Vaccine against Moritella viscosa

Abstract

The present invention provides a vaccine against a new isolate of M.viscosa and a method of using the vaccine.

Description

Technical Field

[0001] The present invention generally relates to the field of aquaculture vaccines.

Background Art

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

[0003] Moritella viscosa (formally Vibrio viscosus) is the main pathogen of winter ulcer disease (Lovoll et al., 2009, Tunsjo et al., 2009, Bjornsson et al., 2011, Karlsen et al., 2017a, Karlsen et al., 2017b). It is a Gram-negative, psychrophilic, facultative anaerobic bacterium capable of both fermentative and respiratory metabolism (Gudmundsdottir and Bjornsdottir, 2007, Tunsjo et al., 2009, Bjornsson et al., 2011). It is oxidase and catalase positive and requires salt for growth; colonies are yellowish translucent and generally viscous (Gudmundsdottir and Bjornsdottir, 2007), although non-viscous M. viscosa strains have also been isolated in recent years.

[0004] Vaccines exist that protect against classical viscous strains of M. viscosa. However, current commercially available vaccines are not effective against emerging strains that differ from classical strains in both genotype and / or phenotype. These emerging strains can be classified as variant types based on the gyrB sequence and classical non-viscous strains based on their non-viscous appearance after culturing on agar plates, in contrast to classical viscous M. viscosa strains that present adhesive colonies forming viscous threads when looped.

[0005] Therefore, there is a need for new vaccines and methods to effectively protect against emerging stocks as well as classical sticky stocks. SUMMARY OF THE INVENTION

[0006] In a first aspect, the present disclosure provides a vaccine for use in protecting fish against infections caused by mutant M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a classical non-sticky M. viscosa strain, thereby addressing these and other needs.

[0007] The vaccine according to the first aspect of the present invention can be used to protect fish against infections caused by mutant M. viscosa and classical non-sticky M. viscosa.

[0008] Also disclosed is a vaccine according to the first aspect of the present invention, wherein the antigen component consists essentially of or consists of an antigen derived from an antigen derived from a classical non-sticky M. viscosa strain.

[0009] Also disclosed is a vaccine according to the first aspect of the present invention, wherein the antigen component consists essentially of or consists of an antigen derived from a classical non-sticky M. viscosa strain and optionally an antigen derived from a classical sticky M. viscosa strain.

[0010] The vaccine according to the first aspect of the present invention can be co-administered with a second vaccine, the second vaccine of this first aspect comprising an antigen derived from a classical sticky M. viscosa strain. Preferably, the second vaccine does not contain an antigen derived from a mutant strain of M. viscosa.

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

[0012] In a second aspect, the present disclosure provides a vaccine comprising an antigenic M. viscosa component for use in protecting fish against infections caused by classical non-viscous M. viscosa, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain.

[0013] The vaccine according to the second aspect of the present invention can be used to protect fish against infections caused by mutant M. viscosa and classical non-viscous M. viscosa.

[0014] Also disclosed is a vaccine according to the second aspect of the present invention, wherein the antigen component consists essentially of or consists of an antigen derived from an antigen derived from a mutant M. viscosa strain.

[0015] Also disclosed is a vaccine according to the second aspect of the present invention, wherein the antigen component consists essentially of or consists of an antigen derived from a mutant M. viscosa strain and, optionally, an antigen derived from a classical viscous M. viscosa strain.

[0016] The vaccine according to the second aspect of the present invention may be co-administered with the second vaccine of the second aspect, and the second vaccine comprises an antigen derived from a classical viscous M. viscosa strain. Preferably, the second vaccine of the second aspect does not contain an antigen derived from a classical non-viscous strain of M. viscosa.

[0017] In this second aspect, a vaccine is also disclosed in which the antigen derived from the mutant M. viscosa strain is an inactivated preparation of the mutant M. viscosa strain. If an antigen derived from a classical viscous M. viscosa strain is present in the vaccine according to the second aspect or the second vaccine of the second aspect, the antigen can be in the form of an inactivated preparation of the classical viscous M. viscosa strain.

[0018] In a third aspect, the present disclosure provides a vaccine comprising an antigenic M. viscosa component for use in protecting fish against infections caused by classical non-viscous M. viscosa and classical viscous M. viscosa, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain.

[0019] The vaccine according to this third aspect can be used to protect fish against infections caused by mutant M. viscosa, classical non-viscous M. viscosa and classical viscous M. viscosa.

[0020] The vaccine according to this third aspect of the present invention does not contain an antigen derived from a classical non-viscous M. viscosa strain and does not contain an antigen derived from a classical viscous M. viscosa strain. In certain embodiments, the M. viscosa component of the vaccine according to this third aspect of the present invention consists essentially of or consists of an antigen derived from a mutant M. viscosa strain.

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

[0022] In a fourth aspect, the present disclosure provides a vaccine comprising an antigenic M. viscosa component for use in protecting fish against infections caused by mutant M. viscosa and classical viscous M. viscosa, wherein the antigenic M. viscosa component comprises an antigen derived from a classical non-viscous M. viscosa strain.

[0023] The vaccine according to this fourth aspect can be used for the protection of fish against infections caused by mutant M. viscosa, classical non-viscous M. viscosa, and classical viscous M. viscosa.

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

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

[0026] The compositions according to the first, second, third, and fourth aspects of the invention further contain non-M. viscosa antigens. In certain embodiments, said one or more non-M. viscosa antigens are selected from the group consisting of IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio (Aliivibrio) salmonicida, 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] All of the above compositions are preferably used in salmonids, most preferably in Atlantic salmon (Salmo salar). In certain embodiments, the weight of said fish is about 15 to about 200 grams at the time of vaccination.

[0029] The compositions disclosed herein are suitable for protecting the fish against infection and include the reduction or elimination of at least one symptom of M. viscosa. In certain embodiments, the at least one symptom is death.

Brief Description of the Drawings

[0030]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0031] For a better understanding of the present invention, the following non-limiting definitions are provided.

[0032] The term "about" or "approximately", when used in relation to a measurable numerical variable, refers to either the indicated value of the variable and all values of the variable that are within the experimental error of the indicated value (e.g., within the 95% confidence interval of the mean) or within 10 percent of the indicated value, whichever is the greater.

[0033] The term "antigenic M. viscosa component" refers to one or more antigens derived from M. viscosa, including classical viscous strains, classical non-viscous strains, and mutant strains.

[0034] "Antigens derived from a pathogen" including classical M. viscosa, classical non - adherent M. viscosa, and mutant M. viscosa refer to desired M. viscosa subtypes including, but not limited to, membrane / cell wall extracts, as well as inactivated preparations of whole bacterial extracts and fractions of the extracts.

[0035] "Classical M. viscosa" also refers to "adherent M. viscosa" or "classical adherent M. viscosa", and refers to M. viscosa strains that form adherent colonies with viscosity when cultured on blood agar at 15 °C for 48 hours at an NaCl concentration of less than 2.5%. M. viscosa usually forms grayish colonies. When the colonies are manipulated with a loop, the colonies of classical adherent M. viscosa form viscous mucus threads.

[0036] "Classical non - adherent M. viscosa" refers to M. viscosa strains that are classified as classical based on the gyrB sequence, but these strains do not form adherent colonies when cultured on blood agar at 15 °C for 48 hours at an NaCl concentration of less than 2.5%.

[0037] Classical isolates of M. viscosa (both adherent and non - adherent) have sequences conserved in their respective gyrB genes. Thus, classical isolates are isolates that contain a subsequence that is at least 96% identical (e.g., at least 97% or at least 98% identical) to SEQ ID NO: 1 in their respective gyrB sequences.

[0038] Two or more vaccines are "co - administered" if they are administered within 15 minutes of each other. 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.

[0039] "M. viscosa" without preceding classical, or mutant, or non - adherent encompasses all three subtypes of M. viscosa.

[0040] The term "pharmaceutically acceptable" refers to substances that are suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic response, etc., within the scope of sound medical judgment, are commensurate with a reasonable benefit / risk ratio, and are effective for their intended uses.

[0041] The term "subject" refers to fish for which administration of the adjuvant composition is desired.

[0042] "Therapeutically effective amount" refers to the amount of an antigen or vaccine that will induce an immune response in a subject that has received the antigen or vaccine and that is sufficient to prevent or reduce the signs or symptoms of a disease, including adverse health effects or complications thereof, caused by infection with a pathogen such as a virus or bacterium. Humoral immunity or cell-mediated immunity, or both humoral and cell-mediated immunity, may be induced. The immunogenic response to a vaccine can be evaluated, for example, indirectly through measurement of antibody titers, lymphocyte proliferation assays, or directly through monitoring of signs and symptoms after challenge with a wild-type strain. The protective immunity conferred by a vaccine can be evaluated, for example, by measuring the reduction in clinical signs such as the mortality, morbidity, overall physical condition, and overall health and performance of the subject.

[0043] "Treating" refers to preventing a disorder, condition, or disease to which such term applies, or preventing or reducing one or more symptoms of such disorder, condition, or disease.

[0044] The term "treatment" refers to the act of "treating" as defined above.

[0045] The term "vaccine" refers to a composition that induces protective immunity in a subject.

[0046] "Protecting against infection caused by M.viscosa" refers to reducing or eliminating at least one clinical sign caused by M.viscosa. The clinical signs include terminal sepsis and skin ulcers that may continue with their combinations. In a particularly preferred embodiment, protection against infection caused by M.viscosa refers to reducing the mortality rate caused by M.viscosa.

[0047] As described above, "mutant M.viscosa" has been determined that classical isolates of M.viscosa (both viscous and non-viscous) have sequences conserved in their respective gyrB genes. Thus, classical isolates are isolates that contain sub-sequences that are at least 98% identical to SEQ ID NO: 1 in their respective gyrB sequences. Conversely, in mutant M.viscosa isolates, each sub-sequence of the gyrB sequence is less than 98% identical to SEQ ID NO: 1. Preferably, in mutant M.viscosa isolates, each sub-sequence is 70-98% identical to SEQ ID NO: 1. In addition to the differences in the gyrB sequences, for the purposes of this application, mutant M.viscosa isolates are not recognized by antibodies produced in salmon against classical viscous M.viscosa strains under the conditions described in Example 1.

[0048] In certain embodiments, mutant M.viscosa isolates are at least 90% identical to SEQ ID NO: 2, preferably at least 95% identical to SEQ ID NO: 2, and have sub-sequences within their gyrB gene sub-sequences, provided that these sub-sequences are no more than 98% identical to SEQ ID NO: 1.

[0049] M.viscosa The inventors have surprisingly discovered that antigens derived from mutant strains of M.viscosa cross-protect against challenge with classical non-viscous strains of M.viscosa, and vice versa. Antigens derived from classical non-viscous strains of M.viscosa cross-protect against challenge with mutant strains of M.viscosa.

[0050] Thus, in a first aspect, the present application provides a vaccine for use in protecting fish against infection caused by mutant M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a classical non-viscous M. viscosa strain. Also disclosed is a vaccine for use in protecting fish against infection caused by mutant M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a classical non-viscous M. viscosa strain, and the vaccine does not contain a mutant M. viscosa antigen. These vaccines can be used to prevent infections caused by mutant M. viscosa and classical non-viscous M. viscosa. In some of these vaccines, the antigenic M. viscosa component consists of an antigen derived from a classical non-viscous M. viscosa strain.

[0051] The vaccine according to this first aspect may be combined with an antigen derived from classical viscous M. viscosa. Such a vaccine can be used against M. viscosa infection, including classical non-viscous M. viscosa, mutant M. viscosa, and classical viscous M. viscosa.

[0052] Alternatively, a vaccine containing an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises (or consists of as described above) an antigen derived from a classical non-viscous M. viscosa strain, can be co-administered with a vaccine containing an antigen derived from a classical viscous strain of M. viscosa. This combination of co-administered vaccines can be used to protect fish in need thereof against infections caused by classical non-viscous M. viscosa, mutant M. viscosa, and classical viscous M. viscosa.

[0053] Antigens derived from classical non - viscous strains of M.viscosa can be provided in the form of inactivated classical non - viscous M.viscosa 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 can be a fraction thereof including, but not limited to, subunits, whole cell extracts of classical non - viscous M.viscosa, or membrane fractions.

[0054] Similarly, antigens derived from classical viscous strains of M.viscosa can be inactivated classical viscous M.viscosa 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 can be a fraction thereof including, but not limited to, subunits, whole cell extracts of classical viscous M.viscosa, or membrane fractions.

[0055] Antigens derived from classical viscous strains of M.viscosa and / or classical non - viscous strains of M.viscosa can be provided in the form of attenuated bacteria. Methods of producing live attenuated bacteria are well - known in the art and include, but are not limited to, serial passage in culture.

[0056] The dosages of the antigenic classical non - viscous M.viscosa component and classical viscous M.viscosa component in the vaccine can vary. Thus, for example, one dose of the vaccine can contain at least 1×10 6 cells / dose of the classical non - viscous M.viscosa component. Without limitation, one dose can contain about 5×10 6 cells / dose, about 1×10 7 cells / dose, about 5×10 7 cells / dose, about 1×10 8 cells / dose, 3×10 8 cells / dose, 5×10 8 cells / dose, 1×10 9 cells / dose. The dose can also be 1×10 6Cells / dose ~ 1×10 7 Cells / dose, or 5×10 6 Cells / dose ~ 5×10 7 Cells / dose, or 1×10 7 Cells / dose ~ 1×10 8 Cells / dose, or 5×10 7 Cells / dose ~ 5×10 8 Cells / dose, or 1×10 8 Cells / dose ~ 1×10 9 It may include Cells / dose.

[0057] Similarly, the amount of the antigenic classical viscous M.viscosa component present in one dose of the vaccine (regardless of whether it is the same vaccine as the vaccine containing the antigenic variant M.viscosa component or a second vaccine) can be at least 1×10 6 Cells / dose of the classical viscous M.viscosa component. Thus, for example, one dose of the vaccine can contain at least 1×10 6 Cells / dose of the classical viscous M.viscosa component. Without limitation, one dose can be about 5×10 6 Cells / dose, about 1×10 7 Cells / dose, about 5×10 7 Cells / dose, about 1×10 8 Cells / dose, 3×10 8 Cells / dose, 5×10 8 Cells / dose, 1×10 9 Cells / dose. The dose can also be 1×10 6 Cells / dose ~ 1×10 7 Cells / dose, or 5×10 6 Cells / dose ~ 5×10 7 Cells / dose, or 1×10 7 Cells / dose ~ 1×10 8 Cells / dose, or 5×10 7 Cells / dose ~ 5×10 8 Cells / dose, or 1×10 8 Cells / dose ~ 1×10 9 It may include Cells / dose.

[0058] In a second aspect, the present application provides a vaccine for use in protecting fish against infections caused by classical non - adherent M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain. Also disclosed is a vaccine for use in protecting fish against infections caused by classical non - adherent M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain, and the vaccine does not contain classical non - adherent M. viscosa antigen. These vaccines can be used to protect against infections caused by classical non - adherent M. viscosa and mutant M. viscosa. In some of these vaccines, the antigenic M. viscosa component consists of an antigen derived from a mutant M. viscosa strain.

[0059] The vaccine according to this second aspect can be combined with an antigen derived from classical adherent M. viscosa. Such a vaccine can be used against M. viscosa infections, including mutant M. viscosa, classical non - adherent M. viscosa, and classical adherent M. viscosa.

[0060] Alternatively, a vaccine containing an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises (or consists of as described above) an antigen derived from a mutant M. viscosa strain, can be co - administered with a vaccine containing an antigen derived from a classical adherent strain of M. viscosa. This combination of co - administered vaccines can be used to protect fish in need thereof from infections caused by mutant M. viscosa, classical non - adherent M. viscosa, and classical adherent M. viscosa.

[0061] Antigens derived from mutant strains of M. viscosa can be inactivated mutant M. viscosa 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 can be the whole cell extract of mutant M. viscosa, or a fraction thereof including, but not limited to, the membrane fraction.

[0062] Similarly, antigens derived from classical viscous strains of M. viscosa can be inactivated classical viscous M. viscosa 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 can be the whole cell extract of classical viscous M. viscosa, or a fraction thereof including, but not limited to, the membrane fraction.

[0063] Antigens derived from classical viscous strains of M. viscosa and / or mutant strains of M. viscosa can be provided in the form of attenuated bacteria. Methods of producing live attenuated bacteria are well known in the art and include, but are not limited to, serial passage in culture.

[0064] The dosage of the antigenic mutant M. viscosa component and classical viscous M. viscosa component in the vaccine can vary. Thus, for example, one dose of the vaccine can contain at least 1x10 6 cells / dose of the mutant M. viscosa component. Without limitation, one dose can contain about 5×10 6 cells / dose, about 1×10 7 cells / dose, about 5×10 7 cells / dose, about 1×10 8 cells / dose, 3×10 8 cells / dose, 5×10 8 cells / dose, 1×10 9 cells / dose. The dose can also be 1×10 6 cells / dose to 1×10 7 cells / dose, or 5×10 6Cells / dosage ~ 5×10 7 Cells / dosage, or 1×10 7 Cells / dosage ~ 1×10 8 Cells / dosage, or 5×10 7 Cells / dosage ~ 5×10 8 Cells / dosage, or 1×10 8 Cells / dosage ~ 1×10 9 May contain cells / dosage.

[0065] Similarly, the amount of the antigenic classical viscous M.viscosa component present in one dose of the vaccine (whether it is the same vaccine as the vaccine containing the antigenic classical non-viscous M.viscosa component or a second vaccine) may be at least 1×10 6 Cells / dosage of the classical viscous M.viscosa component. Thus, for example, one dose of the vaccine may contain at least 1×10 6 Cells / dosage of the classical viscous M.viscosa component. Without limitation, one dose may be about 5×10 6 Cells / dosage, about 1×10 7 Cells / dosage, about 5×10 7 Cells / dosage, about 1×10 8 Cells / dosage, 3×10 8 Cells / dosage, 5×10 8 Cells / dosage, 1×10 9 May contain cells / dosage. The dosage may also be 1×10 6 Cells / dosage ~ 1×10 7 Cells / dosage, or 5×10 6 Cells / dosage ~ 5×10 7 Cells / dosage, or 1×10 7 Cells / dosage ~ 1×10 8 Cells / dosage, or 5×10 7 Cells / dosage ~ 5×10 8 Cells / dosage, or 1×10 8 Cells / dosage ~ 1×10 9 May contain cells / dosage.

[0066] The inventors have also surprisingly discovered that vaccination with the mutant M. viscosa antigen provides cross - protection against classical viscous M. viscosa challenge, but not vice versa (i.e., vaccination with the classical viscous M. viscosa antigen does not protect against mutant M. viscosa challenge). Thus, in a third aspect, the present invention provides a vaccine for use in protecting fish against infections caused by classical viscous M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain.

[0067] As described above, antigens derived from mutant M. viscosa strains can also be used to protect fish against infections caused by classical non - viscous M. viscosa. Thus, the present disclosure also provides an antigenic M. viscosa component vaccine for use in protecting fish against infections caused by classical viscous M. viscosa and infections caused by classical non - viscous M. viscosa strains, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain. Thus, the present disclosure also provides an antigenic M. viscosa component vaccine for use in protecting fish against infections caused by classical viscous M. viscosa and infections caused by classical non - viscous M. viscosa strains, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain and the antigenic M. viscosa component lacks antigens derived from classical viscous M. viscosa strains and classical non - viscous M. viscosa strains.

[0068] In a fourth aspect, the present invention provides a vaccine for use in protecting fish against infections caused by classical viscous M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a classical non - viscous M. viscosa strain.

[0069] As described above, antigens derived from classical non-viscous M. viscosa strains can also be used for the protection of fish against infections caused by mutant M. viscosa. Thus, the present disclosure also provides an antigenic M. viscosa component vaccine for use in protecting fish against infections caused by classical viscous M. viscosa and infections caused by mutant M. viscosa, wherein the antigenic M. viscosa component comprises an antigen derived from a classical non-viscous M. viscosa strain. Thus, the present disclosure also provides an antigenic M. viscosa component vaccine for use in protecting fish against infections caused by classical viscous M. viscosa and infections caused by mutant M. viscosa strains, wherein the antigenic M. viscosa component comprises an antigen derived from a classical non-viscous M. viscosa strain and wherein the antigenic M. viscosa component lacks antigens derived from classical viscous M. viscosa strains and mutant M. viscosa strains.

[0070] The dosages of the classical viscous M. viscosa antigen, classical non-viscous M. viscosa antigen, and mutant M. viscosa antigen of the vaccines described in connection with the first and second aspects are also applicable to the vaccines according to these third and fourth aspects of the invention.

[0071] Additional antigens In all four aspects, each antigenic component of the vaccine (or second vaccine) described herein may contain one or more additional non-M. viscosa antigens, as described below.

[0072] Such antigens can be derived from a bacterial source, viral source, additional parasitic source, and / or fungal source. These additional antigens may be the inactivated organisms listed below, or the antigens may be derived from these organisms including recombinantly prepared antigens.

[0073] Multivalent vaccines containing antigens derived from typical fish pathogens other than M. viscosa are well known in the art and are already commercially available. In addition, representative isolates of relevant fish pathogens are available from various sources.

[0074] In certain embodiments of the present invention, the antigen derived from a bacterial source is selected from the group consisting of live, attenuated, killed bacteria of the 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., Edwarsiella sp., Francisella sp., Pseudomonas sp., Cytophaga sp., Nocardia sp., Mycobacterium sp., parts or subunits of these bacteria, and any combination thereof.

[0075] Such bacterial isolates include, for example, strains of A. salmonicida (ATCC 33658), V. salmonicida (ATCC 43839), V. anguillarum serotype O1 (ATCC 43305), and O2 (ATCC 19264), and are available from the LGC Promochem / American Type Culture Collection ATCC repository and distribution center (ATCC). In addition, cultures of Piscirickettsias salmonis were deposited on June 9, 2006, at the European Collection of Cell Culture (ECACC), Health Protection Agency, Porton Down, Salisbury, Wiltshire (UK), SP4 0JG UK, under the following accession numbers: 06,050,901, 06,050,902, 06,050,903, 07,032,110.

[0076] Another specific embodiment relates to a vaccine, wherein the antigenic substance obtained from a virus source other than the fish virus as defined above is derived 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 pancreas disease virus (SPDV), iridovirus, nodavirus, piscine myocarditis virus (PMCV), and heart and skeletal muscle inflammation virus (HSMIV). These antigens can be included as modified live or inactivated organisms, as a part or subunit of any one of these viruses, as a DNA vaccine, and / or as a combination thereof. Representative strains of such viruses are available to those skilled in the art, 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: USA)); pancreatic necrosis virus; infectious salmon anemia (ISA) virus (ATCC VR-1554, country of origin: Canada). Patent deposits have been made by the applicant previously for the following virus strains: heart and skeletal muscle infection virus (HSMIV, patent deposit number ECACC04050401, country of origin: Norway).

[0077] In a more specific embodiment, the antigenic substance obtained from a virus source other than the fish virus as defined above is derived from the group consisting of the glycoprotein of viral hemorrhagic septicemia virus (VHSV), the nucleoprotein of viral hemorrhagic septicemia virus (VHSV), the glycoprotein of infectious hematopoietic necrosis virus (IHNV), the nucleoprotein structural protein of infectious pancreatic necrosis virus (IPNV), an antigenic fragment of any one of these proteins, and combinations thereof.

[0078] In other embodiments, the antigenic material from additional parasitic sources is derived from a source selected from Lepeophtheirus Sp., Caligus Sp., and Ichthyophthirius Sp., a part of any one of these parasites, and combinations thereof. In yet other embodiments, the antigenic substance is derived from a fungal source selected from the group consisting of Saprolegnia Sp., Branchiomyces sanguinis, Branchiomyces demigrans, and Icthyophonus hoferi.

[0079] In certain embodiments, the additional antigen contained in the vaccine of the present invention and / or the second vaccine containing classical viscous M. viscosa is selected from the group consisting of IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio (Aliivibrio) salmonicida, Yersinia ruckeri O1.

[0080] In other embodiments, the additional antigen contained in the vaccine of the present invention and / or the second vaccine containing classical viscous M. viscosa is selected from the group consisting of IPNV, Aeromonas salmonicida, Vibrio anguillarum serotype 1 and O2, and Vibrio (Aliivibrio) salmonicida.

[0081] Excipients and adjuvants The vaccine of the present invention may further comprise a suitable pharmaceutical carrier and / or adjuvant. The pharmaceutical carrier may be a sterile liquid, such as water or a buffer solution, such as an aqueous saline solution, an aqueous dextrose solution, and an aqueous glycerol solution. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. If desired, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffering agent. Examples of suitable pharmaceutical carriers are described in "Remington’s Pharmaceutical Sciences" by E.W. Martin. The formulation must be compatible with the mode of administration.

[0082] Suitable carriers will be apparent to those skilled in the art and will largely depend on the route of administration. Additional components that may be present in the present invention are adjuvants, preservatives, surfactants, chemical stabilizers, suspending or dispersing agents. Typically, the stabilizers, adjuvants, and preservatives are optimized to determine the best formulation for efficacy in the target subject.

[0083] In presently preferred embodiments, the vaccine comprises an adjuvant. Suitable adjuvants include, but are not limited to, oils. The vaccines disclosed herein may be formulated as water-in-oil emulsions or, more preferably, oil-in-water emulsions. Other formulations such as water-in-oil-in-water (W / O / W) type may also be prepared. In addition, the vaccine may contain one or more suitable surface active compounds or emulsifiers, such as CREMOPHORE®, TWEEN®, and SPAN®. Also, adjuvants such as interleukins, CpG, and glycoproteins may be used.

[0084] The vaccine may also include a "vehicle". A vehicle is a device to which the antigen adheres without covalently binding thereto. Such vehicles are biodegradable nano / micro particles or capsules of PLGA (polylactide-co-glycolic acid), alginate or chitosan, liposomes, niosomes, micelles, multiple emulsions and macrosomes, all of which are known in the art. A special form of such a vehicle in which the antigen is partially embedded in the vehicle is the so-called ISCOM (European patents EP109,942, EP180,564 and EP242,380).

[0085] In certain embodiments, the vaccines described herein are formulated as water-in-oil emulsions. Preferably, the oil is a mineral oil.

[0086] The vaccines described herein can be administered to salmonid fish by a variety of routes including, but not limited to, intraperitoneal, intramuscular, oral, and immersion. Preferably, the vaccine is administered by injection in a microdose such that the volume of a single 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 about 100 μl, or about 50 μl, or about 25 μl.

[0087] The vaccines disclosed herein can be used in protecting multiple salmonid species from infection. Suitable salmonid fish include, but are not limited to, Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), rainbow trout (Oncorhynchus mykiss), sockeye salmon (Oncorhynchus nerka), chinook salmon (Oncorhynchus tshawytscha), and other species.

[0088] Salmonid fish of different ages (or weights) can be vaccinated according to the present invention. In certain embodiments, the salmonid fish is about 15 to about 200 grams in weight at the time of vaccination. Thus, the weight of the salmonid fish at the time of vaccination can be about 25 to about 150 grams, or about 40 to about 110 grams, or about 50 to about 100 grams.

[0089] Here, the present invention will be described with the following exemplary examples.

Example

[0090] Example 1: Mutant and classical non-viscous M.viscosa are not recognized by salmon polyclonal antibodies produced against classical viscous M.viscosa. Materials and Western blot method: Ten strains of M.viscosa were examined using Western blot. The country of origin and year of isolation, gyrB mutant or classical type, and viscosity phenotype regarding viscosity are listed in Table 1. The isolate numbers also correspond to the lane numbers on the Western blot membrane.

Table 1

[0091] Antibodies / conjugates used: 1) Monoclonal mouse anti-mas / salmon IgM antibody, clone 4C10 2) HRP-conjugated polyclonal rabbit anti-mouse (catalog number P0260, Dako) 3) HRP-conjugated Precision Protein STREPTACTIN® (catalog number 161-0380, Bio Rad) 4) Polyclonal salmon a-mutant M.viscosa antibody - generated during the course of this project 5) Polyclonal salmon a-classical M.viscosa antibody - generated during the course of this project 6) Polyclonal rabbit anti-mutant M.viscosa antibody - generated during the course of this project 7) Polyclonal rabbit anti-classical M.viscosa antibody - generated during the course of this project 8) HRP-conjugated swine anti-rabbit (Catalog number P0217, Dako)

Table 2

[0092] Sample preparation For each strain, the bacterial culture was spread on a blood agar plate. The plate was incubated at 15 °C. After 2 days of incubation, colonies from the agar plate were inoculated into 10 ml of growth medium in a 25 cm 2 cell flask and incubated at 15 °C, 100 rpm.

[0093] After incubation, the OD was measured. 2 × 1 ml was centrifuged and the supernatant was pipetted out.

[0094] 10 bacterial pelletized samples were prepared for Western blot. All pellets were lysed in reducing sample buffer, incubated at 100 °C for 10 minutes, and frozen until further analysis. On the day of SDS-PAGE, the samples were diluted / normalized to obtain a theoretical OD2 (calculated from the OD at collection) using freshly prepared sample buffer before use.

[0095] SDS-PAGE and WB: 10 μl of each sample was added to a lane on the gel (6 μl in the molecular weight marker lane). Four parallel gels were run.

[0096] The gel was run at 250 V for approximately 3 minutes and then at 160 V for approximately 50 minutes. Equal volumes of PRECISION PLUS PROTEIN™ All Blue Standard and PRECISION PLUS PROTEIN™ Unstained Standard were combined (3 - 5 μl each) and used as molecular weight standards. Four gels were prepared and four blots were made. Two were for treatment with salmon plasma and two were for treatment with rabbit anti-M. viscosa antibody. The gel was then activated on a gel-doc for 45 seconds, imaged, transferred to a TRANS-BLOT® TURBO™ Transfer Blotting System (BioRad), and a 7-minute turbo program was used. The blotted membrane was immediately transferred to blocking buffer (5% non-fat milk in TBST (TBSTM)). The blot was blocked overnight at 2 - 6 °C.

[0097] Next, as described in Tables 3 and 4, the blots were incubated with antibodies. For visualization of the molecular weight standards, STREPTACTIN® HRP was added together with other HRP-conjugated antibodies. The blots were washed 3×10 minutes with TBST during each incubation (TBS was used in the last wash step before substrate incubation), then incubated with CLARITY™ Western substrate for 5 minutes and then exposed on a gel-doc imaging system (BioRad).

Table 3

Table 4

[0098] The experimental results are shown in Figure 1. This figure shows that classical viscous M.viscosa and mutant M.viscosa are recognized by different antibodies. More specifically, these results show that antibodies produced against classical viscous M.viscosa in salmon do not recognize classical non-viscous M.viscosa and mutant M.viscosa. Surprisingly, it was also shown that classical non-viscous M.viscosa and mutant M.viscosa are recognized by the same antibody.

[0099] Example 2: Vaccination with mutant M.viscosa provides cross-protection against classical non-viscous challenge, but vaccination with classical viscous M.viscosa does not provide cross-protection Materials and Methods The Bath challenge study was a comparative, investigator-blinded, negative control, and randomized laboratory experiment. The study investigated the efficacy of different oil-adjuvanted injectable vaccines for protecting Atlantic salmon against experimental infection with different isolates of M.viscosa. Experimental vaccines containing classical non-viscous M.viscosa and a field isolate of mutant M.viscosa adjuvanted with a water-in-oil (W / O) emulsion were administered intraperitoneally by co-injection with a commercial vaccine according to the manufacturer's instructions. Both of these commercial vaccines contained classical viscous strains of M.viscosa. A negative control group was included. The fish averaged approximately 25 grams at the time of vaccination.

[0100] The fish were exposed to continuous light (24:0) to smoltify before being transferred and challenged in seawater. The start of the light manipulation was initiated approximately 6 weeks prior to challenge with the classical non-viscous strain of M.viscosa. The challenge was performed by immersion 13 weeks after vaccination. Taking into account the biomass, the fish were challenged in two 500 L tanks per challenge isolate, and the results from the same replicate tanks were combined. A total of approximately 60 fish per group (30 fish per tank / group) were challenged per challenge isolate.

[0101] One week before the challenge, the fish were transferred to the disease facility and evenly distributed into two duplicate 500 L tanks for each challenge isolate, and adapted to seawater with a salinity concentration of 34‰, 8 °C, and a 24:0 light regime, which are the environmental parameters during the challenge period. The challenge material was freshly cultured from a frozen bacterial stock by shaking in a yeast extract-based shake flask culture medium at 12 °C for 2 days. The fish were challenged by reducing the water volume in the tanks before directly adding the challenge material into the tanks.

[0102] The challenge was conducted 13 weeks after vaccination. The fish were observed daily after the challenge, and fish with ulcers were euthanized and recorded as dead in the death log. The efficacy was evaluated by a statistical comparison of the mortality and protection against ulcer formation between the vaccinated group and the negative control group after the challenge. The research fish were not vaccinated, had no clinical disease, and had a valid health certificate. Damaged or deformed fish were excluded from the study.

[0103] The results of the experiment are shown in Figure 2.

[0104] As can be seen, the groups treated with PBS and the commercially available vaccine (both containing the classical viscous M. viscosa antigen) resulted in almost 100% mortality 18 days after the fish were challenged with the classical non-viscous M. viscosa strain, thus supporting the validity of the challenge model. There was no statistically significant difference between these three groups.

[0105] In contrast, the groups vaccinated with a composition containing either the classical non-viscous M. viscosa antigen or the mutant antigen showed only about 70% mortality (there was no significant difference between these two groups, but p < 0.0001 compared to the groups treated with PBS or the commercially available vaccine).

[0106] These results suggest that the classical viscous M.viscosa antigen does not provide cross - protection against challenge with classical non - viscous M.viscosa strains. The classical non - viscous antigen provides protection against challenge with classical non - viscous M.viscosa strains. Surprisingly, the mutant M.viscosa antigen provides cross - protection against challenge with classical non - viscous M.viscosa strains.

[0107] Example 3: Vaccination with classical non - viscous M.viscosa provides cross - protection against mutant M.viscosa challenge, but vaccination with classical viscous M.viscosa does not The materials and methods were the same as in Example 2, except that a mutant M.viscosa isolate was used as the challenge strain.

[0108] The results of the experiment are shown in Figure 3.

[0109] As can be seen, the groups treated with PBS and the groups treated with a commercial vaccine (both containing classical viscous M.viscosa antigen) resulted in a mortality rate of approximately 60 - 70% 22 days after the fish were challenged with the classical non - viscous M.viscosa strain, indicating that the challenge model was effective. There was no statistically significant difference between these three groups.

[0110] In contrast, the groups vaccinated with a composition containing either the classical non - viscous M.viscosa antigen or the mutant antigen showed only a mortality rate of approximately 20 - 35% (there was no significant difference between these two groups, but p < 0.0001 compared to the groups treated with PBS or the commercial vaccine).

[0111] These results suggest that the classical viscous M.viscosa antigen does not provide cross - protection against challenge with the mutant M.viscosa strain. The mutant antigen provides protection against challenge with the mutant M.viscosa strain. Surprisingly, the classical non - viscous M.viscosa antigen provides cross - protection against challenge with the mutant M.viscosa strain.

[0112] Example 4: Vaccination with mutant M. viscosa protects against challenge with classical viscous M. viscosa. Materials and methods: The cross-protective efficacy of a monovalent oil adjuvant (W / O emulsion) mutant M. viscosa vaccine was evaluated in a blinded, negative control, and randomized laboratory experiment.

[0113] A monovalent mutant M. viscosa vaccine was administered to one group by intraperitoneal injection. A second group was vaccinated with a vaccine containing classical viscous M. viscosa, and the negative control group was injected with phosphate-buffered saline (PBS). The fish were kept in 500 L tanks of fresh water at 15 °C during the immunization period and exposed to a continuous light regime (24:0 light:dark) for approximately 6 weeks to smoltify before bath challenge into seawater. The challenge material (classical viscous M. viscosa isolate) was freshly cultured from a frozen bacterial stock and shaken for 2 days in a yeast extract-based shaking flask medium at 12 °C. The fish were challenged by reducing the water volume in the tank before directly adding the challenge material to the tank.

[0114] After an immunization period of approximately 9 weeks, challenge was performed by immersion. The challenge was intended to investigate any cross-protective efficacy of the monovalent vaccine containing the inactivated antigen of mutant M. viscosa against the classical viscous M. viscosa isolate. Approximately 1 week before the challenge, the fish were transferred to a disease facility, evenly distributed into parallel 500 L tanks, and gradually acclimated to seawater at 8 °C and a salinity concentration of 34‰. To reduce the biomass density at the time of challenge, groups were challenged in two duplicate tanks per challenge isolate and the results from the two tanks were combined. The fish were observed daily after challenge, and fish with ulcers were euthanized and recorded as dead in the mortality log. Efficacy was evaluated by a statistical comparison of mortality and protection against ulceration between the vaccinated and negative control groups after challenge. The study fish were not vaccinated, had no clinical disease, and had a valid health certificate. Damaged or deformed fish were excluded from the study.

[0115] The results are shown in Figure 4. The group treated with PBS showed a mortality rate of over 40% 22 days after challenge. In contrast, vaccination with the classical M. viscosa antigen or the mutant M. viscosa antigen resulted in a statistically significant decrease in the cumulative mortality rates of approximately 10 and 75, respectively, by day 22 after challenge. These results indicate that vaccination with the mutant M. viscosa antigen can provide cross - protection against challenge with classical M. viscosa. Considering the similar responses of the group vaccinated with the mutant M. viscosa antigen and the group vaccinated with the classical non - adherent M. viscosa antigen, these results also strongly suggest that vaccination with the classical non - adherent M. viscosa antigen can provide cross - protection against challenge with classical M. viscosa.

[0116] All publications, patent publications and non - patent publications listed herein are indicative of the level of skill of those of ordinary skill in the art to which the present invention pertains. All of these publications are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0117] The invention herein has been described with reference to specific embodiments, but it should be understood that these embodiments are merely illustrative of the principles and applications of the invention. Thus, it should be understood that numerous modifications may be made to the exemplary embodiments and other configurations may be devised without departing from the spirit and scope of the invention as defined by the following claims.

Claims

1. A vaccine for use in protecting fish against infections caused by mutant M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a classical non-viscous M. viscosa strain.

2. The vaccine according to claim 1, for use in protecting fish against infections caused by mutant M. viscosa and classical non-viscous M. viscosa.

3. The vaccine according to claim 1 or 2, wherein the vaccine does not contain an antigen derived from a mutant M. viscosa strain.

4. The vaccine according to claim 3, wherein the antigenic M. viscosa component consists of the antigen derived from the classical non-viscous M. viscosa 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 classical viscous M. viscosa strain.

6. The vaccine according to claim 5, wherein the second vaccine does not contain an antigen derived from a mutant M. viscosa strain.

7. A vaccine for use in protecting fish against infections caused by mutant M. viscosa, classical viscous M. viscosa, and classical non-viscous M. viscosa, wherein the M. viscosa antigenic compound further comprises an antigen derived from a classical viscous M. viscosa strain, according to any one of claims 1 to 3.

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

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

10. A vaccine for use in protecting fish against infections caused by classical non-viscous M. viscosa, the vaccine comprising an antigenic M. viscosa component, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain.

11. The vaccine according to claim 10 for use in protecting fish against infection caused by mutant M. viscosa and classical non-viscous M. viscosa.

12. The vaccine according to claim 10 or 11, wherein the vaccine does not contain an antigen derived from a classical non-viscous M. viscosa strain.

13. The vaccine according to claim 12, wherein the antigenic M. viscosa component consists of the antigen derived from the mutant M. viscosa strain.

14. The vaccine according to any one of claims 10 to 12, wherein the vaccine is co-administered with a second vaccine containing an antigen derived from a classical viscous M. viscosa strain.

15. The vaccine according to claim 14, wherein the second vaccine does not contain an antigen derived from a classical non-viscous M. viscosa strain.

16. A vaccine for use in protecting fish against infection caused by mutant M. viscosa, classical viscous M. viscosa, and classical non-viscous M. viscosa, wherein the M. viscosa antigenic compound further comprises an antigen derived from a classical viscous M. viscosa strain, the vaccine according to any one of claims 11 to 13.

17. The vaccine according to claim 16, wherein the antigenic M. viscosa component consists of the antigen derived from the mutant M. viscosa strain and the antigen derived from the classical viscous M. viscosa strain.

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

20. A vaccine containing an antigenic M. viscosa component for use in protecting fish against infection caused by classical non-viscous M. viscosa and classical viscous M. viscosa, wherein the antigenic M. viscosa component comprises an antigen derived from a mutant M. viscosa strain.

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

22. The vaccine does not contain an antigen derived from a classical non-viscous M. viscosa strain, and the vaccine does not contain an antigen derived from a classical viscous M. viscosa strain, the vaccine according to claim 20 or 21.

23. The antigenic M. viscosa component consists of the antigen derived from the mutant M. viscosa strain, the vaccine according to any one of claims 20 to 22.

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

25. A vaccine containing an antigenic M. viscosa component for use in protecting fish against infections caused by mutant M. viscosa and classical viscous M. viscosa, wherein the antigenic M. viscosa component contains an antigen derived from a classical non-viscous M. viscosa strain.

26. The vaccine according to claim 25 for use in protecting fish against infections caused by classical non-viscous M. viscosa.

27. The vaccine does not contain an antigen derived from a mutant M. viscosa strain, and the vaccine does not contain an antigen derived from a classical viscous M. viscosa strain, the vaccine according to claim 25 or 26.

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

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

30. The vaccine according to any one of claims 1 to 29, comprising one or more non-M. viscosa antigens.

31. The second vaccine comprises one or more non-M. viscosa antigens, the vaccine according to claim 30.

32. The one or more non-M. viscosa antigens are selected from the group consisting of IPNV, ISAV, SPDV, Aeromonas salmonicida, Vibrio anguillarum O1, O2, Vibrio (Aliivibrio) salmonicida, Yersinia ruckeri O1, the vaccine according to claim 30 or 31.

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 according to any one of claims 1 to 33, wherein the fish is of the Salmonidae family.

35. The vaccine according to claim 34, wherein the Salmonidae family is Salmo salar.

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

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

38. The protection of the fish against infection comprises reduction or elimination of at least one symptom of M. viscosa. The vaccine according to any one of claims 1 to 37.

39. The protection of the fish against infection comprises a decrease in the mortality rate caused by M. viscosa. The vaccine according to any one of claims 1 to 38.

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