group [tail] Test group [tail] 9.10.3 0 10 10 10.4 1 9 10 10.5 2 9 10 10.6 3 7 10 10.7 4 7 10 10.8 5 6 10 10.9 6 6 10 10.10 7 6 10

[0018]

INDUSTRIAL APPLICABILITY By oral administration of a feed mixed with a eucalyptus extract according to the present invention to fish, it is possible to effectively and safely prevent or suppress the iridvirus disease of marine animals. In particular, the feed containing this eucalyptus extract can be used to reduce or prevent the damage of the above diseases in aquaculture farms.

[Brief description of drawings]

FIG. 1 Comparison of virus-modified cell numbers (CPE)

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 2B005 GA01 GA02 GA04 GA06 KA02                       LB07                 2B104 AA01 AA03 AA07 AA18 BA13                 2B150 AA07 AA08 AB10 DD31 DD42                       DD57                 4C088 AB57 AC05 AC06 BA08 BA10                       NA14 ZC65

Claims (4)

[Claims] 1. An agent for preventing or suppressing a viral disease in aquatic animals, which comprises a eucalyptus extract. 2. The preventive or suppressive agent according to claim 1, wherein the eucalyptus extract is obtained from eucalyptus. 3. The preventive or suppressive agent according to claim 1, wherein the aquatic animal is selected from the group consisting of red sea bream, yellowtail, amberjack, striped horse mackerel, sea bass, stone bream, stone pearl oyster, flounder, eel, sweetfish and prawn. 4. A feed for aquatic animals, comprising the preventive or inhibitory agent according to any one of claims 1 to 3.