Immunogenic composition for the prevention of marine tenacivacramosis caused by Tenacivacram maritimum and Tenacivacram solea in fish, its preparation method and use

Abstract

The present invention discloses an immunogenic composition for preventing marine tenacivaculosis, a disease affecting commercially valuable marine and anadromous fish, comprising Tenacivaculum maritimum strain CECT30394 and Tenacivaculum solea CECT30393. The present invention further includes a method for its preparation and its use against the disease.

Description

[Technical field]

[0001] The present invention relates to the aquaculture industry and comprises a bivalent immunogenic composition for preventing marine tenacibaculosis caused by Tenacibaculum maritimum and Tenacibaculum soleae, its preparation method and its use. [Background technology]

[0002] Marine tenacibaculosis (or marine flexibacteriosis) is one of the most important bacterial diseases that fundamentally affects marine fish worldwide. The disease affects a wide variety of commercially valuable marine and anadromous fishes such as Atlantic salmon, turbot, sole, European sea bass, and gilthead sea bream in Europe, America, Asia, and Oceania (Non-Patent Documents 1, 2, 3, 4). Recent studies (Non-Patent Document 5) have clearly demonstrated that tenacibaculosis, together with vibriosis, is one of the most important diseases affecting marine fish farming in Europe. The disease causes mortality rates that can exceed 20% in the culture of commercially valuable marine species such as turbot, sole, sea bass, gilthead sea bream, Spanish sea bream and Atlantic salmon, with global production reaching 1,985,165 tonnes in recent years and a commercial value of 10,871 million euros (Non-Patent Document 6), which is of great concern to the production sector.

[0003] Characteristic symptoms of the disease are ulcerative lesions on the fish's skin, bleeding, and deterioration of fin tissue, ultimately causing the death or disfigurement of the fish, making them unmarketable (Non-Patent Document 2).

[0004] Until recently, T. maritimum was considered the main causative agent of tenacibacillosis in most species of farmed fish (Non-Patent Document 1, Non-Patent Document 7, Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). However, today, T. maritimum accounts for only 50% of strains isolated in clinical cases (Non-Patent Document 8). Since 2008, other species from the genus Tenacibaculum, such as Tenacibaculum solea, Tenacibaculum discolor, Tenacibaculum dicentrarchi and Tenacibaculum gallaicum, have been described as the causative agent of mortality in turbot and sole farmed in Galicia (Non-patent Document 9, Non-patent Document 10 and Non-patent Document 11, Non-patent Document 12), and Tenacibaculum finnmarkense has been described as the causative agent of tenacibaculosis in farmed salmon in Norway (Non-patent Document 13). Since then, the species T. soleae has been isolated in various regions from other fish species such as sea bass (Non-Patent Document 14), Atlantic salmon, brill, and wedge sole (Non-Patent Document 15), and in Italy from Pacific oysters (Non-Patent Document 16). Today, T. maritimum and T. solea are the main causative species of tenacibacramosis in fish of commercial interest in Spain (sea bass, gilthead sea bream, turbot, and sole) (Non-Patent Document 14, Non-Patent Document 2, Non-Patent Document 4).

[0005] Tenacibaculum is treated by administering antimicrobials in the diet, typically in combination with external disinfectants (Non-Patent Document 17). However, the use of antimicrobials to control fish diseases is limited by the ever-increasing restrictions on the use of antibiotics in aquaculture and the rapid acquisition of drug resistance observed in these bacteria (Non-Patent Document 3, Non-Patent Document 18, Non-Patent Document 19). The most promising alternative to the use of antimicrobials is the prevention of disease through the use of effective vaccines.

[0006] For the prevention of marine tenacivacramosis in fish, several monovalent anti-T. maritimum vaccines have been evaluated at the experimental level (Non-Patent Document 20, Non-Patent Document 21, Non-Patent Document 19). However, only the vaccine developed by our research group, Icthiovac™ (Laboratorios HIPRA) (Non-Patent Document 3, Non-Patent Document 22, Patent Document 1), is commercially available for the prevention of the disease in turbot. The vaccine administered by intraperitoneal injection in turbot confers a level of protection against T. maritimum expressed as a relative survival rate (RPS) of more than 90% (Non-Patent Document 23). Regular use of this vaccine in turbot culture reduced the incidence of tenacivacramosis caused by T. maritimum serovar O2 (Non-Patent Document 24). However, the efficacy of this vaccine in preventing tenacibaculosis caused by T. maritimum in other fish species of commercial interest (gilthead seabream, sea bass, sole or salmon) has not been evaluated, and it is unclear whether the vaccine confers crossed protection against other T. maritimum serovars or other species of tenacibaculum pathogenic to fish. Furthermore, previous serological studies (Non-Patent Document 25) have demonstrated the absence of antigenic relationships between T. maritimum strains of different serovars, as well as between T. maritimum and the species T. solea, T. discolor and T. gallaicum (Non-Patent Document 2, Non-Patent Document 9, Non-Patent Document 25), suggesting that the anti-T. maritimum vaccine (ICTHIOVAC™) does not provide protection against the bacteria.

[0007] These findings clearly demonstrate the need to develop immunogenic compositions for effectively preventing marine tenacibaculosis caused by T. maritimum and T. solea. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] スペインPatent No. 2139549

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[0009]

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[0010] The present invention relates to the aquaculture industry and in particular to a bivalent immunogenic composition comprising the T. maritimum CECT30394 and T. solea CECT30393 strains deposited in the Spanish Type Culture Collection (CECT), for the control and prevention of marine tenacybaculomycosis in fish, its production method and its use.

[0011] The deposited strains are Gram-negative, oxidase- and catalase-positive, nitrate-reducing filamentous bacilli and are unable to produce hydrogen sulfide, characteristics typical of the species Tenacibacrum. Moreover, these strains are negative for the indole and Voges-Proskauer tests. Furthermore, strains of the species T. maritimum show amylase, trypsin, and chymotrypsin activity that is absent in strains of T. solea. T. maritimum and T. solea are homogeneous at the phenotypic level and show serological and genetic variability (Fernandez-Alvarez, 2019; Non-Patent Document 3).

[0012] The composition, diluted 1:1000 in seawater and administered by immersion (two 2-min baths spaced 4 weeks apart) or by injection (a dose of 0.1 mL / tail) confers a high level of protection (RPS = 70%-100%) to fish against marine tenacybaculum disease caused by both pathogenic species.

[0013] This immunogenic composition developed in the present invention represents an advantage over the experimental monovalent vaccines previously described or over the commercial vaccine ICTHIOVAC™, since it allows:

[0014] 1) Improve antigenic coverage by including strains of T. maritimum and T. solea, the major causative agents of tenacivaculosis worldwide.

[0015] 2) To protect against tenacybaculosis caused by T. maritimum and T. solea in fish species of commercial interest.

[0016] In a first aspect, the present invention relates to the strains of T. maritimum and T. solea deposited in the CECT under accession numbers 30394 and 30393, respectively.

[0017] In a second aspect, the present invention relates to an immunogenic composition comprising the inactivated strains CECT30394 and CECT30393. "Inactivated strains" is understood to mean strains that have been subjected to physical or chemical treatments, transforming them into a form incapable of replicating.

[0018] In a preferred embodiment, the immunogenic composition comprises 5×10 9 cells ~5×10 10 Preferably, the immunogenic composition has a concentration of 3×10 10 Contains bacterial cells of the inactive strain at a concentration of 100 cells / mL.

[0019] A third aspect of the invention relates to an immunogenic composition comprising the inactivated strains CECT30394 and CECT30393 and additionally at least one pharma- ceutically acceptable vehicle, preferably an adjuvant, which may be chosen from any of the following: Freund's Complete Adjuvant (FCA), Freund's Incomplete Adjuvant (FIA), oil-based compounds such as Eolane 130, Montanide ISA, and Titermax, inorganic compounds such as alginates, aluminum and potassium salts (aluminum and potassium phosphates, and aluminum hydroxide), PLGA nanoparticles, liposomes, biodegradable microspheres, saponins, immune stimulating complexes (ISCOMs) such as phospholipids, CpG ODN oligodeoxynucleotides, flagellin, pathogen-associated molecular patterns (PAMPs) such as bacterial polysaccharides (LPS), and levamisole, Mycobacterium bovis. bovis, Mycobacterium butyricum, Mycobacterium chenolae, mycobacterial cell wall, chemokines, other immune response modifiers such as chitosan, sorbitan sesquioleate, vitamin C, vitamin E, or any combination thereof.

[0020] In the present specification, an adjuvant is understood to mean any agent that, when incorporated into a vaccine, stimulates the immune system by increasing the magnitude, extent and duration of the immune response.

[0021] The composition may be presented in any clinically acceptable dosage form and therapeutically effective amount. Preferably, administration is by immersion or injection. The therapeutically effective amount administered by injection ranges from 0.05 mL to 0.2 mL per tail. Preferably, the therapeutically effective amount is 0.1 mL per tail. The therapeutically effective dilution administered by immersion ranges from 1:500 to 1:1000. Preferably, the effective therapeutic dilution is 1:1000.

[0022] A fourth aspect of the invention relates to a method for the preparation of a mammalian animal, in particular a fish, preferably Atlantic salmon (Salmo salar), Pacific salmon (Oncorhynchus kisutch), trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus), sole (Solea solea and Solea senegalensis), European sea bass (Dicentrarchus labrax), gilthead sea bream (Sparus aurata), Spanish sea bream (Pagellus bogaraveo), cod (Gadus morpha), and the like. The present invention relates to an immunogenic composition of the invention for its use in catfish (egg, chickweed, turtle, or turtle), mackerel ...

[0023] A fifth aspect of the invention relates to strains CECT30394 and CECT30393 in a pharmaceutical composition of the invention or in a veterinary composition of the invention for use as vaccines in the prevention and control of marine tenacybaculum.

[0024] In another aspect, the present invention relates to strains CECT30394 and CECT30393 in a pharmaceutical composition of the invention or in a veterinary composition of the invention for use in the manufacture of a medicament for the prevention and / or control of marine tenacybaculum.

[0025] Another aspect of the invention relates to antibodies obtained after immunization of an animal with this immunogenic composition for use in the prevention and / or control of marine tenacylcytoma. Preferably, the animal used for immunization is a fish that may be selected from any of the following: Atlantic salmon (Salmo salar), Pacific salmon (Oncorhynchus kisti), trout (Oncorhynchus mykis), turbot (Scoptalmus maximus), sole (Solea solea and Solea senegalensis), European sea bass (Dicentrax labrax), gilthead sea bream (Sparus aurata), Spanish sea bream (Pagerus bogalaveo), cod (Gadus morpha), brill (Scoptalmus lombus), and / or wedge sole (Dicloglossa cuneata).

[0026] Another aspect of the invention relates to a method for producing an immunogenic composition comprising culturing bacterial cells of the T. maritimum and T. solea strains described herein, inactivating the bacterial cells, harvesting the bacterial cells by centrifugation or filtration, and suspending the bacterial cells.

[0027] The culture medium used for culturing the bacterial cells of CECT30394 and CECT30393 may be selected from any of the following: Luria-Bertani (LB) broth and Mueller-Hinton (MH) broth diluted with synthetic seawater, Marine Broth (MB), FMM broth, FMM broth supplemented with 0.5% glucose, FMM broth supplemented with 0.5% sucrose (FMM-S). Preferably, the medium used is FMM-S.

[0028] Inactivation of bacterial cells of CECT30394 and CECT30393 is carried out by any physical or chemical treatment that transforms the strain into a replicable form. Preferably, inactivation is carried out by using formaldehyde and / or heat. Preferably, the formaldehyde concentration is 0.30% to 0.35%. More preferably, the concentration is 0.35%. The time to achieve inactivation using formaldehyde is 120 minutes to 180 minutes. Preferably, the time to achieve inactivation is 180 minutes. The temperature to achieve inactivation is 20°C to 25°C. Preferably, the inactivation temperature is 25°C.

[0029] When heat is used for inactivation, the temperature is preferably 80° C. to 100° C. Preferably, the inactivation temperature is 100° C. The time to achieve inactivation by using heat is 45 minutes to 60 minutes. Preferably, the time to achieve inactivation is 60 minutes.

[0030] Harvesting of the bacterial cells of CECT30394 and CECT30393 can be carried out by centrifugation, microfiltration or ultrafiltration. Preferably, harvesting is carried out by centrifugation.

[0031] The centrifugation speed for recovery is 10,000 rpm to 12,000 rpm. Preferably, the centrifugation speed is 10,000 rpm.

[0032] The filtration method may be selected from microfiltration and tangential filtration. Preferably, the filtration is tangential filtration.

[0033] The suspension of the recovered cells is carried out in any liquid medium that allows preservation. Preferably, the selected medium is a saline solution (0.9% NaCl) containing formaldehyde and phosphate buffered saline (PBS). More preferably, the selected medium is a phosphate buffer containing formaldehyde. The optical density of the suspension measured at 620 nm is 0.5 to 0.8. Preferably, A 620 The optical density at is 0.7.

[0034] Another aspect of the invention relates to a kit for use in inducing an immune response in fish, comprising the immunogenic composition described above, and optionally instructions relating to administration. [Brief description of the drawings]

[0035] [Figure 1] Figure 1. Growth of Tenaxybaculum maritimum CECT30394 and Tenaxybaculum solea CECT30393 in FMM medium without sugar supplementation and supplemented with sugars, namely glucose (FMM-G) and sucrose (FMM-S), showing good growth in FMM-S medium. Results are expressed as cell volume measured using a spectrophotometer (A620) and total cells / mL. [Diagram 2] FIG. 1 shows efficacy of a bivalent vaccine administered by immersion in turbot, sole, gilthead seabream and sea bass, expressed as relative survival rate (RPS). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] The purpose of the examples given below is to illustrate the invention without thereby limiting the scope of the invention. EXAMPLES

[0037] Example 1. Optimization of the culture conditions for Tenaxicabaculum maritimum and Tenaxicabaculum solea To select the optimal conditions for the cultivation of T. maritimum and T. solea, it was assessed whether the incorporation of sugars (glucose or sucrose) in the medium would improve the growth of strains CECT30394 and CECT30393. To this end, the cultivation of T. maritimum CECT30394 and T. solea CECT30393 was started in FMM culture medium and in FMM medium supplemented with glucose (0.5 g / L) (FMM-G) or sucrose (0.5 g / L) (FMM-S) using an inoculum diluted 1 / 100. All growth tests were performed in triplicate. Culture samples (5 mL) were taken at the beginning of the study (t=0) and at regular intervals for 48 h after inoculation. These culture samples were used to evaluate the pH and to measure the pH by spectrophotometer (A 620 ), viable cell number was determined by plate seeding, and total cell number was determined by microscopic counting in a Neubauer chamber (Brand™ Buerker). Results were analyzed using Excel and the SPSS V.22 (IBM) statistical program.

[0038] The results of this assay showed that the optimal culture conditions for T. maritimum CECT30394 and T. solea CECT30393 were incubation for 48 h at 25°C in FMM medium (Laboratorio Conda SA, Madrid, Spain) supplemented with 0.5 g / L sucrose (Sigma Aldrich, Spain) (FMM-S). Under these conditions, T. maritimum CECT30394 cultures reached a total cell count of 8.5 × 10 cells per milliliter. 10 Cell density (A 620 : 0.715) (Figure 1), and T. solea CECT30393 cultures reached a total cell count of 5.6 × 10 10 Cell density (A 620 A cloning rate of 0.01 mg / mL was achieved (Figure 1). The concordant results for both bacteria demonstrate that the conditions and culture media described are ideal for vaccine development.

[0039] Example 2. A method for producing and inactivating bacterial cultures of T. maritimum and T. solea strains constituting a vaccine, comprising the following steps:

[0040] a) Obtaining logarithmic phase inocula of T. maritimum CECT30394 and T. solea CECT30393 by culturing in FMM-S broth (24 h incubation at 25° C. with orbital shaking at 100 rpm).

[0041] b) 30 mL of inoculum of T. maritimum CECT30394 and T. solea CECT30393 is inoculated into a 6-liter flask containing 3 liters of FMM-S broth and incubated at 25° C. under aerobic conditions (0.25 L / min) with orbital shaking (100 rpm) for 48 hours.

[0042] c) The bacterial cultures are inactivated by adding formaldehyde to a final concentration of 0.35% and incubating with shaking at 25° C. for 180 min, after which the cultures are transferred to 4° C. After 24 h at 4° C., the cultures are centrifuged (10,000 rpm for 30 min), the supernatant is discarded, and the cell pellet is suspended in phosphate-buffered saline (PBS, 8 g / L sodium chloride, 0.3 g / L potassium chloride, 0.73 g / L sodium phosphate, 0.2 g / L monopotassium phosphate, pH 7.4) (PBS-0.15) containing formalin at a final concentration of 0.15% (vol / vol).

[0043] d) Equal volumes of suspensions of T. maritimum CECT30394 and T. solea CECT30393 strains are mixed in PBS-0.15 to prepare a bivalent anti-T. maritimum-T. solea vaccine. The vaccine mixture is measured by optical density at a wavelength of 620 nm (absorbance at 620 nm, A 620 The resulting vaccine solution is approximately 3 × 10 10 Contains cells / mL.

[0044] e) Sterility controls of the vaccine are performed by plating the vaccine mixture on FMM-S agar plates, marine agar (MA) plates, and tryptone soy agar (TSA) plates with a final concentration of 1% NaCl (TSA-1), as well as in thioglycolate broth tubes, and incubating at 25°C and 37°C for 72 hours. The vaccine is stored at 4°C until use. Specificity controls are performed by slide agglutination using rabbit antisera specific for strains CECT30394 and CECT30393 and inactivated bacterial cells used in vaccine production as antigen.

[0045] f) Evaluate the safety of the vaccine in fish by intraperitoneal (ip) injection (0.2 mL) or immersion (dilution 1:500) of a double dose of the vaccine and assess its efficacy for 21 days.

[0046] Example 3. Evaluation of the efficacy of a bivalent vaccine prepared using Tenaxybaculum maritimum CECT30394 and Tenaxybaculum solea CECT30393 strains in an animal model Vaccine efficacy was evaluated in experimental assays using flatfish (soil and turbot) and fusiform fish (gilthead seabream and sea bass) as models. Turbot (mean weight 3.06 ± 0.78 g), sole (mean weight 0.71 ± 0.17 g), gilthead seabream (mean weight 7.58 ± 2.26 g), and sea bass (mean weight 3.17 ± 0.96 g) were used in vaccination trials. The numbers of fish recommended in the European Pharmacopoeia 7.0 (2010) were used for these studies.

[0047] Fish were vaccinated by immersion or injection and then experimentally infected with heterologous T. maritimum and T. solea strains.

[0048] a) Administration by immersion: for this, the fish are immersed for 2 minutes in the vaccine diluted 1:1000 in seawater under strong aeration and then returned to the aquaculture tank. After 4 weeks, a booster dose is administered using the same method.

[0049] b) Administration by injection: For this, the fish are anaesthetized with the anesthetic tricaine methanesulfonate (MS-222, Sigma) at a concentration of 60 mg / L and inoculated intraperitoneally with the undiluted vaccine at a dose of 0.1 mL / tail.

[0050] The efficacy of the bath-administered vaccines was evaluated by bath infection, in which fish (vaccinated or control) were immersed for 1 h in a bacterial suspension of the bacteria being evaluated (T. maritimum or T. solea).

[0051] The efficacy of the vaccines administered by injection was evaluated by injecting 0.1 mL of a suspension of the bacterial species being evaluated (T. maritimum or T. solea). For this assay, fish were previously anesthetized with tricaine (MS-222 Sigma Aldrich).

[0052] Relative survival rates were determined at the end of the infection challenge (Revised, 1981; European Pharmacopoeia 7.0, 2010). For this, specific mortality curves were plotted as a function of time since experimental infection for both vaccinated and control groups. These curves were used to interpolate the percentage specific mortality in the vaccinated group to the time (M) at which specific mortality in the control group reached 60% and to calculate the relative survival rate (RPS) using the following formula (European Pharmacopoeia 7.0, 2010): RPS = (1-M / 60) x 100

[0053] Furthermore, the presence of a significant difference in survival between the vaccinated and control groups exposed to experimental infection was confirmed using a chi-square test (χ 2 , p<0.05).

[0054] The vaccine administered by immersion (two 2-min water baths spaced 4 weeks apart) (Figure 2) or injection (0.1 mL dose) confers high levels of protection (RPS = 70%–100%) in fish against marine tenacitybaculum caused by both pathogenic species.

[0055] literature Arnaud M, Moalic PY, Bourgeois F, Carpentier R, Le Breton A. 2018. AQUA 2018, 25-29 August, Montpellier, France. Avendano-Herrera R, Toranzo AE, Magarinos B. 2006a. Dis Aquat Org 71:255–266 Avendano-Herrera R, Magarinos B, Irgang R, Toranzo AE. 2006b. Dis Aquat Org 71:255–266 Avendano-Herrera R, Nunez S. Barja JL, Toranzo AE. 2008. Aquac Int 16(1): 1-11 Burioli E, Varello K, Trancart S, Bozzetta E, Gorla A, Prearo M, Houssin M. 2017. Journal of Fish Diseases. 41. 10.1111 / jfd.12698. Castro N, Balboa S, Nunez S, Toranzo AE, Magarinos B. 2014. Fish Pathol 49(1):16-22 Cepeda C, Santos Y. 2002. Bull Eur Assoc Fish Pathol 22(6):388-392 FAO. 2020. Rome page 348. http: / / www.fao.org / documents / card / en / c / ca9692es. Fernandez-Alvarez C, Santos Y. 2018. Appl Microbiol Biotechnol 102: 9973-9989 Fernandez-Alvarez C, Gonzalez SF, Santos Y. 2019. Aquaculture 498(1):289-296 Garcia-Carballas, 2018. University of Santiago de Compostela Doctoral Thesis (Santiago de Compostela) Le Breton A. 2019. http: / / www.medaid-h2020.eu / index.php / 2019 / 02 / 05 / tenacibaculosis / Lopez JR, Pineiro-Vidal M, Garcia-Lamas N, De La Herran R, Navas Jl, Hachero-Cruzado I, Santos Y. 2010. J Fish Dis 33(3):273-278. Pazos F. 1997. University of Santiago de Compostela Doctoral Thesis (Santiago de Compostela) Pineiro-Vidal M 2008. University of Santiago de Compostela Doctoral Thesis (Santiago de Compostela). Pineiro-Vidal M, Centeno-Sestelo G, Riaza A, Santos Y. 2007. Bull Eur Assoc Fish Pathol 27(1):29-35 Pineiro-Vidal M, Riaza A, Santos Y.2008a. Int J Syst Evol Microbiol 58(1):21-25 Pineiro-Vidal M, Carballas CG, Gomez-Barreiro O, Riaza A, Santos Y. 2008b. Int J Syst Evol Microbiol 58(4):881-885 Pineiro-Vidal M, Gijon D, Zarza C, Santos Y.2012. Int J Syst Evol Microbiol 62(2):425-429 Romalde J, Ravelo C, Lopez-Romalde S, Avendano-Herrera R, Magarinos B, Toranzo A. 2005. Developments in biologicals. 121. 85-95. Santos Y, Pazos F, Barja JL.1999. In: ICES identification leaflets for diseases and parasites of fish and shellfish No. 55, International Council for the Exploration of the Sea. ICES, Denmark, pages 1-6 Smage SB, Brevik OJ, Duesund H, Ottem KF, Watanabe K, Nylund A. 2016. Antonie Van Leeuwenhoek. 2016 Feb; 109(2):273-85. doi: 10.1007 / s10482-015-0630-0. Epub 2015 Dec 11. Santos Y Pazos, 2000. Patent no. ES 2 139549 B1, University of Santiago de Compostela. https: / / consultas2.oepm.es / lnvenesWeb / detalle?referencia=P9801549. Toranzo AE, Magarinos B, Romalde JL. 2005. Aquaculture 246, 37-61. Van Gelderen R, Carson J, Gudkovs N, Nowak B. 2010. J Appl Microbiol 109(5): 1668-1676. European Pharmacopoeia Commission Monograph, 7.0 2010

Claims

**Claim 1** An immunogenic composition, comprising a combination of inactivated bacterial cells of T. maritimum CECT30394 strain and T. solea CECT30393 strain and a buffer solution. **Claim 2** The bacterial cells of T. maritimum CECT30394 and T. solea CECT30393 are both 5×10 ９ cells / mL to 5×10 １０ cells / mL, and the immunogenic composition according to claim 1. **Claim 3** The bacterial cells of CECT30394 and CECT30393 are 3×10 ９ cells / mL to 3×10 １０ cells / mL, and the immunogenic composition according to claim 1 or 2. **Claim 4** The immunogenic composition according to claim 1 or 2, wherein the bacterial cells are inactivated by formaldehyde and / or heat. **Claim 5** The immunogenic composition according to claim 4, wherein the bacterial cells are inactivated by formaldehyde. **Claim 6** The immunogenic composition according to claim 1 or 2, comprising a pharmaceutically acceptable vehicle. **Claim 7** The immunogenic composition according to claim 6, wherein the pharmaceutically acceptable vehicle is an adjuvant. **Claim 8** The immunogenic composition according to claim 7, wherein the adjuvant is selected from the group consisting of Freund's complete adjuvant, Freund's incomplete adjuvant, Eolane 130, Montanide ISA, Titermax, alginate, aluminum salts and potassium salts (aluminum phosphate and potassium phosphate, and aluminum hydroxide), PLGA nanoparticles, liposomes, biodegradable microspheres, saponin, phospholipids, CpG ODN oligodeoxynucleotides, flagellin, bacterial polysaccharides (LPS), levamisole, Mycobacterium bovis, Mycobacterium butyricum, Mycobacterium kelleri, mycobacterial cell wall, chemokines, chitosan, sorbitan sesquioleate, vitamin C, vitamin E, or any combination thereof. **Claim 9** The immunogenic composition according to claim 8, wherein the adjuvant is selected from the list comprising Freund's complete adjuvant, Freund's incomplete adjuvant, Eolane 130, Montanide ISA, Titermax, or any combination thereof. **Claim 10** i. A step of culturing the bacterial cells of CECT30394 strain and CECT30393 strain in a culture medium capable of growing them; ii. A step of inactivating the bacterial cells; iii. A step of recovering the bacterial cells; iv. A step of suspending the bacterial cells; A method for producing the immunogenic composition according to claim 1 or 2, characterized by comprising the above steps. **Claim 11** The method for producing the immunogenic composition according to claim 10, wherein the culture medium is selected from the group consisting of Luria-Bertani (LB) broth and Mueller-Hinton (MH) broth diluted with synthetic seawater, Marine broth (MB), FMM broth, FMM broth supplemented with 0.05% weight / volume glucose (FMM-G), and FMM broth supplemented with 0.05% weight / volume sucrose (FMM-S).

12. The method for producing the immunogenic composition according to claim 11, wherein the culture medium is FMM broth supplemented with 0.05% weight / volume sucrose (FMM-S).

13. The method for producing the immunogenic composition according to claim 10, wherein the inactivation of the bacterial cells is caused by formaldehyde and / or heat.

14. The method for producing the immunogenic composition according to claim 13, wherein the inactivation of the bacterial cells is caused by formaldehyde.

15. The method for producing the immunogenic composition according to claim 13, wherein the inactivation of the bacterial cells is caused by formaldehyde.

16. The method for producing the immunogenic composition according to claim 14, wherein the concentration of the formaldehyde is 0.15% volume / volume to 0.35% volume / volume.

17. The method for producing the immunogenic composition according to claim 15, wherein the concentration of the formaldehyde is 0.35% volume / volume.

18. The method for producing the immunogenic composition according to claim 13, wherein the inactivation time is 2 hours to 3 hours.

19. The method for producing the immunogenic composition according to claim 17, wherein the inactivation time is 3 hours.

20. The method for producing the immunogenic composition according to claim 13, wherein the inactivation temperature is 20°C to 25°C.

21. The method for producing the immunogenic composition according to claim 19, wherein the inactivation temperature is 25°C.

22. The method for producing the immunogenic composition according to claim 13, wherein the heat inactivation temperatures are 80°C and 100°C.

23. The method for producing the immunogenic composition according to claim 21, wherein the heat inactivation temperature is 100°C.

24. The method for producing the immunogenic composition according to claim 21, wherein the inactivation time is 45 minutes to 60 minutes.

25. The method for producing the immunogenic composition according to claim 23, wherein the inactivation time is 60 minutes.

26. The method for producing an immunogenic composition according to claim 10, wherein the recovery of the bacterial cells is carried out by either centrifugation or ultrafiltration.

27. The method for producing an immunogenic composition according to claim 25, wherein the recovery of the bacterial cells is carried out by centrifugation.

28. The method for producing an immunogenic composition according to claim 26, wherein the centrifugation is carried out at 10,000 rpm to 12,000 rpm.

29. The method for producing an immunogenic composition according to claim 27, wherein the centrifugation is carried out at 10,000 rpm.

30. The method for producing an immunogenic composition according to claim 10, wherein the suspension is carried out in an aqueous physiological saline solution containing formaldehyde or phosphate-buffered saline (PBS).

31. The method for producing an immunogenic composition according to claim 29, wherein the suspension is carried out in a buffered aqueous physiological saline solution containing formaldehyde or a phosphate buffer.

32. The method for producing an immunogenic composition according to claim 30, wherein the formaldehyde has a concentration of 0.15% volume / volume to 0.35% volume / volume.

33. The method for producing an immunogenic composition according to claim 31, wherein the formaldehyde has a concentration of 0.15% volume / volume.

34. The method for producing an immunogenic composition according to claim 29, wherein the optical density at 620 nm after suspension is 0.5 to 0.

8.

35. The method for producing an immunogenic composition according to claim 33, wherein the optical density at 620 nm after suspension is 0.

7.

36. The method for producing an immunogenic composition according to claim 10, which contains a pharmaceutically acceptable vehicle.

37. Use of the immunogenic composition according to claim 1 or 2 in the preparation of a pharmaceutical composition or a veterinary composition.

38. Use of the immunogenic composition according to claim 1 or 2 in the preparation of a medicament for the prevention, control and / or treatment of tenacibaculosis.

39. The use according to claim 37, wherein the medicament is a vaccine.

40. The use according to claim 38, wherein the vaccine is for fish.

41. The use according to claim 39, wherein the fish is selected from the group consisting of Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), turbot (Scophthalmus maximus), European plaice (Pleuronectes platessa), European flounder (Platichthys flesus), European sea bass (Dicentrarchus labrax), European seabream (Sparus aurata), Spanish seabream (Pagellus bogaraveo), cod (Gadus morhua), brill (Scophthalmus rhombus), and / or wedge sole (Dicologlossa cuneata).

42. The use according to claim 40, wherein the fish is European seabream (Sparus aurata), European plaice (Pleuronectes platessa), European sea bass (Dicentrarchus labrax) and / or turbot (Scophthalmus maximus).

43. A kit for use in inducing an immune response in fish, comprising the immunogenic composition according to claim 1 or 2 and optionally instructions related to administration.

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