JPH08325158A - Viral disease-preventing and treating agent and feed - Google Patents

Abstract

PURPOSE: To obtain a viral disease-preventing and -treating agent and feed which is effective against viral diseases of Pisces and Crustacea and shows safety and excellent efficacy that it can be used instead of an antibiotic agent. CONSTITUTION: This Pisces and Crustacea viral diseases-preventive and therapeutic agent contains, as an active ingredient, the cells bodies of a bacterium having antiviral activity in Rhodopseudomonas or Acetobacter such as Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides or Acetobacter aceti. These bacteria are cultured, for example, in a medium enriched with carbohydrates, alcohol or organic acids, at a pH of 4.0-9.5 at 20-40 deg.C for 12 hours to 5 days. The culture mixture is transferred to centrifugal tubes, centrifuged at 10,000rpm for 10 minutes to obtain the cells as a precipitate. The therapeutic agent or feed contains the dried cell bodies and the dose is preferably 1-300mg/kg/day in 2-3 portions.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a preventive / therapeutic agent for viral diseases of fish and crustaceans and feed.

[0002]

2. Description of the Related Art The fish farming technology has made rapid progress in recent years, and the aquaculture area (the number of management bodies) is increasing year by year. However, unlike natural fish and crustaceans, many of them are sick because they are raised at a high density in aquaculture. The damage caused by pathogenic viruses is particularly great. For example, the mass mortality of shrimp in Taiwan in 1992 spread to South Korea, China, and Japan the following year, causing great damage to the shrimp aquaculture industry. It is believed that these are due to the secondary infection of baculovirus-infected shrimp with vibriopathies.

Conventionally, antibiotics have been used for infectious diseases of fish and crustaceans, but their use is limited due to the generation of resistant bacteria and the problem of residual in the animal body. . Of these diseases, bacterial components have been found to be highly effective as an alternative to antibiotics for bacterial diseases, and have been developed as highly safe preventive and therapeutic agents, but for viral diseases There are no known effective drugs to replace antibiotics.

The cell wall component of bacteria has long been known as an immunostimulant (adjuvant), and its immunopotentiating activity has been investigated. However, the immune system of fish and invertebrate crustaceans is different from that of mammals, and the mechanism of infection / proliferation is completely different between bacteria and viruses. It is not generally considered to be effective.

Recently, the genus Bacillus, the genus Brevibacterium, the genus Corynebacterium, the genus Escherichia.
Genus, Lactobacillus genus, Streptococcus genus, Streptomyces
genus myces, or Bifidobacteri
um) sterilized product of bacterial cells of genus bacteria, mechanical crushed product of the bacterium, cell crushed product obtained by performing enzymatic decomposition treatment, and cell wall component obtained by fractionating this crushed product A preventive / therapeutic agent for viral diseases of fish and crustaceans, characterized in that at least one of the contained substances is added to the feed, has been reported (JP-A-6-181656).
. However, Rhodopseudomo
There is no mention of using bacteria belonging to the genus nas) or the genus Acetobacter as a preventive / therapeutic agent for viral infections of fish and crustaceans.

[0006]

An object of the present invention is to provide a preventive / therapeutic agent for viral diseases of fish and crustaceans, which has a safe and excellent effect and can substitute for antibiotics, and a feed.

[0007]

Means for Solving the Problems As a result of intensive research to solve the above problems, the present inventors have found that the genus Rhodopseudomonas or the acetobacter (Ac
The inventors have found that bacteria belonging to the genus etobacter) are effective against viral diseases of fish and crustaceans, and have completed the present invention.

That is, the present invention relates to Rhod
genus opseudomonas, or Acetobacter (Acetobacte)
r) a fish, a crustacean viral disease preventive / therapeutic agent which belongs to the genus and contains bacterial cells of bacteria having antiviral infection activity as an active ingredient,
And a feed for fish and crustaceans containing the bacterial cells of the bacterium. Hereinafter, the present invention will be described in detail.

In the present invention, fishes to be prevented or treated include yellowtail, thailand, eel, carp, rainbow trout, sweetfish, coho salmon, striped horse mackerel, tilapia, flounder,
Examples include troughs and crucian carp. In addition, as shellfish, prawns, black tiger, Taisho shrimp,
Examples include white shrimp. The active ingredient of the preventive / therapeutic agent for viral diseases according to the present invention is Rhodopseudomonas.
pseudomonas) or Acetobacter
Bacteria belonging to the genus, specifically Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroid
es), Rhodopseudo
monas palustris), Rhodes pseudomonas viridis (R
hodopseudomonas viridis), Rhodopseudomonas acidophilia, Rhodopseudomonas gelatino
sa), Acetobacter aceti,
Acetobacter liquefaciens
iquefaciens), Acetobacter pasteurianus (Ace
tobacter pasteurianus).

For the culture of the above-mentioned bacteria, any nutrient source which can normally be utilized by these bacteria can be used. For example, glucose, carbohydrates such as sucrose, alcohols such as ethanol and glycerol, organic acids such as acetic acid and propionic acid, soybean oil and the like, or a carbon source of a mixture thereof,
Yeast extract, peptone, meat extract, corn steep liquor, ammonium sulfate, nitrogen-containing inorganic and organic nutrients such as ammonia,
An ordinary medium in which vitamins such as phosphate, magnesium and thiamine are appropriately mixed is used.

As a culture method, for example, p.
Culturing may be carried out at 20 to 40 ° C for 12 hours to 5 days with H in the range of 4.0 to 9.5. After culturing, transfer the culture medium to a centrifuge tube and
Precipitated cells are obtained by centrifugation at 00 rpm for 10 minutes. The microbial cells obtained above are freeze-dried, pulverized, and then thoroughly mixed with an appropriate amount of corn starch, talc, magnesium stearate, etc., and the particle size is adjusted by a conventional method to prepare a formulation. The bacterial cell content in the formulation is not particularly limited, but when added to the feed, it may be a content that can be uniformly mixed in the feed, and when granulating from a powder, depending on the type of fish or crustacean, It may be adjusted according to the growth stage.

On the other hand, in order to prepare the feed, if the feed raw material usually used for aquaculture is appropriately selected and blended according to the target aquatic animal, and the above-mentioned bacterial cells are added, blended and moistened. Good. As feed ingredients, fish meal, shrimp meal, squid meal, wheat flour, corn gluten, brewer's yeast, cholesterol, vitamins and minerals are generally used. When the above-mentioned microbial cells are added to, mixed with and moistened in the feed, the content may be 0.01 to 3% by weight in the total feed.

As the virus (disease) that can be treated or prevented by the preventive and / or therapeutic agent for viral diseases of the present invention, and the feed containing the same, floramelad virus (HRV) is used in flounder, stolon virus is used in trough, and rainbow trout is used in rainbow trout. Infectious pancreatic necrosis virus (IPNV), viral hemorrhagic septicemia (VHS), infectious hematopoietic necrosis virus (IHNV), spring virus disease (SVC) in carp, fin inflammation (SBI), viral disease in catfish ( CCVD), herpes virus in salmon, Pike
(Pike) Viral disease of juvenile, Rod in prawn
Examples include, but are not limited to, shapaed virus-Penaeus japonicus (RV-PJ), and lymphocystis disease virus that infects many seawater and freshwater fish.

The viral disease preventive / therapeutic agent according to the present invention is
It refers to the prevention of the infection and the onset of the above viral diseases, or the treatment of the onset. The dose of the preventive / therapeutic agent for viral diseases or the feed of the present invention, the administration time is the fish to be applied,
Although it may be appropriately changed depending on the type of crustacean and the type of viral disease, for example, 1 to 300 mg / kg body weight of dried cells may be given in 2 to 3 divided doses per day. In addition, a preventive effect can be obtained by giving the preventive / therapeutic agent for viral diseases or the feed of the present invention during the juvenile stage.

[0015]

The present invention will be described in more detail below with reference to examples, but these examples do not limit the scope of the present invention. Example 1 Preparation of viral prophylactic therapeutic agent (1) obtaining polypeptone bacteria (Nissui Pharmaceutical) 10 g, yeast extract (Oriental _{Yeast) 6g, NaCl 5g, MgSO 4} · 7H 2 O 2g, agar (DIFC
o) Dissolve 10 g in 900 ml distilled water and then use 1N NaOH.
The pH was adjusted to 7.0. The medium was filled up to 1 L with distilled water and then sterilized by autoclaving at 124 ° C for 20 minutes.
After sterilization, the medium cooled to about 60 ° C. was sown on a shallow dish to prepare an agar medium.

In the above medium, Rhodopseudomonas capsulata (ATCC 11166),
Rhodopseudomon
as sphaeroides) (ATCC 17023) or Acetobacter aceti (ATCC 15973) by static or liquid culture at 30 ° C for 5 days, transfer the culture medium to a centrifuge tube, and spin at 10,000 rpm. After centrifugation for 10 minutes, the precipitate was freeze-dried to obtain crushed cells.

(2) 200 mg of the bacterial cell obtained in the formulation (1), 150 mg of corn starch
, Talc (80 mg) and magnesium stearate (30 mg) were thoroughly mixed and passed through a 60-mesh wire net to adjust the particle size to obtain the viral disease preventive / therapeutic agent of the present invention.

Example 2 Preparation of Cultured Shrimp Feed (Part 1) 3 wt% of the bacterial cells obtained in Example 1 (1) above was added to a basic prawn for prawns (made by Hayashikane Sangyo Co., Ltd.). The resulting formulation is steamed at 90 ° C or above for 5 minutes to modify the gluten and impart the required shape retention property, then dried at 80 ° C for 8 hours with hot air and pelletized (2 mm) using a pellet machine. (φ x 5 mm) to obtain feed for prawns.

(Part 2) 100 g of dry pellet basic feed (made by Hayashikane Sangyo Co., Ltd.) for prawns was used in Example 1 above.
In the process of (1), add 100 ml of cell suspension (10 ⁶ cells / ml).
After soaking for 30 minutes, it was dried at 30 ° C for 3 hours to prepare a feed containing live bacteria.

[Example 3] Preparation of cultured fish feed (1) Commercially available feed basic powder for troughs [manufactured by Taiyo Feed Co., Ltd.]
3% by weight of the bacterial cells obtained in Example 1 (1) above was added to. Mold the resulting blend using a pellet machine (5
mmφ × 10 mm) and dried at 40 ° C. for 5 hours to obtain dry pellets for trough.

Example 4 Preparation of Cultured Fish Feed (2) Commercially available feed powder for trout fry [manufactured by Taiyo Feed Co., Ltd.]
3% by weight of the bacterial cells obtained in Example 1 (1) above was added to. Mold the resulting compound using a pellet machine (2
mmφ × 10 mm) and dried at 40 ° C. for 5 hours to obtain dry pellets for trout fry.

Example 5 Preparation of Cultured Fish Feed (3) In 100 g of commercially available dry pellet feed basic powder for flounder [manufactured by Taiyo Feed Co., Ltd., 5 mm φ × 5 mm] 100 g of the above Example 1 (1)
100 ml of a suspension of cells (10 ⁶ cells / ml) obtained during the process was sprayed and then dried at 30 ° C for 3 hours to prepare a feed containing live flounder.

[Test Example 1] Shrimp Rod shaped virus-Pen
Preventive effect against aeus japonicus (RV-PJ) virus Infection around the midgut gland of the tiger prawn, which died due to infection with RV-PJ, was excised, and the extract homogenized with 10 volumes of buffer was added to a 0.4 μm pore size filter. When the product filtered in step (2) is injected into the abdominal muscles of healthy shrimp, it dies with the symptoms peculiar to this infection, such as vitiligo on the instep. Furthermore, electron microscopic observation revealed that this infection was caused by a virus that seems to belong to the baculovirus.

[0024] 2 times a day for healthy prawns (average weight 22g)
The feed for cultured shrimp obtained in Example 2 above was given 1% of the body weight per day. On the 14th day, 10 viral prawns were diluted 80 times with the virus solution obtained in the same manner as above.
100 μl each or a buffer was injected as a control, and the survival rate after that was examined. The results are shown in Table 1.

[0025]

[Table 1]

When healthy shrimp were infected with the RV-PJ virus, the mortality rate was 60% after 7 days, but the Rhod of the present invention was used.
By feeding opseudomonas capsulata supplemented feed,
The mortality rate on the 7th day was reduced to 20%, and Rhodopseudomonas sph
Both diets containing aeroides and Acetobacter aceti were able to reduce the mortality rate by 30%. The shrimp that did not infect the virus did not die at all during the test period.

[Test Example 2] Takifugu rubrip
es) for preventing infection of the oral disease by healthy oral troughfish (average body weight 50 g) was fed with the feed for the troughfish obtained in Example 3 above at 10% of the body weight per day. On day 14, ten troughs were inoculated subcutaneously on the dorsal part with 0.1 ml each of 1 × 10 ^3.7 TCID ₅₀ / ml titer of the virus of oral disease, and the survival rate was examined thereafter. Table 2 shows the results.

[0028]

[Table 2]

[0029] Infection of healthy troughfish with the disease of white-cold disease causes a death rate of 80% after 7 days.
Feeding Rhodopseudomonas capsulata-supplemented feed reduced the mortality rate on day 7 to 10%.
Both sphaeroides and Acetobacter aceti-supplemented mortality could be suppressed to less than 30%. It should be noted that the death of the tiger puffer (blank) that does not infect the virus is 1
It was a percentage.

Test Example 3 Rainbow Trout (Salmo gairdner)
Infection preventive effect against infectious pancreatic necrosis virus (IPNV) using i) Healthy rainbow trout (average weight 4 g) was fed with the rainbow trout feed obtained in Example 4 above at 20% of the body weight per day. IPNV with a titer of 5 × 10 ^4.8 TCID ₅₀ / ml on 10 rainbow trouts on day 14
The liquid was intravenously administered at 50 μl each. The results are shown in Table 3.

[0031]

[Table 3]

When a healthy rainbow trout was infected with IPNV, 10
The mortality rate is 80% after a day, but the Rhodopseud of the present invention
By feeding omonas capsulata-supplemented feed, the mortality rate on the 10th day was reduced to 10%, and Rhodopseudomonas sphaeroid
The es and Acetobacter aceti-added feeds were able to reduce the mortality rate to 30% or less. In addition, the rainbow trout (blank) that was not infected with the virus did not die at all during the test period.

Test Example 4 Flounder (Paralichthys oliv)
Aceus) Rhabdovirus (HRV) Preventive Effect The flounder supplemented with the live flounder supplemented feed obtained in Example 5 was fed to flounder to examine the preventive effect against HRV. A healthy flounder (average weight 80 g) was fed with the rainbow trout feed obtained in Example 5 above.
5% of body weight per day was given in two divided doses. 10 on day 14
0.1 ml of HRV solution with a titer of 5 × 10 ^6.8 TCID ₅₀ / ml was added to the tail flounder.
Each was administered intravenously. The results are shown in Table 4.

[0034]

[Table 4]

When healthy flounder is infected with HRV, the mortality rate is 40% after 14 days, but the Rhodopseudom of the present invention
By feeding onas capsulata supplemented feed, the mortality rate on the 14th day was 10%, and Rhodopseudomonas sphaeroide
The mortality rate of s and Acetobacter aceti-containing feeds could be suppressed to 20% or less. The flounder (blank) that was not infected with the virus did not die at all during the test period.

[Test Example 5] Toxicity test A single dose of the above-mentioned three types of bacterial cells was administered to male and female ICR mice. As a control group, 0.7 ml for males,
Females were similarly dosed with 0.6 ml of purified water. The results are shown in Table 5.

[0037]

[Table 5]

As shown in Table 5, no abnormalities or deaths were observed in the test animals in any of the toxicity tests with the above-mentioned three types of bacterial cells, and the lethal dose of a single oral administration of the test substance in mice was 2,000 in both males and females. It was confirmed to be above mg / kg.

[0039]

INDUSTRIAL APPLICABILITY According to the present invention, a prophylactic / therapeutic agent and a feed which are effective and safe against viral diseases of fish and shellfish are provided.

Continuation of front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical display location C12R 1:01) (C12N 1/20 C12R 1:02) (72) Inventor Nobuyuki Sato Wadai, Tsukuba, Ibaraki 16-2 Central Research Institute of Maruha Co., Ltd. (72) Inventor Tsuyoshi Okada Wadai, Tsukuba, Ibaraki Prefecture 16-2 Central Research Institute of Maruha Co., Ltd. (72) Hikari Kimura Rokujo Wakamiya-dori, Shimogyo-ku, Kyoto 81 (72) Inventor Takao Ando 1-122, 1-22 Tsukaguchi-cho, Amagasaki-shi, Hyogo

Claims (2)

[Claims] 1. Rhodopseudomona
s) or a preventive and remedy for viral diseases of fish and crustaceans, which comprises bacterial cells of the genus Acetobacter and has antiviral infection activity as an active ingredient. 2. Rhodopseudomona
s) A feed for fishes and crustaceans, which contains bacterial cells of a bacterium belonging to the genus or the genus Acetobacter and having antiviral infection activity.