AU2008320818A1 - Fish vaccine - Google Patents

Description

WO 2009/056629 PCT/EP2008/064805 Fish vaccine The present invention relates to a combination vaccine for combating bacterial infection in fish, to the use of bacteria for the manufacture of such a vaccine, to methods for the preparation of 5 such a vaccine and to a kit-of-parts. Over the last decades, world-wide a strong increase is seen in the consumption of fish. This equally regards the consumption of cold water fish such as salmon, turbot, halibut and cod, and tropical fish such as Asian sea bass, tilapia, milkfish, yellowtail, amberjack, grouper, snapper and 10 cobia. As a consequence, an increase has been seen in the number and the size of fish farms, in order to meet the increasing needs of the market. As is known from e.g. animal husbandry, large numbers of animals living closely together are 15 vulnerable to all kinds of diseases, even diseases hardly known or seen, or even unknown, before the days of large-scale commercial farming. This is equally the case in fish farming. Bacteria found to be pathogenic to fish belong i.a. to the genus Nocardia, Vibrio, Pasteurella, Photobacterium, Tenacibaculum, Flavobacterium, Flexibacter, Cytophaga, Francisella, Mycobacterium, Streptococcus, Lactococcus or Edwardsiella. 20 Of the Nocardia species, Nocardia seriolae causes chronic problems in warm-water fish. The damage caused in fish farming industry by Nocardial infection has been increasing over the years. In particular yellowtail (Seriolae quinqueradiata), amberjack (Seriolae dumerelli), sea bass (Lateolabraxjaponicus), yellow croacker (Lamitichthys crocea), Pomfret (Pampus 25 argenteus), threadfin (Eleutheronema tetradactylum), snapper (Lutjanus sp), grouper (Epinephelus sp) and trevalli (Caranx sexfasciatus) have been affected by Nocardia infection. The disease, often referred to as marine nocardiosis begins as a silent infection. It develops in fry and juvenile fish. The bacteria multiply within major organs such as spleen, liver and kidney. 30 Because of the low multiplication rate, the bacterium can multiply in fish tissue for a long time before any visual symptoms arise. Therefore, the disease is called chronic. Economic losses are WO 2009/056629 PCT/EP2008/064805 2 significant, if only for the fact that as a consequence of the chronic character, fish weigh often already between 300 and 1000 g before the outbreak becomes manifest. Research indicates that yellowtail sharing tank space with sick juveniles (previously injected with live Nocardia) eventually exhibit internal pathology (granulomas in their spleens) after 3 months 5 of cohabitation. In marine finfish culture, Nocardial infections appear to progress more quickly during the summer months when water temperatures reach 24'C or more, but the mortality due to Nocardia is more commonly experienced in the autumn and early winter months, as the fish has to adapt to the new environmental situation and its immune system wanes. 10 Nocardia seems to be a very poor inducer of immune system against itself, because in spite of the very slow progress of the disease, the immune system does not manage to clear the infection. This may also explain the fact that no efficacious vaccines against Nocardia infection exist. Vaccines comprising live attenuated or inactivated bacteria to a certain extent mimic the natural infection, but if even the natural infection fails to induce an adequate immune response, one 15 would not expect vaccines to perform better. It is clear that efficacious vaccines are highly needed. It is an objective of the present invention to provide efficacious vaccines for combating Nocardia infection. 20 It was surprisingly found now that a combination vaccine comprising not only Nocardia seriolae, but additionally at least bacteria of the species Lactococcus garviae, Photobacterium damselae subspecies piscicidae (= Pasteurellapiscicida) and Vibrio anguillarum provides a high level of protection not only against Lactococcus garviae, Pasteurellapiscicida and Vibrio anguillarum 25 but also an unexpected higher level of protection against Nocardia seriolae infection than that obtained by a monovalent Nocardia seriolae vaccine. Therefore, a first embodiment of the present invention relates to a combination vaccine for combating Nocardia infection in fish, characterised in that said vaccine comprises bacteria of the 30 species Lactococcus garviae, Pasteurellapiscicida, Vibrio anguillarum and Nocardia seriolae and a pharmaceutically acceptable carrier.

WO 2009/056629 PCT/EP2008/064805 3 For the manufacture of such a vaccine, the status of the bacteria; live or inactivated, is not really important. What counts is the fact that the stimulator of Nocardia-specific immunity in fish against Nocardia is still present. This can be i.a. assured by using whole bacterial preparations. As said above, it is not important 5 that the bacteria in the preparation are alive, killed or even fragmented (e.g. by using a French Press). The skilled person will appreciate that the method used for inactivation is not very relevant for the activity of the bacteria. Classical methods for inactivation such as UV-radiation, gamma radiation, treatment with formalin, binary ethylene-imine, thimerosal and the like, all well-known 10 in the art, are applicable. Inactivation of bacteria by means of physical stress, using e.g. a French Press provides an equally suitable starting material for the manufacturing of a vaccine according to the invention. Inactivated bacteria need thus not necessarily be in the form of inactivated whole cells; the cells may be disrupted. Inactivated bacteria have the advantage over live attenuated bacteria that they are very safe. 15 As follows from the Examples below, a Nocardia seriolae bacterin vaccine to be used as part of the combination vaccine according to the invention, can easily be made and works efficaciously. Therefore, in a preferred form of this embodiment, the invention relates to a combination vaccine according to the invention wherein the bacterial species are inactivated. 20 Live attenuated bacteria are also very suitable, because they by definition carry the factor stimulating the cross-specific immunity against Nocardia. And live attenuated bacteria have the advantage over inactivated bacteria that, especially when given without an adjuvant, they are more effective than inactivated bacteria. Moreover they replicate to a certain extent until they are 25 stopped by the immune system, as a result of which a lower number of cells can be given. A live attenuated bacterium is a bacterium that is less pathogenic than its wild-type counterpart, but nevertheless induces an efficacious immune response. Attenuated strains can be obtained along classical routes, long known in the art such as serial passage, temperature-adaptation, chemical mutagenesis, UV-radiation and the like, or by site 30 directed mutagenesis. Therefore, in another preferred form, the invention relates to a combination vaccine according to the invention wherein at least one of the bacterial species is in a live attenuated form.

WO 2009/056629 PCT/EP2008/064805 4 Vaccines according to the invention can be prepared starting from a bacterial culture according to techniques well known to the skilled practitioner. Review articles relating to fish vaccines and their manufacture are i.a. by Sommerset, I., Krossey, 5 B., Biering, E. and Frost, P. in Expert Review of Vaccines 4: 89-101 (2005), by Buchmann, K., Lindenstrem, T. and Bresciani, in J. Acta Parasitologica 46: 71-81 (2001), by Vinitnantharat, S., Gravningen, K. and Greger, E. in Advances in veterinary medicine 41: 539-550 (1999) and by Anderson, D.P. in Developments in Biological Standardization 90: 257-265 (1997). 10 Vaccines according to the invention basically comprise an effective amount of bacteria according to the invention and a pharmaceutically acceptable carrier. The term "effective " as used herein is defined as the amount sufficient to induce an immune response in the target fish that results in a level of pathogenesis that is less that 50% of the pathogenesis seen in non-vaccinated fish under the same conditions, after infection with wild 15 type Nocardia. The amount of cells to be administered will depend i.a. on the amount of bacteria of each antigen used, the condition of the bacteria; attenuated live or inactivated, the presence of an adjuvant and the route of administration. 20 When starting from commercially available vaccines, the manufacturer will provide this information. Otherwise, man skilled in the art finds sufficient guidance in the references mentioned above and in the information given below, especially in the Examples. 25 As said above, vaccines according to the invention can be prepared starting from a bacterial culture according to techniques well known to the skilled practitioner. In the Example-section, examples of the preparation of a vaccine according to the invention are given. 30 Generally spoken, vaccines manufactured according to the invention that are based upon inactivated bacteria can be given in general in a dosage of 10 3 to 1010, preferably 106 to 10 9 , more preferably between 108 and 109 bacteria. A dose exceeding 1010 bacteria, although immunologically suitable, will be less attractive for economical reasons.

WO 2009/056629 PCT/EP2008/064805 5 Vaccines manufactured according to the invention that are based upon live attenuated bacteria can be given in a lower dose, due to the fact that the bacteria will continue replicating for a certain time after administration. Vaccines manufactured according to the invention that are based upon live attenuated bacteria can be given in general in a dosage of 102 to 108, preferably 5 10 3 to 10 5 bacteria Examples of pharmaceutically acceptable carriers that are especially suitable in a vaccine according to the invention are sterile water, saline, aqueous buffers such as PBS and the like. In addition a vaccine according to the invention may comprise other additives such as adjuvants, 10 stabilisers, anti-oxidants and others, as described below. Vaccines manufactured as described in the present invention may in a preferred presentation contain an immunostimulatory substance, a so-called adjuvant. Adjuvants in general comprise substances that boost the immune response of the host in a non-specific manner. A number of 15 different adjuvants are known in the art. Examples of adjuvants frequently used in fish and shellfish farming are muramyldipeptides, lipopolysaccharides, several glucans and glycans and Carbopol(R). An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review paper by Jan Raa (Reviews in Fisheries Science 4(3): 229-288 (1996)). The vaccine may also comprise a so-called "vehicle". A vehicle is a compound to which the 20 bacterium adheres, without being covalently bound to it. Such vehicles are i.a. bio-microcapsules, micro-alginates, liposomes and macrosols, all 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 EP 109.942, EP 180.564, EP 242.380). In addition, the vaccine may comprise one or more suitable surface-active compounds or 25 emulsifiers, e.g. Span or Tween. Thus, in a more preferred form of this embodiment, the combination vaccine according to the invention comprises an adjuvant. 30 For combination vaccines according to the invention, oil adjuvants usually turn out to be somewhat more efficient. Oil adjuvants suitable for use in water-in-oil emulsions are e.g. mineral oils or metabolisable oils. Mineral oils are e.g. Bayol*, Marcol* and Drakeol*.

WO 2009/056629 PCT/EP2008/064805 6 Metabolisable oils are e.g. vegetable oils, such as peanut oil and soybean oil, animal oils such as the fish oils squalane and squalene, and tocopherol and its derivatives. Suitable adjuvants are e.g. w/o emulsions, o/w emulsions and w/o/w double-emulsions Very suitable o/w emulsions are e.g. obtained starting from 5-50% w/w water phase and 95-50% 5 w/w oil adjuvant, more preferably 20-50% w/w water phase and 80-50% w/w oil adjuvant. Thus, in an even more preferred form of this embodiment, the combination vaccine according to the invention comprises an adjuvant, wherein that adjuvant is an oil adjuvant. 10 As said above, oil adjuvants can roughly be divided in adjuvants comprising mineral oil and adjuvants comprising non-mineral oil. Mineral oil may be somewhat less attractive, both from a food safety point of view and due to the lesions it sometimes gives. Therefore, a preferred oil adjuvant comprises a non-mineral oil. 15 A more preferred non-mineral oil is e.g. ISA 763A VG oil as commercially obtainable from SEPPIC France The amount of adjuvant added depends on the nature of the adjuvant itself, and information with respect to such amounts will be provided by the manufacturer. 20 Often, the vaccine is mixed with stabilisers, e.g. to protect degradation-prone proteins from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency. Useful stabilisers are i.a. SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or 25 casein or degradation products thereof, and buffers, such as alkali metal phosphates. Preferably, vaccines as described are presented in a freeze-dried form. In addition, the vaccine may be suspended in a physiologically acceptable diluent. 30 It goes without saying, that other ways of adjuvating, adding vehicle compounds or diluents, emulsifying or stabilizing a protein are also embodied in the present invention.

WO 2009/056629 PCT/EP2008/064805 7 Many ways of administration, all known in the art can be applied. The vaccines as described are preferably administered to the fish via injection such as e.g. intraperitoneal injection or intra muscular injection or through other routes such as immersion, spraying, dipping or per oral. It should be kept in mind however that the route of administration may also depend on the type of 5 vaccine: if the vaccine comprises live attenuated Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae bacteria, it could easily be administered by dipping. If on the other hand the vaccine comprises Lactococcus garviae, Pasteurellapiscicida, Vibrio anguillarum and Nocardia seriolae bacteria in the form of inactivated bacteria, or more generally spoken if the vaccine can be improved by admixing an adjuvant, the preferred way of 10 administration would be the intraperitoneal route. From an immunological point of view, intraperitoneal vaccination is an effective route of vaccination in fish, certainly for inactivated bacteria, especially because it allows the incorporation of adjuvants. A convenient way of making a vaccine according to the invention is, to make use of 15 commercially available vaccines. Lactococcus garviae vaccines, Pasteurellapiscicida vaccines and Vibrio anguillarum vaccines are commercially available, and/or ways to produce them have been described in the literature. For the Nocardia-component of the vaccine, preferably an inactivated Nocardia bacterin, more preferably a Nocardia seriolae bacterin is used. 20 The administration protocol can be optimized in accordance with standard vaccination practice. The age of the fish to be vaccinated is not critical, although clearly one would want to vaccinate against Nocardia infection in an early stage. For many vaccines it goes that they are administered 25 when the fish have a weight of between 10 and 35 grams. This is a very suitable moment for vaccinating against Nocardia as well. For oral administration the vaccine is preferably mixed with a suitable carrier for e.g. oral administration such as cellulose, food or a metabolisable substance such as alpha-cellulose or 30 various oils of vegetable or animals origin. Also an attractive method is administration of the vaccine through bio encapsulation whereby live feed organisms are exposed to high concentrations of the vaccine, followed by the feeding of the live-feed organisms to the fish. Particularly preferred food carriers for oral delivery of the vaccine according to the invention are WO 2009/056629 PCT/EP2008/064805 8 live-feed organisms which are able to encapsulate the vaccine. Suitable live-feed organisms include plankton-like non-selective filter feeders preferably members of Rotifera, Artemia, and the like. Highly preferred is the brine shrimp Artemia sp.. 5 In view of the large number of viruses and organisms pathogenic to fish, it would be beneficial to administer, together with Lactococcus garviae, Pasteurellapiscicida, Vibrio anguillarum and Nocardia seriolae bacteria, also one or more other fish-pathogenic bacteria or viruses, antigens of those bacteria or viruses or genetic material encoding such antigens for the manufacture of a vaccine. 10 Examples of notorious commercially important fish pathogens are the recently found bacterium causing Big Belly syndrome, as described in Thai Patent Application TH 92840, (An example of this novel bacteria (BB E3F 1) has been deposited with the Collection Nationale de Cultures de Microorganisms (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, 15 France, under accession number CNCM 1-3257), Tenacibaculum maritimum, Flavobacterium columnare, Flexibacter maritimus (the old name of Tenacibaculum maritimum), Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae, Edwardsiella tarda, Edwardsiella ictaluri, Mycobacterium maritimum, Francisella sp. as well as viruses such as Nodavirus, Irido virus, Koi herpes virus, channel catfish virus. 20 The advantage of such a combination vaccine is that it not only provides protection against Nocardia, Lactococcus garviae, Pasteurella piscicida and Vibrio anguillarum infection, but also against other diseases. 25 Therefore, in a preferred embodiment, the combination vaccine according to the invention comprises, in addition to bacteria of the species Lactococcus garviae, Pasteurellapiscicida, Vibrio anguillarum and Nocardia seriolae, at least one other microorganism or virus that is pathogenic to fish, or one other antigen of such a microorganism or virus or genetic material encoding said other antigen. 30 In a more preferred embodiment, the at least one other microorganism or virus that is pathogenic to fish, are selected from the group of bacteria consisting of the bacterium causing Big Belly syndrome, Flavobacterium columnare, Tenacibaculum maritimum, Streptococcus iniae, WO 2009/056629 PCT/EP2008/064805 9 Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae, Edwardsiella tarda, Edwardsiella ictaluri, Mycobacterium maritimum, Francisella sp., Nodavirus, Irido virus, Koi herpes virus and channel catfish virus. 5 Still another embodiment of the present invention relates to methods for the preparation of combination vaccines according to the invention for combating Nocardia infection in fish. Such methods comprise the step of mixing of Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae bacteria in a live attenuated or inactivated form and a pharmaceutically acceptable carrier. 10 A preferred form of this embodiment relates to methods that additionally comprise the mixing of an adjuvant. As mentioned earlier, monovalent Lactococcus garviae, Pasteurellapiscicida and Vibrio 15 anguillarum vaccines are currently commercially available. Thus, if for the Lactococcus garviae, Pasteurella piscicida and Vibrio anguillarum vaccine components, ready-to-use vaccines are used, they can be mixed with the Nocardia seriolae vaccine before administration. If they are to be administrated orally, mixing prior to administration would be the preferred choice. If the vaccine is administered by injection, for simultaneous administration the 20 components can be mixed, they can also be administered separately, or sequentially in a series of consecutive injections. Regardless the fact that either commercially available Lactococcus garviae vaccines, Pasteurella piscicida vaccines and Vibrio anguillarum vaccines or vaccines prepared as described in the 25 Examples (such as the Nocardia seriolae vaccine described in the Examples) are used, the skilled person would prefer to use that amount of each of the bacteria that is necessary to induce an immune response against each of the bacterial species. Merely as an example: the amount of Lactococcus garviae bacteria in the combination vaccine according to the invention is sufficient to induce an immune response against Lactococcus garviae infection. 30 The bacteria of the species Lactococcus garviae, Pasteurellapiscicida, Vibrio anguillarum and Nocardia seriolae in the combination vaccine can be administered simultaneously, separately or WO 2009/056629 PCT/EP2008/064805 10 sequentially. They can then, if given within a short interval, nevertheless be considered as a combination vaccine, as is explained below. Simultaneous administration is administration of the Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae bacteria at the same moment in time, preferably 5 injected as a mixture. This would of course be the preferred method of administration, due to ease of handling. Separate administration is administration of the Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae bacteria (partially or fully) separately at two or more different injection sites, preferably at the same moment in time. 10 Sequential administration is administration during which the Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae bacteria are administered at different moments in time. It is clear that if separate or sequential injections are given, these would preferably be given at the same day, more preferably within 12, 10, 8, 6, 4, 2 or 1 hour in that order of preference. Even more preferred is administration within 50, 40, 30, 20, 10 or 5 minutes 15 after each other. If the administration of all vaccines of the combination vaccine would take place within 10 minutes, even better 5 or less than 5 minutes, a single moment for handling of each fish would suffice, and would allow an almost instantaneous full triggering of the immune system. Another embodiment of the present invention relates to the use of bacteria of at least the species 20 Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae for the manufacture of a combination vaccine for combating Nocardia seriolae infection in fish. In a preferred form of this embodiment, the fish in which the Nocardia seriolae infection is to be combated, and for which the vaccine is thus manufactured belong to the species yellowtail 25 (Seriolae quinqueradiata), amberjack (Seriolae dumerelli), sea bass (Lateolabraxjaponicus), yellow croacker (Lamitichthys crocea), Pomfret (Pampus argenteus), threadfin (Eleutheronema tetradactylum), snapper (Lutjanus sp), grouper (Epinephelus sp) and trevalli (Caranx sexfasciatus). 30 In another preferred form of this embodiment, at least one of the bacterial species used for the manufacture is in a live attenuated form.

WO 2009/056629 PCT/EP2008/064805 11 In again another preferred form of this embodiment, the Nocardia species used for the manufacture is inactivated. In still another preferred form of this embodiment, the bacterial species used for the manufacture 5 are inactivated. In a more preferred form of this embodiment, for the manufacture of said vaccine additionally at least one other microorganism or virus that is pathogenic to fish, or one other antigen of such a microorganism or virus or genetic material encoding said other antigen is used. 10 In an even more preferred form of this embodiment said other microorganism or virus is selected from the group of bacteria consisting of the bacterium causing Big Belly syndrome, Tenacibaculum maritimum, Flavobacterium columnare, Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae, Edwardsiella tarda, Edwardsiella 15 ictaluri, Mycobacterium maritimum, Francisella sp., Nodavirus, Irido virus, Koi herpes virus, channel catfish virus. Finally, another embodiment relates to a kit of parts, wherein the kit comprises at least two vaccine vials, and these at least two vials together comprise bacteria of the species Lactococcus 20 garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae and a pharmaceutically acceptable carrier for combating Nocardia seriolae infection in fish. Merely as an example: if the kit comprises two vials together comprising bacteria of the species Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae, this means that each of these four types of bacteria are present in at least one of the vials. As a result the four types of bacteria 25 are all present in the kit. In this example, one vial could comprise Lactococcus garviae, whereas the other vial comprises Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae. Alternatively, one vial could comprise Lactococcus garviae and Pasteurellapiscicida, whereas the other vial could comprise Vibrio anguillarum and Nocardia seriolae. If the kit comprises e.g. four vials, each type of bacteria can be present in one vial. 30 Examples.

WO 2009/056629 PCT/EP2008/064805 12 Example 1. Animal husbandry: Test system 5 Animals Species: Yellowtail (Seriolae quinqueradiata) Source: Wild caught fingerlings Av. weight at start of Exp. approx. 20g 10 Since the fish were wild-caught fingerlings they were free from any previous vaccinations. The fish were placed in a quarantine tank upon arrival until they obtained the correct size for the experiment. Inclusion-exclusion criteria Only healthy animals were used. After vaccination, no treatment of sick animals or exclusion of 15 animals was performed. Water - Salinity: natural sea water 25 - 35 ppt - Temperature: 24'C +/- 2'C after vaccination, 20 26'C +/- 2'C after challenge Feed During the pre-treatment period, feed was given ad libitum several times per day. As soon as a sufficient number of fish reached the desired weight of approximately 20 g and were shown to be 25 free of infections they were transferred to experimental tanks. During the experimental period (after vaccination) the fish were fed at 2-4% of their body weight (adjusted weekly). The amount of feed per Kg body weight was kept as similar as possible for vaccinated and mock-vaccinated fish. At weekly intervals, 10-15 fish per group were weighed in groups to determine the mean fish weight for the recalculation of the feed amount. Fish were starved for 24 hours prior to the vaccination to ensure 30 complete emptying of the gastro-intestinal tract and thereby preventing injury to the internal organs as a result of the injection. After challenge fish were fed 14% BW. Tanks WO 2009/056629 PCT/EP2008/064805 13 At the start of the experiment the fish were grouped, vaccinated or mock-vaccinated and subsequently transferred to 500 L tanks. Groups of fish were separated by means of a net placed vertically in the middle of the tank, dividing the tank in two halves. Tank halves were identified by the tank number and a letter (A or B). After challenge fish were housed in 50 L tanks. 5 Grouping and Dosing Vaccination Vaccination was performed by IP injection on the side of the fish, approximately at the end of the 10 pectoral fin. Small hypodermic, single use needles and single use syringes were used. Control fish were mock vaccinated with SVDB (Standard Vaccine Dilution buffer = PBS). Challenge Preparation ofchallenge material 15 Challenge seed of a wild-type Nocardia seriolae was taken from the <-50'C freezer and allowed to thaw. The contents of the vial was inoculated into Eugon Broth at a rate of 1% (v/v), incubated at 26'C on an orbital shaker with a shaking speed set at 150 RPM for app 64-71h. The OD 66 om of the culture typically was 1.5-1.6 corresponding to an app. viable cell count of 10 7 to 108 CFU per ml. This suspension representing a late log phase was used to prepare the challenge suspension. 20 Challenge suspensions were prepared by performing a dilution of the culture to a final cfu/ml of 3 5x10 7 using 1.5% saline. An appropriate dilution was performed in saline and used for injection. Challenge 25 Challenge was performed by IP injection. From the experimental groups, fish were injected with 0.1 ml of the standardised bacterial suspensions. Each group was anaesthetized in AQUI-S until sedated and injected intra-peritoneally on the left side of the body just behind the tip of the pectoral fin. Immediately after injection the fish were transferred to their allocated tank and recovery was followed. The fish were starved for 24h prior to challenge to ensure the complete emptying of the 30 gastro-intestinal tract. EVALUATION OF RESULTS WO 2009/056629 PCT/EP2008/064805 14 The results of the challenges performed for each of the vaccine formulations was evaluated by calculating the Relative Percentage Survival (RPS) values for each group as compared to the control group the day that control mortality reached 60% or more. 5 In addition, statistical analysis was performed on final confirmed cumulative mortality between treatment groups and respective controls using a 2x2 contingency table and Fisher's exact test (One tailed, Stat Soft Inc (2004), Statistica, data analysis Software system, version 6). The RPS values are calculated according to the following formula: 10 % mortality in vaccinated RPS = 1 - (--------------------) x 100 % mortality in controls 15 The antigen concentration in the Examples will often be given in ODU/ml. The ODU/ml is determined as follows: Antigen Concentration (ODU/ml) = (((OD 66 0 ). 1 + (OD6o). / 0.2118)/0.0018 * DF * 106, wherein (OD 660 ).1 + (OD 6 60 ).2 are the OD 66 0 values of two OD 6 60 measurements and wherein DF is the dilution factor. 20 All monovalent vaccines described here were derived from a formalin inactivated N. seriolae antigens and formulated as oil adjuvanted vaccines. The vaccines VI and V2 were injected using an injection volume of 0.05. The vaccines V3, V4 and V5 were injected after mixing equal volumes of vaccine with vaccine diluent and injection of 0. 1ml. 25 Test articles Vaccine 1: Type: Monovalent N. seriolae /oil adjuvanted Formulation 1.0x10 7 ODU/ml (5.0x10 5 ODU/fish) 30 Vaccine 2: Type: Monovalent N. seriolae / oil adjuvanted WO 2009/056629 PCT/EP2008/064805 15 Formulation 1.0x10 6 ODU/ml (5.0x10 4 ODU/fish) Vaccine 3: Type: Monovalent N. seriolae /oil adjuvanted 5 Formulation 1.0x10 7 ODU/ml (5.0x10 5 ODU/fish) Vaccine 4: Type: Monovalent N. seriolae / oil adjuvanted Formulation 1.Ox106 ODU/ml (5.0x10 4 ODU/fish) 10 Vaccine 5: Type: Monovalent N. seriolae / oil adjuvanted Formulation 1.0x10 5 ODU/ml (5.0x10 3 ODU/fish) Vaccine Diluent 15 Type: Standard Vaccine dilution buffer in oil SVDB Type: Standard vaccine dilution buffer (SVDB) 20 Treatment groups, number of fish for challenges performed at week 3 and week 6. # Noe # fish for # fish for 25 Group fish concentration Challenge Challenge (ODU/fish) Week 3 week 6 Vi 20 5.0 x 105 10 5 V2 20 5.0 x 104 10 5 30 Control 20 SVDB 10 5 V3 35 5..x 1. 15 V4 35 5.0x10 4 15 V5 35 5 Ox 10' 15 35 RESUL TS: RPS VALUES WO 2009/056629 PCT/EP2008/064805 16

RPS

60 values of week 3 and week 6 challenges in different vaccine conditions in the minimum antigen trials. Group RPSo RPSo Wk 3 Wk 6 (n=10-15) (n=5) Vi 33 <0 V2 33 33 Conirol mortalit'0 60% V3 40 V4 30 V5 20 Conirol mortality ? None of the conditions were significant different from controls (One tailed Fisher exact, p<0.05) 5 As follows from this table, the monovalent Nocardia vaccines used in this example do not provide a significant protection against Nocardia challenge. Example 2. 10 Animal husbandry: Test system Animals As for example 1 Inclusion-exclusion criteria 15 As for example 1 Water As for example 1 Feed As for example 1 20 Tanks As for example 1 Grouping and Dosing WO 2009/056629 PCT/EP2008/064805 17 Vaccination As for example 1 Challenge Preparation ofchallenge material 5 As for example 1 Challenge As for example 1 EVALUATION OF RESULTS 10 As for example 1 Test articles: Vaccine 1: Type: Tetravalent V. anguillarum/L. garviae 6.8x108cells/ml, P. piscicida 1.36x109 cells/ml, N. 15 seriolae (1.0x107 ODU/ml)/ oil adjuvanted. All bacteria were formalin inactivated. Injection: 0.05 ml Vaccine 2 20 Type: Tetravalent V. anguillarum/L. garviae 6.8x108 cells/ml, P. piscicida 1.36x1 09 cells/ml, N. seriolae (1.Ox106 ODU/ml)/ oil adjuvanted. All bacteria were formalin inactivated. Injection: 0.05 ml 25 Table: Challenge schedule and numbers of fish used for Noc challenges Ag # fish used for # fish used for concentration/fish Challenge week 3 Challenge week 12 Vaccine 1 5.0x10 Vaccine 2 5.0x10 4 15 10 Control RESUTS: RPS VALUES WO 2009/056629 PCT/EP2008/064805 18 RPS after Ns challenge. (n=10-15) ** indicates statistical differences between vaccinates and controls (One tailed Fisher exact, p<0.05)) Group RPS wk 3 RPS wk 12 Vaccine 1 50** 88** Vaccine 2 83** 63** Com.rol % 80 . m..rta t....... 5 As follows from this table, a combination vaccine comprising a Nocardia vaccine according to Example 1, in combination with a V anguillarum/L. garviae /P. piscicida vaccine provides a surprisingly high level of protection against Nocardia challenge 10 Example 3 Animal husbandry: Test system Animals 15 As for example 1 Inclusion-exclusion criteria As for example 1 Water As for example 1 20 Feed As for example 1 Tanks As for example 1 Grouping and Dosing 25 Vaccination As for example 1 Challenge Preparation of challenge material WO 2009/056629 PCT/EP2008/064805 19 As for example 1 Challenge As for example 1 5 EVALUATION OF RESULTS As for example 1 Test articles 10 Vaccine Type: Tetravalent V. anguillarum/L. garviae 6.8x108 cells/ml, P. piscicida 1.36x1 09 cells/ml, N. seriolae (1.Ox107 ODU/ml)/ oil adjuvanted. All bacteria were formalin inactivated. Injection: 0. 1mI (after mixing)* 15 SVDB-Oil diluent Type: Standard vaccine dilution buffer in ISA 763A VG oil obtained from SEPPIC France 20 SVDB Type: PBS buffer: Standard vaccine dilution buffer (SVDB) *The vaccine was prepared by mixing equal volumes of vaccine with SVDB -Oil diluent. 25 Table: Challenge schedule and numbers of fish used for Noc challenges Condition Concentration Number of fish challenged Ns week 3 (ODU/fish) Vaccinate 5.0x10 4 20 Control 20 RESULTS: RPS VALUES WO 2009/056629 PCT/EP2008/064805 20 RPS after Ns challenge. (n=20) * indicates statistical differences between vaccinates and controls (One tailed Fisher exact, p<0.05)) RPS wk 3 Statistical analysis Vaccinate 100%* p<0.0001 As follows from this table, a combination vaccine comprising a Nocardia vaccine according to 5 V1 or V3 of Example 1, in combination with a V anguillarum/L. garviae /P. piscicida vaccine provides a surprisingly high level of protection against Nocardia challenge This experiment confirms the results of Example 2. 10 Example 4 Animal husbandry: Test system Animals 15 As for example 1 Inclusion-exclusion criteria As for example 1 Water As for example 1 20 Feed As for example 1 Tanks As for example 1 Grouping and Dosing 25 Vaccination As for example 1 Challenge Preparation of challenge material As for example 1 30 Challenge WO 2009/056629 PCT/EP2008/064805 21 As for example 1 EVALUATION OF RESULTS As for example 1 5 Test articles Vaccine: Type: Type: Tetravalent V. anguillarum/L. garviae 6.8x1 08 cells/ml, P. piscicida 1.36x1 09 cells/ml, N. seriolae (1.Ox106 ODU/ml)/ oil adjuvanted. All 10 bacteria were formalin-inactivated. Injection: 0. 1mI (after mixing)* SVDB-Oil diluent Type: Standard vaccine dilution buffer in oil 15 SVDB Type: Standard vaccine dilution buffer (SVDB) *The vaccine was prepared by mixing equal volumes of vaccine with SVDB -Oil diluent. 20 Table: Challenge schedule and numbers of fish used for Noc challenges Concentration # fish used for Group N fish Ns Challenge (ODU/fish) Week 3 Vaccinated 40 5.0x10 4 15 Control 40 - 15 RESULTS: RPS VALUES 25 RPS after Ns challenge. (n=15) * indicates statistical differences between vaccinates and controls (One tailed Fisher exact, p<0.05)) WO 2009/056629 PCT/EP2008/064805 22 Statistical analysis Group RPS Vaccinated 64% p=.34 Cornneltmrtadity 73% 5 As follows from this table, even a combination vaccine comprising a relatively low dose of Nocardia vaccine according to V2 or V4 of Example 1, in combination with a V anguillarum/L. garviae /P. piscicida vaccine provides a surprisingly high level of protection against Nocardia challenge 10 This experiment confirms the results of Example 2.

Claims (1)

1. Claims

   1) Combination vaccine for combating Nocardia infection in fish, characterised in that said vaccine comprises bacteria of the species Lactococcus garviae, Pasteurella piscicida,

   Vibrio anguillarum and Nocardia seriolae and a pharmaceutically acceptable carrier.

   2) Combination vaccine according to claim 1, characterised in that at least one of the bacterial species is in a live attenuated form.

   3) Combination vaccine according to claim 1, characterised in that the bacterial species are inactivated.

   4) Combination vaccine according to claim 1 -3, characterised in that it comprises an adjuvant.

   5) Combination vaccine according to claim 4, characterised in that the adjuvant is an oil adjuvant. 6) Combination vaccine according to claim 5, characterised in that the adjuvant is a non- mineral oil adjuvant.

   7) Combination vaccine according to claim 4-6, characterised in that the adjuvant is ISA 763AVG.

   8) Combination vaccine according to claims 1 -7, characterized in that said vaccine comprises at least one other microorganism or virus that is pathogenic to fish, or one other antigen of such a microorganism or virus or genetic material encoding said other antigen.

   9) Combination vaccine according to claim 8, characterised in that said at least one other microorganism or virus is selected from the group consisting of the bacterium causing Big Belly syndrome, Tenacibaculum maritimum, Flavobacterium columnare,

   Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae, Edwardsiella tarda, Edwardsiella ictaluri, Mycobacterium maritimum, Francisella sp., Nodavirus, Irido virus, Koi herpes virus and channel catfish virus.

   10) Method for the preparation of a combination vaccine according to claim 1 -9, characterised in that said method comprises the steps of mixing bacteria of the species

   Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae and a pharmaceutically acceptable carrier. 11) Use of bacteria of at least the species Lactococcus garviae, Pasteur ella piscicida, Vibrio anguillarum and Nocardia seriolae for the manufacture of a combination vaccine for combating Nocardia seriolae infection in fish.

   12) Use according to claim 9 characterised in that the fish belong to the species yellowtail {Seriolae quinqueradiata), amberjack {Seriolae dumerellϊ), sea bass {Lateolabrax japonicus), yellow croacker {Lamitichthys crocea), Pomfret (Pampus argenteus), threadfin {Eleutheronema tetradactylum), snapper (Lutjanus sp), grouper (Epinephelus sp) or trevalli (Caranx sexfasciatus) .

   13) Use according to claim 11 or 12, characterised in that at least one of the bacterial species is in a live attenuated form.

   14) Use according to claim 11 or 12, characterized in that said bacterial species are inactivated.

   15) Use according to claims 11-14, characterized in that for the manufacture of said vaccine additionally at least one other microorganism or virus that is pathogenic to fish, or one other antigen of such a microorganism or virus or genetic material encoding said other antigen is used.

   16) Use according to claim 15, characterized in that said other microorganism or virus is selected from the group consisting of the bacterium causing Big Belly syndrome, Tenacibaculum maritimum, Flavobacterium columnare, Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae,

   Edwardsiella tarda, Edwardsiella ictaluri, Mycobacterium maritimum, Francisella sp., Nodavirus, Irido virus, Koi herpes virus and channel catfish virus.

   17) Kit of parts characterised in that the kit comprises at least two vaccine vials, said vials together comprising bacteria of the species Lactococcus garviae, Pasteurella piscicida, Vibrio anguillarum and Nocardia seriolae and a pharmaceutically acceptable carrier for combating Nocardia seriolae infection in fish.