JP2583473B2 - Remedies for vibrio infectious disease and treatment and prevention using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a therapeutic agent for infectious diseases using delovibrio, which is effective mainly for fish and shellfish in a culture pond, and a method for treating and preventing the same.

[0002]

[Prior art and its problems] In a special artificial environment in which the balance of the natural natural ecosystem has been disrupted, such as aquaculture ponds, where cultured organisms inhabit at high density and water exchange is small, aquaculture is performed. Water tends to be eutrophic due to excretions and surplus food. In such places, microorganisms adapted to the environment are abnormally generated, and intestinal bacteria including vibrio are not leaked to the examples.

[0003] As a means for treating cultured organisms infected with pathogenic bacteria or preventing the cultured organisms from being infected with pathogenic bacteria, conventionally, an antibiotic such as oxytetracycline is mixed into the feed of the cultured organism. Have been done.
In addition, vaccines are also used for the types of cultured organisms, for example, sweetfish, depending on the type of pathogenic bacteria.

However, in such a case,
Antibiotics have the problem of promoting the emergence of resistant bacteria and lack of safety, and vaccines have the problem that it is difficult to handle them.

[0005]

The problem to be solved by the present invention is to provide an infectious disease therapeutic agent using delovibrio which is safe and easy to handle without the appearance of resistant bacteria. It is an object of the present invention to provide a therapeutic and preventive method, particularly a therapeutic agent for Vibrio infection and a method for treating and preventing Vibrio infection.

[0006]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present invention focuses on gram-negative pathogenic bacteria, in particular, delobibrio, which is parasitic on Vibrio, thereby achieving Gram-negative bacterial infection. Of the disease, in particular, vibrio infections.

[0007] The microorganism according to the present invention may be any microorganism belonging to the genus Derovibrio, and in particular, Derovibrio streppy subspecies BD-4 (B
de-llovibrio stolpii subsp. BD-4) is preferred.

The mycological properties of this microorganism BD-1 are as follows. 1. Morphology The cells are comma-shaped, 0.4 μm in diameter, and 1.0 μm in length. No spores are formed, and both Gram stainability and acid resistance are negative. 2. Growth condition BD-4 D. porphyris sulphate ATCC27111 Transparent plaque-like circular plaques formed on agar medium ++ with host cells suspended therein Transformation ability of host cells into spheroplasts + + -like spherical cells Ability to disrupt host cells 2. Growth +++ E. coli lytic ability +++ Physiological properties BD-4 TEROPERIO STRUCTURE ATCC27111 Nitrate reduction − + catalase − + Attitude to oxygen aerobic aerobic

[0009] BD-4 exhibits almost the same mycological properties as ATCC27111, but differs from ATCC27111 in that it exhibits a negative effect on nitrate reduction and catalase reaction. Therefore,
This fungus is used for Bdellovibrio stolp
ii) was determined to be a subspecies, and Derovibrio strepii subspices BD-4 (Bdellovibriostolpii subsp. BD-
4). The microorganism was deposited with the Institute of Microbial Industry and Technology of the National Institute of Advanced Industrial Science and Technology as Microbiological Laboratory No. 13417.

In order to treat and prevent bacterial infections of fish and shellfish, particularly vibrio infections, using the therapeutic agent according to the present invention, bacteria of the genus Delovibrio are used in a live state. Use forms of microorganisms include culture solutions, viable cell suspensions,
Examples include live bacterial solids, frozen products, freeze-dried products, and the like.

To treat and prevent bacterial infections,
A method in which the present material is administered to a culture pond or mixed into a feed may be mentioned, but any method may be used as long as the microorganisms of the present material can be dispersed in seawater in a viable state.

[0012] Derovibrio is a small gram-negative bacillus, and is widely distributed in the aquatic area and soil as a parasitic bacterium against gram-negative bacteria. Infection of Gram-negative bacteria with delovibrio is performed as follows. When swimming in seawater, Derovibrio invades its host cells when it collides with Gram-negative bacteria. The host bacteria are said to die upon infection. The invading delovibrio grows in the cells of the host bacterium. The sufficiently grown delovibrio is released into water by destroying the cells of the host bacteria. The number is about 200 cells per host bacterial cell. The released derovibrio rapidly kills the host bacteria in the water by newly infecting other host bacteria.

[0013] The host range of Derovibrio is relatively narrow. That is, it is known that a certain kind of derovibrio exhibits parasiticity to a kind of Gram-negative bacterium, Pseudomonas sp., But cannot parasitize the same gram-negative bacterium of the Enterobacteriaceae family. Another kind of delovibrio is
It is known that parasites can be infested by Enterobacteriaceae bacteria and Vibrioceae bacteria, but cannot be parasitized by Pseudomonas bacteria. In addition, none of the derivovrios isolated so far show parasitism for Gram-positive bacteria.

As can be seen from the example of bacteriophage, it is extremely unlikely that an organism exhibiting parasitism in prokaryotes exhibits parasitism in eukaryotes. Therefore, the safety of this material is extremely high. The amount of the therapeutic agent to be introduced and the amount mixed into the feed are not particularly limited. For example, when the cultured organism is prawns and the bait is moist pellets, the amount is 0.1%.

[0015]

EXAMPLE An experiment described below was conducted on prawns reared by the following method. Rearing method of prawns Prawns were obtained from the fish market in Tsukiji. Rearing tank A well-washed sand was spread on the bottom of polycarbonate panlite (30 L), and a circulation filter was buried. Preparation of breeding seawater pH 7.5-8.0, salt concentration 35%. The seawater
It was used as breeding seawater. Selection of test shrimp After shrimp sent in sawdust were sufficiently washed with seawater, shrimp moving actively were put into 30L panlight in units of 10 and left overnight. The next day, the shrimp not diving in the sand was removed, and only the shrimp in the sand were collected, and 10 shrimp were put in each 30L panlight.
The animals were raised at 8 ° C. and used as test shrimp. Feeding and feeding As a rule, the shells on one side of the live clams purchased at the supermarket were removed, and were fed in the evening. On Sundays and holidays, the clams frozen and preserved were similarly removed from the shell on one side before feeding. The fed shellfish was removed the next morning. Exchange of breeding seawater Exchanged once every two to three days.

The progress of the above-mentioned breeding conditions for 5 days is as shown in Table 1. During this period, the prawns dived in the sand during the day, ate food and molted. Therefore, it was determined that this could be used as a basic breeding system for prawns, which are test organisms of the present invention.

[0017]

Table 1 Breeding of prawns Shrimp Panra Number of test shrimp Water temperature (℃) Food consumption (g) Remarks Days No. 1 10 25 12.1 Seawater exchange (25%, pH 7.8) 1 day 2 10 25 11 .4 ３ 3 10 25 13.5 〃 1 10 25 16.2 Number of molts 12 days 2 10 25 16.0 〃 2 3 10 25 17.6 ０ 0 1 10 25 17.0 3 days 2 10 25 15. 7 3 10 25 15.6 1 10 25 18.6 Number of molts 1 Seawater exchange (25%, pH 8.0) 4 days 2 10 25 19.2 Number of molts 0 3 10 25 17.6 １ 1 1 10 25 13. 55 days 2 10 25 14.0 3 10 25 13.5

Establishment of Vibrio Infection System of Kuruma Shrimp at the Aquarium Level Vibrio was cultured in a shrimp meat extract medium (sterilized seawater, 10%
A quantity of kuruma prawn muscle was homogenized and the filtrate was charged with 1.
5% agar) and cultured at 25 ° C for 24 hours.
After being suspended in sterile seawater, prawns (average body weight 14.
5g) in the fourth abdominal muscle, the number of viable bacteria per gram of body weight was 1.
The cells were inoculated so as to have 0x10 cells. When the symptoms were observed and the presence or absence of death was confirmed for 10 days after the inoculation, the results shown in Table 2 were obtained.

[0019]

[Table 2] Vibrio inoculated amount of bacteria Number of test animals Number of dead animals Mortality rate (%) 1.0 × 10 10 10 100 none 100 0

In the above Table 2, the column indicated as "none" is a control group in which sterilized seawater was inoculated in place of Vibrio. In the inoculated group, nine individuals died within 24 hours (one day after) inoculation, and the last one died 10 days later. In the dead individuals, it was observed that the muscles of the sixth abdominal segment had remarkable cloudiness and the muscles of the entire abdominal segment were cloudy. The sixth shrimp died after inoculation with Vibrio.
From the muscles of the abdominal segment, bacteria identical in inoculum and mycological properties were isolated without exception.

Isolation of the Vibrio Parasite Bacteria Derovibrio Experimental Materials and Methods Source: Seawater collected from Yamashita Wharf, Yokohama City, Kanagawa Prefecture, Tokyo Fisheries University Pond, Sakata Seaside Experimental Training Station, Kuruma Prawn Farm, etc. was used. Separation: Vibrio cultured in a shrimp meat extract medium at 25 ° C. for 24 hours is suspended in sterilized seawater, mixed with the collected seawater at a ratio of 1: 1 and then mixed with an equal volume of 0.8% sterilized seawater agar to obtain 25%. C. for 1-2 weeks. Indicator bacteria: Vibrio V-4 isolated from prawns killed by vibrio infection was used. Of the lytic plaques generated in the medium of the indicator of Delovibrio isolation,
Bacteriolytic plaques that were formed 2 days after culturing and increased with the number of days of culturing were used as primary indicators. Pure separation of bacteriolytic bacterium The center of the bacteriolytic bacterium is scraped off with a piece of agar on an agar and suspended in sterile seawater, and then the agar is crushed finely with a sterile pipette to suspend the bacteriolytic plaque forming bacteria in sterile seawater. The suspension is appropriately diluted with sterile seawater, mixed with vibrio cultured in a shrimp meat extract medium, solidified on agar, and cultured. The resulting lysed plaque was treated several times to try pure separation.

Table 3 shows the results based on the materials and methods used in the above experiments.
Shown in

Table 3 Separation of Vibrio parasite bacterium derovibrio Days of culture 1 2 3 4 5 6 7 10 14 Number of flasks 0 0 0 0 (10) (10) 21 43 43

The plaque appeared from day 5 but was very small. The plaque was confirmed from day 7 onward. Since the method for separating derovibrio was established, seawater was collected from the prawn ponds and other parts, and separation of derovibrio was attempted. It has been suggested that derovibrio is involved in the fate of E. coli and the like in seawater. That is, it can be expected that the frequency of detection of derovibrios parasitizing vibrio, which is pathogenic for prawns, differs between the ponds of prawns in which vibrio infectious disease has occurred, the ponds in which prawns have not occurred, and other seawater. Therefore, the number of viable bacteria of Derovibrio in seawater collected from aquaculture ponds and the like was measured. Table 4 shows the measurement results.

[0022]

[Table 4] Localization of derovibrio Sampling site Indicator bacteria Number of plaques Remarks Higashisui University Pond Bd-4 − Tokyo Bay Yamashita Wharf Yokohama City ＋ + 館 Tateyama City, Chiba Prefecture ク ル − Farm A ＋ + Vibrio occurrence every year Ｂ B ＋ + 〃 〃 C ＋ + 〃 Ｄ D 〃 ++ Less vibrio disease occurrence As is clear from Table 4, there is a close relationship between vibrio disease occurrence and viable counts of derivobiosis. It is suggested that there is a relationship.

Examination of treatment method for Klebsiella vivibrio infectious disease with derovibrio Examination of the infectivity of Vibrio of Derovibrio in seawater Bd-4 was suspended in sterilized seawater at 10 cells / mL, and Delovibrio was added thereto at a final cell count of 10 cells / mL. Of the number of viable Bd-4 bacteria over time was examined. The result is shown in FIG. As is clear from FIG. 1, the number of bacteria decreased to about 80% of that of the control group on the first day of culture in the delovibrio-added group, and the number of viable cells decreased to 10% or less on the second day.
After the third day, the viable vibrio count was below the detection limit (<1
0 cells / ml). Derovibrio is B
It was suggested to attack and kill d-4. The reason why the number of viable Bd-4 cells drastically decreased after the second day is considered to be that the vibrio-infected delovibrio matured, flew out of the vibrio cells, and was reinfected.

2. Influence of Vibrio-Derovibrio Mixed Bacterial Solution on Kuma Prawn Abdominal Intramuscular Injection From the result of 1 above, it was confirmed that the separated Derovibrio attacked Vibrio in seawater, so the bacterial suspension and Vibrio suspension in plaque were removed. The effects of the bacterial solution on the prawns were examined by mixing and injecting the prawns intramuscularly into the abdomen. Table 5 shows the results.

[0025]

Table 5 Effect of Vibrio and Derivovrio Mixed Bacterial Solution on Injection into Kuma Prawn Abdominal Region Days Control Group Vibrio bacteria Derovibrio Vibrio + Intramuscular Injection Intramuscular Injection Delovibrio Intramuscular Injection 0 5 5 5 5 5 1 5 0 5 3 3 5 5 33 35 53 45 45 55 55 53

In the experiments shown in Table 5, prawns having a size of 11 to 13 g were used. Table 5
, Sterilized seawater used for suspending Vibrio and delovibrio was intramuscularly injected into shrimp in an amount equivalent to that of the treated section to obtain a control section. 0.5 ml of a sterile seawater suspension (10 cells / ml) of Vibrio or plaque
By intramuscular injection into the abdominal segment, a vibrio intramuscular injection or a derovibrio intramuscular injection was performed. Furthermore, the vibrio and derovibrio suspensions prepared as described above were mixed at a ratio of 9: 1, and 0.5 ml of the suspension was intramuscularly injected into the fourth abdominal node of the shrimp to obtain a vibrio + derivovrio intramuscular injection. did.

When put into the seawater in the panlight, there were a few shrimp laid sideways in each section,
I got up within a minute and started to dive in the sand. On the following day, all the individuals died on the bottom sand in the Vibrio intramuscular injection plot, whereas three fish survived in the sand in the Vibrio + Derovibrio intramuscular plot. All individuals in the control plot and the derovibrio intramuscular plot survived in the sand. The bait is made of frozen clams, shelled on one side, and shrimp 1
Each animal was fed one by one, but on the day after the treatment, little leftover food was observed in the control group, the intramuscular injection of derovibrio, and the intramuscular injection of vibrio + derovibrio. In the Vibrio treatment area, all the shellfish were eaten little by little, and almost no food was left. After the second day of treatment, almost no food left was observed in the control group, the intramuscular injection of derovibrio, and the intramuscular injection of vibrio + derovibrio. From the above results, it was found that the bacteria in the plaque suppressed vibrio infectious disease without showing toxicity to prawns under the conditions tested.

【The invention's effect】

As described above, according to the present invention, a therapeutic agent for vibrio infectious disease is used as a therapeutic agent for vibrio infectious disease using devivibrio which is a parasitic bacterium of vibrio. Therefore, resistant bacteria are produced by utilizing microorganisms existing in nature. And treat Vibrio infections without compromising safety. Further, according to the present invention, since the parasitic bacteria are used as described above, the handling thereof is extremely easy, and there is an advantage that the cost can be reduced as compared with the conventional types and substances.

[Brief description of the drawings]

FIG. 1 is a diagram showing experimental results obtained by using a method according to one embodiment of the present invention for prawns.

Claims (6)

(57) [Claims] 1. A microorganism comprising a microorganism belonging to the genus Delovibrio,
A therapeutic agent for Vibrio infection. 2. A method for treating and preventing vibrio infectious disease, which comprises spraying a therapeutic agent for vibrio infectious disease comprising a microorganism of the genus Delovibrio into water of a fish and fish culture pond. 3. A method for treating and preventing vibrio infectious diseases, comprising mixing a therapeutic agent for vibrio infectious disease comprising a microorganism of the genus Delovibrio into feed and administering the same to fish and shellfish in a pond for cultivation. 4. The therapeutic agent for Vibrio infectious disease according to claim 1, wherein the microorganism is Derovibrio streppy. 5. The therapeutic agent according to claim 1, wherein the microorganism is Derovibrio streppies subspecies BD-4 (No. 13417, provided by NIKKEI). 6. The method for treating and preventing vibrio infectious diseases according to claim 2 or 3, wherein the microorganism is Derovibrio streppy subspecies BD-4.