The present invention relates to a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium causing a new, deadly, disease in fish, to a microbiological culture comprising said bacterium, to a vaccine comprising said bacterium and methods for the preparation of such a vaccine, to antibodies reactive with said bacterium, to diagnostic test kits and to the use of said bacterium.

Description of the Invention

SUMMARY OF THE INVENTION

Recently, the inventor found the causative agent of a recently found disease of hitherto unknown origin, further referred to also as Cod's Syndrome in Atlantic cod (Gadus morhua). It has now been determined that the causative agent of this disease is a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium. It is an objective of the present invention to provide the causative agent of this enigmatic disease as well as vaccines aiming at combating and preventing the disease. Moreover, it is an objective of the present invention to provide means to detect and identify the causative agent.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Atlantic cod suffering from this new disease show loss of appetite, reduced swimming performance, and dark pigmentation. There are few other external signs of disease, but white spots may be found on gills and in the mouth cavity. It has been observed that the disease spreads within cod farms. Acute infection can result in mortality within 10 days after challenge, but with few gross clinical signs of disease. The most prominent clinical sign is a swollen and liquefied kidney. Cod suffering from chronic disease may have swollen spleen, kidney and heart. These blood rich organs will in most cases contain white cyst-like inclusions/nodules. The cyst-like structures may contain a transparent liquid. Microscope sections of these tissues show degeneration and proliferation of cells. A few intracellular bacteria may be observed in cells from these tissues. White cyst-like structures may also be found on and in the liver.

The causative agent of this disease is a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium. An example of the DNA sequence of the 16S rRNA gene, the intergenic spacer and the 23S rRNA gene of this novel bacterium is depicted in FIG. 1 and SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3 respectively (see Original Patent). SEQ ID NO 1 represents the 16S rRNA gene except for the first 65 nucleotides which are not reproduced here. SEQ ID NO 2 represents the 16S-23S intergenic spacer. SEQ ID NO 3 represents the 23S rRNA gene, except for the last 880 nucleotides which are not reproduced here.

Comparison of these sequences with a genome databank unexpectedly revealed that the bacterium bears a relatively high level of resemblance to a so-called Rickettsia-like organism (RLO) found earlier in tropical fish species such as Tilapia.

Therefore, it is now assumed that there might be some evolutionary relationship between the newly identified RLO according to the invention and the RLO described in tropical fish species such as Tilapia. This relationship is surprising, if only because of the differences in growth temperature. Thus, the moment of diversification of the newly identified pathogen and the Tilapia RLO must be early in evolution.

The novel bacterium can be discriminated from the RLO as found in Tilapia on the basis of its 16S rRNA and its 23S rRNA. The 16S rRNA is depicted in SEQ ID NO: 1 and the 23S rRNA is depicted in SEQ ID NO: 3. It turned out that the 16S rRNA of the novel bacterium according to the invention and the Tilapia RLO have a 99% sequence identity, whereas the respective 23S rRNA's have a 96% sequence identity

A sequence comparison of the 16S, spacer and 23S rRNA's of the novel bacterium according to the invention and the Tilapia RLO is presented in FIG. 1. This comparison shows the differences in sequence and therefore allows to develop primers that are specific for the novel bacterium found in cod.

The Tilapia RLO has been described i.a. by Chern, R. S. and Chao, C. B. in Fish Pathology 29: 61-71, (1994).

The novel bacterium can i.a. be characterized on the basis of its 16S rRNA or on the basis of its 23S rRNA, and finally it can be characterized by the fact that it specifically reacts with a unique set of primers, as will be explained below.

SEQ ID NO 1 shows a typical example of the nucleotide sequence of by far most of the 16S rRNA gene of a bacterium according to the invention. Natural variations leading to minor changes in the 16S rRNA sequence (or spacer sequence or 23S rRNA sequence) are however found.

It is therefore considered that a rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, of which the nucleotide sequence of the region of the 16S rRNA gene corresponding to the 16S rRNA gene as depicted in SEQ ID NO 1 has a level of identity of at least 99.1%, preferably 99.2%, more preferably 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% in increasing order of preference and most preferably 100% with the nucleotide sequence as depicted in SEQ ID NO 1, belongs to the novel bacterium according to the invention.

Thus, a first embodiment of the invention relates to a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, of which the nucleotide sequence of the region of the 16S rRNA gene corresponding to the 16S rRNA gene as depicted in SEQ ID NO 1 has a level of identity of at least 99.1%, preferably 99.2%, more preferably 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% in increasing order of preference and most preferably 100% to the nucleotide sequence as depicted in SEQ ID NO 1.

With a level of identity is of course meant the level of identity of the sequence of SEQ ID NO 1 and the corresponding region of the 16S rRNA gene of the bacterium of which the level of identity has to be determined.

Another, alternative way to characterize a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium according to the invention relates to the sequence of the 23S rRNA of the bacterium.

SEQ ID NO 3 shows a typical example of the nucleotide sequence of the 23S rRNA gene of a bacterium according to the invention. Natural variations leading to minor changes in the 23S rRNA sequence are however found.

It is therefore considered that a rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, of which the nucleotide sequence of the region of the 23S rRNA gene corresponding to the 23S rRNA gene as depicted in SEQ ID NO 3 has a level of identity of at least 96.0%, preferably 96.5%, more preferably 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.2%, 99.4%, 99.6%, 99.8%, 99.9% in increasing order of preference and most preferably 100% identical to the nucleotide sequence as depicted in SEQ ID NO 3, belongs to the novel bacterium according to the invention.

Thus, another form of this first embodiment of the invention relates to a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, of which the nucleotide sequence of the region of the 23S rRNA gene corresponding to the 23S rRNA gene as depicted in SEQ ID NO 3 has a level of identity of at least at least 96.0%, preferably 96.5%, more preferably 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.2%, 99.4%, 99.6%, 99.8%, 99.9% in increasing order of preference and most preferably 100% to the nucleotide sequence as depicted in SEQ ID NO 3.

Still another, alternative, way to characterize the novel rod-shaped pleiomorphic non-motile Gram-negative bacterium according to the invention depends on a PCR-test using primer sets that are specific for the 16S rRNA gene sequence of bacteria according to the invention. These primer sets, of which the sequence is depicted in SEQ ID NO 4-7, were selected for their specific selectivity for the novel bacterium. They specifically react with the 16S rRNA gene of the novel bacterium but not with that of closely related Tilapia RLO that does not belong to the bacterium according to the invention. The test, which is described in more detail in the Examples section, is a standard PCR test.

It is therefore considered that a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, of which the 16S rRNA gene reacts in a PCR reaction with primers as depicted in SEQ ID NO.: 4 (CSF-1) or SEQ ID NO.:5 (CSF-2) on the one hand, and SEQ ID NO.: 6 (CSR-1) or SEQ ID NO.: 7 (CSR-2) on the other hand, to give a PCR product of 567+/-10 base pairs (CSF1+CSR1), 523+/-10 base pairs (CSF2+CSR1), 283+/-10 base pairs (CSF1+CSR2) or 239+/-10 base pairs (CSF2+CSR2) is considered to belong to the novel bacterium of the invention.

Thus, again another form of the first embodiment also relates to a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, of which the 16S rRNA gene reacts in a PCR reaction with a primer as depicted in SEQ ID NO 4 or 5, and with a primer as depicted in SEQ ID NO 6 or 7 to give a PCR product of 567+/-10 base pairs (CSF1+CSR1), 523+/-10 base pairs (CSF2+CSR1), 283+/-10 base pairs (CSF1+CSR2) or 239+/-10 base pairs (CSF2+CSR2).

A preferred form of this embodiment relates to a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, of which the nucleotide sequence of the region of the 16S rRNA gene corresponding to the 16S rRNA gene as depicted in SEQ ID NO 1 has a level of identity of at least 99.1%, preferably 99.2%, more preferably 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% in increasing order of preference and most preferably 100% to the nucleotide sequence as depicted in SEQ ID NO 1 and of which the nucleotide sequence of the region of the 23S rRNA gene corresponding to the 23S rRNA gene as depicted in SEQ ID NO 3 has a level of identity of at least at least 96.0%, preferably 96.5%, more preferably 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.2%, 99.4%, 99.6%, 99.8%, 99.9% in increasing order of preference and most preferably 100% to the nucleotide sequence as depicted in SEQ ID NO 3.

A more preferred form of this embodiment relates to a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, of which the nucleotide sequence of the region of the 16S rRNA gene corresponding to the 16S rRNA gene as depicted in SEQ ID NO 1 has a level of identity of at least 99.1%, preferably 99.2%, more preferably 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% in increasing order of preference and most preferably 100% to the nucleotide sequence as depicted in SEQ ID NO 1 and of which the nucleotide sequence of the region of the 23S rRNA gene corresponding to the 23S rRNA gene as depicted in SEQ ID NO 3 has a level of identity of at least at least 96.0%, preferably 96.5%, more preferably 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.2%, 99.4%, 99.6%, 99.8%, 99.9% in increasing order of preference and most preferably 100% to the nucleotide sequence as depicted in SEQ ID NO 3 and of which the 16S rRNA gene reacts in a PCR reaction with a primer as depicted in SEQ ID NO 4 or 5, and with a primer as depicted in SEQ ID NO 6 or 7 to give a PCR product of 567+/-10 base pairs (CSF1+CSR1), 523+/-10 base pairs (CSF2+CSR1), 283+/-10 base pairs (CSF1+CSR2) or 239+/-10 base pairs (CSF2+CSR2).

In an even more preferred form of this embodiment, the bacterium according to the invention is in an inactivated form, for reasons which will be explained below.

In another more preferred form of this embodiment, the bacterium according to the invention is in a live attenuated form, for reasons which will be explained below.

It is one of the merits of the present invention that the hitherto unknown causative agent of the disease has now been unambiguously determined, confirmed by Koch's postulates. Now that the cause of the disease has been found and could be demonstrated to be of bacterial origin, the disease could deliberately be induced and the typical signs of the disease described were seen as expected.

Up till now, the disease is seen in cod. Surprisingly however, it was found that the disease can also be induced in salmon.

After intraperitoneal infection of Atlantic salmon these fish die without any clear clinical signs of disease. Microscopy of the blood rich organs spleen, kidney and heart reveals much less bacteria than observed for cod in the acute phase of infection.

In co-habitants the development of the disease was different from that observed in fish challenged by an intraperitoneal injection. Some of the co-habitants survived for up to 4 months developing more and more signs of disease before dying with distinct pathological changes in the pseudobranchs, gills, kidneys and spleen.

These results demonstrate the ability of this pathogen to seriously affect the two most economical valuable species in the Norwegian fish farming industry.

It is probably only a matter of time before the disease will be demonstrated in other cultured aquatic species.

The newly discovered causative agent of the syndrome is now determined to be a novel rod-shaped pleiomorphic non-motile Gram-negative bacterium, as mentioned above. The novel bacterium is found both freely in/between the tissues and intracellular inside host cell, apparently in vacuoles. The novel bacterium is highly pleiomorphic. The coccoid stages are around 0.5-0.8 .mu.m (these are found when culturing the bacteria on blood agar), while the elongate stages (commonly found in fish and in cell cultures) are generally spoken 1-2 .mu.m long and 0.5-0.9 .mu.m wide.

An example of the novel bacteria has been deposited with the Collection Nationale de Cultures de Microorganisms (CNCM), Institut Pasteur, 25 Rue du Docteur Roux, F-75724 Paris Cedex 15, France, under accession number CNCM I-3511.

As can be seen in the dendrogram below, the novel bacterium according to the invention, the cause of Cod's Syndrome, as exemplified by the deposited strain forms a distinguished species, which is closely related to Francisella philomiragia and the Tilapia RLO described above.

The meaning of those numbers that are not explained in the dendrogram is as follows: AY 375394, AY 375395, AY 375396; Francisella endosymbiont of Dermacentor albipictus, AF001077; Francisella endosymbiont of Dermacentor andersoni, AY375402; Francisella endosymbiont of Dermacentor occidentalis, AY375405; Francisella endosymbiont of Dermacentor variabilis, AB001522: Ornithodoros moubata symbiote, AY375407: Francisella endosymbiont of Amblyomma maculatum, WLBRRBSA; Wolbachia persica -- see Original Patent.

Basically, a typical PCR-based test suitable for the discrimination between the novel bacterium according to the invention and other pathogens could be based upon the followin -- see Original Patent.

The primer sets to be used in the test are CSF1 or CSF2 as forward primer and CSR1 or CSR2 as backward primer (see below) -- see Original Patent.

A small portion of a colony, but at least 10 cells, preferably 10.sup.3 cell (volume about 1 .mu.l), is to be picked from a suitable agar plate (see Examples) and transferred to a tube containing puReTaq Ready-To-Go PCR beads dissolved in 23 .mu.l of double distilled water (Amersham Biosciences Cat no. 27-9558-01). Subsequently, 0.5 .mu.l of each of the primers BBF-1 with BBR-4 (10 .mu.l stock solution) is added. The sample is subsequently run using a thermal cycler with the following settings -- see Original Patent.

PCR-techniques are extensively described in text books such as Dieffenbach & Dreksler; PCR primers, a laboratory manual. ISBN 0-87969-447-5 (1995).

If analysis of the PCR-product reveals a PCR product of approximately 567 base pairs (using primers F1 and R1), approximately 523 base pairs (using primers F2 and R1), approximately 283 base pairs (using primers F1 and R2) or approximately 239 base pairs (using primers F2 and R2), this unequivocally demonstrates that the analysed bacterium belongs to the novel bacterium according to the invention. A PCR product of approximately 567 base pairs is a PCR product with a length of 567+/-10 base pairs, and thus with a length of between 557 and 587 base pairs.

In principle, the length of the PCR fragments is more likely to be the expected length+/-2 nucleotides, or even exactly the expected length. The +/-10 margin is mainly mentioned here because some variation in the region between the spacer regions may exist in variants of the novel bacterium according to the invention.

As is known to the skilled artisan, due to slight changes in salt concentration or temperature of the PCR reaction, non-specific PCR-fragments of the same length as indicated above may occur, if the test described above is performed with other bacteria not belonging to the novel species according to the invention. Therefore, the following should be take into account when using a PCR test to determine if a bacterium belongs to the novel bacterium according to the invention or not: the PCR test must include a positive control PCR reaction mix and a negative control PCR reaction mix. These reaction mixes differ only in one respect from the reaction mix of the bacterium to be tested: the negative control mix comprises a Tilapia RLO 16S rRNA gene and the positive control mix comprises a 16S rRNA gene of a bacterium according to the invention. If the positive reaction mix gives a PCR-product of the expected size, and the negative reaction mix gives no PCR product, it can be assumed that the PCR-conditions for the reaction mix of the bacterium to be tested are right.

As will be discussed in extenso in the Examples below, another of the merits of the present invention is, that a suitable growth medium for the bacterium according to the invention has now been found. Bacteria according to the invention can now be grown in vitro.

The skilled person finds in the Examples below a method for the isolation of the bacterium according to the invention from diseased fish, as well as for further growth of the bacterium on suitable medium.

Therefore, another embodiment of the present invention relates to a microbial culture comprising a bacterium according to the invention.

It is again one of the merits of the present invention that, the causative agent being known now, the development of vaccines became feasible. The strong immune response triggered in infected survivor fish (fish that survive a first infection), which leads to the induction of immunity against a second infection with the bacterium according to the invention is in itself already an indication that vaccination is feasible. The problem encountered in the natural course of the disease however is, that the onset of an adequate immune response usually is too slow. An adequate immune response, i.e. a response that suppresses the infection to at least a level that enables the fish to survive the infection, takes time to build up. Under natural conditions, this time is usually not available due to the very rapid progress of the disease: 90% of the infected fish die within days. Because the pathogen causing the disease has now been identified, a vaccine based upon this pathogen solves this problem because after vaccination, the immunological defense against the bacterium can build up before a natural infection strikes.

Thus, another embodiment of the present invention relates to a vaccine for combating the disease; Cod's Syndrome, as caused by the novel bacterium, wherein said vaccine comprises a bacterium according to the invention and a pharmaceutically acceptable carrier.

The vaccine according to the invention may comprise the bacteria in attenuated live or inactivated form. Attenuated live vaccines, i.e. vaccines comprising the bacterium according to the invention in a live attenuated form, have the advantage over inactivated vaccines that they best mimic the natural way of infection. In addition, their replicating abilities allow vaccination with low amounts of bacteria; their number will automatically increase until it reaches the trigger level of the immune system. From that moment on, the immune system will be triggered and will finally eliminate the bacteria. A minor disadvantage of the use of live attenuated bacteria however might be that inherently there is a certain level of virulence left. This need not be a real disadvantage as long as the level of virulence is acceptable, i.e. as long as the vaccine at least prevents the fish from dying. Of course, the lower the rest virulence of the live attenuated vaccine is, the less influence the vaccination has on weight gain during/after vaccination.

Therefore, one preferred form of this embodiment of the invention relates to a vaccine comprising a bacterium according to the invention in a live attenuated form.

A live attenuated bacterium is a bacterium that has a decreased level of virulence when compared to field strains. As mentioned above, the virulence of field strains of the novel bacterium according to the invention is very high: mortality typically exceeds 70% of all infected fish. A bacterium having a decreased level of virulence is considered a bacterium that only induces disease to the extent that mortality does not exceed 10%, and 90% of all infected fish survive the infection. Bacteria often behave attenuated as a result of a decreased growth rate. If such bacteria are used as the basis of an attenuated live vaccine, contrary to the situation described earlier, the immune system is triggered to the level necessary to suppress the disease before the fish die. As a result, the fish will not only survive but additionally, they build up immunity against future infections with a fully virulent field strain. Attenuated strains can e.g. be obtained by growing the bacteria according to the invention in the presence of a mutagenic agent. Many of such agents are known in the art and methods for the attenuation of bacteria using such agents have been known in the art for decades. Another way of obtaining mutated bacteria is to subject them to growth under temperatures exceeding the temperature of their natural habitat. Yet another way of mutating bacteria well-known in the art is transposon-mutagenesis.

Selection methods for slow-growing mutants or for temperature sensitive mutants (Ts-mutants) are also well-known in the art. Merely as an example: a suitable method for selection of slow-growing mutants simply relies on the plating of bacteria treated with a mutagen followed, after incubation, by visual selection of small colonies. Such colonies are slow-growing and thus they form the desired live attenuated bacteria. Selection for Ts-mutants is equally easy: replica-plating of bacteria treated with a mutagen followed by incubation at a sub-optimal (2-4 degrees below native growth temperature) or the optimal temperature, followed by visual selection of those colonies that did grow normal speed under sub-optimal temperature but did grow slower at the optimal temperature.

Inactivated vaccines are, in contrast to their live attenuated counterparts, inherently safe, because there is no rest virulence left. In spite of the fact that they usually comprise a somewhat higher dose of bacteria compared to live attenuated vaccines, they may e.g. be the preferred form of vaccine in fish that are suffering already from other diseases. Fish that are kept under sub-optimal conditions, such as incomplete nutrition or sub-optimal temperatures, would also benefit from inactivated vaccines.

Therefore, another preferred form of this embodiment relates to a vaccine comprising a bacterium according to the invention in an inactivated form.

Many physical and chemical methods of inactivation of bacteria are nowadays known in the art. Examples of physical inactivation are UW-radiation, X-ray radiation, gamma-radiation and heating. Examples of inactivating chemicals are .beta.-propiolactone, glutaraldehyde, binary ethylene-imine and formaldehyde. The skilled person would undoubtedly know how to apply these methods. Preferably the strain is inactivated with .beta.-propiolactone, glutaraldehyde, ethylene-imine or formaldehyde. Of these, .beta.-propiolactone and ethylene-imine are the most preferred. It is obvious that other ways of inactivating the bacteria are also embodied in the present invention. Vaccines comprise the bacterium according to the invention in an attenuated live and/or killed form, and in addition they comprise a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be as simple as e.g. distilled water or a physiological salt solution. It can also be e.g. a buffer solution.

Vaccines according to the present invention may in a preferred presentation also contain an immunostimulatory substance, a so-called adjuvant. Adjuvants in general comprise substances that boost the immune response of the host in a non-specific manner. A number of different adjuvants are known in the art. Examples of adjuvants frequently used in fish and shellfish farming are muramyldipeptides, lipopolysaccharides, several glucans and glycans and Carbopol.RTM. (a homopolymer). 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 (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.RTM., Marcol.RTM. and Drakeol.RTM.. 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 o/w 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.

The amount of adjuvant added depends on the nature of the adjuvant itself, and information with respect to such amounts will be provided by the manufacturer.

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

Preferably, vaccines according to the invention are stored/presented in a freeze-dried form.

In addition, the vaccines 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 are also embodied in the present invention.

Vaccines based upon inactivated bacteria can in principle be administered in doses between 10.sup.3 and 10.sup.9 CFU bacteria. Doses below 10.sup.3 bacteria might, depending i.a. on the method of inactivation, not be sufficiently immunogenic, and doses that exceed 10.sup.9 bacteria would from a commercial point of view not be very attractive. Although suitable amounts would range between 10.sup.3 and 10.sup.9 CFU bacteria, amounts between 10.sup.5 and 10.sup.8 CFU are preferred amounts.

Vaccines based upon live attenuated bacteria can in principle be administered in lower doses, because the bacteria multiply themselves during the infection. Therefore, although suitable amounts would range between 10.sup.3 and 10.sup.9 CFU bacteria, amounts between 10.sup.3 and 10.sup.6 CFU are preferred amounts.

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. Injection is more labor-intensive, and is primarily applied for the administration of inactivated vaccines. Administration by immersion, dipping or per oral is the most preferred way of administration for live attenuated vaccines because it is quick and allows mass application, and is very suitable for very young/small fish, for which injection is not practical.

The administration protocol can be optimized in accordance with standard vaccination practice. Preferably the vaccine is administered via immersion or per oral, especially in case of the use of vaccines in commercial aquaculture farms. For oral administration the vaccine is preferably mixed with a suitable carrier for oral application i.e. cellulose, food or a metabolisable substance such as alpha-cellulose or different oils of vegetable or animals origin. Also an attractive way is administration of the vaccine to high concentrations of live-feed organisms, followed by feeding the live-feed organisms to the target animal, e.g. 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. Suitable live-feed organisms include plankton-like non-selective filter feeders preferably members of Rotifera, Artemia, and the like. Highly preferred is the brine shrimp Artemia sp.

The administration protocol can be optimized in accordance with standard vaccination practice. A recent overview of fish vaccination, written by Bowden et al., (Fisheries Research Service Marine Laboratory, Aberdeen, Scotland) is available from www.intrafish.com.

The age of the fish to be vaccinated is not critical, although clearly one would want to vaccinate against Cod's Syndrome in an early stage. In principle, it would be tempting to vaccinate fish preferably at 0.2 grams, but certainly before 5 grams of weight. Fish having a weight of <0.5 grams however are assumed to be insufficiently immune competent. Therefore, in practice, one would vaccinate fish from 0.5 upwards. Since it is one of the merits of the present invention that it is now possible to perform early diagnosis of the bacterium and of the disease, control measurements such as sanitation can be developed in order to postpone outbreaks until fish have been vaccinated.

It would be beneficial to add to a vaccine, together with bacteria according to the invention, 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.

Examples of notorious commercially important fish pathogens are Vibrio anguillarum, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscose, Vibrio ordalii, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Piscirickettsia salmonis, SPD virus, SD virus, VNN virus, IPN virus and iridoviruses.

The advantage of such a combination vaccine is that it not only provides protection against Cod's Syndrome, 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.

Thus, in a more preferred form of this embodiment, the other microorganism or virus is selected from the following group of notorious commercially important fish pathogens: Vibrio anguillarum, Aeromonas salmonicidae, Vibrio salmonicidae, Moritella viscose, Vibrio ordalii, Flavobacterium sp., Flexibacter sp., Streptococcus sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Piscirickettsia salmonis, SPD virus, SD virus, VNN virus, IPN virus and iridoviruses.

All vaccines described above contribute to active vaccination, i.e. they trigger the host's defense system.

Alternatively, antibodies can be raised against the bacterium according to the invention in e.g. rabbits or can be obtained from antibody-producing cell lines as described below. Such antibodies can then be administered to the fish. This method of vaccination, passive vaccination, is the vaccination of choice when an animal is already infected, and there is no time to allow the natural immune response to be triggered. It is also the preferred method for vaccinating animals that are prone to sudden high infection pressure. The administered antibodies reactive with the bacterium according to the invention can in these cases interfere with the bacterium according to the invention and thus suppress Cod's Syndrome. This approach has the advantage that it decreases or stops Cod's Syndrome development, independent of the fish' immune status.

Therefore, one other form of this embodiment of the invention relates to a vaccine for combating Cod's Syndrome that comprises antibodies reactive with bacteria according to the invention and a pharmaceutically acceptable carrier.

Still another embodiment of this invention relates to antibodies reactive with bacteria according to the invention.

Antibodies or antiserum against bacteria according to the invention can be obtained quickly and easily by vaccination of e.g. pigs, poultry or e.g. rabbits with inactivated bacteria according to the invention in e.g. a water-in-oil suspension followed, after about four weeks, by bleeding, centrifugation of the coagulated blood and decanting of the sera. Such methods of raising antibodies are well-known in the art for decades.

Another source of antibodies is the blood or serum of e.g. cod or salmon that have been naturally infected with bacteria according to the invention. Other methods for the preparation of antibodies, which may be polyclonal, monospecific or monoclonal (or derivatives thereof) are well-known in the art. If polyclonal antibodies are desired, techniques for producing and processing polyclonal sera are well-known in the art (e.g. Mayer and Walter, eds. Immunochemical Methods in Cell and Molecular Biology, Academic Press, London, 1987). Monoclonal antibodies, reactive against the novel bacterium according to the invention can be prepared by immunizing inbred mice by techniques also known in the art (Kohler and Milstein, Nature, 256, 495-497, 1975).

A vaccine can also be prepared using antibodies prepared from eggs of chickens that have been vaccinated with a vaccine according to the invention (IgY antibodies).

Preferably a vaccine for oral administration of the antibodies is prepared, in which the antibodies are mixed with an edible carrier such as fish food.

Still another embodiment relates to a method for the preparation of a vaccine according to the invention that comprises the admixing of a bacterium according to the invention and a pharmaceutically acceptable carrier.

Still another embodiment relates to a method for the preparation of a vaccine according to the invention that comprises the admixing of antibodies reactive with a bacterium according to the invention and a pharmaceutically acceptable carrier.

Again another embodiment of the present invention relates to bacteria according to the invention for use in a vaccine.

Still another embodiment of the present invention relates to the use of a bacterium according to the invention for the manufacture of a vaccine for combating Cod's Syndrome.

As mentioned above, lethality after bacterial infection can easily be up to 70-90% and can even reach 100%. In addition to this, disease strikes at a dramatically high speed. Thus, for efficient protection against disease, a quick and correct diagnosis of Cod's Syndrome is important.

Therefore it is another objective of this invention to provide diagnostic tools suitable for the detection of Cod's Syndrome.

A diagnostic test kit based upon the detection of a bacterium according to the invention or antigenic material of that bacterium and therefore suitable for the detection of bacterial infection may i.a. comprise a standard ELISA test. In one example of such a test the walls of the wells of an ELISA plate are coated with antibodies directed against the bacterium. After incubation with the material to be tested, labeled antibodies reactive with the bacterium are added to the wells. A color reaction then reveals the presence of antigenic material of the bacterium. Therefore, still another embodiment of the present invention relates to diagnostic test kits for the detection of a bacterium according to the invention or antigenic material of that bacterium. Such test kits comprise antibodies reactive with a bacterium according to the invention or antigenic material thereof. Antigenic material of the bacterium is to be interpreted in a broad sense. It can be e.g. the bacterium in a disintegrated form, or bacterial envelope material comprising bacterial outer membrane proteins, just to name a few. As long as the material of the bacterium reacts with antiserum raised against the bacterium, the material is considered to be antigenic material.

A diagnostic test kit based upon the detection in serum of antibodies reactive with the bacterium according to the invention and therefore suitable for the detection of Cod's Syndrome may also i.a. comprise a standard ELISA test. In such a test the walls of the wells of an ELISA plate can e.g. be coated with the bacterium according to the invention or antigenic material thereof. After incubation with the material to be tested, labeled antibodies reactive with the bacterium according to the invention are added to the wells. A lack of color reaction then reveals the presence of antibodies reactive with he bacterium according to the invention.

Therefore, still another embodiment of the present invention relates to diagnostic test kits for the detection of antibodies reactive with the bacterium. Such test kits comprise the bacterium according to the invention, or antigenic material thereof.

The design of the immunoassay may vary. For example, the immunoassay may be based upon competition or direct reaction. Furthermore, protocols may use solid supports or may use cellular material. The detection of the antibody-antigen complex may involve the use of labeled antibodies; the labels may be, for example, enzymes, fluorescent-, chemoluminescent-, radio-active- or dye molecules.

Suitable methods for the detection of antibodies reactive with a bacterium according to the present invention in the sample include the enzyme-linked immunosorbent assay (ELISA), immunofluorescense test (IFT) and Western blot analysis.

A very quick and easy diagnostic test for diagnosing the presence or absence of a bacterium according to the invention is a PCR test as described above, comprising PCR-primer CSF1 or CSF2 and primer CSR1 or CSR2 as depicted in SEQ ID NO 4-7.

It goes without saying, that more primers can be used than the four primers identified above. The present invention provides for the first time the unique sequence of the 16S rRNA, the Spacer rRNA and the 23S rRNA gene of Cod's Syndrome strains. This allows the skilled person to select without any additional efforts, other selective primers in addition to the four primers shown there. By simple computer-analysis of the rRNA gene sequences provided by the present invention with the, known, rRNA gene sequences of other bacteria, the skilled person is able to develop other specific PCR-primers for diagnostic tests for the detection of a Cod's Syndrome strain and/or the discrimination between Cod's Syndrome strain and other bacterial (fish) pathogens.

PCR-primers that specifically react with the 16S rRNA, the Spacer rRNA or the 23S rRNA gene of Cod's Syndrome strains are understood to be those primers that react only with the 16S rRNA, the Spacer rRNA or the 23S rRNA gene of Cod's Syndrome strains and not with the 16S rRNA, the Spacer rRNA or the 23S rRNA gene of another (fish) pathogenic bacterium, or group of (fish) pathogenic bacteria.

Thus, another embodiment relates to a diagnostic test kit for the detection of a bacterium according to the invention, which test has as a characteristic feature that it comprises PCR-primers that specifically react with the 16S rRNA, the spacer rRNA or the 23S rRNA gene of Cod's Syndrome strains.

A preferred form of this embodiment relates to test kits comprising the specific PCR-primers CSF1, SCF2, CSF3 and CSF4 as depicted in SEQ ID NO 4-7.
 

Claim 1 of 10 Claims

1. An isolated rod-shaped pleiomorphic non-motile Gram-negative bacterium causing Cod's Syndrome in fish, wherein a 16S rRNA gene in said bacteria comprises the nucleic acid sequence of SEQ ID NO: 1.
 

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