Novel microorganism, anti-white spot virus agent containing novel microorganism, method for producing the same, and method for controlling white spot virus

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

[Technical Field]

[0001] The present invention relates to a novel microorganism, an anti-white spot virus agent containing the novel microorganism, etc., a method for producing the same, and Niho This invention relates to a method for controlling white spot virus. [Background technology]

[0002] In recent years, disease control has become the biggest challenge in the farming or breeding of aquatic animals. In shrimp farming both in Japan and overseas, drugs such as antibiotics have traditionally been used as a measure to prevent pathogenic bacteria and viral infections. However, the use of these drugs has the problem of promoting the emergence of resistant bacteria and affecting other biological species in nature, raising safety concerns when aquatic organisms treated with these drugs are to be consumed.

[0003] Therefore, research is being conducted on materials using useful microorganisms as an alternative to the above-mentioned drugs. For example, Patent Document 1 describes the antiviral activity of Bacillus subtilis strains against White Spot Syndrome Virus (hereinafter referred to as "WSSV"). Patent Document 2 describes the "early stage" of the disease caused by pathogenic Vibrio bacteria. death The effectiveness of a photosynthetic bacterium against acute hepatopancreatic necrosis syndrome / acute hepatopancreatic necrosis disease (EMS / AHPHD) has been described. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japan Special Publication No. 2020-506872 [Patent Document 2] Japanese Patent Publication No. 2019-97530 Summary of the Invention [Problem to be solved by the invention]

[0005] 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 low at around 20%. Patent Document 2 does not disclose the effectiveness of the microorganism against WSSV. Therefore, there is a need for an anti-white spot virus agent that is naturally derived and has a strong control effect against WSSV.

[0006] One aspect of the present invention is a novel microorganism ,new Anti-white spot virus agent containing a novel microorganism, etc., and its manufacturing method, Niho The object of the present invention is to provide a method for controlling white spot virus. [Means for solving the problem]

[0007] An anti-white spot virus agent containing ingredients derived from Rhodovulum sp.

[0008] A method for producing an anti-white spot virus agent, comprising a step of culturing Rhodovulum sp.

[0009] Rhodovulum sp. OKHT3 strain (NITE BP-03498). [Effects of the Invention]

[0010] According to one aspect of the present invention, an anti-white spot virus agent having excellent control effect against WSSV can be provided. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the results of measuring the body weight of the vannamei shrimp used in Example 1, where (A) shows the measurement results 2 weeks after the start of the test, (B) shows the measurement results 4 weeks after the start of the test, and (C) shows the change in body weight over the test period. [Figure 2]1 shows the results of a WSSV infection test in Example 2, where (A) shows the results of an infection test using a low-concentration virus solution, and (B) shows the results of an infection test using a high-concentration virus solution. [Figure 3] In Example 3, the results of a WSSV inactivation test using a mixture of culture supernatant and cell extract of the OKHT3 strain are shown. [Figure 4] Example 4 shows the results of a WSSV inactivation test using a culture supernatant of the OKHT3 strain, a bacterial cell extract, and a mixture thereof. [Figure 5] In Example 4, the results of a WSSV inactivation test were shown, which was carried out using a fraction obtained by fractionating a mixture of culture supernatant and cell extract of the OKHT3 strain, or a heat-treated product of the mixture. [Figure 6] In Example 5, the results of a WSSV inactivation test performed using the OKHT16 strain under conditions 1 and 2 are shown. DETAILED DESCRIPTION OF THE INVENTION

[0012] 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 various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention.

[0013] [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.

[0014] As mentioned above, Patent Document 1 describes the effectiveness of Bacillus bacteria against WSSV, but the survival rate of shrimp in infection tests was only about 20% at most. WSSV is a virus that causes white spot disease, and damage has been reported at shrimp farms around the world. It is highly pathogenic, and infected shrimp are said to die within 3 to 10 days. Furthermore, Patent Document 2 describes the effectiveness of photosynthetic bacteria against Vibrio bacteria, but does not disclose its effectiveness against WSSV.

[0015] The present inventors have confirmed that components derived from Rhodovulum sp. are highly effective against white spot virus disease in aquatic organisms. Rhodovulum sp. is a marine photosynthetic bacterium.

[0016] "Components derived from Rhodovulum sp." refer to components derived from bacteria belonging to Rhodovulum sp. The components may be, for example, the bacterial cells themselves of a strain belonging to Rhodovulum sp. In this specification, the bacterial cells may be live or dead. Furthermore, the components may be, for example, extracellular components produced by the strain and released outside the bacterial cells, or bacterial cell extracts extracted from the bacterial cells of the strain.

[0017] Here, "sp." is generally known to mean a species or a species for which the species name cannot be identified. Therefore, "bacteria belonging to Rhodovulum sp." may refer to bacteria belonging to the genus Rhodovulum, whether the species is identified or not. 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, and Rhodovulum marinum.

[0018] When the components contain the cells of the strain, for example, the culture solution of the cells can be used. The culture solution can be obtained by, for example, adding 1×10 cells to the medium (pH 6.8) used in Example 1 described later. 3 For example, a culture medium is added to give a concentration of 100 cells / mL, and the mixture is cultured with shaking at 25 to 28°C under aerobic and bright conditions for 5 days.

[0019] The extracellular components may be, for example, the culture supernatant of the strain. The culture supernatant can be obtained, for example, by removing the bacterial cells from the culture medium of the bacterial cells by centrifugation at 4,000 × g for 10 minutes.

[0020] The culture supernatant prepared by the above method or the like can be used as it is as a component of an anti-white spot virus agent.

[0021] The bacterial cell extract can be obtained, for example, by preparing a suspension of the bacterial strain, disrupting the bacterial cells using a French press, a homogenizer, a mortar, or the like, removing insoluble matter by centrifugation, and recovering the supernatant.

[0022] More specifically, for example, a bacterial cell extract is preferably prepared by adding distilled water to the bacterial cell pellet obtained by removing the culture supernatant from the bacterial cell culture solution, crushing the bacterial cell pellet in a mortar, and centrifuging the mixture at 20,000 × g for 10 minutes at 4°C to separate the supernatant, which is then recovered and filtered through a filter with a pore size of 0.7 μm.

[0023] The component derived from Rhodovulum sp. is preferably a culture supernatant of Rhodovulum sp. According to this configuration, the anti-white spot virus agent can be easily ingested by aquatic organisms by soaking food in the culture supernatant and feeding the food soaked in the culture supernatant to the aquatic organisms. Furthermore, as shown in the examples described below, an excellent inactivation effect against WSSV can be obtained.

[0024] It is more preferable that the components contain the culture supernatant and the bacterial cell extract, and although the mechanism is unclear, as described in the Examples below, a more excellent inactivation effect can be obtained than when only the culture supernatant is used as the component.

[0025] 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 to the bacterial cell extract can be, for example, a volume ratio of 1: 1. The mixing of the culture supernatant and the bacterial cell extract can be carried out by any method, and is not particularly limited.

[0026] The molecular weight of the component is preferably 100 kDa or more. As shown in the Examples below, when the molecular weight of the component is 100 kDa or more, a superior inactivation effect against WSSV can be obtained compared to when a component with a molecular weight of less than 100 kDa is used.

[0027] Furthermore, it is preferable that the components exhibit the same inactivation effect even when heated at 60°C for 2 hours as when not heated. This configuration can 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 that is excellent in heat stability and storage stability.

[0028] The anti-white spot virus agent according to one embodiment of the present invention may contain other components in addition to the component derived from Rhodovulum sp. Examples of the other components include sterilized artificial seawater.

[0029] As shown in the Examples below, the anti-white spot virus agent according to one embodiment of the present invention not only inactivates WSSV, but also activates the immune system in aquatic organisms when the agent is mixed into feed. Thus, the anti-white spot virus agent according to one embodiment of the present invention can also be used as an immunostimulant for aquatic organisms.

[0030] The Rhodovulum sp. is not particularly limited, but is preferably at least one 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, and more preferably at least one selected from the group consisting of OKHT3 strain, OKHT16 strain, Rhodovulum imhoffii, and Rhodovulum viride.

[0031] The present inventors isolated a bacterial strain from seawater collected from coastal sediments in Wakayama Prefecture, Japan, which they named OKHT3. They performed draft genome analysis using the MiSeq (registered trademark, Illumina) next-generation sequencer, compared the 16S rRNA of OKHT3 with that of the known Rhodovulum sp. NI22 strain (https: / / pubmed.ncbi.nlm.nih.gov / 25614575 / ), and performed maximum likelihood phylogenetic tree analysis.

[0032] The OKHT3 strain was found to belong to the marine photosynthetic bacterium genus Rhodovulum and to be the same species as, but different from, the Rhodovulum sp. NI22 strain. The OKHT3 strain has been deposited at the National Institute of Technology and Evaluation (NITE BP-03498, accession date: July 28, 2021, transfer date: July 12, 2022) at the Patent Microorganism Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan).

[0033] The OKHT16 strain is one of the strains that can be confirmed by the draft genome analysis to be the same species as the Rhodovulum sp. NI22 strain, but different from the NI22 strain and the OKHT3 strain. The OKHT16 strain has been deposited at the National Institute of Technology and Evaluation (NITE BP-03499, accession date: July 28, 2021, transfer date: July 12, 2022) at the Patent Microorganism Deposit Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan).

[0034] 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 culturing Rhodovulum sp. The Rhodovulum sp. is preferably at least one 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.

[0035] In this step, for example, 1 × 10 cells of a strain belonging to Rhodovulum sp. were added to the medium (pH 6.8) used in Example 1 described below. 3 For example, a culture medium is added to give a concentration of 100 cells / mL, and the mixture is cultured with shaking at 25 to 28°C under aerobic and bright conditions for 5 days.

[0036] The culture medium obtained through the above steps contains bacterial cells and a culture supernatant. Both the bacterial cells and the culture supernatant correspond to the "components derived from Rhodovulum sp." described above in [1. Anti-white spot virus agent]. Therefore, the culture medium can be used as a component of an anti-white spot virus agent according to one embodiment of the present invention.

[0037] For example, as described above in [1. Anti-white spot virus agent], the culture medium obtained through the above steps can be centrifuged at 4,000 × g for 10 minutes to remove the bacterial cells, thereby preparing a culture supernatant. The culture supernatant can then be used as a component of the anti-white spot virus agent according to one embodiment of the present invention.

[0038] Furthermore, as described above in [1. Anti-white spot virus agents], a mixture obtained by preparing a bacterial cell extract and mixing the bacterial cell extract with the culture supernatant can also be used as a component of an anti-white spot virus agent according to one embodiment of the present invention. In this case, the mixing ratio of the culture supernatant to the bacterial cell extract is as described above in [1. Anti-white spot virus agents].

[0039] As described above in [1. Anti-white spot virus agents], the molecular weight of the components of these anti-white spot virus agents is preferably 100 kDa or more. Components of anti-white spot virus agents having a molecular weight of 100 kDa or more can be obtained by fractionating the molecular size of the components using, for example, an ultrafiltration filter.

[0040] [3 .Ho How to control white spot virus According to one embodiment of the present invention Ruho A method for controlling white spot virus is a method comprising a step of causing an aquatic organism to ingest the anti-white spot virus agent according to one embodiment of the present invention.

[0041] An anti-white spot virus agent according to one embodiment of the present invention can be applied to aquatic organisms that may be affected by WSSV, including various aquatic animals such as fish, crustaceans, and shellfish.

[0042] The aquatic organism is preferably a shrimp, since it is an aquatic organism particularly susceptible to WSSV disease. In particular, the method can be suitably used to control the disease in shrimp of the Penaeidae family, including kuruma prawn, vannamei shrimp, and Penaeus monodon (black tiger shrimp).

[0043] The anti-white spot virus agent can be ingested by aquatic organisms by, for example, mixing it with the feed of the aquatic animals, thereby allowing the agent to exert its effects.

[0044] When the anti-white spot virus agent is mixed into the feed, it is preferable to mix 0.3 mL to 0.5 mL of culture medium of Rhodovulum sp. per 1 g of feed. The preparation of the culture medium is as described above.

[0045] By controlling diseases using the anti-white spot virus agent according to one embodiment of the present invention, it is possible to contribute to achieving and realizing Goal 2 "Zero Hunger" and Goal 14 "Life below water" of the Sustainable Development Goals (SDGs).

[0046] [4. Rhodovulum sp. OKHT3 strain (NITE BP-03498)] As described above, the Rhodovulum sp. OKHT3 strain was isolated by the present inventors from bacteria in seawater, and is a novel strain belonging to the genus Rhodovulum.

[0047] [5. Summary] The present invention includes the following aspects. <1> An anti-white spot virus agent containing ingredients derived from Rhodovulum sp. <2> The anti-white spot virus agent according to <1>, wherein the Rhodovulum sp. is at least one 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. <3> The anti-white spot virus agent according to <1> or <2>, wherein the component is a culture supernatant of Rhodovulum sp. <4> The anti-white spot virus agent according to <3>, further comprising a fungal extract of Rhodovulum sp. as the ingredient. <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 a step of culturing Rhodovulum sp. <7> The method for producing an anti-white spot virus agent according to <6>, wherein the Rhodovulum sp. is at least one 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. <8> A method comprising a step of ingesting the anti-white spot virus agent according to any one of <1> to <5> to an aquatic organism. , Ho How to control white spot virus. <9> The aquatic organism is a crustacean, as described in <8>. Noho How to control white spot virus. <10> The crustacean is a shrimp, as described in <9>. Noho How to control white spot virus. <11> Rhodovulum sp. OKHT3 strain (NITE BP-03498). [Example]

[0048] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0049] Example 1 A feeding test was conducted 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.

[0050] Vannamei shrimp weighing an average of 0.26g were raised at a rate of 200 shrimp per 100L in a circulating, filtered 100L tank (water temperature around 27°C) containing artificial seawater. A diet of standard shrimp feed used in Thailand soaked in a culture solution of the OKHT16 strain was supplied three times a day at a rate of 5% by mass of the shrimp's body weight (this is called the treatment group).

[0051] The culture medium used was the medium described below (pH 6.8) containing 3% (W / V) sodium chloride, and the cells of the OKHT16 strain were cultured at a concentration of 1 × 10 3 The culture solution was added to the medium to give a concentration of 1000 cells / mL, and the mixture was cultured with shaking at 25 to 28°C under aerobic and bright conditions for 5 days to obtain the desired cell suspension.

[0052] The medium contains components ranging from L-sodium glutamate monohydrate to biotin. below The amount of the solution in mg indicated on the label was weighed into a container, 30 g of sodium chloride was added, and then sterilized water was added to bring the volume to 1 L.

[0053] The composition of the medium is shown below. Units other than sodium chloride are mg / L. Monosodium L-glutamate monohydrate 3800 DL-Malic acid 2700 yeast extract 2000 (NH4)2HPO4800 KH2PO4 (anhydrous) 500 K2HPO4 (anhydrous) 500 MgSO4·7H2O 200 CaCl2·2H2O 53 MnSO4·5H2O 1.2 Thiamine Hydrochloride 5 Nicotinic Acid 5 Biotin 0.05 30g sodium chloride The shrimp feed was prepared by immersing the shrimp feed in the culture solution and allowing the culture solution to fully permeate the shrimp feed.

[0054] As a control, vannamei shrimps were reared in another 100 L tank and were fed with food that had not been soaked in the culture solution three times a day in an amount of 5% by mass of the shrimps' body weight (this was referred to as the control group).

[0055] Two and four weeks after the start of the test, 20 animals were randomly selected from each test group and weighed. The weight measurement results are shown in Figure 1.

[0056] FIG. 1 shows the results of measuring the body weight of the vannamei shrimp used in Example 1. (A) in FIG. 1 shows the results of measurement two weeks after the start of the test, and (B) shows the results of measurement four weeks after the start of the test. In (A) and (B) in FIG. 1, the horizontal axis indicates the type of test plot in which the shrimp were reared, and the vertical axis indicates the shrimp body weight (g), with values ​​expressed as mean ± standard deviation. (C) in FIG. 1 shows the change in shrimp body weight over the test period, with the horizontal axis indicating the number of days elapsed since the start of the test was set at 0, and the vertical axis indicating the shrimp body weight (g), with values ​​expressed as mean ± standard error.

[0057] As shown in Figure 1, no significant effect of the feed containing the OKHT16 strain on the body weight of vannamei shrimp was confirmed. Therefore, it was confirmed that the feed containing the OKHT16 strain does not affect the growth of vannamei shrimp.

[0058] Example 2 An infection test was conducted to confirm resistance to WSSV. Specifically, of the vannamei shrimp used in the feeding test described in Example 1 above, 20 shrimp were randomly selected from each test plot two weeks after the start of the feeding test, and 25 shrimp were randomly selected from each test plot four weeks later. A low-concentration (0.4 mL / 10 L) or high-concentration (4.0 mL / 10 L) virus solution, prepared by using a homogenate of WSSV-infected shrimp as the stock solution, was used.

[0059] The infection test was carried out by immersion infection of shrimp in the virus solution for 3 hours, and the progress was observed for 15 days from the day of immersion infection (the start of the test). The survival rate of shrimp was calculated using the following formula. Survival rate = ((initial population size - number of dead individuals) / initial population size) x 100 The results of the WSSV infection test are shown in Figure 2 and Table 2.

[0060] The virus solution concentration was determined by a preliminary test in advance: shrimp from the control group in Example 1 were immersed in the virus solution of the low or high concentration for 3 hours, and the concentration was determined by confirming that the shrimp died in a proportion to the concentration.

[0061] In addition, shrimp that had died in the early stages were confirmed to be infected with WSSV using a shrimp virus detection kit, Shrimple (Fujikura Kasei).

[0062] Figure 2 shows the results of the WSSV infection test in which vannamei shrimp were subjected to the test two weeks after the start of the feeding test. Figure 2 (A) shows the results of the infection test using a low-concentration virus solution, and (B) shows the results of the infection test using a high-concentration virus solution. The horizontal axis of the figure indicates the number of days since the start of the test, with the day being set as 0, and the vertical axis indicates the survival rate (%) of shrimp. An asterisk in the figure indicates that the survival rate of shrimp in the treatment group was significantly higher than that of the control group. In (A), the P value was less than 0.0001, and in (B), the P value was 0.0002.

[0063] [Table 1] Table 1 shows the survival rates of shrimp 2 and 4 weeks after the start of the WSSV infection test. The numerator is the number of surviving individuals, and the denominator is the initial population.

[0064] As shown in Figure 2 and Table 1, it was confirmed that feeding food containing a culture medium of the OKHT16 strain reduced mortality due to WSSV infection. This indicates that the culture medium has an anti-WSSV disease effect (also referred to as an "infection control effect"). In other words, the culture medium corresponds to an anti-white spot virus agent according to one embodiment of the present invention.

[0065] Example 3 An inactivation test was conducted to confirm the inactivation effect of the OKHT3 strain on WSSV. The test was conducted using vannamei shrimp (10 shrimp per test group) weighing approximately 1 g, reared in a 15 L submerged filtration tank containing artificial seawater at a temperature of approximately 28°C. The shrimp were fed twice a day using shrimp feed commonly used in Thailand.

[0066] For the OKHT3 strain, 0.1 mL of the culture supernatant, 0.1 mL of a diluted solution of WSSV, 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 allow the reaction to occur.

[0067] The culture supernatant was prepared by the following method. On the ground , OKHT3 strain, 1 x 10 3 The culture medium was added to a concentration of 1000 cells / mL and cultured with shaking at 25-28°C under aerobic and light conditions for 5 days. After the culture with shaking was completed, the resulting culture medium was centrifuged at 4,000 × g for 10 minutes to obtain the culture supernatant.

[0068] The dilution ratio of the WSSV diluted solution was determined by a preliminary experiment. The preliminary experiment was performed as follows: A homogenate of WSSV-infected whitenamei shrimp was centrifuged to remove shrimp tissues and other precipitates. The supernatant was then collected and filtered using a polyethersulfone membrane (Merck Millipore) with 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 increments to obtain diluted solutions of each dilution ratio, and 50 μl of each diluted solution was injected into the tail of a whitenamei shrimp. It was confirmed that shrimp died when diluted with dilutions up to the power of 10, and the dilution ratio used in the examples was determined to be 10 to the power of 4.

[0069] After the reaction, 50 μL of the mixture was injected into the tail of each whiteleg shrimp, and 50 μL of the mixture prepared using 0.1 mL of the medium described in Example 1 instead of the culture supernatant (hereinafter referred to as the "control mixture") was injected into the tail of each control whiteleg shrimp. The number of surviving shrimp was counted over 11 days from the start of the test (the day of injection). Shrimp that had died in the early stages were confirmed to be infected with WSSV using Shrimp (Fujikura Kasei).

[0070] The control mixture was prepared by mixing 0.1 mL of the medium described in Example 1, 0.1 mL of a diluted solution of WSSV, and 0.8 mL of sterilized artificial seawater, and allowing the resulting mixture to stand at 25°C for 1 hour.

[0071] The results of the WSSV inactivation test using a mixture containing the culture supernatant of the OKHT3 strain are shown in Table 2 and Figure 3.

[0072] [Table 2] Table 2 shows the number of surviving shrimp over time for 11 days from the start of the test ("Test Date" in the table) in the test of inactivation of WSSV by the OKHT3 strain according to Example 3 of the present invention.

[0073] Figure 3 is a graph of the results of Table 2. The horizontal axis shows the number of days elapsed since the start of the test, with the day being set as 0, and the vertical axis shows the number of surviving shrimp (pieces).

[0074] As shown in Table 2 and FIG. 3, it was confirmed that the culture supernatant of the OKHT3 strain exhibited a high inactivation effect against WSSV.

[0075] Example 4 For the OKHT3 strain, whose WSSV inactivation effect was confirmed in the inactivation test (Example 3), further WSSV inactivation tests were performed using the culture supernatant, bacterial extract, and a mixture thereof. The culture supernatant used was the same as that prepared for the OKHT3 strain in Example 3.

[0076] The bacterial cell extract was prepared by adding distilled water to the bacterial cell pellet obtained when preparing the culture supernatant in Example 3, crushing the pellet in a mortar, centrifuging the mixture at 20,000 × g for 10 minutes at 4°C, recovering the separated supernatant, and filtering it through a filter with a pore size of 0.7 μm.

[0077] A mixture of the culture supernatant and the bacterial cell extract was prepared by mixing the culture supernatant and the bacterial cell extract at a volume ratio of 1:1.

[0078] For the OKHT3 strain, the culture supernatant, the bacterial cell extract, or a mixture of the culture supernatant and the bacterial cell extract (0.1 mL each) was mixed with 0.1 mL of the diluted WSSV solution used in Example 3 and 0.8 mL of sterilized artificial seawater, and the resulting mixtures were allowed to stand at 25°C for 1 hour to react.

[0079] After the reaction, 50 μL of the mixture was injected into the tail of each vannamei shrimp, and 50 μL of the control mixture used in Example 3 was injected into the tail of control vannamei shrimp. The number of surviving shrimp was counted over 11 days from the start of the test (the day of injection). Shrimp that had died in the early stages were confirmed to be infected with WSSV using Shrimp (Fujikura Kasei). The results are shown in Table 3 and Figure 4.

[0080] [Table 3] Table 3 shows the results of a WSSV inactivation test using the culture supernatant, bacterial extract, and a mixture of these from the OKHT3 strain, and shows the number of surviving shrimp over time for 11 days from the start of the test (labeled "Test Date" in the table).

[0081] Figure 4 is a graph of the results of Table 3. The horizontal axis indicates the number of days elapsed since the start of the test, with the day being set as 0, and the vertical axis indicates the number of surviving shrimp. In the figure, "supernatant" refers to the culture supernatant, "extract" refers to the bacterial extract, and "mixture" refers to the mixture.

[0082] As shown in Table 3 and Figure 4, it was confirmed that the use of the culture supernatant of the OKHT3 strain had a superior WSSV inactivation effect compared to the use of the bacterial cell extract and the control, and that the use of a mixture of the culture supernatant and the bacterial cell extract had an even superior WSSV inactivation effect.

[0083] Therefore, in order to investigate the molecular weight of the substance that contributes to the inactivation of WSSV in a mixture of culture supernatant and bacterial cell extract of the OKHT3 strain, the WSSV inactivation test described above in this example was performed using fractions obtained by fractionating the mixture using an ultrafiltration filter.

[0084] The mixture was fractionated using an ultrafiltration filter (Merck Millipore, product number Amicon Ultra) into molecular weight fractions of ≥100 kDa, <100 kDa, <30 kDa, or <10 kDa. In addition to the ultrafiltration fractions, WSSV inactivation tests were also performed using the filtrate obtained by filtering the mixture through a 0.22 μm pore size polyethersulfone membrane (Merck Millipore) (0.22 μm filtrate) and the mixture after heat treatment at 60°C for 2 hours (hereinafter referred to as the "heat-treated product"). The results are shown in Table 4 and Figure 5.

[0085] The heat treatment was carried out by placing a mixture of the culture supernatant and cell extract of the OKHT3 strain in an incubator set at 60° C. and heating for 2 hours.

[0086] [Table 4] Table 4 shows the results of a WSSV inactivation test conducted using fractions or heat-treated products obtained by fractionating a mixture of culture supernatant and bacterial extract of the OKHT3 strain, and shows the number of surviving shrimp over time for 8 days from the start of the test ("Test Date" in the table).

[0087] Figure 5 is a graph of the results of Table 4. The horizontal axis shows the number of days elapsed since the start of the test, with the day being set as 0, and the vertical axis shows the number of surviving shrimp (pieces).

[0088] In Table 4 and Figure 5, "Mixture" represents the fraction with a molecular weight of 100 kDa or greater when a mixture of culture supernatant and cell extract from the OKHT3 strain is fractionated using an ultrafiltration filter. "100 kDa" represents the fraction with a molecular weight of less than 100 kDa, "30 kDa" represents the fraction with a molecular weight of less than 30 kDa, and "10 kDa" represents the fraction with a molecular weight of less than 10 kDa. "0.22 μm" represents the filtrate obtained by filtering the mixture of culture supernatant and cell extract from the OKHT3 strain using the polyethersulfone membrane. "Mixture 60°C" represents the heat-treated product. "Control" represents the case where the fraction or heat-treated product of the mixture was not used.

[0089] As shown in Table 4 and Figure 5, no WSSV inactivation effect was observed with a molecular weight of less than 100 kDa. On the other hand, it was confirmed that the WSSV inactivation effect was not completely lost by heat treatment at 60°C. In other words, it was revealed that the substances involved in the WSSV inactivation effect are high molecular weight substances with a molecular weight of 100 kDa or more, and that the WSSV inactivation effect of these substances is not completely lost by heat treatment at 60°C.

[0090] Example 5 In order to investigate in more detail the WSSV inactivation effect of the OKHT16 strain, the effectiveness of which was confirmed in the WSSV infection test (Example 2), a WSSV inactivation test was carried out under conditions 1 and 2.

[0091] In condition 2, a mixture of culture supernatant and bacterial cell extract prepared using the OKHT16 strain instead of the OKHT3 strain in Example 3 was used, and an experiment similar to that in Example 3 was conducted, except for the test period. In condition 1, the same method as in condition 2 was conducted, except that a sample of the mixture stored at 4°C for approximately six months was used instead of the mixture used in condition 2. As a control in this example, a control mixture prepared in the same manner as in Example 3 was used, and an experiment similar to that in Example 3 was conducted, except for the test period.

[0092] [Table 5] Table 5 shows the number of surviving shrimp over time for 7 days from the start of the test ("Test Date" in the table) in the WSSV inactivation test for the OKHT16 strain under conditions 1 and 2.

[0093] Figure 6 is a graph of the results of Table 5. The horizontal axis shows the number of days elapsed, with the test start date set as 0, and the vertical axis shows the number of surviving shrimp (pieces).

[0094] As shown in Table 5 and Figure 6, the inactivation effect of WSSV was confirmed under condition 2. When a sample of the mixture used under condition 2 that had been stored at 4°C for a long period of time was used under condition 1, it was confirmed that the inactivation effect of WSSV was reduced more than under condition 2. From these results, it is considered preferable to store the anti-white spot virus agent according to one embodiment of the present invention under conditions such as -80°C.

[0095] Example 6 To confirm the inactivation effect of strains belonging to Rhodovulum sp. other than OKHT3 and OKHT16 on WSSV, inactivation tests were performed using culture supernatants of the following strains. Rhodovulum sp. OKHT3 strain (same as in the above example) Rhodovulum imhoffii (RIKEN JCM No. 13589) ·Rhodovulum viride (NBRC's 109122) The culture supernatant of each of the above strains was prepared in the same manner as in Example 3. As in Example 3, 0.1 mL of the obtained culture supernatant, 0.1 mL of a diluted solution of WSSV, 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.

[0096] 50 μL of the mixture was injected into the tail of 10 vannamei shrimps weighing an average of 3.5 g. The vannamei shrimps were reared for 11 days from the day of injection, and the number of surviving vannamei shrimps in each test group was counted.

[0097] As a result, it was confirmed that all vannamei shrimp used survived not only the Rhodovulum sp. OKHT3 strain, but also Rhodovulum imhoffii and Rhodovulum viride, confirming the inactivation effect on WSSV.

[0098] Example 7 In order to confirm the resistance of strains belonging to Rhodovulum sp. other than the OKHT3 and OKHT16 strains to WSSV, a resistance confirmation test was carried out using the culture medium of the following strains. Rhodovulum sp. OKHT3 strain (same as in the above example) Rhodovulum imhoffii (RIKEN JCM No. 13589) ·Rhodovulum viride (NBRC's 109122) Rhodovulum strictum (RIKEN JCM No. 9221) Rhodovulum marinum (RIKEN JCM No. 13300) The culture medium for each of the above strains was obtained in the same manner as in Example 1.

[0099] Twenty whiteleg shrimp with an average weight of 1.0 g were placed in a 100 L recirculating tank. A common shrimp feed used in Thailand was soaked in the culture solution of the above strain, and the shrimp were fed with food in an amount of 5% by mass of their body weight three times a day.

[0100] The feed was prepared by adsorbing 300 μL of the culture solution of the above strain per 1.0 g of the shrimp feed, and allowing the culture solution to thoroughly permeate the shrimp feed.

[0101] On the eighth day after the start of feeding, an infection test was conducted by immersion infection of the shrimp in the same manner as in Example 2. After rearing for 11 days from the day of immersion infection (the start day of the test), the survival rate of the shrimp in each test group was confirmed.

[0102] It was confirmed that when the Rhodovulum sp. OKHT3 strain was fed with the culture medium for 8 days, WSSV infection was suppressed. In other words, disease resistance against WSSV was confirmed. Furthermore, as shown in Table 6, disease resistance against WSSV was confirmed not only for the Rhodovulum sp. OKHT3 strain, but also for Rhodovulum imhoffii, Rhodovulum viride, Rhodovulum strictum, and Rhodovulum marinum. Note that "medium used in Example 1" in the table refers to the case where the feed was supplemented with only the medium used in Example 1 instead of the culture medium.

[0103] [Table 6] Example 8 To confirm the effect of Rhodovulum sp. OKHT3 on the host defense function of vannamei shrimp, we observed changes in the expression levels of immune-related genes in the gills of vannamei shrimp.

[0104] For test group 1, 30 whiteleg shrimp weighing 4.0±1.25 g were placed in a 100 L circulating, filtered tank containing artificial seawater maintained at 28°C. A diet of shrimp food commonly used in Thailand soaked in culture solution was fed to the shrimp three times a day for eight days, at a rate of 5% by mass of the shrimp's body weight. The culture solution used was prepared in the same manner as in Example 1. The diet was prepared by adsorbing 300 μL of culture solution per 1.0 g of the shrimp food and allowing the culture solution to thoroughly soak into the shrimp food.

[0105] In addition, for test group 2, 30 vannamei shrimp weighing 4.0±1.25 g were placed in another 100 L tank, and food soaked in the culture supernatant instead of the culture solution was fed to the shrimp at a rate of 5% by mass of their body weight, three times a day for 8 days. The culture supernatant used was prepared in the same manner as in Example 3. The food was prepared by adsorbing 300 μL of the culture supernatant per 1.0 g of the shrimp food and allowing it to thoroughly soak into the shrimp food.

[0106] Furthermore, as a control group, 30 whiteleg shrimp weighing 4.0±1.25 g were placed in another 100 L aquarium, and a diet prepared by adding only the medium used in Example 1 was fed to the shrimp three times a day for 7 days at a rate of 5% by mass of their body weight. The diet was prepared by adsorbing 300 μL of the medium used in Example 1 per 1.0 g of the shrimp diet and allowing it to thoroughly soak into the shrimp diet.

[0107] Three hours after feeding, five shrimp were randomly selected from each experimental and control group to obtain gill samples, and three of the five gill samples were randomly selected for global gene expression analysis.

[0108] In the present example, comprehensive gene expression analysis was performed using a Miseq system (manufactured by Illumina) as a sequencer. Then, differentially expressed genes were detected according to the following procedure. 1. Using RSEM, we estimated the expression levels of mRNA obtained from comprehensive gene expression analysis. 2. Using the statistical software R, a two-group comparison (DESeq2) was performed to detect genes whose expression levels differed by more than two-fold between the gill samples of shrimp in the control area and those in the gill samples of shrimp in experimental area 1.

[0109] [Table 7] Table 7 shows the genes detected in 2 above. It was confirmed that the expression of penaeidin-3a-like was increased by more than twofold in the gill samples of shrimp from Test Area 1 compared to the gill samples of shrimp from the control area. 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).

[0110] 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 activating immunity in the shrimp body. [Industrial Applicability]

[0111] One aspect of the present invention can be used in the field of feed additives in aquatic animal farming. [Accession number]

[0112] NITE BP-03498 NITE BP-03499

Claims

1. An anti-white spot virus agent for application to crustaceans or shellfish, comprising a culture supernatant of Rhodovulum sp. as an ingredient derived from Rhodovulum sp.

2. The anti-white agent according to claim 1, wherein the Rhodovulum sp. is at least one 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. Tospot virus agent.

3. 2. The anti-white spot virus agent according to claim 1, further comprising a fungal extract of Rhodovulum sp. as an ingredient.

4. The anti-white spot virus agent according to claim 1, wherein the molecular weight of the component is 100 kDa or more.

5. A method for producing an anti-white spot virus agent for application to crustaceans or shellfish, comprising the step of culturing Rhodovulum sp. and obtaining a culture supernatant.

6. The anti-white agent according to claim 5, wherein the Rhodovulum sp. is at least one 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. A method for manufacturing a Tospot virus agent.

7. A method for controlling white spot virus, comprising a step of having a crustacean or shellfish ingest the anti-white spot virus agent according to any one of claims 1 to 4.

8. A method for controlling white spot virus, comprising a step of having crustaceans ingest an anti-white spot virus agent described in any one of claims 1 to 4.

9. The method for controlling white spot virus according to claim 8, wherein the crustacean is shrimp.

10. Rhodovulum sp. OKHT3 strain (NITE BP-03498).

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