Attenuated bacterial strain, live attenuated vaccine, control method, and production method

Abstract

Provided by the present invention is an attenuated bacterial strain of Nocardia seriolae, in which there is a mutation in at least a part of the MtrA gene and the amino acid 179 Val is substituted by Gly in at least part of the amino acid sequence encoded by the MtrA gene.

Description

Attenuated bacterial strain, live attenuated vaccine, control method, and production method

[0001] The present invention relates to attenuated bacterial strains, live attenuated vaccines, control methods, and production methods.

[0002] Nocardiosis is a serious fish disease in yellowtail aquaculture. Nocardiosis is an infectious disease of yellowtail, including yellowtail, amberjack, and kingfish, caused by infection with Nocardia seriolae, a type of actinomycete. In fish infected with nocardiosis, ulcers on the body surface and nodules on the gills, kidneys, and spleen are observed from the mid-stage of infection onward. Therefore, even if fish survive, the commercial value of fish infected with nocardiosis and developing ulcers or nodules is significantly reduced. Nocardia seriolae is sensitive to antibiotics such as erythromycin or spiramycin, or synthetic antibacterial agents such as sulfamonomethoxine or sulfisozole sodium, and these antibiotics or synthetic antibacterial agents are effective. However, the extensive use of antibiotics or synthetic antibacterial agents leads to the emergence of drug-resistant strains, and therefore the development of a vaccine is strongly desired.

[0003] Patent Document 1 describes a vaccine against fish infectious diseases caused by Edwardsiella tarda, which contains an antigen derived from Edwardsiella tarda that is substantially non-pathogenic to the target fish species, a method for producing a vaccine against fish infectious diseases caused by Edwardsiella tarda, which is characterized by using Edwardsiella tarda that is substantially non-pathogenic to the target fish species, and a method for preventing fish infectious diseases caused by Edwardsiella tarda, which includes a step of administering the vaccine to fish.

[0004] Patent Document 2 describes a method for producing a pharmacological composition, which comprises a culture step of culturing Nocardia seriolae in a medium containing a divalent cation chelating agent, a pharmacological composition obtained by the method for producing a pharmacological composition, and a fish vaccine containing the pharmacological composition.

[0005] Patent Document 3 describes a combination vaccine for combating Nocardia infection in fish, which is characterized by containing bacteria of the species Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum, and Nocardia seriolae, and a pharmaceutically acceptable carrier.

[0006] In an attempt to develop a vaccine against Nocardia seriolae, closely related species N. soli, N. fluminea, or N. uniformis were injected into yellowtail (Seriola quinqueradiata). A low-pathogenic strain of N. seriolae was also administered to model the concept of live vaccine use. Fish injected with live cells of N. soli and N. fluminea showed slight resistance to artificial challenge with N. seriolae. Meanwhile, fish that survived N. seriolae infection showed complete resistance to N. seriolae challenge. These results suggest that a protective immune response against N. seriolae can be induced in yellowtail (NPL 1).

[0007] Nocardia seriolae, a Gram-positive facultative intracellular pathogen, has been identified as the causative agent of fish nocardiosis, causing significant mortality and morbidity in a wide range of fish species. Therefore, an effective vaccine against this pathogen is urgently needed to prevent significant losses in aquaculture. To develop a live vaccine, mutagenic N. seriolae strains S-250 and U-20 were derived from the wild-type strain ZJ0503 by serial passage and UV irradiation. Furthermore, the biological properties, virulence, stability, mediation of immune responses, and protective efficacy against hybrid snakehead fish (Canna maculata (female) × C. argus (male)) were investigated. The results showed that the U-20 strain exhibited dramatically changed morphological characteristics and significantly reduced pathogenicity to hybrid snakeheads compared to the ZJ0503 strain, whereas the S-250 strain showed no significant differences compared to the ZJ0503 strain. When the ZJ0503, S-250, and U-20 strains were intraperitoneally injected into hybrid snakeheads at subclinical doses, nonspecific immune parameters (serum LYZ, POD, ACP, AKP, and SOD activity), specific antibody (IgM) titer production, and expression of immune-related genes (CC1, CC2, IL-1β, IL-8, TNFα, IFNγ, MHC1α, MHCIIα, CD4, CD8α, TCRα, and TCRβ) were increased, indicating that they could induce humoral and cellular immune responses. Furthermore, the relative survival rates (RPS) of hybrid snakeheads vaccinated with the ZJ0503, S-250, and U-20 strains were 28.85%, 56.89%, and 89.65%, respectively. These results suggest that the S-250 and U-20 strains can induce a strong immune response against N. seriolae and provide protection to hybrid snakeheads, making them candidates for live vaccines to control nocardiosis in farmed fish (Non-Patent Document 2).

[0008] Nocardia seriolae is the major pathogen of fish nocardiosis. Our previous study identified alanine dehydrogenase as a potential virulence factor of N. seriolae. Based on this fact, we knocked out the alanine dehydrogenase gene (NsAld) of N. seriolae and established the ΔNsAld strain for vaccine development against fish nocardiosis. LD of the ΔNsAld strain ５０ is 3.90 x 10 ５ CFU / animal, and the wild-type strain (5.28 × 10 ４ The ΔNsAld strain was used as a live vaccine and given to hybrid snakeheads (Channa maculata (female) × Channa argus (male)) at 2.47 × 10 ５ Intraperitoneal injection of CFU / fish induced increases in nonspecific immune indices (LZM, CAT, AKP, ACP, and SOD activity), specific antibody titers (IgM), and several immune-related genes (CD4, CD8α, IL-1β, MHC1α, MHCIIα, and TNFα) in different tissues, indicating that this vaccine can induce humoral and cellular immune responses. Furthermore, the survival rate (RPS) of the ΔNsAld vaccine was 76.48%. These results suggest that the ΔNsAld strain may be a potential candidate for the development of a live vaccine to control nocardiosis in farmed fish (Non-Patent Document 3).

[0009] JP 2007-238505 A JP 2013-184916 A JP 2011-502972 A

[0010] Itano, T. 3 others, “Live vaccine trials against nocardiosis in yellowtail Seriola quinqueradiata”, Aquaculture, 2006, Vol. 261, p. 1175-1180Li, B. 7 others, “Induction of attenuated Nocardia seriolae and their use as live vaccine trials against fish nocardiosis”, Fish and Shellfish Immunology, 2022, Volume 131, p. 10-20 Liu, Y. 6 others, “Construction of an alanine dehydrogenase gene deletion strain for vaccine development against Nocardia seriolae in hybrid snakehead (Channa maculata ♀ × Channa argus ♂)”, Fish and Shellfish Immunology, 2023, Volume 138, 108827

[0011] Inoculation of yellowtail with N. seriolae cells inactivated by formalin or heat treatment did not provide any protective effect, and no vaccine highly effective in preventing nocardiosis in yellowtail has yet been developed.

[0012] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an attenuated bacterial strain and a live attenuated vaccine that are useful for producing a live attenuated vaccine for controlling nocardiosis in yellowtail.

[0013] As a result of extensive research to solve the above problems, the present inventors have found that there is a mutation in a part of the MtrA gene, and part of the amino acid sequence encoded by the MtrA gene is V. １７９ →G １７９ The present inventors have found that the use of a mutated N. seriolae strain as a live attenuated vaccine can effectively control nocardiosis in yellowtail, and have completed the present invention.

[0014] [1] An attenuated bacterial strain of Nocardia seriolae, which has a mutation in at least a portion of the MtrA gene, and in at least a portion of the amino acid sequence encoded by the MtrA gene, the 179th amino acid is substituted from Val to Gly. [2] The attenuated bacterial strain according to [1], wherein the nucleotide sequence having the mutation in a portion of the MtrA gene is given by SEQ ID NO: 1. [3] The attenuated bacterial strain according to [1] or [2], wherein the amino acid sequence encoded by the MtrA gene is given by SEQ ID NO: 3. [4] The attenuated bacterial strain according to any of [1] to [3], which is Nocardia seriolae FPC1073. [5] The attenuated bacterial strain according to any of [1] to [4], which is an isolated bacterial strain. [6] A live attenuated vaccine comprising the attenuated bacterial strain according to any of [1] to [5]. [7] Use of the attenuated bacterial strain according to any one of [1] to [5] as a live attenuated vaccine for controlling nocardiosis in fish of the genus Seriola. [8] A method for controlling nocardiosis in fish of the genus Seriola, comprising administering the live attenuated vaccine according to [6] to Seriola. [9] A method for producing fish of the genus Seriola, comprising administering the live attenuated vaccine according to [6] to Seriola.

[0015] According to the present invention, an attenuated bacterial strain and a live attenuated vaccine useful for producing a live attenuated vaccine for controlling nocardiosis in yellowtail can be provided.

[0016] Figure 1 is a graph showing the infection protective effect of N. seriolae FPC1073 on yellowtail (S. quinqueradiata) (Experimental Example 1). Figure 2 is a diagram showing the results of comparing a portion of the MtrA gene base sequence on the genome of the attenuated N. seriolae strain FPC1073 and the virulent N. seriolae strain 024013 (Experimental Example 2). Figure 3 is a graph showing the infection protective effect of N. seriolae FPC1073 on amberjack (S. dumerili) (Experimental Example 3). Figure 4 is a graph showing the infection protective effect of N. seriolae FPC1073 on yellowtail (S. lalandi) (Experimental Example 4).

[0017] Patent Documents 1 to 3 are silent about the presence of a mutation in a portion of the MtrA gene of the N. seriolae FPC1073 strain, the substitution of the 179th amino acid in a portion of the amino acid sequence of the MtrA gene from Val to Gly, and the FPC1073 strain itself.

[0018] In the present invention, "yellowtail" and "fish of the genus Seriola" include yellowtail (scientific name: Seriola quinqueradiata), amberjack (scientific name: Seriola lalandi), amberjack (scientific name: Seriola dumerili), and long-finned amberjack (scientific name: Seriola rivoliana). However, if the scientific name has been changed due to taxonomic reorganization of fish, the species will be included in "yellowtail" and "fish of the genus Seriola" if the scientific name before the change is as described above. For example, Seriola lalandi is included in Seriola aureovittata, Seriola dorsalis, and Seriola When the three species Seriola lalandi are reclassified into three species, all three species are included in the "Seriola" and "Seriola genus." Furthermore, molecular phylogenetic studies (Premachandra, HKA, et al. "Genomic DNA variation confirmed Seriola lalandi comprises three different populations in the Pacific, but with recent divergence." Scientific Reports 7.1 (2017): 9386.) have shown that S. zonata and S. fasciata are closely related to yellowtail (S. quinqueradiata), amberjack (S. dumerili), and long-finned amberjack, and therefore S. zonata and S. fasciata are also included in the "Seriola" and "Seriola genus."

[0019] The following describes in detail the embodiments of the present invention. However, the present invention is not limited to the embodiments described below, and various modifications are possible without departing from the gist of the present invention.

[0020] [Attenuated Bacterial Strain] The attenuated bacterial strain of this embodiment is an attenuated bacterial strain of Nocardia seriolae that has a mutation in at least a part of the MtrA gene, and in at least a part of the amino acid sequence encoded by the MtrA gene, the 179th amino acid is substituted from Val to Gly.

[0021] The presence of at least a partial mutation in the MtrA gene, resulting in a substitution of Val to Gly at amino acid 179 in the encoded amino acid sequence, can be determined, for example, by determining the DNA nucleotide sequence of the MtrA gene of the strain, or by determining the nucleotide sequence of the transcription product (mRNA) or complementary DNA (cDNA) of the MtrA gene of the strain.

[0022] A preferred embodiment of the attenuated bacterial strain of this embodiment is Nocardia seriolae FPC 1073. The attenuated bacterial strain of this embodiment may be a culture of Nocardia seriolae FPC 1073.

[0023] The attenuated bacterial strain of this embodiment is preferably an isolated bacterial strain.

[0024] Nocardia seriolae FPC1073 has been deposited as NITE BP-04013 at the National Institute of Technology and Evaluation, National Patent Microorganism Depositary (NPMD) (Room 122, Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (Deposit date: November 15, 2023).

[0025] [Live Attenuated Vaccine] The live attenuated vaccine of this embodiment is a live attenuated vaccine containing the above-described attenuated bacterial strain.

[0026] The attenuated live vaccine of this embodiment may further contain a pharmaceutically acceptable liquid or solid carrier in addition to the attenuated bacterial strain described above. Examples of liquid carriers include water, phosphate buffered saline (PBS), and physiological saline. Examples of solid carriers include excipients such as talc and sucrose. The form of the attenuated live vaccine of this embodiment is not particularly limited and may be an injection, oral formulation, or immersion formulation. However, it is preferable to adopt an injection form, which has long-lasting efficacy with a small dose. Furthermore, when the vaccine is in the form of an oral formulation, the attenuated bacterial strain may be mixed with ordinary fish feed.

[0027] An injectable vaccine can be prepared by suspending live cells of the attenuated bacterial strain in sterilized physiological saline for fish, etc. In addition to the live cells and physiological saline, the injectable vaccine can also contain suspending agents, stabilizers, emulsifiers, buffers, preservatives, solubilizers, or other appropriate additives that are commonly used in injections.

[0028] Furthermore, adjuvants have traditionally been used to improve the immune effects of vaccines. The attenuated live vaccine of this embodiment can achieve sufficient immune effects without the need for such adjuvants. However, the attenuated live vaccine of this embodiment does not limit the use of adjuvants, and an adjuvant can be incorporated in addition to the above components.

[0029] Adjuvants are generally substances that nonspecifically enhance the immune response of a host, and many different adjuvants are known in the art. Examples of adjuvants include, but are not limited to, minerals, plant and animal fats and oils, oil-soluble vitamins such as vitamin E, and surfactants for adding these, alum, aluminum compounds, bentonite, muramyl dipeptide derivatives, interleukins, and endotoxins.

[0030] The amount of live bacterial cells contained in the attenuated live vaccine of this embodiment is not particularly limited, but is preferably 1.0 × 10 ２ ~1.0 x 10 ４ CFU / 100 μL, more preferably 1.0 × 10 ３~1.0 x 10 ４ CFU / 100 μL The amount of the attenuated bacterial strain contained in the vaccine is preferably 0.1 to 1.0 μg in terms of wet cells per administration.

[0031] The effective amount can be adjusted appropriately by increasing or decreasing the dose (μL) of the vaccine administered to the fish, and therefore the content of the attenuated bacterial strain in the live attenuated vaccine of this embodiment is not limited to the above. When using the prepared live attenuated vaccine, it is desirable to test the safety and efficacy of the vaccine in accordance with, for example, the Yellowtail Alpha-Hemolytic Streptococcus Inactivated Vaccine (Injectable) in the Animal Biological Product Testing Standards, and to confirm and guarantee its quality.

[0032] The desirable dose when the vaccine of the present invention is administered intraperitoneally to fish varies depending on various factors such as the season and water temperature, the type, age, and weight of the fish, and cannot be generally determined. However, it is preferable to use a vaccine prepared by further diluting the vaccine, for example, at 0.1 g / mL wet cell weight by 10,000 times, and to intraperitoneally inject about 50 to 500 μL into a fish weighing 30 to 500 g, depending on the body weight.

[0033] The vaccine of the present invention can be used for fish of the genus Seriola, for example, fish of the genus Seriola that normally weigh 30 g or more, specifically 30 g to 5 kg, preferably 30 g to 200 g, and more preferably 50 g to 200 g. To utilize the vaccine more effectively, it is preferable to administer it to fish before they become infected with nocardiosis, for example, at the juvenile stage. It is particularly preferable to administer 0.05 to 0.5 mL, more preferably 0.1 to 0.2 mL, of the vaccine of the present invention intraperitoneally when the fish are 3 to 7 months old, preferably 4 to 5 months old.

[0034] The attenuated live vaccine of this embodiment has a duration of action lasting for one year or more with a single administration, and can exert a protective effect against nocardiosis infection over a long period of time. The vaccine of the present invention may be administered once as long as its effect is sustained, but it may also be administered multiple times, for example, 2 to 5 times, at intervals of 1 to 30 days.

[0035] The fish to which the live attenuated vaccine of this embodiment is to be administered are not limited to fish of the genus Seriola (yellowtail). In particular, in the aquaculture of yellowtail, the vaccine of this embodiment is thought to be highly useful because it can reduce the mortality rate due to nocardiosis during aquaculture and increase the yield.

[0036] [Control Method] The control method of this embodiment is a method for controlling nocardiosis in fish of the genus Seriola, which comprises administering an attenuated live vaccine to Seriola.

[0037] In the control method of this embodiment, the live attenuated vaccine administered is the live attenuated vaccine described above.

[0038] In the control method of this embodiment, the attenuated live vaccine can be administered to fish of the genus Yellowtail by injection, immersion, oral administration, etc. Examples of each administration method are described below.

[0039] (A) Injection method: Inject 1.0 x 10 ３ ~1.0 x 10 ４ 0.05 to 0.1 mL of the attenuated live vaccine solution prepared to CFU / 100 μL is administered intraperitoneally to each fish. The injection solution may further contain a pharmaceutically acceptable liquid carrier in addition to the attenuated bacterial strain. Examples of liquid carriers include water, phosphate buffered saline (PBS), and physiological saline.

[0040] (B) Immersion method: For fish weighing 30 g or more, the number of attenuated bacterial strains is 1.0 x 10 ７ ~1.0 x 10 ８ The fish are immersed in a solution containing the bacteria adjusted to CFU / L for 10 to 60 minutes. Note that the immersion method is thought to be less effective in preventing infection than the injection method, so a booster immunization may be performed as necessary.

[0041] (C) Oral Method In the oral method, live cells of the attenuated bacterial strain were mixed with the feed at a dose of 1.0 x 10 per fish. 3 ~1.0 x 10 7 When this method is employed, booster immunization may be performed as necessary.

[0042] In the control method of this embodiment, the various administration methods described above can be appropriately selected and adopted depending on the characteristics, morphology, etc. of the fish species to be treated, but in order to further enhance the infection prevention effect, it is preferable to adopt intraperitoneal injection as performed in the examples described below.

[0043] According to the control method of this embodiment, nocardiosis in fish of the genus Seriola can be more efficiently prevented.

[0044] [Production method] The production method of this embodiment is a method for producing fish of the genus Seriola, which comprises administering the above-mentioned live attenuated vaccine to yellowtail. The method for administering the live attenuated vaccine to yellowtail is the same as the above-mentioned method for controlling nocardiosis in fish of the genus Seriola.

[0045] The present invention will be explained in more detail below using experimental examples, but the present invention is not limited to the examples described below.

[0046] Experimental Example 1: Investigation of a live attenuated vaccine using Nocardia seriolae FPC1073 (FPC, Fisheries Technology National Institute) with a mutation in part of the MtrA gene, resulting in a substitution of Val at position 179 with Gly in part of the amino acid sequence encoded by the gene. N. seriolae 024013, a virulent strain of nocardiosis provided by Oita Prefecture, was passaged to obtain a novel attenuated strain, N. seriolae FPC1073, with a pathogenic mutation.

[0047] 10 ４ CFU / 100 μL of N. seriolae FPC1073 was inoculated into the body cavity of yellowtail, and the yellowtail were reared in seawater (running water) at 25° C. for one month. To artificially infect the inoculated yellowtail, a highly virulent strain of N. seriolae KGN1266 (KGN, Kagoshima Prefectural Fisheries Technology Research and Development Center) was added to the rearing water for 10 min. ７ CFU / L and immersed for 10 minutes. After infection, the mice were reared in running seawater at 25°C and observed for 60 days. The survival rate was calculated, and as shown in Figure 1, the mice showed a high infection prevention effect.

[0048] <Evaluation of live attenuated vaccines using other Nocardia species> 10 ８CFU / 100 μL of N. fluminea JCM11440, N. salmonicida subsp. cumidelens JCM11441, or N. uniformis JCM3224 was inoculated into the body cavity of yellowtail, and the fish were reared in seawater (running water) at 25°C for 14 days. To artificially infect the inoculated yellowtail, 100 μL of the highly virulent strain N. seriolae 024013 was added to the rearing water. ８ CFU / L and immersed for 10 minutes. After infection, the cells were reared in running seawater at 24°C and observed for 37 days, after which the survival rate was calculated. As shown in Figure 1, no protective effect against infection was observed. JCM is an acronym for Japan Collection of Microorganisms (RIKEN BioResource Research Center, 3-1-1 Takanodai, Tsukuba, Ibaraki Prefecture, Japan).

[0049] <Results> Figure 1 is a graph showing the infection prevention effect of N. seriolae FPC1073 on yellowtail (S. quinqueradiata). Example 1: N. seriolae FPC1073 Comparative Example 1-1: N. fluminea JCM11440 Comparative Example 1-2: N. salmonicida subsp. cumidelens JCM11441 Comparative Example 1-3: N. uniformis JCM3224 Comparative Example 1-0: PBS-T: PBS (phosphate buffered saline) with 0.5% Tween 80 added *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001, Fisher's exact test, Holm's method.

[0050] [Experimental Example 2] <Mutation Analysis by Comparison of N. seriolae FPC1073 with Other N. seriolae Strains> When the whole genomes of N. seriolae 024013, a highly virulent strain of nocardiosis provided by Oita Prefecture, and a novel attenuated strain of N. seriolae FPC1073 whose pathogenicity has mutated through subculture, were compared, focusing on a mutation in the MtrA gene (a transcription factor that couples with kinase (MtrB) and transmits various signals) gene, which is presumed to be involved in pathogenicity, out of three SNPs (single nucleotide polymorphisms), it was revealed that there was also a mutation (p.Val179Gly) at the amino acid level. Various highly virulent N. seriolae strains owned by the inventors and N. seriolae strains in public databases were compared. When the MtrA genes of S. seriolae and related species were compared, no mutation in the amino acid sequence (p.Val179Gly) was confirmed in any strains other than FPC 1073. Figure 2 shows the nucleotide sequence of the MtrA gene, including the portion where the mutation occurred, as a waveform output from a DNA sequencer.

[0051] Table 1 shows a partial alignment of the amino acid sequences of the MatrA gene products (proteins) of seven strains of Nocardia seriolae, bacteria of the genus Nocardia other than N. seriolae (five species, six strains), and high-GC-content Gram-positive bacteria of the genus Mycobacterium (two species, four strains). The results suggest that the MtrA mutation (p.Val179Gly) is a characteristic of attenuated strains of N. seriolae with high live vaccine efficacy.

[0052]

[0053] [Experimental Example 3] <Study on infection control effect in amberjack (S. dumerili)> N. seriolae FPC1073 was inoculated into the abdominal cavity of amberjack. The experimental method was the same as that for yellowtail.

[0054] <Results> Figure 3 is a graph showing the infection protective effect of N. seriolae FPC1073 on amberjack (S. dumerili). Example 3: N. seriolae FPC1073 Comparative Example 3: PBS-T: PBS (phosphate buffered saline) with 0.5% Tween 80 added The survival rate was calculated in the same manner as in Experimental Example 1, and as shown in Figure 3, a high infection protective effect was observed.

[0055] Experimental Example 4 <Study on infection control effect in yellowtail (S. lalandi)> Yellowtail was inoculated with N. seriolae FPC1073 by the immersion method. The experimental method was the same as that for yellowtail.

[0056] <Results> Figure 4 is a graph showing the infection protective effect of N. seriolae FPC1073 on yellowtail kingfish (S. lalandi). Example 3: N. seriolae FPC1073 Comparative Example 3: PBS-T: PBS (phosphate-buffered saline) containing 0.5% Tween 80 **, p<0.01 Fisher's exact test The survival rate was calculated in the same manner as in Experimental Example 1, and as shown in Figure 4, a high infection protective effect was observed.

[0057] [Experimental Example 5] PCR was performed to amplify MtrA using extracted genomic DNA from Nocardia seriolae strain FPC1073 or 024013 as a template. Subsequently, to decipher the nucleotide sequence of the resulting amplified product, DNA sequencing was performed by the Sanger method using the amplified product as a template. Oligonucleotides designed for the reverse complementary sequence downstream of the open reading frame of the MtrA gene were used as primers. The reverse complementary sequence of the MtrA gene obtained by this sequencing was converted to its complementary sequence using sequence assembly software.

[0058] Table 2 shows the DNA sequences of the MtrA genes obtained.

[0059]

[0060] The amino acid sequences of the obtained MtrA gene products are shown in Table 3. However, these amino acid sequences are deduced from the DNA base sequence of the MtrA gene.

[0061]

[0062] The attenuated bacterial strain of the present invention can be used as a live vaccine useful for controlling nocardiosis in fish of the genus Seriola. Because the live attenuated vaccine of the present invention is highly effective in controlling nocardiosis, it can prevent losses due to nocardiosis in farmed fish of the genus Seriola, and is expected to be economically beneficial.

[0063] NPMD NITE BP-04013 NPMD is an acronym for the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan).

Claims

1. An attenuated bacterial strain of Nocardia seriolae, which has a mutation in at least a portion of the MtrA gene, and in at least a portion of the amino acid sequence encoded by the MtrA gene, the 179th amino acid is substituted from Val to Gly.

2. The attenuated bacterial strain according to claim 1, wherein the base sequence of the MtrA gene having a mutation in a portion thereof is given by SEQ ID NO:

1.

3. The attenuated bacterial strain of claim 1, wherein the amino acid sequence encoded by the MtrA gene is given in SEQ ID NO:

3.

4. The attenuated bacterial strain according to claim 1, which is Nocardia seriolae FPC1073.

5. The attenuated bacterial strain according to any one of claims 1 to 4, which is an isolated bacterial strain.

6. A live attenuated vaccine comprising the attenuated bacterial strain according to any one of claims 1 to 4.

7. Use of the attenuated bacterial strain according to any one of claims 1 to 4 as a live attenuated vaccine for controlling nocardiosis in fish of the genus Seriola.

8. A method for controlling nocardiosis in fish of the genus Seriola, which comprises administering the live attenuated vaccine according to claim 6 to Seriola.

9. A method for producing fish of the genus Seriola, which comprises administering the live attenuated vaccine according to claim 6 to Seriola.

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