MX2008016248A

MX2008016248A - Combination vaccine against streptococcus.

Info

Publication number: MX2008016248A
Application number: MX2008016248A
Authority: MX
Inventors: Luc Grisez; Chow Yong Ng
Original assignee: Intervet Int Bv
Priority date: 2006-07-06
Filing date: 2007-07-05
Publication date: 2009-03-05
Also published as: CR10490A; ECSP099066A; WO2008003734A1; HN2009000005A

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

VACU NA COM BI NADA ESTREPTOCOCICA Field of the Invention This invention relates to a combined vaccine for the protection of fish against streptococcal infections and to the preparation of methods for the combination of such vaccines. Background of the Invention Several species of the genus of streptococcal bacteria is known to produce infections in fish today, more specifically in fish kept in aquaculture. Examples of such species are Streptococcus iniae, S. difficile, S. agalactiae, S. dysgalactiae and S. phocae. Lately there have been some changes of opinion on the correct nomenclature of Streptococcus difficile and Streptococcus agalactiae. Vandamme et al. (Int.J. Syst. Bacteriology 47: 81-85 (1,995)) have suggested that Streptococcus difficile is actually a non-hemolytic Streptococcus agalactiae (Streptococcus agalactiae is a β-hemolytic bacteria). For reasons of convenience, the traditional nomenclature is used here: Streptococcus iniae, S. difficile, S. agalactiae. Streptococcus iniae is frequently found in Tilapia, rainbow trout, European sea bass, sea bream, Asian sea bass, ocellar corvinon, rabbit fish, Japanese flounder, flounder flounder and yellowtail turbot and striped bass hybrid. The annual impact of Streptococcus infections iniae in aquaculture exceeds 1 billion US dollars. Streptococcus difficile is frequently found in Asian sea bass, tilapia, a species of edible fish and in the cat-fish. Streptococcus agalactiae currently affects mainly found in Tilapia. Vaccines to combat streptococcal infection in fish are known technique. Many of the streptococcal vaccines are based on dead whole cells. Streptococcus agalactiae vaccines are, among others, described in the application of Pat. US No. 2005/0208077. Vaccines against Streptococcus iniae are described, inter alia, in Pat. US No. 6,379,677. Streptococcus iniae vaccines are also commercially available. An example is Norvax Strep Si, a vaccine against Streptococcus iniae marketed by I ntervet I nt. B .V. Eldar et al. have described (Vaccine, 1 3: 867-870, (1,995)) a vaccine against Streptococcus difficile from dead whole cells. Due to the fact that Streptococcus iniae and Streptococcus difficile are frequently the cause of diseases in fish, more specifically in Tilapia and Asian sea bass, it is attractive to combine vaccines against Streptococcus iniae and Streptococcus difficile during administration.

However, it was unexpectedly found that when the vaccines against Streptococcus iniae and Streptococcus difficile were administered in combination, the level of protection against Streptococcus iniae is highly impaired. But if the vaccine against Streptococcus iniae without Streptococcus difficile is administered intraperitoneally (IP), the protection against Streptococcus iniae is as expected. It seems that the presence of Streptococcus difficile in a combined vaccine is, highly surprising, of the development of the anti-Streptococcus Iniae immune response for reasons that are not known. An objective of this invention is to find a solution to this problem. It has now been discovered by surprise that if a combined vaccine for the protection of fish against streptococcal infection comprises an amount of Streptococcus difficile cell immunizer and an amount of Streptococcus iniae cell immunizer in which the proportion of Streptococcus difficile cells to the Streptococcus iniae cells is equal to or greater than 40 to 10, this problem of suppressing the development of an auto-immune response to Streptococcus iniae occurs at any acceptable level, or is not seen at all. As stated above, the presence of an amount of Streptococcus difficile cell immunizer and an amount of Streptococcus inae cell immunizer is, of course, necessary to induce an immune response to each of the bacterial species. The number of Streptococcus difficile cells in the vaccine must be at least four times the number of Streptococcus iniae cells. Thus, a first embodiment of the present invention relates to a combined vaccine for the protection of fish against streptococcal infection which has as a characteristic a combined vaccine comprising an amount of Streptococcus iniae cell immunizer in both the proportion of cells of Streptococcus difficile to Streptococcus iniae cells is equal to or greater than 40 to 1 0. As would be clear from the Examples, a combined vaccine in which the number of Streptococcus difficile cells in the vaccine is four times greater than the amount of Streptococcus iniae cells even less suppression of the development of an anti-immune response against Streptococcus iniae. Therefore, a combination vaccine where the ratio of Streptococcus difficile cells to Streptococcus iniae cells is equal to or greater than 40 to 8 is preferred, and a ratio is equal to or greater than 40 to 6, 40 to 4, 40 to 2 and 40 to 1, in that order is more progressively preferred. Thus, preferred forms of this embodiment relate to a combined vaccine, according to the invention, wherein the ratio is equal to or greater than 40 to 8, more preferably equal to or greater than 40 to 6, even more preferably equal to or greater than 40 to 4. , still even more preferably equal to or greater than 4 to 2, and more preferably 40 to 1. Traditionally, many Asian vaccines for fish, if possible in all, determinant as a water base vaccine. They did not understand a phase of the oil. However, there is a current tendency to use emulsions of water in oil, water in oil in water, or oil in water. Such vaccines-emulsions have the advantage of being more effective than their base-water counterparts. On the other hand, the emulsion vaccines allow to use smaller amounts of antigen. Therefore, another preferred form of this embodiment relates to a combined vaccine according to the invention wherein the vaccine is a water-in-oil vaccine. Yet another preferred form of this embodiment relates to a combined vaccine according to the invention which is an oil-in-water vaccine. Again another preferred form of this embodiment refers to a combined vaccine according to the invention wherein the vaccine is a water-in-oil-in-water vaccine. The problem to solve, for example the problem is the presence of Streptococcus difficile in a combination vaccine is highly repressive of the development of an immune response against Streptococcus iniae, it is found both in water-based vaccines and emulsions based on water and oil. The solution presented above is a solution to the problem.

In case of an emulsion of water and oil for the vaccine, another feasible solution was found, possibly in combination with the solution given above. Now it was surprisingly found, that if the Streptococcus difficile and Streptococcus iniae cells are present in separate water droplets in a water-in-oil emulsion, the problem is less significant until negligible. In that case, initially there is no need to maintain the ratio of Streptococcus difficile cells to Streptococcus iniae cells equal to or greater than 40 to 8. A water-in-oil emulsion in which the Streptococcus iniae and Streptococcus difficile cells are present. in separate water droplets in the emulsion is relatively easy to make. The normal procedure for making a water-in-oil basic emulsion is based on the mixture of a single amount of an aqueous phase, an oil phase and a quantity of bacterial cells. The aqueous phase can be, for example, no more water, a water-based storage or the like. The oil phase can be a mineral or non-mineral oil (see below). Additionally, one or more emulsifiers can be added and detergerte. Methods for making such emulsions and components for use in such emulsions are well known in the art. This present method differs from the common method in that instead of a single aqueous phase two aqueous phases are used; one that includes Streptococcus iniae cells and the other that comprises Streptococcus difficile cells. Each of the two aqueous phases is emulsified with an amount of oil phase and, after making the emulsions separated in water, the two water-in-oil emulsions are mixed. A variant of this method is, for example; making a water-in-oil emulsion comprising one or two types of streptococcal cells and adding the other type of cells in aqueous phase, for example during the emulsifying process. By doing this, most of the other streptococcal cells will automatically be integrated into new water droplets in the water-in-oil emulsion. It goes without saying that water-in-oil emulsions in which Streptococcus iniae and Streptococcus difficile cells are present in water droplets separated from the emulsion always fall within the scope of the invention no matter how they are made. Then, another embodiment of this invention relates to a combined vaccine for the protection of fish against streptococcal infections where the vaccine is a water-in-oil vaccine comprising an amount of Streptococcus iniae and Streptococcus difficile cell immunizer in which the Streptococcus iniae and Streptococcus difficile cells are present in water droplets separated from such water-in-oil emulsion. Another possibility is to make use of a water emulsion in oil in water, wherein the amount of Streptococcus iniae and Streptococcus difficile cell immunizer is present in separate droplets of such water-in-oil-in-water emulsion. Such an emulsion comprises aqueous droplets, present within small droplets of oil, which are in turn surrounded by an aqueous phase. Then, another form of this embodiment involves a combined vaccine for the protection of fish against streptococcal infections which is characterized as a water-in-oil-in-water vaccine comprising an amount of Streptococcus iniae and Streptococcus difficile immunizer in which the component Streptococcus iniae and the component Streptococcus difficile are present in water droplets separated from such an emulsion water in oil in water. Another possibility to keep the cells separated from Streptococcus iniae and the Streptococcus difficile cells in a water-in-oil-in-water emulsion, is first to make a water-in-oil emulsion in which the aqueous 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 an aqueous phase comprising another of the two bacterial components, ie the Streptococcus difficile cells or the Streptococcus iniae cells. Another form of this modality refers to a combined vaccine for the protection of fish against infections of streptococcus, wherein such a vaccine is a water-in-oil-in-water vaccine comprising an amount of Streptococcus iniae cell immunizer and Streptococcus difficile cells where the Streptococcus iniae cells are present water droplets encapsulated in oil such an emulsion water in oil in water , and the Streptococcus difficile cells are present in the water-free phase of such water-in-oil-in-water emulsion. Thus, another form of this modality relates to a combined vaccine for the protection of fish against streptococcal infection, where it indicates a vaccine for being a water-in-oil-in-water vaccine comprising an immunizing amount of Streptococcus iniae and Streptococcus difficile cells. wherein the Streptococcus difficile cells are present in the water droplets encapsulated in the oil of such water-in-oil emulsion in water, and the Streptococcus iniae cells are present in the water-in-oil-in-water free emulsion aqueous phase. Such emulsions as water in oil and water in oil in water described above may have, after a certain time, a tendency to settle, or a creamy consistency, or both, and in the worst case, to be cut. This phenomenon depends, among other things, on the size, time and temperature of the storage droplets, the emulsifiers used, etc. Then, the best results will be obtained with vaccines combined in which the ratio of Streptococcus difficile cells to Streptococcus iniae cells is greater than or equal to 40 to 8 and additionally the two bacterial components are additionally separated into different droplets, or in different aqueous phases as indicated above. Such vaccines have a double security: even if the emulsion were broken, the proportion of Streptococcus difficile cells to Streptococcus iniae cells safeguards the effectiveness of the vaccine. Therefore, another embodiment of this invention relates to a combined vaccine for the protection of fish against streptococcal infection characterized in that such a vaccine comprises an amount of Sfrepfococcus difficile cell immunizer and Streptoccccus iniae cells and the proportion of Streptococcus cells. Difficile to Streptococcus iniae cells is greater than or equal to 40 to 10, 40 to 8, 40 to 6, 40 to 4, 40 to 2 or 40 to 1, in this order of preference, and in which the Streptococcus difficile cells and the Streptococcus iniae cells are present in water droplets separated from such water-in-oil or water-in-oil-in-water emulsion. Another form of this embodiment refers to water in oil in water a combined vaccine for the protection of fish against streptococcal infection, characterized by a vaccine comprising an immunizing amount of Streptococcus difficile cells and Streptoccccus iniae cells where the proportion of Streptococcus difficile cells to Streptococcus iniae cells is greater than or equal to 40 to 10, 40 to 8, 40 to 6, 40 to 4, 40 to 2 or 40 to 1, in this order of preference, and this modality is also characterized because the combined vaccine for the protection of fish against streptococcal infection in which the Streptococcus iniae cells are present in the droplets of the aqueous phase encapsulated in oil of such emulsion water in oil in water, and the cells of Streptococcus difficile are present in the free aqueous phase of such emulsion water in oil in water. Another form of embodiment refers to a combined water-in-oil-in-water vaccine for the protection of fish against streptococcal infection, characterized such a vaccine comprises an Immunizing amount of Streptococcus difficile cells and an amount of Streptococcus iniae cell immunizer where the proportion of Streptoccccus difficile cells to Streptococcus iniae cells is May or equal to 40 to 10, 40 to 8, 40 to 6, 40 to 4, 40 to 2 or 40 a, in this order of preference, and in which the vaccine combined for the protection of fish against streptococcal infection in which the Streptococcus difficile cells are present in the droplets of the aqueous phase encapsulated in oil of such water-in-oil emulsion in water and the Streptococcus iniae cells are present in the aqueous phase water-in-oil-in-water emulsion free. Combined vaccines according to this invention may contain also in a preferred embodiment an immunostimulating substance, called an adjuvant. Adjuvants in general comprise substances that promote the immune response of the host in a non-specific manner. A different number of adjuvants are known in this art. Examples of adjuvants frequently used in the breeding of fish and crustaceans are muramylipeptides, lipopolysaccharides, various glycans and glycans and Carbopol® (a homopolymer). An extensive review of adjuvants suitable for vaccines for fish and crustaceans is given in a paper by Jan Raa (Review in Fisheries Science 4 (3): 229-288 (1996) .The vaccine may also comprise a so-called vehicle, it is a compound which the bacterium adheres, without having a covalent link with that substance, Such vehicles are, among others, bio-microcapsules, micro-alginates, liposomes and macrosols, all known in the art. which antigen is partially embedded in the vehicle, is the so-called ISCOM (PE 109.942, PE 180,564, PE 242,380) In addition, the vaccine may comprise one or more suitable surfactant compounds or emulsifiers, for example span or between. Use in emulsions water in oil, water in oil in water or oil in water are, for example, mineral oils or metabolizable oils. minerals are, p. eg, Bayo®, Marcol® and Drakeol®. Metabolizable oils are, for example, vegetable oils, such as peanut oil and soybean oil, animal oils such as squalene and squalene fish oils, and tocopherol and its derivatives. The amount of adjuvant added depends on the nature of the adjuvant itself, and information regarding such amounts will be provided by the manufacturer. Often, the vaccine is mixed with stabilizers, for example to protect the bacteria against degradation, to enhance the period of validity of the vaccine, or to improve the efficiency of the drying process under freezing. Useful stabilizers are, among others, SPGA, carbohydrates for example: sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose; proteins such as albumin or casein or degradation products thereof, and intermediates such as alkali metal phosphates. As said before, the anti-streptococcal vaccine can be administered, for example, by injection, usually intraperitoneal injection (referred to as IP injection). From the point of view of protection, IP injection is preferred because it gives a good and lasting protection. This is especially when it comes to inactivated vaccines. The vaccines of this invention basically comprise a quantity of an immunizer of a bacterium and a transporter pharmaceutically acceptable. The term "immunizer", 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 an intermediate or an emulsion. The amount of cells administered will depend on the streptococcal species used, the presence of an adjuvant, the route of administration, the time of administration, the age of the fish to be vaccinated, the general health status, water temperature and diet. When the vaccines begin to be commercialized, the manufacturer will provide this information. Generally speaking, the vaccines to be used according to the invention are based on the bacteria, in general doses of 103 to 1 0 0, preferably 106 to 1 9, more preferably between 1 08 and 109 bacteria can be given. A dose exceeding 101 0 bacteria, although immunologically adequate, will be less attractive for commercial reasons. The vaccines according to the invention are based on live attenuated bacteria, they can be given in lower doses because the bacteria continue to replicate for a certain time after the administration. In general, vaccines for use according to this invention, will preferably be vaccines that protect against streptococcal infection and, in addition, against one or more of a virus or microorganism pathogenic to fish.

Thus, it would be beneficial to use, then the streptococcal bacteria for the manufacture of the vaccine, also at least one other virus or pathogenic microorganism for the fish, an antigen of such a virus or microorganism or genetic material encoding such an antigen in a combined vaccine. The advantage of such a vaccine is that it provides protection not only against streptococcus but also against other diseases of fish. Accordingly, a preferred form of this embodiment refers to a vaccine wherein said vaccine comprises at least one or the other virus or microorganism that is pathogenic to the fish, or one or another antigen or genetic material encoding another antigen, and where another antigen or genetic material is derived from a virus or pathogenic microorganism for fish. Examples of commercially important pathogens in tropical and / or Mediterranean fish are Vibrio anguillarum, Photobacteritun damselae subspecies piscicida, Tenacibaculum maritimum, Fiavobacterium sp., Flexibacter sp, Lactococcus garviae, Edwardsiella tarda, E. ictaluri, viral necrosis virus, iridovirus, Spring Virennia of carp and Koi herpesvirus. Examples of commercially important pathogens in cold-water fish are Vibrio anguillarum, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Fiavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Piscirickettsia salmonis, pancreatic salmon disease virus, sleeping sickness virus, viral necrosis virus, infectious pancreatic necrosis virus, hematopoietic necrosis virus Infectious, salmon infectious anemia virus, salmonella virus viral hemorrhagic septicemia and iridovirus. Parasites that infect salmonids are, among others, Lepeophterlus salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis and Kudoa thyrsites. A parasite that infects freshwater fish is, for example; Ichthyophthirius muttifiliis. Parasites of Tilapia are, for example, Dactylogyrus spp. and Trichodina spp. Marine fish may suffer from the parasite Benedenia seriolae, among others. Then, in a more preferred embodiment of the invention, the other viruses or microorganism is selected from the group consisting of the following fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp. , Flexibacter sp. , Lactococcus garviae, Edwardsiella tarda, E. Ictaluri, viral necrosis virus, carp spring viraemia virus, iridovirus, infectious hematopoietic necrosis virus, infectious salmon anemia virus ", septicemia virus Viral haemorrhagic fever and Koi herpesvirus, Aeromonas salmoniciclae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Piscirickettsia salmonis, salmon pancreatic disease virus, sleep, viral necrosis nerve virus, infectious pancreatic necrosis virus, iridovirus, Lepeophterius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis, Kudoa thyrsites, Ichthyophthirius multifiliis Dactylogyrus spp., Trichodina spp. and Benedenia seriolae. Furthermore, the invention relates to methods of preparing a combined vaccine for the protection of fish against streptococcal infection, which is a water-in-oil or water-in-oil-in-water vaccine comprising an amount of Streptococcus iniae cell immunizer and of Streptococcus difficile cells, and where Streptococcus iniae and Streptococcus difficile cells are present in water droplets separated from such emulsion water in oil or water in oil in water, where the method comprises the steps of making a water emulsion in oil or a water-in-oil-in-water emulsion comprising an amount of Streptococcus iniae cell immunizer and a water-in-oil emulsion or a water-in-oil-in-water emulsion containing an amount of Streptococcus difficile cell immunizer followed by the step of Mix two emulsions. The invention also relates to methods for the preparation of a combined vaccine for the protection of fish against streptococcal infection, wherein said vaccine is a water-in-oil-in-water emulsion comprising an amount of Streptococcus iniae cell immunizer and Streptococcus difficile cells, the Streptococcus iniae cells being present in water droplets encapsulated by the water-in-oil in water emulsion oil and the Streptococcus difficile cells are in the water-free phase of such water-in-oil-in-water emulsion, comprising such a method the steps of making a water-in-oil emulsion in which the aqueous phase comprises only the Streptococcus iniae cells or the Streptococcus difficile cells, followed by this step by emulsifying this water-in-oil emulsion in an aqueous phase comprising the another of the two bacterial components, ie the Streptococcus iniae or Streptococcus difficile cells. EXAMPLES Example 1 Evaluation of the efficacy of the antigens of S. iniae and S. difficile formulated in variable proportion in vaccines for Tilapia. Varieties faced The opposing varieties were a wild variety of S. difficile and a wild variety of S. inae. Vaccines All bacterial cultures used for the preparation of these vaccines were inactivated with formalin and are derived from wild varieties of S. difficile and S. iniae. As an oil adjuvant for the preparation of vaccines ISA 736A VG is used commercially available Six different vaccines were tested in which the proportion of the two antigens varied. The composition of the vaccines is given in Table 1. Table 1. Details of the composition of the vaccines and composition of each antigen used in various formulations Experimental design Grouping A total of 315 fish of similar size were used for this experiment and were divided into 7 groups each consisting of 45 fish. Vaccination All fish were kept without food for 48 hours prior to vaccination, to ensure that the gastrointestinal tract was empty. Six groups of 45 fish each they were vaccinated with one of the vaccines each and a group of 45 fish was not treated to serve as control. Each group of fish was anesthetized and injected by hand with the designated vaccines at a rate of 0.05 ml per fish. Each fish was injected peritoneally on the side of the body just behind the tip of the pectoral fin. The post-vaccination observation lasted for 24 days. Challenge to grow Streptococcus niae A jar of the challenge seed was taken from the freezer at less than or equal to 55 ° C, thawed and the content was inoculated into 1000 μm of tryptic soy extract. The culture is incubated at 32 ° C, put on an orbital shaker with a speed of 1 50 rpm. After 23 hours of incubation, the culture produced an OD66 or 0.804. A bacterial suspension was prepared with OD6eo of 0.71 5 using a 0.9% N aCL solution and left at room temperature 2 hours before using as a challenge material. Streptococcus difficile A bottle of the challenge seed was taken from the freezer at less than or equal to 55 ° C, thawed and the content was reduced to 1,00, 0 ml of mean streptococcal development. It was placed in an orbital shaker with a speed of 1 50 rpm. After 24 hours of incubation, the culture produced an OD6e value of 0.176. A suspension was prepared bacterial with OD660 of 0.133 using a portion of the culture and 9% NaC1 solution, and then diluted 100 times, using such suspension as challenge material. Challenge All fish were deprived of feed for 48 hours before inoculation ensuring complete emptying of the gastrointestinal tract. The inoculation was carried out by means of IP injection. All the fish were sedated and injected peritoneally just behind the tip of the pectoral fin with 0.1 ml of the standardized bacterial suspension. After the inoculation all the fish were transferred to their designated tanks and their recovery from the anesthetic treatment is initiated.

Table 2 shows the viable counts of the challenge suspensions -RESULTS Safety Mortality was not observed in the 24-day periods following vaccination. The average weight gain of the fish in each of the groups in the 24-day periods following vaccination is shown in Table 3. Table 3. Average weight gain per fish in the 24-day period following vaccination.

Prototype Weight Weight Weight Weight Weight gain Difference before before after prom. In 24 vaccination gain inoculation days after weight between vaccination groups A 13.5 21.4 6.4 B 13.0 20.2 6.2 C 12.9 21.1 6.5 D 12.9 22.7 6.7 + 0.3 g E 13.1 22.0 6.8 F 12.7 20.2 6.5 Control 13.6 21.4 6.3 Efficacy of vaccines The efficacy of the 6 bivalent vaccines is evaluated and expressed in terms of% PRS (Relative percentage of survival). Table 4 shows data of% RPS for each variety of infection in each of the vaccinated groups. The mortality recorded for all infected fish is based on positive re-isolations of the challenged bacteria. Table 4 shows the% PRS for each challenge strain in each of the vaccinated groups and the percentage mortality in the control groups challenged with a specific pathogen.

S. difficUe 5 inlae Prototype A 0 71.43 B 64.29 71.43 c 71.43 71.43 D 64.29 57.14 E 71.43 42.86 F 50.00 14.29% Control in mortality 9333 46.67 Figures 1 and 2 show the PRS values of each of the vaccines with respect to the results obtained after infection with a specific variety of bacteria at 24 days after vaccination. Fig. 1 shows the efficacy of vaccines loaded with S. difficile. Mortality associated with the positive isolation of the bacteria used in the 93% control group. All bivalent vaccines with oil adjuvant showed protection against S. difficile. No protection against S. difficile was observed in aqueous bivalent vaccines. This is most likely due to the absence of an oil adjuvant. Vaccines formulated with oil adjuvant in the proportion (S. difficile: S. iniae) of 40: 2 and 40: 8 gave 71% PRS against S. difficile, while vaccines with a ratio of 40: 1 and 40: 4 gave values PRS lower, 64%. The lowest RPS value obtained with bivalent vaccines with oil adjuvant came from a 40:40 formulation (S. difficile: S. iniae) with 50% PRS. Fig. 2 shows the efficacy of bivalent vaccines containing S. iniae. All bivalent vaccines showed protection against S. iniae with 46% mortality and positive isolation of the bacteria used in the control group. No difference was observed in the level of protection between formulated aqueous vaccines with the proportion of (S. difficile: S. iniae) 40: 1, oily vaccines formulated in a ratio of 40: 1 and 40: 2, giving both the same PRS value of 71%. However, a decline in PRS values is observed as the proportion of S. dlfficile: S. iniae in vaccines formulated with oil adjuvant is reduced from 40: 4 to 40: 8 and 40:40. The PRS values in the cited oily adjuvant vaccines were 57%, 42% and 14% respectively. CONCLUSION The results obtained from this study show that at the end of the post vaccination period there was no apparent difference between the average weight of the fish in any vaccinated group and the control group. Therefore, all bivalent vaccines formulated and administered in the dose of 0.05 ml per fish were safe for use by IP vaccination in Tilapia. The results obtained from S. difficile cultures showed that an adjuvant is required, preferably an oily adjuvant, for bivalent vaccines (S. difficile and S. iniae) against S. difficile to be effective. A mortality of 93% was observed with positive isolation of bacteria used in the control group and it was observed that all the vaccines with oil adjuvant gave protection with variable intensity: the level more than 50% PRS, was observed with the formulation 40:40 (S. difficile: S. iniae). S. iniae used as inoculants in the control group gave 46% mortality with re-isolation of the bacterium inoculated It was observed that the bivalent aqueous vaccine formulated with 40: 1 (S. difficile, S. iniae) a proportion and all the bivalent vaccines with oil adjuvant gave protection against S. iniae. However, the lowest PRS value of 14% was again observed in the oil adjuvanted vaccine formulated with a 40:40 ratio of S. difficile: S. iniae. In this study it is noted that when an antigen of S. difficile was formulated 40 or 20 times higher than the antigen of S. iniae (on the base of cell: cell), values of 64% PRS or 71% PRS against S. iniae, and 71% PRS against S. iniae. Legend of the figures Figure 1: efficacy of bivalent vaccines using cultures of S. difficile. Figure 2: efficacy of bivalent vaccines using S. iniae cultures.

Claims

REVIVAL DICTION EN

1 . Combined vaccine for the protection of fish against the infection of streptococcus which has as characteristic a combined vaccine comprising an amount of Streptococcus iniae cell immunizer while the proportion of cells of Streptococcus difficile to Streptococcus iniae cells is equal to or greater 40 to 10.

2. Combined vaccine according to claim 1, characterized in that said ratio is equal to or greater than 40 to 8, preferably equal to or greater than 40 to 6, more preferably equal to or greater than 40 to 4, even more. preferably equal to or greater than 40 to 2 and most preferably equal to or greater than 40 to 1.

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

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

5. Combined vaccine for the protection of fish against streptococcal infection characterized by being a water-in-oil vaccine comprising an amount of Streptococcus iniae and Streptococcus difficile cell immunizer where the cells of Srrepfococcus iniae and Streptococcus difficile are present in separate droplets of the aqueous phase of such water-in-oil emulsion.

6. Combined vaccine for the protection of fish against Streptococcal infection characterized by being a water-in-oil-in-water vaccine comprising an amount of Streptococcus iniae and Streptococcus difficile cell immunizer and wherein the Streptococcus iniae and Streptococcus difficile cells are present in separate droplets of the aqueous phase of such emulsion water in oil in water.

7. Combined vaccine for the protection of fish against streptococcal infection characterized by being a water-in-oil-in-water vaccine comprising an amount of Streptococcus iniae and Streptococcus difficile cell immunizer and where the Streptococcus iniae cells are present in droplets Aqueous encapsulated in the oil of such water-in-oil emulsion in water and the Streptococcus difficile cells are present in the water-free phase of such water-in-oil-in-water emulsion.

8. Combined vaccine for the protection of fish against streptococcal infection characterized by being a water-in-oil-in-water vaccine comprising an amount of Streptococcus difficile cell immunizer and the Streptococcus iniae cells in which the Streptococcus difficile cells are located. present in aqueous droplets encapsulated in the oil of such water-in-oil emulsion in water and the Streptococcus iniae cells are present in the water-free phase of such water-in-oil-in-water emulsion.

9. Combined vaccine according to claim 1 to 2, characterized in that the vaccine is a water-in-oil vaccine according to claim 5 or a water-in-oil-in-water vaccine according to claims 6-8.

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

11. The combined vaccine according to claims 1-10, characterized in that said combined vaccine comprises at least one or another pathogenic microorganism or virus for the fish, or one or another antigen or genetic material that encodes another antigen where another antigen or genetic material is derived from a virus or pathogenic microorganism for fish.

12. Combined vaccine according to claim 10, characterized in that the other microorganism or virus is selected from the group of the following fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibacuium maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, virus of viral necrosis, iridovirus, Spring Virennia of carp, Kol herpesvirus, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Piscirickettsia salmonis, pancreatic salmon disease virus, virus of the sleeping sickness, viral necrosis virus, infectious pancreatic necrosis virus, infectious hematopoietic necrosis virus, infectious salmon anemia virus, viral hemorrhagic septicemia virus, irdovirus, Lepeophterius salmonis, Caligus elongatus, Cryptobia salmositica. Myxobolus cerebralis, Kudoa thyrsites, lchthyophthirius multifiliis, Dactylogyrus spp., Trichodina spp. and Benedenia seriolae.

13. Method for the preparation of the vaccine according to claim 5, characterized by comprising the step of making a water-in-oil emulsion comprising an immunizing amount of Streptococcus iniae cells and a water-in-oil emulsion comprising an amount of cell immunizer of Streptococcus difficile and then the stage of mixing both emulsions.

14. Method for the preparation of the vaccine according to claim 6, characterized in comprising the step of making a water-in-oil-in-water emulsion comprising an immunizing amount of Streptococcus iniae cells and a water-in-oil-in-water emulsion comprising an amount of immunity of Streptococcus difficile cells following by the stages of mixing 2 emulsions.

15. Method for the preparation of the vaccine according to claim 7, characterized in that it comprises such methods of the step of making a water-in-oil emulsion in which the aqueous phase comprises only the cells of. Streptococcus iniae, followed by the step of emulsifying this water-in-oil emulsion with an aqueous phase comprising the Streptococcus difficile cells.

16. Method for the preparation of the vaccine according to claim 8, characterized by comprising the step of making a water-in-oil emulsion in which the aqueous phase comprises only the Streptococcus difficile cells, followed by the step of emulsifying this water-in-oil emulsion with an aqueous phase comprising Streptococcus iniae cells .