JP2526733B2 - Agent for preventing and treating bacterial diseases of fish and crustaceans - Google Patents

Description

TECHNICAL FIELD The present invention relates to a preventive / therapeutic agent for bacterial diseases of fish and crustaceans, and a fish feed containing the same.

[Conventional technology]

The fish farming technology in Japan has made rapid progress in recent years, and the amount of aquaculture production has been increasing year by year. However, unlike natural fish, farmed fish are bred at a high density, so many fish suffer from diseases. Especially, there are many damages caused by fish diseases caused by pathogenic bacteria.

For example, there are streptococcal disease of yellowtail, nodular disease, vibrio disease of Thailand, paracolo disease of eel, vibrio disease of rainbow trout, and vibrio disease of prawns.

Antibiotics have been used to prevent and treat these bacterial diseases, but the use of antibiotics has recently been restricted due to the development of resistant bacteria and the problem of antibiotic retention on fish bodies. is there.

Therefore, fish farmers are demanding new preventive / therapeutic agents that are safe and have an excellent effect instead of antibiotics.

On the other hand, bacterial cell wall components are immunostimulants (adjuvants)
It has been known for a long time as to have been prepared from the bacterial wall of the bacterium Mycobacterium bovis, and its efficacy as an immunopotentiating activity and a cancer immunotherapeutic agent has been studied (Cancer, Vol. 66, 493-505). (Page 1974) In addition, it was reported that the product prepared from the cell wall of the bacterium Bifidobacterium thermophilum was used as a preventive and / or therapeutic agent for livestock diarrhea (Jpn. J. Vet.
Sci. 49, pp. 235-243, 1987, JP-A-62-265231).
However, the preventive and therapeutic effects of these immunostimulants against bacterial diseases of fish and crustaceans are not known.

[Problems to be Solved by the Invention]

It is an object of the present invention to provide a preventive and / or therapeutic agent for bacterial diseases of fish and crustaceans, which have a safe and excellent effect which can replace antibiotics.

[Means for solving the problem]

As a result of repeated intensive studies in view of the above circumstances, the present inventors belong to the genus Bacillus, Brevibacterium, Corynebacterium, Acercia, Lactobacillus, Streptococcus, Streptomyces, or Bifidobacterium. The present inventors have found that a cell wall component-containing material of bacteria has an effect of preventing and treating a bacterial disease of fish and crustaceans, and thus completed the present invention.

That is, the present invention is Bacillus, Brevibacterium, Corynebacterium, Aceria, Lactobacillus, Streptococcus, Streptomyces or Bifidobacterium sterilized bacterial cells, fungi of the bacterium. A fish characterized by containing at least one of a cell lysate obtained by subjecting the body to mechanical crushing treatment or enzymatic degradation treatment and a cell wall component-containing product obtained by fractionating the cell crushed product. The present invention relates to a bacterial disease of crustaceans, a preventive and a therapeutic agent, and a feed containing these agents.

Examples of the bacterium used in the preventive or therapeutic agent of the present invention include bacteria belonging to the genus Bacillus such as Bacillus subtilis AT CC 13952, Brevibacterinm lactofermentum ATCC 1386.
Corynebacterium glutamicum ATCC 1 such as Brevibacterium bacteria such as 9
Corynebacterium such as 3060, Escherichia coli ATCC 8739, and Lactobacillus acidophilus
acidophilus) ATCC 4356 and other bacteria belonging to the genus Lactobacillus, Streptococcus thermophilus (Streptococcus th
ermophilus) ATCC 19987 etc., Streptomyces tanaciensis (Streptomyc)
es tanashiensis) ATCC 15238 and other Streptomyces bacteria, Bifidobacterium thermophilum ATCC
Examples include Bifidobacterium bacteria such as 25525.

For the cultivation of these bacteria, any nutrient source that can normally be utilized by these bacteria can be used. For example, carbohydrates such as glucose, sucrose, alcohols such as ethanol and glycerol, acetic acid, organic acids such as propionic acid, carbon sources of soybean oil and the like or a mixture thereof, yeast extract, peptone, meat extract, corn steep liquor, ammonium sulfate, Nitrogen-containing inorganic organic nutrient source such as ammonia, phosphate,
An ordinary medium in which inorganic nutrients such as magnesium, iron, manganese, and potassium and vitamins such as biotin and thiomine are appropriately mixed is used. As a culturing method, the nutrient medium may be cultivated at 20 to 40 ° C. for 12 hours to 5 days in a pH range of 4.0 to 9.5.

The bacterial cells obtained by the culture can be used as they are after being separated from the culture and sterilized by heat treatment or the like. However, it is preferable that the cells have been crushed. The cells to be crushed may be live cells or sterilized products. The crushing method may be either a mechanical method or a method utilizing an enzyme. As a mechanical method, for example, using a French press or the like, about 800 to 2000 k
The cells may be disrupted at a pressure of g / cm ² , or the cells may be disrupted using an ultrasonic disruptor or the like.
When the bacterial cells are disrupted using an enzyme, the cultured bacterial cells or a mechanically disrupted product of the cultured bacterial cells are suspended in physiological saline or the like, and a cell wall lysing enzyme is added thereto to decompose the cell walls of the bacterial cells. Any enzyme may be used as long as it has the ability to decompose the cell wall, and lysozyme protease is a typical example. Enzyme treatment conditions may be according to known methods. In both the mechanical method and the enzymatic method, the cell crushing rate is preferably about 20% or more, more preferably about 60% or more. The crushing rate can be measured by the turbidity reduction rate of the suspension at a wavelength of 660 nm. Further, it is preferable to use a mechanical method and an enzymatic method in combination in order to increase the crushing rate.

It is also possible to fractionate the crushed product of the microbial cells to separate the cell wall component-containing product for use. The fractionation method may be performed by simply removing insoluble matter by centrifugation, and known methods such as ultrafiltration and gel filtration can be used for molecular weight fractionation of proteins and the like.

As a method of using the preventive / therapeutic agent of the present invention, the prepared bacterial cell, its crushed product, or the cell wall component-containing material can be directly given or orally added to feed.

The administration time is not limited, but if given from the larval stage, it has a preventive effect.

The dose is 0.01 to 5% as dry matter in the feed, preferably
It is recommended to add 0.05-1%.

As the feed, the feed raw materials usually used for aquaculture may be blended according to the object.

Generally, fish meal, bone meal, skim milk, cottonseed meal, wheat flour, wheat germ, rice bran, brewer's yeast, vitamins and the like are used.

In the present invention, the fishes / crustaceans include yellowtail, Thailand, eel, carp, rainbow trout, sweetfish, coho salmon, horse mackerel, tilapia, flounder, crucian carp, prawns, black tiger and the like.

 Hereinafter, detailed description will be given with reference to examples.

〔Example〕

Example 1 (1) Preparation of bacterial cells Glucose 1.0 g / dl, yeast extract 1.0 g / dl, peptone 1.
0g / dl, (NH ₄ ) ₂ SO ₄ 0.5g / dl, K ₂ HPO ₄ 0.3g / dl, KH ₂ PO ₄ 0.1g
/ dl, and 50 the medium (pH 7) containing MgSO ₄ · 7H ₂ O0.05g / dl
50 ml was placed in a 0 ml flask and sterilized at 115 ° C. for 15 minutes. Bacillus subtilis ATCC 13952, Previbacterium lactofermentum ATCC 13869, Corynebacterium glutamicum ATCC 13060, Escherichia coli ATCC 8739, Lactobacillus acidophilus ATCC 4356, and Streptococcus were cultured in broth agar medium at 30 ° C for 1 day. -One platinum loop of each of Thermophilus ATCC 19987 and Streptomyces tanaciensis ATCC 15238 was inoculated and shake-cultured at 30 ° C for 24 hours. After completion of the culture, each culture was centrifuged to collect the cells. Each of the cells was suspended in the same amount of physiological saline as the culture solution, heated at 100 ° C. for 10 minutes, and collected again by centrifugation.

(2) Machine-treated product 25 mM for each bacterial cell (wet bacterial cell) prepared in (1)
10% by weight was suspended in the phosphate buffer solution (pH 7.0). Put this cell suspension in a stainless steel bottle (50 ml volume), ultrasonic crusher (UR-200P type, Tommy Seiko Co., Ltd.)
Manufactured by K.K.) and processed at an oscillation frequency of 20 kHz and 200 W output for 15 minutes. After the treatment, the contents contained in the cell wall fraction were further fractionated by centrifugation.

(3) Enzyme-decomposed product 0.01% by weight of egg white lysozyme (manufactured by Sigma) in 25 mM phosphate buffer (pH 7.0) containing 10% by weight of the mechanically processed product of cells prepared in (2) as a solid matter. 0.02% by weight of actinase (70000 units manufactured by Kaken Pharmaceutical Co., Ltd.) was added and treated at 37 ° C. for 12 hours. Thereafter, the enzyme was inactivated by heat treatment at 100 ° C. for 2 minutes.

(4) Immunostimulatory effect using mouse spleen cells RPMI 1640 medium containing 10% fetal bovine inactivated serum, 5 × 10 ⁻⁵
M 2 mercaptoethanol was added, and the mixture was sterilized by filtration.
Spleen-floating cells prepared from the spleen of a 10-week-old female DBA / 2 strain mouse were suspended therein at 2.5 × 10 ⁶ cells / ml. In addition, bacterial cells of the bacteria prepared in (1) and (3) and enzymatic degradation products or Kluyvera citophi (Kluyvera citophi) prepared by the same method as in (1) and (3).
lia) IFO 8193, Beijerinkya Indica
rinckia indica) ATCC 9037 bacterial cells and enzymatically decomposed products were added to each concentration, and the cells were cultured at 37 ° C. in a 5% CO ₂ incubator for 4 days. After culturing, culture supernatant is 1%
After diluting 1000 times with 0.01M Tris-HCl buffer (pH8.1) containing bovine serum albumin, the total amount of mouse IgM produced in the culture solution was measured by ELISA method to prepare each additive preparation. The immunostimulatory effect was measured. As a standard substance for assaying the immunostimulatory effect, lipopolysaccharide (LPS) derived from pathogenic Escherichia coli was used.

 The results are shown in FIG.

FIG. 1 shows the relationship between the sterilized cells of each genus and the enzyme-decomposed products of the same, and the production of mouse IgM / antibody. In the figure, the abscissa indicates the added concentration (μg / ml), and the ordinate indicates the concentration (μg / ml) of total mouse IgM produced in the culture of spleen cultured cells. The broken line shows the case where sterilized cells were added, and the solid line shows the case where enzyme-decomposed products were added.

As is apparent from FIG. 1, Bacillus subtilis ATCC 13952, Brevibacterium lactofermentum ATCC 13869, Corynebacterium glutamicum of the present invention.
ATCC 13060, E. Coli ATCC 8739, Lactobacillus acidophilus ATCC 4356, Streptococcus thermophilus ATCC 19987, Streptomyces
The antibody-producing ability of the Tanachiensis ATCC 15238 enzyme-treated product tended to be higher than that of the bactericidal cells, and LP
It shows high activity exceeding the maximum value of antibody production in S (150 μg / ml), indicating that it has an excellent immunostimulatory effect.

Example 2 (1) Preparation of Feed Containing Bacterial Disease Preventive / Therapeutic Agent A blended feed for rainbow trout fry was blended with the composition shown in Table-1,
Sterilization and mechanical decomposition of Brevibacterium lactofermentum cells prepared in Example-1, enzymatic decomposition treatment product.
1, 1 wt% was added. The obtained mixture was molded into pellets by a conventional method to obtain a rainbow trout fry feed.

(2) Effect of rainbow trout on vibrio disease Fertilized rainbow trout (average weight 2.0 g) is sterilized and mechanically decomposed with the bacterial cells prepared in the preceding paragraph, and the enzyme-decomposed product-containing feed contains Vibrio anguillarum (Vibrio anguillarum) 5 × 10 ⁷ / feed 100 g was added and fed, and the plant was bred at a water temperature of 20 ° C. under aeration for 30 days.

The rainbow trout juveniles were bred in a 500-liter aquarium as 100 animals each, and as a comparative example, a feed containing Vibrio spp.

 The survival rate after 30 days of breeding is shown in Table-2.

It was clearly shown that the rainbow trout has a preventive / therapeutic effect on Vibrio disease by giving the decomposed product of the bacterial cells.

Example 3 (1) Preparation of a feed containing a prophylactic / therapeutic agent for shrimp bacterial disease A compound feed for black tiger was blended in the composition shown in Table 3 to prepare Bacillus subtilis cells and Brevibacterium lacto prepared in Example 1. 0.01 to 1% by weight of a sterilized, mechanically decomposed and enzymatically decomposed product of famentum cells was added.
The obtained mixture was molded into pellets by a conventional method to obtain a black tiger feed.

(2) Effect of black tiger on Pibrio's disease Black tiger (average body weight 1.1 g) is sterilized and mechanically decomposed with the bacterial cells prepared in the preceding paragraph, and 5 × 10 ⁷ Vibrio anguillarum is added to the feed containing the enzymatic degradation product. / Feed 100
g was added and fed, and the animals were raised at a water temperature of 27 ° C. under aeration for 8 weeks.

15 black tigers were housed in a 45-liter aquarium, each containing 15 fish. As a comparative example, a feed containing Vibrio sp.

 The survival rate after 8 weeks of breeding is shown in Table-4.

By giving various decomposition products of bacterial cells, the preventive and therapeutic effects of black tiger against Vibrio disease were clearly shown.

〔The invention's effect〕

The sterilized product, mechanically crushed or enzyme-processed product of the bacterial cell of the present invention is useful as a prophylactic / therapeutic agent for bacterial diseases of fish and crustaceans.

[Brief description of drawings]

FIG. 1 is a graph showing the relationship between sterilized cells of each genus and mouse IgM / antibody production of the enzymatically degraded products.

Claims (2)

(57) [Claims] 1. A genus Bacillus, a genus Brevibacterium, a genus Corynebacterium, a genus Ethericia, a genus Lactobacillus,
Streptococcus, Streptomyces genus or Bifidobacterium genus bacteria sterilized product,
It is characterized by containing at least one of a cell lysate obtained by subjecting the bacterial cells of the bacterium to mechanical crushing treatment or enzymatic decomposition treatment and a cell wall component-containing product obtained by fractionating the cell crushed product. A preventive and / or therapeutic agent for bacterial diseases of fish and crustaceans. 2. A feed for fish and crustaceans containing the preventive / therapeutic agent for bacterial diseases according to claim 1.