WO2008074783A1

WO2008074783A1 - Immune stimulant against fish pathogenic bacteria

Info

Publication number: WO2008074783A1
Application number: PCT/EP2007/064082
Authority: WO
Inventors: Luc Grisez; Laura Labrie
Original assignee: Intervet International BV
Current assignee: Intervet International BV
Priority date: 2006-12-18
Filing date: 2007-12-17
Publication date: 2008-06-26
Anticipated expiration: 2009-06-18
Also published as: TW200843789A; CL2007003660A1

Abstract

The present invention relates to the use of a bacterium of the genus Nocardia for the manufacture of a vaccine for combating non-Nocardial bacterial infection in fish.

Description

IMMUNE STIMULANT AGAINST FISH PATHOGENIC BACTERIA

Over the last decades, world- wide a strong increase is seen in the consumption of fish. This equally regards the consumption of cold water fish such as salmon, turbot, halibut and cod, and tropical fish such as Asian sea bass, tilapia, milkfish, yellowtail, amberjack, grouper and cobia. As a consequence, an increase has been seen in the number and the size of fish farms, in order to meet the increasing needs of the market.

As is known from e.g. animal husbandry, large numbers of animals living closely together are vulnerable to all kinds of diseases, even diseases hardly known or seen, or even unknown, before the days of large-scale commercial farming. This is equally the case in fish farming. Bacteria found to be pathogenic to fish belong i.a. to the genus Vibrio, Photobacterium damselae, Tenacibaculum, Flavobacterium, Flexibacter, Cytophaga, Streptococcus, Lactococcus or Edwardsiella.

Examples of notorious commercially important fish pathogens are the recently found bacterium causing Big Belly syndrome, as described in Thai Patent Application TH 92840, (An example of this novel bacteria (BB E3F1) 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 1-3257), Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium columnare, Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae, Lactococcus garviae, Edwardsiella tarda and Edwardsiella ictaluri. These bacteria are pathogenic to at least one of the following fish species: yellowtail, amberjack, red sea bream, Asian sea bass, grouper, snapper, tilapia, catfish and carp.

Against some of these pathogens, vaccines are available, whereas against others there are no vaccines. In such cases, treatment with antibiotics is the only treatment against infection. By far most of the antibiotic 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 vaccines against fish- pathogenic bacteria. Moreover, it would be even more desirable to have a multivalent vaccine that is capable of protecting against several bacterial species.

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

It was surprisingly found now, that bacteria of the genus Nocardia are capable of providing a significant level of protection against non-Nocardial fish-pathogenic Gram-negative bacteria in fish.

This protection against non-Nocardial fish-pathogenic bacteria in fish is even induced by vaccines comprising Nocardia bacteria in the absence of non-Nocardial fish-pathogenic bacteria.

Moreover, this protection is conferred by Nocardia bacteria when given as a bacterin and/or when given in a live attenuated form.

An attractive aspect is the fact that this protection is also obtained if the bacteria are administered orally or by means of immersion vaccination.

Even more surprising, a significant level of protection is even obtained if such an immersion vaccine comprises Nocardia in an inactivated form. In general, immersion vaccination with inactivated bacteria is significantly less effective compared to immersion vaccination with live attenuated bacteria.

The effects of Nocardia as an adjuvant, i.e. as a general but non-specific stimulator of the immune system as such have been described in the art, i.a. NL 7306964, DT 2713-680, BE- 861-782. However, first of all, this has only be described in mammals. In fish, this phenomenon has to our knowledge never been described, most likely because the immune system of mammals and fish are very different systems.

Secondly, even in mammals, for the purpose of protecting against a specific antigen e.g. a bacterium, such general but non-specific stimulation induced by Nocardial components is only useful in combination with that specific antigen. In that respect Nocardial components are merely another form of the classical adjuvant.

Merely as an example, when Nocardial components and a vaccine against a bacterial disease are administered together, the Nocardial components could be expected to improve the immune response to the vaccine. The Nocardial components could of course not be expected to protect against the bacterial disease in the absence of the vaccine, i.e. to replace the vaccine.

The working mechanism behind the unexpected finding that bacteria of the genus Nocardia are capable of providing a significant level of protection against non-Nocardial fish- pathogenic bacteria is currently unknown. It is assumed however that a component present on or attached to the cell surface, and common to all Nocardia bacteria is a powerful stimulator of cross-specific immunity in fish against other bacteria. Cross-specific immunity in fish is the most significant against Gram-negative bacteria, such as Vibrio, Photobacterium damselae, Tenacibaculum, Flavobacterium, Flexibacter or Edwardsiella. Cross-specific in this respect means: induced by Nocardia and providing protection against non-Nocardial bacterial species.

Thus, the invention relates to the use of a bacterium of the genus Nocardia for the manufacture of a vaccine for combating non-Nocardial Gram-negative bacterial infection in fish.

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 non-Nocardial fish-pathogenic bacteria 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).

Live attenuated bacteria are very suitable, because they by definition carry the factor stimulating the cross-specific immunity against non-Nocardial fish-pathogenic bacteria. 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 a bacterium of the genus Nocardia according to the invention wherein the Nocardia is a live attenuated Nocardia bacterium.

On the other hand, the factor stimulating the cross-specific immunity against non-Nocardial fish-pathogenic bacteria 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 an equally preferred form, the invention relates to the use of a bacterium of the genus Nocardia according to the invention wherein the Nocardia is in the form of a bacterin.

There are several species of the genus Nocardia known in the art, e.g.: N. asteroides, N salmonicida, N. crassostreae, N. brasiliensis, N. rhodocrans, N. opaca, N. rubra and N. seriolae (formerly known as N. kampachϊ).

In a preferred form, the Nocardia species for use according to the invention is Nocardia seriolae.

Preferably the vaccine is manufactured for combating non-Nocardial bacterial infection in fish wherein the bacterium causing said non-Nocardial bacterial infection is a bacterium causing Big Belly syndrome or a fish-pathogenic bacterium of the genus Vibrio, Photobacterium damselae, Tenacibaculum, Flavobacterium, Flexibacter or Edwardsiella.

More preferably the vaccine is manufactured for combating non-Nocardial bacterial infection in fish wherein the bacterium causing said non-Nocardial bacterial infection is a bacterium causing Big Belly syndrome, Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium columnare, Flexibacter maritimus, Edwardsiella tarda or Edwardsiella ictaluri.

Most preferably the vaccine is manufactured for combating non-Nocardial bacterial infection in fish wherein the bacterium causing said non-Nocardial bacterial infection is a bacterium causing Big Belly syndrome or Flavobacterium columnare.

Vaccines manufactured 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, inJ. 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 manufactured 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 Nocardia cells administered will depend on the route of administration, the presence of an adjuvant and the moment of administration.

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 manufactured according to the invention that are based upon bacterins can be administered by injection 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. For the amount of bacteria in a vaccine manufactured according to the invention and for oral application, the examples below will provide ample guidance.

In the case of Nocardia it is however difficult to accurately determine the number of Nocardia cells in a volume by total cell count, due to the branching character of the cells. Therefore, as will also show from the Examples, the antigen concentration will be given in ODU/ml. The ODU/ml is determined as follows: Antigen Concentration (ODU/ml) = (((OD₆₆O)-I + (OD₆₆₀).2)/2)-0.2118)/0.0018 * DF * 10⁶, wherein (OD₆₆₀).l + (OD₆₆₀).2 are the OD₆6o values of two OD₆6o measurements and wherein DF is the dilution factor.

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

Often, the vaccine is mixed with stabilisers, e.g. to protect degradation-prone proteins from being degraded, to enhance the she If- 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 Nocardia vaccine for use according to the invention are preferably administered to the fish via injection, immersion, dipping or per oral.

Merely as an example, if the Nocardia bacterium now used for the manufacture of a vaccine for combating non-Nocardial bacterial infection in fish 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 Nocardial bacterium now used for the manufacture of a vaccine for combating non-Nocardial bacterial infection in fish is in the form of a bacterin, oral application and e.g. intraperitoneal application are attractive ways of administration. Especially in the case of intraperitoneal application, the presence of an adjuvant would be preferred.

Generally spoken: if the vaccine can be improved by adding 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.

Immersion vaccination, even in the absence of an adjuvant, and using inactivated bacteria has turned out to provide a surprising level of immunity against non-Nocardial fish-pathogenic Gram-negative bacteria.

This route of administration is preferred for its ease of administration of the vaccine.

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 non-Nocardial fish-pathogenic bacteria in as early a stage as possible, i.e. prior to possible exposure to the pathogen.

For the vaccines based upon the Nocardia bacteria described above, generally spoken these would be administered at the moment they would normally be administered in order to protect against infection with the non-Nocardia Gram-negative bacteria. In practice, this means that the fish would be vaccinated in as early a stage as possible.

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, if necessary or desired, 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 Nocardia bacterium for use according to the invention is of the species seriolae.

It would be beneficial to use, in addition to Nocardia bacteria, also at least one non-Nocardial fish-pathogenic microorganism or virus, an antigen of such microorganism or virus or genetic material encoding such an antigen, for the manufacture of the vaccine.

The combined use of Nocardia bacteria and at least one non-Nocardial fish-pathogenic bacterium for the manufacture of the vaccine would have the benefit that the specific protection that builds up against said non-Nocardial fish-pathogenic bacterium, lasts longer.

The combined use of Nocardia bacteria and at least one fish-pathogenic virus for the manufacture of the vaccine would have the benefit that protection is obtained against both bacterial infection and infection with said fish-pathogenic virus.

Therefore, a preferred form of this embodiment relates to the use of Nocardia bacteria and at least one non-Nocardial fish-pathogenic microorganism or a fish-pathogenic virus, an antigen of such microorganism or virus or genetic material encoding such an antigen, for the manufacture of the vaccine.

Examples of commercially important fish pathogens in tropical and/or Mediterranean fish are Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae, Viral Haemorrhagic Septicaemia virus, Viral Necrosis virus, iridovirus and Koi Herpesvirus.

Thus, in a more preferred form of this embodiment, the other microorganism or virus is selected from the following group of fish pathogens: Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae, Viral Haemorrhagic Septicaemia virus, Viral Necrosis virus, iridovirus, Spring viremia of Carp and Koi Herpesvirus.

The Examples given below provide broad guidance of how to use bacteria of the genus Nocardia for the manufacture of a vaccine for combating non-Nocardial bacterial infection in fish.

Examples.

Example 1:

Big Belly infection (also referred to as BB infection, and see reference above), caused by the species tested is one of the major infections in juvenile Asian sea bass farming. Outbreaks of BB- disease occur predominantly at early stages of the culture process.

BB-culture

A culture of BB-bacteria grown on Chocolate agar and harvested in tryptone/yeast broth was inactivated with 0.5% formalin and the OD₆6o was measured to be 0.5. No total cell count or other means of quantifying the cells was performed mainly because of the strongly pleomorphic nature of the cells in question.

N. seriolae culture

As the immune stimulating antigen, formalin inactivated whole cell fermentor produced N. seriolae at 9.1 x 10⁸ ODU/ml was used.

The N. seriolae antigen used is derived from a field strain of N. seriolae. The antigen concentration of the N. seriolae antigen was determined as follows:

Antigen Concentration (ODU/ml) = (((OD₆₆₀).! + (OD₆₆₀).2)/2)-0.2118)/0.0018 * DF * 10⁶ wherein (OD₆6o)- 1 + (OD₆6o)-2 are different batches of the same N. seriolae bacteria, and DF is the dilution factor.

The antigenic strength of this batch was 9.1 x 10⁸ ODU/ml.

The N. seriolae is further also referred to as the Immune Stimulant (IS).

The BB-culture and the Nocardia-cxύture are termed "antigen" in the following.

Animals used

Asian sea bass (Lates calcarifer) having an average weight at the start of the experiment of 0.5 g. The fish were kept in full strength sea water of 26 ± 2°C with a density of maximum 20 kg/m fish tank volume.

Three groups of 110 fish were collected as they came at hand from the quarantine tanks and assigned to the respective groups sequentially. Fish were immersed and placed in the allocated tank-half. Fish were not marked individually.

Immersion treatment

The fish were starved for 24 h prior to vaccination. Groups of 110 fish were caught and transferred to the respective treatments. The treatments given, the tank allocation and designation of treatments is given in Table 1. Both antigen solutions were diluted approximately 10 and 100 fold for the BB-antigen and Nocardia respectively.

Fish were immersed for 15 minutes in these final suspensions during which period pH, O₂, and temperature were monitored. After immersion the fish were transferred to their designated tanks as indicated in Table 1.

Table 1 : Treatments and tank allocations used

Preparation of challenge culture A BB-strain was sub-cultured on Chocolate agar and incubated at 26°C overnight. Subsequently, the growth was collected in 0.22 μm sterile filtered sea water. The resulting suspension was brought to an OD660 of 1.573 and this suspension was used as highest challenge suspension. In addition 4 more ten- fold dilutions were made from this initial suspension in sterile seawater which were all used to inject fish.

Challenge

At 3 weeks post immersion treatment groups of 10 to 12 fish per condition (BB; Nocardia or control) were netted, anaesthetized in Aqui-S and injected intra-peritoneal with 0.1 ml of the prepared dilutions. Immediately after injection, fish were transferred to their designated tank-part and the recovery from the anaesthesia was followed.

Per challenge dilution, all three groups were placed in the same tank, i.e. a group of 10 BB- immersed fish, a group of 10 Nocαrafø-immersed fish and a group of 10 or 12 control fish, all injected with the same challenge suspension were placed in the same tank in which the three groups were kept separate by means of vertical separation screens placed in the tanks.

A total of 5 challenge concentrations were used ranging from an un-diluted suspension (termed "neat") to a 10,000 fold diluted neat suspension.

Observations of mortality Fish were observed daily over a 3 week period and dead or obviously moribund fish were collected at least once a day. A representative number of fish was examined for the occurrence of typical Big-Belly disease signs and Giemsa-staining from the internal organs - which typically are lumped together in a BB-infected fish - was performed to confirm the causative organism. At the end of the 21 day observation period, all remaining fish were collected and evaluated for the occurrence of typical BB-disease signs. If present these fish were included as "positive for BB" in the evaluation of the results.

RESULTS Interpretation of results The mortality in the two treatment groups (BB and Nocardia) as compared to the controls was evaluated by calculating the relative percent survival (RPS), according to the following formula:

f % mortality in vaccinated ]

RPS = \ 1 - ( ) \ x 100 I % mortality in controls J Mortality after challenge

The observed mortality per day in the different treatment groups and in the control groups per challenge dose is presented in Figure 1 (5 sequential figures).

The sequential graphs unambiguously show a clear relation between challenge dose and occurrence of first mortality. In addition, the higher doses applied yield 100% mortality whereas in the lower doses, at least in some groups some fish survive the challenge. A third observation from these graphs is that in most cases the BB-immersed group displays the highest peak mortality and this peak occurs in general earlier in the BB-group then in the other groups. All dead fish clearly displayed typical Big Belly disease signs and the occurrence of BB-bacteria was apparent in all fish sampled.

Efficacy evaluation

Based on the mortality figures obtained, the RPS values were calculated per condition. The results are expressed in Figure 2

The results show that protection was obtained in the two lowest challenge concentrations applied where cumulative control mortality ranged from 58.3-83.3%. (cumulative control mortality in the 3 highest doses resulted in 100% mortality in the controls). Furthermore, the BB-treated groups show negative RPS values indicating that the treatment with BB-antigen through immersion had a negative effect on subsequent challenge.

The immersion with Nocardia seriolae antigen unambiguously induced protection against BB challenge. This is remarkable since the immune stimulation was performed three weeks prior to the challenge and since the stimulation was performed through immersion, moreover with inactivated Nocardia seriolae, and the challenge was performed through injection resulting in a substantial mortality in the controls.

Example 2: "Columnaris disease" caused by F. columnare is a major diseases causing mortality in most cultured freshwater fish species. Tilapia is a highly susceptible species and was used as the target species in this Example.

As the non-specific immune stimulator (also referred to as the immune stimulant), formalin inactivated whole cell fermentor produced N. seriolae at 9x10⁸ ODU/ml was used. Challenge strains

F. columnare field isolate isolated from channel catfish, Mississippi USA. F. columnare field isolate isolated from Tilapia, Indonesia.

Table 2: Immune stimulant concentration, number of fish used, application time and distribution after immune stimulation.

The experiments were done in Tilapia (Oreochromis sp.) having an average weight of approximately 5 g at the start of the experiment.

Water conditions during the experiment

^■ Salinity: 4-6ppt after immune stimulation*/Oppt after challenge

^■ Temperature: 26°C +/- 3°C

^■ Tanks: 500L after immune stimulation/50L after challenge

* One day prior to the first challenge all tanks were changed to freshwater and kept in freshwater until all challenges were finalised.

Experimental design

Assignment of animals to treatment groups

A total of 240 fish were selected as they came at hand.

Immune stimulation schedule

Fish were starved for at least 24h before immune stimulation to assure complete emptying of the intestinal tract and thereby reducing the stress caused by the immersion procedure. The experiment included 3 groups (2 immune stimulant groups and 1 control group), each consisting of 80 fish. Therefore, 2 groups of 80 fish were immune stimulated through a 15 min immersion exposure and 80 control fish were mock exposed. All fish from the same immuno group were immersed in the immune stimulant bath for 15 min. The inactivated N. seriolae culture was diluted 1 : 100 prior to use. The total immune stimulation bath volume was 5L. Control fish were bathed in 5L of freshwater for the same duration. Immune stimulation was performed according to Table 2. Water quality (pH, temperature and DO) was recorded during the 15 min exposure time. Immediately after immersion, fish were transferred to their allocated tanks and the recovery was followed.

Preparation of challenge inoculum

F. columnare challenge strains

The F. columnare USA strain was reactivated by using 1 vial (ImI) of a <-50°C stock culture. 0.5ml of the vial was collected and a ten fold dilution was made in distilled water. 1% was inoculated into 100ml Cytophaga broth. The broth was incubated at 26°C for 22h. When an OD₆6o of 0.433 was obtained the culture was used to inoculate a larger volume of Cytophaga broth. The inoculum size was 1% of the culture volume. Cultures were incubated at 26°C for app. 17h, aeration was provided using a stirrer bar. An OD₆6o of 0.405 was obtained and the culture was used for challenge. The number of colony forming units in the challenge culture was determined by standard spread plating of 100 μl aliquots of ten- fold diluted bacterial suspensions on Cytophaga agar and subsequent incubation at 26°C for 24-48 hours.

The F. columnare Asian strain was reactivated by streaking a portion of a <-50°C stock culture on Cytophaga agar. Plates were incubated at 26⁰C for app. 24h. Growth was collected in DIH₂O until an OD₆6o of app. 0.18-0.20 was reached. The collected growth was subsequently diluted 10 fold in distilled water and inoculated into modified Cytophaga broth containing trace elements. The inoculum size was 1% of the culture volume. The broth was incubated at 26°C for 16-2Oh. Aeration was provided using a stirrer bar. An OD₆6o of approximately 0.350-0.395 was obtained. The number of colony forming units in the challenge culture was determined by standard spread plating of 100 μl aliquots of ten- fold diluted bacterial suspensions on Cytophaga agar and subsequent incubation at 26°C for 24-48 hours.

Challenge

Challenge was performed by immersion with both challenge strains at day 6 and day 1 post immune-stimulation. For the first challenge, 40 fish from each treated group were collected and divided into 2 groups of 20 fish each. These fish were either immersion challenged for 15 min (USA strain) or 30 min (Asian strain). There were 2 treated groups and one control group and therefore a total of 3x20 fish were used for immersion challenge with each challenge strain. The challenge suspension was 5L and 2L for USA strain and Asian strain respectively. For the repeat challenge on day 8 and 3 and on week 3 and 2 (immuno-1 and immuno-2 respectively) only the Asian strain was used and the same procedure was followed as before for this strain. The challenge was performed by transferring the fish from the treatment room to the challenge containers. After challenge, fish were transferred into the challenge tanks. An overview of the distribution of the different challenge groups is indicated in table 3.

Table 3 : Distribution of different challenge groups after challenge.

NA: not applicable, ND: not done

Observation after challenge

Fish were observed for 2-10 days after challenge and the occurrence of mortality was recorded. Dead fish were removed from the tanks and subjected to examination. The number of dead fish was noted per day.

External examination consisted of the indication of the presence of gill and/or skin lesions on a special recording sheet. From a representative number of fish the gill/skin lesions were sampled for bacteriological analysis and plated on Cytophaga agar. Plates were incubated for 24h-48h at 26°C and evaluated for the presence of typical F. columnare growth (flat, rhizoid, swarming, adherent, and yellow). It was concluded that the observed mortality was caused by F. columnare when typical gill/skin lesions were observed and/or this organism was re- isolated from the lesion.

Efficacy Efficacy of the immune stimulation was expressed as relative percent survival. The formula stated below was used.

RPS = (1- ^{% mOrtaUty VaCdnateS} X 100) [ % mortality controls J

RESULTS

Safety

No mortality was observed during or immediately after immune stimulation. No mortality was observed during the 3 weeks observation period.

Challenge concentrations

The concentrations of the challenge cultures used for preparation of the challenge baths as well as the effective challenge concentrations used for wk3, wk 3 repeat and wk 4 challenges are given in table 4. All challenge suspensions were pure when used.

Table 4: CFU determinations for challenge cultures and challenge baths used.

NA: not applicable, D: day, Wk: week

* time points for immuno-1 and immuno-2 groups respectively Mortality after challenge

No immediate (within 2 hours post immersion) mortality was observed after challenge. However the challenge model used induced a very quick onset of mortality. In all challenge concentrations first mortality could be observed 4-6 hours after challenge. Fish would present typical swollen, haemorrhaging gills and all fish would showed different degrees of gill necrosis. From about 6 hours onwards, typical skin and fin lesions could be seen. Mortality always started the day of challenge and peaked the next day. Thereafter very few to no mortality was seen. All fish that died showed typical disease signs and almost all of the re-isolations were positive (unless overgrown as gills were plated). Since all fish presented clinical signs all mortality was considered to be due to the challenge organism. The cumulative mortality caused by the F. columnare challenges at the different time points and with the different challenge strains (USA strain and Asian strain) are represented in figures 3 to 6.

Efficacy Both immuno groups showed significantly lower mortality than the control groups (2 tailed Fisher exact, p <0.05) at all challenge times over the whole trial period regardless of the challenge strain used. An RPS value of 100% was obtained at day 6 and day 1 post immune stimulation when fish were challenged with the US challenge strain (60% mortality in controls). RPS values of the different immuno groups in time challenged with the Asian strain is given in figure 7. When challenged with the Asian challenge strain, the RPS value was high for immuno- 1 (day 6 post stimulation, RPS = 80) and lower for immuno-2 (day 1 post stimulation, RPS = 25) with 100% mortality in the controls. However, both groups were significantly different than the control group (p<0.05). When challenge was performed on day 3 and day 8, RPS values were good in both groups (RPS = 60 with 85% mortality in the controls). The lower RPS value at day 1 with immuno-2 appears indicative of the early onset of the non specific immune stimulation. When challenge was performed at 2 and 3 weeks post immune stimulation, RPS values in both groups were good (RPS = 73.3 and 60.0 for immuno-1 (wk 3) and immuno-2 (wk 2) respectively.

CONCLUSION

Overall, excellent RPS values were obtained up to 3 weeks post stimulation in Tilapia challenged with F. columnare, using an inactivated Nocardia seriolae vaccine manufactured according to the invention, that was administered by immersion. Legend to the figures:

Figure 1 : Daily mortality per vaccine condition over time after BB challenge performed 3 weeks post stimulation using different challenge doses. BB vax: Big Belly vaccinated, Immuno-S: N. seriolae immune stimulated

Figure 2: RPS values per treatment condition as compared to the controls for each BB challenge concentration. Vacc: Big Belly vaccinated. IS: N. seriolae immune stimulated

Figure 3: % cumulative mortality for challenge time 1 (day 6 and day 1 post immune stimulation) induced by F. columnare Asian challenge strain (n=20).

Figure 4: % cumulative mortality for challenge time 2 (day 8 and day 3 post immune stimulation) induced by F. columnare Asian challenge strain (n=20).

Figure 5: % cumulative mortality for challenge time 3 (wk 3 and wk 2 post immune stimulation) induced by F. columnare Asian challenge strain (n=20).

Figure 6: % cumulative mortality for challenge time 1 (day 6 and day 1 post immune stimulation) induced by challenge F. columnare USA challenge strain (n=20).

Figure 7: RPS values obtained with the immune stimulant at the different challenge time points using the F. columnare Asian challenge strain (n=20).

Claims

Claims:

1) Use of a bacterium of the genus Nocardia for the manufacture of a vaccine for combating non-Nocardial Gram-negative bacterial infection in fish. 2) Use according to claim 1, characterized in that said bacterium of the genus Nocardia is a live attenuated bacterium

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

4) Use according to claims 1-3, characterized in that said bacterium of the genus Nocardia is of the species N. asteroides, N Salmonicida, N. crassostreae, N. brasiliensis, N rhodocrans, N opaca, N rubra or N. seriolae.

5) Use according to claim 4, characterized in that said bacterium of the genus Nocardia is of the species Nocardia seriolae.

6) Use according to claims 1-5, characterized in that the bacterium causing said non- Nocardial bacterial Gram-negative infection is a bacterium causing Big Belly syndrome or a fish-pathogenic bacterium of the genus Vibrio, Photobacterium damselae, Tenacibaculum, Flavobacterium, Flexibacter or Edwardsiella.

7) Use according to claim 6, characterized in that the bacterium causing said non- Nocardial bacterial infection is a bacterium causing Big Belly syndrome or is Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium columnare, Flexibacter maritimus, Edwardsiella tarda or Edwardsiella ictaluri.

8) Use according to claim 7, characterized in that the bacterium causing said non- Nocardial bacterial infection is a bacterium causing Big Belly syndrome or is Flavobacterium columnare.

9) Use according to claims 1-8, characterized in that for the manufacturing of said vaccine at least one other microorganism or virus is used that is pathogenic to fish, or one other antigen or genetic material encoding said other antigen is used, wherein said other antigen or genetic material is derived from a virus or microorganism pathogenic to fish.

10) Use according to claim 9, characterized in that said other microorganism or virus is selected from the group consisting of Vibrio anguillarum, Photobacterium damselae subspecies piscicida, Tenacibaculum maritimum, Flavobacterium sp., Flexibacter sp., Streptococcus iniae, Streptococcus difficile, Streptococcus agalactiae, Streptococcus dysgalactiae, Lactococcus garviae, Edwardsiella tarda, E. ictaluri, Viral

Haemorrhagic Septicaemia virus, Viral Necrosis virus, iridovirus, Spring viremia of Carp virus and Koi Herpesvirus