Novel streptococcus agalactiae serotype ia bacteria and vaccines thereof

Abstract

The present invention relates to the field of aquaculture, in particular to tilapia farming, and more particularly, the present invention relates to a novel bacterium of Streptococcus agalactiae serotype Ia (Sa Ia) which is much more pathogenic than the Sa Ia isolates known so far and which can break the protection provided by the existing Sa Ia vaccine. In particular, the present invention relates to a novel breaking Sa Ia bacterium which is defined by a number of unique characteristics and which is deposited at the CNCM in Paris. Furthermore, the present invention relates to preparations, compositions and vaccines of the novel Sa Ia bacterium, to diagnostic kits, and to the (medical) use and manufacturing process of the preparations, compositions and vaccines of the novel Sa Ia bacterium.

Description

Technical Field

[0001] This invention relates to the field of aquaculture, and more particularly to tilapia farming. Specifically, this invention relates to a novel agalactiae streptococcal serotype Ia (Sa Ia). Furthermore, this invention relates to preparations, compositions, and vaccines of this novel Sa Ia bacterium or its progeny, to diagnostic kits, and to the (medical) uses and methods of preparings, compositions, and vaccines of this novel Sa Ia bacterium. Background of the Invention Streptococcus is a genus within the order Lactobacillus, consisting of Gram-positive, non-motile cocci. Its genome size is approximately 2 Mb, and based on its 16S rRNA sequence, Streptococcus is divided into six groups according to phylogenetic parameters.

[0003] Previously, the phenotypic classification of Streptococcus species was based on their hemolytic properties. A taxonomic system based on the so-called "Lancefield grouping," which is based on the carbohydrate composition of cell wall antigens, was also applied, (currently) dividing them into 15 groups.

[0004] Further subtyping is based on serological testing of capsular polysaccharides, which are type-specific and important virulence factors. Ten serotypes have been described to date: Ia, Ib, and II through IX. Such subtyping can also be performed using different types of genotyping, such as by polymerase chain reaction (PCR) detection of capsular polysaccharide synthesis genes.

[0005] Streptococcus agalactiae (Sa) is taxonomically classified under the group of Streptococcus pyogenes. It is catalase-negative and a facultative anaerobe.

[0006] Streptococcus spp. (S.) is subdivided into Lancefield group B, hence its common name "Group B Streptococcus" (GBS). S. is a pathogenic commensal bacterium that typically lives in a symbiotic form, but can become pathogenic in certain circumstances and cause invasive infections and diseases in humans and many animals; this can lead to sepsis and may affect several organs.

[0007] Sa is named for its important role in bovine mastitis. However, Sa can also be pathogenic to other animals; for example, Sa is a major pathogen in fish (e.g., tilapia), especially in large-scale aquaculture. Of the known Sa serotypes, four (Ia, Ib, II, and III) have been found in fish so far, and these are zoonotic, making Sa a foodborne human pathogen. Zang (2001, Pathogens, vol. 10, p. 558) reviewed this. Serotype Ib of Sa is non-hemolytic (also known as γ-hemolytic), while Sa Ia may be α-hemolytic or γ-hemolytic.

[0008] Tilapia are fish belonging to the family Cichlidae; among them, the most economically valuable is the common tilapia, previously taxonomically classified under the genus *Tilapia*, but now reclassified into the genera *Coptodon*, *Oreochromis*, and *Sarotherodon*. Nile tilapia (*Oreochromis niloticus*; formerly known as *Tilapia nilotica*) and its various hybrids are the most widely farmed. Warm freshwater tilapia aquaculture is highly developed in several countries in Central America, South America, Africa, and Asia. In these aquaculture operations, *Sarcophaga* infection can lead to mass mortality and significant economic losses; see: Evans et al. (2006, *Aquaculture Health Int.*, no. 7, pp. 10-14).

[0009] The disease in fish caused by Sa infection is called streptococcal disease. Environmental factors (especially temperature) play an important role in the development of this disease: a significant increase in mortality usually occurs during the hot season, while as the water temperature decreases, a low but persistent mortality level may occur.

[0010] Common clinical signs of streptococcal infection include abnormal swimming posture, exophthalmos, and hemorrhage, inflammation, and ascites in affected organs. In addition to its serious impact on animal welfare, the disease's main effects are economic: acute illness and death lead to lost income and increased costs.

[0011] In aquaculture, adjusting aquaculture management practices is often insufficient to overcome these negative effects, and the use of antibiotics can be controversial. Therefore, vaccination is the preferred method for preventing infections and diseases caused by Sa. Different types of vaccines against Sa have been developed and proven to be highly effective; Liu et al. (2016, Dis. Aq. Org., vol. 122, p.163-170) reviewed this.

[0012] Well-known streptococcal vaccines used for fish are based on bacterin antigens (bacterial cells killed by formalin) emulsified with an oil adjuvant. Eldar et al. (1995, Vaccine, vol. 13, pp. 867-870) described a vaccine for *Streptococcus difficile*, which has now been reclassified as serotype Ib *Streptococcus agalactiae*. Such vaccines can be administered by injection (e.g., intraperitoneal injection). Examples of commercially available vaccines against Sa are from MSD Animal Health: AQUAVAC® Strep Sa-Si, for combating Streptococcus agalactiae serotype Ib and Streptococcus iniae infection; and AQUAVAC® Strep Sa1, for combating Sa serotypes Ia and III, containing formalin-inactivated bacteria (vaccine) of two Sa serotypes: Ia (strain TI 1422) and III (strain TI 1428). This vaccine is a water-in-oil emulsion using a commercially available non-mineral oil adjuvant: Montanide™ ISA 763AVG. See also WO 2011 / 048041.

[0013] WO 2008 / 003734 and WO 2011 / 048041 describe the current development of streptococcal vaccines.

[0014] With global climate change, such as warming and acidification of freshwater habitats, the risk of Sa infection and disease in aquaculture is increasing (Phuoc et al., 2021, Aquaculture, vol. 534, 736256). Therefore, there is an ongoing need to provide more effective Sa vaccines.

[0015] Therefore, one object of the present invention is to overcome the deficiencies in the prior art and to meet the needs of the field by providing a vaccine against novel Sa Ia bacteria. Detailed Implementation

[0016] Surprisingly, it has been found that this objective can be achieved by providing a novel Sa Ia bacterium with serotype Ia, overcoming one or more drawbacks of the prior art. This bacterium is more pathogenic than previous Sa Ia isolates and can bypass the protection against Sa Ia infection provided by existing vaccines. This novel Sa Ia bacterium can now be used in a new, highly effective vaccine for streptococcal disease in fish.

[0017] In freshwater lakes in Chiapas, Mexico, several large tilapia farms regularly vaccinated juvenile fish against serotypes Sa Ia, Ib, and III. However, in July 2021, a sudden and large-scale mortality occurred, with a fish mortality rate as high as 65%. Autopsies revealed intestinal lesions in the infected fish, resulting in severe gastric and intestinal hemorrhage, accompanied by intussusception (also known as "cylindrical changes") as a prominent sign. Toxicological studies of the feed and water were conducted, but no abnormalities were found. Over a three-month period, symptoms first appeared in older fish, then spread to juveniles, and also to downstream aquaculture areas. This indicates that the disease is contagious.

[0018] The symptoms observed in the aquaculture environment did not directly point to streptococcal disease; therefore, researchers isolated tissue samples from various organs of dead and diseased fish: brain, gills, heart, prokidney, liver, spleen, intestine, and stomach. The new isolates were then characterized using multiple assays.

[0019] Based on initial histopathological observations, infection with Tilapia Lake Virus (TiLV) was suspected. However, even after testing several mixed tissue samples with TiLV-specific nested PCR, no positive results for TiLV were found in the test samples from the outbreak in Mexico.

[0020] The farm's temporary use of antibiotics (florfenicol-Aquaflor®, MSD Animal Health) immediately reduced mortality during the peak of the outbreak. Therefore, the primary cause was believed to be bacterial infection.

[0021] In the fall of 2022, a similar vaccine breakthrough event occurred at a tilapia farm in a freshwater reservoir in Cortés Province, Honduras, leading to a large-scale tilapia mortality rate. Of the dead fish tested, 27% exhibited intussusception. Sa Ia bacteria, with characteristics substantially identical to the Mexican breakthrough isolate, were obtained from tissues of the affected fish.

[0022] Surprisingly, samples from these outbreaks were found to contain a novel Sa serotype Ia bacterium, which is more pathogenic than known Sa Ia isolates. Furthermore, existing vaccines against Sa Ia only provide moderate immunity. However, when these new isolates are used as vaccines themselves, they offer excellent protection against both homologous challenge (i.e., against the novel strain itself) and heterologous challenge (using previously known Sa Ia isolates).

[0023] When compared with early Sa Ia isolates used in Sa vaccines and early Sa Ia isolates used to date for challenge infection, the novel breakthrough isolate exhibits substantially the same behavior in several aspects: during its in vitro culture, in tests for serological classification (latex agglutination), and in studies for biochemical characterization (API 20™ STREP, bioMerieux SA) and for protein characterization (SDS-PAGE and Western blot).

[0024] Interestingly, the new isolate differs significantly from known Sa Ia vaccine and challenge strains: it showed up in unimmunized fish (na) The pathogenicity in *Sa Ia* fish was significantly higher, and its serological cross-protection showed weak antibody recognition against existing Sa Ia vaccine strains. However, conversely, vaccination of fish with a vaccine containing the new isolate did provide protection against known Sa Ia strains. Specific details are described in the examples below.

[0025] Interestingly, when tilapia were tested for infection using the novel Sa Ia isolate under laboratory conditions, the infected fish did indeed exhibit typical signs of streptococcal disease, such as spiral swimming, bulging eyes, meningoencephalitis, and petechiae on the gill covers and fins. Furthermore, when the fish were fed after challenge infection, intussusception, as observed in aquaculture environments, appeared in over 25% of cases.

[0026] Several bacterial isolates from the 2021 outbreak in Mexico and the 2022 outbreak in Honduras were purified and cultured for further study. One isolate from Mexico, TI 2893, was used to prepare the bacterial strain for the vaccination study. In a competitive inhibition ELISA, this isolate showed low inhibitory levels against antiserum produced against existing vaccine strains, although slightly higher than other isolates from Mexico in 2021.

[0027] A representative sample of the strain derived from isolate TI 2893 has been submitted to the French National Center for Microbial Collections (CNCM) at the Pasteur Institute in Paris, France, with accession number CNCM I-5929, and deposited on December 15, 2022. The sample has been received and confirmed to be viable, as evidenced by the receipt form and the accompanying viability declaration.

[0028] The exact mechanisms or reasons for the increased pathogenicity of novel Sa Ia bacteria, and why they are able to overcome the immune response induced by existing Sa Ia vaccines, remain unclear. This makes these novel Sa Ia isolates not readily apparent from any existing technology, and the Sa Ia bacteria that differ from known Sa Ia isolates in certain properties, as described herein, were previously unknown.

[0029] Therefore, one aspect of the present invention relates to a Streptococcus agalactiae (Sa) bacterium of serotype Ia (Sa Ia) that has the characterization features of a bacterium deposited at the French National Center for Microbial Collections (CNCM) at the Pasteur Institute in Paris, France, with accession number CNCM I-5929.

[0030] "Streptococcus" is a well-known genus of bacteria. Detailed information on its characterization and classification can be found, for example, in well-known manuals such as Bergey's Manual of Bacteriological Identification and Bergey's Manual of Systematic Bacteriology.

[0031] “Agalactotropic” species (i.e., Lancefield Group B) can be easily identified in a similar manner. In addition, numerous commercially available tests are available for the necessary biochemical and serological identification.

[0032] As described in the CDC (www.cdc.gov / streplab / groupb-strep / index.html), the determination of serotypes (such as serotype Ia) of Sa Ia bacteria is well-known, and several commercial vendors offer convenient test kits, for example, using PCR. Preferably, one of several rapid agglutination assays is used to determine the Ia serotype, employing latex particles coated with serotype-specific antibodies. One example is the "ImmuLex™ Streptococcus Group B Ia Serological Assay Kit" (SSI Diagnostica, Denmark).

[0033] As is also known in the art, the classification of microorganisms within a particular taxonomic group is based on their combination of characteristics. Therefore, the present invention also includes variants of Sa Ia species subdivided in any way, such as subspecies, strains, isolates, genotypes, variants, subtypes, or subgroups. Furthermore, it will be apparent to those skilled in the art that while a particular Sa Ia bacterium of the present invention may currently be classified into that species or subtype, such taxonomic classification may change over time, as new insights may lead to its reclassification into new or different taxa. For example, some Sa serotype Ib groups were previously classified as *Streptococcus difficile* (*S. difficile* / difficilis).

[0034] However, since this does not change the bacteria themselves or their antigenic repertoire, but only their scientific name or classification, such reclassified bacteria are still within the scope of this invention.

[0035] The Sa Ia bacteria according to the present invention are contained in or on a suitable vector.

[0036] For the purposes of this invention, the carrier may be a liquid or a (semi)solid, such as a liquid such as water, glycerol, a buffer solution or a culture medium; a semi-solid such as a gel; a solid such as a freeze-dried body; or a physical structure such as paper or a polymer sheet, or a molded article.

[0037] The Sa Ia bacteria according to the invention can be obtained directly or indirectly from the preserved bacteria of the invention: direct acquisition is by obtaining the preserved sample; indirect acquisition is by obtaining the bacteria from the progeny of the preserved bacteria of the invention (as defined herein). Such progeny and indirectly obtained bacteria retain the characterization features of the preserved bacteria.

[0038] Bacteria obtained directly or indirectly can be amplified through one or more in vivo or in vitro passages.

[0039] In addition to being infectious and pathogenic to fish (especially tilapia), the preserved bacteria also share many characteristics with known Sa bacteria, such as being serotype Ia. This serotype can be determined using various well-known tests, such as latex agglutination of bacterial culture samples or (multiplex) PCR testing of the capsular antigen gene in the bacterial genetic material.

[0040] Furthermore, after incubation at 26°C for 3 days on sheep blood agar plates, this novel Sa Ia bacterium was found not to exhibit hemolytic activity, therefore it is γ-hemolytic, as is the case with many Sa Ia bacteria.

[0041] Furthermore, as determined by multi-site sequence typing (MLST) using standard procedures, the new isolate was identified as "Sequence Type" (ST) 7; see Examples for details. This is noteworthy because many known Sa Ia strains are also ST7, including existing Sa Ia vaccine strains. Therefore, this is not a discriminative indicator of serological and pathological variation in Sa Ia bacteria.

[0042] For the purposes of this invention, the “characterizing features” of the preserved bacteria refer to various elements and properties of the genotype and phenotype of the preserved bacteria, such as morphological and genomic characteristics, as well as biological characteristics such as their physiological, biochemical, immunological and / or pathological behaviors, which distinguish them from known Sa Ia bacteria.

[0043] For future Sa bacteria isolates, those skilled in the art can readily determine whether their characterization features match or differ from those of the preserved samples using conventional methods and procedures.

[0044] One characterizing feature that distinguishes the novel bacteria according to the invention from known Sa Ia bacteria is that the novel bacteria induce pathological signs in tilapia at much lower inoculum doses: as described herein, at doses 4,000 times lower than known Sa Ia isolates, the novel bacteria still caused at least the same level of mortality in comparative inoculation tests.

[0045] Another characteristic feature of the novel Sa Ia bacteria according to the invention is that they are able to overcome vaccine protection induced by existing Sa Ia vaccines: as described herein, existing vaccines protect only 20%–50% of vaccinated fish against challenge infection with this novel bacterium. On the other hand, and in the reverse case: as described herein, vaccines based on the novel Sa Ia protect at least 81% of fish from infection and disease caused by known Sa Ia challenge strains.

[0046] Furthermore, other characterizing features of this novel Sa Ia bacterium according to the invention can be observed by testing its genetic material in a PCR assay using specific primer sets as defined herein. These primer sets will produce PCR products with nucleotide fragment sizes significantly different from those obtained when testing the genetic material of known Sa Ia bacteria using the same primer sets. This even applies to Sa Ia isolated from the same region of Mexico several years ago. Detailed information is provided in the examples and Table 1 below.

[0047] These combined properties of novel Sa Ia bacteria work together and complement each other to characterize the bacteria according to the invention: preserved samples, descriptions of the biological, immunological and genetic properties of the bacteria, pathological symptoms caused by their infection, vaccine breakthrough, and PCR fragments of unique size generated from their genetic material using specific primer sets.

[0048] These properties together provide a complete overview for those skilled in the art, characterizing the novel Sa Ia bacteria according to the invention in a comprehensive manner and distinguishing them from known Sa Ia bacteria.

[0049] The Sa Ia bacteria according to the invention can be readily amplified under suitable conditions by in vivo or in vitro culture, followed by one or more passages, using methods as described herein and well known to those skilled in the art. In vivo culture involves inoculation into susceptible animals such as fish (as defined below). For in vitro culture, a variety of suitable culture media and culture containers are available. Suitable culture media include, for example, agar or tryptone soybean broth, or brain heart infusion, containing 0 to 5.5% w / v NaCl. Suitable in vitro culture conditions include, for example, incubation at 25 to 37°C for 16 to 24 hours under facultative anaerobic conditions, and (for flask culture) shaking at 100–160 rpm.

[0050] Details of embodiments and further aspects of the present invention will be described below.

[0051] In one embodiment, the Sa Ia bacteria according to the invention are contained in a vector.

[0052] The "carrier" used in this invention can be a solid or a liquid, whether at room temperature or below zero. For example, it can be a liquid containing Sa Ia bacteria according to the invention. The liquid can be, for example, water, glycerol, physiological buffer, culture medium, or stabilizer. The carrier may contain other excipients, such as proteins, sugars, amino acids, or polymers, to stabilize the bacteria.

[0053] When it is a (semi)solid, the carrier can be, for example, a gel, a frozen liquid or a freeze-dried body, a semi-solid such as a gel; a solid such as a freeze-dried body; or a physical structure such as paper or polymer sheet, or a molded article.

[0054] In one embodiment, the carrier according to the invention is selected from one or more of the following: physiological buffer, glycerol, stabilizer and preservative.

[0055] Stabilizers used in this invention are selected from, for example, polysaccharides, glucose polymers, chemicals (e.g., DMSO), and proteins (e.g., skim milk or serum).

[0056] In one embodiment, the Sa Ia bacteria according to the invention are isolated bacteria.

[0057] For the purposes of this invention, "isolated" means: taken from its natural environment and free from (including partially and substantially free from) certain contaminants. For the purposes of this invention, "isolated" does not preclude the use of carriers as described above for the purposes of this invention. For example, samples collected from infected fish or fish tissue, samples collected from water in which infected fish are present, or samples collected from bacterial cultures in liquid, semi-solid, or solid culture media can constitute isolated SaIa bacteria according to the present invention.

[0058] Therefore, one of the characterizing features of the novel Sa Ia bacteria according to the invention becomes apparent when its genetic material is tested in a PCR assay using a specific set of primers. Thus, the Sa Ia bacteria according to the invention can be distinguished from known Sa Ia bacteria.

[0059] Therefore, in one embodiment, the Sa Ia bacteria according to the invention are characterized in that, in a polymerase chain reaction (PCR) using genetic material from said bacteria, nucleotide (nt) fragments of a specific size are produced when a specific set of primers is used as follows: - Using the primer set of SEQ ID NO: 1 and SEQ ID NO: 2, generate a 500 nucleotide (nt) fragment; or - Using the primer set of SEQ ID NO: 3 and SEQ ID NO: 4, a 1672 nt fragment was generated; or - Using the primer set of SEQ ID NO: 5 and SEQ ID NO: 6, a 1772 nt fragment was generated; or - Using the primer set of SEQ ID NO: 7 and SEQ ID NO: 8, a 1912 nt fragment was generated; or - Using the primer set of SEQ ID NO: 9 and SEQ ID NO: 10, a 2016 nt fragment was generated; or - Using the primer set of SEQ ID NO: 11 and SEQ ID NO: 12, an 876 nt fragment was generated.

[0060] In a preferred embodiment, the Sa Ia bacterium according to the invention is characterized in that, in PCR using genetic material from said bacterium, the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 is used to produce a nucleotide (nt) fragment of 500 ± 5 nt.

[0061] In another preferred embodiment, the Sa Ia bacterium according to the invention is characterized in that, in PCR using genetic material from said bacterium, the primer set of SEQ ID NO: 3 and SEQ ID NO: 4 is used to produce an nt fragment of 1672 ± 5 nt.

[0062] In another preferred embodiment, the Sa Ia bacterium according to the invention is characterized in that, in PCR using genetic material from said bacterium, the primer set of SEQ ID NO: 5 and SEQ ID NO: 6 is used to produce an nt fragment of 1772 ± 5 nt.

[0063] In yet another preferred embodiment, the Sa Ia bacterium according to the invention is characterized in that, in PCR performed with genetic material from said bacterium, the primer set of SEQ ID NO: 7 and SEQ ID NO: 8 is used to produce an nt fragment of 1912 ± 5 nt.

[0064] In even another preferred embodiment, the Sa Ia bacterium according to the invention is characterized in that, in PCR performed with genetic material from said bacterium, the primer set of SEQ ID NO: 9 and SEQ ID NO: 10 is used to produce an nt fragment of 2016 ± 5 nt.

[0065] In yet another preferred embodiment, the Sa Ia bacteria according to the invention are characterized in that, in PCR performed with genetic material from said bacteria, the primer set of SEQ ID NO: 11 and SEQ ID NO: 12 is used to produce an nt fragment of 876 ± 5 nt.

[0066] The size of the PCR product of the bacteria according to the invention is obtained using the indicated primer set against genomic DNA, which is derived not only from the preserved bacteria (isolate TI 2893), but also from other co-isolates from Mexico in 2021, and from isolates from the Sa Ia outbreak in Honduras in 2022.

[0067] For this invention, PCR can be performed using common methods and materials, such as those for sample preparation, cycling conditions, and PCR reagents. Specific details and preferred PCR conditions for each primer set to be used will be described in the examples.

[0068] Those skilled in the art will understand that the type of PCR and the type of bacterial genetic material used in this invention are not critical. For example, the PCR can be endpoint PCR performed on DNA or reverse transcriptase PCR (RT-PCR) performed on RNA. Furthermore, the PCR can be qualitative or quantitative. Quantitative PCR (qPCR) is also known as real-time PCR; these are well known in the art.

[0069] Preferably, the PCR used in this invention is an endpoint PCR. Preferably, the genetic material is bacterial genomic DNA.

[0070] As the inventors have discovered, each of these primer sets will produce PCR products of the indicated size for Sa Ia bacteria according to the invention, which are significantly different in size from those obtained when using the same primer sets on genetic material from known Sa Ia bacteria.

[0071] For the present invention, the “size” of the nt fragment obtained by PCR can be precisely determined by computer analysis of digital sequence data, specifically by computer-simulated PCR analysis of long read sequences of genomic DNA from Sa Ia bacteria. However, and as those skilled in the art will understand, when such size determination is performed under wet-lab conditions, the size cannot always be determined with such precision up to the last nucleotide number indicated by the present invention.

[0072] Therefore, the length of the nucleotide (nt) fragment indicated by this invention should be interpreted as: adding or subtracting 25 nt from the indicated size value; preferably, adding or subtracting 20, 15, 10, 5, 4, 3, 2, or 1 nt from the indicated size value in the order of preference. Most preferably, no nt is added or subtracted from the indicated size value.

[0073] Therefore, for example, a 500 nt PCR product obtained by using the primer set of SEQ ID NO: 1 and SEQ ID NO: 2 may be detected as having a size of 475 to 525 nucleotides, preferably 480 to 520 nt, etc.

[0074] Even considering this very limited level of error, the Sa Ia bacteria according to the invention can still be clearly distinguished from known Sa Ia isolates using the PCR test described herein with reference to the invention.

[0075] To compare with existing technologies, primer sets as defined herein for this invention were tested on a variety of Sa Ia samples isolated from tilapia suffering from streptococcal disease between 2001 and 2022, covering multiple countries in Asia and Central America.

[0076] Table 1 shows the comparison between the PCR product size of the preserved isolate TI 2893 and the PCR results of known Sa Ia isolates.

[0077] As shown in Table 1, using primer sets of SEQ ID NO 9 and 10 or SEQ ID NO 11 and 12 for Sa Ia bacteria other than those according to the present invention can produce PCR fragments of different sizes, depending on the specific isolate tested. However, for bacteria according to the present invention, these primer sets yielded fragments of clearly distinguishable sizes.

[0078] Table 1: PCR Comparison Results

[0079] For the purposes of this invention, the last column of Table 1 describes the (presumed) gene regions from which the PCR fragments in the TI 2893 genome originate; the genomic sequence of TI 2893 is presented in SEQ ID NO: 13 herein. The indicated names refer to the corresponding (presumed) genes, which can be annotated by various analyses and comparisons with known (Sa) bacterial genomes: - pepN: A well-known bacterial gene encoding an aminopeptidase, which is a virulence factor in some streptococci. In SEQ ID NO: 13, it is located in the nt 1034500 region; - DUF1310: Encodes a conserved domain present in a presumed family of proteins approximately 125 amino acids in size, which appear to be specific to Listeria and Streptococcus species. The function of this family is unknown. In the InterPro database, DUF1310 is also known as PF07006. It is located in the nt 1935000 region of SEQ ID NO: 13; - DUF3307: Encodes a conserved protein domain of unknown function within a bacterial protein family. DUF3307 is also known as PF11750. It is located in the nt. 1028000 region of SEQ ID NO: 13; - yjdM: Encodes a protein initially thought to be involved in alkylphosphonate uptake. In *E. coli*, the yjdM gene is regulated by a promoter containing an SOS box. It is located in the nt 991000 region of SEQ ID NO: 13; - abiH: A gene associated with abortion infection and stress responses such as phage infection. It is located in the nt 1274000 region of SEQ ID NO:13; and - fbsA: Encodes fibrinogen-binding protein, a known virulence factor. It is located in the nt 1094000 region of SEQ ID NO:13.

[0080] As is well known in the art, "DUF" stands for "Unknown Function Domain" and refers to a conserved protein domain encoded by a (presumed) gene whose function has not yet been assigned. These domains are derived from multiple alignments of a large number of known sequences. A list of DUF domains is publicly available from the protein domain databases Pfam and InterPro, and can be accessed, for example, through the EMBL-EBI website: www.ebi.ac.uk.

[0081] In a more preferred embodiment, the Sa Ia bacteria according to the invention are characterized in that, in PCR using genetic material from said bacteria, two or more primer sets as defined in the invention are used to produce nt fragments of the size defined in the invention.

[0082] In one embodiment of two or more primer sets as defined in this invention, the two or more primer sets are selected from: - SEQ ID NO: 1 and 2, with SEQ ID NO: 3 and 4; - SEQ ID NO: 1 and 2, with SEQ ID NO: 5 and 6; - SEQ ID NO: 1 and 2, with SEQ ID NO: 7 and 8; - SEQ ID NO: 3 and 4, with SEQ ID NO: 5 and 6; - SEQ ID NO: 3 and 4, with SEQ ID NO: 7 and 8; - SEQ ID NO: 5 and 6, with SEQ ID NO: 7 and 8; - SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, and SEQ ID NO: 5 and 6; - SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, and SEQ ID NO: 7 and 8; - SEQ ID NO: 1 and 2, SEQ ID NO: 5 and 6, and SEQ ID NO: 7 and 8; - SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6, and SEQ ID NO: 7 and 8; and - SEQ ID NO: 1 and 2, SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 6; with SEQ ID NO: 7 and 8.

[0083] In another embodiment of two or more primer sets as defined in this invention, the two or more primer sets are selected from the primer sets of SEQ ID NO: 9 and 10, and from one or more primer sets selected from: - SEQ ID NO: 1 and 2; - SEQ ID NO: 3 and 4; - SEQ ID NO: 5 and 6; - SEQ ID NO: 7 and 8; and - SEQ ID NO: 11 and 12.

[0084] In yet another embodiment, the two or more primer sets as defined in this invention are selected from the primer sets of SEQ ID NO: 11 and 12, and one or more primer sets selected from: - SEQ ID NO: 1 and 2; - SEQ ID NO: 3 and 4; - SEQ ID NO: 5 and 6; - SEQ ID NO: 7 and 8; and - SEQ ID NO: 9 and 10.

[0085] In one or even more preferred embodiment, the Sa Ia bacteria according to the invention are characterized in that, in PCR using genetic material from said bacteria, all six primer sets as defined in the invention are used to produce nt fragments of the size defined in the invention.

[0086] In a more or even preferred embodiment, the Sa Ia bacteria according to the invention are characterized in that the bacteria are those deposited with accession number CNCM I-5929, or are descendants of said deposited bacteria.

[0087] In this invention, the "descendants" of the preserved bacteria are SaIa bacteria obtained from the preserved bacteria through subculturing. This subculturing can be carried out in vitro or in vivo, and can be performed once or more.

[0088] As is well known, bacterial "passage" refers to incubating the bacteria under conditions favorable to their proliferation for a certain period of time. In this context, this duration is the time of at least one generation of the bacteria (also known as a division cycle or generation), but preferably, the passage duration exceeds one generation. For the novel Sa Ia bacteria according to the invention, one generation is approximately 30 minutes.

[0089] The offspring according to the invention possess all the characterization features of the Sa Ia bacteria preserved according to the invention.

[0090] In one embodiment, the Sa Ia bacteria or its progeny according to the invention have a genome that is 90% identical to the sequence of SEQ ID NO: 13.

[0091] SEQ ID NO: 13 presents 2.1 million nucleotides of the bacterial genome of isolate TI 2893, deposited as CNCM I-5929. The isolation, sequencing, and analysis of this genome are described in the examples.

[0092] In a preferred embodiment, the Sa Ia bacteria or its progeny according to the invention have a genome with 91% nucleotide sequence identity to the sequence of SEQ ID NO: 13; more preferably, they have genomes with 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% nucleotide sequence identity to the sequence of SEQ ID NO: 13 in the order of preference.

[0093] In one embodiment, the Sa Ia bacteria or its progeny according to the invention have a genome containing the sequence of SEQ ID NO: 13.

[0094] In one embodiment, the Sa Ia bacteria or its progeny according to the invention have a genome consisting of the sequence of SEQ ID NO: 13.

[0095] For this invention, the percentage of nucleotide sequence identity relative to the sequence of SEQ ID NO, as disclosed in this application, is calculated by nucleotide sequence alignment using the NCBI Blast™ computer program (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), wherein the “blastn” and “Align two or more sequences” options are selected and standard settings and default parameters are used. In these alignments, the sequence from SEQ ID NO of this application is used as the comparison sequence (also known as the target).

[0096] In our analysis of the Sa Ia bacteria according to the invention, the inventors found that the bacteria can contain plasmids. SEQ ID NO: 14 presents the 4.4 kb sequence of this extrachromosomal plasmid as detected in isolate TI 2893, which is deposited as CNCM I-5929.

[0097] In one embodiment, the Sa Ia bacteria or its progeny according to the invention may contain a plasmid comprising the sequence of SEQ ID NO: 14.

[0098] Preferably, the Sa Ia bacteria or its progeny contain a plasmid consisting of the sequence of SEQ ID NO: 14.

[0099] The Sa Ia bacteria or their progeny according to the invention can be advantageously used in a wide range of medical and non-medical applications, including vaccination and diagnosis. However, for vaccination, the live wild-type form of such bacteria is pathogenic to fish, therefore, in further uses, methods, and compositions, they are preferably used in attenuated or inactivated (i.e., non-live) form. Mixtures of attenuated live bacteria and non-live bacteria may also be used.

[0100] The use as an attenuated live vaccine can also refer to the use of the wild-type live bacteria according to the invention as a vaccine for target animals that are less susceptible to pathogenicity than tilapia.

[0101] Therefore, in another aspect of the invention, there is a composition comprising a preparation of Sa Ia bacteria or its progeny according to the invention.

[0102] The “composition” of the present invention may be a solid, a semi-solid or a liquid, and may contain a carrier as described herein with respect to the present invention.

[0103] As used herein, the term “comprising” (and variations such as “including,” “containing,” and “having”) is intended to refer to all elements contemplated for the invention and any possible combinations thereof that are covered or included in any part of the text, paragraph, claim, etc., where the term is used, even if such elements or combinations are not expressly stated; and does not exclude any such elements or combinations.

[0104] Therefore, any such text portion, paragraph, claim, etc. may also relate to one or more embodiments, wherein the term "comprising" (or variations thereof) is replaced with terms such as "consisting of," "formed by," or "substantially composed of."

[0105] The “preparation” of the Sa Ia bacteria or their descendants according to the invention refers to a form of such bacteria that has been treated (e.g., chemically, biologically, physically, or mechanically). Examples of preparations of the invention include extracts, lysates, and acoustic treatments; all of which are well known in the art.

[0106] Treatments applied to the preparation of the present invention may result in partial or complete inactivation of the Sa Ia bacteria or their progeny according to the invention. Such (partial) inactivation may be an expected or unexpected result of the treatment.

[0107] Therefore, in one embodiment, the composition according to the invention is characterized in that the preparation comprises an inactivated form of the Sa Ia bacteria or its progeny according to the invention.

[0108] Compositions containing inactivated bacteria are commonly known as: vaccines.

[0109] In this invention, "inactivation" means that the product cannot be replicated even under optimal conditions; it also means that the product is not active.

[0110] Inactivation can be partial or complete, so in the composition according to the invention, many live cells, some live cells, or no live cells are retained from the live cells of the Sa Ia bacteria or their progeny present before the inactivation treatment is applied.

[0111] In a preferred embodiment of the composition according to the invention, inactivation is complete inactivation, i.e., there are no longer any living cells of the Sa Ia bacteria or their progeny according to the invention.

[0112] Widely known treatments for bacterial inactivation include chemical, physical, biological, and mechanical methods. Examples of physical and mechanical inactivation include heating, high shear, high pressure, freeze-thaw cycles, or exposure to ionizing radiation such as ultraviolet, X-rays, or gamma rays. One example of biological inactivation is incubation with enzymes. Examples of chemical inactivation include exposure to high or low pH, detergents, organic or inorganic solvents, chaotropic agents, formaldehyde, lactones, or aziridine.

[0113] In one embodiment of the composition according to the invention comprising an inactivated form of the Sa Ia bacteria or its progeny, the bacteria are inactivated using formaldehyde.

[0114] The formaldehyde (methanal) used in this invention is preferably a compound with CAS registration number 50-00-0. Formaldehyde is usually and more conveniently used in aqueous solution (i.e., formalin).

[0115] Conditions for formalin inactivation of Sa bacteria are well known and include, for example, treatment with 0.01-5% v / v formalin for 30 minutes to several hours at a certain temperature (e.g., room temperature), depending on the inactivation incubation temperature.

[0116] The Sa Ia bacteria, progeny, and / or compositions according to the invention can be advantageously used for a variety of purposes, particularly for veterinary health purposes in aquaculture, such as in diagnosis and vaccination.

[0117] Therefore, in another aspect of the invention, the use of Sa Ia bacteria, progeny, or compositions according to the invention in diagnostic testing is discussed.

[0118] Diagnostic testing is well known in the art, and for the purposes of this invention, diagnostic testing may refer, for example, to the use of the bacteria or preparations thereof in a test to determine whether an animal has specific antibodies against the Sa Ia bacteria or its progeny according to the invention.

[0119] Alternatively, antibodies against the Sa Ia bacteria or progeny or composition according to the invention can be generated, and these antibodies can be used, for example, in detection to determine whether the animal contains the Sa Ia bacteria or progeny (antigen) according to the invention.

[0120] As is well known, "specific" antibodies are immunoglobulins capable of binding to Sa Ia bacteria or their progeny according to the invention, or to a portion of or preparation of a composition according to the invention, and the strength of such binding (i.e., affinity and / or cohesion) is greater than that against another antigen. Such specific binding can be readily distinguished from any nonspecific binding or background binding, for example, in in vitro binding assays, by progressively diluting the antibody or antigen; nonspecific binding typically disappears rapidly, for example at 1:10 or 1:100 dilutions, while specific binding persists even at higher dilutions.

[0121] In another aspect of the invention, there is a diagnostic test kit, characterized in that the kit comprises a container containing Sa Ia bacteria, progeny, or a combination thereof according to the invention.

[0122] In another aspect of the invention, there is a diagnostic test kit, characterized in that the kit comprises a container containing antibodies that specifically bind to Sa Ia bacteria or their progeny according to the invention.

[0123] As described, the bacteria of the present invention are particularly advantageous in the method and use of vaccinating fish against streptococcal disease.

[0124] Therefore, in another aspect of the invention, there is a use of an antibody that specifically binds to the Sa Ia bacteria or its progeny according to the invention as a vaccine against streptococcal disease in fish.

[0125] In another aspect of the invention, there is a connection to the Sa Ia bacteria, progeny, or composition according to the invention, which are used as a vaccine against streptococcal disease in fish.

[0126] In another aspect, the present invention relates to a vaccine against streptococcal disease in fish, characterized in that the vaccine comprises Sa Ia bacteria, progeny or a combination thereof according to the present invention, and a pharmaceutically acceptable carrier.

[0127] As is well known, a vaccine is an immunogenic composition containing an immunogenic compound in a pharmaceutically acceptable carrier. Vaccines can induce a protective immune response against a pathogen in the target immune system by specifically and / or specifically activating a humoral and / or cellular immune response to the target.

[0128] The vaccine according to the invention, and its use as a vaccine, is used to induce protection against Sa bacteria infection, particularly Sa Ia bacteria infection, and / or diseases caused by said infection in inoculated fish. This protection is achieved by preventing or reducing the colonization or proliferation of such Sa bacteria in target organs. This is accomplished, for example, by reducing the bacterial load or interfering with bacterial amplification in the inoculated target. This, in turn, leads to a reduction in the number, intensity, and / or severity of clinical signs of streptococcal disease in the target fish.

[0129] For Sa Ia infection, the main signs of infection and disease in fish include unilateral or bilateral exophthalmos (also known as "proptosis"), abdominal distension, petechiae on the gill covers and fins, and abnormal swimming posture (spiral swimming). Necropsy reveals meningoencephalitis, and the affected organs show hemorrhage, inflammation, and ascites.

[0130] The application of the vaccine according to the invention and its use as a vaccine in the vaccination of fish against infections and / or diseases caused by Sa Ia bacteria is entirely within the skill range of a conventional practitioner.

[0131] Similarly, the efficacy of the vaccine of the present invention is significantly demonstrated by the immune response after vaccination, for example, by means of the reduction of clinical symptoms or mortality after challenge infection in an experimental setting, scoring disease signs, clinical scores, serological parameters of the vaccinated fish, or by re-isolation of the challenge pathogen; and by comparing these results with the response of unvaccinated animals to the same challenge.

[0132] With respect to the present invention, the protective effect against streptococcal disease provided by the vaccine according to the invention and its use as a vaccine improves the health and economic performance of the target fish after vaccination. This can be assessed, for example, by parameters such as improved welfare levels, survival rates and growth rates, reduced feed conversion ratios, reduced veterinary care costs, and improved economic efficiency.

[0133] For the purposes of this invention, the "protective effect" was determined in a tilapia vaccination-challenge experiment and expressed as a "relative survival percentage" (RPS) value, calculated by dividing the mortality percentage of the vaccinated group by the mortality percentage of the control group. In this system, a higher RPS value indicates a better protective effect. Details of the challenge model to be used and the RPS calculation are also described in WO 2011 / 048041.

[0134] The vaccine according to the invention can induce protection against Sa Ia infection and disease, with a protection of at least 50% RPS; preferably at least 60%, 70%, 80% or 90% RPS; more preferably at least 95% RPS.

[0135] The following describes the vaccine according to the invention, its use as a vaccine, and various implementation schemes, preferences, and examples of vaccination.

[0136] "Pharmaceutically acceptable carriers" are designed to aid in the stabilization and administration of vaccines while being harmless to and well-tolerated by the target. Such carriers can be, for example, sterile water or sterile saline solutions. In more complex forms, for example, the carrier can be a buffer solution, which may contain other additives such as stabilizers or preservatives.

[0137] In one embodiment, the vaccine according to the invention and its use as a vaccine comprises an inactivated form of the Sa Ia bacteria or its progeny according to the invention.

[0138] In one embodiment, the vaccine according to the invention and its use as a vaccine includes an adjuvant.

[0139] In a preferred embodiment, the vaccine according to the invention and its use as a vaccine comprises an inactivated form of the Sa Ia bacteria or its progeny according to the invention, and contains an adjuvant.

[0140] "Adjuvants" are a well-known component of vaccines that stimulate a non-specific immune response to a target. Many different adjuvants are known in the art. Examples of adjuvants include: complete or incomplete Freund's adjuvants, vitamin E or α-tocopherol, nonionic block polymers and polyamines such as dextran sulfate, Carbopol™, pyrans, and saponins such as Quil A™ or Q-vac™. Saponins and vaccine components can be combined in ISCOM™. Additionally, there are aluminum salts, such as aluminum phosphate or aluminum hydroxide, which are available, for example, as Alhydrogel™ (Brenntag Biosector), Rehydragel™ (Reheis), and Rehsorptar™ (Armour Pharmaceutical).

[0141] A common adjuvant is an oil, such as a mineral oil like light (white) mineral (paraffin) oil; or a non-mineral oil such as squalene; squalane; vegetable oils or their derivatives, such as ethyl oleate. Combination products such as ISA™ (Seppic) or DiluvacForte™ and Xsolve™ (both from MSD Animal Health) can also be used advantageously.

[0142] The manual on adjuvants and their uses and effects is: "Vaccine adjuvants" (Methods inmolecular medicine, vol. 42, D. O'Hagan ed., 2000, Humana press, NJ, ISBN:0896037355).

[0143] The adjuvant may be included in or used as a vaccine according to the invention in several ways. When the adjuvant contains an oil, the vaccine may be provided in an aqueous form and may be formulated with the oil in different ways as an emulsion: water-in-oil (W / O), oil-in-water (O / W), or a biemulsion (W / O / W or O / W / O).

[0144] An "emulsion" is a mixture of at least two immiscible liquids, one dispersed in the other. Typically, the dispersed phase consists of very small droplets, in the micrometer range or smaller. Procedures and equipment for preparing emulsions of any scale are well known in the art. To stabilize the emulsion, one or more emulsifiers may be used.

[0145] An emulsifier is an amphiphilic molecule, possessing both hydrophobic and hydrophilic sides. Many emulsifiers with different properties are known in the art. Most are readily available commercially and are available in several purity grades. Commonly used emulsifiers for vaccines include sorbitan monooleate (Span® 80) and polyoxyethylene-sorbitan monooleate (polysorbate 80 or Tween® 80). In addition, emulsion stabilizers may be added; examples include benzyl alcohol and triethanolamine.

[0146] In a preferred embodiment of the vaccine comprising an adjuvant according to the present invention and its use as a vaccine, the adjuvant comprises an oil. In one embodiment, the oil is a non-mineral oil, for example, a commercially available product: Montanide™ ISA 763A or 763B.

[0147] More preferably, the oil is a mineral oil. Even more preferably, the mineral oil comprises a light (or white) liquid paraffin oil. Examples of light liquid paraffin oils used in vaccine adjuvants include Drakeol® 6VR (Penreco), Marcol® 52 (Exxon Mobile), and Klearol® (Sonneborn).

[0148] In a preferred embodiment of the vaccine according to the invention, which also includes an adjuvant and wherein said adjuvant comprises an oil, and for use as a vaccine, the vaccine is formulated as a water-in-oil emulsion.

[0149] The "fish" in this invention refers to an aquatic organism with fins and gills, which may be a cartilaginous fish or a bony fish, and may come from freshwater, brackish water or saltwater habitats.

[0150] Preferably, the target fish for the vaccine and its use in this invention is tilapia.

[0151] Tilapia belong to the Cichlidaceae family. They are no longer classified as belonging to their own genera, but are now divided into several genera according to taxonomy. The most relevant of these are: *Tilapia spp.*, *Tilapia spp.*, and *Tilapia spp.*

[0152] Therefore, the embodiment of the Sa Ia bacteria, progeny, or composition used in accordance with the invention and the vaccine according to the invention is characterized in that the fish is selected from the genera: *Carassius*, *Carassius*, and *Carassius*.

[0153] In a preferred embodiment, the fish from the genera *Coptodon*, *Oreochromis*, or *Oreochromis* are characterized in that they are selected from the following species: *Coptodon zillii*, *Coptodonguineensis*, *Oreochromis niloticus*, *Oreochromisaureus*, *Oreochromis mossambicus*, *Oreochromishornorum*, and *Sarotherodon melanotheron*; or hybrids of one or more of these species.

[0154] Notable examples of tilapia hybrids include the following hybrids: Oreochromis tilapia × Mozambique tilapia; Oreochromis tilapia × Nile tilapia; and Nile tilapia × (tail scale) thrush tilapia.

[0155] For the purposes of this invention, these fish names are interpreted in the same way as those for bacteria, i.e., they are taxonomic classifications that may change over time as new insights may lead to their reclassification into new or different taxa. However, since this does not change the fish itself, but only its scientific name or classification, such reclassified fish remain within the scope of this invention. This also includes any subtypes, variants, hybrids, or hybrids of these fish of this invention.

[0156] For fish vaccination, it is advantageous to use combined vaccines containing multiple antigens from various pathogens. This not only saves time and effort, improving operational economics, but also reduces stress on vaccinated fish, as they do not require multiple treatments and vaccinations.

[0157] Therefore, in one embodiment of the vaccine according to the invention and its use as a vaccine, the vaccine contains at least one other immunogen from a fish pathogen.

[0158] The other antigens may be derived from fish pathogens in any suitable manner, for example, as attenuated “live” antigens, inactivated antigens, or subunit antigens from microorganisms pathogenic to fish. Examples of tilapia pathogens include: bacteria: Streptococcus agalactiae serotypes Ia (known strains), Ib, and III, Streptococcus dolphinii; and / or bacteria from the following genera: Francisella, Edwardsiella, Aeromonas, Vibrio, Flavobacterium, Lactococcus, Balloonella, Pseudomonas, Mycobacterium, and Chlamydia; and / or viruses: Tilapia lake virus, tilapia juvenile encephalitis virus, iridovirus, iridovirus-like virus, Bohle virus, infectious pancreatic necrosis virus, parvovirus, herpesvirus, aquatic animal double RNA virus, Noda virus type B, frog virus, cytomegalovirus, and lymphocystis virus.

[0159] In a preferred embodiment of the vaccine according to the invention and its use as a vaccine, the other immunogen is derived from one or more of the following: Streptococcus agalactiae Ia, Streptococcus agalactiae Ib, Streptococcus agalactiae III, and Streptococcus dolphinus.

[0160] The vaccine according to the invention can be prepared from the Sa Ia bacteria, progeny, and / or compositions according to the invention by means of the method described herein, which can be readily applied by those skilled in the art. For example, the bacteria are cultured at 32°C with stirring for at least 6 hours in a medium containing yeast extract and 0.5% w / v NaCl, and then harvested by centrifugation. Next, the bacteria are inactivated by incubation with formalin and formulated into a water-in-oil (W / O) emulsion with mineral oil (i.e., light liquid paraffin oil).

[0161] General techniques and precautions applicable to the manufacture of vaccines according to well-known pharmaceutical manufacturing standards are described in, for example, government directives and regulations (Pharmacopoeia, 9 CFR) and in well-known handbooks such as: "Remington: the science and practice of pharmacy" (2000, Lippincott, USA, ISBN: 683306472) and "Veterinary vaccinology" (P. Pastoret et al., ed., 1997, Elsevier, Amsterdam, ISBN 0444819681). Vaccines are typically prepared aseptically using pharmaceutical-grade excipients.

[0162] Vaccine manufacturing typically involves microbiological testing for sterility and freedom from exogenous agents; and may include in vivo or in vitro studies to confirm inactivation, vaccine efficacy, and safety. Once testing for quality, quantity, sterility, safety, and efficacy is completed, the vaccine can be marketed. All of these are well known to those skilled in the art.

[0163] Therefore, in another aspect of the invention, there is a method for preparing a vaccine according to the invention, the method comprising the step of mixing the Sa Ia bacteria, progeny or composition according to the invention with a pharmaceutically acceptable carrier.

[0164] In one embodiment, the method for preparation according to the invention is characterized in that the method includes the step of inactivating the Sa Ia bacteria or its progeny according to the invention.

[0165] In another aspect of the invention, there is a connection to the use of the Sa Ia bacteria, progeny, or composition according to the invention for the manufacture of a vaccine according to the invention.

[0166] The vaccine according to the invention can be administered to fish via various routes. In one embodiment, the vaccine according to the invention is administered via a parenteral route, i.e., through the skin, for example, intramuscularly or intraperitoneally. Alternative routes include via mucosal routes, immersion routes, or oral routes.

[0167] The preferred route of administration is intraperitoneal.

[0168] The volume of each dose of the vaccine according to the invention can be selected based on the characteristics of the specific vaccine used, the characteristics of the target, and the intended route of administration. The commonly used dose for parenteral injection in fish is 0.01-1 ml / target. Preferably, the dose is 0.01 to 0.1 ml / target dose. More preferably, the dose is selected from 10, 25, and 50 μL / target dose.

[0169] The vaccine according to the invention can be used as both a preventative and therapeutic treatment because it interferes with the establishment and progression of streptococcal infection in fish.

[0170] The vaccine according to the invention can be used as an effective primary immunization, which can be followed up and amplified by booster vaccination with the same or different vaccines.

[0171] The method, timing, dosage, and volume of the vaccine according to the present invention can be adjusted and optimized according to the specific species of fish to be vaccinated; at the same time, the time and life stage at which the fish may be exposed to streptococcal infection should also be taken into account.

[0172] For tilapia, the administration of the vaccine according to the invention is preferably carried out as early as possible to prevent potential infection from the aquaculture environment; therefore, vaccination is preferably carried out in the environment of a hatchery or nursery and before transfer to facilities for adult rearing. For tilapia, vaccination by injection can practically be performed on fish weighing approximately 5 grams. Feeding is typically suspended the day before vaccination, and the fish are anesthetized shortly before vaccination. These are well known in the art.

[0173] The administration regimen for administering the vaccine according to the invention to target fish can be a single-dose or multi-dose regimen, which should be compatible with the vaccine formulation and take into account the aquaculture characteristics of the target fish, and the dosage should be an immunologically effective amount.

[0174] Preferably, the vaccine according to the invention is administered only once, i.e., it is a single-dose vaccine.

[0175] Ideally, the regimen for administering the vaccine according to the invention is integrated into existing vaccination schedules for other vaccines that the target fish may require, also to reduce stress and cost. These other vaccines may be administered simultaneously, in parallel, or sequentially, or through so-called “combined use”; preferably, these combinations are applied in a manner compatible with the approved use of these vaccines.

[0176] Those skilled in the art are fully capable of optimizing vaccines according to the invention by adjusting their use or composition. For example, this may involve fine-tuning the efficacy or safety of the vaccine. This can be achieved by adjusting the vaccine dosage, quantity, frequency, route of administration, by using vaccines in other forms or formulations, or by adjusting the excipients of the vaccine (e.g., stabilizers, preservatives, or adjuvants).

[0177] The amount of antigen in each animal dose of the vaccine according to the invention can be readily determined by testing which amounts are immunologically effective relative to different levels of challenge infection.

[0178] In one embodiment, the amount of inactivated Sa Ia bacteria according to the invention per ml of vaccine according to the invention corresponds to 1 x 10^6 - 1 x 10^10 bacteria / ml as present before inactivation. Preferably, the amount of Sa Ia bacteria according to the invention corresponds, in order of preference, to 1 x 10^7 - 5 x 10^9, 5 x 10^7 - 5 x 10^9, or even 1 x 10^8 - 1 x 10^9 bacteria per ml of vaccine.

[0179] Sa bacteria can be counted on a standard blood agar plate using the standard plate count method to determine the amount in colony forming units (cfu).

[0180] At a dose volume of 50 µl / strip, the preferred dose of the vaccine according to the invention contains 1 x 10^7 - 5 x 10^7 cells / dose of Sa Ia bacteria according to the invention.

[0181] As described above and illustrated below, the vaccine according to the invention can be advantageously used to prevent or reduce fish infections of known types and novel non-lactate streptococci Ia as disclosed herein, as well as diseases caused by said infections.

[0182] Therefore, another aspect of the present invention relates to a method for protecting fish from streptococcal disease, the method comprising the step of administering to the fish a vaccine according to the invention, or a vaccine obtainable by the method according to the invention, or a vaccine obtainable by the use according to the invention.

[0183] Similarly, in other aspects, the present invention relates to: - The use of the vaccine according to the invention for the prevention or reduction of Sa bacterial infection, preferably Sa Ia bacterial infection and related disease symptoms.

[0184] - A method for preventing or reducing Sa bacterial infection, preferably Sa Ia bacterial infection and related disease symptoms, the method comprising administering a vaccine according to the invention to fish.

[0185] - A method for vaccinating fish to prevent or reduce Sa bacteria infection, preferably Sa Ia bacteria infection and related disease symptoms, the method comprising the step of vaccinating said fish with a vaccine according to the invention.

[0186] Another beneficial effect of the vaccine according to the invention in reducing bacterial load is the prevention or reduction of shedding, and thus the prevention or reduction of the spread of Sa bacteria in the aquaculture environment, including both vertical transmission to offspring and horizontal transmission within the population and within specific geographical areas. Therefore, the use of the vaccine according to the invention results in a reduction in the prevalence of Sa, preferably Sa Ia.

[0187] Therefore, further aspects of the present invention include: - The use of the fish vaccine according to the invention for reducing the prevalence of Sa bacteria, preferably Sa Ia bacteria, in a population or in a specific geographical area.

[0188] - The fish vaccine according to the invention is used to reduce the prevalence of Sa bacteria, preferably SaIa bacteria, in a population or in a specific geographical area.

[0189] The present invention has been described herein in various aspects and embodiments. It should be understood that any combination of these aspects and embodiments is considered to be within the scope of the invention. However, for the sake of brevity only, not every possible combination has been exhaustively listed herein.

[0190] The invention will now be further described through the following non-limiting embodiments.

[0191] Example Example 1: Sample Separation and Purification In the summer of 2021, an outbreak of acute disease and mass mortality occurred at tilapia farms in Mexico, affecting fish of all sizes despite regular vaccination against streptococcal disease. Clinical signs observed in these fish included: pale coloration (corresponding to gastrointestinal bleeding); intussusception; lethargy; and acute death.

[0192] Samples were collected from various tissues of the diseased fish, such as the brain, gills, heart, prokidney, liver, spleen, intestines and stomach, and used for various preliminary tests to identify the main cause of the disease.

[0193] Preliminary purification can be achieved through methods such as those described in N. Buller's manual (2014, Bacteria and Fungi from Fish and Other Aquatic Animals; A Practical Identification Manual, 2). nd The procedure was performed as described in ed., ISBN: 978-1-84593-805-5. Tissue samples from individual fish were inoculated onto blood agar plates for primary isolation, and colonies were identified by Gram staining. Secondary isolation was also performed on blood agar plates. After incubation for 2 days, selected colonies were streaked onto microbial agar columns for transport, and these columns were sent to our laboratory.

[0194] Upon receipt, all bacterial samples were immediately streaked onto trypsin-soy agar (TSA) plates. Single colonies were picked from 10 original isolates, resuspended in TSB broth with added glycerol, and frozen for preservation.

[0195] Isolates from Mexico in 2021 were designated as TI 2889 - TI 2898.

[0196] Example 2: Overall Characterization of Epidemic Samples Serum grouping: Serological typing of isolated bacterial samples was performed using a commercial kit (ImmuLex™ Group B Streptococcus Ia; SSI Diagnostica) via latex agglutination with a specific polyclonal antibody against capsular polysaccharide serotype Ia, following the manufacturer's instructions. All 10 isolates from Mexico in 2021 were identified as Sa-Ia.

[0197] It is worth noting that the aquaculture farm where this outbreak occurred has been routinely using commercial vaccines containing Sa Ia antigen for vaccination.

[0198] Hemolytic: No α-hemolysis or β-hemolysis was observed in TI 2893 after incubation at 26°C for 3 days on blood agar plates; therefore, it is γ-hemolysis.

[0199] Sa Ia typing by PCR Two strains (samples TI 2893 and TI 2898) isolated from Mexico in 2021 were subjected to specific PCR detection for Sa Ia strains. The PCR primers used were obtained from GenBank accession number: AB028896, based on the study by Kong et al. (2005, J. Med. Micro., vol. 54, p. 1133-1138). Isolates TI1422 and TI 1580, both known Sa Ia strains, were also detected, isolated from diseased tilapia in Thailand in 2005.

[0200] In this assay, the results of the two epidemic isolates were completely consistent with those of TI 1422 and TI 1580, and were therefore identified as Sa of serotype Ia.

[0201] Genotypic analysis: MLST genotyping was performed as described by Jones et al. (2003, J. Clin. Microbiol., vol. 41, p. 2530-2536), using a modified protocol as described by Jolley et al. (2018, Wellcome Open Res., vol. 3:124). In short, the gene sequences of a selected number of housekeeping genes were determined using the PCR primer sequences described by Jones et al.: alcohol dehydrogenase (adhP), phenylalanine-tRNA synthetase (pheS), glutamine transporter (atr), glutamine synthetase (glnA), serine dehydratase (sdhA), glucokinase (glcK), and transketolase (tkt). The discovered sequences were then compared using the public "Streptococcus agalactiae typing database" PubMLST. For simplicity, you can paste the discovered sequence into a FASTA sequence file, then upload it to the website: https: / / pubmlst.org / bigsdb?db=pubmlst_sagalactiae_seqdef and compare it with known Sa isolate sequences.

[0202] The allele profile of the TI 2893 isolate was found to be a perfect match for the known allele profile of the ST7 genotype Sa Ia strain.

[0203] Biochemical analysis: Biochemical characterization of TI 2893 isolate from Mexico in 2021 was performed using the commercial API 20™ STREP kit (bioMerieux) according to the manufacturer's instructions. Simultaneously, two Sa Ia isolates from Thailand in 2005, TI 1422 and TI 1580, were re-analyzed.

[0204] ABI 20 Strep assay results showed that isolate TI 2893 responded identically to previous SaIa isolates on all parameters.

[0205] Therefore, in terms of their basic biochemical characteristics, these new epidemic strains are not fundamentally different from known Sa Ia isolates.

[0206] Protein characterization: Whole-cell protein and supernatant samples were prepared from small-scale cultures of the epidemic isolate TI 2893 and the known Sa Ia strain TI 1580 for comparison. In short: cultures were centrifuged at 12,100 xg for 30 minutes at 4–8°C.

[0207] Secreted proteins were collected and concentrated from the culture medium using ammonium sulfate. The final protein precipitate was resuspended in HEPES buffer and desalted using a commercially available desalting column.

[0208] The cell pellet was resuspended in PBS and OD was adjusted. Cells were lysed in sample buffer, followed by heat treatment and then acoustic treatment. Centrifugation was used to remove insoluble proteins from the cell lysates.

[0209] Protein concentrations were measured using the BCA method, with secreted proteins adjusted to 30 µg / µl and cell lysates adjusted to 10 µg / µl.

[0210] Both samples were separated by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and parallel gels were subjected to Western blotting. Coomassie Brilliant Blue was used to stain the gels. For this blotting, the primary antibody used was serum from tilapia vaccinated with a commercial Sa Ia vaccine containing strain TI 1422. Next, a secondary antibody (mouse anti-tilapia antibody) was added to the blot, followed by a trivalent antibody (anti-mouse HRP conjugated antibody).

[0211] The result is Figure 1 The results were presented in the images. Notably, it was observed that the protein banding patterns of TI2893 were completely identical to those of isolate TI 1580 for both SDS-PAGE and Western blot analysis.

[0212] in conclusion: Therefore, the differences between the strain of the 2021 Mexican outbreak and known Sa Ia strains cannot be directly reflected by standard genetic, biochemical, and serological characterization.

[0213] Example 3: Characterization of serological differences To investigate the potential serological reasons behind the 2021 Mexican vaccination breakthrough, we used antiserum from tilapia vaccinated with the Sa Ia isolate TI 1422 monovalent vaccine as the primary antibody for this inhibition and performed competitive ELISA detection on the isolates.

[0214] In short: ELISA plates were coated with TI 1422 bacterial vaccine antigen, followed by casein blocking of the wells. Then, a mixture of bacterial isolate test antigen or PBS control with primary antibody was added to the wells and incubated. Next, enzyme-conjugated secondary antibody (anti-tilapia antibody) was added, followed by the chromogenic substrate. The optical density reading of the control wells was taken as 100% binding, and the percentage of inhibition was calculated. Using this experimental setup, isolates serologically related to the Sa Ia vaccine strain are expected to bind to the primary antibody, thus exhibiting the inhibition shown by the control group's binding level.

[0215] The results are presented in Figure 2 The vertical axis represents the relative percentage of inhibition. The horizontal axis represents the 2021 Mexican COVID-19 samples, numbered TI 2889 - TI 2898. Negative controls used were *Streptococcus dolphinus* (Si) and *Tenacibaculum maritimum* (Tmar). Positive controls were bacteria from the Sa Ia “vaccine strain” TI 1422 and Sa Ia strain TI 1580.

[0216] The results showed that the positive and negative inhibition responses of various control samples were as expected. For various 2021 Mexican isolates, the inhibition response was significantly reduced to only 2% to 15%; far lower than the 44-49% inhibition of the "classic" Sa Ia isolates TI 1422 and TI 1580.

[0217] This clearly demonstrates that antibodies produced against known Sa Ia bacteria (such as those induced by commercial Sa vaccines) are indeed significantly less capable of recognizing isolates from this outbreak, which explains why these vaccines offered virtually no protection against the 2021 outbreak in Mexico.

[0218] Furthermore, in a similar inhibition ELISA, Sa Ia bacteria isolated from diseased tilapia in the same region of Mexico in 2018 were tested. These isolates scored more closely than the 2005 Thai isolates, showing an inhibition level of 35–40%. Therefore, a novel Sa Ia bacterium did indeed emerge in Mexico by 2021.

[0219] Example 4: Preparation of TI 2893 strain A strain of isolate TI 2893 was prepared for use in various animal experiments and for preservation in the CNCM.

[0220] The TI 2893 strain was revived from a frozen glycerol stock solution, inoculated onto TSA plates, and subsequently incubated at 26–32°C for 3 days. Well-isolated colonies were selected from the TSA plates, transferred to fresh TSA plates, and incubated overnight. Colonies on the plates were swabbed and collected in sterile physiological saline. This preparation was inoculated into yeast extract / NaCl medium and incubated at 26–32°C. After overnight incubation, 30% glycerol was added, and the mixture was filled into cryovials and stored below -60°C. Twelve of these cryovials were sent to the CNCM.

[0221] Example 5: Characterization by PCR Unique primers were designed to identify the novel Sa Ia bacteria according to the present invention.

[0222] Primers were ordered from an outsourced company and delivered in purified and lyophilized form. The primers were reconstituted in UltraPure™ water to prepare a 100 µM stock solution and then frozen.

[0223] To prepare DNA samples, TI 2893 bacteria were inoculated onto agar plates and incubated overnight. The plates were then wiped with 500 µl of 40 mM PBS. 100 µl of this suspension was taken and genomic DNA was extracted using the QIAamp® DNA Mini Kit (Qiagen) according to the manufacturer's instructions. Finally, the TI 2893 genomic DNA was resuspended in AE buffer and stored.

[0224] PCR assays were performed using a Proflex™ PCR instrument (Thermo Fisher) with the default ramp rate.

[0225] Prepare a PCR reaction premix containing, for each reaction: 2.5 µl of each of the two 10 µM primers used for assay; 19 µl of ultrapure water; and 1 µl of DNA; up to a total volume of 25 µl per reaction. Add the premix to a pre-filled single-dose cup equipped with lyophilized PCR beads (illustra PuReTaq™ Ready-To-Go PCR Bead; Cytiva) and use the cups according to the manufacturer's instructions.

[0226] Sequence comparisons were performed using isolates of Sa Ia and Sa III, with the negative control containing no DNA.

[0227] The cycling conditions used were basically the same, except that for the primer sets of SEQ ID NO: 3 and 4, some "touchdown" cycles were included.

[0228] Therefore, for the primer sets SEQ ID NO: 1 and 2 (pepN gene region), 5 and 6 (DUF3307 gene region), and 7 and 8 (yjdM gene region), the cycling conditions are as follows: - 95℃ for 5 minutes - Less than 35 cycles: • 95℃ for 1 minute • 58℃ for 1 minute • 72℃ for 2 minutes - 72℃ for 10 minutes - Keep at 4°C.

[0229] For the primer sets of SEQ ID NO: 9 and 10 (abiH gene region), the cycling conditions are as follows: - 95℃ for 5 minutes - Less than 30 cycles: • 95℃ for 1 minute • 63℃ for 1 minute • 72℃ for 2 minutes - 72℃ for 10 minutes - Keep at 4°C.

[0230] For the primer sets of SEQ ID NO: 3 and 4 (DUF1310 gene region), the cycling conditions are: - 94℃ for 5 minutes - Less than 12 cycles: • 94℃ for 1 minute • The first cycle is 66℃ for 1 minute, but for each of the next 11 cycles, the temperature is reduced by 0.5℃. • 72℃ for 2 minutes - Less than 20 cycles: • 94℃ for 1 minute • 60℃ for 1 minute • 72℃ for 2 minutes - 72℃ for 10 minutes - Keep at 4°C.

[0231] Similarly, for the primer sets SEQ ID NO: 11 and 12 (fbsA gene region), the cycling conditions are: - 95℃ for 5 minutes - Less than 30 cycles: • 95℃ for 1 minute • 63℃ for 1 minute • 72℃ for 1 minute - 72℃ for 10 minutes - Keep at 4°C.

[0232] PCR products can be frozen until loading. Then, take 10 µl from each PCR tube and load it onto a pre-prepared 1% agarose gel. Perform electrophoresis on an E-Gel™ system (Thermo Fisher) using standard settings for 13–18 minutes, depending on the gel size. Use the 1 Kb Plus™ DNA ladder (ThermoFisher) as a marker, following the manufacturer's instructions.

[0233] As described above (see Table 1), the nucleotide fragments produced by these PCRs of the 2021 Mexican isolates (TI 2889-2898) are significantly different in size from those obtained by PCRs of nucleic acids from previously known Sa Ia isolates using the same primers and conditions.

[0234] Furthermore, when these PCR protocols were used to detect isolates from the 2022 Honduras outbreak (isolates numbered TI 2925-2934), the PCR fragments produced by these isolates were the same size as those from the 2021 Mexican isolates, and therefore also met the conditions for Sa Ia bacteria according to the present invention.

[0235] This also suggests that the 2022 Honduras outbreak was caused by the same SaIa bacteria that caused the 2021 Mexico outbreak, which may have somehow contaminated water bodies in other countries.

[0236] Example 6: Characterization of Pathological Differences To characterize the pathogenicity of the novel Mexican isolate, comparative challenge infection was performed in tilapia.

[0237] Overnight small-scale cultures of isolates TI 2893 and TI 1580 were prepared and quantified by measuring the optical density (OD) at 660 nm using a spectrophotometer. Tilapia were intraperitoneally inoculated using these cultures, and the number of dead fish was counted after incubation.

[0238] Death was observed as early as day 1 post-challenge, peaking on day 2. The pathological features observed were identical for both isolates tested; therefore, under laboratory conditions, the overall pathology of TI 2893 showed clinical features similar to those of known Sa Ia isolates (but far more acute, as shown below).

[0239] The pathological differences lie in the number of bacteria required to cause severe illness. To test this, the number of colony-forming units (CFU) inoculated with TI2893 was set much lower than that of TI 1580. Nevertheless, the results were still impressive: • For TI 1580, 100,000 cfu / fish was intraperitoneally inoculated, and 16 out of 25 inoculated fish were observed to die. The corresponding mortality rate was 64%.

[0240] • For TI 2893, only 25 cfu / fish were inoculated, but 20 out of 25 inoculated fish died; the mortality rate was 80%.

[0241] Therefore, the pathogenicity of TI 2893 appears to be at least 4000 times higher than that of known pathogenic Sa Ia isolates.

[0242] Example 7: Vaccination-challenge test and cross-protection efficacy of single and combination vaccines 7.1 Introduction In a carefully designed vaccination-challenge experiment conducted on juvenile tilapia, the protective efficacy of existing vaccines against infection by challenge with known (isolated strain TI 1580) or novel (isolated strain TI 2893) Sa Ia bacteria was tested. Furthermore, the vaccine was modified by adding a vaccine derived from the novel bacteria, and the (cross-protective) characteristics of the novel bacteria against both challenge types in the vaccine were tested separately.

[0243] All vaccines used were water-in-oil (W / O) emulsions containing formalin-inactivated streptococcal bacteria.

[0244] 7.1.1 Experimental Design This experiment used a total of 330 tilapia. The fish were randomly divided into 6 equal groups. Five groups of fish were intraperitoneally (ip) injected with one of the different test vaccines: - 'Sa1': AQUAVAQ Strep Sa1 - 'Strep-4': AQUAVAC Strep-4 - Sa1 + TI 2893 vaccine - Strep-4 + TI 2893 vaccine, and - TI 2893 vaccine.

[0245] Group 6 fish were injected with standard vaccine dilution buffer (SVDB) as an unvaccinated control group. SVDB is a phosphate-buffered saline solution.

[0246] Three weeks after vaccination, each group of fish was challenged with either isolate TI 2893 (Sa Ia, Mexico, 2021) or isolate TI 1580 (Sa Ia, Thailand, 2005). Post-challenge observation (mortality and clinical signs) was conducted for 14 days post-challenge, during which dead and dying fish were collected daily, and visceral samples were inoculated onto non-selective culture media to determine the presence of the challenge bacteria.

[0247] Note: Because Streptococcus agalactiae may cause zoonotic diseases in humans and other mammals, appropriate health and biosafety measures must be taken throughout the experiment.

[0248] 7.2 Materials and Methods 7.2.1 Vaccines As described in this article, AQUAVAC® Strep Sa1 (MSD Animal Health) (Sa1 in this article) is a commercial bivalent vaccine of Sa bacteria, formalin-inactivated from Sa Ia isolate TI 1422 and Sa III isolate TI 1428; formulated with Montanide™ ISA 763A VG.

[0249] AQUAVAC® Strep-4 (MSD Animal Health) (Strep-4 in this article) is a commercially available quadrivalent vaccine of formalin-inactivated Streptococcus: Sa Ia isolate TI 1422, Sa Ib isolate 513, Sa III isolate TI 1428 and Dolphin Streptococcus isolate SB430; formulated in light liquid paraffin oil.

[0250] "TI 2893 vaccine" is a W / O emulsion of a novel bacterium according to the present invention, inactivated by formalin and formulated in light liquid paraffin oil.

[0251] In short: As described above, the TI 2893 isolate was revived, inoculated, swabbed, and prepared into a suspension, which was then cultured in yeast extract / NaCl medium. Next, the culture was inactivated overnight with 0.5% v / v formalin at 26–32°C and then stored at 4°C until vaccine preparation was complete.

[0252] For emulsification, formalin-killed bacterial culture TI 2893 was mixed with paraffin oil at a ratio of 45% water:55% oil, using Span™ and Tween80™ as surfactants. The composition was then emulsified for a total of 3 minutes at 11,000 rpm using an IKA T25 Ultrathorax; droplet size in the aqueous phase was examined by microscopy. 25 ml of the vaccine was then filled into each of four 100 ml glass vials and stored at 4°C until use. The bacterial count in the TI 2893 vaccine was 5 x 10^8 cells / ml vaccine.

[0253] The dual vaccine was administered via intraperitoneal injection as a continuous dose: Sa1 + TI 2893 and Strep-4 + TI2893.

[0254] 7.2.2 Antiviral materials The TI 2893 strain was cryopreserved in 1.5 ml vials at -60°C at a concentration of 6.5 x 10^9 cfu / ml.

[0255] The TI 1580 strain was stored in 1 ml vials at -60°C at a concentration of 5.2 x 10^8 cfu / ml.

[0256] For TI 1580, inoculate 1 ml of the bacterial culture into 100 ml of yeast extract / NaCl medium and incubate overnight with shaking at 26-32°C.

[0257] For TI 2893, as described above, the stock solution was revived, inoculated, wiped off, prepared into a suspension, and cultured in yeast extract / NaCl medium.

[0258] Both final cultures were then diluted separately with 0.9% w / v NaCl for use in challenge. To control the challenge dose, each challenge suspension was serially diluted and inoculated in duplicate onto TSA plates to determine the viable count for each challenge dose. Viable counts were determined after inoculation of the challenge suspensions.

[0259] 7.2.3 Experimental Animals Tilapia (Oreochromis spec.) fry produced at a hatchery, sourced from Temasek Life Sciences Laboratory, Singapore. Their average weight upon arrival was 2 g / fish. The fish were acclimatized in isolated tanks, with an average weight of 10 g / fish at the start of the experiment.

[0260] Only healthy fish were used, a total of 330.

[0261] The fish were not individually tagged, but different groups were kept in separate, tagged tanks.

[0262] 7.2.4 Water quality conditions - The salinity is 0.2% after vaccination and becomes freshwater after challenge.

[0263] - The water temperature is 28℃ ± 2℃ after vaccination and 30℃ ± 2℃ after challenge.

[0264] - The aquarium size was 500 L after vaccination and 70 L after challenge.

[0265] Daily monitoring of water quality conditions includes: O2, NH4, NO2 and NO3 concentrations; salinity; water temperature; and pH.

[0266] 7.2.5 Feeding After vaccination, feed the fish daily at 2-4% of their body weight, and adjust the diet weekly for each group. After challenge with the virus, the fish are allowed free access to feed.

[0267] However, the fish should be fasted for at least 12 hours before performing operations such as transferring them to the aquarium and weighing them. Additionally, the fish should be fasted for at least 48 hours before vaccination and challenge therapy.

[0268] 7.2.5 Processing Vaccination When the average weight of the fish reaches 10 grams, they are removed from the isolation tank and randomly divided into 6 groups.

[0269] The fish were anesthetized with AQUI-S®. Then, the vaccine was administered via intraperitoneal injection at approximately halfway between the base and tip of the pelvic fin.

[0270] Vaccines “Sa1” and TI 2893 were administered intraperitoneally at a dose of 50 µl; Strep-4 vaccine was administered at a dose of 100 µl. Mock-vaccination using buffer was administered intraperitoneally at a dose of 150 µl.

[0271] Combined vaccination: Sa1 + TI 2893 and Strep-4 + TI 2893 were each administered in two consecutive doses: 50 and 50 µl, respectively; and 100 and 50 µl, respectively.

[0272] attacking poison Challenge was performed 3 weeks after vaccination; at this time, the average weight of the fish was 15.5 grams. Prior to challenge, the fish were fasted for at least 48 hours to ensure complete emptying of the gastrointestinal tract, thus preventing damage to internal organs due to injection. The fish were then anesthetized. The challenge was administered intraperitoneally at a dose of 100 µl, using either TI 1580 or TI 2893 bacteria as the inoculum.

[0273] The attack composition used is as follows: - TI 2893: 253 cfu / ml - TI 1580: 1x10^6 cfu / ml Immediately after inoculation, the fish were transferred to 12 separate temporary holding tanks for virus treatment.

[0274] monitor Monitor fish after vaccination and after challenge to observe any direct effects of sedation. Thereafter, observe fish at least once daily. Post-challenge monitoring continues for 14 days. Typical clinical signs include abnormal swimming behavior (spiral or circling motion) and protruding eyes. Dissect and examine internal organs of dead fish. For bacteriological sampling, collect brain and / or internal organ samples and inoculate onto TSA plates. Incubate the plates at 26–32°C for 1–3 days, then score them.

[0275] 7.2.6 Results Evaluation Cumulative mortality rate The mortality rate after the challenge was expressed as a cumulative mortality percentage and compared.

[0276] The calculation is as follows: Cumulative mortality rate % = (Total number of dead fish / Total number of fish in the group) x 100% Vaccine efficacy The efficacy of the vaccine in protecting fish from challenge was determined as the relative survival percentage (RPS). The RPS value for the vaccinated group was calculated using the following formula: RPS = {1 - (mortality rate in the vaccinated group % / mortality rate in the control group %)} × 100 This is also mentioned on pages 16-17 of WO 2011 / 048041.

[0277] 7.3 Results The experiment proceeded smoothly without any unexpected events that led to interruptions, unintended infections, or deaths. Furthermore, the vaccination and challenge treatments themselves did not cause any problems, and the challenge infection had the expected effects on the unvaccinated fish.

[0278] 7.3.1 Mortality rate Figure 3 and Figure 4 The cumulative mortality rates after challenge with TI 1580 or TI 2893 isolates are presented in the figures.

[0279] The vertical axis represents the observed cumulative mortality rate (%), which is a function of the number of days post-challenge, represented by the horizontal axis. Different experimental groups are represented by different symbols indicated in the legend.

[0280] It can be readily observed that isolate TI 1580 caused a lower mortality rate in unvaccinated fish than isolate TI 2893: 64% vs. 80%. This was still the case even when the challenge dose of TI 2893 was 4000 times lower than that of TI 1580. This confirms the results observed in the experiments disclosed above in Example 6.

[0281] All types of vaccinations administered, including the TI 2893 vaccine, significantly reduced mortality from TI 1580 challenge. The classic Sa vaccines, Sa1 and Strep-4, were less effective in reducing mortality from TI 2893 challenge. This confirms observations made during the 2021 outbreak in Mexico.

[0282] When the TI 2893 vaccine is used, death from challenge is almost completely avoided, whether the TI 2893 vaccine is used alone or in combination with one of the classic vaccines, which is even more effective.

[0283] Similar effects to mortality rates have been observed in vaccination outcomes.

[0284] 7.3.1 Vaccine efficacy Table 2: Results of vaccination against two given types of viral challenge

[0285] Table 2 presents the efficacy of various vaccines administered and tested against two types of viral infection. Vaccination efficacy is presented as % RPS.

[0286] The "classic" vaccines Sa1 and Strep-4 showed good protection against TI 1580 challenge, with RPS values ​​of 81% and 100%, respectively. However, these two vaccines provided much less protection against TI 2893 challenge, with RPS values ​​of 20% and 50%, respectively.

[0287] The TI 2893 vaccine, even as a monovalent vaccine, showed good protective efficacy against both homologous challenge (TI 2893) and heterologous challenge (TI1580), with RPS of 95% and 81%, respectively.

[0288] When used in combination with the classic Sa Ia vaccine, the TI 2893 vaccine can improve protection against TI 2893 to a very high level, increasing RPS from 20%-50% to 85%. Furthermore, adding the TI 2893 vaccine to the Sa1 and Strep-4 vaccines did not actually affect the protective effect against TI 1580 challenge.

[0289] Note: In this system where efficacy is assessed using % RPS, the mortality rate in the negative control group is set as the benchmark for calculating the RPS values ​​of other groups.

[0290] 7.4 Conclusion Vaccines targeting known Sa Ia isolates were found to be minimally effective against infection with the novel Sa Ia bacterium (isolated strain TI2893) according to the present invention. This corroborates the severe outbreak observed at a tilapia farm in Mexico during the summer of 2021.

[0291] However, it is advantageous that vaccines prepared from this highly pathogenic isolate have been found to effectively prevent not only death caused by the same TI 2893 isolate, but also death induced by challenge with the known Sa Ia isolate (TI 1580).

[0292] Alternatively, the TI 2893 vaccine could be added to existing Sa vaccines to supplement their protective efficacy against TI 2893.

[0293] Therefore, the present invention enables the preparation and use of fish vaccines that provide good protection against both known Sa Ia bacteria and the novel outbreak Sa Ia bacteria according to the present invention.

[0294] Example 8: Genome Sequence Analysis Sample preparation: As described above, isolate TI 2893 (corresponding to the preserved sample) was cultured in a 50 ml volume. The cell pellet was harvested, frozen at -20°C, and sent to a CRO (BaseClear, Leiden, Netherlands) for DNA sequencing. Cells were mechanically lysed using ZR BashingBead™ lysis tubes and vortexed for 5 minutes. Genomic DNA was then extracted using the ZymoBIOMICS™ DNA miniprep kit according to the manufacturer's instructions. The extracted genomic DNA was quantified and normalized based on measurements taken using the Qubit™ Broad Range kit.

[0295] DNA sequencing Long-read sequencing was performed using Oxford Nanopore™ technology as follows: libraries were constructed using 1D ligation, and sequence reads were generated using the Oxford Nanopore GridION™ system equipped with an R9.4.1 flowcell. Base identification was performed using Guppy™ v. 5.0.13. ONT sequencing statistics are presented in Table 2.

[0296] Table 2: ONT sequencing statistics

[0297] For short-read sequencing, libraries were constructed using the Illumina Nextera™ XT DNA Library Construction Kit. Sequencing of the libraries was then performed on the Novaseq™ 6000 system using the Illumina PE150 kit. Paired-end sequencing results were used to generate FASTQ read sequence files using bcl2fastq v. 2.20 (Illumina). Initial quality assessment was based on data filtered using Illumina Chastity™. Subsequently, reads containing Phix control signals were removed using a filtering scheme (developed by the CRO). Additionally, reads containing (partial) adapters were trimmed to a minimum read length of 50 bp. A second quality assessment was performed based on the remaining reads using the FASTQC quality control tool v. 0.11.8. Illumina sequencing statistics are provided in Table 3.

[0298] In addition to the genome, a 4441 bp plasmid was assembled on a full set of Illumina reads using the plasmidSPAdes script of SPAdes v. 3.15.3 (Antipov et al., 2016, Bioinformatics, vol. 32, p. 3380-3387).

[0299] Table 3: Illumina sequencing statistics

[0300] Genome assembly and annotation Using the obtained ONT reads, a genome assembly draft was generated using Flye 2.9 (M. Kolmogorov et al., 2019, Nature Biotechnology, vol. 540, p. 540-546), followed by a correction step using Medaka v.1.4.3. A final version of the genome assembly was produced using subsequent correction steps with Illumina paired-end reads (Pilon, v. 1.23). Assembly statistics are provided in Table 4.

[0301] Table 4: Genome assembly statistics

[0302] Subsequently, prokaryotic genome annotation was performed on the assembled contigs using a Prokka-based annotation workflow (T. Seemann, 2014, Bioinformatics, vol.30, p. 2068-2069). This workflow includes the following procedures: - Prokaryotic gene prediction, via Prodigal (Hyatt et al., 2010, Bioinformatics, vol. 11, p. 119) v. 2.6.3, - rRNA detection was performed using BARRNAP (Seemann, 2014, same source) v. 0.8. - tRNA prediction, via Aragorn (Laslett et al., 2004, Nucleic Acids Res., vol.32, p. 11-16) v. 1.2.38, and - pCDS physicochemical properties (BaseClear, Leiden, Netherlands).

[0303] And use the following public databases: - Uniprot-Swissprot, v. 2019_08; and - Rfam (Kalvari et al., 2017, Nucleic Acids Res., vol. 49, D1), v. 14.1.

[0304] Proteins are inferred from homology using: - EC number, from UniProt BLAST best hit (Apweiler et al., 2004, Nucleic AcidsRes., vol. 32, D115-9; Pundir et al., 2016, doi.org / 10.1002 / 0471250953.bi0129s53); - Signal peptides and cellular localization were derived from SignalP (Petersen et al., 2011, Nat. Methods, vol. 8, p. 785-786) v. 4.1; - CAZY numbers and function annotations are derived from UniProt BLAST best hits (Apweiler et al., 2004; Pundir et al., 2016; sources are the same as above); and - Conservative structural domain, via HMMER-3 (Eddy et al., 2011, Nucleic Acids Res., vol.39, W29-37).

[0305] As is well known in the art, when a protein is likely to exist because of the presence of a clear ortholog in a closely related species, it can be identified by "homology inference".

[0306] Genome analysis SEQ ID NO: 13 presents the complete 2.1 Mb genomic DNA sequence of the bacteria deposited as isolate CNCM I-5929. The sequence is presented with the first nucleotide of the dnaA gene designated as nt number 1. Several other annotated genes are indicated herein by their location regions in the genome as presented in SEQ ID NO: 13.

[0307] Based on the genome sequence of the *Sa Ia* bacterium according to the present invention, several genetic determinants were analyzed, such as: 16S rRNA genes; multisite sequence typing genes; mobile genetic elements; prophage typing; and molecular serotyping. The results have been described above. Other relevant features of the genome organization found in SEQ ID NO: 13 include: - The six rRNA cluster regions are located at nt 15400 to 440300.

[0308] - MLST sequence typing was performed based on the sequences of seven housekeeping genes: adhP (approximately 72430 nt); tkt (approximately 298000 nt); glcK (approximately 516380 nt); atr (approximately 539580 nt); pheS (approximately 923670 nt); glnA (approximately 1760150 nt); and sdhA (approximately 2082200 nt).

[0309] - The cpsIa gene cluster is located in the nt region from 1194000 to 1213000.

[0310] - The CRISPR array is located near nt 964400.

[0311] - No prophages were detected in the genome of isolate TI 2893.

[0312] By comparing its genome sequence with that of known Sa Ia isolates using OrthoVenn analysis (Xu et al., 2019, Nucleic Acids Res., vol. 47, W52-W58) and by using LastZ alignment v. 1.02.00 (Geneious™ Prime suite, v. 2022.1.1), marker genes specific to highly virulent Sa Ia bacteria according to the present invention were identified. As described in detail above, a set of selective PCR primers was developed to identify the Sa Ia bacteria-specific genetic markers according to the present invention.

[0313] Another example of this type of analysis is a comparison with the genome sequence of isolate TFJ0901, a Sa Ia strain isolated in 2009 from tilapia without obvious disease symptoms. Its sequence is publicly available in GenBank under accession number NZ_CP034315. Both TI 2893 and TFJ0901 are Sa Ia strains, both are genotype 7, neither encodes a prophage, and both possess the following characteristics regarding the integrated mobile genetic elements: 4 copies of IS 1161; 1 copy of IS 1501; and 6 copies of IS 702.

[0314] However, TI 2893 also contains 5 copies of IS 6110 and 5 copies of IS 630, while TFJ0901 does not have any copies of IS 6110 or IS 630. Since the insertion of such mobile elements can affect gene expression or regulation, they may be associated with alterations in bacterial replication, virulence, or pathogenicity.

[0315] Specifically: In TI 2893, the four marker genes identified in this invention contain mobile genetic elements: - The yjdM gene, located at nt position 991000 on the genome, contains the IS 630 element. - The DUF3307 gene, located near position 1028000, contains the IS 6110 element on its 5' side. Similarly, the abiH gene, located near position 1274000, contains the IS 6110 element on its 5' side, and - The DUF1310 gene, located near position 1935000, contains the IS 6110 element.

[0316] All of these mobile genetic elements are known in the art: IS 1161 is an insertion element encoding a putative transposase similar to the aa sequence UniProtKB:P37245.

[0317] IS 1501 is an insertion element encoding an uncharacterized 19.7 kDa protein similar to the aa sequence UniProtKB:P60046.

[0318] - IS 702 is an insertion element encoding a 128 aa putative transposase similar to the aa sequence UniProtKB:Q00462 and a 130 aa putative transposase similar to the aa sequence UniProtKB:Q00462.

[0319] - IS 630 is an insertion element containing a presumed gene that encodes a 169 aa protein similar to the aa sequence UniProtKB: P16943 and an uncharacterized 39 kDa protein.

[0320] IS 6110 is an insertion element containing the Rv0795 gene, which encodes an uncharacterized 12 kDa protein similar to the aa sequence UniProtKB: P9WKH5. IS 6110 is often associated with IS 904, which encodes a transposase similar to the aa sequence UniProtKB: P35878.

[0321] SEQ ID NO: 14 presents the complete DNA sequence of the plasmid found in the Sa Ia bacterium according to the invention: isolate TI 2893, which is deposited as CNCM I-5929.

[0322] The plasmid detected in TI 2893 is also found in certain known Sa Ia isolates previously obtained from several other countries, which induce only “conventional” Sa Ia pathology in tilapia. Nevertheless, the conservation of this plasmid in the bacteria according to the invention suggests that it is clearly important for the bacteria. Attached Figure Description

[0323] Figure 1 Image of an SDS-PAGE gel stained with Coomassie Brilliant Blue (inset A) and image of a Western blot stained with primary antibody from fish vaccinated with Sa Ia vaccine (inset B). See Example 2 for details.

[0324] In the two insets: Lane 1: Molecular weight marker - Mw labeled on the left; Lane 2: Whole-cell protein lysate of isolate TI 1580 (Thailand, 2005); Lane 3: Whole-cell protein sample from isolate TI 2893 from the 2021 Mexican outbreak; Lane 4: Supernatant of TI 1580 microculture; Lane 5: Supernatant of TI 2893 microculture.

[0325] Figure 2 The results of an inhibitory ELISA using tilapia serum produced against the Sa Ia vaccine as the primary antibody and inhibitory antibody against ten Mexican isolates from 2021 are illustrated. See Example 3 for details.

[0326] The vertical axis represents the inhibition level relative to the no-antigen control. The horizontal axis represents the various isolates tested: TI 2889-TI 2898 are ten isolates from the 2021 Mexican outbreak; the "vaccine strain" is Sa Ia TI 1422; TI 1580 is another known Sa Ia isolate; "Si" and "Tmar" are negative control antigens from *Streptococcus dolphinus* (Si) and *Mycobacterium marineum*, respectively. "ND" indicates: Not detected.

[0327] Figure 3 and Figure 4 A graph showing the cumulative mortality rate % observed in the vaccination-challenge experiment disclosed in Example 7. Figure 3 Challenge the virus using the "classic" Sa Ia isolate TI 1580; Figure 4 Challenge with the novel Sa Ia bacteria according to the present invention: TI 2893.

Claims

1. A serotype Ia agalactiae streptococcus (Sa Ia) having the characterization features of a bacterium deposited at the French National Center for Microbial Collections (CNCM) at the Pasteur Institute in Paris, France, accession number CNCM I-5929.

2. The Sa Ia bacteria according to claim 1, characterized in that, In polymerase chain reaction (PCR) using genetic material from the bacteria, nucleotide (nt) fragments of a specific size are produced when a specific set of primers is used as follows: - Using the primer set of SEQ ID NO: 1 and SEQ ID NO: 2, a 500 nt fragment was generated; or - Using the primer set of SEQ ID NO: 3 and SEQ ID NO: 4, a 1672nt fragment was generated; or - Using the primer set of SEQ ID NO: 5 and SEQ ID NO: 6, a 1772nt fragment was generated; or - Using the primer set of SEQ ID NO: 7 and SEQ ID NO: 8, a 1912nt fragment was generated; or - Using the primer set of SEQ ID NO: 9 and SEQ ID NO: 10, a 2016nt fragment was generated; or - Using the primer set of SEQ ID NO: 11 and SEQ ID NO: 12, an 876nt fragment was generated.

3. The Sa Ia bacteria according to claim 1 or 2, characterized in that, In PCR using genetic material from the bacteria, two or more primer sets as defined in claim 2 are used to produce an nt fragment of the size defined in claim 2.

4. The Sa Ia bacteria according to any one of claims 1-3, characterized in that, In the PCR using genetic material from the bacteria, all six primer sets as defined in claim 2 are used to produce nt fragments of the size defined in claim 2.

5. The Sa Ia bacteria according to any one of claims 1-4, characterized in that, The bacteria are those deposited with accession number CNCM I-5929, or are descendants of the deposited bacteria.

6. The Sa Ia bacterium according to any one of claims 1-5 or the progeny according to claim 5, characterized in that, The bacteria have a genome that is 90% identical to the sequence in SEQ ID NO:

13.

7. A composition comprising a preparation of Sa Ia bacteria or its progeny according to any one of claims 1-6.

8. The composition according to claim 7, characterized in that, The preparation comprises an inactivated form of Sa Ia bacteria or its progeny according to any one of claims 1-6.

9. A diagnostic test kit, characterized in that, The kit comprises a container containing Sa Ia bacteria or progeny according to any one of claims 1-6 and / or a composition according to claim 7 or 8.

10. The Sa Ia bacteria or its progeny according to any one of claims 1-6 and / or the composition according to claim 7 or 8, used as a vaccine against streptococcal disease in fish.

11. A vaccine for fish against streptococcal disease, characterized in that, The vaccine comprises Sa Ia bacteria or progeny according to any one of claims 1-6 and / or the composition according to claim 7 or 8, and a pharmaceutically acceptable carrier.

12. The vaccine of claim 11, further comprising an adjuvant.

13. The Sa Ia bacteria, progeny, or composition used according to claim 10, or the vaccine according to claim 11 or 12, characterized in that, The fish are selected from the following genera: *Carassius*, *Carassius*, and *Carassius*.

14. The Sa Ia bacteria, progeny, or composition used according to claim 13, or the vaccine, characterized in that, The fish is selected from the following species: tilapia guilloché, tilapia guinea, tilapia nigra, tilapia oligra, tilapia mozambique, tilapia thrush, and tilapia nigra; or one or more hybrids of these species.

15. A method for preparing a vaccine according to claim 11 or 12, the method comprising the steps of mixing a Sa Ia bacterium or its progeny according to any one of claims 1-6 with a pharmaceutically acceptable carrier and / or mixing a composition according to claim 7 or 8 with a pharmaceutically acceptable carrier.

16. The method according to claim 15, characterized in that, The method includes the step of inactivating the Sa Ia bacteria or its progeny according to any one of claims 1-6.

17. Use of the Sa Ia bacteria or its progeny according to any one of claims 1-6 and / or the composition according to claim 7 or 8 for the preparation of the vaccine according to claim 11 or 12.

18. A method for protecting fish from streptococcal disease, the method comprising the step of administering to the fish a vaccine according to claim 11 or 12, or a vaccine obtainable by the method according to claim 15 or 16, or a vaccine obtainable by the use according to claim 17.

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