Vaccines against subsp. Mucilaginosa

Abstract

The present invention provides vaccines against newly occurring isolates of Mortiella mucilaginosa and methods of using the vaccines.

Description

Technical Field

[0001] The present invention is generally in the field of aquaculture vaccines. Background Art

[0002] Winter canker affects both Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) and causes increased mortality and significant economic losses due to downgrading of fish at slaughter.

[0003] Moritella viscosa (formally known as Vibrioviscosus) is the main cause of winter canker ( et al., 2009; et al., 2009; et al., 2011; Karlsen et al., 2017a; Karlsen et al., 2017b). It is a Gram-negative, psychrophilic, facultative anaerobic bacterium capable of both fermentation and respiratory metabolism (Gudmundsdóttir and 2007; et al., 2009; et al., 2011). It is oxidase and catalase positive and requires salt for growth; colonies are yellowish translucent and often sticky (Gudmundsdóttir and 2007), but in recent years non-viscous strains of M viscosa have also been isolated.

[0004] Existing vaccines prevent the classical, viscoelastic strains of M. viscosus. However, current commercial vaccines are not effective against emerging strains that differ genotypically and / or phenotypically from the classical strains. These emerging strains can be classified as variant strains based on the gyrB sequence and can be classified as classical, non-viscous strains based on their non-viscous appearance after culture on agar plates, whereas classical, viscoelastic M. viscosus strains present as adherent colonies that form a sticky thread when manipulated with a loop.

[0005] Therefore, new vaccines and approaches are needed to effectively protect against newly emerging strains as well as classically virulent strains. Summary of the invention

[0006] In a first aspect, the present disclosure addresses these and other needs by providing a vaccine comprising an antigenic M. viscosus component comprising antigens derived from a classical non-viscous strain of M. viscosus for protecting fish from infection caused by variant M. viscosus.

[0007] The vaccine according to the first aspect of the invention may be used to protect fish from infection caused by M. viscosus variants and M. viscosus classically non-viscosus.

[0008] According to the first aspect of the present invention, there is also disclosed a vaccine, wherein said antigenic component consists essentially of or consists of said antigen derived from said antigen, said antigen being derived from said classical non-viscous M. viscosus strain.

[0009] According to the first aspect of the present invention, there is also disclosed a vaccine, wherein the antigenic component consists essentially of or consists of the antigen derived from the classical non-viscous M. viscosus strain and optionally an antigen derived from the classical viscosus M. viscosus strain.

[0010] The vaccine according to the first aspect of the invention may be co-administered with a second vaccine, wherein the second vaccine of the first aspect comprises antigens derived from a classical strain of M. viscosus. Preferably, the second vaccine does not contain antigens derived from a variant strain of M. viscosus.

[0011] In the vaccine according to the first aspect of the present invention, the antigen derived from the classical non-viscous M. viscosus strain is an inactivated preparation of the classical non-viscous M. viscosus strain. If the antigen derived from the classical viscosus M. viscosus strain is present in the vaccine according to the first aspect, or in the second vaccine of the first aspect, the antigen may be in the form of an inactivated preparation of the classical viscosus M. viscosus strain.

[0012] In a second aspect, the present disclosure provides a vaccine comprising an antigenic M. viscosus component, wherein the antigenic M. viscosus component comprises an antigen derived from a variant M. viscosus strain, and the vaccine is used to protect fish from infection caused by classical non-viscous M. viscosus.

[0013] The vaccine according to the second aspect of the invention may be used to protect fish from infection caused by M. viscosus variants and M. viscosus classically non-viscosus.

[0014] According to a second aspect of the present invention, there is also disclosed a vaccine, wherein the antigenic component consists essentially of or consists of the antigen derived from the variant M. viscosus strain.

[0015] According to a second aspect of the present invention, there is also disclosed a vaccine, wherein the antigenic component consists essentially of or consists of the antigens derived from the variant M. viscosus strain and optionally an antigen derived from a classical M. viscosus strain.

[0016] The vaccine according to the second aspect of the invention may be co-administered with the second vaccine of the second aspect, wherein the second vaccine comprises antigens derived from a classically viscous strain of M. viscosus. Preferably, the second vaccine of the second aspect does not contain antigens derived from a classically non-viscous strain of M. viscosus.

[0017] In this second aspect, a vaccine is also disclosed, wherein the antigen derived from the variant M. viscosus strain is an inactivated preparation of the variant M. viscosus strain. If the antigen derived from the classical M. viscosus strain is present in the vaccine according to the second aspect, or in the second vaccine of the second aspect, the antigen may be in the form of an inactivated preparation of the classical M. viscosus strain.

[0018] In a third aspect, the present disclosure provides a vaccine comprising an antigenic M. viscosus component, wherein the antigenic M. viscosus component comprises an antigen derived from a variant M. viscosus strain, and the vaccine is used to protect fish from infection caused by classical non-viscous M. viscosus and classical M. viscosus.

[0019] The vaccine according to this third aspect may be used to protect fish from infection caused by M. viscosus variants, M. viscosus classically non-viscous and M. viscosus classically.

[0020] The vaccine according to this third aspect of the invention does not include antigens derived from a classical non-viscous strain of M. viscosus and does not include antigens derived from a classical viscosus strain of M. viscosus. In certain embodiments, the M. viscosus component of the vaccine according to this third aspect of the invention consists essentially of or consists of the antigens derived from the variant M. viscosus strain.

[0021] In the vaccine according to this third aspect of the invention, the antigen derived from the variant M. viscosus strain is an inactivated preparation of the variant M. viscosus strain.

[0022] In a fourth aspect, the present disclosure provides a vaccine comprising an antigenic M. viscosus component, wherein the antigenic M. viscosus component comprises an antigen derived from a classical non-viscous M. viscosus strain, and the vaccine is used to protect fish from infection caused by variant M. viscosus and classical M. viscosus.

[0023] The vaccine according to this fourth aspect may be used to protect fish from infection caused by M. viscosus variants, M. viscosus classically non-viscous and M. viscosus classically.

[0024] The vaccine according to this fourth aspect of the invention does not include antigens derived from variant M. viscosus strains and does not include antigens derived from classical M. viscosus strains. In certain embodiments, the M. viscosus component of the vaccine according to this fourth aspect of the invention consists essentially of or consists of said antigens derived from classical non-viscous M. viscosus strains.

[0025] In the vaccine according to this fourth aspect of the invention, the antigen derived from the classical non-adhesive M. viscosus strain is an inactivated preparation of the classical non-adhesive M. viscosus strain.

[0026] The composition according to the first, second, third and fourth aspects of the invention further contains a non-viscous M. aegypti antigen. In certain embodiments, the one or more non-viscous M. aegypti antigens are selected from the group consisting of: IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio (Aliivibrio) salmononicida, Yersinia ruckeri O1.

[0027] The compositions according to the first, second, third and fourth aspects of the invention are provided as water-in-oil emulsions.

[0028] Any of the compositions described above are preferably used in salmonids, most preferably Atlantic salmon (Salmo salar). In certain embodiments, the weight of the fish at the time of vaccination is about 15-200 grams.

[0029] The compositions disclosed herein are suitable for protecting the fish from infection, comprising reducing or eliminating at least one symptom of M. viscosus. In certain embodiments, the at least one symptom is mortality. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1is a photograph of a Western blot showing that different antibodies recognize classically viscose M. viscosus on the one hand and classically non-viscous M. viscosus and variant M. viscosus on the other hand.

[0031] Figure 2 Cumulative survival of fish vaccinated with classical viscosus, classical non-viscosus, and variant viscosus M. viscosus after challenge with classical non-viscosus M. viscosus is shown.

[0032] Figure 3 Cumulative survival of fish vaccinated with classical M. viscosus, classical M. non-viscosus, and M. variabilis after challenge with M. variabilis is shown.

[0033] Figure 4 Cumulative survival of fish vaccinated with classical and variant M. viscosus after challenge with classical M. viscosus is shown. DETAILED DESCRIPTION

[0034] In order to better understand the present invention, the following non-limiting definitions are provided:

[0035] When used in conjunction with a measurable numerical variable, the term "about" refers to the indicated value of the variable and all values ​​of the variable that are within the experimental error range of the indicated value (e.g., within a 95% confidence interval for the mean) or within 10% of the indicated value, whichever is greater.

[0036] The term "antigenic M. viscosus component" refers to one or more antigens derived from M. viscosus, including classically viscous strains, classically non-viscous strains, and variant strains.

[0037] "Antigens derived from pathogens including classical M. viscosus, classical non-m. viscosus, and variant M. viscosus" refers to inactivated preparations of the desired M. viscosus subtype, as well as whole bacterial extracts and fractions of extracts including, but not limited to, membrane / cell wall extracts.

[0038] "Classical M. viscidae" also known as "M. viscidae" or "classical M. viscidae" refers to strains of M. viscidae that form sticky adherent colonies when cultured on blood agar at 15°C for 48 hours in the presence of NaCl concentrations below 2.5%. M. viscidae typically forms gray colonies. Colonies of classical M. viscidae form sticky slime threads when the colonies are manipulated with a loop.

[0039] "Classical non-adhesive M. viscosus" refers to strains that are classified as classical M. viscosus based on the gyrB sequence, but these strains do not form sticky adherent colonies when cultured on blood agar at 15°C for 48 hours in the presence of NaCl concentrations below 2.5%.

[0040] Classical isolates of M. viscosus (both viscosus and non-viscosus) have conserved sequences in their corresponding gyrB genes. Thus, a classical isolate is an isolate that contains a subsequence in its corresponding gyrB sequence that is at least 96% identical (e.g., at least 97% or at least 98% identical) to SEQ ID NO: 1.

[0041] If two or more vaccines are administered within 15 minutes of each other, the two or more vaccines are "co-administered". Preferably, the two or more vaccines are administered within 10 minutes, or within 5 minutes, or within 4 minutes, or within 3 minutes, or within 2 minutes, or within 1 minute of each other.

[0042] "Mottia viscosus" without the preceding classic, variant, or non-viscous covers all three subtypes of Mottia viscosus.

[0043] The term "pharmaceutically acceptable" refers to a substance that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of subjects without excessive toxicity, irritation, allergic response, etc., commensurate with a reasonable benefit-risk ratio, and effective for its intended use.

[0044] The term "subject" refers to a fish to which administration of an adjuvant composition is desired.

[0045] The phrase "therapeutically effective amount" refers to the amount of an antigen or vaccine that will induce an immune response in a subject receiving the antigen or vaccine, which is sufficient to prevent or reduce the signs or symptoms of a disease caused by infection with a pathogen such as a virus or bacteria, including adverse health effects or complications thereof. Humoral immunity or cell-mediated immunity, or both humoral and cell-mediated immunity, can be induced. The immunogenic response to the vaccine can be assessed indirectly by measuring antibody titers, lymphocyte proliferation assays, or directly by monitoring signs and symptoms after stimulating with a wild-type strain. The protective immunity conferred by the vaccine can also be assessed by measuring the reduction of clinical signs, such as mortality, morbidity, overall physical condition, and overall health and performance of the subject.

[0046] The terms "treat," "treat," or "treating" refer to preventing the disorder, condition, or disease to which such term applies or preventing or reducing one or more symptoms of such disorder, condition, or disease.

[0047] The term "treatment" refers to the act of "treating" as defined above.

[0048] The term "vaccine" refers to a composition that elicits protective immunity in a subject.

[0049] "Preventing infection caused by M. viscosus" means reducing or eliminating at least one clinical sign caused by M. viscosus. The clinical sign includes skin ulceration, which may be followed by late-stage sepsis, or a combination thereof. In a particularly preferred embodiment, preventing infection caused by M. viscosus means reducing the mortality rate caused by M. viscosus.

[0050] "Variant M. viscosus":, as previously described, it has been determined that classical isolates of M. viscosus (both viscose and non-viscose) have conserved sequences in their corresponding gyrB genes. Thus, a classical isolate is an isolate that contains a subsequence in its corresponding gyrB sequence that is at least 98% identical to SEQ ID NO: 1. In contrast, in variant M. viscosus isolates, the corresponding subsequence of the gyrB sequence is less than 98% identical to SEQ ID NO: 1. Preferably, in variant M. viscosus isolates, the corresponding subsequence is 70-98% identical to SEQ ID NO: 1. Apart from the differences in the gyrB sequence, for the purposes of this application, antibodies raised in salmon against classical viscose M. viscosus strains under the conditions described in Example 1 do not recognize variant M. viscosus isolates.

[0051] In certain embodiments, the variant M. viscosus isolate has subsequences in its gyrB gene sequence that are at least 90% identical to SEQ ID NO:2, preferably at least 95% identical to SEQ ID NO:2, provided that these subsequences are no more than 98% identical to SEQ ID NO:1.

[0052] Motia viscosus

[0053] The inventors surprisingly found that antigens from variant strains of M. viscosus cross-protect against challenge with the classical non-viscosifying strain of M. viscosus and vice versa: antigens from the classical non-viscosifying strain of M. viscosus cross-protect against challenge with variant strains of M. viscosus.

[0054] Therefore, in a first aspect, the present application provides a vaccine comprising an antigenic M. viscosus component, the antigenic M. viscosus component comprising an antigen derived from a classical non-viscous M. viscosus strain, and the vaccine is used to protect fish from infection caused by variant M. viscosus. A vaccine is also disclosed, the vaccine comprising an antigenic M. viscosus component, the antigenic M. viscosus component comprising an antigen derived from a classical non-viscous M. viscosus strain, and the vaccine is used to protect fish from infection caused by variant M. viscosus, wherein the vaccine does not contain any variant M. viscosus antigens. These vaccines can be used to prevent infections caused by variant M. viscosus and classical non-viscous M. viscosus. In some of these vaccines, the antigenic M. viscosus component consists of antigens derived from a classical non-viscous M. viscosus strain.

[0055] The vaccine according to this first aspect may be combined with an antigen derived from classical viscosus M. viscosus. Such a vaccine may be used to prevent infection with M. viscosus, including classical non-viscous M. viscosus, M. viscosus variants and classical viscosus M. viscosus.

[0056] Alternatively, a vaccine containing an antigenic M. viscosus component comprising (or consisting of) an antigen derived from a classical non-viscous strain of M. viscosus may be co-administered with a vaccine containing an antigen derived from a classical viscosus strain of M. viscosus. This combination of co-administered vaccines may be used to protect fish in need thereof from infection caused by classical non-viscous M. viscosus, M. viscosus variants, and classical viscosus M. viscosus.

[0057] Antigens derived from a classical non-viscous strain of M. viscosus may be provided in the form of an inactivated classical non-viscous M. viscosus preparation, such as an inactivated whole organism. Methods of bacterial inactivation are well known and include, but are not limited to, incubation with formalin, BEI and / or beta propiolactone (BPL). Alternatively, the antigen may be a subunit, a whole cell extract of the classical non-viscous M. viscosus, or a fraction thereof, including but not limited to a membrane fraction.

[0058] Similarly, antigens derived from strains of the classical viscosus of M. viscosus may be inactivated classical viscosus M. viscosus preparations, such as inactivated whole organisms. Methods of bacterial inactivation are well known and include, but are not limited to, incubation with formalin, BEI and / or beta propiolactone (BPL). Alternatively, the antigen may be a subunit, a whole cell extract of the classical viscosus M. viscosus or a fraction thereof, including but not limited to a membrane fraction.

[0059] Antigens derived from a classical viscous strain of M. viscosus and / or a classical non-viscous strain of M. viscosus may be provided in the form of attenuated bacteria. Methods for preparing live attenuated bacteria are well known in the art and include, but are not limited to, culture passaging.

[0060] The doses of the antigens in the vaccine, the classical non-viscous M. viscosus component and the classical viscosus M. viscosus component, may vary. Thus, for example, one dose of the vaccine may contain at least 1x10 6 cells / dose of the classic non-viscous Morpholipids fraction. Without restriction, one dose could contain approximately 5x10 6 cells / dose, about 1x10 7 Cells / dose, about 5x10 7 cells / dose, about 1x10 8 cells / dose, 3x10 8 cells / dose, 5x10 8 cells / dose, 1x10 9 cells / dose. Doses can also contain 1x10 6 cells / dose up to 1x10 7 cells / dose, or 5x10 6 cells / dose up to 5x10 7 cells / dose, or 1x10 7 cells / dose up to 1x10 8 cells / dose, or 5x10 7 cells / dose up to 5x10 8 cells / dose, or 1x10 8 cells / dose up to 1x10 9 cells / dose.

[0061] Similarly, the amount of the antigenically classically viscose M. viscosus component present in one dose of a vaccine (whether the same vaccine as the vaccine containing the antigenically variant M. viscosus component, or a second vaccine) may be at least 1 x 10 6 cells / dose of the classically viscoelastic M. viscosus component. Thus, for example, a dose of vaccine may contain at least 1x10 6 cells / dose of the classically sticky M. viscosa fraction. Without restriction, one dose could contain approximately 5x10 6 cells / dose, about 1x10 7 Cells / dose, about 5x10 7 cells / dose, about 1x10 8 cells / dose, 3x10 8 cells / dose, 5x108 cells / dose, 1x10 9 cells / dose. Doses can also contain 1x10 6 cells / dose up to 1x10 7 cells / dose, or 5x10 6 cells / dose up to 5x10 7 cells / dose, or 1x10 7 cells / dose up to 1x10 8 cells / dose, or 5x10 7 cells / dose up to 5x10 8 cells / dose, or 1x10 8 cells / dose up to 1x10 9 cells / dose.

[0062] In a second aspect, the present application provides a vaccine comprising an antigenic viscosus component, the antigenic viscosus component comprising an antigen derived from a variant viscosus strain, and the vaccine is used to protect fish from infection caused by the classical non-viscous viscosus. A vaccine is also disclosed, the vaccine comprising an antigenic viscosus component, the antigenic viscosus component comprising an antigen derived from a variant viscosus strain, and the vaccine is used to protect fish from infection caused by the classical non-viscous viscosus, wherein the vaccine does not contain any classical non-viscous viscosus antigens. These vaccines can be used to prevent infections caused by classical non-viscous viscosus and variant viscosus. In some of these vaccines, the antigenic viscosus component consists of antigens derived from a variant viscosus strain.

[0063] The vaccine according to this second aspect may be combined with an antigen derived from classical viscosus M. viscosus. Such a vaccine may be used to prevent infection with M. viscosus, including M. viscosus variants, M. viscosus classical non-viscous and M. viscosus classical viscosus.

[0064] Alternatively, a vaccine containing an antigenic M. viscosus component comprising (or consisting of) an antigen derived from a strain of M. viscosus variant, as described above, may be co-administered with a vaccine containing an antigen derived from a strain of M. viscosus classically viscosus. This combination of co-administered vaccines may be used to protect fish in need thereof from infection caused by M. viscosus variant, M. viscosus classically non-viscosus, and M. viscosus classically viscosus.

[0065] The antigen derived from a variant strain of M. viscosus may be an inactivated variant M. viscosus preparation, such as an inactivated whole organism. Methods of bacterial inactivation are well known and include, but are not limited to, incubation with formalin, BEI and / or beta propiolactone (BPL). Alternatively, the antigen may be a whole cell extract of M. viscosus variant or a fraction thereof, including but not limited to a membrane fraction.

[0066] Similarly, the antigen derived from the classical viscosus strain of M. viscosus can be an inactivated classical viscosus M. viscosus preparation, such as an inactivated whole organism. Methods of bacterial inactivation are well known and include, but are not limited to, incubation with formalin, BEI and / or beta propiolactone (BPL). Alternatively, the antigen can be a whole cell extract of classical viscosus M. viscosus or a fraction thereof, including but not limited to a membrane fraction.

[0067] Antigens derived from the classical viscosus strain of M. viscosus and / or variant strains of M. viscosus may be provided in the form of attenuated bacteria. Methods for preparing live attenuated bacteria are well known in the art and include, but are not limited to, culture passaging.

[0068] The doses of the antigenically variable M. viscosus component and the classical M. viscosus component in the vaccine may vary. Thus, for example, a dose of the vaccine may contain at least 1x10 6 cells / dose of the variable viscosus component. Without limitation, one dose can contain approximately 5x10 6 cells / dose, about 1x10 7 Cells / dose, about 5x10 7 cells / dose, about 1x10 8 cells / dose, 3x10 8 cells / dose, 5x10 8 cells / dose, 1x10 9 cells / dose. Doses can also contain 1x10 6 cells / dose up to 1x10 7 cells / dose, or 5x10 6 cells / dose up to 5x10 7 cells / dose, or 1x10 7 cells / dose up to 1x10 8 cells / dose, or 5x10 7 cells / dose up to 5x10 8 cells / dose, or 1x10 8 cells / dose up to 1x10 9 cells / dose.

[0069] Similarly, the amount of the antigenic classically viscose M. viscosus component present in a dose of a vaccine (whether the same vaccine as the vaccine containing the antigenic classically non-viscous M. viscosus component, or a second vaccine) may be at least 1 x 10 6 cells / dose of the classically viscoelastic M. viscosus component. Thus, for example, a dose of vaccine may contain at least 1x10 6 cells / dose of the classically sticky M. viscosa fraction. Without restriction, one dose could contain approximately 5x10 6 cells / dose, about 1x10 7 Cells / dose, about 5x10 7 cells / dose, about 1x10 8 cells / dose, 3x10 8 cells / dose, 5x10 8 cells / dose, 1x10 9 cells / dose. Doses can also contain 1x10 6 cells / dose up to 1x10 7 cells / dose, or 5x10 6 cells / dose up to 5x10 7 cells / dose, or 1x10 7 cells / dose up to 1x10 8 cells / dose, or 5x10 7 cells / dose up to 5x10 8 cells / dose, or 1x10 8 cells / dose up to 1x10 9 cells / dose.

[0070] The inventors have also surprisingly found that vaccination with variant M. viscosus antigens provides cross protection against challenge with classical M. viscosus, but not vice versa (i.e. vaccination with classical M. viscosus antigens does not protect against challenge with variant M. viscosus). Thus, in a third aspect, the present invention provides a vaccine comprising an antigenic M. viscosus component comprising antigens derived from a strain of M. viscosus variant, the vaccine being useful for protecting fish from infection caused by classical M. viscosus.

[0071] As discussed above, antigens derived from variant strains of M. viscosus may also be used to protect fish from infections caused by classical non-viscous M. viscosus. Thus, the present disclosure also provides a vaccine comprising an antigenic M. viscosus component comprising antigens derived from variant strains of M. viscosus, the vaccine being useful for protecting fish from infections caused by classical M. viscosus and from infections caused by classical non-viscous strains of M. viscosus. Thus, the present disclosure also provides a vaccine comprising an antigenic M. viscosus component comprising antigens derived from variant strains of M. viscosus, the vaccine being useful for protecting fish from infections caused by classical M. viscosus and from infections caused by classical non-viscous strains of M. viscosus, wherein the antigenic M. viscosus component lacks antigens derived from classical and classical non-viscous strains of M. viscosus.

[0072] In a fourth aspect, the present invention provides a vaccine comprising an antigenic M. viscosus component, wherein the antigenic M. viscosus component comprises an antigen derived from a classical non-viscous M. viscosus strain, and the vaccine is used to protect fish from infection caused by classical non-viscous M. viscosus.

[0073] As discussed above, antigens derived from a classical non-adhesive strain of M. viscosus may also be used to protect fish from infection caused by variant M. viscosus. Thus, the present disclosure also provides a vaccine comprising an antigenic M. viscosus component comprising antigens derived from a classical non-adhesive strain of M. viscosus, the vaccine being useful for protecting fish from infection caused by classical M. viscosus and from infection caused by variant M. viscosus strains. Thus, the present disclosure also provides a vaccine comprising an antigenic M. viscosus component comprising antigens derived from a classical non-adhesive strain of M. viscosus, the vaccine being useful for protecting fish from infection caused by classical M. viscosus and from infection caused by variant M. viscosus strains, wherein the antigenic M. viscosus component lacks antigens derived from classical and variant M. viscosus strains.

[0074] The dosages of classically viscose, classically non-viscous and variant viscose M. aureus antigens for use in vaccines described in conjunction with the first and second aspects are also applicable to vaccines according to these third and fourth aspects of the invention.

[0075] Additional antigens

[0076] In all four aspects, the corresponding antigenic component of the vaccine described herein (or the secondary vaccine) may contain one or more additional non-mucosa antigens, as described below.

[0077] Such antigens may be derived from bacterial sources, viral sources, other parasitic sources and / or fungal sources. These additional antigens may be inactivated organisms as described below, or the antigens may be derived from these organisms, including recombinantly prepared antigens.

[0078] Multivalent vaccines containing antigens from classical fish pathogens other than M. viscosus are well known in the art and are commercially available. In addition, representative isolates of relevant fish pathogens are available from a variety of sources.

[0079] In a specific embodiment of the invention, the antigen from a bacterial source is selected from the group consisting of the following live, attenuated or killed bacterial species: Piscirickettsias sp., Aeromonas sp., Vibrio sp., Aliivibrio sp., Listonella sp., Tenacibaculum sp., Pasteurella sp., Photobacterium sp., Flavobacterium sp., Yersinia sp., Renibacterium sp., Streptococcus sp., Lactococcus sp., Leuconostoc sp., Bifidobacterium sp., Pediococcus sp., Brevibacterium sp., Edwardsiella sp. sp.), Francisella sp., Pseudomonas sp., Cytophaga sp., Nocardia sp., Mycobacterium sp., parts or subunits of these bacteria, and any combination thereof.

[0080] Isolates of such bacteria are available, for example, from the LGC Promochem / American Type Culture Collection ATCC Storage and Distribution Center (ATCC), including strains of Aeromonas salmonicida (ATCC 33658), Vibrio salmonicida (ATCC 43839), Vibrio anguillarum serotypes O1 (ATCC 43305) and O2 (ATCC 19264). In addition, cultures of Piscirickettsias salmonids have been deposited at the Health Protection Agency European Collection of Cell Cultures (ECACC), Porton Down, Salisbury, Wiltshire (UK), SP4 0JG UK, on ​​June 9, 2006, with the following accession numbers: 06050901, 06050902, 06050903 and 07032110.

[0081] Other specific embodiments relate to a vaccine in which the antigenic material obtained from a viral source other than a fish virus as defined above is from a virus selected from the group consisting of: Viral Hemorrhagic Septicemia Virus (VHSV), Infectious Hematopoietic Necrosis Virus (IHNV), Infectious Pancreatic Necrosis Virus (IPNV), Infectious Salmon Anemia Virus (ISAV), Salmon Pancreatic Disease Virus (SPDV), Iridovirus, Nodavirus, Fish Myocarditis Virus (PMCV) and Heart and Skeletal Muscle Inflammation Virus (HSMIV). These antigens may include modified live or inactivated organisms, parts or subunits of any of these viruses, DNA vaccines and / or combinations thereof. Representative species of such viruses are available to the skilled artisan, for example from the following deposits: infectious pancreatic necrosis virus (IPNV, ATCC VR-1318, country of origin: unknown), viral hemorrhagic septicemia virus (VHSV, ATCC VR-1389, country of origin: Denmark); infectious hematopoietic necrosis virus (IHNV, ATCC VR-1392, country of origin: United States (USA)); pancreatic necrosis virus; infectious salmon anemia (ISA) virus (ATCC VR-1554,, country of origin: Canada). The applicant has previously made patent deposits for the following virus species: heart and skeletal muscle infection virus (HSMIV, patent deposit number ECACC 04050401, country of origin: Norway).

[0082] In a more specific embodiment, the antigenic material obtained from a viral source other than a fish virus as defined above is from the group consisting of: a glycoprotein of viral hemorrhagic septicemia virus (VHSV), a nucleoprotein of viral hemorrhagic septicemia virus (VHSV), a glycoprotein of infectious hematopoietic necrosis virus (IHNV), a nucleoprotein structural protein of infectious pancreatic necrosis virus (IPNV), an antigenic fragment of any of these proteins, and combinations thereof.

[0083] In other embodiments, the antigenic material from another parasitic source is from a source selected from Lepeophtheirus Sp., Caligus Sp., and Ichthyophthirius Sp, parts of any of these parasites, and combinations thereof. In yet other embodiments, the antigenic material is from a fungal source selected from the group consisting of Saprolegnia Sp., Branchiomyces sanguinis, Branchiomyces demigrans, and Icthyophonus hoferi.

[0084] In certain embodiments, the additional antigens included in the vaccines of the invention and / or the second vaccine containing the classical viscoelastic Agrobacterium are selected from the group consisting of IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio salmonicida (Alternaria salmonicida), Yersinia ruckeri O1.

[0085] In other embodiments, the additional antigens included in the vaccine of the invention and / or the second vaccine containing the classical viscosus A. viscosus are selected from the group consisting of IPNV, Aeromonas salmonicida, Vibrio anguillarum serotypes 1 and 02, and Vibrio salmonicida (Alternaria salmonicida).

[0086] Excipients and adjuvants

[0087] Vaccine of the present invention may further comprise suitable pharmaceutical carriers and / or adjuvants. Pharmaceutical carriers may be sterile liquids, such as water or buffered solutions, such as physiological saline and glucose and glycerol aqueous solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talcum, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. If desired, compositions may also contain a small amount of wetting agent or emulsifier or pH buffer. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EWMartin. Formulations should be suitable for mode of administration.

[0088] Suitable carriers will be apparent to those skilled in the art and will depend to a great extent on the route of administration. Other components that may be present in the present invention are adjuvants, preservatives, surfactants, chemical stabilizers, suspending agents or dispersants. Typically, stabilizers, adjuvants and preservatives are optimized to determine the formulation that is most effective in the target subject.

[0089] In currently preferred embodiments, the vaccine comprises an adjuvant. Suitable adjuvants include, but are not limited to, oils. The vaccines disclosed herein can be formulated as an oil-in-water emulsion, or more preferably, a water-in-oil emulsion. Other formulations can also be prepared, such as water-in-oil-in-water (W / O / W). In addition, the vaccine can comprise one or more suitable surface-active compounds or emulsifiers, such as and Adjuvants such as interleukins, CpG, and glycoproteins may also be used.

[0090] Vaccines can also include "vehicles". Vehicles are devices to which antigens adhere without being covalently bound. Such vehicles are biodegradable nano / micro particles or capsules, liposomes, vesicles, micelles, multiple emulsions and macromolecular sols (macrosol) of PLGA (poly-lactide-co-glycolic acid), alginate or chitosan, all of which are known in the art. A special form of such vehicles is so-called ISCOMs, in which the antigen is partially embedded in the vehicle (European Patents EP 109.942, EP 180.564 and EP 242.380).

[0091] In certain embodiments, the vaccines described herein are formulated as water-in-oil emulsions. Preferably, the oil is mineral oil.

[0092] The vaccines described herein can be administered to salmonids by a variety of routes, including but not limited to intraperitoneal, intramuscular, oral, and by immersion. Preferably, the vaccine is administered in microdoses by injection, such that the volume of one dose is less than 500 μl, or less than 400 μl, or less than 300 μl, or less than 200 μl or about 100 μl, or less than 100 μl, or about 50 μl or about 25 μl.

[0093] Vaccines disclosed herein can be used to protect a variety of salmonid species from infection. Suitable salmonids include, but are not limited to, Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus nerka), sockeye salmon (Oncorhynchus nerka), Chinook salmon (Oncorhynchus tshawytscha), and other species.

[0094] Salmonids of different ages (or weights) can be vaccinated according to the present invention. In certain embodiments, the weight of the salmonids at the time of vaccination is between about 15 and about 200 grams. Thus, the weight of the salmonids at the time of vaccination can be between about 25 and about 150 grams, or between about 40 and about 110 grams, or between about 50 and about 100 grams.

[0095] The invention will now be described in the following non-limiting examples.

[0096] Examples

[0097] Example 1: Salmonella polyclonal antibodies raised against classically viscose M. viscosus do not recognize variant and classically non-viscose M. viscosus.

[0098] Materials and Methods Western Blot:

[0099] Ten strains of M. viscosus were studied using Western blotting. The origin and year of the isolates, gyrB variant or classical type, and viscosity phenotype associated with viscosity are listed in Table 1. The isolate number also corresponds to the lane number in the Western blot membrane.

[0100] Table 1. Overview of M. viscosus strains included in the study. Bacteria were not inactivated prior to sample preparation under reducing conditions.

[0101]

[0102] * When grown on blood agar at 15°C, 2% NaCl for 48 hours.

[0103] # Adapted from Grove et al., Dis Aquat Org, 93:51-61, 2010.

[0104] Antibodies / conjugates used:

[0105] 1) Monoclonal mouse anti-trout / salmon IgM antibody, clone 4C10

[0106] 2) Polyclonal rabbit anti-mouse HRP conjugated (Catalog No. P0260, Dako)

[0107] 3) Conjugated Precision Protein HRP (Catalog No. 161-0380, Bio Rad)

[0108] 4) Polyclonal salmonid a-variant M. viscosa antibodies - generated during the project

[0109] 5) Polyclonal salmonid A-classical M. viscosa antibodies - generated during the course of the project

[0110] 6) Polyclonal rabbit anti-Motiella muciniphila antibody - generated during the project

[0111] 7) Polyclonal rabbit anti-classical M. viscosus antibody - generated during the project

[0112] 8) Conjugated pig anti-rabbit HRP (Catalog No. P0217, Dako)

[0113] Table 2. Materials list.

[0114]

[0115] Sample preparation:

[0116] Bacterial cultures of each strain were spread on blood agar plates. The plates were incubated at 15°C. After two days of incubation, colonies from the agar plates were inoculated into 25 cm 2 Place cells in 10 ml growth medium in flask and incubate at 15 °C and 100 rpm.

[0117] After incubation, measure OD, centrifuge 2 x 1 ml and aspirate supernatant.

[0118] Prepare 10 bacterial pellet samples for Western blotting. All pellets were dissolved in reducing sample buffer, incubated at 100°C for 10 minutes and frozen until further analysis. On the day of SDS-PAGE, samples were diluted / normalized using freshly prepared sample buffer to obtain a theoretical OD of 2 (calculated based on the OD at harvest) before use.

[0119] SDS-PAGE and WB:

[0120] Add 10 μl of each sample to a lane on the gel (6 μl for molecular weight marker-lane). Run four parallel gels.

[0121] The gel was run at 250 V for approximately 3 minutes and then at 160 V for approximately 50 minutes. An equal volume of PRECISION PLUS PROTEIN TM All blue standards and PRECISION PLUS PROTEIN TM Unstained standards were combined (3-5 μl each) and used as molecular weight standards. Four gels were prepared to prepare four blots; two treated with salmon plasma and two with rabbit anti-M. viscosus antibodies. The gels were then activated on gel-doc for 45 seconds, imaged and transferred to TURBO TM Transfer blotting system (Bio-Rad) using a 7-minute turbo program. Immediately transfer the blot to blocking buffer (5% skim milk in TBST (TBS™)). Block overnight at 2-6°C.

[0122] The blots were then incubated with antibodies as described in Tables 3 and 4. HRP was added along with other HRP-conjugated antibodies to visualize molecular weight standards. The blots were washed 3x 10 min with TBST between each incubation (TBS was used in the final wash step before substrate incubation) and then washed with CLARITY TM Western substrate was incubated for 5 min and finally exposed on a gel-doc imaging system (Bio-Rad).

[0123] Table 3. Antibodies, dilutions and incubation times for blots incubated with salmon antibodies. RT = room temperature

[0124]

[0125] Table 4. Antibodies, dilutions and incubation times for blots incubated with rabbit antibodies. RT = room temperature

[0126]

[0127] The experimental results are Figure 1 . The figure shows that classical viscoelastic M. viscosus and variant M. viscosus are recognized by different antibodies. More specifically, these results show that antibodies raised in salmon against classical viscoelastic M. viscosus do not recognize classical non-viscous M. viscosus and variant M. viscosus. Surprisingly, it was also shown that classical non-viscous M. viscosus and variant M. viscosus are recognized by the same antibody.

[0128] Example 2: Inoculation with variant M. viscosus produces cross-protection against challenge with classical non-viscous M. viscosus, whereas inoculation with classical viscosus M. viscosus does not produce cross-protection

[0129] Materials and methods

[0130] Bath challenge studies were comparative, investigator-blind, negative-controlled, and randomized laboratory experiments. The study investigated the efficacy of different injectable vaccines containing oil adjuvants in protecting Atlantic salmon from experimental infection with different isolates of M. viscosus. Experimental vaccines containing field isolates of classical non-viscous M. viscosus and M. viscosus variants adjuvanted with water-in-oil (W / O) emulsions were administered intraperitoneally by co-injection with commercial vaccines according to the manufacturer's instructions. Both commercial vaccines contained classical viscosus strains of M. viscosus. A negative control group was included. The average weight of the fish was approximately 25 g at the time of vaccination.

[0131] Before the fish were transferred to seawater and challenged, they were exposed to continuous illumination (24:0) for silvering. Light manipulation was started about 6 weeks before the challenge with the classic non-viscous strain of M. viscosus. 13 weeks after vaccination, the challenge was carried out by immersion. For biomass considerations, each challenge isolate was challenged in two 500L fish tanks, and the results from the same repeated fish tanks were combined. Each group had a total of about 60 fish (30 fish / group per fish tank) to receive the challenge of each challenge isolate.

[0132] One week prior to challenge, fish were transferred to the disease facility and equally distributed into two replicate 500 L aquaria for each challenge isolate and acclimated to seawater of 34‰ salinity, 8°C, 24:0 light regime, which were the environmental parameters during the challenge period. Challenge material was freshly cultured from frozen bacterial stocks in shake flask medium based on yeast extract at 12°C for two days. Fish were challenged by reducing the water volume in the aquaria before adding the challenge material directly to the aquaria.

[0133] Challenge was performed 13 weeks after vaccination. Fish were observed daily after challenge and fish that developed ulcers were euthanized and recorded as mortality in the mortality log. Efficacy was evaluated by statistical comparison of protection against mortality and ulcers between the vaccinated group and the negative control group after challenge. The study fish were unvaccinated, free of clinical disease, and had a valid health certificate. Injured or deformed fish were excluded from the study.

[0134] The experimental results are Figure 2 Displayed in.

[0135] It can be seen that ten days after the challenge of the fish with the classical non-adhesive M. viscosus strain, the group treated with PBS as well as the group treated with the commercial vaccine (both containing the classical adhesive M. viscosus antigens) resulted in almost 100% mortality, thus supporting the validity of the challenge model. There were no statistically significant differences between the three groups.

[0136] In contrast, groups vaccinated with a composition comprising either the classical non-adhesive M. viscosa antigen or the variant antigens showed only about 70% mortality (no significant difference between the two groups, but p<0.0001 compared to groups vaccinated with PBS or commercial vaccine).

[0137] These results indicate that the classically viscoelastic M. viscosus antigens do not cross-protect against challenge with the classically non-viscous M. viscosus strain. The classically non-viscous antigens protect against challenge with the classically non-viscous M. viscosus strain. Surprisingly, the variant M. viscosus antigens cross-protect against challenge with the classically non-viscous M. viscosus strain.

[0138] Example 3: Inoculation with classical non-adhesive M. viscosus provides cross-protection against challenge with variant M. viscosus, whereas inoculation with classical adhesive M. viscosus does not provide cross-protection

[0139] Materials and methods were the same as in Example 2, except that the M. viscosus isolate was used as the challenge strain.

[0140] The experimental results are Figure 3 Displayed in.

[0141] It can be seen that 22 days after the challenge of the fish with the classical non-adhesive M. viscosus strain, the group treated with PBS as well as the group treated with the commercial vaccine (both containing the classical adhesive M. viscosus antigens) resulted in about 60 to 70% mortality, indicating that the challenge model is effective. There were no statistically significant differences between the three groups.

[0142] In contrast, groups vaccinated with a composition comprising either the classical non-adhesive M. viscosa antigen or the variant antigens showed only about 20-35% mortality (no significant difference between the two groups, but p<0.0001 compared to groups vaccinated with PBS or commercial vaccine).

[0143] These results indicate that the classically viscoelastic M. viscosus antigens do not cross-protect against challenge with variant M. viscosus strains. Variant antigens protect against challenge with variant M. viscosus strains. Surprisingly, the classically non-viscous M. viscosus antigens cross-protect against challenge with variant M. viscosus strains.

[0144] Example 4: Vaccination with variant M. viscosus to prevent challenge with classic M. viscosus.

[0145] Materials and methods:

[0146] The cross-protective efficacy of a monovalent oil-adjuvanted (W / O emulsion) M. viscosus variant vaccine was evaluated in an investigator-blind, negative-controlled, randomized laboratory experiment.

[0147] One group was administered a monovalent variant M. viscosus vaccine by intraperitoneal injection. The second group was vaccinated with a vaccine containing the classical M. viscosus, and the negative control group was injected with phosphate buffered saline (PBS). During the immunization period, the fish were kept in a 500 L fish tank under 15°C fresh water and exposed to a continuous light regimen (24:0 light: dark) for approximately 6 weeks to silver before seawater bath challenge. The challenge material (classical M. viscosus isolate) was freshly cultured from frozen bacterial stocks and shaken in a shake flask medium based on yeast extract at 12 degrees Celsius for two days. The fish were challenged by reducing the water volume in the fish tank before adding the challenge material directly to the fish tank.

[0148] After an immunization period of approximately 9 weeks, challenges were performed by immersion. Challenges were designed to investigate the cross-protective efficacy of a monovalent vaccine containing inactivated antigens of variant M. viscosus against classical M. viscosus isolates. Approximately one week prior to challenge, fish were transferred to the disease facility and groups were equally matched to parallel 500 L aquaria and gradually adapted to seawater at 8 °C and 34‰ salinity. To reduce biomass density at challenge, each challenge isolate of each group was challenged in two replicate aquaria and the results from both aquaria were combined. Fish were observed daily after challenge and fish that developed ulcers were euthanized and recorded as mortality in the mortality log. Efficacy was evaluated by statistical comparison of protection against mortality and ulcers between vaccinated groups and negative controls after challenge. Study fish were not vaccinated, had no clinical disease, and held valid health certificates. Injured or deformed fish were excluded from the study.

[0149] The results are Figure 4 The groups treated with PBS showed a mortality rate of over 40% 22 days after challenge. In contrast, vaccination with either the classical M. viscosus antigen or the variant M. viscosus antigen resulted in a statistically significant decrease in cumulative mortality by day 22 after challenge, to approximately 10 and 75, respectively. These results indicate that vaccination with the variant M. viscosus antigens may cross-protect against challenge with the classical M. viscosus. Given that the responses of the groups vaccinated with the variant M. viscosus antigens were similar to those of the groups vaccinated with the classical non-viscous M. viscosus antigens, these results also strongly suggest that vaccination with the classical non-viscous M. viscosus antigens may cross-protect against challenge with the classical M. viscosus.

[0150] All publications, both patent publications and non-patent publications cited in this specification are indicative of the technical level of those skilled in the art to which the invention pertains. All of these publications are incorporated herein by reference in their entirety to the same extent as if each individual publication was expressly and individually indicated to be incorporated by reference.

[0151] Although the present invention has been described herein with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present invention. Therefore, it should be understood that many modifications may be made to the illustrative embodiments and other arrangements may be designed without departing from the spirit and scope of the present invention as defined by the following claims.

Claims

1. A vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises antigens derived from a classical non-viscous M. viscosa strain, wherein the vaccine is useful for protecting fish from infection caused by variant M. viscosa.

2. The vaccine according to claim 1, for protecting fish against infections caused by variant M. viscosus and by classical non-viscous M. viscosus.

3. The vaccine according to claim 1 or 2, wherein the vaccine does not comprise antigens derived from a strain of M. viscosus variants.

4. The vaccine of claim 3, wherein the antigenic M. viscosus component consists of the antigens derived from the classically non-viscous M. viscosus strain.

5. The vaccine according to any one of claims 1 to 3, wherein the vaccine is co-administered with a second vaccine comprising an antigen derived from a classically viscoelastic strain of M. viscosus.

6. The vaccine of claim 5, wherein the second vaccine does not contain antigens derived from a strain of M. viscosus variants.

7. The vaccine according to any one of claims 1 to 3, for protecting fish from infection caused by variant M. viscosus, classical M. viscosus and classical non-viscous M. viscosus, wherein the M. viscosus antigenic compound further comprises an antigen derived from a classical M. viscosus strain.

8. The vaccine according to claim 7, wherein the antigenic M. viscosus component consists of the antigens derived from the classical non-viscous M. viscosus strain and the antigens derived from the classical viscosus M. viscosus strain.

9. The vaccine according to any one of claims 1 to 8, wherein the antigen derived from the classical non-adhesive M. viscosus strain is an inactivated preparation of the classical non-adhesive M. viscosus strain.

10. A vaccine comprising an antigenic M. viscosus component comprising antigens derived from a variant M. viscosus strain, for protecting fish from infection caused by classical non-viscous M. viscosus.

11. The vaccine according to claim 10, for use in protecting fish against infections caused by M. viscosus variants and by M. viscosus classically non-viscosus.

12. The vaccine of claim 10 or 11, wherein the vaccine does not comprise antigens derived from a classically non-viscous strain of M. viscosus.

13. The vaccine of claim 12, wherein the antigenic M. viscosus component consists of the antigens derived from the variant M. viscosus strain.

14. The vaccine according to any one of claims 10 to 12, wherein the vaccine is co-administered with a second vaccine comprising an antigen derived from a classically viscoelastic strain of M. viscosus.

15. The vaccine of claim 14, wherein the second vaccine does not contain antigens derived from a classically non-viscous strain of M. viscosus.

16. The vaccine according to any one of claims 11 to 13, for use in protecting fish from infection caused by M. viscosus variants, M. viscosus classically and M. viscosus classically non-, wherein the M. viscosus antigenic compound further comprises an antigen derived from a strain of M. viscosus classically.

17. The vaccine of claim 16, wherein the antigenic M. viscosus component consists of the antigens derived from the variant M. viscosus strain and the antigens derived from the classical M. viscosus strain.

18. The vaccine according to any one of claims 10 to 17, wherein the antigen derived from the variant M. viscosus strain is an inactivated preparation of the variant M. viscosus strain.

19. The vaccine according to any one of claims 5 to 9 or 14 to 18, wherein the antigen derived from the classical M. viscosus strain is an inactivated preparation of the classical M. viscosus strain.

20. A vaccine comprising an antigenic M. viscosus component comprising antigens derived from a variant M. viscosus strain, for protecting fish from infection caused by both classical non-viscous M. viscosus and classical M. viscosus.

21. The vaccine according to claim 20, for use in protecting fish against infection caused by M. variabilis.

22. A vaccine according to claim 20 or claim 21, wherein the vaccine does not include antigens derived from a classically non-viscous strain of M. viscosus and the vaccine does not include antigens derived from a classically viscous strain of M. viscosus.

23. The vaccine according to any one of claims 20 to 22, wherein the antigenic M. viscosus component consists of the antigens derived from the variant M. viscosus strain.

24. The vaccine according to any one of claims 20 to 23, wherein the antigen derived from the variant M. viscosus strain is an inactivated preparation of the variant M. viscosus strain.

25. A vaccine comprising an antigenic M. viscosus component comprising antigens derived from a classical non-viscous strain of M. viscosus, for protecting fish from infection caused by M. viscosus variants and M. viscosus classically.

26. The vaccine according to claim 25, for use in protecting fish against infection caused by classical non-adhesive M. viscosus.

27. A vaccine according to claim 25 or claim 26, wherein the vaccine does not include antigens derived from a variant M. viscosus strain and the vaccine does not include antigens derived from a classical M. viscosus strain.

28. The vaccine according to any one of claims 25 to 27, wherein the antigenic M. viscosus component consists of the antigens derived from the classical non-viscous M. viscosus strain.

29. The vaccine according to any one of claims 25 to 28, wherein the antigen derived from the classical non-viscous M. viscosus strain is an inactivated preparation of the variant M. viscosus strain.

30. A vaccine according to any one of claims 1 to 29 comprising one or more non-viscous M.

31. The vaccine of claim 30, wherein the second vaccine comprises one or more non-viscous M.

32. The vaccine of claim 30 or 31, wherein the one or more non-viscous M. salmonicida antigens are selected from the group consisting of IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum 01, 02, Vibrio (Aliivibrio) salmononicida, Yersinia ruckeri 01.

33. The vaccine according to any one of claims 1 to 32, wherein the vaccine is in the form of a water-in-oil emulsion.

34. The vaccine of any one of claims 1 to 33, wherein the fish is a salmonid.

35. The vaccine of claim 34, wherein the salmonid is Atlantic salmon (Salmo salar).

36. The vaccine according to any one of claims 1 to 35, wherein the fish weighs 15-200 grams.

37. The vaccine of any one of claims 1 to 36, wherein the vaccine is administered peritoneally.

38. The vaccine of any one of claims 1 to 37, wherein protecting the fish from infection comprises reducing or eliminating at least one symptom of M. viscosus.

39. The vaccine of any one of claims 1 to 38, wherein protecting the fish from infection comprises reducing mortality caused by M. viscosus.

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