JP2011506577A - Fish vaccine - Google Patents

Abstract

  The present invention relates to a nocardia-free combination vaccine for the treatment of bacterial infections in fish, the use of bacteria for the production of such a vaccine, a kit comprising a method and parts for the preparation of such a vaccine (kit- of-parts).

Description

  The present invention relates to a nocardia-free combination vaccine for the treatment of bacterial infections in fish, the use of bacteria for the production of such a vaccine, a kit comprising methods and parts for the preparation of such a vaccine (Kit-of-parts).

  In the last decade, there has been a marked increase in fish consumption worldwide. This also takes into account the consumption of cold sea fish such as salmon, turbot, halibut and cod and tropical fish such as asia sea bas, tilapia, mackerel, yellowtail, amberjack, grouper, snapper and cedar.

  As a result, the number and scale of fish farms is expanding to meet growing market needs.

  A large number of closely bred animals, for example as known from livestock, are rarely known or rarely seen for all kinds of diseases before large-scale commercial livestock emerges Or even vulnerable to unknown diseases. This also applies to fish farming. Bacteria found to be pathogenic to fish include, among others, Nocardia, Vibrio, Pasteurella, Photobacterium, Tenashibacrum, Flavobacterium, Flexibacter, Cytophaga, Francisella, Mycobacterium, Streptococcus, Lactococcus Or belongs to the genus Edward Ziera.

  Of the genus Nocardia, Nocardia seriolee cause chronic problems in warm water fish. The damage caused by the Nocardia infection in the fish farming industry has been increasing over the years. In particular, yellowtail (Seriolee quinqueradiata), campachi (Seriolee dumerelli), perch (Lateolabrax japonicus), cicchi (la Pampus argenteus), Swallows white (Eleutheronema tetradactylum), snapper (Lutjanus sp.), Grouper genus (E. Tous (Caranx sexfacciatus) has been affected by Nocardia infection.

  This disease, often called marine nocardiosis, begins as a static infection. This is expressed in fry and juveniles. This bacterium grows in major organs such as the spleen, liver and kidney.

  Due to the low growth rate, the bacteria can grow in fish tissue for extended periods before any visible symptoms appear. The disease is therefore said to be chronic. As a result, the economic loss is significant only by the fact that the fish weight is often between 300 and 1000 g when the outbreak becomes obvious.

  Studies show that yellowtails that use the same tank space as diseased juveniles (previously injected with raw nocardia) eventually show lesions in the body (white spots in their spleen) 3 months after cohabitation. In marine fish farming, nocardia infection appears to progress more rapidly in the summer when the water temperature reaches 24 ° C or higher, but deaths due to nocardia are more common in autumn and early winter, which may lead to fish being in a new environmental situation and its immune system weakening. Because it has to adapt.

  Nocardia appears to be very weak as an inducer of the immune system itself because the immune system is unable to eliminate this infection despite the very slow progression of the disease. This can also explain the fact that there is no effective vaccine against Nocardia infection. Vaccines containing live attenuated or inactive bacteria mimic natural infection to some extent, but even if natural infection cannot induce a proper immune response, it is unlikely that the vaccine will work better.

  Clearly there is a great need for an effective vaccine.

  The object of the present invention is to provide a better vaccine for treating Nocardia infection.

  Surprisingly, this time also known as Lactococcus garviae, Pasteurella piscida (Photobacterium damselae) subspecies Piscidae V. (Currently also known as Listonella anguillarum) Nocardia-free mixed vaccines, including species of bacteria, as expected, Lactococcus garviae, Pasteurella piscida (Pasteurella piscida) ) And Vibrio Anne Rarumu (Vibrio anguillarum) not only provides protection against infection, completely unexpectedly, Nocardia Seriore (Nocardia seriolae) was found to provide even significant level of protection against infection. Most surprisingly, this level of protection against Nocardia seriolee infection is significantly higher than the level of protection obtained with a simple Nocardia seriolee vaccine.

  Accordingly, a first embodiment of the present invention comprises bacteria of the species Lactococcus garviae, Pasteurella piscida and Vibrio angillarum and a pharmaceutically acceptable carrier. In particular, it relates to a nocardia-free combination vaccine for treating Nocardia infection in fish.

  The mechanism of action behind this unexpected finding is currently unknown. However, it is presumed that the components present or linked to the bacterial cell surface used are potent stimulators of cross-specific immunity in fish against Nocardia. Cross-specificity in this respect means that it is not induced by Nocardia but nevertheless provides protection against Nocardia.

  For the production of such a vaccine, the bacterial state; live or inactive is not really important. Importantly, there are still stimulators of cross-specific immunity in fish against Nocardia.

  This can be ensured, inter alia, by using whole bacterial preparations. As mentioned above, it is not important whether the bacteria in the formulation are alive, killed or fragmented (eg by compressing it through French Press).

  As will be appreciated by those skilled in the art, the method used for inactivation has little to do with bacterial activity. Classical methods for inactivation, such as treatment with ultraviolet radiation, gamma radiation, formalin, binary ethyleneimine, thimerosal, etc., well known in the art can be utilized. Bacterial inactivation by physical stress, for example using a French press, provides likewise suitable starting materials for the production of the vaccine according to the invention. Thus, inactivated bacteria need not necessarily be in the form of inactivated whole cells, and the cells can be destroyed. Inactivated bacteria have the advantage over live attenuated bacteria that they are very safe.

  Accordingly, in a preferred form of this embodiment, the present invention relates to a combination vaccine according to the present invention in which the bacterial species are inactivated.

  This is also highly preferred because live attenuated bacteria inherently have factors that stimulate cross-specific immunity against Nocardia. Live attenuated bacteria have the advantage over inactivated bacteria that they are more effective than inactivated bacteria, especially when given without an adjuvant. Furthermore, they replicate to some extent until they are arrested by the immune system, so that a small number of cells can be given. Live attenuated bacteria are bacteria that are less pathogenic than their wild type, but nevertheless induce an effective immune response.

  Attenuated strains can be obtained along classical pathways long known in the art, such as serial passage, temperature adaptation, chemical mutagenesis, ultraviolet radiation, or by site-directed mutagenesis .

  Accordingly, 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.

  The vaccine according to the invention can be prepared starting from bacterial culture according to techniques well known to those skilled in the art.

  Review articles on fish vaccines and their production can be found, inter alia, in Somerset, I., in Expert Review of Vaccines 4: 89-101 (2005). Krossoy, B .; Biering, E .; And Frost, P .; According to J. Acta Parasitology 46: 71-81 (2001), Buchmann, K .; Lindenstrom, T .; And by Vescantani, Advances in vegetarian medicine 41: 539-550 (1999). Gravningen, K .; And Greger, E .; And by Anderson, D. in Developments in Biological Standardization 90: 257-265 (1997). P. Is due to.

  The vaccine according to the invention basically comprises an effective amount of the bacterium according to the invention and a pharmaceutically acceptable carrier.

  As used herein, the term “effective” results in an onset level that is less than 50% of the onset seen in unvaccinated wild-type Nocardia-infected fish under the same conditions. , Defined as the amount that is sufficient to induce an immune response in the target fish.

  The amount of cells to be administered depends, inter alia, on the amount of various bacteria used, the state of the bacteria; live attenuated or inactivated, the presence of an adjuvant and the route of administration.

  If starting from a commercial vaccine, the manufacturer provides this information.

  Otherwise, those skilled in the art will find sufficient guidance in the above references and in the information given below, especially in the examples.

  As mentioned above, the vaccine according to the invention can be prepared starting from bacterial culture according to techniques well known to those skilled in the art. In the Examples section an example of the preparation of a vaccine according to the invention is given.

Generally speaking, vaccines produced in accordance with the present invention based on inactivated bacteria will generally contain 10 ³ to 10 ¹⁰ , preferably 10 ⁶ to 10 ⁹ , more preferably 10 ⁸ to 10 ⁹ bacteria. It can be given in between doses. A dose above 10 ¹⁰ bacteria is immunologically appropriate, but less attractive for economic reasons. Vaccines produced according to the present invention based on live attenuated bacteria can be given at lower doses since the bacteria will continue to replicate for a period of time after administration. Vaccines produced according to the invention, based on live attenuated bacteria, can generally be given at a dose of 10 ² to 10 ⁸ , preferably 10 ³ to 10 ⁵ bacteria.

  Examples of pharmaceutically acceptable carriers that are particularly suitable in vaccines according to the present invention are sterile water, saline, aqueous buffers such as PBS, and the like. Furthermore, the vaccine according to the present invention may contain other additives such as adjuvants, stabilizers, antioxidants, etc. as described below.

The vaccine produced as described in the present invention may contain immunostimulants, so-called adjuvants, in a preferred formulation. Adjuvants generally include substances that non-specifically promote host immune responses. Many different adjuvants are known in the art. Examples of adjuvants frequently used in farming fish and shellfish, muramyl dipeptide, lipopolysaccharides are some glucans and glycans and Carbopol ^(R). A detailed review of suitable adjuvants for fish and shellfish vaccines is given in a review by Jan Raa (Summary in Fishers Science 4 (3): 229-288 (1996)). The vaccine may also include a so-called “vehicle”. A vehicle is a compound that bacteria adhere to without covalently binding to it. Such vehicles are, among others, biomicrocapsules, microalginates, 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 EP1099.942, EP180.564, EP242.380). In addition, the vaccine may contain one or more suitable surface active compounds or emulsifiers, such as Span or Tween.

  Thus, in a more preferred form of this embodiment, the combination vaccine according to the invention comprises an adjuvant.

It can be seen that oil adjuvants are usually somewhat more effective for combination vaccines according to the present invention. Oily adjuvants suitable for use in water-in-oil emulsions are, for example, mineral oils or metabolizable oils. Mineral oils are, for ^{example, Bayol (R), Marcol (R} ) and Drakeol ^(R). Metabolizable oils are, for example, vegetable oils such as peanut oil and soybean oil, animal oils such as fish oil squalane and squalene, and tocopherols and derivatives thereof. Suitable adjuvants are, for example, w / o emulsions, o / w emulsions and w / o / w double emulsions. Very suitable o / w emulsions are for example 5-50% w / w aqueous phase and 95-50% w / w oily adjuvant, more preferably 20-50% w / w aqueous phase and 80-50% w. / W starting from an oily adjuvant.

  Thus, in an even more preferred form of this embodiment, the combination vaccine according to the invention comprises an adjuvant, in which case the adjuvant is an oily adjuvant.

  As mentioned above, oily adjuvants can be broadly classified into adjuvants containing mineral oils and adjuvants containing non-mineral oils. Mineral oil can be less attractive from a food safety perspective and due to the damage it can cause (from both). Accordingly, preferred oily adjuvants include non-mineral oils.

  A more preferred non-mineral oil is, for example, ISA 763A VG oil as commercially available from SEPPIC France.

  The amount of adjuvant added depends on the nature of the adjuvant itself, and information regarding such amounts is provided by the manufacturer.

  Often the vaccine is mixed with a stabilizer, for example to prevent degradation of easily degradable proteins, to extend the shelf life of the vaccine, or to improve lyophilization efficiency. Useful stabilizers include, among others, SPGA (Bovarnik et al .; J. Bacteriology 59: 509 (1950)), carbohydrates such as sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein, etc. Or it is a buffer solution, such as its degradation product and alkali metal phosphate.

  Preferably, the vaccine as described is given in lyophilized form.

  In addition, the vaccine can be suspended in a physiologically acceptable diluent. It goes without saying that other methods of emulsifying or stabilizing proteins are also encompassed by the invention, adding vehicle compounds or diluents.

  Many methods of administration (all known in the art) can be utilized. Vaccines as described are preferably administered to fish via injection, such as intraperitoneal injection, immersion, spraying, dipping or orally. However, it should be noted that the route of administration may also depend on the type of vaccine: the vaccine is attenuated live Lactococcus garviae, Pasteurella piscida and Vibrio angillarum. If it contains bacteria, it can be easily administered by dipping. On the other hand, if the vaccine comprises Lactococcus garviae, Pasteurella piscida and Vibrio anguillarum bacteria in the form of inactivated bacteria, or more generally, an adjuvant, Is preferred, the preferred method of administration is the intraperitoneal route. From an immunological point of view, intraperitoneal vaccination is indeed an effective vaccination route in fish in the case of inactivated bacteria, especially because it allows the incorporation of adjuvants.

  A convenient way to make a vaccine according to the present invention is to use a commercially available vaccine. Lactococcus garviae vaccines, Pasteurella piscida vaccines and Vibrio anguillarum vaccines are commercially available and / or methods for making them are described in the literature. Has been.

  The administration protocol can be optimized according to standard vaccination practices.

  The age of the fish to be vaccinated is not important, but obviously it would be desirable to vaccinate against Nocardia infection at an early stage. For many fish vaccines, they are administered when the fish weighs between 10 and 35 grams. This is also a very appropriate time to vaccinate against Nocardia.

  For oral administration, the vaccine is preferably mixed with a carrier suitable for oral administration, ie cellulose, food or metabolizable substances such as α-cellulose or various vegetable or animal derived oils. Administration of the vaccine through bioencapsulation (which exposes live food to the high concentration of the vaccine and subsequently feeds the live food to the fish) is also an attractive method. A particularly preferred food carrier for oral delivery of a vaccine according to the present invention is a live bait organism capable of encapsulating the vaccine. Suitable live organisms include plankton-like non-selective filter eaters, preferably members such as annelids, Artemia sp. Brine shrimp Artemia sp. Is highly preferred.

  In view of a number of viruses and organisms that are pathogenic to fish, Lactococcus garviae, Pasteurella piscida and Vibrio anguillarum, Vibrio anguillarum, for the production of vaccines It is beneficial to administer with these other non-Nocardia fish pathogenic bacteria or viruses, antigens of these bacteria or viruses, or genetic material encoding such antigens.

  An example of a well-known commercially important fish pathogen is an example of a recently discovered bacterium (this novel bacterium (BB E3F1) that causes Big Berry syndrome as described in Thai patent application TH92840. Collection Nationale de Cultures de Microorganisms (CNCM), Institute Pasteur, 25 Rue du Doctour Roux, F-75724 Paris Cedex 15, France, with the accession number CNCM1-3257. Flavobacterium columnare, Lexical Arthrobacter Maritimusu (Flexibacter maritimus) (formerly known as Tenakibakuramu-Maritimamu (Tenacibaculum maritimum)), Streptococcus iniae (Streptococcus iniae), Streptococcus difficile (Streptococcus difficile), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus THIS galactosamine Chie (Streptococcus dysgalactiae, Edwardsiella tarda, Edwardsiella ictaluri, Micobacterium maritimum (M) cobacterium maritimum), Francisella sp (Francisella sp.) as well as the virus, for example Noda virus, iridoviruses, koi herpes virus, such as the United States catfish virus, it is.

  The advantages of such a combined vaccine are that it not only protects against Lactococcus garviae, Pasteurella piscida and Vibrio angularum infections, as well as other nocardia infections Is to protect against other diseases.

  Thus, in a preferred embodiment, the nocardia-free mixed vaccine according to the invention is in addition to the bacteria of the species Lactococcus garviae, Pasteurella piscida and Vibrio anguillarum species, It includes at least one other microorganism or virus that is pathogenic to, or some other antigen of such microorganism or virus, or genetic material that encodes this other antigen.

In a more preferred embodiment, at least one other microorganism that is pathogenic to fish, or some other antigen of such a microorganism, or genetic material encoding this other antigen, is Big Berry syndrome. causing bacteria, Flavobacterium Korumunare (Flavobacterium columnare), Tena Fang crumb Mali tee Mumm (Tenacibaculum maritimum), Streptococcus iniae (Streptococcus iniae), Streptococcus difficile (Streptococcus difficile), Streptococcus agalactiae (Streptococcus agalactiae), Streptococcus Disgarak Streptococcus dysgalactiae, Edwardsiella tarda, Edwardsiella ichtaluri, Mycobacteriaum sp. Selected from the group of bacteria consisting of viruses, American catfish virus.

  Yet another embodiment of the invention relates to a method for the manufacture of a vaccine for treating Nocardia infection in fish. Such methods include attenuated live or inactivated forms of Lactococcus garvia, Pasteurella piscida and Vibrio anguillarum bacteria and pharmaceutically acceptable carriers. Including a stage of mixing.

  A preferred form of this embodiment relates to a method further comprising admixture of an adjuvant.

  If Lactococcus garviae, Pasteurella piscida and Vibrio anguillarum vaccine components are used immediately, these can be mixed prior to administration.

  If these are to be administered orally, mixing before administration is a preferred choice. When the vaccine is administered by injection, the components can be mixed for simultaneous administration, and they can also be administered individually or sequentially in a series of continuous infusions.

  The fact that either commercially available Lactococcus garviae vaccines, Pasteurella piscida vaccines and Vibrio angillarum vaccines or vaccines prepared as described in the examples are used. Regardless, one skilled in the art prefers to use the amount of each bacterium that is necessary to induce an immune response against each bacterial species. By way of example only, the amount of Lactococcus garviae bacteria in a nocardia-free vaccine according to the present invention is preferably sufficient to induce an immune response against Lactococcus garviae infection.

  Bacteria of Lactococcus garviae, Pasteurella piscida and Vibrio anguillarum species in a combination vaccine can be administered simultaneously, individually or sequentially. Then, if within a short interval, these can still be considered mixed vaccines, as explained below. Co-administration is the administration of Lactococcus garviae, Pasteurella piscida and Vibrio angillarum at the same moment, preferably as a mixture. Needless to say, this is a preferred method of administration because it facilitates manipulation. Individual administration may involve (partially or completely) Lactococcus garviae, Pasteurella piscida and Vibrio angillarum bacteria (partially or completely) preferably at two or more different injection sites. It is to administer at the same time.

  Continuous administration is administration in which Lactococcus garviae, Pasteurella piscida and Vibrio angillarum bacteria are administered at different times. It will be appreciated that when individual or continuous infusions are performed, these are preferably performed in the desired order, on the same day, more preferably within 12, 10, 8, 6, 4, 2 or 1 hour. Even more preferred is administration within 50, 40, 30, 20, 10 or 5 minutes after each. If administration of the entire vaccine of the combined vaccine takes place within 10 minutes (even more preferably less than 5 minutes is even more preferred), a moment is sufficient for each fish operation, which System invocation begins almost immediately.

  Another embodiment of the present invention is at least Lactococcus garviae, Pasteurella piscida for the manufacture of a nocardia-free combination vaccine for treating Nocardia seriolee infections in fish. ) And bacteria of the species of Vibrio angillarum.

  In a preferred form of this embodiment, the fish to be treated for Nocardia seriolee infection and thus the fish from which the vaccine is produced is yellowtail (Seriolee quinqueradiata), amberjack (Seriole domelliri ( Seriolae dumerelli), Suzuki (Lateolabrax japonicus), Kiguchi (Lamitichis crosea), Managatsuo (Pampus Argenteus) tetradactylum)), Feda (Rutsujanusu genus (Lutjanus sp.)), Grouper (Epineferusu genus (Epinephelus sp.)) And belonging to the species of bigeye trevally (Karankusu-Sexton fascia-to-scan (Caranx sexfasciatus)).

  In another preferred form of this embodiment, at least one of the bacterial species used for production is an attenuated live form.

  In yet another preferred form of this embodiment, the bacterial species used for production is inactivated.

  In a more preferred form of this embodiment, for the production of this vaccine, at least one other microorganism or virus that is pathogenic to fish or some other antigen of such microorganism or virus or this other Genetic material encoding the antigen is used.

  In an even more preferred form of this embodiment, the other microorganism or virus is a bacterium that causes Big Belly Syndrome, Tenacibacumum maritimum, Flavobacterium columnare, Flavobacterium columnum, Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus diagalactiae (Streptococcus edifice) Talda (Edwardsiella tarda), Edwardsiella ictaluri, Mycobacterium maritimum, Francisella sp., Virus, Idavirus, Idavirus Selected from the group.

  Finally, another embodiment relates to a kit of parts, the kit comprising at least two vaccine vials, which together are combined with Nocardia in fish. Lactococcus garviae, Pasteurella piscida and Vibrio anguillarum pharmaceutically acceptable carriers of Vibrio anguillarum and Vibrio anguillarum for treating seriole (Nocardia seriolae) infection . Merely by way of example, if this kit contains two vials together of bacteria of the species Lactococcus garviae, Pasteurella piscida and Vibrio angillarum, Each of these three types of bacteria means present in at least one of these vials. As a result, all three types of bacteria are present in the kit. In this example, one vial may contain Lactococcus garviae while the other vial contains Pasteurella piscida and Vibrio angillarum. Alternatively, one vial can contain Lactococcus garviae and Pasteurella piscida, while the other vial can contain Vibrio angillarum. If the kit contains, for example, 3 vials, each type of bacteria can be in one vial.

  Example

Animal breeding:
Test system Animal Species: Yellowtail (Seriola quinqueradiata)
Source: Wild catch larvae Average weight at start of experiment: Approximately 20 g (18-27 g)
Since the fish were wild catch larvae, they had not been vaccinated before. These fish were placed in isolation tanks upon arrival until they were appropriately sized for the experiment.

Exclusion criteria Only healthy animals were used. After vaccination, sick fish were not treated or animals were excluded.

Water salinity: natural seawater 25-35ppt
-Temperature: 24 ° C +/- 2 ° C after vaccination, 26 ° C +/- 2 ° C after infection induction
During the pre-treatment period, food was fed at a discretion several times a day. As soon as a sufficient number of fish reached the desired weight of approximately 20 g and showed no infection, they were transferred to the experimental tank. During the experimental period (after vaccination), these fish were fed to 2-3% of their body weight (BW) (weekly adjustment). Feeding per kg body weight was kept as similar as possible to vaccinated and mock vaccinated fish. At weekly intervals, 10-15 fish per group were weighed in groups to determine the average fish weight for recalculation of feed. The fish were starved for 24 hours prior to vaccination to ensure that the gastrointestinal tract was completely emptied so that it did not damage the internal organs as a result of the injection. After induction of infection, the fish were fed with 1-3% BW.

Tank At the start of the experiment, the fish were grouped and vaccinated or mock vaccinated and then transferred to a 500 L tank. A group of fish was divided by installing a net vertically in the center of the tank and dividing the tank into two. Each half of the tank was identified by tank number and letter (A or B). After infection induction, fish were raised in 50 L tanks.

Grouping and administration Vaccination Vaccination was performed by IP injection on the side of the fish, near the end of the fin. A small hypodermic injection needle and disposable syringe were used. Control fish were sham-vaccinated with SVDB (standard vaccine dilution buffer = PBS).

Induction of infection Preparation of infection-inducing material Wild-type Nocardia seriole infection-inducing seeds were removed from the <-50 ° C freezer and thawed. The contents of the vial were inoculated into Yugon broth at a rate of 1% (v / v) and incubated at 26 ° C. on an orbital shaker with a shaking speed of 150 RPM for approximately 67-71 hours. The OD _{660 nm} of the culture was typically 1.5-1.6, corresponding to an approximate viable cell number of 10 ⁸ CFU / mL. This suspension, which applies to the late log phase, was used to prepare the infection-induced suspension. Appropriate dilutions were made in saline and used for injection.

Induction of infection Infection was induced by IP injection. From the experimental group, 0.1 mL of standard bacterial suspension was injected into the fish. Each group was anesthetized in AQUI-S until sedated and injected intraperitoneally immediately after the tip of the fin on the left side of the body. Immediately after injection, the fish were transferred to the assigned tank and then allowed to recover. The fish was starved for 24 hours prior to challenge to ensure that the digestive tract was completely emptied.

Evaluation of Results On the day when the control mortality reached 50% or higher, the relative survival rate (RPS) value for each group when compared to the control group was calculated for each vaccine formulation. The results of the infection induction performed were evaluated.

  In addition, using the 2x2 contingency table and Fisher's exact test (one-sided, Stat Soft Inc (2004), Statistica, data analysis software system, version 6), the final confirmed cumulative death between treatment groups and individual controls. Statistical analysis was performed on rates.

  The RPS value is calculated according to the following formula.

The antigen concentration in the examples is often given in ODU / mL. Calculating the ODU / mL as follows: Antigen Concentration _{(ODU / mL) = ((} (OD 660) .1+ (OD 660) .2) / 2) -0.2118) /0.0018 * DF * 10 ⁶ (where (OD ₆₆₀ ) .1+ (OD ₆₆₀ ) .2 is the OD ₆₆₀ value of two OD ₆₆₀ measurements, where DF is the dilution factor).

  All plain vaccines described herein are formalin inactivated N.V. It was derived from seriole antigen and formulated as an oil-based adjuvant vaccine. Vaccines V1 and V2 were injected using an injection volume of 0.05. After mixing equal volumes of vaccine with 0.1 mL vaccine dilution and injection solution, vaccines V3, V4 and V5 were injected.

Test substance Vaccine 1: Type: plain N.I. Seriole / Oil-based adjuvant Formula 1.0 × 10 ⁷ ODU / mL (5.0 × 10 ⁵ ODU / fish)
Vaccine 2: Type: plain N.I. Seriole / Oil-based adjuvant Formula 1.0 × 10 ⁶ ODU / mL (5.0 × 10 ⁴ ODU / fish)
Vaccine 3: Type: plain N.I. Seriole / Oil-based adjuvant Formula 1.0 × 10 ⁷ ODU / mL (5.0 × 10 ⁵ ODU / fish)
Vaccine 4: Type: plain N. Seriole / Oil-based adjuvant Formula 1.0 × 10 ⁶ ODU / mL (5.0 × 10 ⁴ ODU / fish)
Vaccine 5: Type: plain N.I. Seriole / Oil-based adjuvant Formula 1.0 × 10 ⁵ ODU / mL (5.0 × 10 ³ ODU / fish)
Vaccine dilution type: Standard vaccine dilution buffer in oil SVDB
Type: Standard vaccine dilution buffer (SVDB)
The vaccine formulations tested are listed in Table 1. This vaccine is a water-in-oil vaccine formulated as a microcomponent and is mixed with the ISA 763A VG oil-vaccine dilution prior to use.

  RPS value

  From Table 2 as follows, the survival rate of vaccinated fish was not significantly different from the control (one-sided Fisher exact test, p <0.05). The relative% of survival and mortality rates for both the vaccinated and control groups are not statistically different.

Animal husbandry:
Test system Animals Same as for Example 1 Exclusion criteria Same as for Example 1 Water Same as for Example 1 Feed Same as for Example 1 Tank Same as for Example 1 Grouping and administration Vaccination As for Example 1 Induction of infection Preparation of infection-inducing material As for Example 1 Induction of infection Same as for Example 1 Evaluation of results Same as for Example 1 Test substance Vaccine Vaccine:
Type: Trivalent V.V. V. anguillarum / L. L. garvieae 6.8 × 10 ⁸ cells / mL, P. cerevisiae P. picicida 1.36 × 10 ⁹ cells / mL
Injection: 0.1mL
The vaccine formulations tested are listed in Table 3. This vaccine is a water-in-oil vaccine formulated as a microcomponent and was mixed with ISA 763A VG oil vaccine diluent prior to use. Antigens used for these vaccines are P.P. P. picicida, L. L. garvieae and V. It is derived from a normal production strain of V. anguillarum.

N. RPS after induction of seriole infection (n = 15). The asterisk ^* indicates a statistical difference between the vaccinated group and the control (one-sided Fisher exact test, p <0.05)). The control mortality obtained when calculating the RPS was 73%.

  From this experiment, Table 4, the relative% survival of the vaccinated group was 73%, while at that time> 60% of the fish died in the control group (actually 72% mortality in the control group). Result. Therefore, P.I. Piccida, L.C. Garbier and V. It can be concluded that vaccines containing angiralum can protect fish from Nocardia infection.

Claims (17)

  Nocardia infection in fish characterized in that it comprises bacteria of Lactococcus garviae, Pasteurella piscida and Vibrio anguillarum species and a pharmaceutically acceptable carrier. Nocardia-free mixed vaccine for.   The nocardia-free combination vaccine according to claim 1, characterized in that at least one of the bacterial species is in an attenuated live form.   2. The nocardia-free combination vaccine according to claim 1, wherein the bacterial species is inactivated.   The nocardia-free combination vaccine according to claims 1 to 3, characterized in that it comprises an adjuvant.   The nocardia-free combination vaccine according to claim 4, wherein the adjuvant is an oil-based adjuvant.   The nocardia-free combination vaccine according to claim 4 or 5, wherein the adjuvant is a non-mineral oil adjuvant.   The nocardia-free mixed vaccine according to claims 4 to 6, characterized in that the adjuvant is ISA 763A VG.   Claim 1 to claim 3, characterized in that it comprises at least one other microorganism or virus that is pathogenic to fish or some other antigen of such microorganism or virus or genetic material encoding said other antigen. 8. Nocardia-free mixed vaccine according to 7.   The at least one other microorganism or virus is a bacterium causing Big Berry syndrome, Tenacibacumum maritimum, Flavobacterium columnare, Streptococcus s.・ Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiee, Edward Sierra Tardda (ellward) a tarda), Edwardsiella ictaluri, Mycobacterium maritimum, Francisella sp., nodavirus, iridovirus, koi herpes virus, or American herpes virus The nocardia-free mixed vaccine according to claim 8, wherein   Mixing bacteria of a species of Lactococcus garviae, Pasteurella piscida and Vibrio anguillarum and a pharmaceutically acceptable carrier, characterized in that it comprises the steps of: A method for the preparation of a nocardia-free combination vaccine according to claims 1-9.   At least Lactococcus garviae, Pasteurella piciida u, and Vibrio bilum biliga for the production of a nocardia-free combination vaccine for treating Nocardia seriolee infections in fish ) Use of seed bacteria.   The fish is yellowtail (Seriolae quinqueradiata), amberjack (Seriole dumerelli), perch (Lateolabrax japonicus (Lateolabrax japonicus) or cigula cereal). 12. Use according to claim 11, characterized in that it belongs to a species.   Use according to claim 11 or claim 12, characterized in that at least one of the bacterial species is in an attenuated live form.   13. Use according to claim 11 or claim 12, characterized in that the bacterial species are inactivated.   For the production of the vaccine, at least one other microorganism or virus that is pathogenic to fish or other antigens of such microorganisms or viruses or genetic material encoding the other antigens is used Use according to claims 11 to 14, characterized in that   The other microorganism or virus is a bacterium that causes Big Berry syndrome, Tenacibacumum maritimum, Flavobacterium columnare, Streptococcus pytoscus cc Streptococcus difficile), Streptococcus agalactiae, Streptococcus dysgalactiae, Edward Sierra tarda (Edwardsell) The group consisting of Edwardsiella iktaluri, Mycobacterium maritimum, Francisella sp., Nodavirus, iridovirus, koi herpesvirus and American catfish virus The use according to claim 15.   To treat Nocardia seriolee infections in fish, it includes at least two vaccine vials, which together are Lactococcus garviae, Pasteurella piscicida and Bibliumral A kit of parts (kit-of-parts), characterized in that it comprises a bacterium of the species (Vibrio angillarrum) and a pharmaceutically acceptable carrier.