WO2007138036A1

WO2007138036A1 - Vaccine against rickettsia-like organisms

Info

Publication number: WO2007138036A1
Application number: PCT/EP2007/055167
Authority: WO
Inventors: Luc Grisez; Chow Yong Ng
Original assignee: Intervet International BV
Current assignee: Intervet International BV
Priority date: 2006-05-30
Filing date: 2007-05-29
Publication date: 2007-12-06
Anticipated expiration: 2008-11-30
Also published as: CR10457A; MX2008015164A; ECSP088907A

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 Rickettsia-likc organisms.

Infection of several salmonid and non-salmonid fish with Rickettsial Rickettsia-Wkς, organism has been reported for decades, the first report dating from 1939 when a Rickettsia-likc organism was found in Tetrodon fahaka from the river Nile. It took however until 1975 before a first observation of the presence of a Rickettsia-Hkc organism in stained tissue cells was done by Ozel M. and Schwanz-Pfitzner, L, (Zentralblatt fur Bacteriologie, Microbiologic und Hygiene, I abt Originate A 1975; 230: 1-14 (1975)).

The real importance of Piscirickettsia salmonis as a pathogen became dramatically clear in 1989, when in Chile 1.5. million market-size Coho salmon died of a then unknown infectious agent. (Bravo, S. and Campos, M. FHS/AFS Newsletter 17:3 (1989), Cvitanich et al, FHS/AFS Newsletter 18:1-2 (1990), Fryer et al., Fish Pathol. 25: 107-1114 (1990)). This unknown infectious agent was found to be Piscirickettsia salmonis.

Piscirickettsia salmonis infection of white sea bass has recently been shown by Arkush, K.D. et al., in Diseases of Aquatic Organisms 64: 107-119 (2005).

Rickettsia-likc organisms, also referred to further as RLOs have now been described i.a. in Tilapia (Chern, R.S. and Chao, C.B., Fish Pathology 29: 61-71 (1994), Chen, S.C. 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)), grouper, (Chen, S.C. et al., Journ. Fish Dis. 23: 415-418 (2000)), blue-eyed plecostomus (Khoo, L. et al., J. Fish Diseases 18:35-48 (1995)) and Sea bass (Comps, M. et al., Bull.

Europ. Ass. Fish Pathol. 16, 30-33 (1996)). These fish species are tropical or Mediterranean fish. Recently, RLOs were shown to infect Atlantic species as well. It was shown by Nylund et al., that RLOs cause mortality in Norwegian cod.

The differences between the real Rickettsia, i.e. Piscirickettsia salmonis as described by

Fryer, JX et al., (Int. J. Syst. Bacterid 42: 120-126 (1992)) and the Rickettsia-like organisms such as the Tilapia-RLO described over a decade ago by Chern (see above) have sufficiently been explained. In their review, Mauel and Miller once more describe the differences (Veterinary Microbiology 87: 279-289 (2002)).

In spite of the efforts so far, no really efficacious Rickettsia- or RLO -based vaccine against Rickettsia or RLO exists. The vaccines currently in use are experimental, and they share as a common characteristic that they offer only a moderate protection. In addition, their level of protection is inconsistent (Mauel & Miller, see above).

Only two Patent Applications, WO 01/68865 and CA 2,281,913, relating to Piscirickettsia salmonis vaccines have been published. They claim several Piscirickettsia subunit vaccines. A field trial with a vaccine on the basis of a Piscirickettsia salmonis bacterin, in Coho salmon has been described by Smith et al., (Bull. Eur. Ass. Fish Pathol. 15(4): 137-141 (1995)). So far, however, results with Piscirickettsia salmonis bacterins have been variable, not very consistent and often below acceptable vaccine protection standards. These problems are reflected by the fact that currently no fully effective commercial Rickettsia- or RLO -based vaccine against Rickettsia or Rickettsia-likc organisms is on the market.

Currently, treatment with antibiotics, specifically quinolones, is the only treatment against RLO infection. By far most of the antibiotics use is therapeutic. Prophylactic treatment i.a. includes i.p. injection of brood stock before spawning, and incorporation of antibiotics in the water during hardening of the eggs.

The use of antibiotics is however not the preferred method from an ecological point of view, if only due to the fact that resistance against various antibiotics has been reported.

It is clear that there really is a need for novel, more efficacious RLO vaccines.

It is an objective of the present invention to provide such vaccines.

It was surprisingly found now, that bacteria of the genus Streptococcus, i.e. non-RLO Gram- positive bacteria, are capable of providing a high level of long-lasting protection against RLO infection. This protection is conferred by Streptococcal bacteria when given as a bacterin and/or when given in a live attenuated form.

The working mechanism behind this unexpected finding is currently unknown. It is assumed however that a component present on or attached to the cell surface, and common to all Streptococcal bacteria is a powerful stimulator of cross-specific immunity in fish against RLO. Cross-specific in this respect means: not induced by RLO but nevertheless providing protection against RLO.

Thus, the invention relates to the use of a bacterium of the genus Streptococcus for the manufacture of a vaccine for combating RLO infection. For the manufacture of such a vaccine, the status of the bacterium; live or inactivated, is not really important. What is important is the fact that the stimulator of cross-specific immunity in fish against RLO is still present. This can be assured by using whole bacterial preparations. As said above, it is not important if the bacterium in the preparation is alive, killed or even fragmented (e.g. by pressing it through a French Press). What is important, is that the components making up the bacterium are still present in the vaccine.

Live attenuated bacteria are very suitable, because they by definition carry the factor stimulating the cross-specific immunity against RLO. And live attenuated bacteria have the advantage over bacterins, that they can easily be given without an adjuvant. Moreover they self-replicate to a certain extent until they are stopped by the immune system, as a result of which a lower number of cells can be given.

Therefore, in a preferred form, the invention relates to the use of live attenuated Streptococcal bacteria for the manufacture of a vaccine for combating Rickettsia-like organism infection according to the invention.

On the other hand, the factor stimulating the cross-specific immunity against RLO is also present on bacteria when these bacteria are in the form of a bacterin. Bacterins have the advantage over live attenuated bacteria that they are very safe. Therefore, in another preferred form, the invention relates to the use of a Streptococcal bacterin for the manufacture of a vaccine for combating Rickettsia-like organism infection according to the invention.

The genus Streptococcus comprises i.a. Streptococcus iniae, Streptococcus difficile and Streptococcus agalactiae.

Vaccines for use according to the invention can be prepared starting from a bacterial culture according to techniques well known to the skilled practitioner.

Review articles relating to fish vaccines and their manufacture are i.a. by Sommerset, L, Krossøy, B., Biering, E. and Frost, P. in Expert Review of Vaccines 4: 89-101 (2005), by

Buchmann, K., Lindenstrøm, T. and Bresciani, in J. Acta Parasitologica 46: 71-81 (2001), by Vinitnantharat, S., Gravningen, K. and Greger, E. in Advances in veterinary medicine 41 : 539-550 (1999) and by Anderson, D.P. in Developments in Biological Standardization 90: 257-265 (1997).

A live attenuated bacterium is a bacterium that is less pathogenic than its wild-type counterpart, while nevertheless inducing an efficacious immune response. Attenuated strains can be obtained along classical routes, long known in the art such as chemical mutagenesis, UV -radiation and the like, or by site-directed mutagenesis.

A bacterin is defined here as bacteria in an inactivated form. The method used for inactivation appears to be not relevant for the activity of the bacterin. Classical methods for inactivation such as heat-treatment, treatment with formalin, binary ethylene imine, thimerosal and the like, all well-known in the art, are equally applicable. Inactivation of bacteria by means of physical stress, using e.g. a French Press provides an equally suitable starting material for the manufacturing of a vaccine according to the invention.

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 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.

Otherwise, man skilled in the art finds sufficient guidance in the references mentioned above and in the information given below, especially in the Examples.

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.

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 may comprise other additives such as adjuvants, stabilisers, anti-oxidants and others, as described below.

Vaccines for use according to the present invention, especially the vaccines comprising a bacterin, 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 CarbopolW. An extensive overview of adjuvants suitable for fish and shellfish vaccines is given in the review paper by Jan Raa (Reviews in Fisheries Science 4(3): 229-288 (1996)).

The vaccine may also comprise a so-called "vehicle". A vehicle is a compound to which the bacterium adheres, without being covalently bound to it. Such vehicles are i.a. bio- microcapsules, micro-alginates, liposomes and macrosols, all known in the art. A special form of such a vehicle, in which the antigen is partially embedded in the vehicle, is the so-called ISCOM (European Patents EP 109.942, EP 180.564, EP 242.380). In addition, the vaccine may comprise one or more suitable surface-active compounds or emulsifiers, e.g. Span or Tween.

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

An example of a non-mineral oil adjuvants is e.g. Montanide-ISA-763-A

Metabolisable oils are e.g. vegetable oils, such as peanut oil and soybean oil, animal oils such as the fish oils squalane and squalene, and tocopherol and its derivatives. Suitable adjuvants are e.g. w/o emulsions, o/w emulsions and w/o/w double-emulsions

Very suitable w/o emulsions are e.g. obtained starting from 5-50% w/w water phase and 95-

50% w/w oil adjuvant, more preferably 20-50% w/w water phase and 80-50% w/w oil adjuvant.

An example of a water-based nano-particle adjuvant is e.g. Montanide-IMS-2212.

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 degradation-prone proteins from being degraded, to enhance the shelf- life of the vaccine, or to improve freeze-drying efficiency. Useful stabilisers are i.a. SPGA (Bovarnik et al; J. Bacteriology 59: 509 (1950)), carbohydrates e.g. sorbitol, mannitol, trehalose, starch, sucrose, dextran or glucose, proteins such as albumin or casein or degradation products thereof, and buffers, such as alkali metal phosphates. In addition, the vaccine may be suspended in a physiologically acceptable diluent. It goes without saying, that other ways of adjuvating, adding vehicle compounds or diluents, emulsifying or stabilizing a protein are also embodied in the present invention.

Many ways of administration, all known in the art can be applied. The vaccines according to the invention are preferably administered to the fish via injection, immersion, dipping or per oral. It should be kept in mind however that the route of administration highly depends on the way the Streptococcal vaccine for use according to the invention is usually given. Merely as an example, if the Streptococcus bacterium now used for the manufacture of a vaccine for combating RLO infection is e.g. a live attenuated bacterium, the vaccine could i.a. be administered by immersion or bath vaccination, due to the ease of administration. Such vaccines are often applied by immersion vaccination.

If on the other hand the Streptococcus bacterium now used for the manufacture of a vaccine for combating RLO infection is in the form of a bacterin, oral application and e.g. intraperitoneal application are attractive ways of administration.

Generally spoken: if the vaccine can be improved by admixing an adjuvant, the way 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 in accordance with standard vaccination practice. The skilled artisan would know how to do this, or he would find guidance in the papers mentioned above.

The age of the fish to be vaccinated is not critical, although clearly one would want to vaccinate against the RLO in as early a stage as possible, i.e. prior to possible exposure to the pathogen.

For the vaccines based upon the Streptococcal bacteria described above, generally spoken these would be administered at the moment they would normally be administered in order to protect against Streptococcal infection. From that moment on, the fish would then additionally be protected against the RLO.

Immersion vaccination would be the vaccination of choice especially when fish are still small, e.g. between 2 and 5 grams. Fish from 5 grams and up can also be vaccinated by means of injection. For oral administration the vaccine is preferably mixed with a suitable carrier for oral administration i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin. Also an attractive method is administration of the vaccine to high concentrations of live-feed organisms, followed by feeding the live-feed organisms to the fish. Particularly preferred food carriers for oral delivery of the vaccine according to the invention are live-feed organisms which are able to encapsulate the vaccine.

Preferably, the Streptococcal 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.

In general, the vaccines for use according to the invention will preferably be vaccines that protect against RLO and additionally against more than one non-RLO pathogenic microorganism. 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 RLO and against Streptococcal infection, but also against other 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 piscicidae, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garvieae, Edwardsiella tarda, E. ictaluri, Viral Necrosis virus, iridovirus 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 garvieae, Edwardsiella tarda, E. ictaluri, Piscirickettsia salmonis, Salmon Pancreatic Disease virus, Sleeping Disease virus, Viral Nervous Necrosis virus, Infectious Pancreatic Necrosis 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 piscicidae, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garvieae, 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, iridoviruses, Lepeophtherius salmonis, Caligus elongatus, Cryptobia salmositica, Myxobolus cerebralis, Kudoa thyrsites, Ichthyophthirius multifiliis, Dactylogyrus spp. , Trichodina spp. and Benedenia seriolae.

The Examples given below provide broad guidance of how to use bacteria of the genus Streptococcus for the manufacture of a vaccine for combating Rickettsia-like organism infection.

Examples.

Example 1: In this Example, a comparison is given between the efficacy of RLO-based vaccines, Streptococcus -based vaccines and a non-Streptococcal Gram-negative vaccine

Vaccines:

1) RLO-based vaccines A RLO-strain originally isolated from diseased tilapia, cultured in Indonesia showing typical RLO-disease signs was used both as vaccine strain and as challenge strain. This strain was sub-cultured on Chocolate agar and subsequently inoculated and grown in a liquid broth on an orbital shaker at 26°C - 150 RPM. After approximately 24 h incubation the culture reached an OD₆₆o_nm 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. Sixty ml of overnight inactivated culture was centrifuged at 3,000 RPM for 30 minutes at 4°C and the pellet was re-suspended in 20 ml supernatant resulting in an effective cell concentration of 1.11 x 10¹⁰ cells per ml. This antigen was used for vaccine preparation.

T) Vibrio anguillarum based vaccines

The V. anguillarum vaccines were prepared starting from a V. anguillarum serotype 02 A strain. Standard V. anguillarum growth methods were applied. Vaccines comprising 2.4 x 10⁹ and 1.0 xlO¹⁰ cells were prepared.

3) Streptococcus iniae/S. difficile based vaccines

The S. iniae culture and the S. difficile culture were mixed with standard buffer and brought to a total count of 5 x 10⁹ cells per ml each.

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 whereas an oil-adjuvanted vaccine was prepared to a final cell concentration of 2.4 x 10⁹ cells per ml vaccine. An overview of the vaccines used is presented in Table 1.

Challenge strain

The RLO used for challenge was grown as described under preparation of challenge material.

Vaccination and challenge schedule

Vaccination was done by IP-injection of tilapia with 0.1 ml of the respective vaccines (See table 1). Fish were maintained for 3 weeks after vaccination and subsequently challenged with a viable RLO culture. Challenged fish were kept separate and mortality was followed over a 1 week period post-challenge. The efficacy was evaluated by expression the relative percent survival of vaccinated fish as compared to the controls.

Table 1: Vaccine composition with regard to number of cells and presence of adjuvant.

Preparation of challenge material A vial of RLO seed glycerol stock was thawed and inoculated onto Chocolate agar and incubated at 26°C for 24 hours. Growth from the plates was collected and inoculated as solid growth into 100 ml of liquid broth and incubated on an orbital shaker (150 RPM) at 26°C. After approximately 24 hours of growth the purity of the culture was checked and the OD₆₆₀ was measured. A challenge suspension was prepared corresponding to OD₆₆₀ = 0.250 / 10 with sterile 0.9% (w/v) saline. This suspension was used directly for challenge. Purity of the challenge suspension was evaluated by streaking a portion of the challenge suspension onto Chocolate agar and 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. Fish were IP-challenged by injection with 0.1 ml of the suspension. The same procedure was followed for all groups. After challenge fish were transferred to different tanks and kept for observation and the occurrence of mortality.

Observations of mortality

After challenge, fish were checked twice daily and dead fish were removed from the tank, weight and length was recorded and internal organs, notably kidney and spleen were evaluated for the presence of typical granulomas. From a representative number of fish, spleen samples were plated onto Chocolate agar to confirm the presence of the challenge organism and thereby verifying the cause of mortality.

RESULTS

Mortality after challenge

Mortality started in all challenged groups on day 2 post challenge and increased rapidly in most groups. The fish that died during the first 3 days all displayed accumulation of fluid in the body cavity and generalized liquefaction of the internal organs. Towards the end of the observation period however, some fish showed the typical disease signs associated with RLO infections as seen in the field, i.e. granuloma formation in kidney and spleen. When observing the mortality on day 3 post challenge it is apparent that a distinction can be made between most water based vaccines and the controls as compared to all oil-based vaccines. Oil-adjuvanted vaccines display a delayed mortality as compared to the water based vaccines, however, PBS-oil without bacteria does not show this delayed mortality.

The observed mortality per group was used for the calculation of RPS values. An overview of the RPS values, as expressed against the PBS control group is given in Figure 1. The results show that: a) Surprisingly, a vaccine containing Streptococcal antigens is capable of protecting fish against RLO infection. b) A RLO vaccine containing the challenge strain, i.e. a homologous vaccine, is strikingly less efficient then vaccines based on Streptococcal antigens. c) Oil-adjuvanted vaccines perform better then water based vaccines d) Even a water-based Streptococcal vaccine provides better protection then an oil based vaccine based on RLO 's e) As expected a randomly chosen Gram-negative but non-Streptococcal vaccine, in this case the fish pathogenic Vibrio anguillarum 02 A strain does not provide protection against RLO infection.

CONCLUSIONS

The vaccines tested were found to be safe. A challenge of vaccinated fish was performed 3 weeks after the IP-vaccination with RLO 's grown in artificial media. The disease as well as the typical disease signs could be reproduced and the challenge organism could be re-isolated from dead fish.

It follows directly from figure 1 , that Tilapia can be protected against RLO infections through IP-injection vaccination with both oil- and water -based Streptococcal vaccines. Surprisingly, the performance of even the water-based Streptococcal vaccines is strikingly better than the performance of the oil -based RLO vaccines. Moreover, oil-adjuvanted Streptococcal vaccines outperform non-adjuvanted vaccines.

The relatively low RPS values in this experiment are the result of the very high RLO challenge dose applied in this specific experiment.

Example 2:

The objective of this experiment was to evaluate whether protection against RLO infections is obtained when tilapia are vaccinated with oil-based bivalent vaccines containing Streptococcus iniae and S. difficile. Also, the relation between the level of protection and the number of bacteria present in the Streptococcus -based vaccine was determined in this Example. Two different vaccines, containing both Streptococcus iniae and S. difficile at different concentrations in a non-mineral water-in-oil emulsion were IP-injected in juvenile tilapia. The antigen levels in the vaccines were 6.8 x 10⁸ and 1.7 x 10⁸ cells of each antigen per ml of vaccine. Three weeks after vaccination, fish from each vaccine group and non- vaccinated control groups were challenged with a suspension of Rickettsia-Hkc bacteria.

Experimental design Groups of tilapia originating from the same batch were vaccinated with one out of two experimental oil-adjuvanted vaccines. The vaccines tested contained S. iniae and S. difficile but the two vaccines differed in the concentration of the cells. One group of fish was not vaccinated and was kept as control. Three weeks after vaccination, all groups were challenged with a standardized challenge inoculum of RLO-bacteria and fish were kept for observation for a period of 2 weeks after challenge. Daily, dead fish were removed and the causative bacterium was re-isolated from a representative number of fish that had died. The efficacy of the vaccines was expressed as Relative percent survival (RPS) in the vaccinated groups as compared to the controls.

Vaccines were prepared starting 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, i.e. after mixing with the PBS/oil emulsion.

Vaccination

The fish were starved for 48 h prior to vaccination to assure complete emptying of the intestinal tract and thereby reducing the possibility of damaging the internal organs when injecting. The 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 from the vaccine groups was injected with 0.1 ml of vaccine according to Table 2. Control fish were not injected.

Preparation of challenge culture

A vial of an RLO glycerol stock seed was thawed and inoculated onto Chocolate agar and incubated at 26°C for 48 hours. Growth from the plates was collected and inoculated as solid growth into 100 ml of liquid broth and incubated on 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 starting from this culture by dilution the culture to an OD₆₆₀ = 0.027 with sterile 0.9% (w/v) saline. This suspension was then used directly for challenge. Purity of the challenge suspension was evaluated by streaking a portion of the challenge suspension onto Chocolate agar and subsequent incubation at 26°C.

Challenge

Challenge was performed by injection. The fish were starved for 48 hours prior to the challenge to ensure complete emptying of the gastro-intestinal tract. From all vaccine groups and from the control group, 20 fish were injected with 0.1 ml of a standardised bacterial suspension. For this each group was netted, anaesthetised in Aqui-S until sedated and injected intra-peritoneal on the side of the body just behind the tip of the pectoral fin.

Immediately after injection the fish were transferred to their allocated tank and the recovery was followed.

Observations of mortality After challenge, fish were checked twice daily and dead fish were removed from the tank, weight and length was recorded and internal organs, notably kidney and spleen were evaluated for the presence of typical granulomas. From a few fish, spleen samples were plated onto Chocolate agar to confirm the presence of the challenge organism and thereby verifying the cause of mortality.

RESULTS

Mortality after challenge

The observed mortality in the different vaccine groups and in the control groups is presented in

Figure 2 and figure 3. The vaccines are represented in the graph in relation to the concentration (cell/ml) of each antigen in the final vaccine.

Re-isolation was performed from the fish that died after challenge. These fish showed clear positive re-isolation. Efficacy evaluation

Based on the mortality figures obtained the RPS values were calculated per condition. The results are expressed in Figure 2 and 3. Fish, IP -vaccinated with a bivalent oil-adjuvanted streptococcus vaccine containing S. iniae and S. difficile show protection against RLO-challenges. Moreover, the results suggest a dose response type of reaction in which a high dose vaccine provides better protection than a low dose vaccine. An RPS of 73% was obtained with a vaccine containing 6.8 x 10⁸ cells of S. iniae and 6.8 x 10⁸ cells of S. difficile. Mortality in the non-vaccinated control fish was 75%.

Example 3:

In this experiment the efficacy of a monovalent Streptococcus difficile (Sd) vaccine in a non- mineral water-in- oil emulsion was tested. Groups of tilapia were vaccinated with a monovalent Streptococcus difficile vaccine comprising 6.8 x 10⁷ Sd cells in a non-mineral water-in-oil emulsion, or left un- vaccinated as a control group.

Vaccines were prepared as described in Examples 1 and 2. The following challenges with RLO were performed:

a) At 10 days post vaccination a co-habitation challenge was performed. The ten days challenge moment was chosen since this represents the normal field-situation. Co-habitation was chosen since this model showed the development of typical disease signs at the appropriate time as seen in field outbreaks in a pre-challenge experiment. b) At 6 weeks post vaccination an injection challenge was performed.

Overall conclusion:

Cohabitation challenge done at 10 days PV and injection challenge performed at 6 weeks PV both show that a monovalent oil-adjuvanted 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 post-vaccination with a monovalent Streptococcus difficile vaccine followed by injection challenge, cumulative mortality in control fish reached 90%, whereas of the vaccinated fish only 20% died. This results in a RPS of 78%. Legend to the figures.

Figure 1 : RPS values per vaccine group as calculated compared to the PBS- mock vaccinated control group

Figure 2: Cumulative percentage mortality over time, in relation to the vaccine dose given. The effect of the number of cells in the vaccine on the relative percentage survival after RLO -challenge is given.

Figure 3 : Final cumulative percentage survival (compare figure 2), in relation to the vaccine dose given.

Figure 4: Relative percentage of survival of fish vaccinated with a monovalent

Streptococcus difficile vaccine comprising 6.8 x 10⁷ Sd cells in a non-mineral water-in- oil emulsion.

Claims

Claims:

1) Use of a bacterium of the genus Streptococcus for the manufacture of a vaccine for combating Rickettsia-like organism infection.

2) Use according to claim 1 , characterized in that said bacterium is a live attenuated bacterium

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

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

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

6) Use according to claims 1-5, characterized in that said 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.

7) Use according to claim 6, characterized in that said 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 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.