This invention relates to a novel variant of Serratia liquefaciens, which is useful for the design and preparation of vaccines for use in the prevention and treatment of diseases in fish, especially in salmonids, and for the development of compositions and kits useful for the detection, identification and treatment of such diseases.

Aquaculture is currently one of the fastest growing food-producing industries and is becoming increasingly important to resolve the current and projected global shortfalls in aquatic foods and seafood availability. Salmon and trout are amongst the most intensively cultured fish and culture conditions in fish farms and hatcheries may involve poor water quality, often due to increased organic material load from overcrowding, increased water temperature and/or lowered oxygen content. These conditions cause external stress, which in turn can cause outbreak of diseases.

One of such diseases which is triggered by external stress is yersiniosis or enteric redmouth disease (ERM), caused by the Yersinia ruckeri bacterium. Y. ruckeri is a member of the family Enterobacteriaceae. They are Gram-negative rod-shaped organisms with rounded ends. Stressed or nutrient- starved cells of Y. ruckeri may enter a dormant state and can survive for long periods at low salinity, which makes it difficult to control ERM in freshwater salmonids. ERM clinically manifests itself by lethargy, anorexia and hemorrhages in and around the oral cavity. Hemorrhages are also common on the body surface, the base of the fins and in the head region. Internal hemorrhages occur in certain organs, the musculature and the swim bladder.

The disease can be treated by antibiotics, however, resistances have been observed. Moreover, since customers have become increasingly concerned about consuming farmed fish from fish farms and hatcheries employing antibiotics, many fish farmers rather euthanize affected fish then using antibiotics. To keep financial loss at bay, the industry demanded effective vaccines.

Vaccine development started in the late seventies resulted in the provision of attenuated bacteria. More recent vaccines were based on subunits, e.g. Yrpl, a serralysin metalloprotease involved in pathogenesis. After initial success of vaccination, fish farmers in Norway - one of the world’s largest producers of farmed salmon - have nowadays increased problems and face economic losses due to ERM. As of today there are no commercially available vaccines that effectively prevent ERM outbreaks in salmonids and the demand of the industry for such vaccines is high.

In the context of assessing biological variation of Yersiniaceae in fish farms in Norway, the inventors of the present invention obtained water samples from salmon farms seemingly affected with ERM. The samples were analyzed with the intention to isolate/identify Y. ruckeri and carry out whole-genome sequencing of isolates which yielded a positive result using the standard Y. ruckeri identification method described by J Carson and T Wilson, Australia and New Zealand Standard Diagnostic Procedure, 2009, page 14, which is based on amplification and sequencing of the 16S rRNA gene using primers developed for Y. ruckeri , i.e. the primer pair with SEQ ID NO: 1/SEQ ID NO: 2.

Surprisingly, the inventors of the present invention found that while the isolates yielded a positive result with the standard Y. ruckeri identification method described above, genome sequencing revealed that the isolates were in fact neither Y. ruckeri , nor a member of the genus Yersinia, but belonged to Serratia liquefaciens, which seems to be the causative agent of the disease that had affected the fish in the fish farms where the water samples were obtained from.

D. McIntosh et al., J Fish Biol 36, 1990, 765-772, reported that S. liquefaciens, is the causative agent of a disease in Atlantic salmon in Scotland. The authors disclosed that the identified organism exhibited characteristics intermediate between Aeromonas veronii and Serratia liquefaciens and only after having obtained advice from the Centers for Disease Control, Atlanta, USA, the authors decided on equating the organism with S. liquefaciens. The organism was not sequenced and the distinction between A. veronii and S. liquefaciens was based on biochemical tests available at the time of publication rather than by (whole genome) sequencing.

C.E. Starliper, J Fish Dis 24, 2001, 53-56, reported S. liquefaciens to be the causative agent of a disease in Arctic char. It is disclosed that the strains found in Arctic char differed on several criteria compared to those found in Atlantic salmon. Again, characterization of the strains was carried out by biochemical tests rather than genome sequencing.

The main objective of the present invention is to provide a novel variant of S. liquefaciens , i.e. a novel strain, that seems to be the causative agent in a disease in salmonids which manifests itself by symptoms which are similar/identical with those observed in ERM. Such a variant may be used to design and prepare vaccines for use in the prevention and treatment of said diseases in fish and for the development of therapeutic and diagnostic compositions and methods to treat, detect and identify such diseases.

The present invention provides a variant of Serratia liquefaciens, wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4.

In another embodiment, the present invention provides a variant of Serratia liquefaciens , wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core genes tlhyp and/or yegE.

The variant of the invention comprises the core gene tlhyp, which encodes a protein of unknown function and whose nucleic acid sequence is 94% or greater identical to the nucleic acid sequence identified as SEQ ID NO: 9, e.g. 95% greater or 96% or greater, 97% or greater, 98% or greater or 99% or greater. In one embodiment, the species of the invention comprises the gene tlhyp with the nucleic acid sequence identified in SEQ ID NO: 9.

The variant of the invention comprises the core gene yegE, which encodes a diguanylate cyclase, an enzyme participating in the formation of the ubiquitous second messenger, cyclic-di-GMP, involved in bacterial biofilm formation and persistence, and whose nucleic acid sequence is 94% or greater identical to the nucleic acid sequence identified as SEQ ID NO: 10, e.g. 95% greater or 96% or greater, 97% or greater, 98% or greater or 99% or greater. In one embodiment, the species of the invention comprises the gene yegE with the nucleic acid sequence identified in SEQ ID NO: 10.

Thus, in one embodiment, the present invention provides a variant of Serratia liquefaciens , wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core genes tlhyp and/or yegE whose nucleic acid sequences are 94% or greater identical to the nucleic acid sequence identified as SEQ ID NO: 9 (for tlhyp) and SEQ ID NO: 10 (for yegE), respectively.

In another embodiment, the present invention provides a variant of Serratia liquefaciens, wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core gene tlhyp with the nucleic acid sequence identified in SEQ ID NO: 9 and/or the core gene yegE with the nucleic acid sequence identified in SEQ ID NO: 10.

In yet another embodiment, the present invention provides a variant of Serratia liquefaciens , wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8.

In one embodiment, the variant of Serratia liquefaciens according to the invention comprises the core gene tlhyp. In another embodiment, the variant comprises the core gene yegE. In yet another embodiment, the variant comprises the core genes tlhyp and yegE.

The assessment of the aforementioned isolates obtained from water samples from salmon farms comprised whole-genome sequencing and pan-genome comparison of the sequences of said isolates, i.e. comparing all genes of all isolate with each other. The aim of the pan-genome analysis is to identify core genes, i.e. genes that are present in all isolates, softcore genes, i.e. genes that are present in a large fraction of the isolates, clade genes, i.e. genes which are present in a specific clade of the isolates and complementary as well as mobile genetic elements which vary at the strain level, i.e. which can be different in two strains of the same clade within the same variant of bacteria. In addition to the aforementioned core genes tlhyp and yegE, various other core genes were identified which are unique for the Serratia liquefaciens variant of the invention.

In one embodiment, such other core gene is the gene traC which encodes a DNA primase, an enzyme involved in the replication of DNA, and whose nucleic acid sequence is 94% or greater identical to the nucleic acid sequence identified as SEQ ID NO: 11, e.g. 95% greater or 96% or greater, 97% or greater, 98% or greater or 99% or greater. In one embodiment, the variant of the invention comprises the gene traC with the nucleic acid sequence identified in SEQ ID NO: 11.

In another embodiment, such other core gene is the gene chbP which encodes a N,N'-diacetylchitobiose phosphorylase, an enzyme that catalyzes the reversible phosphorolysis of chitobiose, and whose nucleic acid sequence is 94% or greater identical to the nucleic acid sequence identified as SEQ ID NO: 12, e.g. 95% greater or 96% or greater, 97% or greater, 98% or greater or 99% or greater. In one embodiment, the variant of the invention comprises the gene chbP with the nucleic acid sequence identified in SEQ ID NO: 12.

In yet another embodiment, such other core gene is the gene rfbX which encodes an O-antigen transporter, a protein involved in the pathway LPS O-antigen biosynthesis, which is part of bacterial outer membrane biogenesis, and whose nucleic acid sequence is 94% or greater identical to the nucleic acid sequence identified as SEQ ID NO: 13, e.g. 95% greater or 96% or greater, 97% or greater, 98% or greater or 99% or greater. In one embodiment, the variant of the invention comprises the gene traC with the nucleic acid sequence identified in SEQ ID NO: 13.

Thus, in one embodiment, the present invention provides a variant of Serratia liquefaciens , wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises one or more of the core genes selected from the group consisting of tlhyp, yegE, traC, chbP and rfbX.

In another embodiment, the present invention provides a variant of Serratia liquefaciens , wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises one or more of the core genes selected from the group consisting of tlhyp, yegE, traC, chbP and rfbX whose nucleic acid sequences are 94 % or greater identical to the nucleic acid sequence identified as SEQ ID NO: 9 (tlhyp), SEQ ID NO: 10 (yegE), SEQ ID NO: 11 (traC), SEQ ID NO: 12 (chbP) and SEQ ID NO: 13 (rfbX), respectively.

In yet another embodiment, the present invention provides a variant of Serratia liquefaciens, wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises one or more of the core genes selected from the group consisting of tlhyp with the nucleic acid sequence identified in SEQ ID NO: 9, yegE with the nucleic acid sequence identified in SEQ ID NO: 10, traC with the nucleic acid sequence identified in SEQ ID NO: 11, chbP with the nucleic acid sequence identified in SEQ ID NO: 12, and rfbX with the nucleic acid sequence identified in SEQ ID NO: 13.

In another embodiment, the present invention provides a variant of Serratia liquefaciens , wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8, and wherein said variant comprises one or more core genes selected from the group consisting of traC, chbP and rfbX.

In yet another embodiment, the present invention provides a variant of Serratia liquefaciens , wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8, and wherein said variant comprises one or more core genes selected from the group consisting of traC, chbP and rfbX, whose nucleic acid sequences are 94 % or greater identical to the nucleic acid sequence identified as SEQ ID NO: 11 (traC), SEQ ID NO: 12 (chbP) and SEQ ID NO: 13 (rfbX), respectively.

In yet another embodiment, the present invention provides a variant of Serratia liquefaciens, wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8, and wherein said variant comprises one or more of the core genes selected from the group consisting traC with the nucleic acid sequence identified in SEQ ID NO: 11, chbP with the nucleic acid sequence identified in SEQ ID NO: 12, and rfbX with the nucleic acid sequence identified in SEQ ID NO: 13.

In another embodiment, the present invention provides a variant of Serratia liquefaciens , wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8 and wherein said variant comprises the core gene traC which can be amplified by PCR using the primer pair with SEQ ID NO: 14/SEQ ID NO: 15, and/or the core gene chbP which can be amplified by PCR using the primer pair with SEQ ID NO: 16/SEQ ID NO: 17 and/or the core gene rfbX which can be amplified by PCR using the primer pair with SEQ ID NO: 17/SEQ ID NO: 18.

The terms“can be amplified by PCR” or“cannot be amplified by PCR” in the context of the invention relates to a PCR carried out with standard PCR reactants according to a standard protocol. Standard PCR reactants are a DNA template that contains the DNA target region to be amplified, a DNA polymerase; an enzyme that polymerizes new DNA strands; two DNA primers that are complementary to the 3' ends of each of the sense and anti-sense strands of the DNA target; deoxynucleoside triphosphates, or dNTPs (sometimes called "deoxynucleotide triphosphates";

nucleotides containing triphosphate groups), the building blocks from which the DNA polymerase synthesizes a new DNA strand; a buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA polymerase; bivalent cations and monovalent cations. Standard PCR reactants are commercially available, e.g. in the form of a kit which includes several of the reactants. The basic steps of a PCR are well known in the art and a standard protocol for carrying out these steps is commonly available together with the reactants or the kit from commercial suppliers. The standard protocol requires the inclusion of certain controls, i.e. a positive control, a negative control and a no-template control. In the context of the invention, the isolate deposited under the Budapest Treaty with the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 26 October 2018 under the accession number DSM 32941 may serve as a positive control while common E. coli laboratory strains like K12, BL25 or DH5a may serve as a negative control. With the standard PCR reagents, the standard PCR protocol and the aforementioned controls, a person skilled in the art is able to determine whether a gene comprised in the variant of the invention“can be amplified by PCR” or“cannot be amplified by PCR”.

According to one embodiment, the variant of Serratia liquefaciens according to the invention is a fish pathogen, preferably a salmonids pathogen, i.e. a pathogen that causes diseases in salmonids including fish from the genus Salmo, Oncorhynchus, Salvelinus and Thymallus, e.g. Atlantic salmon, coho salmon, chinook salmon, chum salmon, sockeye salmon, pink salmon, amago salmon, masu salmon, trout, rainbow trout, brook trout, brown trout, common grayling, Arctic grayling and Arctic char. In one embodiment, the disease manifests itself by symptoms which are similar/identical with those observed in ERM.

According to another embodiment, the present invention provides an isolate deposited under the Budapest Treaty with the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) on 26 October 2018 under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate. The term“having substantially the same genotypic and phenotypic

characteristics as said isolate” shall be taken to encompass variants of the isolate specified by accession number DSM 32941 having at least 95 % identity (see:

Stackebrandt & Goebel, 1994, Int. J. Syst. Bacteriol. 44:846-849). In one embodiment, said variant has at least 95 % identity with the 16S rRNA sequence of the isolate specified by accession number DSM 32941, e.g. at least 96% identity, at least 97% identity, at least 98 % identity, at least 99 % identity or more than 99% identity.

According to yet another embodiment, the present invention provides an isolate deposited with the DSMZ under the accession number DSM 32941.

Thus, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said isolate can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4.

In one embodiment, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said isolate can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said isolate comprises the core genes tlhyp and/or yegE.

In another embodiment, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said variant comprises the core gene tlhyp with the nucleic acid sequence identified in SEQ ID NO: 9 and/or the core gene yegE with the nucleic acid sequence identified in SEQ ID NO: 10.

In yet another embodiment, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said variant can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said isolate comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8.

In yet another embodiment, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said isolate can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said isolate comprises one or more of the core genes selected from the group consisting of tlhyp, yegE, traC, chbP and rfbX.

In yet another embodiment, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said isolate can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said isolate comprises one or more of the core genes selected from the group consisting of tlhyp with the nucleic acid sequence identified in SEQ ID NO: 9, yegE with the nucleic acid sequence identified in SEQ ID NO: 10, traC with the nucleic acid sequence identified in SEQ ID NO: 11, chbP with the nucleic acid sequence identified in SEQ ID NO: 12, and rfbX with the nucleic acid sequence identified in SEQ ID NO: 13.

In yet another embodiment, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said isolate can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said isolate comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8, and wherein said isolate comprises one or more core genes selected from the group consisting of traC, chbP and rfbX.

In yet another embodiment, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said isolate can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said isolate comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8, and wherein said isolate comprises one or more of the core genes selected from the group consisting of traC with the nucleic acid sequence identified in SEQ ID NO: 11, chbP with the nucleic acid sequence identified in SEQ ID NO: 12, and rfbX with the nucleic acid sequence identified in SEQ ID NO: 13.

In yet another embodiment, the present invention provides the isolate deposited with the DMSZ under the accession number DSM 32941, wherein the 16S rRNA of said isolate can be amplified by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2 but cannot be amplified using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4 and wherein said isolate comprises the core gene tlhyp which can be amplified by PCR using the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and/or yegE which can be amplified using the primer pair with SEQ ID NO: 7/SEQ ID NO: 8 and wherein said isolate comprises the core gene traC which can be amplified by PCR using the primer pair with SEQ ID NO: 14/SEQ ID NO: 15, and/or the core gene chbP which can be amplified by PCR using the primer pair with SEQ ID NO: 16/SEQ ID NO: 17 and/or the core gene rfbXwhich can be amplified by PCR using the primer pair with SEQ ID NO: 17/SEQ ID NO: 18.

According to another embodiment, the present invention provides an isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, which is a fish pathogen, preferably a salmonids pathogen.

According to another embodiment, the present invention provides an isolate deposited with the DSMZ under the accession number DSM 32941 which is a fish pathogen, preferably a salmonids pathogen.

According to yet another embodiment, the invention provides an isolated fraction derived from the variant of Serratia liquefaciens according to the invention. In one embodiment, the invention provides an isolated fraction derived from the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate. In another embodiment, the invention provides an isolated fraction derived from the isolate deposited with the DSMZ under the accession number DSM 32941.

The isolated fraction may be or comprise a gene, a part of a gene such as a plasmid or a nucleotide sequence, a gene product such as RNA or a protein, an intracellular structure such as organelles, ribosomes or other multiprotein complexes or extracellular structures such as the cell envelope, cell wall, plasma membrane or parts thereof, e.g. a surface protein, and the like. In one embodiment, the isolated fraction may comprise a gene, part of a gene or a gene product which is responsible for the pathogenicity in fish, preferably in salmonids.

In another embodiment, the invention provides an isolated fraction derived from the variant of Serratia liquefaciens according to the invention, comprising one or more of a gene, a part of a gene, a gene product, an intracellular structure or an extracellular structure which is responsible for or involved in the pathogenicity in fish. In one embodiment, said variant is the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate. In another embodiment, said variant is the isolate deposited with the DSMZ under the accession number DSM 32941.

According to yet another embodiment, the present invention provides a vaccine against a disease in fish, preferably in salmonids, caused by the variant of Serratia liquefaciens according to the invention.

In one embodiment, the present invention provides a vaccine against a disease in fish, preferably in salmonids, caused by the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate.

In another embodiment, the present invention provides a vaccine against a disease in fish, preferably in salmonids, caused by the isolate deposited with the DSMZ under the accession number DSM 32941.

The term“vaccine” denotes a biological agent in a form suitable for administration to a fish, preferably to salmonids, which is capable of stimulating the immune system of the recipient fish to recognize the agent as a threat, destroy it, and to further recognize and destroy any of the microorganisms associated with that agent that it may encounter in the future.

According to yet another embodiment the invention provides a vaccine comprising the variant of Serratia liquefaciens according to the invention and optionally a pharmaceutical carrier. The vaccine may comprise said variant in live, attenuated or inactivated form.

In one embodiment, the invention provides a vaccine comprising the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate and optionally a pharmaceutical carrier. The vaccine may comprise said isolate in live, attenuated or inactivated form.

In another embodiment, the invention provides a vaccine comprising the isolate deposited with the DSMZ under the accession number DSM 32941 and optionally a pharmaceutical carrier. The vaccine may comprise said isolate in live, attenuated or inactivated form.

According to yet another embodiment, the invention provides a vaccine comprising one or more isolated fractions derived from the variant of Serratia liquefaciens according to the invention and optionally a pharmaceutical carrier.

In one embodiment, the invention provides a vaccine comprising one or more isolated fractions of the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate and optionally a pharmaceutical carrier.

In another embodiment, the invention provides a vaccine comprising one or more isolated fractions of the isolate deposited with the DSMZ under the accession number DSM 32941 and optionally a pharmaceutical carrier.

The isolated fraction may be a suitable protein such as a (inactivated) toxin or a surface protein.

In one embodiment, the vaccines of the invention comprise a pharmaceutical carrier, e.g. an aqueous carrier such as water or an aqueous buffer. The vaccines of the invention may further comprise other pharmaceutical excipients such as adjuvants, i.e. immuno stimulating agents, stabilizers, viscosity modifiers and the like. Suitable adjuvants include mineral oil or other adjuvants which are commonly used in fish vaccines, e.g. mannide oleate in a mineral oil solution. Suitable mineral oils to be included are e.g. white mineral oil, i.e. sorbitan oleate, or polyoxyethylene sorbitan monooleate. Synthetic mineral oils, such as those commercially available under the name Montanide may also be used. Other useful adjuvants include squalene, muramyl peptides, lipopolysaccharides, glycans, glucans or high molecular weight, crosslinked polyacrylic acid polymers such as Carbopol®. Useful inorganic adjuvants include aluminium hydroxide and calcium phosphates.

The vaccines of the invention may be preventive vaccines or therapeutic vaccines, i.e. vaccines treating a disease. The term“treat” or“treatment” in the context of the invention includes alleviating symptoms of a disease and ameliorating and curing a disease.

In one embodiment, the vaccines of the invention are autogenous vaccines. In general, autogenous vaccines are prepared from cultures of microorganisms obtained from a fish, e.g. a (sick) fish of a particular fish farm, and then used to immunize the same fish and other fish from said particular fish farm to treat the disease caused by said microorganisms or to prevent the disease in animals which are not yet infected. In one embodiment, the vaccines of the invention are autogenous vaccines comprising the variant of Serratia liquefaciens according to the invention in inactivated form, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941.

In the context of the invention, autogenous vaccines are prepared from cultures of the variant of Serratia liquefaciens according to the invention obtained from fish of a particular fish farm and then used to immunize the same fish and other fish from said particular fish farm to treat, i.e. alleviate symptoms or ameliorate or cure a disease caused by the variant and to prevent the disease in animals which are not yet infected.

The vaccines of the invention may be manufactured by methods well known in the art, e.g. by a method that comprises admixing an immunologic ally effective amount of the variant of Serratia liquefaciens according to the invention, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate or one or more isolated fractions of the variant of the invention, e.g. an isolated fraction of the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, with the optional pharmaceutical carrier and optionally with other pharmaceutical excipients.

For a preventive vaccine, the term“immunologically effective amount” is to be understood as an amount capable of stimulating the immune system of a recipient fish sufficiently to at least reduce the negative effects of a post-vaccination challenge by the variant of the invention.

For a therapeutic vaccine, the term is to be understood as being an amount capable of stimulating the immune system of a recipient fish to attack the variant of the invention and destroy it to such a degree that the disease is ameliorated or cured.

The vaccines according to the invention may be administered to fish by methods known in the art. Such methods include injection, e.g. intramuscular or intraperitoneal injection, oral ingestion or immersion of the fish in water comprising the vaccines.

The isolates belonging to the variant according to the invention were obtained from water of fish farms farming Atlantic salmon. The vaccine of the invention may thus be useful for salmonids in general, including fish from the genus Salmo,

Oncorhynchus, Salvelinus and Thymallus, e.g. Atlantic salmon, coho salmon, chinook salmon, chum salmon, sockeye salmon, pink salmon, amago salmon, masu salmon, trout, rainbow trout, brook trout, brown trout, common grayling, Arctic grayling and Arctic char.

The vaccines according to the invention may be polyvalent vaccines, i.e.

vaccines comprising the variant of Serratia liquefaciens according to the invention, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate or an isolated fraction of the variant of the invention, e.g. an isolated fraction of the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate may also be included in polyvalent vaccines, and one or more additional agents which target the same or a different disease than the disease caused by the aforementioned variant. A non-limiting list of additional agents that may be included in a polyvalent vaccine of the invention - in live, attenuated or inactivated form - are fish virus selected from the group consisting of infectious hematopoietic necrosis virus (IHNV), infectious pancreatic necrosis virus (IPNV), infectious salmon anemia virus (ISAV), salmon pancreatic vims (SAV), viral hemorrhagic septicemia virus (VHSC), cardiomyopathy syndrome vims (CMSV) and/or bacteria selected from the group consisting of Aeromonas sp., Bifidobacterium sp., Brevibacterium sp., Cytophaga sp., Edwarsiella sp., Flavobacterium sp., Francisella sp., Factococcus sp., Feuconostoc sp., Fistonella sp. Moritella viscosa, Mycobacterium sp. Nocardia sp., Pediococcus sp., Photobacterium damsel, Pseudumonas sp., Renibacterium sp., Rhodococcus sp., Streptococcus sp., Vibrio sp., Rahnella sp., Serratia sp other than S. liquefaciens , Serratia liquefaciens variants other than the variant of the invention and Yersinia sp..

In one embodiment, the vaccines of the inventions are polyvalent vaccines which further comprise Yersinia ruckeri in live, attenuated or inactivated form. In one embodiment, the vaccines of the invention are polyvalent vaccines which comprise the variant of Serratia liquefaciens of the invention and Yersinia ruckeri in inactivated form. In one embodiment, such vaccines are autogenous vaccines. In another embodiment, the vaccines of the inventions as described herein are polyvalent vaccines which further comprise one or more isolated fractions of Yersinia ruckeri.

In another embodiment, the vaccines of the inventions are polyvalent vaccines which further comprise Rahnella sp., preferably the Rahnella species disclosed in the Norwegian patent application number 20181043, more preferably the isolate deposited with the DSMZ under the accession number 32809 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, in live, attenuated or inactivated form. In another embodiment, the vaccines of the inventions as described herein are polyvalent vaccines which further comprise one or more isolated fractions of Rahnella sp., preferably one or more isolated fractions of the Rahnella species disclosed in the Norwegian patent application number 20181043, more preferably the one or more isolated fractions of the isolate deposited with the DSMZ under the accession number 32809 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate. In yet another embodiment, the vaccines of the inventions are polyvalent vaccines which further comprise a Serratia species other than S. liquefaciens, preferably the Serratia sp. disclosed in the International patent application number PCT/N02019/050160, more preferably the isolate deposited with the DSMZ under the accession number 32810 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, in live, attenuated or inactivated form or the Serratia sp. disclosed in the International patent application number

PCT/NO2019/050198, more preferably the isolate deposited with the DSMZ under the accession number 32808 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, in live, attenuated or inactivated form.

In yet another embodiment, the vaccines of the inventions are polyvalent vaccines which further comprise one or more isolated fractions of a Serratia species other than S. liquefaciens , preferably one or more isolated fractions of the Serratia sp. disclosed in the International patent application number PCT/N02019/050160, more preferably the isolate deposited with the DSMZ under the accession number 32810 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate and/or one or more isolated fractions of or the Serratia sp. disclosed in the International patent application number PCT/N02019/050198, more preferably the isolate deposited with the DSMZ under the accession number 32808 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate.

In yet another embodiment, the vaccines of the inventions are polyvalent vaccines which further comprise - in live, attenuated form or inactivated form - one or more of Yersinia ruckeri, Serratia species other than Serratia liquefaciens and

Rahnella sp..

In yet another embodiment, the vaccines of the inventions are polyvalent vaccines which further comprise - in live, attenuated form or inactivated form - one or more of Yersinia ruckeri, and/or Rahnella sp., preferably the Rahnella sp. disclosed in the Norwegian patent application number 20181043, more preferably the isolate deposited with the DSMZ under the accession number 32809 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, and/or a Serratia species other than S. liquefaciens, preferably the Serratia sp. disclosed in the International patent application number PCT/N02019/050160, more preferably the isolate deposited with the DSMZ under the accession number 32810 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, and/or the Serratia sp. disclosed in the International patent application number PCT/N02019/050198, more preferably the isolate deposited with the DSMZ under the accession number 32808 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate.

In yet another embodiment, the vaccines of the invention as described herein are polyvalent vaccines which further comprise one or more isolated fractions of Yersinia ruckeri and/or one or more isolated fractions a Rahnella sp., preferably the Rahnella sp. disclosed in the Norwegian patent application number 20181043, more preferably the isolate deposited with the DSMZ under the accession number 32809 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, and/or one or more isolated fractions of a Serratia species other than S. liquefaciens, preferably the Serratia sp. disclosed in the International patent application number PCT/N02019/050160, more preferably the isolate deposited with the DSMZ under the accession number 32810 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, and/or one or more isolated fractions of the Serratia sp. disclosed in the International patent application number PCT/N02019/050198, more preferably the isolate deposited with the DSMZ under the accession number 32808 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate.

According to yet another embodiment, the invention provides a composition for use in preventing a disease in fish, preferably in salmonids, wherein said composition comprises an immunologically effective amount of the variant according to the invention, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate or one or more isolated fractions of the variant according to the invention, e.g. an isolated fraction of the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, and optionally a

pharmaceutical carrier. According to another embodiment, the invention provides a method for preventing a disease in fish, preferably in salmonids, comprising the step of administering either an immunologic ally effective amount of a vaccine according to the invention or a composition for preventing a disease in fish according to the invention, to a fish in need thereof.

According to yet another embodiment, the invention provides a composition for use in ameliorating or treating a disease in fish, preferably in salmonids, wherein said composition compries an immunologically effective amount of the variant according to the invention, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate or one or more isolated fractions of the variant of Serratia liquefaciens according to the invention, e.g. an isolated fraction of the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate and optional a pharmaceutical carrier.

According to yet another embodiment, the invention provides a method for ameliorating or treating a disease in fish, preferably in salmonids comprising the step of administering either an immunologically effective amount of a vaccine according to the invention or a composition for use in treating a disease in fish according to the invention, to a fish in need thereof.

According to another embodiment, the invention provides a kit for detecting the variant of Serratia liquefaciens according to the invention, wherein said kit comprises one or more primer pairs suitable for amplifying one or more genes being unique for said variant by PCR. In one embodiment, the kit comprises one or more primer pairs suitable for amplifying one gene. In another embodiment, the kit comprises more than one primer pair suitable for amplifying more than one gene.

In one embodiment, the invention provides for a kit for detecting the variant according to the invention comprising one or more primer pairs suitable for amplifying by PCR the gene tlhyp. In one embodiment, the aforementioned kit comprises the primer pair with SEQ ID NO: 5/SEQ ID NO: 6.

In another embodiment, the invention provides for a kit for detecting the variant according to the invention comprising one or more primer pairs suitable for amplifying by PCR the gene yegE. In one embodiment, the aforementioned kit comprises the primer pair with SEQ ID NO: 7/SEQ ID NO: 8.

In yet another embodiment, the invention provides for a kit for detecting the variant according to the invention comprising one or more primer pairs suitable for amplifying by PCR the gene tlhyp and one or more primer pairs which are suitable for amplifying by PCR the gene yegE. In one embodiment, the aforementioned kit comprises the primer pair with SEQ ID NO: 5/SEQ ID NO: 6 and the primer pair with SEQ ID NO: 7/SEQ ID NO: 8.

In yet another embodiment, the invention provides a kit for detecting the variant according to the invention comprising one or more primer pairs suitable for amplifying by PCR one or more of the genes selected from the group consisting of traC, chbP and rfbX.

In one embodiment, the invention provides a kit for detecting the variant according to the invention comprising one or more primer pairs suitable for amplifying by PCR the gene traC. In one embodiment, the aforementioned kit comprises the primer pair with SEQ ID NO: 14/SEQ ID NO: 15.

In one embodiment, the invention provides a kit for detecting the variant according to the invention comprising one or more primer pairs suitable for amplifying by PCR the gene chbP. In one embodiment, the aforementioned kit comprises the primer pair with SEQ ID NO: 16/SEQ ID NO: 17.

In one embodiment, the invention provides a kit for detecting the variant according to the invention comprising one or more primer pairs suitable for amplifying by PCR the gene rfbX. In one embodiment, the aforementioned kit comprises the primer pair with SEQ ID NO: 18/SEQ ID NO: 19.

In yet another embodiment, the invention provides a kit for detecting the variant according to the invention comprising one or more primer pairs suitable for amplifying by PCR one or more of the genes selected from the group consisting of tlhyp, yegE, traC, chbP and rfbX.

In yet another embodiment, the invention provides a kit for detecting the variant according to the invention comprising one or more primers selected from the group consisting of the primer pair with SEQ ID NO: 5/SEQ ID NO: 6, the primer pair with SEQ ID NO: 7/SEQ ID NO: 8, the primer pair with SEQ ID NO: 14/SEQ ID NO: 15, the primer pair with SEQ ID NO: 16/SEQ ID NO: 17 and the primer pair with SEQ ID NO: 18/SEQ ID NO: 19.

In one embodiment, the aforementioned kits of the invention are for detecting the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate. In another embodiment, the aforementioned kits of the invention are for detecting the isolate deposited with the DSMZ under the accession number DSM 32941.

The kits according to the invention may further comprise instructions how to use the one or more primer pairs.

According to yet another embodiment, the invention provides a method for detecting the variant of Serratia liquefaciens according to the invention, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, using a kit according to the invention.

According to yet another embodiment, the invention provides a method for detecting the variant of Serratia liquefaciens according to the invention, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, said method comprises: a) obtaining a sample comprising the variant according to the invention, b) cultivating the sample, c) isolating the DNA from the sample and d) using one or more primer pairs suitable for amplifying a gene by PCR which is unique for said variant. Suitable genes and primer pairs for amplifying said genes have been discussed in detail above.

The present invention furthermore includes an oligonucleotide sequence comprising a sequence of at least 10 nucleotides, wherein said oligonucleotide sequence hybridizes to a nucleic acid sequence comprised in genes, e.g. core genes, comprised in the Serratia liquefaciens variant of the invention, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate.

In one embodiment, the invention provides an oligonucleotide sequence comprising a sequence of at least 10 nucleotides, wherein said oligonucleotide sequence hybridizes to a nucleic acid sequence comprised in the nucleic acid sequence of the core genes tlhyp or yegE.

In one embodiment, such oligonucleotide sequences are those with SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

In one embodiment, such oligonucleotide sequences are those with SEQ ID NO: 5 and SEQ ID NO: 6. Said oligonucleotide sequences hybridize to a nucleic acid sequence comprised in the nucleic acid sequence of the core gene tlhyp.

In one embodiment, such oligonucleotide sequences are those with SEQ ID NO: 7 and SEQ ID NO: 8. Said oligonucleotide sequences hybridize to a nucleic acid sequence comprised in the nucleic acid sequence of the core gene yegE.

In yet another embodiment, the invention provides an oligonucleotide sequence comprising a sequence of at least 10 nucleotides, wherein said oligonucleotide sequence hybridizes to a nucleic acid sequence comprised in the nucleic acid sequence of the core genes traC or chbP or rfbX.

In one embodiment, the oligonucleotide sequence is a sequence selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

In yet another embodiment, such oligonucleotide sequences are those with SEQ ID NO: 14 and SEQ ID NO: 15. Said oligonucleotide sequences hybridize to a nucleic acid sequence comprised in the nucleic acid sequence of the core gene traC.

In yet another embodiment, such oligonucleotide sequences are those with SEQ ID NO: 16 and SEQ ID NO: 17. Said oligonucleotide sequences hybridize to a nucleic acid sequence comprised in the nucleic acid sequence of the core gene chbP.

In yet another embodiment, such oligonucleotide sequences are those with SEQ ID NO: 18 and SEQ ID NO: 19. Said oligonucleotide sequences hybridize to a nucleic acid sequence comprised in the nucleic acid sequence of the core gene rfbX.

In another embodiment, the invention provides an oligonucleotide sequence comprising a sequence of at least 10 nucleotides, wherein said oligonucleotide sequence hybridizes to a nucleic acid sequence comprised in genes comprised in the Serratia liquefaciens variant of the invention, wherein said oligonucleotide sequence is selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19.

The aforementioned oligonucleotide sequences can be used for the detection of the Serratia liquefaciens variant of the invention, e.g. the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, e.g. by using them as primers for amplifying a gene comprised in the Serratia liquefaciens variant of the invention. The aforementioned oligonucleotide sequences may thus be included in the kits of the invention.

According to yet another embodiment, the invention provides a bacteriophage for infecting the variant of Serratia liquefaciens according to the invention, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of a bacterium belonging to the variant of Serratia liquefaciens according to the invention.

In one embodiment, the invention provides a bacteriophage for infecting a bacterium of the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of said bacterium.

Such bacteriophages can be obtained by methods known in the art, e.g. plaque assays (see for instance Kropinski A.M.et ah, Methods Mol Biol 501, 2009, 69-76).

In another embodiment, the invention provides a bacteriophage for infecting a bacterium of the isolate deposited with the DSMZ under the accession number DSM 32941, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of said bacterium.

In yet another embodiment, the invention provides a composition comprising a bacteriophage for use in the treatment of a disease in fish caused by the variant of Serratia liquefaciens according to the invention, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of a bacterium of said the variant. In one embodiment, the invention provides a composition comprising a bacteriophage for use in the treatment of a disease in fish caused by the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of a bacterium belonging to said isolate.

In one embodiment, the invention provides a composition comprising a bacteriophage for use in the treatment of a disease in fish caused by the isolate deposited with the DSMZ under the accession number DSM 32941, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of a bacterium belonging to said isolate.

The composition comprising said bacteriophage may further comprise a pharmaceutical carrier, e.g. an aqueous carrier such as water or an aqueous buffer, and other suitable pharmaceutical excipients.

The composition comprising said bacteriophage can be manufactured by methods known in the art, e.g. a method that comprises admixing a therapeutically effective amount of the bacteriophage with a pharmaceutical carrier and optionally other pharmaceutical excipients.

The composition comprising said bacteriophage according to the invention can be administered to fish by any of the suitable methods known in the art. Such methods include oral ingestion or immersion, i.e. absorption through skin by immersion of the fish in the composition.

The composition comprising said bacteriophage may be useful for treating diseases caused by the species of the invention or a strain thereof in salmonids in general, including fish from the genus Salmo, Oncorhynchus, Salvelinus and

Thymallus, e.g. Atlantic salmon, coho salmon, chinook salmon, chum salmon, sockeye salmon, pink salmon, amago salmon, masu salmon, trout, rainbow trout, brook trout, brown trout, common grayling, Arctic grayling and Arctic char.

According to yet another embodiment, the invention provides a method for treating a disease in fish comprising the steps of administering a therapeutically effective amount of the composition comprising a bacteriophage according to the invention to a fish in need thereof. In yet another embodiment, the invention provides a composition comprising a bacteriophage for use in decreasing the number of bacteria belonging to the variant of Serratia liquefaciens according to the invention in water used in fish farms, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of said bacteria.

In one embodiment, the invention provides a composition comprising a bacteriophage for use in decreasing the number of bacteria belonging to the isolate deposited with the DSMZ under the accession number DSM 32941 or an isolate having substantially the same genotypic and phenotypic characteristics as said isolate in water used in fish farms, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of said bacteria.

In one embodiment, the invention provides a composition comprising a bacteriophage for use in decreasing the number of bacteria belonging to the isolate deposited with the DSMZ under the accession number DSM 32941 in water used in fish farms, wherein said bacteriophage comprises structural elements for attaching itself to a surface receptor being present at the surface of said bacteria.

The composition comprising said bacteriophage may further comprise a carrier, e.g. an aqueous carrier such as water or an aqueous buffer and other suitable excipients.

The composition may be manufactured by methods known in the art, e.g. a method that comprises admixing an effective amount of the bacteriophage with a carrier and optionally other excipients.

The composition according to the invention is added to water used in fish farms, preferably fish farms where used water is re-cycled and re-used.

The composition comprising said bacteriophage may be useful to treat water in fish farms for salmonids farming in general, including farming of fish from the genus Salmo, Oncorhynchus, Salvelinus and Thymallus, e.g. Atlantic salmon, coho salmon, chinook salmon, chum salmon, sockeye salmon, pink salmon, amago salmon, masu salmon, trout, rainbow trout, brook trout, brown trout, common grayling, Arctic grayling and Arctic char.

According to yet another embodiment, the invention provides a method for decreasing the number of bacteria belonging to the variant of the invention in water used in fish farms by adding an effective amount of the composition comprising a bacteriophage according to the invention to such water.

The invention will be illustrated by the following non-limiting examples. Examples

Table 1: Primers used for PCR analyses/amplification in the Examples

The genes in bold in Table 1 above are comprised in the novel variant of Serratia liquefaciens according to the invention

Example 1: Isolation, cultivation and preservation of the Serratia liquefaciens variant of the invention

Water samples were collected at various Atlantic salmon farms on the coast of Norway, where salmon seemingly affected with ERM had been reported.

100 mL of such water samples was filtered through a 0.22 mhi filter (Merck, Norway) and the filters were inoculated in 1 mL of ROD medium (Rodgers, C. J Fish Diseases 15(3), 1992, 243-254) for 24 hrs at 4 °C before plating on LB-agar and subsequent incubation at 25 °C for 24 hrs.

Colonies were picked and screened by PCR using the 16S rRNA Yersinia ruckeri specific primer pair with SEQ ID NO: 1/SEQ ID NO: 2. Colonies with a positive result in the PCR were subsequently plated for a minimum of 3 passages and analyzed using PCR (with the aforesaid primer pair) daily to ensure purity of the sample. Finally, the samples were stored at -80 °C in LB supplemented with 20% glycerol. One isolate was provisionally named LAA and eventually deposited under the Budapest Treaty with the Leibniz-Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DMSZ) under the accession number DSM 32941.

Example 2: Further testing for Yersinia ruckeri

A new passage was used to screen the LAA isolate further by PCR using the Yersinia ruckeri specific primer pair with SEQ ID NO: 3/SEQ ID NO: 4. This primer pair was designed to amplify a 409 bp fragment of Y. ruckeri 16S rDNA, while not amplifying or being genetically related to Yersinia or a wide variety of other aquatic or piscine bacteria (J. T. LeJeune et ah, J Vet Diagn Investig 12, 2000, 558-561). The isolate could, however, not be amplified with this primer pair. Based on the contradicting results of Examples 1 and 2, it was decided to sequence the isolate.

Example 3: Genotypic characterization of the isolates

Genomic DNA from the LAA isolate was extracted from 1 mL overnight culture using the DNeasy Blood & Tissue Kit (Qiagen) as instructed in cultured cells protocol included in the kit. DNA libraries were made using the Nextera Flex Sample Prep kit (Illumina) according to manufacturer’s instructions and sequenced with Illumina MiSeq (Illumina) using V3 chemistry.

Raw sequences were adapter trimmed, quality filtered (Q>20), de novo assembled using SPAdes V3.12.0 (A. Bankevich et ah, J Comput Biol 19(5), 2012, 455-477) and annotated using the Prokka pipeline (T. Seemann, Bioinformatics, 30(14), 2014. 2068-2069). Contigs shorter than 1000 bp or with lower than 5x coverage were removed from each assembly prior to gene annotation. To identify the closest relative to the isolate, a pan-genome analysis was performed. The protein coding sequences of whole-genome sequenced isolates were compared to all existing genome sequences under the Yersiniaceae family on the National Center for Biotechnology Information (NCBI) using a BLASTP all-against- all approach (C. Camacho et al., BMC Bioinformatics 2009, 10, 421) and grouped into orthologous clusters using Roary 3.12.0 (A. J. Page et al., Bioinformatics 31(22), 2015, 3691-3693). A presence/absence matrix for each orthologous gene cluster and each genome was constructed for the pan-genome before statistical and clustering analysis of the matrix was performed in R (http://www.r-proiect.org/). The pan-genome analysis showed that the isolate belongs to S. liquefaciens. sharing 3935 orthologous gene clusters. The second closest relative was S. proteomaculans, sharing 2013 orthologous gene clusters, a number that is also shared by most of Serratia species.

The new S. liquefaciens variant contains 347 unique orthologous gene clusters that are not found in other S. liquefaciens , demonstrating that the variant is significantly different from the existing S. liquefaciens isolates.

Example 4: Development of specific primer for PCR amplification of core genes of the variant of Serratia liquefaciens according to the invention

Specific primer pairs were designed for the PCR amplification of the following core genes of the LAA isolate: tlhyp, encoding a protein of unknown function, yegE, a diguanylate cyclase, traC, a DNA primase, chbP, a chitobiose phosphorylase and rfbX, an o-antigen transporter (see Table 1). With these primers, a PCR-product was successfully amplified using genomic DNA from the LAA isolate.

Example 5: In silico analysis of whole genome sequencing

An in silico analysis of the results of the whole genome sequencing of the LAA isolate was conducted with the aim to identify virulence factors which could support the hypothesis that the isolate is a fish pathogen and explain the occurrence of ERM in the fish farm from which the isolate was obtained. A number of genes were found which relate to biosynthesis of flagella (required for motility), adhesins (required for adhesion to cell surfaces), invasins which enable the variant of the invention to pass through cells or cell-membranes, hemolysins which break down blood cells for iron- capture, genes for uptake and capture of iron, genes related to biofilm synthesis, and a number of toxin/antitoxin systems. All of these genes are typical virulence factors.