WO2023022080A1 - Novel microorganism, agent against white spot syndrome virus containing novel microorganism or like, method for producing same, and controlling method against white spot syndrome virus - Google Patents

Novel microorganism, agent against white spot syndrome virus containing novel microorganism or like, method for producing same, and controlling method against white spot syndrome virus Download PDF

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WO2023022080A1
WO2023022080A1 PCT/JP2022/030514 JP2022030514W WO2023022080A1 WO 2023022080 A1 WO2023022080 A1 WO 2023022080A1 JP 2022030514 W JP2022030514 W JP 2022030514W WO 2023022080 A1 WO2023022080 A1 WO 2023022080A1
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rhodovulum
white spot
strain
shrimp
spot virus
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PCT/JP2022/030514
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French (fr)
Japanese (ja)
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聡 田中
博史 奥畑
育生 廣野
秀裕 近藤
開 伊藤
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関西電力株式会社
国立大学法人東京海洋大学
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Priority to JP2023542366A priority Critical patent/JPWO2023022080A5/en
Publication of WO2023022080A1 publication Critical patent/WO2023022080A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K61/00—Culture of aquatic animals
    • A01K61/50—Culture of aquatic animals of shellfish
    • A01K61/59—Culture of aquatic animals of shellfish of crustaceans, e.g. lobsters or shrimps
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
    • Y02A40/81—Aquaculture, e.g. of fish

Definitions

  • the present invention relates to novel microorganisms, anti-white spot virus agents containing novel microorganisms, etc., methods for producing the same, and methods for controlling anti-white spot viruses.
  • Patent Literature 1 describes the antiviral activity of Bacillus subtilis strain against White Spot Syndrome Virus (hereinafter referred to as "WSSV").
  • WSSV White Spot Syndrome Virus
  • WO 2005/010201 describes the effect of microbes, photosynthetic bacteria, on "Early Fatty Syndrome/Acute Hepatopancreatic Necrosis” (EMS/AHPHD) caused by pathogenic Vibrio.
  • Patent Document 1 Although the microorganism described in Patent Document 1 has been confirmed to have antiviral activity against WSSV, the survival rate of shrimp in infection tests is as low as about 20%. Patent document 2 does not show the effect of microorganisms against WSSV. Therefore, there is a demand for an anti-white spot virus agent that is naturally derived and has a strong control effect against WSSV.
  • One aspect of the present invention realizes a novel microorganism, an anti-white spot virus agent containing the novel microorganism, a novel microorganism, an anti-white spot virus agent containing the novel microorganism, etc., a method for producing the same, and a method for controlling the anti-white spot virus.
  • a method for producing an anti-white spot virus agent including the step of culturing Rhodovulum sp.
  • Rhodovulum sp. OKHT3 strain (NITE BP-03498).
  • FIG. 1 is a diagram showing the measurement results of the body weight of vannamei shrimp used in Example 1, (A) shows the measurement results after 2 weeks from the start of the test, (B) shows the measurement results after 4 weeks from the start of the test, ( C) shows changes in body weight during the test period.
  • FIG. 2 shows the results of WSSV infection tests in Example 2, where (A) shows the results of infection tests with low-concentration virus solutions, and (B) shows the results of infection tests with high-concentration virus solutions.
  • Example 3 the result of subjecting the mixture of the culture supernatant of the OKHT3 strain and the cell extract to the WSSV inactivation test is shown.
  • Example 4 the results of subjecting the culture supernatant of OKHT3 strain, the cell extract, and a mixture thereof to the WSSV inactivation test are shown.
  • 4 shows the results of a WSSV inactivation test carried out in Example 4 using a fraction obtained by fractionating a mixture of a culture supernatant of OKHT3 strain and a cell extract or a heat-treated mixture.
  • Example 5 the results of the WSSV inactivation test performed on the OKHT16 strain under conditions 1 and 2 are shown.
  • An anti-white spot virus agent contains a component derived from Rhodovulum sp.
  • Patent Document 1 describes the effect of bacteria of the genus Bacillus on WSSV, but the survival rate of shrimp in infection tests is only about 20% at maximum.
  • WSSV is a virus that causes white spot disease, and damage has been reported in shrimp farms around the world. Its pathogenicity is extremely high, and infected shrimp are said to die within 3 to 10 days.
  • Patent Document 2 describes the effect of photosynthetic bacteria on Vibrio, but does not show the effect on WSSV.
  • Rhodovulum sp. exhibit excellent effects on white spot virus disease in aquatic organisms.
  • Rhodovulum sp. is a marine photosynthetic bacterium.
  • “Ingredients derived from Rhodovulum sp.” refer to ingredients derived from bacteria belonging to Rhodovulum sp.
  • Said component may be, for example, the cells themselves of a strain belonging to Rhodovulum sp.
  • the microbial cells may be viable microbial cells or dead microbial cells.
  • the component may be, for example, an extracellular component produced by the strain and released outside the cells, or a bacterial cell extract extracted from the cells of the strain.
  • sp is generally known to mean a species or a species whose species name cannot be specified. Therefore, "a bacterium belonging to Rhodovulum sp.” may be either a strain whose species is specified or a strain whose species is not specified, as long as it is a bacterium belonging to the genus Rhodovulum.
  • examples of bacteria belonging to Rhodovulum sp. include OKHT3 strain, OKHT16 strain, Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, Rhodovulum marinum, and the like. is mentioned.
  • a culture solution of the cells can be mentioned.
  • the culture solution obtained by adding the cells to the medium (pH 6.8) used in Example 1 described later so as to have a concentration of 1 ⁇ 10 3 cells/mL, Shaking culture for 5 days under aerobic conditions at 25 to 28° C. can be mentioned.
  • the culture supernatant of the strain can be used as the extracellular component.
  • a method for obtaining the culture supernatant for example, a method of removing the cells from the above-mentioned culture solution of the cells by centrifuging under conditions of 4,000 ⁇ g and 10 minutes can be mentioned.
  • the culture supernatant prepared by the above method can be used as it is as a component of the anti-white spot virus agent.
  • the bacterial cell extract is obtained, for example, by preparing a suspension of the bacterial strain, crushing the bacterial cells using a French press, homogenizer, mortar, etc., removing insoluble matter by centrifugation, and recovering the supernatant.
  • a French press for example, crushing the bacterial cells using a French press, homogenizer, mortar, etc., removing insoluble matter by centrifugation, and recovering the supernatant.
  • distilled water is added to the cell pellet obtained by removing the culture supernatant from the cell culture solution, and the cell pellet is crushed in a mortar at 20,000 ⁇ g for 10 minutes and 4 times. It is preferable to prepare a cell extract by collecting the supernatant separated by centrifugation at °C conditions and filtering using a filter with a pore size of 0.7 ⁇ m.
  • the component derived from Rhodovulum sp. is preferably the culture supernatant of Rhodovulum sp. According to this configuration, the aquatic organisms can be easily ingested with the anti-white spot virus agent by immersing the bait in the culture supernatant and providing the aquatic organism with the bait permeated with the culture supernatant. In addition, as shown in Examples described later, an excellent inactivation effect against WSSV can be obtained.
  • the component more preferably contains the culture supernatant and the bacterial cell extract.
  • the inactivation effect can be obtained even more excellent than in the case of using only the culture supernatant as the component, as described later in Examples.
  • the mixing ratio of the culture supernatant prepared as described above and the bacterial cell extract is, for example, 1:1 by volume. can do.
  • Mixing of the culture supernatant and the cell extract may be performed by any method, and is not particularly limited.
  • the molecular weight of the component is preferably 100 kDa or more. As shown in Examples described later, when the molecular weight of the component is 100 kDa or more, a superior inactivation effect against WSSV can be obtained as compared with the case where the component with a molecular weight of less than 100 kDa is used.
  • the anti-white spot virus agent according to one embodiment of the present invention may contain other components in addition to components derived from Rhodovulum sp.
  • Other components include, for example, sterilized artificial seawater.
  • the anti-white spot virus agent according to one embodiment of the present invention has the effect of inactivating WSSV, and in addition to the action of inactivating WSSV, by mixing the anti-white spot virus agent into feed, as shown in the examples described later. It has the effect of stimulating immunity in the body. Therefore, the anti-white spot virus agent according to one embodiment of the present invention can also be used as an immunostimulator against aquatic organisms.
  • Rhodovulum sp. is not particularly limited, but OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum, and more preferably at least one selected from the group consisting of OKHT3 strain, OKHT16 strain, Rhodovulum imhoffii, and Rhodovulum viride.
  • the present inventors isolated one strain from among the bacteria in seawater collected from the coastal sediment in Wakayama Prefecture, and named it the OKHT3 strain.
  • the present inventors performed draft genome analysis using the next-generation sequencer MiSeq (registered trademark, manufactured by Illumina), and analyzed the 16S rRNA of the OKHT3 strain with the known Rhodovulum sp. NI22 strain (https:// pubmed.ncbi.nlm.nih.gov/25614575/) and compared with 16S rRNA, maximum likelihood molecular phylogenetic tree analysis was performed.
  • the OKHT3 strain belongs to the genus Rhodoblum, a marine photosynthetic bacterium, and is the same species as the Rhodovulum sp. NI22 strain, but is a different strain.
  • the OKHT3 strain has been deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (acceptance number is NITE BP-03498 (acceptance date: July 28, 2021, transfer date: July 12, 2022)).
  • the OKHT16 strain is one of the strains that can be confirmed by the draft genome analysis that it is the same species as the Rhodovulum sp. NI22 strain and that it is different from the NI22 strain and the OKHT3 strain. be.
  • the OKHT16 strain has been deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (Room 122, Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture) (acceptance number is NITE BP-03499 (acceptance date: July 28, 2021, transfer date: July 12, 2022)).
  • a method for producing an anti-white spot virus agent according to one embodiment of the present invention includes a step of culturing Rhodovulum sp.
  • the Rhodovulum sp. is selected from the group consisting of OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum. At least one is preferred.
  • step for example, cells of a strain belonging to Rhodovulum sp. are added to the medium (pH 6.8) used in Example 1 described later so as to have a concentration of 1 ⁇ 10 3 cells/mL.
  • a method of culturing with shaking the culture solution added to the above at 25 to 28° C. under aerobic conditions for 5 days can be exemplified.
  • the culture solution obtained through the above process contains bacterial cells and culture supernatant. Both the bacterial cells and the culture supernatant were prepared according to [1. anti-white spot viral agent]. Therefore, the culture medium can be used as a component of the anti-white spot virus agent according to one embodiment of the present invention.
  • the above [1. anti-white spot virus agent] the culture solution obtained through the above steps is centrifuged at 4,000 x g for 10 minutes to remove the cells, to prepare the culture supernatant. can do. Then, the culture supernatant can be used as a component of the anti-white spot virus agent according to one embodiment of the present invention.
  • the above [1. anti-white spot virus agent] a mixture obtained by preparing a bacterial cell extract and mixing the bacterial cell extract with the culture supernatant is also an anti-white spot virus agent according to an embodiment of the present invention. It can be used as a component of spot virus agents. At this time, the mixing ratio of the culture supernatant and the bacterial cell extract is the same as in [1. anti-white spot virus agent].
  • the molecular weights of the components of these anti-white spot virus agents are as described in [1. anti-white spot virus agent], it is preferably 100 kDa or more.
  • a component of an anti-white spot virus agent having a molecular weight of 100 kDa or more can be obtained by fractionating the molecular size of the component using, for example, an ultrafiltration filter.
  • a method for controlling an anti-white spot virus according to an embodiment of the present invention is a method comprising the step of ingesting an anti-white spot virus agent according to an embodiment of the present invention to an aquatic organism.
  • the anti-white spot virus agent according to one embodiment of the present invention can be applied to aquatic organisms that are susceptible to disease caused by WSSV.
  • aquatic organisms include various aquatic animals such as fish, crustaceans, and shellfish.
  • the aquatic organisms are preferably shrimps because they are aquatic organisms that are particularly susceptible to WSSV-induced disease.
  • it can be suitably used for controlling the above-mentioned diseases of prawns belonging to the prawn family including kuruma prawn, vannamei prawn, and bull prawn (black tiger).
  • the anti-white spot virus agent can be ingested by aquatic organisms by, for example, mixing it into the food of aquatic animals, and can exert its effect.
  • the anti-white spot virus agent When the anti-white spot virus agent is mixed in the feed, it is preferable to mix the culture solution of Rhodovulum sp. in an amount of 0.3 mL to 0.5 mL per 1 g of the feed. . Preparation of the culture solution is as described above.
  • the Sustainable Development Goals (SDGs) Goal 2 "Zero hunger” and Goal 14 "Abundant seas” We can contribute to the achievement and realization of "Let's protect”.
  • Rhodovulum sp. OKHT3 strain (NITE BP-03498)
  • Rhodovulum sp. OKHT3 strain was isolated from bacteria in seawater by the present inventors, and is a novel strain belonging to Rhodovulum sp.
  • the present invention includes the following aspects.
  • the Rhodovulum sp. is a group consisting of OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum
  • the anti-white spot virus agent according to ⁇ 1> which is at least one selected from the above.
  • ⁇ 3> The anti-white spot virus agent according to ⁇ 1> or ⁇ 2>, wherein the component is the culture supernatant of Rhodovulum sp.
  • ⁇ 4> The anti-white spot virus agent according to ⁇ 3>, further comprising the Rhodovulum sp. bacterial cell extract as the component.
  • ⁇ 5> The anti-white spot virus agent according to any one of ⁇ 1> to ⁇ 4>, wherein the component has a molecular weight of 100 kDa or more.
  • a method for producing an anti-white spot virus agent comprising the step of culturing Rhodovulum sp.
  • ⁇ 7> The Rhodovulum sp.
  • the method for producing the anti-white spot virus agent according to ⁇ 6> which is at least one selected from ⁇ 8>
  • a method for controlling an anti-white spot virus comprising the step of causing an aquatic organism to ingest the anti-white spot virus agent according to any one of ⁇ 1> to ⁇ 5>.
  • the method for controlling an anti-white spot virus according to ⁇ 9>, wherein the crustacean is shrimp.
  • Rhodovulum sp. OKHT3 strain NITE BP-03498).
  • Example 1 In order to confirm the effect of the anti-white spot virus agent according to one embodiment of the present invention on the growth of Vannamei shrimp, a diet test was conducted.
  • the culture medium contains 3% (W / V) sodium chloride and is described below (pH 6.8).
  • the culture solution added to the medium to give the concentration was obtained by culturing with shaking at 25-28° C. under aerobic conditions for 5 days.
  • the medium was prepared by weighing the components from sodium L-glutamate hydrate to biotin in mgs listed in Table 1 into a container, adding 30 g of sodium chloride thereto, and then adding sterilized water to the volume. to 1 L.
  • the composition of the medium is described below. Units other than sodium chloride are mg/L.
  • the food was prepared by immersing the shrimp food in the culture solution and allowing the culture solution to sufficiently permeate the shrimp food.
  • control group 5% by mass of the body weight of the shrimp was supplied to the vannamei shrimp bred in another 100 L water tank three times a day with the food not impregnated with the culture solution (this is called a control group).
  • FIG. 1 shows the results of body weight measurements.
  • FIG. 1 is a diagram showing the measurement results of the body weight of the vannamei shrimp used in Example 1.
  • A) of FIG. 1 shows the measurement results two weeks after the start of the test, and (B) shows the measurement results four weeks after the start of the test.
  • the horizontal axis indicates the type of test plot in which the shrimp were bred
  • the vertical axis indicates the weight (g) of the shrimp
  • the values are shown as mean ⁇ standard deviation.
  • C) of FIG. 1 shows the change in the weight of shrimp during the test period
  • the horizontal axis indicates the number of days elapsed from the test start date
  • the vertical axis indicates the weight of shrimp (g), and the values are averages. Shown as ⁇ s.e.m.
  • Example 2 An infection test was performed to confirm resistance to WSSV. Specifically, among the vannamei shrimp subjected to the dietary test described in Example 1, 20 animals were randomly selected in each test group two weeks after the start of the dietary test, and 25 animals were randomly selected in each test group after 4 weeks. Shrimp were submitted to the infection test. As the virus solution, low-concentration (0.4 mL/10 L) or high-concentration (4.0 mL/10 L) virus solutions prepared from WSSV-infected shrimp homogenates were used.
  • the infection test was performed by immersion infection by immersing the shrimp in the virus solution for 3 hours, and the progress was observed for 15 days from the day of the immersion infection (test start date).
  • the results of the WSSV infection test are shown in FIG. 2 and Table 2.
  • the concentration of the virus solution was determined by performing a preliminary test in advance. That is, the shrimp in the control group of Example 1 were immersed in the low-concentration or high-concentration virus solution for 3 hours, and it was confirmed that death of the shrimp was observed approximately proportional to the concentration, and the concentration was determined.
  • the shrimp virus detection kit was used to confirm that the shrimp were infected with WSSV in the early stages of death.
  • FIG. 2 shows the results when vannamei shrimp were subjected to the WSSV infection test two weeks after the start of the diet test.
  • (A) of FIG. 2 shows the infection test results with a low-concentration virus solution
  • (B) shows the infection test results with a high-concentration virus solution.
  • the horizontal axis of the figure indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the shrimp survival rate (%).
  • Asterisks in the figure indicate that the survival rate of shrimp was significantly higher in the administration group than in the control group.
  • the P value was less than 0.0001
  • Table 1 shows the shrimp survival rate two weeks and four weeks after the start of the WSSV infection test.
  • the numerator is the number of living individuals and the denominator is the initial number of individuals.
  • the culture medium has anti-pathogenicity (also referred to as "infection control effect") against WSSV. That is, the culture solution corresponds to the anti-white spot virus agent according to one embodiment of the present invention.
  • Example 3 An inactivation test was performed to confirm the inactivation effect of the OKHT3 strain on WSSV. The test was carried out using vannamei shrimp weighing about 1 g (10 in each test plot), which were kept in a 15 L throw-in filtration type water tank containing artificial seawater at a water temperature of about 28°C. Feeding was carried out twice a day, and the common shrimp feed used in Thailand was used as the feed.
  • 0.1 mL of culture supernatant, 0.1 mL of WSSV diluent, and 0.8 mL of sterilized artificial seawater were mixed, and the resulting mixture was allowed to stand at 25°C for 1 hour to react.
  • the culture supernatant was prepared by the following method. That is, the culture solution obtained by adding the OKHT3 strain to the medium used in Example 1 (the medium described in Table 1) so as to have a concentration of 1 ⁇ 10 3 cells/mL was heated at 25 to 28° C. under aerobic conditions. Shaking culture was carried out for 5 days under these conditions. After shaking culture was completed, the resulting culture solution was centrifuged at 4,000 ⁇ g for 10 minutes to obtain a culture supernatant.
  • the dilution ratio of the WSSV diluent was determined by conducting a preliminary experiment in advance. The preliminary experiment was conducted in the following procedure. A homogenate of WSSV-infected vannamei shrimp was centrifuged to remove shrimp tissue and the like as a precipitate. Then, the supernatant was recovered and filtered using a polyethersulfone membrane (manufactured by Merck Millipore) having a pore size of 0.22 ⁇ m, and the resulting solution was used as a stock solution. The stock solution was diluted in 10-fold steps to obtain dilutions of each dilution rate, and 50 ⁇ l of the dilutions were injected into the tail of vannamei shrimp. It was confirmed that shrimp injected with diluents up to the fifth power of 10 died, and the dilution rate used in the examples was determined to be the fourth power of 10.
  • control mixture 50 ⁇ L of the mixed solution after the reaction was injected into the tail of the Vannamei shrimp, and the mixed solution prepared using 0.1 mL of the medium described in Example 1 instead of the culture supernatant (hereinafter referred to as “control mixture”) was injected at 50 ⁇ L.
  • control mixture the mixed solution prepared using 0.1 mL of the medium described in Example 1 instead of the culture supernatant
  • control mixture was prepared by mixing 0.1 mL of the culture medium described in Example 1, 0.1 mL of the WSSV diluent, and 0.8 mL of sterilized artificial seawater, and the resulting mixture was allowed to stand at 25°C for 1 hour. prepared by placing
  • Table 2 and Figure 3 show the results of the WSSV inactivation test using a mixed solution containing the culture supernatant of the OKHT3 strain.
  • Table 2 shows the number of survival shrimp over time for 11 days from the test start date (“test date” in the table) in the OKHT3 strain to WSSV inactivation test according to Example 3 of the present invention.
  • Figure 3 is a graph of the results in Table 2.
  • the horizontal axis indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the number of surviving shrimp (individuals).
  • Example 4 A WSSV inactivation test was further performed on the OKHT3 strain, which was confirmed to have a WSSV inactivation effect in the inactivation test (Example 3), using culture supernatants, cell extracts, and mixtures thereof.
  • the culture supernatant used was the same as that prepared in Example 3 for the OKHT3 strain.
  • the bacterial cell extract was obtained by adding distilled water to the bacterial cell pellet obtained when the culture supernatant was prepared in Example 3, crushing it in a mortar, and subjecting it to 20,000 x g for 10 minutes at 4°C.
  • the supernatant separated by centrifugation at was collected and prepared by filtration using a filter with a pore size of 0.7 ⁇ m.
  • a mixture of the culture supernatant and the cell extract was prepared by mixing the culture supernatant and the cell extract at a volume ratio of 1:1.
  • the WSSV diluent used in Example 3 was added to the culture supernatant, the bacterial cell extract, or the mixture of the culture supernatant and the bacterial cell extract (0.1 mL each). 0.1 mL and 0.8 mL of sterilized artificial seawater were mixed, and each resulting mixture was allowed to stand at 25° C. for 1 hour to react.
  • Table 3 shows the results of subjecting the culture supernatant of the OKHT3 strain, the cell extract, and the mixture thereof to the WSSV inactivation test, respectively. survival over time.
  • Figure 4 is a graph of the results in Table 3.
  • the horizontal axis indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the number of surviving shrimp (individuals).
  • “supernatant” refers to the culture supernatant
  • extract refers to the bacterial cell extract
  • mixture refers to the mixture.
  • the molecular weight of the mixture was fractionated to 100 kDa or more, less than 100 kDa, less than 30 kDa, or less than 10 kDa with an ultrafiltration filter (manufactured by Merck Millipore, product number Amicon Ultra).
  • an ultrafiltration filter manufactured by Merck Millipore, product number Amicon Ultra
  • a filtrate (0.22 ⁇ m filtrate) obtained by filtering the mixture using a polyethersulfone membrane (manufactured by Merck Millipore) having a pore size of 0.22 ⁇ m A WSSV inactivation test was performed using the mixture (hereinafter referred to as “heat-treated product”) that had been heat-treated at 60° C. for 2 hours. Results are shown in Table 4 and FIG.
  • the heat treatment was carried out by placing a mixture of the culture supernatant of the OKHT3 strain and the cell extract in a heat incubator set at 60°C and heating for 2 hours.
  • Table 4 shows the results of the WSSV inactivation test conducted using the fraction obtained by fractionating the mixture of the culture supernatant of the OKHT3 strain and the cell extract or the heat-treated product. The survival number of shrimp for 8 days from "test day”) is shown over time.
  • Fig. 5 is a graph of the results of Table 4. The horizontal axis indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the number of surviving shrimp (individuals).
  • mixture represents a fraction with a molecular weight of 100 kDa or more when a mixture of the culture supernatant of the OKHT3 strain and the cell extract is fractionated with an ultrafiltration filter.
  • 100 kDa represents a fraction with a molecular weight of less than 100 kDa
  • 30 kDa represents a fraction with a molecular weight of less than 30 kDa
  • 10 kDa represents a fraction with a molecular weight of less than 10 kDa.
  • Example 5 A WSSV inactivation test was conducted under conditions 1 and 2 in order to examine in more detail the WSSV inactivation effect of the OKHT16 strain, which was confirmed to be effective in the WSSV infection test (Example 2).
  • Condition 2 used the mixture prepared by using the OKHT16 strain instead of the OKHT3 strain in Example 3 as the mixture of the culture supernatant and the cell extract, and conducted the same experiment as in Example 3 except for the test period. I have been there. Condition 1 was performed in the same manner as Condition 2, except that instead of the mixture used in Condition 2, a sample obtained by storing the mixture at 4°C for about half a year was used. For the control of this example, a control mixture prepared in the same manner as in Example 3 was used, and the same experiment as the control in Example 3 was performed except for the test period.
  • Table 5 shows the chronological survival of shrimp for 7 days from the test start date (“test date” in the table) in the WSSV inactivation test for conditions 1 and 2 of the OKHT16 strain.
  • Figure 6 is a graph of the results of Table 5.
  • the horizontal axis indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the number of surviving shrimp (individuals).
  • condition 1 when a sample obtained by storing the mixture used in condition 2 at 4° C. for a long period of time was used, it was confirmed that the WSSV inactivation effect was attenuated more than under condition 2. From this result, it is considered preferable to store the anti-white spot virus agent according to one embodiment of the present invention, for example, under conditions of -80°C.
  • Example 6 In order to confirm the effect of inactivating strains belonging to Rhodovulum sp. on WSSV other than the OKHT3 and OKHT16 strains, an inactivation test was performed using the culture supernatants of the following strains. ⁇ Rhodovulum sp. OKHT3 strain (same as above example) ⁇ Rhodovulum imhoffii (RIKEN JCM No. 13589) - Rhodovulum viride (NBRC 109122) The culture supernatant of each of the above strains was prepared in the same manner as in Example 3.
  • Example 3 In the same manner as in Example 3, 0.1 mL of the obtained culture supernatant, 0.1 mL of WSSV diluent, and 0.8 mL of sterilized artificial seawater were mixed, and the resulting mixture was allowed to stand at 25° C. for 1 hour. reacted.
  • Rhodovulum sp. OKHT3 strain but also Rhodovulum imhoffii and Rhodovulum viride were confirmed to be viable in all of the vannamei shrimp used, confirming their inactivation effect on WSSV.
  • Example 7 In order to confirm the resistance to WSSV of strains other than the OKHT3 and OKHT16 strains belonging to Rhodovulum sp., a culture solution of the following strains was used to perform a disease resistance confirmation test.
  • ⁇ Rhodovulum sp. OKHT3 strain (same as above example)
  • ⁇ Rhodovulum imhoffii (RIKEN JCM No. 13589)
  • NBRC 109122) Rhodovulum strictum
  • RKEN JCM No. 9221 ⁇ Rhodovulum marinum (RIKEN JCM No. 13300)
  • a culture solution of each of the above strains was obtained in the same manner as in Example 1.
  • the food was prepared by adsorbing 300 ⁇ L of the culture solution of the above strain per 1.0 g of the shrimp food, and allowing the culture solution to sufficiently permeate the shrimp food.
  • Rhodovulum sp. OKHT3 strain it was confirmed that WSSV infection was suppressed when the culture solution was added to the feed and fed for 8 days. In other words, the disease resistance against WSSV could be confirmed. Furthermore, as shown in Table 6, not only the Rhodovulum sp. OKHT3 strain, but also Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, and Rhodovulum marinum were confirmed to be resistant to WSSV.
  • the term "medium used in Example 1" in the table refers to the case where only the medium used in Example 1 was added instead of the culture solution.
  • Example 8 In order to confirm the effect of the Rhodovulum sp. OKHT3 strain on the biological defense function of the Vannamei shrimp, changes in the expression levels of immune-related genes in the gills of the Vannamei shrimp were observed.
  • test area 1 30 vannamei shrimp weighing 4.0 ⁇ 1.25 g were put into a 100 L circulation filtration water tank containing artificial seawater kept at a temperature of 28°C.
  • a common shrimp feed used in Thailand impregnated with a culture solution was fed to the shrimp 3 times a day for 8 days at 5% by weight of the body weight.
  • the culture medium used was prepared in the same manner as in Example 1.
  • the feed was prepared by adsorbing 300 ⁇ L of the culture solution per 1.0 g of the shrimp feed, and allowing the shrimp feed to sufficiently permeate the culture solution.
  • test group 2 30 vannamei shrimp weighing 4.0 ⁇ 1.25 g were added to another 100 L water tank, and the food impregnated with the culture supernatant instead of the culture solution was fed 3 times a day, 8shrimp were fed 5% by weight of body weight for days.
  • the culture supernatant used was prepared in the same manner as in Example 3. The feed was prepared by adsorbing 300 ⁇ L of the culture supernatant per 1.0 g of the shrimp feed and permeating the shrimp feed sufficiently.
  • Example 1 As a control group, 30 vannamei shrimp weighing 4.0 ⁇ 1.25 g were added to another 100 L water tank, and the food prepared by adding only the medium used in Example 1 was fed three times a day. , fed shrimp at 5% by mass of body weight for 7 days. The feed was prepared by adsorbing 300 ⁇ L of the culture medium used in Example 1 per 1.0 g of the shrimp feed, and thoroughly impregnating the shrimp feed.
  • Feeding was performed once on the 8th day, and 3 hours after feeding, 5 shrimp were randomly selected in each test group and control group to obtain gill samples. Furthermore, 3 of the 5 gill samples were randomly selected for comprehensive gene expression analysis.
  • Table 7 shows the above 2. shows genes detected in . It was confirmed that the expression of penaeidin-3a-like in the shrimp gill sample of Test Group 1 was enhanced by two times or more compared to the shrimp gill sample of the control group.
  • penaeidin-3a-like is a well-known immune-related gene (reference: Shih-Hu Ho, Yu-Chan Chao, Hsiao-Wei Tsao, Masahiro Sakai, Hong-Nong Chou and Yen-Ling Song (2004) Molecular Cloning and Recombinant Expression of Tiger Shrimp Penaeus monodon Penaeidin. Fish Pathology, 39(1), 15-23, 2004.3).
  • the anti-white spot virus agent according to one embodiment of the present invention has the effect of stimulating immunity in the body of shrimp.
  • One aspect of the present invention can be used in fields such as feed additives in aquaculture.

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Abstract

The present invention achieves an agent against white spot syndrome virus that has an excellent effect of controlling white spot syndrome virus. An agent against white spot syndrome virus according to one embodiment of the present invention contains an ingredient derived from Rhodovulum sp.

Description

新規微生物、新規微生物等を含有する抗ホワイトスポットウイルス剤およびその製造方法、並びに抗ホワイトスポットウイルスの防除方法Novel microorganism, anti-white spot virus agent containing novel microorganism, etc., method for producing the same, and method for controlling anti-white spot virus
　本発明は、新規微生物、新規微生物等を含有する抗ホワイトスポットウイルス剤およびその製造方法、並びに抗ホワイトスポットウイルスの防除方法に関する。 The present invention relates to novel microorganisms, anti-white spot virus agents containing novel microorganisms, etc., methods for producing the same, and methods for controlling anti-white spot viruses.
　近年、水生動物の養殖または飼育において、病害対策が最大の課題となっている。国内外のエビの養殖においては、病原菌およびウイルス感染に対する防除対策として、従来、抗生物質等の薬剤が使用されてきた。しかしながら、前記薬剤の使用は、耐性菌等の出現を促進させ、自然界の他の生物種に影響を与えるという問題があり、前記薬剤を投与した水生生物を食用とする場合には安全性が懸念されている。 In recent years, disease control has become the biggest issue in the cultivation or breeding of aquatic animals. In shrimp farming at home and abroad, conventionally, drugs such as antibiotics have been used as control measures against pathogenic bacteria and viral infections. However, the use of the drug promotes the emergence of resistant bacteria, etc., and has the problem of affecting other species in the natural world. It is
　そこで、前記薬剤に代わるものとして、有用微生物を用いた資材についての研究等が行われている。例えば、特許文献１には、バチルス・サブチリス株菌によるホワイトスポットウイルス（Ｗｈｉｔｅ　Ｓｐｏｔ　Ｓｙｎｄｒｏｍｅ　Ｖｉｒｕｓ、以下、「ＷＳＳＶ」）に対する抗ウイルス活性が記載されている。特許文献２には、病原性のビブリオ菌によって引き起こされる「早期脂肪症候群／急性肝膵臓壊死病」（ＥＭＳ／ＡＨＰＨＤ）に対する、光合成細菌である微生物の効果が記載されている。 Therefore, as an alternative to the above drugs, research is being conducted on materials using useful microorganisms. For example, Patent Literature 1 describes the antiviral activity of Bacillus subtilis strain against White Spot Syndrome Virus (hereinafter referred to as "WSSV"). WO 2005/010201 describes the effect of microbes, photosynthetic bacteria, on "Early Fatty Syndrome/Acute Hepatopancreatic Necrosis" (EMS/AHPHD) caused by pathogenic Vibrio.
日本国特表２０２０－５０６８７２号公報Japanese special table 2020-506872 日本国特開２０１９－９７５３０号公報Japanese Patent Application Laid-Open No. 2019-97530
　しかしながら、特許文献１に記載の微生物は、ＷＳＳＶに対する抗ウイルス活性を有することが確認されているが、感染試験におけるエビの生存率は２割程度と低い。特許文献２には、微生物のＷＳＳＶに対する効果は示されていない。そのため、天然に由来し、かつ、ＷＳＳＶに対する強い防除効果を備えた抗ホワイトスポットウイルス剤が求められている。 However, although the microorganism described in Patent Document 1 has been confirmed to have antiviral activity against WSSV, the survival rate of shrimp in infection tests is as low as about 20%. Patent document 2 does not show the effect of microorganisms against WSSV. Therefore, there is a demand for an anti-white spot virus agent that is naturally derived and has a strong control effect against WSSV.
　本発明の一態様は、新規微生物、新規微生物を含有する抗ホワイトスポットウイルス剤および新規微生物、新規微生物等を含有する抗ホワイトスポットウイルス剤およびその製造方法、並びに抗ホワイトスポットウイルスの防除方法を実現することを目的とする。 One aspect of the present invention realizes a novel microorganism, an anti-white spot virus agent containing the novel microorganism, a novel microorganism, an anti-white spot virus agent containing the novel microorganism, etc., a method for producing the same, and a method for controlling the anti-white spot virus. intended to
　ロドブラム・エスピー（Rhodovulum sp.）に由来する成分を含有する、抗ホワイトスポットウイルス剤。 An anti-white spot virus agent containing components derived from Rhodovulum sp.
　ロドブラム・エスピー（Rhodovulum sp.）を培養する工程を含む、抗ホワイトスポットウイルス剤の製造方法。 A method for producing an anti-white spot virus agent, including the step of culturing Rhodovulum sp.
　ロドブラム・エスピー（Rhodovulum sp.）ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）。 Rhodovulum sp. OKHT3 strain (NITE BP-03498).
　本発明の一態様によれば、ＷＳＳＶに対する防除効果に優れる抗ホワイトスポットウイルス剤を提供することができる。 According to one aspect of the present invention, it is possible to provide an anti-white spot virus agent with excellent control effect against WSSV.
実施例１に供試したバナメイエビの体重の測定結果を示す図であり、（Ａ）は試験開始２週間後の測定結果を示し、（Ｂ）は試験開始４週間後の測定結果を示し、（Ｃ）は試験期間中の体重の推移を示す。1 is a diagram showing the measurement results of the body weight of vannamei shrimp used in Example 1, (A) shows the measurement results after 2 weeks from the start of the test, (B) shows the measurement results after 4 weeks from the start of the test, ( C) shows changes in body weight during the test period. 実施例２におけるＷＳＳＶ感染試験の結果を示す図であり、（Ａ）は低濃度のウイルス液での感染試験結果を示し、（Ｂ）は高濃度のウイルス液での感染試験結果を示す。FIG. 2 shows the results of WSSV infection tests in Example 2, where (A) shows the results of infection tests with low-concentration virus solutions, and (B) shows the results of infection tests with high-concentration virus solutions. 実施例３において、ＯＫＨＴ３菌株の培養上清と菌体抽出物との混合物をＷＳＳＶの不活化試験に供した結果を示す。In Example 3, the result of subjecting the mixture of the culture supernatant of the OKHT3 strain and the cell extract to the WSSV inactivation test is shown. 実施例４において、ＯＫＨＴ３菌株の培養上清、菌体抽出物、およびこれらの混合物をＷＳＳＶの不活化試験に供した結果を示す。In Example 4, the results of subjecting the culture supernatant of OKHT3 strain, the cell extract, and a mixture thereof to the WSSV inactivation test are shown. 実施例４において、ＯＫＨＴ３菌株の培養上清と菌体抽出物との混合物を分画した分画物または前記混合物の加熱処理物を用いて実施した、ＷＳＳＶの不活化試験の結果を示す。4 shows the results of a WSSV inactivation test carried out in Example 4 using a fraction obtained by fractionating a mixture of a culture supernatant of OKHT3 strain and a cell extract or a heat-treated mixture. 実施例５において、ＯＫＨＴ１６菌株を条件１および２について実施した、ＷＳＳＶの不活化試験の結果を示す。In Example 5, the results of the WSSV inactivation test performed on the OKHT16 strain under conditions 1 and 2 are shown.
　本発明の一実施形態について、以下に説明するが、本発明はこれに限定されるものではない。本発明は、以下に説明する各構成に限定されるものではなく、請求の範囲に示した範囲で種々の変更が可能であり、異なる実施形態にそれぞれ開示された技術的手段を適宜組み合わせて得られる実施形態に関しても本発明の技術的範囲に含まれる。 An embodiment of the present invention will be described below, but the present invention is not limited to this. The present invention is not limited to the configurations described below, and can be modified in various ways within the scope of the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. Any embodiment is also included in the technical scope of the present invention.
　〔１．抗ホワイトスポットウイルス剤〕
　本発明の一実施形態に係る抗ホワイトスポットウイルス剤は、ロドブラム・エスピー（Rhodovulum sp.）に由来する成分を含有する。 [1. Anti-white spot virus agent]
An anti-white spot virus agent according to one embodiment of the present invention contains a component derived from Rhodovulum sp.
　前述したように、特許文献１には、バチルス属細菌によるＷＳＳＶへの効果が記載されているが、感染試験におけるエビの生存率は最大でも２割程度にしかならない。ＷＳＳＶは、ホワイトスポット病の原因となるウイルスであり、世界中のエビ類養殖場で被害が報告されている。その病原性は非常に高く、感染したエビは３～１０日で死亡すると言われている。また、特許文献２には、光合成細菌によるビブリオ菌への効果が記載されているが、ＷＳＳＶへの効果は示されていない。 As mentioned above, Patent Document 1 describes the effect of bacteria of the genus Bacillus on WSSV, but the survival rate of shrimp in infection tests is only about 20% at maximum. WSSV is a virus that causes white spot disease, and damage has been reported in shrimp farms around the world. Its pathogenicity is extremely high, and infected shrimp are said to die within 3 to 10 days. Moreover, Patent Document 2 describes the effect of photosynthetic bacteria on Vibrio, but does not show the effect on WSSV.
　本発明者らは、ロドブラム・エスピー（Rhodovulum sp.）に由来する成分が水生生物のホワイトスポットウイルス病に優れた効果を示すことを確認した。ロドブラム・エスピー（Rhodovulum sp.）は、海洋性の光合成細菌である。 The present inventors have confirmed that components derived from Rhodovulum sp. exhibit excellent effects on white spot virus disease in aquatic organisms. Rhodovulum sp. is a marine photosynthetic bacterium.
　「ロドブラム・エスピー（Rhodovulum sp.）に由来する成分」とは、ロドブラム・エスピー（Rhodovulum sp.）に属する細菌に由来する成分を言う。前記成分は、例えば、ロドブラム・エスピー（Rhodovulum sp.）に属する菌株の菌体自体であってもよい。本明細書において、前記菌体は、生菌体であってもよいし、死菌体であってもよい。また、前記成分は、例えば、前記菌株によって産生され、菌体外に放出された菌体外成分であってもよいし、前記菌株の菌体内から抽出した菌体抽出物であってもよい。 "Ingredients derived from Rhodovulum sp." refer to ingredients derived from bacteria belonging to Rhodovulum sp. Said component may be, for example, the cells themselves of a strain belonging to Rhodovulum sp. In the present specification, the microbial cells may be viable microbial cells or dead microbial cells. Further, the component may be, for example, an extracellular component produced by the strain and released outside the cells, or a bacterial cell extract extracted from the cells of the strain.
　ここで、「エスピー（sp.）」とは、一般的に、種、または種名の特定できない１種を意味することが知られている。よって、「ロドブラム・エスピー（Rhodovulum sp.）に属する細菌」は、ロドブラム属に属する細菌であれば、種が特定されている菌株、および種が特定されていない菌株のいずれであってもよい。本発明は特に限定されるものではないが、ロドブラム・エスピー（Rhodovulum sp.）に属する細菌としては、例えば、ＯＫＨＴ３菌株、ＯＫＨＴ１６菌株、Rhodovulum imhoffii、Rhodovulum viride、Rhodovulum strictum、Rhodovulum lacipunicei、またはRhodovulum marinum等が挙げられる。 Here, "sp." is generally known to mean a species or a species whose species name cannot be specified. Therefore, "a bacterium belonging to Rhodovulum sp." may be either a strain whose species is specified or a strain whose species is not specified, as long as it is a bacterium belonging to the genus Rhodovulum. Although the present invention is not particularly limited, examples of bacteria belonging to Rhodovulum sp. include OKHT3 strain, OKHT16 strain, Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, Rhodovulum marinum, and the like. is mentioned.
　前記成分として、前記菌株の菌体自体を含む場合としては、例えば、前記菌体の培養液を挙げることができる。前記培養液を得る方法としては、例えば、後述する実施例１で用いた培地（ｐＨ６．８）に、前記菌体を１×１０３ｃｅｌｌｓ／ｍＬの濃度となるように加えた培養液を、２５～２８℃、好気明条件下で５日間、振盪培養する方法を挙げることができる。 In the case of containing the cells of the strain itself as the component, for example, a culture solution of the cells can be mentioned. As a method for obtaining the culture solution, for example, the culture solution obtained by adding the cells to the medium (pH 6.8) used in Example 1 described later so as to have a concentration of 1×10 3 cells/mL, Shaking culture for 5 days under aerobic conditions at 25 to 28° C. can be mentioned.
　前記菌体外成分としては、例えば、前記菌株の培養上清を用いることができる。当該培養上清を得る方法としては、例えば前述した前記菌体の培養液から、４，０００×ｇおよび１０分間の条件で遠心分離することにより、菌体を除去する方法を挙げることができる。 For example, the culture supernatant of the strain can be used as the extracellular component. As a method for obtaining the culture supernatant, for example, a method of removing the cells from the above-mentioned culture solution of the cells by centrifuging under conditions of 4,000×g and 10 minutes can be mentioned.
　前記方法等によって調製した培養上清は、そのまま抗ホワイトスポットウイルス剤の成分として用いることができる。 The culture supernatant prepared by the above method can be used as it is as a component of the anti-white spot virus agent.
　前記菌体抽出物は、例えば、前記菌株の懸濁液を調製し、フレンチプレス、ホモジナイザー、乳鉢等を用いて菌体を破砕し、遠心分離によって不溶物を除去し、上清を回収することによって得ることができる。 The bacterial cell extract is obtained, for example, by preparing a suspension of the bacterial strain, crushing the bacterial cells using a French press, homogenizer, mortar, etc., removing insoluble matter by centrifugation, and recovering the supernatant. can be obtained by
　より具体的には、例えば、前記菌体の培養液から培養上清を除いて得られた菌体ペレットに蒸留水を加えて乳鉢にて破砕し、２０，０００×ｇ、１０分間、および４℃の条件で遠心分離することにより分離した上清を回収し、孔径０．７μｍのフィルターを用いてろ過することによって、菌体抽出物を調製することが好ましい。 More specifically, for example, distilled water is added to the cell pellet obtained by removing the culture supernatant from the cell culture solution, and the cell pellet is crushed in a mortar at 20,000×g for 10 minutes and 4 times. It is preferable to prepare a cell extract by collecting the supernatant separated by centrifugation at ℃ conditions and filtering using a filter with a pore size of 0.7 μm.
　前記ロドブラム・エスピー（Rhodovulum sp.）に由来する成分は、前記ロドブラム・エスピー（Rhodovulum sp.）の培養上清であることが好ましい。当該構成によれば、前記培養上清に餌を浸漬させ、前記培養上清を浸透させた餌を水生生物に与えることにより、水生生物に容易に抗ホワイトスポットウイルス剤を摂取させることができる。また、後述する実施例に示すように、ＷＳＳＶに対する優れた不活化効果を得ることができる。 The component derived from Rhodovulum sp. is preferably the culture supernatant of Rhodovulum sp. According to this configuration, the aquatic organisms can be easily ingested with the anti-white spot virus agent by immersing the bait in the culture supernatant and providing the aquatic organism with the bait permeated with the culture supernatant. In addition, as shown in Examples described later, an excellent inactivation effect against WSSV can be obtained.
　前記成分は、前記培養上清と、前記菌体抽出物とを含有することがより好ましい。この場合、メカニズムは不明であるが、後述する実施例に記載するように、前記成分として前記培養上清のみを用いる場合よりも、さらに優れた前記不活化効果を得ることができる。 The component more preferably contains the culture supernatant and the bacterial cell extract. In this case, although the mechanism is unknown, the inactivation effect can be obtained even more excellent than in the case of using only the culture supernatant as the component, as described later in Examples.
　前記成分として、前記培養上清と、前記菌体抽出物とを用いる場合、前述したように調製した培養上清と、菌体抽出物との混合比は、例えば、体積比で１：１とすることができる。前記培養上清と菌体抽出物との混合は、任意の方法によって行えばよく、特に限定されるものではない。 When the culture supernatant and the bacterial cell extract are used as the components, the mixing ratio of the culture supernatant prepared as described above and the bacterial cell extract is, for example, 1:1 by volume. can do. Mixing of the culture supernatant and the cell extract may be performed by any method, and is not particularly limited.
　前記成分の分子量は、１００ｋＤａ以上であることが好ましい。後述する実施例に示すように、前記成分の分子量が１００ｋＤａ以上である場合、前記分子量が１００ｋＤａ未満の成分を用いた場合よりも、ＷＳＳＶに対する優れた不活化効果を得ることができる。 The molecular weight of the component is preferably 100 kDa or more. As shown in Examples described later, when the molecular weight of the component is 100 kDa or more, a superior inactivation effect against WSSV can be obtained as compared with the case where the component with a molecular weight of less than 100 kDa is used.
　また、前記成分は、６０℃で２時間加熱した場合であっても、当該加熱を行わない場合と同等の前記不活化効果を奏することが好ましい。当該構成によれば、本発明の一実施形態に係る抗ホワイトスポットウイルス剤に十分な耐熱性を付与することができる。そのため、熱に対する安定性に優れ、保存性に優れる抗ホワイトスポットウイルス剤を提供することができる。 In addition, even when the component is heated at 60°C for 2 hours, it is preferable that the same inactivation effect as when the heating is not performed is achieved. According to this configuration, it is possible to impart sufficient heat resistance to the anti-white spot virus agent according to one embodiment of the present invention. Therefore, it is possible to provide an anti-white spot virus agent with excellent heat stability and storage stability.
　本発明の一実施形態に係る抗ホワイトスポットウイルス剤は、ロドブラム・エスピー（Rhodovulum sp.）に由来する成分以外に、他の成分を含んでいてもよい。他の成分としては、例えば、滅菌人工海水等を挙げることができる。 The anti-white spot virus agent according to one embodiment of the present invention may contain other components in addition to components derived from Rhodovulum sp. Other components include, for example, sterilized artificial seawater.
　本発明の一実施形態に係る抗ホワイトスポットウイルス剤は、後述する実施例に示すように、ＷＳＳＶを不活化させる作用に加え、前記抗ホワイトスポットウイルス剤を餌に混入させることにより、水生生物の体内において免疫を賦活化させる作用がある。よって、本発明の一実施形態に係る抗ホワイトスポットウイルス剤は、水生生物に対する免疫賦活剤としても使用できる。 The anti-white spot virus agent according to one embodiment of the present invention has the effect of inactivating WSSV, and in addition to the action of inactivating WSSV, by mixing the anti-white spot virus agent into feed, as shown in the examples described later. It has the effect of stimulating immunity in the body. Therefore, the anti-white spot virus agent according to one embodiment of the present invention can also be used as an immunostimulator against aquatic organisms.
　前記ロドブラム・エスピー（Rhodovulum sp.）は、特に限定されるものではないが、ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）、ＯＫＨＴ１６菌株（ＮＩＴＥ　ＢＰ－０３４９９）、Rhodovulum imhoffii、Rhodovulum viride、Rhodovulum strictum、Rhodovulum lacipunicei、およびRhodovulum marinumからなる群より選ばれる少なくとも１つであることが好ましく、ＯＫＨＴ３菌株、ＯＫＨＴ１６菌株、Rhodovulum imhoffii、およびRhodovulum virideからなる群より選ばれる少なくとも１つであることがより好ましい。 The Rhodovulum sp. is not particularly limited, but OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum, and more preferably at least one selected from the group consisting of OKHT3 strain, OKHT16 strain, Rhodovulum imhoffii, and Rhodovulum viride.
　本発明者らは、和歌山県の沿岸域底質より採取した海水中の細菌の中から、１種類の菌株を単離し、これをＯＫＨＴ３菌株と命名した。本発明者らは、次世代シーケンサＭｉＳｅｑ（登録商標、イルミナ製）を用いてドラフトゲノム解析を行い、ＯＫＨＴ３菌株の１６Ｓ　ｒＲＮＡを、既知のロドブラム・エスピー（Rhodovulum sp.）ＮＩ２２株(https:/ /pubmed.ncbi.nlm.nih.gov/25614575/)の１６Ｓ　ｒＲＮＡと比較し、最尤法分子系統樹解析を行った。 The present inventors isolated one strain from among the bacteria in seawater collected from the coastal sediment in Wakayama Prefecture, and named it the OKHT3 strain. The present inventors performed draft genome analysis using the next-generation sequencer MiSeq (registered trademark, manufactured by Illumina), and analyzed the 16S rRNA of the OKHT3 strain with the known Rhodovulum sp. NI22 strain (https:// pubmed.ncbi.nlm.nih.gov/25614575/) and compared with 16S rRNA, maximum likelihood molecular phylogenetic tree analysis was performed.
　その結果、ＯＫＨＴ３菌株は、海洋性の光合成細菌であるロドブラム属に属し、ロドブラム・エスピー（Rhodovulum sp.）ＮＩ２２株と同一種であるが、異なる菌株であることが分かった。ＯＫＨＴ３菌株は、独立行政法人製品評価技術基盤機構特許微生物寄託センター（千葉県木更津市かずさ鎌足２－５－８　１２２号室）に寄託されている（受託番号は、ＮＩＴＥ　ＢＰ－０３４９８（受託日：２０２１年７月２８日、移管日：２０２２年７月１２日）である）。 As a result, it was found that the OKHT3 strain belongs to the genus Rhodoblum, a marine photosynthetic bacterium, and is the same species as the Rhodovulum sp. NI22 strain, but is a different strain. The OKHT3 strain has been deposited at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) (acceptance number is NITE BP-03498 (acceptance date: July 28, 2021, transfer date: July 12, 2022)).
　ＯＫＨＴ１６菌株は、前記ドラフトゲノム解析により、前記ロドブラム・エスピー（Rhodovulum sp.）ＮＩ２２株と同一種であることが確認でき、かつ、ＮＩ２２株およびＯＫＨＴ３菌株とは異なることが確認できる菌株の一つである。ＯＫＨＴ１６菌株は、独立行政法人製品評価技術基盤機構特許微生物寄託センター（千葉県木更津市かずさ鎌足２－５－８　１２２号室）に寄託されている（受託番号は、ＮＩＴＥ　ＢＰ－０３４９９（受託日：２０２１年７月２８日、移管日：２０２２年７月１２日）である）。 The OKHT16 strain is one of the strains that can be confirmed by the draft genome analysis that it is the same species as the Rhodovulum sp. NI22 strain and that it is different from the NI22 strain and the OKHT3 strain. be. The OKHT16 strain has been deposited at the National Institute of Technology and Evaluation Patent Microorganism Depositary Center (Room 122, Kazusa Kamatari 2-5-8, Kisarazu City, Chiba Prefecture) (acceptance number is NITE BP-03499 (acceptance date: July 28, 2021, transfer date: July 12, 2022)).
　〔２．抗ホワイトスポットウイルス剤の製造方法〕
　本発明の一実施形態に係る抗ホワイトスポットウイルス剤の製造方法は、ロドブラム・エスピー（Rhodovulum sp.）を培養する工程を含む。前記ロドブラム・エスピー（Rhodovulum sp.）は、ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）、ＯＫＨＴ１６菌株（ＮＩＴＥ　ＢＰ－０３４９９）、Rhodovulum imhoffii、Rhodovulum viride、Rhodovulum strictum、Rhodovulum lacipunicei、およびRhodovulum marinumからなる群より選ばれる少なくとも１つであることが好ましい。 [2. Method for producing anti-white spot virus agent]
A method for producing an anti-white spot virus agent according to one embodiment of the present invention includes a step of culturing Rhodovulum sp. The Rhodovulum sp. is selected from the group consisting of OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum. At least one is preferred.
　前記工程としては、例えば、後述する実施例１で用いた培地（ｐＨ６．８）に、ロドブラム・エスピー（Rhodovulum sp.）に属する菌株の菌体を１×１０３ｃｅｌｌｓ／ｍＬの濃度となるように加えた培養液を、２５～２８℃、好気明条件下で５日間、振盪培養する方法を挙げることができる。 As the step, for example, cells of a strain belonging to Rhodovulum sp. are added to the medium (pH 6.8) used in Example 1 described later so as to have a concentration of 1×10 3 cells/mL. A method of culturing with shaking the culture solution added to the above at 25 to 28° C. under aerobic conditions for 5 days can be exemplified.
　前記工程を経て得られた培養液には、菌体および培養上清が含有されている。菌体および培養上清は、共に、前記〔１．抗ホワイトスポットウイルス剤〕で述べた、「ロドブラム・エスピー（Rhodovulum sp.）に由来する成分」に該当する。よって、前記培養液を、本発明の一実施形態に係る抗ホワイトスポットウイルス剤の成分として用いることができる。 The culture solution obtained through the above process contains bacterial cells and culture supernatant. Both the bacterial cells and the culture supernatant were prepared according to [1. anti-white spot viral agent]. Therefore, the culture medium can be used as a component of the anti-white spot virus agent according to one embodiment of the present invention.
　例えば、前記〔１．抗ホワイトスポットウイルス剤〕で述べたように前記工程を経て得られた培養液から、４，０００×ｇおよび１０分間の条件で遠心分離して菌体を除去することにより、培養上清を調製することができる。そして、当該培養上清を、本発明の一実施形態に係る抗ホワイトスポットウイルス剤の成分として用いることができる。 For example, the above [1. anti-white spot virus agent], the culture solution obtained through the above steps is centrifuged at 4,000 x g for 10 minutes to remove the cells, to prepare the culture supernatant. can do. Then, the culture supernatant can be used as a component of the anti-white spot virus agent according to one embodiment of the present invention.
　さらに、前記〔１．抗ホワイトスポットウイルス剤〕で述べたように、菌体抽出物を調製し、当該菌体抽出物を前記培養上清と混合して得られた混合物も、本発明の一実施形態に係る抗ホワイトスポットウイルス剤の成分として用いることができる。このとき、培養上清と、菌体抽出物との混合比は、前記〔１．抗ホワイトスポットウイルス剤〕で述べたとおりである。 Furthermore, the above [1. anti-white spot virus agent], a mixture obtained by preparing a bacterial cell extract and mixing the bacterial cell extract with the culture supernatant is also an anti-white spot virus agent according to an embodiment of the present invention. It can be used as a component of spot virus agents. At this time, the mixing ratio of the culture supernatant and the bacterial cell extract is the same as in [1. anti-white spot virus agent].
　これらの抗ホワイトスポットウイルス剤の成分の分子量は、前記〔１．抗ホワイトスポットウイルス剤〕で述べたように、１００ｋＤａ以上であることが好ましい。分子量が１００ｋＤａ以上である抗ホワイトスポットウイルス剤の成分は、例えば限外ろ過フィルターを用い、前記成分の分子サイズを分画することによって得ることができる。 The molecular weights of the components of these anti-white spot virus agents are as described in [1. anti-white spot virus agent], it is preferably 100 kDa or more. A component of an anti-white spot virus agent having a molecular weight of 100 kDa or more can be obtained by fractionating the molecular size of the component using, for example, an ultrafiltration filter.
　〔３．抗ホワイトスポットウイルスの防除方法〕
　本発明の一実施形態に係る抗ホワイトスポットウイルスの防除方法は、本発明の一実施形態に係る抗ホワイトスポットウイルス剤を水生生物に摂取させる工程を含む方法である。 [3. Anti-white spot virus control method]
A method for controlling an anti-white spot virus according to an embodiment of the present invention is a method comprising the step of ingesting an anti-white spot virus agent according to an embodiment of the present invention to an aquatic organism.
　本発明の一実施形態に係る抗ホワイトスポットウイルス剤は、ＷＳＳＶによる病害が発生しうる水生生物に適用することができる。前記水生生物としては、魚類、甲殻類、貝類等の種々の水生動物を挙げることができる。 The anti-white spot virus agent according to one embodiment of the present invention can be applied to aquatic organisms that are susceptible to disease caused by WSSV. Examples of the aquatic organisms include various aquatic animals such as fish, crustaceans, and shellfish.
　前記水生生物は、ＷＳＳＶによる病害が特に顕著な水生生物であるため、エビであることが好ましい。また、特に、クルマエビ、バナメイエビ、ウシエビ（ブラックタイガー）を始めとするクルマエビ科のエビの前記病害の防除に好適に使用することができる。 The aquatic organisms are preferably shrimps because they are aquatic organisms that are particularly susceptible to WSSV-induced disease. In particular, it can be suitably used for controlling the above-mentioned diseases of prawns belonging to the prawn family including kuruma prawn, vannamei prawn, and bull prawn (black tiger).
　前記抗ホワイトスポットウイルス剤は、水生動物の餌に混入させること等によって、水生生物に摂取させることができ、その効果を発揮させることができる。 The anti-white spot virus agent can be ingested by aquatic organisms by, for example, mixing it into the food of aquatic animals, and can exert its effect.
　前記抗ホワイトスポットウイルス剤を餌に混入させる場合は、ロドブラム・エスピー（Rhodovulum sp.）の菌体の培養液が、餌１ｇ当たりに０．３ｍＬ～０．５ｍＬとなるように混入させることが好ましい。前記培養液の調製については、前述したとおりである。 When the anti-white spot virus agent is mixed in the feed, it is preferable to mix the culture solution of Rhodovulum sp. in an amount of 0.3 mL to 0.5 mL per 1 g of the feed. . Preparation of the culture solution is as described above.
　本発明の一実施形態に係る抗ホワイトスポットウイルス剤を使用して病害を防除することにより、持続可能な開発目標（ＳＤＧｓ）の目標２「飢餓をゼロに」および目標１４「海の豊かさを守ろう」の達成・実現に貢献することができる。 By controlling diseases using the anti-white spot virus agent according to one embodiment of the present invention, the Sustainable Development Goals (SDGs) Goal 2 "Zero hunger" and Goal 14 "Abundant seas" We can contribute to the achievement and realization of "Let's protect".
　〔４．ロドブラム・エスピー（Rhodovulum sp.）ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）〕
　ロドブラム・エスピー（Rhodovulum sp.）ＯＫＨＴ３菌株は、前述したように、本発明者らが海水中の細菌から単離したものであり、ロドブラム・エスピー（Rhodovulum sp.）に属する新規な菌株である。 [4. Rhodovulum sp. OKHT3 strain (NITE BP-03498)]
As described above, the Rhodovulum sp. OKHT3 strain was isolated from bacteria in seawater by the present inventors, and is a novel strain belonging to Rhodovulum sp.
　〔５．まとめ〕
　本発明には、以下の態様が含まれる。
〈１〉ロドブラム・エスピー（Rhodovulum sp.）に由来する成分を含有する、抗ホワイトスポットウイルス剤。
〈２〉前記ロドブラム・エスピー（Rhodovulum sp.）は、ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）、ＯＫＨＴ１６菌株（ＮＩＴＥ　ＢＰ－０３４９９）、Rhodovulum imhoffii、Rhodovulum viride、Rhodovulum strictum、Rhodovulum lacipunicei、およびRhodovulum marinumからなる群より選ばれる少なくとも１つである、〈１〉に記載の抗ホワイトスポットウイルス剤。
〈３〉前記成分が、前記ロドブラム・エスピー（Rhodovulum sp.）の培養上清である、〈１〉または〈２〉に記載の抗ホワイトスポットウイルス剤。
〈４〉前記成分として、前記ロドブラム・エスピー（Rhodovulum sp.）の菌体抽出物をさらに含有する、〈３〉に記載の抗ホワイトスポットウイルス剤。
〈５〉前記成分の分子量が１００ｋＤａ以上である、〈１〉から〈４〉のいずれか１つに記載の抗ホワイトスポットウイルス剤。
〈６〉ロドブラム・エスピー（Rhodovulum sp.）を培養する工程を含む、抗ホワイトスポットウイルス剤の製造方法。
〈７〉前記ロドブラム・エスピー（Rhodovulum sp.）は、ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）、ＯＫＨＴ１６菌株（ＮＩＴＥ　ＢＰ－０３４９９）、Rhodovulum imhoffii、Rhodovulum viride、Rhodovulum strictum、Rhodovulum lacipunicei、およびRhodovulum marinumからなる群より選ばれる少なくとも１つである、〈６〉に記載の抗ホワイトスポットウイルス剤の製造方法。
〈８〉〈１〉から〈５〉のいずれか１つに記載の抗ホワイトスポットウイルス剤を水生生物に摂取させる工程を含む、抗ホワイトスポットウイルスの防除方法。
〈９〉前記水生生物は甲殻類である、〈８〉に記載の抗ホワイトスポットウイルスの防除方法。
〈１０〉前記甲殻類はエビである、〈９〉に記載の抗ホワイトスポットウイルスの防除方法。
〈１１〉ロドブラム・エスピー（Rhodovulum sp.）ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）。 [5. summary〕
The present invention includes the following aspects.
<1> An anti-white spot virus agent containing a component derived from Rhodovulum sp.
<2> The Rhodovulum sp. is a group consisting of OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum The anti-white spot virus agent according to <1>, which is at least one selected from the above.
<3> The anti-white spot virus agent according to <1> or <2>, wherein the component is the culture supernatant of Rhodovulum sp.
<4> The anti-white spot virus agent according to <3>, further comprising the Rhodovulum sp. bacterial cell extract as the component.
<5> The anti-white spot virus agent according to any one of <1> to <4>, wherein the component has a molecular weight of 100 kDa or more.
<6> A method for producing an anti-white spot virus agent, comprising the step of culturing Rhodovulum sp.
<7> The Rhodovulum sp. is a group consisting of OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum The method for producing the anti-white spot virus agent according to <6>, which is at least one selected from
<8> A method for controlling an anti-white spot virus, comprising the step of causing an aquatic organism to ingest the anti-white spot virus agent according to any one of <1> to <5>.
<9> The method for controlling an anti-white spot virus according to <8>, wherein the aquatic organisms are crustaceans.
<10> The method for controlling an anti-white spot virus according to <9>, wherein the crustacean is shrimp.
<11> Rhodovulum sp. OKHT3 strain (NITE BP-03498).
　以下に実施例により本発明をさらに詳しく説明するが、本発明はこれらに限定されるものではない。 The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these.
　〔実施例１〕
　本発明の一実施形態に係る抗ホワイトスポットウイルス剤によるバナメイエビの成長への影響を確認するために、食餌試験を行った。 [Example 1]
In order to confirm the effect of the anti-white spot virus agent according to one embodiment of the present invention on the growth of Vannamei shrimp, a diet test was conducted.
　人工海水を収容した循環濾過式の１００Ｌ水槽（水温２７℃前後）で、平均体重０．２６ｇのバナメイエビを２００匹／１００Ｌで飼育した。タイで使用されている一般的なエビ餌にＯＫＨＴ１６菌株の培養液を染み込ませた餌を、１日３回、エビの体重の５質量％供給した（これを投与区とよぶ）。 　In a circulating filtration type 100L water tank containing artificial seawater (water temperature around 27℃), 200 vannamei shrimp with an average weight of 0.26g were bred per 100L. A common shrimp feed used in Thailand impregnated with a culture solution of the OKHT16 strain was supplied to the shrimp three times a day at 5% by mass of the body weight of the shrimp (this is referred to as a dosing group).
　前記培養液は、培地として、３％（Ｗ／Ｖ）の塩化ナトリウムを含有する、以下に記載した培地（ｐＨ６．８）を用い、ＯＫＨＴ１６菌株の菌体を、１×１０３ｃｅｌｌｓ／ｍＬの濃度となるように前記培地に加えた培養液を、２５～２８℃、好気明条件下で５日間、振盪培養することによって得た。 The culture medium contains 3% (W / V) sodium chloride and is described below (pH 6.8). The culture solution added to the medium to give the concentration was obtained by culturing with shaking at 25-28° C. under aerobic conditions for 5 days.
　なお、前記培地は、Ｌ－グルタミン酸ナトリウム－水和物からビオチンまでの成分を表１に記載されたｍｇ数だけ容器に秤量し、そこに塩化ナトリウムを３０ｇ加えた後、滅菌水を加えて体積を１Ｌとすることによって調製した。 The medium was prepared by weighing the components from sodium L-glutamate hydrate to biotin in mgs listed in Table 1 into a container, adding 30 g of sodium chloride thereto, and then adding sterilized water to the volume. to 1 L.
　以下に、前記培地の組成を記載する。塩化ナトリウム以外の単位はｍｇ／Ｌである。
Ｌ－グルタミン酸ナトリウム一水和物　３８００
ＤＬ－リンゴ酸　２７００
酵母抽出物　２０００
　（ＮＨ４）２ＨＰＯ４　８００
ＫＨ２ＰＯ４（無水）　５００
Ｋ２ＨＰＯ４（無水）　５００
ＭｇＳＯ４・７Ｈ２Ｏ　２００
ＣａＣｌ２・２Ｈ２Ｏ　　５３
ＭｎＳＯ４・５Ｈ２Ｏ　１．２
チアミン塩酸塩　５
ニコチン酸　５
ビオチン　０．０５
塩化ナトリウム　３０ｇ
　餌の調製は、前記エビ餌を前記培養液に浸漬させ、前記培養液を前記エビ餌に十分に染み込ませることによって行った。 The composition of the medium is described below. Units other than sodium chloride are mg/L.
Sodium L-glutamate monohydrate 3800
DL-malic acid 2700
Yeast extract 2000
( NH4 ) 2HPO4 800
KH2PO4 ( anhydrous ) 500
K2HPO4 ( anhydrous ) 500
MgSO4.7H2O200 _
CaCl2.2H2O53 _
MnSO4.5H2O 1.2
Thiamine hydrochloride 5
nicotinic acid 5
Biotin 0.05
30 g sodium chloride
The food was prepared by immersing the shrimp food in the culture solution and allowing the culture solution to sufficiently permeate the shrimp food.
　また、対照として、別の１００Ｌ水槽で飼育されているバナメイエビに、前記培養液を染み込ませていない餌を、１日に３回、エビの体重の５質量％供給した（これを対照区とよぶ）。 In addition, as a control, 5% by mass of the body weight of the shrimp was supplied to the vannamei shrimp bred in another 100 L water tank three times a day with the food not impregnated with the culture solution (this is called a control group). ).
　試験開始の２週間後および４週間後に、各試験区でランダムに２０匹の体重を測定した。体重の測定結果を図１に示す。 　20 weeks and 4 weeks after the start of the test, the weight of 20 animals was measured randomly in each test group. FIG. 1 shows the results of body weight measurements.
　図１は、実施例１に供試したバナメイエビの体重の測定結果を示す図である。図１の（Ａ）は試験開始２週間後の測定結果を示し、（Ｂ）は試験開始４週間後の測定結果を示す。図１の（Ａ）および（Ｂ）の横軸はエビが飼育された試験区の種類を示し、縦軸はエビの体重（ｇ）を示し、値は平均±標準偏差で示されている。図１の（Ｃ）は、試験期間中のエビの体重の推移を示し、横軸は試験開始日を０とした経過日数を示し、縦軸はエビの体重（ｇ）を示し、値は平均±標準誤差で示されている。 FIG. 1 is a diagram showing the measurement results of the body weight of the vannamei shrimp used in Example 1. (A) of FIG. 1 shows the measurement results two weeks after the start of the test, and (B) shows the measurement results four weeks after the start of the test. In (A) and (B) of FIG. 1, the horizontal axis indicates the type of test plot in which the shrimp were bred, the vertical axis indicates the weight (g) of the shrimp, and the values are shown as mean±standard deviation. (C) of FIG. 1 shows the change in the weight of shrimp during the test period, the horizontal axis indicates the number of days elapsed from the test start date, and the vertical axis indicates the weight of shrimp (g), and the values are averages. Shown as ± s.e.m.
　図１に示すように、ＯＫＨＴ１６菌株を含有する餌による、バナメイエビの体重への有意な影響は確認されなかった。よって、ＯＫＨＴ１６菌株を含有する餌は、バナメイエビの生育に影響を及ぼさないことが確認された。 As shown in Figure 1, no significant effect on the body weight of vannamei shrimp was confirmed by feed containing the OKHT16 strain. Therefore, it was confirmed that the feed containing the OKHT16 strain did not affect the growth of Vannamei shrimp.
　〔実施例２〕
　ＷＳＳＶに対する抗病性を確認するために、感染試験を実施した。具体的には、上記実施例１に記載の食餌試験に供したバナメイエビのうち、食餌試験開始日の２週間後に各試験区においてランダムに２０匹、４週間後に各試験区においてランダムに２５匹のエビを感染試験に供試した。ウイルス液としては、ＷＳＳＶに感染したエビのホモジネートを原液とした低濃度（０．４ｍＬ／１０Ｌ）または高濃度（４．０ｍＬ／１０Ｌ）のウイルス液を使用した。 [Example 2]
An infection test was performed to confirm resistance to WSSV. Specifically, among the vannamei shrimp subjected to the dietary test described in Example 1, 20 animals were randomly selected in each test group two weeks after the start of the dietary test, and 25 animals were randomly selected in each test group after 4 weeks. Shrimp were submitted to the infection test. As the virus solution, low-concentration (0.4 mL/10 L) or high-concentration (4.0 mL/10 L) virus solutions prepared from WSSV-infected shrimp homogenates were used.
　感染試験は、エビをウイルス液に３時間浸漬させることによる浸漬感染により行い、浸漬感染を行った日（試験開始日）から１５日間、経過を観察した。エビの生存率は、以下の式で算出した。
生存率＝（（初期個体数－斃死個体数）／初期個体数）×１００
ＷＳＳＶ感染試験の結果を図２および表２に示す。 The infection test was performed by immersion infection by immersing the shrimp in the virus solution for 3 hours, and the progress was observed for 15 days from the day of the immersion infection (test start date). The survival rate of shrimp was calculated by the following formula.
Survival rate = ((initial number of individuals - number of dead individuals) / initial number of individuals) x 100
The results of the WSSV infection test are shown in FIG. 2 and Table 2.
　なお、前記ウイルス液の濃度は、事前に予備試験を行って決定した。すなわち、実施例１の対照区のエビを、前記低濃度または高濃度のウイルス液に３時間浸漬させ、濃度に略比例したエビの斃死が見られることを確認し、前記濃度を決定した。 The concentration of the virus solution was determined by performing a preliminary test in advance. That is, the shrimp in the control group of Example 1 were immersed in the low-concentration or high-concentration virus solution for 3 hours, and it was confirmed that death of the shrimp was observed approximately proportional to the concentration, and the concentration was determined.
　また、斃死初期のエビについては、エビウイルス検出キットであるShrimple（藤倉化成）を使用し、ＷＳＳＶに感染していることを確認した。 In addition, the shrimp virus detection kit, Shrimple (Fujikura Kasei), was used to confirm that the shrimp were infected with WSSV in the early stages of death.
　図２は、食餌試験開始日の２週間後にバナメイエビを前記ＷＳＳＶ感染試験に供した場合の結果を示す。図２の（Ａ）は低濃度のウイルス液での感染試験結果を示し、（Ｂ）は高濃度のウイルス液での感染試験結果を示す。図の横軸は、試験開始日を０とした経過日数を示し、縦軸はエビの生存率（％）を示す。図に記載されるアスタリスクは、エビの生存率が投与区において対照区と比較して有意に高かったことを示す。（Ａ）において、Ｐ値は０．０００１未満であり、（Ｂ）において、Ｐ値は０．０００２であった。 Fig. 2 shows the results when vannamei shrimp were subjected to the WSSV infection test two weeks after the start of the diet test. (A) of FIG. 2 shows the infection test results with a low-concentration virus solution, and (B) shows the infection test results with a high-concentration virus solution. The horizontal axis of the figure indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the shrimp survival rate (%). Asterisks in the figure indicate that the survival rate of shrimp was significantly higher in the administration group than in the control group. In (A) the P value was less than 0.0001 and in (B) the P value was 0.0002.
Figure JPOXMLDOC01-appb-T000001
 　表１は、前記ＷＳＳＶ感染試験開始から２週間後および４週間後のエビの生存率を示す。分子は生存している個体の数、分母は初期個体数である。
Figure JPOXMLDOC01-appb-T000001
 Table 1 shows the shrimp survival rate two weeks and four weeks after the start of the WSSV infection test. The numerator is the number of living individuals and the denominator is the initial number of individuals.
　図２および表１に示すように、ＯＫＨＴ１６菌株の培養液を含有する餌の給餌によって、ＷＳＳＶ感染症による斃死が軽減されることが確認された。よって、前記培養液にＷＳＳＶに対する抗病性（「感染防除効果」ともいう）があることが示された。つまり前記培養液は、本発明の一実施形態に係る抗ホワイトスポットウイルス剤に該当する。 As shown in Figure 2 and Table 1, it was confirmed that the feeding of the food containing the culture solution of the OKHT16 strain reduced mortality due to WSSV infection. Therefore, it was shown that the culture medium has anti-pathogenicity (also referred to as "infection control effect") against WSSV. That is, the culture solution corresponds to the anti-white spot virus agent according to one embodiment of the present invention.
　〔実施例３〕
　ＯＫＨＴ３菌株のＷＳＳＶへの不活化効果を確認するために不活化試験を行った。試験は、人工海水を収容し、水温が２８℃前後の投げ込み濾過式の１５Ｌ水槽で飼育された、体重約１ｇのバナメイエビ（各試験区で１０匹）を使用して行った。給餌は１日２回行い、餌はタイで使用されている一般的なエビ餌を用いた。 [Example 3]
An inactivation test was performed to confirm the inactivation effect of the OKHT3 strain on WSSV. The test was carried out using vannamei shrimp weighing about 1 g (10 in each test plot), which were kept in a 15 L throw-in filtration type water tank containing artificial seawater at a water temperature of about 28°C. Feeding was carried out twice a day, and the common shrimp feed used in Thailand was used as the feed.
　ＯＫＨＴ３菌株について、培養上清０．１ｍＬ、ＷＳＳＶの希釈液０．１ｍＬ、および滅菌人工海水０．８ｍＬを混合させ、得られた混合液を２５℃で１時間静置し反応させた。 For the OKHT3 strain, 0.1 mL of culture supernatant, 0.1 mL of WSSV diluent, and 0.8 mL of sterilized artificial seawater were mixed, and the resulting mixture was allowed to stand at 25°C for 1 hour to react.
　前記培養上清は、以下の方法によって調製した。すなわち、実施例１で用いた培地（表１に記載の培地）に、ＯＫＨＴ３菌株を、１×１０３ｃｅｌｌｓ／ｍＬの濃度となるように加えた培養液を、２５～２８℃、好気明条件下で５日間、振盪培養した。振盪培養終了後、得られた培養液を４，０００×ｇおよび１０分間の条件で遠心分離し、培養上清を得た。 The culture supernatant was prepared by the following method. That is, the culture solution obtained by adding the OKHT3 strain to the medium used in Example 1 (the medium described in Table 1) so as to have a concentration of 1×10 3 cells/mL was heated at 25 to 28° C. under aerobic conditions. Shaking culture was carried out for 5 days under these conditions. After shaking culture was completed, the resulting culture solution was centrifuged at 4,000×g for 10 minutes to obtain a culture supernatant.
　前記ＷＳＳＶの希釈液の希釈倍率は、事前に予備実験を行って決定した。前記予備実験は以下の手順で行った。ＷＳＳＶに感染したバナメイエビのホモジネートを遠心分離し、エビの組織などを沈殿物として取り除いた。次いで、上清を回収して孔径０．２２μｍのポリエーテルスルホンメンブレン（メルクミリポア製）を用いてろ過し、得られた溶液を原液とした。前記原液を１０倍段階で希釈して各希釈倍率の希釈液を得、前記希釈液５０μｌをバナメイエビの尾部に注射した。希釈倍率が１０の５乗までの希釈液を注射したエビで斃死が見られることを確認し、実施例で使用する希釈倍率を１０の４乗に決定した。 The dilution ratio of the WSSV diluent was determined by conducting a preliminary experiment in advance. The preliminary experiment was conducted in the following procedure. A homogenate of WSSV-infected vannamei shrimp was centrifuged to remove shrimp tissue and the like as a precipitate. Then, the supernatant was recovered and filtered using a polyethersulfone membrane (manufactured by Merck Millipore) having a pore size of 0.22 μm, and the resulting solution was used as a stock solution. The stock solution was diluted in 10-fold steps to obtain dilutions of each dilution rate, and 50 μl of the dilutions were injected into the tail of vannamei shrimp. It was confirmed that shrimp injected with diluents up to the fifth power of 10 died, and the dilution rate used in the examples was determined to be the fourth power of 10.
　反応後の前記混合液をバナメイエビの尾部に５０μＬ注射し、対照のバナメイエビの尾部には、培養上清の代わりに、実施例１で述べた培地０．１ｍＬを用いて調製した前記混合液（以下、「対照混合液」という）を５０μＬ注射した。試験開始日（注射を行った日）から１１日間、エビの生存数を経時的に確認した。斃死初期のエビについては、Shrimple（藤倉化成）を使用し、ＷＳＳＶに感染していることを確認した。 50 μL of the mixed solution after the reaction was injected into the tail of the Vannamei shrimp, and the mixed solution prepared using 0.1 mL of the medium described in Example 1 instead of the culture supernatant (hereinafter referred to as , referred to as “control mixture”) was injected at 50 μL. For 11 days from the test start date (the day of injection), the survival number of shrimp was confirmed over time. Shrimp in the early stages of death were confirmed to be infected with WSSV using Shrimple (Fujikura Kasei).
　なお、前記対照混合液は、実施例１で述べた培地０．１ｍＬ、ＷＳＳＶの希釈液０．１ｍＬ、および滅菌人工海水０．８ｍＬを混合させ、得られた混合液を２５℃で１時間静置することによって調製した。 The control mixture was prepared by mixing 0.1 mL of the culture medium described in Example 1, 0.1 mL of the WSSV diluent, and 0.8 mL of sterilized artificial seawater, and the resulting mixture was allowed to stand at 25°C for 1 hour. prepared by placing
　ＯＫＨＴ３菌株の培養上清を含む混合液を使用したＷＳＳＶ不活化試験の結果を、表２および図３に示す。 Table 2 and Figure 3 show the results of the WSSV inactivation test using a mixed solution containing the culture supernatant of the OKHT3 strain.
Figure JPOXMLDOC01-appb-T000002
 　表２は、本発明の実施例３に係る、ＯＫＨＴ３菌株のＷＳＳＶへの不活化試験における、試験開始日（表中の「試験日」）から１１日間のエビの生存数を経時的に示す。
Figure JPOXMLDOC01-appb-T000002
 Table 2 shows the number of survival shrimp over time for 11 days from the test start date (“test date” in the table) in the OKHT3 strain to WSSV inactivation test according to Example 3 of the present invention.
　図３は、表２の結果をグラフ化したものである。横軸は試験開始日を０とした経過日数を示し、縦軸はエビの生存数（匹）を示す。 Figure 3 is a graph of the results in Table 2. The horizontal axis indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the number of surviving shrimp (individuals).
　表２および図３に示すように、ＯＫＨＴ３菌株の培養上清は、ＷＳＳＶに対し、高い不活化効果を示すことが確認された。 As shown in Table 2 and Figure 3, it was confirmed that the culture supernatant of the OKHT3 strain exhibited a high inactivation effect against WSSV.
　〔実施例４〕
　前記不活化試験（実施例３）でＷＳＳＶの不活化効果が確認されたＯＫＨＴ３菌株につき、培養上清、菌体抽出物、およびこれらの混合物を用いて、さらにＷＳＳＶ不活化試験を行った。培養上清は、ＯＫＨＴ３菌株について実施例３で調製したものと同じものを用いた。 [Example 4]
A WSSV inactivation test was further performed on the OKHT3 strain, which was confirmed to have a WSSV inactivation effect in the inactivation test (Example 3), using culture supernatants, cell extracts, and mixtures thereof. The culture supernatant used was the same as that prepared in Example 3 for the OKHT3 strain.
　菌体抽出物は、実施例３で培養上清を調製した際に得られた菌体ペレットに蒸留水を加えて乳鉢にて破砕し、２０，０００×ｇ、１０分間、および４℃の条件で遠心分離することにより分離した上清を回収し、孔径０．７μｍのフィルターを用いてろ過することによって調製した。 The bacterial cell extract was obtained by adding distilled water to the bacterial cell pellet obtained when the culture supernatant was prepared in Example 3, crushing it in a mortar, and subjecting it to 20,000 x g for 10 minutes at 4°C. The supernatant separated by centrifugation at , was collected and prepared by filtration using a filter with a pore size of 0.7 μm.
　培養上清と菌体抽出物との混合物は、前記培養上清と、前記菌体抽出物とを、体積比１：１で混合することによって調製した。 A mixture of the culture supernatant and the cell extract was prepared by mixing the culture supernatant and the cell extract at a volume ratio of 1:1.
　試験は、ＯＫＨＴ３菌株について、前記培養上清、前記菌体抽出物、もしくは前記培養上清と前記菌体抽出物との混合物（各０．１ｍＬ）に、実施例３で用いたＷＳＳＶの希釈液０．１ｍＬ、および滅菌人工海水０．８ｍＬを混合させ、得られた各混合液を２５℃で１時間静置し反応させた。 In the test, for the OKHT3 strain, the WSSV diluent used in Example 3 was added to the culture supernatant, the bacterial cell extract, or the mixture of the culture supernatant and the bacterial cell extract (0.1 mL each). 0.1 mL and 0.8 mL of sterilized artificial seawater were mixed, and each resulting mixture was allowed to stand at 25° C. for 1 hour to react.
　反応後の前記混合液を、それぞれ、バナメイエビの尾部に５０μＬ注射し、対照のバナメイエビの尾部には実施例３で用いた対照混合液を５０μＬ注射した。試験開始日（注射を行った日）から１１日間、エビの生存数を経時的に確認した。斃死初期のエビについては、Shrimple（藤倉化成）を使用し、ＷＳＳＶに感染していることを確認した。結果を表３および図４に示す。 50 μL of the mixed solution after the reaction was injected into the tail of the vannamei shrimp, and 50 μL of the control mixed solution used in Example 3 was injected into the tail of the control vannamei shrimp. For 11 days from the test start date (the day of injection), the survival number of shrimp was confirmed over time. Shrimp in the early stages of death were confirmed to be infected with WSSV using Shrimple (Fujikura Kasei). Results are shown in Table 3 and FIG.
Figure JPOXMLDOC01-appb-T000003
 　表３は、ＯＫＨＴ３菌株の培養上清、菌体抽出物、およびこれらの混合物をＷＳＳＶ不活化試験に供した結果をそれぞれ示し、試験開始日（表中の「試験日」）から１１日間のエビの生存数を経時的に示す。
Figure JPOXMLDOC01-appb-T000003
 Table 3 shows the results of subjecting the culture supernatant of the OKHT3 strain, the cell extract, and the mixture thereof to the WSSV inactivation test, respectively. survival over time.
　図４は、表３の結果をグラフ化したものである。横軸は試験開始日を０とした経過日数を示し、縦軸はエビの生存数（匹）を示す。また、図中、「上清」は前記培養上清、「抽出物」は前記菌体抽出物、「混合物」は前記混合物をそれぞれ指す。 Figure 4 is a graph of the results in Table 3. The horizontal axis indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the number of surviving shrimp (individuals). In the figure, "supernatant" refers to the culture supernatant, "extract" refers to the bacterial cell extract, and "mixture" refers to the mixture.
　表３および図４が示すように、ＯＫＨＴ３菌株の培養上清を使用した場合に、菌体抽出物を使用した場合および対照と比較して、ＷＳＳＶの不活化効果が優れることが確認され、培養上清と菌体抽出物との混合物を使用した場合にＷＳＳＶの不活化効果がさらに優れることが確認された。 As shown in Table 3 and FIG. 4, it was confirmed that when the culture supernatant of the OKHT3 strain was used, the WSSV inactivation effect was superior to when the cell extract was used and when compared with the control. It was confirmed that the WSSV inactivation effect was more excellent when the mixture of the supernatant and the cell extract was used.
　そこで、ＯＫＨＴ３菌株の培養上清と菌体抽出物との混合物について、ＷＳＳＶの不活化に寄与する物質の分子量を調べるため、前記混合物を限界濾過フィルターで分画した分画物を使用して、本実施例にて前述したＷＳＳＶ不活化試験を行った。 Therefore, in order to examine the molecular weight of a substance that contributes to the inactivation of WSSV in a mixture of the culture supernatant of the OKHT3 strain and the cell extract, a fraction obtained by fractionating the mixture with an ultrafiltration filter was used. The WSSV inactivation test described above in this example was performed.
　前記混合物の分子量は、限界濾過フィルター（メルクミリポア製、品番Amicon Ultra）で１００ｋＤａ以上、１００ｋＤａ未満、３０ｋＤａ未満、または１０ｋＤａ未満に分画した。また、前記限界濾過フィルターによる分画物の他に、前記混合物を、孔径０．２２μｍのポリエーテルスルホンメンブレン（メルクミリポア製）を用いて濾過することにより得たろ液（０．２２μｍ濾過物）、および６０℃で２時間の熱処理を行った前記混合物（以下、「加熱処理物」）を用いて、ＷＳＳＶ不活化試験を行った。結果を表４および図５に示す。 The molecular weight of the mixture was fractionated to 100 kDa or more, less than 100 kDa, less than 30 kDa, or less than 10 kDa with an ultrafiltration filter (manufactured by Merck Millipore, product number Amicon Ultra). In addition to the fractions obtained by the ultrafiltration filter, a filtrate (0.22 μm filtrate) obtained by filtering the mixture using a polyethersulfone membrane (manufactured by Merck Millipore) having a pore size of 0.22 μm, A WSSV inactivation test was performed using the mixture (hereinafter referred to as “heat-treated product”) that had been heat-treated at 60° C. for 2 hours. Results are shown in Table 4 and FIG.
　なお、前記熱処理は、ＯＫＨＴ３菌株の培養上清と菌体抽出物の混合物を、６０℃にセットした保温器に入れ、２時間加熱することにより行った。 The heat treatment was carried out by placing a mixture of the culture supernatant of the OKHT3 strain and the cell extract in a heat incubator set at 60°C and heating for 2 hours.
Figure JPOXMLDOC01-appb-T000004
 　表４は、ＯＫＨＴ３菌株の培養上清と菌体抽出物との混合物を分画した分画物または加熱処理物を用いて実施したＷＳＳＶ不活化試験に結果を示し、試験開始日（表中の「試験日」）から８日間のエビの生存数を経時的に示す。
Figure JPOXMLDOC01-appb-T000004
 Table 4 shows the results of the WSSV inactivation test conducted using the fraction obtained by fractionating the mixture of the culture supernatant of the OKHT3 strain and the cell extract or the heat-treated product. The survival number of shrimp for 8 days from "test day") is shown over time.
　図５は、表４の結果をグラフ化したものである。横軸は試験開始日を０とした経過日数を示し、縦軸はエビの生存数（匹）を示す。 Fig. 5 is a graph of the results of Table 4. The horizontal axis indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the number of surviving shrimp (individuals).
　表４および図５において、「混合物」は、ＯＫＨＴ３菌株の培養上清と菌体抽出物との混合物を限外ろ過フィルターにより分画した場合の、分子量が１００ｋＤａ以上の画分を表す。また、「１００ｋＤａ」は、分子量が１００ｋＤａ未満の画分、「３０ｋＤａ」は、分子量が３０ｋＤａ未満の画分、「１０ｋＤａ」は、分子量が１０ｋＤａ未満の画分をそれぞれ表す。そして、「０．２２μｍ」は、ＯＫＨＴ３菌株の培養上清と菌体抽出物との混合物を、前記ポリエーテルスルホンメンブレンを用いて得たろ液、「混合物６０℃」は、前記加熱処理物をそれぞれ表す。「対照」は、前記混合物の分画物または加熱処理物を用いなかった場合である。 In Table 4 and FIG. 5, "mixture" represents a fraction with a molecular weight of 100 kDa or more when a mixture of the culture supernatant of the OKHT3 strain and the cell extract is fractionated with an ultrafiltration filter. In addition, "100 kDa" represents a fraction with a molecular weight of less than 100 kDa, "30 kDa" represents a fraction with a molecular weight of less than 30 kDa, and "10 kDa" represents a fraction with a molecular weight of less than 10 kDa. And "0.22 μm" is a mixture of the culture supernatant of the OKHT3 strain and the bacterial cell extract, the filtrate obtained using the polyethersulfone membrane, and "mixture 60 ° C." is the heat-treated product. show. A "control" is when no fraction or heat treatment of the mixture was used.
　表４および図５が示すように、分子量が１００ｋＤａ未満ではＷＳＳＶの不活化効果は見られなかった。一方で、６０℃の熱処理でＷＳＳＶの不活化効果は完全には消失しないことが確認された。つまり、ＷＳＳＶの不活化効果に関与する物質は、分子量が１００ｋＤａ以上の高分子の物質であり、前記物質のＷＳＳＶの不活化効果は、６０℃の熱処理で完全には失活しないことが明らかとなった。 As shown in Table 4 and FIG. 5, no WSSV inactivation effect was observed when the molecular weight was less than 100 kDa. On the other hand, it was confirmed that the 60° C. heat treatment did not completely eliminate the WSSV inactivation effect. In other words, it is clear that the substance involved in the WSSV inactivation effect is a macromolecular substance with a molecular weight of 100 kDa or more, and the WSSV inactivation effect of said substance is not completely deactivated by heat treatment at 60°C. became.
　〔実施例５〕
　前記ＷＳＳＶ感染試験（実施例２）で効果が確認されたＯＫＨＴ１６菌株のＷＳＳＶの不活化効果をより詳細に調べるために、条件１と条件２についてＷＳＳＶ不活化試験を行った。 [Example 5]
A WSSV inactivation test was conducted under conditions 1 and 2 in order to examine in more detail the WSSV inactivation effect of the OKHT16 strain, which was confirmed to be effective in the WSSV infection test (Example 2).
　条件２は、培養上清と菌体抽出物との混合物として、実施例３でＯＫＨＴ３菌株の代わりにＯＫＨＴ１６菌株を用いて調製した混合物を用い、試験期間を除いて実施例３と同様の実験を行ったものである。条件１は、条件２で用いた前記混合物の代わりに、当該混合物を４℃で約半年の期間保存した試料を用いたこと以外は、条件２と同じ方法を行ったものである。本実施例の対照は、実施例３と同じ方法で調製した対照混合液を用い、試験期間を除いて実施例３の対照と同様の実験を行ったものである。 Condition 2 used the mixture prepared by using the OKHT16 strain instead of the OKHT3 strain in Example 3 as the mixture of the culture supernatant and the cell extract, and conducted the same experiment as in Example 3 except for the test period. I have been there. Condition 1 was performed in the same manner as Condition 2, except that instead of the mixture used in Condition 2, a sample obtained by storing the mixture at 4°C for about half a year was used. For the control of this example, a control mixture prepared in the same manner as in Example 3 was used, and the same experiment as the control in Example 3 was performed except for the test period.
Figure JPOXMLDOC01-appb-T000005
 　表５は、ＯＫＨＴ１６菌株の条件１および条件２についてのＷＳＳＶ不活化試験における、試験開始日（表中の「試験日」）から７日間のエビの生存数を経時的に示す。
Figure JPOXMLDOC01-appb-T000005
 Table 5 shows the chronological survival of shrimp for 7 days from the test start date (“test date” in the table) in the WSSV inactivation test for conditions 1 and 2 of the OKHT16 strain.
　図６は、表５の結果をグラフ化したものである。横軸は試験開始日を０とした経過日数を示し、縦軸はエビの生存数（匹）を示す。 Figure 6 is a graph of the results of Table 5. The horizontal axis indicates the number of days elapsed since the test start date was set to 0, and the vertical axis indicates the number of surviving shrimp (individuals).
　表５および図６が示すように、条件２において、ＷＳＳＶの不活化効果が確認された。条件１では、条件２で用いた混合物を４℃で長期間保存した試料を用いたところ、ＷＳＳＶの不活化効果が、条件２よりも減衰することが確認された。この結果から、本発明の一実施形態に係る抗ホワイトスポットウイルス剤は、例えば、－８０℃の条件で保存することが好ましいと考えられる。 As shown in Table 5 and FIG. 6, the WSSV inactivation effect was confirmed under condition 2. In condition 1, when a sample obtained by storing the mixture used in condition 2 at 4° C. for a long period of time was used, it was confirmed that the WSSV inactivation effect was attenuated more than under condition 2. From this result, it is considered preferable to store the anti-white spot virus agent according to one embodiment of the present invention, for example, under conditions of -80°C.
　〔実施例６〕
　ＯＫＨＴ３菌株およびＯＫＨＴ１６菌株以外でロドブラム・エスピー（Rhodovulum sp.）に属する菌株のＷＳＳＶへの不活化効果を確認するために、下記菌株の培養上清を用いて不活化試験を行った。
・Rhodovulum sp.ＯＫＨＴ３菌株（上記の実施例と同じ）
・Rhodovulum imhoffii（理化学研究所のJCM No. 13589）
・Rhodovulum viride（NBRCの109122）
　上記の各菌株の培養上清は、実施例３と同様の方法で調製した。実施例３と同様に、得られた培養上清０．１ｍＬ、ＷＳＳＶの希釈液０．１ｍＬ、および滅菌人工海水０．８ｍＬを混合させ、得られた混合液を２５℃で１時間静置し反応させた。 [Example 6]
In order to confirm the effect of inactivating strains belonging to Rhodovulum sp. on WSSV other than the OKHT3 and OKHT16 strains, an inactivation test was performed using the culture supernatants of the following strains.
・ Rhodovulum sp. OKHT3 strain (same as above example)
・Rhodovulum imhoffii (RIKEN JCM No. 13589)
- Rhodovulum viride (NBRC 109122)
The culture supernatant of each of the above strains was prepared in the same manner as in Example 3. In the same manner as in Example 3, 0.1 mL of the obtained culture supernatant, 0.1 mL of WSSV diluent, and 0.8 mL of sterilized artificial seawater were mixed, and the resulting mixture was allowed to stand at 25° C. for 1 hour. reacted.
　前記混合液を、平均体重３．５ｇのバナメイエビ１０匹の尾部に５０μＬずつ注射した。注射を行った日から１１日間、前記バナメイエビを飼育した後、各試験区におけるバナメイエビの生存数を確認した。 50 μL of the mixed solution was injected into the tails of 10 vannamei shrimps with an average weight of 3.5 g. After breeding the vannamei shrimp for 11 days from the day of injection, the survival number of the vannamei shrimp in each test group was confirmed.
　その結果、Rhodovulum sp.ＯＫＨＴ３菌株だけではなく、Rhodovulum imhoffiiおよびRhodovulum virideについても、使用した全てのバナメイエビが生存していることが確認され、ＷＳＳＶへの不活化効果を確認できた。 As a result, not only the Rhodovulum sp. OKHT3 strain, but also Rhodovulum imhoffii and Rhodovulum viride were confirmed to be viable in all of the vannamei shrimp used, confirming their inactivation effect on WSSV.
　〔実施例７〕
　ＯＫＨＴ３菌株およびＯＫＨＴ１６菌株以外で、ロドブラム・エスピー（Rhodovulum sp.）に属する菌株のＷＳＳＶに対する抗病性を確認するために、下記菌株の培養液を用いて、抗病性確認試験を行った。
・Rhodovulum sp.ＯＫＨＴ３菌株（上記の実施例と同じ）
・Rhodovulum imhoffii（理化学研究所のJCM No. 13589）
・Rhodovulum viride（NBRCの109122）
・Rhodovulum strictum（理化学研究所のJCM No. 9221）
・Rhodovulum marinum（理化学研究所のJCM No. 13300）
　上記の各菌株の培養液は、実施例１と同様の方法で得た。 [Example 7]
In order to confirm the resistance to WSSV of strains other than the OKHT3 and OKHT16 strains belonging to Rhodovulum sp., a culture solution of the following strains was used to perform a disease resistance confirmation test.
・ Rhodovulum sp. OKHT3 strain (same as above example)
・Rhodovulum imhoffii (RIKEN JCM No. 13589)
- Rhodovulum viride (NBRC 109122)
・Rhodovulum strictum (RIKEN JCM No. 9221)
・Rhodovulum marinum (RIKEN JCM No. 13300)
A culture solution of each of the above strains was obtained in the same manner as in Example 1.
　循環式の１００Ｌ水槽に平均体重１．０ｇのバナメイエビを２０匹投入した。タイで使用されている一般的なエビ餌に上記菌株の培養液を染み込ませた餌を、１日３回、エビの体重の５質量％供給した。 　20 vannamei shrimp with an average weight of 1.0g were put into a circulating 100L water tank. A common shrimp feed used in Thailand impregnated with the culture solution of the above strain was fed three times a day at 5% by mass of the body weight of the shrimp.
　餌の調製は、前記エビ餌１．０ｇ当たり、上記菌株の培養液３００μＬを吸着させ、前記培養液を前記エビ餌に十分に染み込ませることにより行った。 The food was prepared by adsorbing 300 μL of the culture solution of the above strain per 1.0 g of the shrimp food, and allowing the culture solution to sufficiently permeate the shrimp food.
　給餌を開始して８日目に、実施例２と同様の方法でエビを浸漬感染させることにより感染試験を行った。浸漬感染を行った日（試験開始日）から１１日間飼育した後、各試験区におけるエビの生存率を確認した。 On the 8th day after the start of feeding, an infection test was conducted by immersing the shrimp in the same manner as in Example 2. After breeding for 11 days from the day of immersion infection (test start date), the survival rate of the shrimp in each test plot was confirmed.
　Rhodovulum sp.ＯＫＨＴ３菌株について、前記培養液を餌に添加し、８日間給餌すると、ＷＳＳＶの感染が抑制されることが確認できた。つまり、ＷＳＳＶに対する抗病性が確認できた。さらに、表６に示すように、Rhodovulum sp.ＯＫＨＴ３菌株だけではなく、Rhodovulum imhoffii、Rhodovulum viride、Rhodovulum strictum、Rhodovulum marinumについてもＷＳＳＶに対する抗病性が確認できた。なお、表中の「実施例１で用いた培地」とは、前記培養液の代わりに、実施例１で用いた培地のみを添加した餌を与えた場合のことである。 Regarding the Rhodovulum sp. OKHT3 strain, it was confirmed that WSSV infection was suppressed when the culture solution was added to the feed and fed for 8 days. In other words, the disease resistance against WSSV could be confirmed. Furthermore, as shown in Table 6, not only the Rhodovulum sp. OKHT3 strain, but also Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, and Rhodovulum marinum were confirmed to be resistant to WSSV. The term "medium used in Example 1" in the table refers to the case where only the medium used in Example 1 was added instead of the culture solution.
Figure JPOXMLDOC01-appb-T000006
 　〔実施例８〕
　Rhodovulum sp.ＯＫＨＴ３菌株が、バナメイエビの生体防御機能に与える影響を確認するために、バナメイエビの鰓における免疫関連遺伝子の発現量の変化を観察した。
Figure JPOXMLDOC01-appb-T000006
 [Example 8]
In order to confirm the effect of the Rhodovulum sp. OKHT3 strain on the biological defense function of the Vannamei shrimp, changes in the expression levels of immune-related genes in the gills of the Vannamei shrimp were observed.
　試験区１として、温度を２８℃に保った人工海水を収容した循環濾過式の１００Ｌ水槽に、体重４．０±１．２５ｇのバナメイエビを３０匹投入した。タイで使用されている一般的なエビ餌に培養液を染み込ませた餌を、１日３回、８日間、エビに体重の５質量％供給した。使用した培養液は実施例１と同様の方法で調製した。餌の調製は、前記エビ餌１．０ｇ当たり、培養液３００μＬを吸着させ、前記培養液を前記エビ餌に十分に染み込ませることにより行った。 As test area 1, 30 vannamei shrimp weighing 4.0 ± 1.25 g were put into a 100 L circulation filtration water tank containing artificial seawater kept at a temperature of 28°C. A common shrimp feed used in Thailand impregnated with a culture solution was fed to the shrimp 3 times a day for 8 days at 5% by weight of the body weight. The culture medium used was prepared in the same manner as in Example 1. The feed was prepared by adsorbing 300 μL of the culture solution per 1.0 g of the shrimp feed, and allowing the shrimp feed to sufficiently permeate the culture solution.
　また、試験区２として、別の１００Ｌ水槽に、体重４．０±１．２５ｇのバナメイエビを３０匹投入し、培養液の代わりに培養上清を染み込ませた餌を、１日３回、８日間、エビに体重の５質量％供給した。使用した培養上清は、実施例３と同様の方法で調製した。餌の調製は、前記エビ餌１．０ｇ当たり、培養上清３００μＬを吸着させ、前記エビ餌に十分に染み込ませることにより行った。 In addition, as test group 2, 30 vannamei shrimp weighing 4.0 ± 1.25 g were added to another 100 L water tank, and the food impregnated with the culture supernatant instead of the culture solution was fed 3 times a day, 8 Shrimp were fed 5% by weight of body weight for days. The culture supernatant used was prepared in the same manner as in Example 3. The feed was prepared by adsorbing 300 μL of the culture supernatant per 1.0 g of the shrimp feed and permeating the shrimp feed sufficiently.
　さらに、対照区として、別の１００Ｌ水槽に、体重４．０±１．２５ｇのバナメイエビを３０匹投入し、実施例１で用いた培地のみを添加することにより調製した餌を、１日３回、７日間、エビに体重の５質量％供給した。餌の調製は、前記エビ餌１．０ｇ当たり、実施例１で用いた培地３００μＬを吸着させ、前記エビ餌に十分に染み込ませることにより行った。 Furthermore, as a control group, 30 vannamei shrimp weighing 4.0 ± 1.25 g were added to another 100 L water tank, and the food prepared by adding only the medium used in Example 1 was fed three times a day. , fed shrimp at 5% by mass of body weight for 7 days. The feed was prepared by adsorbing 300 μL of the culture medium used in Example 1 per 1.0 g of the shrimp feed, and thoroughly impregnating the shrimp feed.
　８日目に給餌を１回行い、給餌から３時間後に、各試験区および対照区においてランダムに５匹のエビを選び、鰓のサンプルを得た。さらに、５匹分の鰓のサンプルのうち３匹分の鰓のサンプルをランダムに選び、網羅的遺伝子発現解析を行った。 Feeding was performed once on the 8th day, and 3 hours after feeding, 5 shrimp were randomly selected in each test group and control group to obtain gill samples. Furthermore, 3 of the 5 gill samples were randomly selected for comprehensive gene expression analysis.
　本願実施例では、シーケンサとしてＭｉｓｅｑシステム（イルミナ社製）を使用して網羅的遺伝子発現解析を行った。その後、発現変動遺伝子の検出を以下の手順で行った。
１．ＲＳＥＭを使用して、網羅的遺伝子発現解析で得られたｍＲＮＡの発現量の推定を行った。
２．統計ソフトウェアＲを使用して二群間比較（ＤＥＳｅｑ２）を行い、対照区のエビの鰓サンプルと、試験区１のエビの鰓サンプルとで発現量が２倍以上異なる遺伝子を検出した。 In the examples of the present application, comprehensive gene expression analysis was performed using the Miseq system (manufactured by Illumina) as a sequencer. After that, the expression-variable gene was detected by the following procedure.
1. RSEM was used to estimate the expression level of mRNA obtained by the global gene expression analysis.
2. A two-group comparison (DESeq2) was performed using statistical software R, and genes whose expression levels differed by 2-fold or more between the shrimp gill samples of the control group and the shrimp gill samples of the test group 1 were detected.
Figure JPOXMLDOC01-appb-T000007
 　表７は、上記２．で検出された遺伝子を示す。試験区１のエビの鰓サンプルにおいて、対照区のエビの鰓サンプルと比較して、penaeidin-3a-likeの発現が２倍以上増強されていることが確認できた。penaeidin-3a-likeは、免疫関連遺伝子として周知の遺伝子である（参考文献：Shih-Hu Ho, Yu-Chan Chao, Hsiao-Wei Tsao, Masahiro Sakai, Hong-Nong Chou and Yen-Ling Song (2004) Molecular Cloning and Recombinant Expression of Tiger Shrimp Penaeus monodon Penaeidin. Fish Pathology,39(1),15-23,2004.3）。
Figure JPOXMLDOC01-appb-T000007
 Table 7 shows the above 2. shows genes detected in . It was confirmed that the expression of penaeidin-3a-like in the shrimp gill sample of Test Group 1 was enhanced by two times or more compared to the shrimp gill sample of the control group. penaeidin-3a-like is a well-known immune-related gene (reference: Shih-Hu Ho, Yu-Chan Chao, Hsiao-Wei Tsao, Masahiro Sakai, Hong-Nong Chou and Yen-Ling Song (2004) Molecular Cloning and Recombinant Expression of Tiger Shrimp Penaeus monodon Penaeidin. Fish Pathology, 39(1), 15-23, 2004.3).
　この結果から、本発明の一実施形態に係る抗ホワイトスポットウイルス剤は、エビの体内において免疫を賦活化させる作用があることが確認された。 From these results, it was confirmed that the anti-white spot virus agent according to one embodiment of the present invention has the effect of stimulating immunity in the body of shrimp.
　本発明の一態様は、水生動物の養殖における飼料添加剤等の分野で使用可能である。 One aspect of the present invention can be used in fields such as feed additives in aquaculture.
　ＮＩＴＥ　ＢＰ－０３４９８
　ＮＩＴＥ　ＢＰ－０３４９９ NITE BP-03498
NITE BP-03499

Claims (11)

  1. 　ロドブラム・エスピー（Rhodovulum sp.）に由来する成分を含有する、抗ホワイトスポットウイルス剤。 An anti-white spot virus agent containing components derived from Rhodovulum sp.
  2. 　前記ロドブラム・エスピー（Rhodovulum sp.）は、ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）、ＯＫＨＴ１６菌株（ＮＩＴＥ　ＢＰ－０３４９９）、Rhodovulum imhoffii、Rhodovulum viride、Rhodovulum strictum、Rhodovulum lacipunicei、およびRhodovulum marinumからなる群より選ばれる少なくとも１つである、請求項１に記載の抗ホワイトスポットウイルス剤。 The Rhodovulum sp. is selected from the group consisting of OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum The anti-white spot virus agent of claim 1, which is at least one.
  3. 　前記成分が、前記ロドブラム・エスピー（Rhodovulum sp.）の培養上清である、請求項１または２に記載の抗ホワイトスポットウイルス剤。 The anti-white spot virus agent according to claim 1 or 2, wherein the component is the culture supernatant of Rhodovulum sp.
  4. 　前記成分として、前記ロドブラム・エスピー（Rhodovulum sp.）の菌体抽出物をさらに含有する、請求項３に記載の抗ホワイトスポットウイルス剤。 The anti-white spot virus agent according to claim 3, further comprising the Rhodovulum sp. bacterial cell extract as the component.
  5. 　前記成分の分子量が１００ｋＤａ以上である、請求項１～４のいずれか１項に記載の抗ホワイトスポットウイルス剤。 The anti-white spot virus agent according to any one of claims 1 to 4, wherein the component has a molecular weight of 100 kDa or more.
  6. 　ロドブラム・エスピー（Rhodovulum sp.）を培養する工程を含む、抗ホワイトスポットウイルス剤の製造方法。 A method for producing an anti-white spot virus agent, including the step of culturing Rhodovulum sp.
  7. 　前記ロドブラム・エスピー（Rhodovulum sp.）は、ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）、ＯＫＨＴ１６菌株（ＮＩＴＥ　ＢＰ－０３４９９）、Rhodovulum imhoffii、Rhodovulum viride、Rhodovulum strictum、Rhodovulum lacipunicei、およびRhodovulum marinumからなる群より選ばれる少なくとも１つである、請求項６に記載の抗ホワイトスポットウイルス剤の製造方法。 The Rhodovulum sp. is selected from the group consisting of OKHT3 strain (NITE BP-03498), OKHT16 strain (NITE BP-03499), Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, Rhodovulum lacipunicei, and Rhodovulum marinum The method for producing the anti-white spot virus agent according to claim 6, which is at least one.
  8. 　請求項１から５のいずれか１項に記載の抗ホワイトスポットウイルス剤を水生生物に摂取させる工程を含む、抗ホワイトスポットウイルスの防除方法。 A method for controlling an anti-white spot virus, comprising the step of ingesting the anti-white spot virus agent according to any one of claims 1 to 5 to an aquatic organism.
  9. 　前記水生生物は甲殻類である、請求項８に記載の抗ホワイトスポットウイルスの防除方法。 The method for controlling an anti-white spot virus according to claim 8, wherein the aquatic organisms are crustaceans.
  10. 　前記甲殻類はエビである、請求項９に記載の抗ホワイトスポットウイルスの防除方法。 The method for controlling an anti-white spot virus according to claim 9, wherein the crustacean is shrimp.
  11. 　ロドブラム・エスピー（Rhodovulum sp.）ＯＫＨＴ３菌株（ＮＩＴＥ　ＢＰ－０３４９８）。 Rhodovulum sp. OKHT3 strain (NITE BP-03498).
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