WO2008003734A1

WO2008003734A1 - Combination vaccine against streptococcus

Info

Publication number: WO2008003734A1
Application number: PCT/EP2007/056789
Authority: WO
Inventors: Luc Grisez; Chow Yong Ng
Original assignee: Intervet International BV
Current assignee: Intervet International BV
Priority date: 2006-07-06
Filing date: 2007-07-05
Publication date: 2008-01-10
Anticipated expiration: 2009-01-06
Also published as: MX2008016248A; ECSP099066A; HN2009000005A; CR10490A

Abstract

The present invention relates to combination vaccines for the protection of fish against streptococcal infection, and to methods for the preparation of such combination vaccines.

Description

COMBINATION VACCINE AGAINST STREPTOCOCCUS

The present invention relates to a combination vaccine for the protection of fish against streptococcal infection, and to methods for the preparation of such a combination vaccine.

Several species of the bacterial genus Streptococcus are currently known to cause infections in fish, more specifically in fish that are kept in aquaculture. Examples of such streptococcal species are Streptococcus iniae, S. difficile, S. agalactiae, S. dysgalactiae and S. phocae.

There have recently been some changes in opinion about the correct nomenclature of Streptococcus difficile and Streptococcus agalactiae. Vandamme et al (Int. J. Syst. Bacteriology 47: 81-85 (1995)) have suggested that Streptococcus difficile is in fact a non-hemolytic Streptococcus agalactiae {Streptococcus agalactiae is a β-hemolytic bacterium). For reasons of convenience, the traditional nomenclature: Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae is used here.

Streptococcus iniae is frequently found in Tilapia, Rainbow trout, European sea bass and bream, Asian sea bass, Red drum, Rabbit fish, Japanese flounder, Yellowtail and hybrid striped bass. The annual impact of Streptococcus iniae infection to aquaculture exceeds 100 million US-dollars.

Streptococcus difficile is frequently found in Asian sea bass, Tilapia, snapper and catfish.

Streptococcus agalactiae is currently mainly found to infect Tilapia.

Vaccines for combating Streptococcal infection in fish are known in the art. Many of the Streptococcus vaccines are based upon killed whole cells. Streptococcus agalactiae vaccines are i.a. described in the US-Patent Application US 2005/0208077. Streptococcus iniae vaccines are i.a. described in US-Patent US 6,379,677. Vaccines for combating Streptococcus iniae infection are also commercially available. An example is Norvax Strep Si, a vaccine against Streptococcus iniae infection and sold by Intervet Int. B.V. Eldar et al (Vaccine 13: 867-870 (1995)) have described a vaccine against Streptococcus difficile on the basis of killed whole cells. Due to the fact that i.a. Streptococcus iniae and Streptococcus difficile are frequently found as the cause of disease in fish, more specifically in tilapia and Asian sea bass, it seems attractive to combine the vaccines against Streptococcus iniae and Streptococcus difficile during administration.

However it was unexpectedly found that when Streptococcus iniae and Streptococcus difficile were administered as a combination vaccine, the level of protection against Streptococcus iniae is highly impaired. If on the other hand a Streptococcus iniae vaccine is administered intraperitoneal (IP) without Streptococcus difficile, the protection against Streptococcus iniae is as expected. It appeared that the presence of Streptococcus difficile in a combination vaccine is, for unknown reasons, highly suppressive to the development of an anti-Streptococcus iniαe immune response.

It is an objective of the present invention to provide a solution to this problem.

It was surprisingly found now, that if a combination vaccine for the protection of fish against streptococcal infection comprises an immunogenic amount of Streptococcus difficile cells and an immunogenic amount of Streptococcus iniαe cells wherein the ratio of Streptococcus difficile cells to Streptococcus iniαe cells is more than or equal to (>) 40 to 10, this problem of suppression of the development of an anti-immune response to Streptococcus iniαe either occurs to an acceptable level, or is not seen at all.

As said above, the presence of an immunogenic amount of Streptococcus difficile cells and an immunogenic amount of Streptococcus iniαe cells is of course necessary to induce an immune response to each of the bacterial species. The amount of Streptococcus difficile cells in the vaccine should however be at least four times the amount of Streptococcus iniαe cells.

Thus, a first embodiment of the present invention relates to a combination vaccine for the protection of fish against streptococcal infection that has as a characteristic that this combination vaccine comprises an immunogenic amount of Streptococcus difficile cells and an immunogenic amount of Streptococcus iniαe cells wherein the ratio of Streptococcus difficile cells to Streptococcus iniαe cells is > 40 to 10. As will become clear from the Examples, a combination vaccine wherein the amount of Streptococcus difficile cells in the vaccine is more than four times the amount of Streptococcus iniae cells gives even less suppression of the development of an anti-immune response against Streptococcus iniae.

Therefore, a combination vaccine wherein the ratio of Streptococcus difficile cells to Streptococcus iniae cells is > 40 to 8 is preferred and ratios of > 40 to 6, > 40 to 4, > 40 to 2 and > 40 to 1 are in that order increasingly more preferred.

Thus, preferred forms of this embodiment relate to a combination vaccine according to the invention wherein that ratio is > 40 to 8, more preferably > 40 to 6, even more preferably > 40 to 4, still even more preferably > 40 to 2 and most preferably > 40 to 1.

Traditionally, many Asian fish vaccines were, if possible at all, given as water-based vaccines. They comprised no oil phase. However currently there is a tendency to use water-in-oil emulsions, water- in- oil-in-water emulsions or oil-in- water emulsions. Such emulsion-vaccines have the advantage that they are more efficacious than their water-based counterparts. Moreover, emulsion vaccines allow for lower amounts of antigen to be used.

Therefore, another preferred form of this embodiment relates to a combination vaccine according to the invention wherein the vaccine is a water-in-oil vaccine.

Still another preferred form of this embodiment relates to a combination vaccine according to the invention wherein the vaccine is an oil-in-water vaccine.

Again another preferred form of this embodiment relates to a combination vaccine according to the invention wherein the vaccine is a water-in-oil-in- water vaccine.

The problem to be solved, i.e. the problem that the presence of Streptococcus difficile in a combination vaccine is highly suppressive to the development of an anti-Streptococcus iniαe immune response, is encountered both in water-based vaccines and in emulsions on the basis of water and oil. The solution as provided above is one solution to the problem. In case an emulsion on the basis of water and oil is used for the vaccine, another solution was found to be feasible, possibly in combination with the solution given above.

It was surprisingly found now, that if the Streptococcus iniae cells and Streptococcus difficile cells are present in separate water droplets of a water- in-oil emulsion, the problem is less significant or even insignificant. In that case, there is in principle even no need to keep the ratio Streptococcus difficile cells to Streptococcus iniae cells > 40 to 8.

A water-in-oil emulsion wherein the Streptococcus iniae cells and Streptococcus difficile cells are present in separate water droplets of the emulsion can relatively easy be made. The standard procedure for making a basic water-in-oil emulsion is based upon the mixing of an amount of a single watery phase, a single oil phase and an amount of bacterial cells. The watery phase can e.g. be just water, a water-based buffer and the like. The oil phase can be a mineral or non-mineral oil (see below). Additionally, one or more emulsifϊers and/or detergents can be added. The methods for making such emulsions, and the components for use in such emulsions are well-known in the art. The present method differs from the standard method in that instead of a single watery phase two watery phases are used; one comprising Streptococcus iniae cells and the other one comprising Streptococcus difficile cells. Each of the two watery phases is now emulsified with an amount of oil phase and after the separate water-in-oil emulsions are made, the two water-in-oil emulsions are mixed. A variant to this method is e.g. to make a water-in-oil emulsion comprising one of the two Streptococcal cell types and to add the other Streptococcal cell type in watery phase e.g. during the emulsifϊcation process. By doing this, (most of) the second Streptococcal cell type will automatically become integrated in new water droplets in the water-in-oil emulsion. It goes without saying that water-in-oil emulsions wherein the Streptococcus iniae cells and

Streptococcus difficile cells are present in separate water droplets of the emulsion always fall within the scope of the invention, regardless the way they are made.

Therefore, another embodiment of the present invention relates to a combination vaccine for the protection of fish against streptococcal infection wherein said vaccine is a water-in-oil vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the Streptococcus iniae cells and the Streptococcus difficile cells are present in separate water droplets of said water- in-oil emulsion.

Another possibility is, to make use of a water- in-oil- in- water emulsion, wherein the immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells are present in separate water droplets of said water- in-oil-in-water emulsion.

Such an emulsion comprises watery droplets, present inside small oil droplets, which in turn are surrounded by a watery phase.

Therefore, another form of this embodiment relates to a combination vaccine for the protection of fish against streptococcal infection, wherein said vaccine is a water-in-oil-in- water vaccine comprising an immunogenic amount of Streptococcus iniae and Streptococcus difficile wherein the Streptococcus iniae component and the Streptococcus difficile component are present in separate water droplets of said water-in-oil-in- water emulsion.

Still another possibility to keep the Streptococcus iniae cells and the Streptococcus difficile cells separated in a water-in-oil-in- water emulsion is, to first make a water-in-oil emulsion wherein the watery phase comprises only the Streptococcus iniae cells or the Streptococcus difficile cells. In a next step, this water-in-oil emulsion can then be emulsified in a watery phase that comprises the other of the two bacterial components, i.e. the Streptococcus difficile cells or the Streptococcus iniae cells.

Thus, still another form of this embodiment relates to a combination vaccine for the protection of fish against streptococcal infection, wherein said vaccine is a water-in-oil-in- water vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the Streptococcus iniae cells are present in the oil- encapsulated water droplets of said water-in-oil-in- water emulsion and the Streptococcus difficile cells are present in the free watery phase of said water- in-oil- in-water emulsion.

Thus, still another form of this embodiment relates to a combination vaccine for the protection of fish against streptococcal infection, wherein said vaccine is a water-in-oil-in- water vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the

Streptococcus difficile cells are present in the oil-encapsulated water droplets of said water-in-oil- in- water emulsion and the Streptococcus iniae cells are present in the free watery phase of said water-in- oil-in-water emulsion.

Emulsions such as the water-in-oil and water-in-oil-in- water emulsions described above may have, after some time, a certain tendency towards sedimentation (w/o) or creaming (w/o/w) in the worst case followed by breaking. This phenomenon is i.a. depending on droplet size, time and temperature of storage, emulsifϊers used and so on.

Therefore, the best results will be obtained with combination vaccines in which both the ratio Streptococcus difficile cells to Streptococcus iniae cells > 40 to 8 and additionally the two bacterial components are additionally separated over different droplets, or different watery phases as indicated above. Such vaccines have a two-step safety: even if the emulsion breaks, the ratio of the Streptococcus difficile cells to Streptococcus iniae cells safeguards the efficacy of the vaccine.

Therefore, another embodiment of the present invention relates to a water-in-oil or a water-in-oil- in- water combination vaccine for the protection of fish against streptococcal infection, wherein the vaccine comprises an immunogenic amount of Streptococcus difficile cells and an immunogenic amount of

Streptococcus iniae cells wherein the ratio of Streptococcus difficile cells to Streptococcus iniae cells is > 40 to 10, > 40 to 8, > 40 to 6, > 40 to 4, > 40 to 2 or > 40 to 1, in that order of preference, and wherein the Streptococcus iniae cells and the Streptococcus difficile cells are present in separate water droplets of said water-in-oil or water-in-oil-in- water emulsion.

Another form of this embodiment relates to a water- in- oil-in-water combination vaccine for the protection of fish against streptococcal infection, wherein the vaccine comprises an immunogenic amount of Streptococcus difficile cells and an immunogenic amount of Streptococcus iniae cells wherein the ratio of Streptococcus difficile cells to Streptococcus iniae cells is > 40 to 10, > 40 to 8, > 40 to 6, > 40 to 4, > 40 to 2 or > 40 to 1, in that order of preference, and wherein the combination vaccine for the protection of fish against streptococcal infection wherein the Streptococcus iniae cells are present in the oil-encapsulated watery phase droplets of said water- in-oil-in-water emulsion and the Streptococcus difficile cells are present in the free watery phase of said water-in-oil-in- water emulsion.

Still another form of this embodiment relates to a water-in-oil- in- water combination vaccine for the protection of fish against streptococcal infection, wherein the vaccine comprises an immunogenic amount of Streptococcus difficile cells and an immunogenic amount of Streptococcus iniae cells wherein the ratio of Streptococcus difficile cells to Streptococcus iniae cells is > 40 to 10, > 40 to 8, > 40 to 6, > 40 to 4, > 40 to 2 or > 40 to 1, in that order of preference, and wherein the combination vaccine for the protection of fish against streptococcal infection wherein the Streptococcus difficile cells are present in the oil-encapsulated watery phase droplets of said water- in-oil-in-water emulsion and the Streptococcus iniae cells are present in the free watery phase of said water-in-oil-in-water emulsion.

Combination vaccines according to the present invention may in a preferred presentation also 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 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(^) (a homopolymer). An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in a 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 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 (EP 109.942, EP 180.564, EP 242.380) In addition, the vaccine may comprise one or more suitable surface-active compounds or emulsifϊers, e.g. Span or Tween.

Oil adjuvants suitable for use in water-in-oil, water-in-oil-in-water and oil-in-water emulsions are e.g. mineral oils or metabolisable oils. Mineral oils are e.g. Bayol^®, Marcol^® and Drakeol^®. 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.

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.

Often, the vaccine is mixed with stabilisers, e.g. to protect the bacteria from being degraded, to enhance the shelf-life of the vaccine, or to improve freeze-drying efficiency. Useful stabilisers are i.a. SPGA, carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates.

As said above, streptococcal vaccines can e.g. be administered by injection, usually as intraperitoneal injection (also referred to as IP injection). From a point of view of protection the IP -vaccination is the preferred vaccination, because it gives a good and long-lasting protection. This is especially true for inactivated vaccines.

Vaccines according to the invention basically comprise an immunogenic amount of a bacterium and a pharmaceutically acceptable carrier. The term "immunogenic" as used herein is defined as the amount sufficient to induce an immune response in the target fish. A pharmaceutically acceptable carrier can be as simple as water or a buffer, or an emulsion.

The amount of cells administered will depend on the Streptococcus species used, the presence of an adjuvant, the route of administration, the moment of administration, the age of the fish to be vaccinated, general health, water temperature and diet. When starting from commercially available vaccines, the manufacturer will provide this information.

Generally spoken, vaccines for use according to the invention that are based upon bacterins can be given in general in a dosage of 10³ to 10¹⁰, preferably 10⁶ to 10⁹, more preferably between 10⁸ and

10⁹ bacteria. A dose exceeding 10¹⁰ bacteria, although immunologically suitable, will be less attractive for commercial reasons.

Vaccines 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.

In general, the vaccines for use according to the invention will preferably be vaccines that protect against streptococcal infection and additionally against one or more than one other fish-pathogenic virus or microorganism.

Thus, it would be beneficial to use, next to streptococcal bacteria for the manufacture of the vaccine, also at least one other fish-pathogenic microorganism or virus, an antigen of such microorganism or virus or genetic material encoding such an antigen, in a combination-vaccine. The advantage of such a vaccine is that it not only provides protection against streptococcal infection, but also against other fish diseases. Therefore, a preferred form of this embodiment relates to a vaccine wherein that vaccine comprises at least one other microorganism or virus that is pathogenic to fish, or one other antigen or genetic material encoding said other antigen, wherein said other antigen or genetic material is derived from a virus or microorganism pathogenic to fish.

Examples of commercially important fish pathogens in tropical and/or Mediterranean fish are Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Viral Necrosis virus, iridovirus, Spring Viremia of Carp virus and Koi Herpesvirus.

Examples of commercially important cold water fish pathogens are Vibrio anguillarum, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Piscirickettsia salmonis, Salmon Pancreatic Disease virus, Sleeping Disease virus, Viral Nervous Necrosis virus, Infectious Pancreatic Necrosis virus, Infectious haematopoietic necrosis virus, Infectious Salmon Anaemia virus, Viral Haemorrhagic Septicaemia virus and iridoviruses.

Parasites infecting Salmonids are i.a. Lepeophtherius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis and Kudoa thyrsites. A parasite infecting freshwater fish is e.g. Ichthyophthirius multifiliis. Tilapia parasites are e.g. Dactylogyrus spp. and Trichodina spp. Marine fish may suffer i.a from the parasite Benedenia seriolae

Thus, in a more preferred form of this embodiment, the other microorganism or virus is selected from the following group of fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Viral Necrosis virus, Spring Viremia of Carp virus, iridovirus, Infectious haematopoietic necrosis virus, Infectious Salmon Anaemia virus, Viral Haemorrhagic Septicaemia virus and Koi Herpesvirus, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Piscirickettsia salmonis, Salmon Pancreatic Disease virus, Sleeping Disease virus, Viral Nervous Necrosis virus, Infectious Pancreatic Necrosis virus, iridoviruses, Lepeophtherius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis, Kudoa thyrsites, Ichthyophthirius multifiliis, Dactylogyrus spp., Trichodina spp. and Benedenia seriolae. Furthermore, the invention relates to methods for the preparation of a combination vaccine for the protection of fish against streptococcal infection wherein said vaccine is a water- in-oil vaccine or a water-in-oil-in- water vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the Streptococcus iniae cells and the Streptococcus difficile cells are present in separate water droplets of said water-in-oil or water-in-oil-in- water emulsion, wherein the method comprises the steps of making a water-in-oil or water-in-oil- in- water emulsion comprising an immunogenic amount of Streptococcus iniae cells and a water-in-oil or water-in-oil-in-water emulsion comprising an immunogenic amount of Streptococcus difficile cells followed by the step of mixing the two emulsions.

The invention also relates to methods for the preparation of a combination vaccine for the protection of fish against streptococcal infection, wherein said vaccine is a water-in-oil-in-water vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the Streptococcus iniae cells are present in the oil- encapsulated water droplets of said water-in-oil-in-water emulsion and the Streptococcus difficile cells are present in the free watery phase of said water-in-oil- in-water emulsion, said method comprising the steps of making a water-in-oil emulsion wherein the watery phase comprises only the Streptococcus iniae cells or the Streptococcus difficile cells, followed by the step of emulsifying this water-in-oil emulsion in a watery phase that comprises the other of the two bacterial components, i.e. the Streptococcus difficile cells or the Streptococcus iniae cells.

Examples

Example 1.

Evaluation of the efficacy of S. difficile and S. iniae antigens formulated in varying ratio in vaccines for Tilapia.

Challenge strains

The challenge strains used in this experiment were a S. difficile wild-type strain and S. iniae wild-type strain.

Vaccines All bacterial cultures used for the preparation of these vaccines were inactivated with formalin and were derived from S. difficile and S. iniae wild-type strains. The commercially available ISA 736A VG was used as oil-adjuvant for vaccines preparations.

Six different vaccines in which the ratio of the 2 antigens is varied were tested. The composition of each vaccine is given in Table 1.

Table 1 showing the details on vaccines composition and concentration of each antigen used for the various formulations.

Experimental design

Grouping A total of 315 fish of similar size was used for this experiment and divided into 7 groups each consisting of 45 fish.

Vaccination

All fish were starved for 48 hours prior to vaccination, to ensure complete empting of gastro-intestinal tract. Six groups of 45 fish each were vaccinated with one of the vaccines and a group of 45 fish that served as control was not treated. Each group of vaccinated fish was anaesthetised and injected by hand with one of the designated vaccines, m a volume of 0 05 ml per fish Each fish was injected mtra-pentoneal on the side of the body just behind the tip of the pectoral fin Post-vaccmation observation lasted for 24 days

Challenge cultures Streptococcus iniae

A challenge seed vial was taken from the < -5O⁰C freezer, thawed and its content inoculated into 100 0 ml of Tryptic Soy Broth The culture was incubated at 32⁰C, placed on an orbital shaker with shaking speed set at 150 RPM After 23 hours incubation, the culture yielded an OD₆₆₀ of 0 804 Using 0 9% NaCl, a bacterial suspension with OD₆₆₀ of 0 715 was prepared and left at room temperature for 2 hours prior to use as challenge material Streptococcus difficile

A challenge seed vial was taken from the < -5O⁰C freezer, thawed and its content inoculated into 100 0 ml of Streptococcal Growth Medium The culture was incubated at 32⁰C, placed on an orbital shaker with shaking speed set at 150 RPM After 24 hours incubation, the culture yielded an OD₆₆₀ value of 0 176 Using 0 9% NaCl portion of the culture was used to prepare a bacterial suspension with OD₆₆₀ of 0 133, followed by a further 100-fold dilution and the said suspension was used as challenge material

Challenge

All fish were starved for 48 hours prior to the challenge, ensuring complete emptying of the gastro- intestinal tract Challenge was performed by IP-mjection All fish were sedated and injected intraperitoneal just behind the tip of the pectoral fin, with a 0 1 ml standardized bacterial suspension After challenge, all fish were transferred into their designated tanks and recovery from anesthetic treatment followed

Table 2 showing the viable counts of the challenge suspensions

RESULTS

Safety

No mortality was observed during the 24 days post- vaccination period. The average weight gain per fish in each of the groups over the 24 days post-vaccination period are shown below in Table 3.

Table 3 showing average weight gain per fish over the post-vaccination period of 24 days.

No apparent difference in average weight gain per fish between the various experimental groups was observed.

Efficacy of vaccines The efficacy of the 6 bivalent vaccines were evaluated and expressed in terms of % RPS (Relative percentage survival). Data on % RPS for each challenge strain in each of the vaccinated group are shown in Table 4. Mortality recorded for all challenged fish were based on positive re-isolations of the challenged bacteria. Table 4 showing the % RPS for each challenge strain in each of the vaccinated groups and percentage mortality in the control groups challenged with a specific pathogen.

The figures 1 and 2 show the RPS values of each of the vaccines with respect to the results obtained after challenge with a specific bacterial strain at 24 days post- vaccination.

Figure 1 shows the efficacy of bivalent vaccines challenged with S. difficile.

Mortality in connection with positive re-isolation of the challenge bacterium in the control group was 93%. All oil-adjuvant bivalent vaccines showed protection against S. difficile. No protection against S. difficile was observed in the water-base formulated bivalent vaccine. This most likely has to do with the fact that no adjuvant was present at all. Oil adjuvant vaccines formulated in the ratio of (S. difficile : S. iniae) 40:2 and 40:8 yielded 71% RPS against S. difficile while both 40:1 and 40:4 vaccines yielded lower RPS values of 64%. The lowest RPS value obtained in the oil-adjuvant bivalent vaccines was observed from the 40:40 (S. difficile : S iniae) formulation with 50% RPS .

Figure 2 shows the efficacy of bivalent vaccines challenged with S. iniae.

All bivalent vaccines showed protection against S. iniae with 46% mortality and positive re-isolation of the challenge bacterium in the control group. No difference in protection level was observed between water- based vaccines formulated in the ratio of (S. difficile : S. iniae) 40:1, oil-base vaccines formulated in the ratio 40: 1 and 40:2, each giving the same RPS value of 71 %. However, a decline in RPS values was observed as the ratio of S. difficile : S. iniae formulated in oil- adjuvant vaccines reduce from 40:4 down to 40:8 and 40:40. The RPS values in each of the mentioned oil- adjuvant vaccines were 57%, 42% and 14%, respectively.

CONCLUSION

Results obtained from this study showed that at the end of the post -vaccination period, there was no apparent difference between the fish average weight from any vaccinated group as compared to the control group. Therefore, all bivalent vaccines formulated and administered in dosage of 0.05 ml per fish were safe to be used for IP -vaccination into Tilapia.

The results obtained from S. difficile challenge showed that an adjuvant, preferably an oil adjuvant is required for bivalent (S. difficile and S. iniae) vaccines to be efficacious against S. difficile. A 93% mortality with positive re- isolation of the challenge bacterium was observed in the control group and protection was observed in all oil-adjuvant vaccines with varying protection levels: the lowest RPS value of 50.0% was observed in the 40:40 (S. difficile : S. iniae) vaccine formulation.

S. iniae challenge gave 46% mortality with positive re-isolation of the challenge bacterium in the control group. It was also observed that water-based bivalent vaccine formulated with a 40: 1 (S. difficile : S. iniae) ratio and all oil-adjuvant bivalent vaccines showed protection against S. iniae. However, the lowest RPS value of 14% was again observed in the 40:40 (S. difficile : S. iniae) oil-adjuvant vaccine formulation.

In this study it was noted that when S. difficile antigen was formulated at a level that is 40 or 20 times higher than S. iniae antigen (on a cell : cell basis), 64% RPS or 71% RPS against S. difficile were observed, and a RPS value of 71% RPS against S. iniae was observed.

Legend to the figures. Figure 1 : efficacy of bivalent vaccines challenged with S. difficile. Figure 2: efficacy of bivalent vaccines challenged with S. iniae.

Claims

1) Combination vaccine for the protection of fish against streptococcal infection, characterized in that said vaccine comprises an immunogenic amount of Streptococcus difficile cells and an immunogenic amount of Streptococcus iniae cells wherein the ratio of Streptococcus difficile cells to Streptococcus iniae cells is > 40 to 10.

2) Combination vaccine according to claim 1, characterized in that said ratio is > 40 to 8, preferably > 40 to 6, more preferably > 40 to 4, even more preferably > 40 to 2 and most preferably > 40 to l.

3) Combination vaccine according to claim 1 or 2, characterized in that it is a water-in-oil vaccine.

4) Combination vaccine according to claim 1 or 2, characterized in that it is a water- in-oil-in- water vaccine.

5) Combination vaccine for the protection of fish against streptococcal infection, characterized in that said vaccine is a water-in-oil vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the Streptococcus iniae cells and the Streptococcus difficile cells are present in separate watery phase droplets of said water-in-oil emulsion.

6) Combination vaccine for the protection of fish against streptococcal infection, characterized in that said vaccine is a water-in-oil- in- water vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the Streptococcus iniae cells and the Streptococcus difficile cells are present in separate watery phase droplets of said water-in-oil- in- water emulsion.

7) Combination vaccine for the protection of fish against streptococcal infection, characterized in that said vaccine is a water-in-oil- in- water vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the Streptococcus iniae cells are present in the oil-encapsulated watery phase droplets of said water-in-oil- in-water emulsion and the Streptococcus difficile cells are present in the free watery phase of said water-in-oil-in- water emulsion.

8) Combination vaccine for the protection of fish against streptococcal infection, characterized in that said vaccine is a water-in-oil-in- water vaccine comprising an immunogenic amount of Streptococcus iniae cells and Streptococcus difficile cells wherein the Streptococcus difficile cells are present in the oil-encapsulated water droplets of said water-in-oil-in- water emulsion and the Streptococcus iniae cells are present in the free watery phase of said water-in-oil-in-water emulsion.

9) Combination vaccine according to claim 1 or 2, characterised in that the vaccine is a water- in- oil based vaccine according to claim 5, or a water-in-oil-in-water vaccine according to claims 6-8.

10) Combination vaccine according to claims 1-9, characterized in that said combination vaccine comprises an adjuvant.

11) Combination vaccine according to claims 1-10, characterized in that said combination vaccine comprises at least one other microorganism or virus that is pathogenic to fish, or one other antigen or genetic material encoding said other antigen, wherein said other antigen or genetic material is derived from a virus or microorganism pathogenic to fish.

12) Combination vaccine according to claim 10, characterized in that the other microorganism or virus is selected from the following group of fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Viral Necrosis virus, iridovirus and Koi Herpesvirus, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Piscirickettsia salmonis, Salmon Pancreatic Disease virus, Sleeping Disease virus, Viral Nervous Necrosis virus, Infectious Pancreatic Necrosis virus, Infectious haematopoietic necrosis virus, Infectious Salmon Anaemia virus, Viral Haemorrhagic Septicaemia virus, Spring Viremia of Carp virus, iridoviruses, Lepeophtherius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis, Kudoa thyrsites, Ichthyophthirius multifiliis, Dactylogyrus spp., Trichodina spp. and Benedenia seriolae. 13) Method for the preparation of a combination vaccine according to claim 5, characterized in that said method comprises the steps of making a water-in-oil emulsion comprising an immunogenic amount of Streptococcus iniae cells and a water-in-oil emulsion comprising an immunogenic amount of Streptococcus difficile cells followed by the step of mixing the two emulsions.

14) Method for the preparation of a combination vaccine according to claim 6, characterized in that said method comprises the steps of making a water-in-oil-in-water emulsion comprising an immunogenic amount of Streptococcus iniae cells and a water-in-oil-in-water emulsion comprising an immunogenic amount of Streptococcus difficile cells followed by the step of mixing the two emulsions.

15) Method for the preparation of a combination vaccine according to claim 7, characterized in that said method comprises the steps of making a water-in-oil emulsion wherein the watery phase comprises only the Streptococcus iniae cells, followed by the step of emulsifying this water-in-oil emulsion in a watery phase that comprises the Streptococcus difficile cells.

16) Method for the preparation of a combination vaccine according to claim 8, characterized in that said method comprises the steps of making a water-in-oil emulsion wherein the watery phase comprises only the Streptococcus difficile cells, followed by the step of emulsifying this water-in-oil emulsion in a watery phase that comprises the Streptococcus iniae cells.