MX2008015164A - Vaccine against rickettsia-like organisms. - Google Patents

Abstract

The present invention relates to the use of bacteria of the genus Streptococcus for the manufacture of a vaccine for combating Rickettsia-like organism infection.

Description

VACCINE AGAINST ORGANISMS OF TYPE RICKETTSIA Description of the Invention The present invention relates to the use of bacteria of the genus Streptococcus for the manufacture of a vaccine to combat infection by Rickettsia type organisms. The infection of several salmonid and non-salmonid fish with Rickettsial / Rickettsia type organisms has been reported for decades, the first report originates from 1939 where a Rickettsia type organism was found in Tetrodon fahaka of the Nile River. However, it was not until 1975 that a first observation was made of the presence of a Rickettsia-like organism in tissue cells stained by Ozel M. and Schwanz-Pfitzner, I., (Zentralblatt fur Bacteriologie, Microbiologic und Hygiene, I abt Originate A 1975; 230: 1- 14 (1975)). The real importance of Piscirickettsia salmonis as a pathogen has been dramatically clarified in 1989, when in Chile 1.5. millions of commercial-sized Coho salmon died at that time an unknown infectious agent. (Bravo, S. and Campos, M. FHS / AFS Newsletter 17: 3 (1989), Cvítanich et al., FHS / AFS Newsletter 18: 1-2 (1990), Fryer et al., Físh Pathol. -1114 (1990)). It was found that infectious agent was Piscirickettsia salmonis. Piscirickettsia salmonis infection of white bass has recently been shown by Arkush, K.D. et al., in Diseases of Aquatic Organisms 64: 107-119 (2005). Rickettsia-like organisms, also referred to below as RLOs, have now been described among others in Tilapia (Chern, RS and Chao, CB, Fish Pathology 29: 61-71 (1994), Chen, SC et al., J. Fish Diseases 17: 591-599 (1994), Mauel et al., Diseases of aquatic organisms, 53: 249-255 (2003), Fryer, JX and Mauel, MJ., Emerging Infectious Diseases 3: 137-144 (1997)) , mere, (Chen, SC et al., Journ.Fish Dis. 23: 415-418 (2000)), pleco blue eye (Khoo, L. et al., J. Fish Diseases 18: 35-48 (1995) ) and bass (Comps, M. et al., Bull, Europ. Ass. Fish Pathol., 16, 30-33 (1996)). These fish species are tropical or Mediterranean fish. Recently, it was shown that RLOs also infected Atlantic species. It was shown by Nylund et al., That RLOs cause mortality in Norwegian cod. The differences between true Rickettsia, ie Piscirickettsia salmonis as described by Fryer, JX et al., (Int.J. Syst. Bacterid 42: 120-126 (1992)) and Rickettsia type organisms have been sufficiently explained. such as Tilapia-RLO described a decade ago by Chern (see above). In their review, Mauel and Miller once again describe the differences (Veterinary Microbiology 87: 279-289 (2002)). Despite previous efforts, there is no vaccine based on Rickettsia or RLO that is really effective against Rickettsia or RLO. The vaccines currently used are experimental, and share as a common characteristic that they offer only moderate protection. In addition, their level of protection is inconsistent (Mauel and Miller, see above). Only two patent applications have been published, WO 01/68865 and CA 2,281,913, concerning the Piscirickettsia salmonis vaccines. They require several subunit Piscirickettsia vaccines. A field trial with a bacterium-based vaccine of Piscirickettsia salmonis, in Coho salmon, has been described by Smith et al. (Bull. Eur. Ass. Fish Pathol. 15 (4): 137-141 (1995)) . However, until now the results with Piscirickettsia salmonis bacterins have been variable, not very consistent and are frequently below acceptable vaccine protection standards. These problems are reflected by the fact that currently no commercial vaccine based on Rickettsia or RLO is completely effective against Rickettsia or Rickettsia type organisms in the market. Currently, treatment with antibiotics, specifically quinolones, is the only treatment against RLO infection. To a large extent, most of the use of antibiotics is therapeutic. Prophylactic treatment among others includes i.p. of the fish before spawning, and the incorporation of antibiotics in the water during the hardening of the eggs. The use of antibiotics, however, is not the preferred method from an ecological point of view, due to the fact that the resistance against several antibiotics has been reported. It is clear that there is really a need for new and more effective RLO vaccines. It is an object of the present invention to provide such vaccines. It was now surprisingly found that bacteria of the genus Streptococcus, ie non-RLO gram-positive bacteria, are capable of providing a high level of durable protection against RLO infection. This protection is conferred by the Streptococcus type bacteria when administered as a bacterin and / or when administered in a live attenuated form. The working mechanism behind this unexpected finding is currently unknown. However, it is assumed that a component present in or attached to the cell surface, and common to all Streptococcus bacteria, is a potent stimulator of inter-specific immunity in fish against RLO. The inter-specificity in this respect means that it is not induced by RLO but nevertheless provides protection against RLO. Therefore, the invention relates to the use of a bacterium of the genus Streptococcus for the manufacture of a vaccine to combat RLO infection. For the manufacture of such vaccine, the state of the bacterium; live or inactivated, is not really important. The important thing is the fact that the stimulator of inter-specific immunity in fish against RLO is still present. This can be ensured by using all bacterial preparations. As mentioned above, it is not important if the bacteria in the preparation is alive, mute or even fragmented (for example by pressing it through a French Press). The important thing is that the components that make up the bacteria are still present in the vaccine. Attenuated live bacteria are very convenient, because by definition it contains the factor that stimulates inter-specific immunity against RLO. And the attenuated bacteria have an advantage over the bacterins, in that they can be easily administered without an adjuvant. On the other hand they reproduce themselves to the point where the immune system stops them, they can be administered as a result of their lower number of cells. Therefore, in a preferred form, the invention relates to the use of alive attenuated Streptococcus type bacteria for the manufacture of a vaccine to combat infection by Rickettsia type organism according to the invention. On the other hand, the factor that stimulates inter-specific immunity against RLO is also present in bacteria when these bacteria are in the form of bacterin. Bacterins have an advantage over live attenuated bacteria, in that they are very safe.

Therefore, in another preferred form, the invention relates to the use of a Streptococcus type bacterin for the manufacture of a vaccine to combat infection by Rickettsia type organisms according to the invention. The genus Streptococcus comprises, among others, Streptococcus iniae, Streptococcus difficile and Streptococcus agalactias. Vaccines for use according to the invention can be prepared from a bacterial culture according to techniques well known to the skilled artisan. The reviews concerning fish vaccines and their manufacture are among others by Sommerset, L, Krossoy, B., Biering, E. and (2005), by Buchmann, K., Lindenstrom, T. and Bresciani, in J. Acta Parasitological 46: 71-81 (2001), by Vinitnantharat, S., Gravningen, K. and Greger, E. in Advances in veterinary medicine 41: 539-550 (1999) and by Anderson, DP in Developments in Biological Standardization 90: 257-265 (1997). A live attenuated bacterium is a bacterium that is less pathogenic than its wild type counterparts, although they nevertheless induce an effective immune response. Attenuated strains can be obtained through classical routes, i.e. through the technique such as chemical mutagenesis, UV radiation and the like, or by site-site mutagenesis. A bacterin is defined herein as bacteria in an inactive form. The method used for inactivation seems to be irrelevant to the activity of the bacterin. Classical methods for inactivation such as heat treatment, formalin treatment, binary ethylenimine, thimerosal and the like, all well known in the art, are equally applicable. Inactivation of bacteria by means of physical tension, using for example a French Press provides an equally convenient raw material for the manufacture of a vaccine according to the invention. The vaccines according to the invention basically comprise an effective amount of a bacterium for use according to the invention and a pharmaceutically acceptable carrier. The term "effective" as used herein is defined as the amount sufficient to induce an immune response in the target fish. The amount of cells administered will depend on the species of Streptococcus used, presence of an adjuvant, route of administration, time of administration, 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. Otherwise, the person skilled in the art will find sufficient guidance in the references mentioned above and in the information provided below, especially in the examples.

Generally speaking, vaccines for use according to the invention that are based on bacterins can generally be administered at a dosage of 10 3 to 10 10, preferably 10 6 to 10 9, more preferably between 10 8 and 10 9 bacteria. A dose exceeding 1010 bacteria, although immunologically convenient, will be less attractive for commercial reasons. Vaccines according to the invention that are based on live attenuated bacteria can be administered at a lower dose, due to the fact that the bacteria will continue to reproduce for some time after administration. Examples of pharmaceutically acceptable carriers that are suitable for use in a vaccine for use according to the invention are sterile water, saline, aqueous buffers such as PBS and the like. In addition, a vaccine according to the invention can comprise other additives such as adjuvants, stabilizers, antioxidants and others, as described below. Vaccines for use according to the present invention, especially vaccines comprising a bacterin, may in a preferred embodiment also contain an immunostimulatory substance, a so-called adjuvant. Adjuvants in general comprise substances that elevate the host immune response in a non-specific manner. A number of various adjuvants are known in the art. Examples of adjuvants frequently used in the culture of fish and crustaceans are muramildipeptides, lipopolysaccharides, various glycans and glycans and Carbopol®. An extensive description of suitable adjuvants for fish and crustacean vaccines is given in the Jan Raa review document ((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 covalent binding Such vehicles are bio-microcapsules, among others, micro-alginates, liposomes and macrosols, all known in the art.A special form of such a vehicle, in which the antigen is partially integrated into the vehicle, it is called ISCOM (European Patents EP 109,942, EP 180,564, EP 242,380) In addition, the vaccine may comprise one or more suitable surfactant compounds or emulsifiers, for example Span or Tween. Suitable oil adjuvants for use in water-in-oil emulsions are mineral oils or metabolizable oils, for example Bayol®, Marcol® and Drakeol®. Non-mineral adjuvants of an oil is for example Montanide-ISA-763- ?. The metabolizable oils are for example vegetable oils, such as peanut oils and soybean oil, animal oils such as squalane of fish oil and squalene, and tocopherol and its derivatives. Suitable adjuvants are for example w / o emulsions and double w / o / w emulsions. Very convenient w / o emulsions for example are obtained from 5-50% w / w of water phase and 95-50% w / w of adjuvant oil, more preferably 20-50% w / w of water phase and 80-50% p / p oil adjuvant. An example of a water-based nanoparticle adjuvant is for example Montanide-IMS-2212. The amount of adjuvant added depends on the nature of the adjuvant by itself, and information regarding such amounts will be provided by the manufacturer.

Frequently, the vaccine is mixed with stabilizers, for example to protect the proteins prone to degradation against degradation, to improve the period of validity of the vaccine, or to improve the efficiency of dehydration by freezing. Useful stabilizers are among others SPGA (Bovarnik et al, J. Bacteriology 59: 509 (1950)), carbohydrates for example sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or their products of degradation, and buffers, such as alkali-metal phosphates. In addition, the vaccine can be suspended in a physiologically acceptable diluent. It is evident that other forms of adjuvants, adding vehicle compounds or diluents, emulsifying or stabilizing a protein are also incorporated in the present invention.

Many administration methods, all known in the art, can be applied. The vaccines according to the invention are preferably administered to fish via injection, immersion, submerging or orally. It should be kept in mind, however, that the route of administration highly depends on the manner in which the streptococcal vaccine for use according to the invention is generally administered. Just as an example, if the Streptococcus type bacterium now used for the manufacture of a vaccine to fight the infection by RLO is for example a live attenuated bacterium, the vaccine could among others be administered by immersion bath or vaccination, due to the ease of administration. Such vaccines are frequently applied by immersion vaccination. If on the one hand the Streptococcus-type bacterium now used for the manufacture of a vaccine to combat RLO infection is in the form of bacterin, oral use and for example intraperitoneal use are attractive administration forms. Generally speaking, if the vaccine can be improved by mixing an adjuvant, the manner of administration would preferably be the intraperitoneal route. From an immunological point of view, intraperitoneal vaccination with a bacterin is a very effective route of vaccination, especially because it allows the incorporation of adjuvants. The administration protocol can be optimized according to standard vaccination practice. The expert would know in what way to do this, or would find the guidance in the documents mentioned above. The age of the fish to be vaccinated is not critical, although it is clearly desired to vaccinate them against RLO as soon as possible, that is before possible exposure to the pathogen. For vaccines based on Streptococcus type bacteria described above, generally spoken these are administered at the time they would normally be administered for protection against Streptococcus infection. From that moment, the fish would then be protected additionally against RLO. Vaccination by immersion would be the vaccination of choice especially when the fish are still small, for example between 2 and 5 grams. Fish of 5 grams and more can also be vaccinated by injection. For oral administration the vaccine is preferably mixed with a convenient carrier for oral administration ie cellulose, feed or a metabolizable substance such as alpha-cellulose or various oils of vegetable or animal origin. Also an attractive method is the administration of vaccine at high concentrations of live feeding organisms, followed by feeding live food organisms to the fish. Particularly preferred carriers of food for the oral delivery of the vaccine according to the invention are living food organisms that can encapsulate the vaccine. Preferably, the Streptococcus type bacterium for use according to the invention is of the species Streptococcus iniae, agalactiae or difficile. More preferably, the bacterium for use according to the invention is of the species Streptococcus difficile. Generally, vaccines for use according to the invention will preferably be vaccines that protect against RLO and also against more than one non-RLO pathogenic microorganism. It would be beneficial to use, in addition to the Streptococcus type bacteria for the manufacture of the vaccine, also at least one other microorganism or pathogenic fish 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 RLO and against Streptococcus type infection, but also against other diseases. Therefore, a preferred form of this embodiment relates to a vaccine wherein the vaccine comprises at least one other microorganism or virus that is a fish pathogen, or another antigen or genetic material that encodes the other antigen, wherein the other antigen or genetic material is derived from a virus or pathogenic fish organism. Examples of commercially important fish pathogens in tropical and / or Mediterranean fish are Vibrio anguillarum, Photobacterium damselae, subspecies piscicidae, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, viral necrosis virus. , iridovirus and herpes virus Koi. The examples of water fish pathogen. cold commercially important are Vibrio anguillarum, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, Piscirickettsia salmonis, salmon pancreatic disease virus , sleeping sickness virus, viral necrosis virus, infectious pancreatic necrosis virus and iridovirus. The parasites of infection of salmonids are among others Lepeophtherius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis and Kudoa thyrsites. A parasite that infects freshwater fish is for example Ichthyophthirius multifiliis. Tilapia parasites are for example Dactylogyrus spp. and Trichodina spp. The marine fish can suffer among others of the parasite Benedenia seriolae. Therefore, 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 piscicidae, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, viral necrosis virus, iridovirus and Koi herpes virus, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Piscirickettsia salmonis, pancreatic salmon disease virus, sleeping sickness virus, viral necrosis virus, virus of infectious pancreatic necrosis, iridovirus, Lepeophtherius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis, Kudoa thyrsites, Ichthyophthirius multifiliis, Dactylogyrus spp. , Trichodina spp. and Benedenia seriolae. The examples given below provide a broad guide on how to use bacteria of the genus Streptococcus for the manufacture of a vaccine to fight infection by Rickettsia-like organisms. Examples Example 1: This example provides a comparison between the efficacy of RLO-based vaccines, Streptococcus-based vaccines, and a Gram-negative non-Streptococcus Vaccine type vaccine: 1) RLO-based vaccines An RLO strain originally isolated from diseased tilapia , cultivated in Indonesia that showed common signs of disease by RLO, was used as a vaccine strain and as a challenge strain. This strain was subcultured on chocolate agar and subsequently inoculated and grown in a liquid broth in an orbital shaker at 26 ° C - 150 rpm. After incubation for approximately 24 h the culture reached an OD660nm of 0.674 and was inactivated by the addition of 0.5% formalin and transferred to a new vessel. The total amount of bacterial cells in the inactivated culture was 3.7 x 109 cells / ml. 60 ml of inactivated culture overnight were centrifuged at 3,000 rpm for 30 minutes at 4 ° C and the pellet was resuspended in 20 ml of supernatant resulting in an effective cell concentration of 1.11 x 10 10 cells per ml. This antigen was used for the vaccine preparation. 2) Vaccines based on Vibrio anguillarum Vaccinations of V. anguillarum were prepared from a strain of serotype 02 A of V. anguillarum Standard methods of development of V. anguillarum were applied. Vaccines comprising 2.4 x 109 and 1.0 x 1010 cells were prepared. 3) Vaccines based on Streptococcus iniae / S. difficile The culture of S. iniae and the culture of S. difficile were mixed with the standard buffer and were carried out at a total count of 5 x 109 cells per ml. Two separate vaccines were prepared from each initial mixture, the water-based vaccine was derived from the above mixtures by diluting it to 1.0 x 1010 cells per ml with standard buffer while an oil adjuvant vaccine was prepared at a final cell concentration of 2.4. x 109 cells per ml of vaccine. A description of the vaccines used is presented in Table 1. Challenge strain The RLOs used for the challenge were developed as described in the preparation of the challenge material. Vaccination schedule and challenge Vaccination was done by IP injection of Tilapia with 0.1 ml of the respective vaccines (see table 1). The fish were maintained for 3 weeks after vaccination and challenged with a viable RLO culture. The challenged fish were kept separate and the mortality was followed for a 1 week period after the challenge. Efficacy was assessed by the relative survival expression percentage of vaccinated fish with respect to controls. Table 1: Vaccine composition with respect to the number of cells and the presence of adjuvant.

Preparation of challenge material An RLO vessel seeded with glycerol solution was thawed and inoculated onto chocolate agar and incubated at 26 ° C for 24 hours. The growth of the plates was collected and inoculated as solid growth in 100 ml of liquid broth and incubated in an orbital shaker (150 rpm) at 26 ° C. After approximately 24 hours of development the purity of the culture was checked and the OD6eo was measured. A challenge suspension was prepared in correspondence to OD660 = 0.250 / 10 with 0.9% sterile (weight / volume) of saline. This suspension was used directly for the challenge. The purity of the challenge suspension was evaluated by sowing a portion of the challenge suspension on chocolate agar, followed by a subsequent incubation at 26 ° C. Challenge Groups of approximately 15 fish were taken from the tanks and injected with the challenge suspension as described above. The fish were challenged IP by injection with 0.1 ml of suspension. The same procedure was followed for all groups. After the challenge the fish were transferred to various tanks and were kept for observation and to determine the occurrence of mortality. Mortality observations After the challenge, the fish were monitored twice a day and the dead fish were removed from the tank, the weight and length were recorded and the internal organs, notably the kidney and spleen were evaluated to determine the presence of common granulomas . From a representative number of fish, the spleen samples were plated on chocolate agar to confirm the presence of the challenge organism and thereby verify the cause of mortality. Results Mortality after challenge Mortality started in all groups challenged on day 2 after the challenge and increased rapidly in most groups. The fish that died during the first 3 days all exhibited an accumulation of fluid in the body cavity and generalized the liquefaction of the internal organs. Towards the end of the observation period, however, some fish showed common signs of disease associated with RLO infections as observed in the field, ie granuloma formation in the kidney and spleen. By observing mortality on day 3 after challenge it is clear that a distinction can be made between most water-based vaccines and controls with respect to all oil-based vaccines. The oil adjuvant vaccines exhibit a delayed mortality with respect to water-based vaccines, however, PBS-oil without the bacteria does not show this delayed mortality.

The mortality observed per group was used for the calculation of the RPS values. A description of the RPS values, as expressed against the PBS control group is given in Table 1. The results show that: a) Surprisingly, a vaccine containing Streptococcus antigens is capable of protecting fish from infection by RLO. b) An RLO vaccine containing the challenge strain, ie a homologous vaccine, is surprisingly less efficient than vaccines based on Streptococcus antigens. c) Oil adjuvant vaccines work better than water-based vaccines d) Even a water-based Streptococcus vaccine provides better protection than an oil-based vaccine based on RLOs e) As expected a random choice It is a Gram-negative vaccine but not Streptococcus, in this case the 02 A pathogenic strain of Vibrio anguillarum of the fish does not provide protection against RLO infection. Conclusions It was found that the vaccines tested were safe. A challenge of vaccinated fish was made 3 weeks after IP vaccination with RLOs developed in artificial media. The disease as well as the common samples of the disease could be reproduced and the challenge organism could be isolated again from the dead fish. Following directly to Figure 1, Tilapia can be protected against RLO infections with vaccination by IP injection with oil-Sfrepfococcus and water-based vaccines. Surprisingly, the operation even of water-based Streptococcus type vaccines is astonishingly better than the operation of oil-RLO vaccines. On the other hand, Streptococcus vaccines with oil adjuvant outperform vaccines without adjuvant. The relatively low values of RPS in this experiment are the result of the very high dose of RLO challenge applied in this specific experiment. Example 2: The objective of this experiment was to assess whether protection against RLO infections is obtained when Tilapia is vaccinated with the bivalent oil-based vaccines containing Streptococcus iniae and S. difficile. Also, the relationship between the level of protection and the number of bacteria present in the Streptococcus-based vaccine was determined in this example. Two different vaccines, which contain Streptococcus iniae and S. difficile at different concentrations in a water-in-oil non-mineral emulsion were injected IP into juvenile tilapia. The antigen levels in the vaccines were 6.8 x 108 and 1.7 x 108 cells of each antigen per my vaccine. Three weeks after vaccination, the fish from each vaccinated group and the unvaccinated control groups were challenged with a suspension of Rickettsia type bacteria. Experimental design Tilapia groups that originated from the same lot were vaccinated with one of two experimental oil adjuvant vaccines. The vaccines tested contained S. iniae and S. difficile but the two vaccines differed in cell concentration. A group of fish was not vaccinated and was kept as a control. Three weeks after vaccination, all groups were challenged with a standard challenge inoculation of RLO bacteria and the fish were kept for observation for a period of 2 weeks after the challenge. Daily the dead fish were removed and the causative bacteria was isolated again from a representative number of fish that had died. The efficacy of the vaccines was expressed as the relative percentage survival (RPS) in the vaccinated groups with respect to the controls.

The vaccines were prepared from a S. iniae fermentor culture and a S. difficile fermentor culture. All vaccine formulations were prepared in a non-mineral water-in-oil adjuvant. The vaccines used in the study are presented in Table 2.

Table 2: Vaccines used in the study The antigen concentrations specified in Table 2 represent the final vaccine concentration, ie after mixing with the PBS / oil emulsion. Vaccination The fish were starved for 48 h before vaccination to ensure complete emptying of the intestinal tract and thereby reducing the possibility of damaging the internal organs in the injection. Vaccination was performed by IP injection. The experiment included 3 experimental groups (2 vaccine groups and 1 control group), each consisting of 25 fish. Each fish of the vaccine groups was injected with 0.1 ml of vaccine according to table 2. The control fish was not injected. Challenge Culture Preparation One vial of an RLO glycerol solution seed was thawed and inoculated onto chocolate agar and incubated at 26 ° C for 48 hours. The development of the plates was collected and inoculated as solid development in 100 ml of liquid broth and incubated in an orbital shaker (150 rpm) at 26 ° C. After approximately 48 hours of growth the purity of the culture was checked and the OD660 was measured. Challenge suspensions were prepared from this culture by dilution of the culture to OD660 = 0.027 with 0.9% (w / v) sterile saline. This suspension was then used directly for the challenge. The purity of the challenge suspension was evaluated by sowing a portion of the challenge suspension on chocolate agar and followed by subsequent incubation at 26 ° C. Challenge The challenge was made by injection. The fish were subjected to starvation for 48 hours before challenge to ensure complete emptying of the gastrointestinal tract. Of all the vaccine groups and the control group, 20 fish were injected with 0.1 ml of a standardized bacterial suspension. For this, each group was obtained, anesthetized in Aqui-S until they were sedated and injected intraperitoneally in the body just behind the tip of the tip of the pectoral fin. Immediately after the injection the fish were transferred to their assigned tank and recovery was followed. Mortality observation After the challenge, fish were monitored twice a day and dead fish were removed from the tank, weight and length were recorded and internal organs, notably kidney and spleen were evaluated to determine the presence of common granulomas. For some fish, spleen samples were plated on chocolate agar to confirm the presence of the challenge organism and thereby prove the cause of mortality. Results Mortality after challenge The mortality observed in the various vaccine groups and in the control groups is presented in figure 2 and figure 3. The vaccines are plotted in the graph with reference to the concentration (cells / ml) of each antigen in the final vaccine. The re-isolation was performed on the fish that died after the challenge. These fish showed clear positive re-isolation. Efficacy assessment According to the figures of mortality obtained, the RPS values were calculated by the condition. The results are expressed in figure 2 and 3. IP vaccinated fish with a bivalent oil adjuvant streptococcal vaccine containing S. iniae and S. difficile showed protection against the challenges of RLO. On the other hand, the results suggest a type of reaction at a certain dose of reaction in which a high-dose vaccine provides better protection than a low-dose vaccine. A RPS of 73% was obtained with a vaccine that contained 6.8 x 108 cells of S. iniae and 6.8 x 108 cells of S. difficile. Mortality in unvaccinated control fish was 75%. Example 3: In this experiment the efficacy of a monovalent Streptococcus difficile vaccine in a non-mineral water-in-oil emulsion was tested. Tilapia groups were vaccinated with a monovalent Streptococcus difficile vaccine comprising 6.8 x 10 7 Sd cells in a water-in-oil non-mineral emulsion, or were left unvaccinated as a control group. The vaccines were prepared as described in examples 1 and 2. The following challenges with RLO were performed: a) 10 days after the vaccination a cohabitation challenge was performed. The ten-day challenge moment was chosen since it represents the normal field situation. Cohabitation was chosen since this model showed the development of the common signs of the disease at the appropriate time as seen in field shoots in the pre-challenge experiment. b) In the vaccination after 6 weeks a challenge was made by injection. Overall conclusion: The challenge of cohabitation done at 10 days PV and the injection challenge performed at 6 weeks PV, showed that a monovalent oil adjuvant vaccine containing Streptococcus difficile provides protection against RLO infection. The RPS value after vaccination in the cohabitation challenge experiment was 89%.

Figure 4 shows that at 6 weeks after vaccination with a monovalent Streptococcus difficile vaccine followed by the challenge of injection, cumulative mortality in control fish reached 90%, while vaccinated fish only 20% died. This results in a RPS of 78%.

Legend of the figures. Figure 1: RPS values per vaccine group as calculated in comparison to control group vaccinated with PBS-placebo. Figure 2: Cumulative percentage mortality in a certain period of time, with reference to the given dose of vaccine. The effect of the number of cells in the vaccine on the relative percentage survival after the RLO challenge occurs. Figure 3: Final cumulative percentage survival (compare figure 2), with reference to the dose of vaccine given.

Figure 4: Relative percentage survival of the vaccinated fish with a monovalent Streptococcus difficile vaccine comprising 6.8 x 10 7 Sd cells in a water-in-oil non-mineral emulsion.

Claims (7)

1. Use of a bacterium of the genus Streptococcus for the manufacture of a vaccine to combat infection by Rickettsia type organisms.

2. Use according to claim 1, characterized in that the bacterium is a live attenuated bacterium.

3. Use according to claim 1, characterized in that the bacterium is in the form of bacterin.

4. Use according to claims 1-3, characterized in that the bacterium of the genus Streptococcus is of the species Streptococcus iniae, agalactiae or difficile.

5. Use according to claims 1-3, characterized in that the bacterium of the genus Streptococcus is of the species Streptococcus difficile.

6. The use according to claims 1-5, characterized in that the vaccine comprises at least one other microorganism or virus that is a fish pathogen, or another antigen or genetic material that encodes the other antigen, wherein the other antigen or Genetic material is derived from a virus or fish pathogen microorganism. The use according to claim 6, characterized in that the other microorganism or virus is selected from the group consisting of Vibrio anguillarum, Photobacterium damselae subspecies piscicidae, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, viral necrosis virus, iridovirus, Koi herpes virus, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscosa, Vibrio ordalii, Piscirickettsia salmonis, pancreatic salmon disease virus, sleeping sickness virus, necrosis virus viral, infectious pancreatic necrosis virus, iridovirus, Lepeophtherius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis, Kudoa thyrsites, Ichthyophthirius multifiliis, Dactylogyrus spp., Trichodina spp. and Benedenia seriolae.