METHODS AND COMPOSITIONS TO TREAT AQUATIC ORGANISMS

Field of the Invention

The invention relates generally to the field of aquaculture, it provides methods and compositions for controlling and/or preventing diseases in culture of aquatic organisms, in particular in controlling and/or preventing disease in aquaculture of marine invertebrates such as crustaceans and bivalves or larvae of low vertebrates such as fish larvae More specifically, the invention provides the use of heat shock proteins in methods and compositions of controlling and/or preventing diseases in aquaculture of aquatic organisms, In one embodiment, the compositions relate to the enrichment of life food organisms including microalgae; rotifers, such as for example Brachionus; Artemia; zooplankton including copepods, nematodes, cladocerans and trochophora larvae; and other with heat shock proteins, or fragments thereof.

The compositions and methods of the present invention are particularly useful to enhance the resistance of organisms, which are at the moment of treatment not able to make antibodies, to opportunistic or obligate pathogenic micro- organisms. More specifically the invention describes the anti-infectious effect of feeding heat shock proteins (HSPs) , or fragments thereof, and in particular of the bacterial HSPs provided in Table 1 below.

Background to the Invention

Cultivation of (aquatic) animals in high density tends to

cause problem because of high microbial interference, leading to reduced but especially unpredictable survival values under industrial circumstances. Especially for animals that cannot be vaccinated, such as invertebrates and larval stages of vertebrates (hereafter these kind of animals are called Antibody Negative Animals or ANA' s : they comprise invertebrates, such as crustaceans and bivalves and certain early life stages of vertebrates, such as larval fish) , there is a need for a general prophylactic treatment that will increase their ability to deal with pathogens. Vaccination is the preferential technique in vertebrate organisms, but as stated before, this can not be applied on ANA's. In addition, a vaccine is directed towards a particular pathogen, which precludes its use for preventative treatment against uncharacterised pathogens.

Negative microbial interactions with animals that are unable to make antibodies can be controlled by a series of techniques . The overall hygiene can be controlled, keeping microbial numbers low. This common practice aims at reducing the possibility that pathogens or opportunistic pathogens are coming into contact with the target organisms. Yet keeping microbial densities low at all times is often an unrealistic goal .

Aquatic ANA's, such as marine invertebrates and marine vertebrate larvae, are very vulnerable to the presence of microbes in general. Therefore under industrial circumstances it is still common practice to use antibiotics. However, because of general public health concerns, the prophylactic use of antibiotics is strongly discouraged.

Alternative techniques that exclude the use of antibiotics are widely investigated now. The use of immunostimulants (such as, lipopolysaccharides, beta-glucans, nucleotides) and nonspecific immune enhancers, and the use of probiotics are strategies with a variable degree of success. Hence the cultivation of ANA' s requires additional strategies and/or compounds to deal with pathogenic micro-organisms.

The method in the present invention describes the phenomenon that increasing the concentration of heat shock proteins in the feed of ANA' s can enhance the resistance of ANA' s against opportunistic or obligate pathogenic microorganisms .

Heat shock proteins have previously shown to render proteins highly antigenic in mammals when they are bound to these proteins (either covalently or not) (US patent (5830464) and they have been shown to behave as adjuvants during vaccination to a particular pathogen. Heat-shock proteins (HSPs) are also expressed at high levels by bacterial pathogens during adaptation to intracellular survival or are expressed by the host upon an invasion by a pathogen. Both host and pathogen heat-shock proteins contribute to immunity in mammals by receptor-mediated activation of the innate immune response and by participation in the presentation of antigens for the adaptive immune response. "The mammalian immune system uses its own HSPs as a danger signal for stressed cells. Mammalian HspβO, Hsp70, Hsp90 and Gp96, released from damaged cells, have been proposed to interact with immune cells through a variety of cell-surface signalling receptors: the a-macroglobulin receptor CD91 with Hsp70, Hsp90 and Gp96; CD36 with Gp96 and, CD40, CD14 and

toll-like receptors (TLR) -2 and -4 with Hsp70. HspβO might also signal through CD14, TLR-2 and TLR-4 but probably binds to a different molecule. HSP signalling has been shown to induce the production of proinflammatory molecules such as the cytokines TNF (tumour necrosis factor) -α, IL-Ib, IL-12 and GM-CSF (granulocyte-macrophage colony-stimulating factor) , several chemokines and nitric oxide, and to stimulate the activation and maturation of dendritic cells (DCs) . Similarly, the HSPs that are produced by bacterial pathogens during infection have been reported to induce the production of a variety of cytokines and chemokines, and to influence maturation of immune cells" (Stewart and Young, 2004) . In invertebrates similar kind of receptors has been described but their interaction with HSPs is less well or not documented.

In this respect, it was recently shown that exposure to a non-lethal heat shock stress shielded larvae of a particular ANA (namely Artemia) against infection by pathogenic Vibrio. A combined hypothermic/hyperthermic shock followed by recovery at ambient temperature induced Hsp70 synthesis in Artemia larvae. Thermotolerance was also increased as was protection against infection by Vibrio campbellii, showing reduced mortality and lower bacterial load in challenge tests. The data suggest a causal link between Hsp70 accumulation induced by abiotic stress and enhanced resistance to infection by V. campbellii, perhaps via stimulation of the Artemia immune system. It was the first time that this increase in resistance through the effect of heat shock proteins could be demonstrated in a zootechnical test.

According to Singh and Aballay (2006) , exposure to increased temperature in the invertebrate Caenorhabd.itis elegans, results in the activation of a conserved pathway involving the heat-shock (HS) transcription factor (HSF)-I that enhances immunity. The HSF-I defense response requires a system of chaperones including small and 90-kDa inducible HS proteins. In addition, HSF-I is needed for the effects of the DAF-2 insulin-like pathway in defense to pathogens, indicating that interacting pathways control stress response, aging, and immunity. The results also show that HSF-I is required for C. elegans immunity against Pseudomonas aeruginosa, Salmonella enterica, Yersinia pestis, and Enterococcus faecalis.

Compared to the above, it has now surprisingly been found that feeding an ANA with a food composition supplemented with HSPs, and in particular the bacterial HSP provided in table 1 below, even more in particular HSP70 including its bacterial homologs, can protect this organism against a subsequent attack by a pathogenic organism. This is also surprising as HSPs delivered to the host via the gastrointestinal tract run the risk of being degraded (e.g. by peptidase such as trypsin) before it can reach the target.

It is accordingly an object of the present invention to provide a composition supplemented with heat shock proteins, or fragments thereof and its use in controlling and /or preventing disease in aquaculture of aquatic organisms, in particular in controlling and/or preventing disease in aquaculture of ANA's, such as crustaceans and bivalves or larvae of aquatic organisms, including fish larvae.

In a further aspect, the present invention provides a method of controlling and/or preventing diseases in aquatic organisms, comprising feeding the aquatic organisms a life food organisms enriched with heat shock proteins, or fragments thereof.

Summary of the Invention

This invention is based on the finding that feeding ANA' s with feed compositions enriched with heat shock proteins results in a prophylactic protection against opportunistic or obligate pathogens in ANA's.

In a first objective the present invention provides a composition supplemented with heat shock proteins, or fragments thereof for controlling and/or preventing diseases in aquatic organisms, in particular a feed composition. In one embodiment, the compositions are for use in controlling and/or preventing diseases in aquatic ANA' s such as crustaceans and bivalves or larvae of aquatic organisms, including fish larvae.

Feed compositions as used herein include, for example;

• Life food organisms including microalgea; nematodes; zooplankton including rotifers (such as for example

Brachionus) , Artemia, and copepods;

• Artificial diets such as flakes; pellets; sticks; granules; particulated feed; enrichment products such as dried unicellular algae, algal pastes, yeast, emulsified preparations, micro-particulated feed, compound diets, self-emulsifying concentrates; and other forms of artificial feed (diets) such as the

complete feed for fish larvae described in PCT publication WO0064273; • Formulated feed (protein) , such as for example protein cross-linked microcapsules (described in British Patents 79437454 and 2103568); particles consisting of a matrix material containing water- soluble nutrients encapsulated with phospholipids/biomembranes (described in US Patent 6,623,776); lipogel particles whereby a nutritional or pharmacologically active component is entrapped in liposomes and the liposomes are encapsulated in a hydrocolloid matrix (described in PCT patent Publication WO 87/01587); feed formulations wherein the nutritional or pharmacologically active component is encapsulated in coated particles that allow for colon specific hydrolysis (Yang L., Journal of Controlled release 125: 77-86 (2008)); lipid-wall capsules contained within complex, cross-linked protein microcapsules (described in US Patent 5,776,490); and other forms of formulated feed (diets) .

In one embodiment the feed compositions are enriched with purified heat shock proteins or fragments thereof, including recombinantly obtained HSPs, such as for example by mixing the protein directly with the feed or by supplementing the feed compositions with a formulated heat shock protein such as the formulated feed (protein) compositions mentioned hereinbefore, e.g. the HSP or fragments thereof (infra) is formulated in the cross- linked microcapsules; the lipogel particles; the coated heat shock protein; or the encapsulated liposomes

mentioned hereinbefore. In a particular embodiment the formulated heat shock protein consist of the HSPs or fragments thereof (infra) encapsulated in coated particles that allow for colon specific hydrolysis.

In an even further embodiment the composition consists essentially of life food organisms enriched with heat shock proteins or fragments thereof, such as for example by submersion in a suspension of said purified HSP or fragments thereof (infra) , or by submersion in a suspension of micro-organism such as yeast cells or bacterial cells that were previously exposed to a non- lethal heat shock. The organisms used in this embodiment of the present invention, particularly consist of micro-organisms such as yeast cells and bacteria, that have the GRAS (GRAS: generally regarded as safe) status, such as for example Saccharomyces cerevisiae, Lactobacillus acidophilus, Lactobacillus lactis, and Pediococcus acidilacticis .

In a further embodiment the composition comprises a therapeutically effective amount of a heat shock protein, or a fragment thereof, i.e. an amount sufficient to prevent and/or control diseases in aquatic organisms. In particular, an amount sufficient to enhance the resistance of aqueous organisms against opportunistic or obligate pathogenic micro-organisms, such as for example Gram negative bacteria, including but not limited to species of Vibrio, Aeromonas, Photobacterium, Yersinia, Edwarsiella, Renibacterium, Flavobacterium; Gram positive bacteria, including but not limited to species of Streptococcus, Lactococcus; viruses including but not limited to

baculoviridae; and parasites including but not limited to Ceratomyxa shasta, Ichthyophthirius multifillius, Cryptobia salmositica, Lepeophtheirus salmonis, Tetrahymena species, Trichodina species and Epistylus species.

In a second objective, the present invention provides the use of a composition as provided herein, in preventing and/or controlling disease in aquaculture of aqueous organisms, i.e. in enhancing the resistance of aqueous organisms against opportunistic or obligate pathogenic micro-organisms. In particular for the use of a composition as provided herein, in preventing and/or controlling disease in aquaculture of ANA's.

In a further objective, the present invention provides a method for controlling and/or preventing disease in aquaculture of aqueous organisms, in particular for controlling and/or preventing disease in aquaculture of crustaceans and bivalves or of larvae of aquatic organisms, including fish larvae. In a first embodiment the method of controlling and/or preventing disease in aquaculture, comprises feeding the aquatic organisms a composition supplemented with heat shock proteins, or fragments thereof. In a further embodiment, feeding the aquatic organisms with life food organisms enriched with HSPs either by enrichment with heat-shocked non-pathogenic micro-organisms or by enrichment with the HSPs directly, optionally with the HSPs in a formulated form.

Brief Description of the Drawings

Figure 1 Western blotting of protein extracts of the bacterial strains LVS2, LVS3, LVS8, GR8 and GRlO, resolved on 10% SDS polyacrylamide gels, and transferred to polyvinylidene fluoride membranes, using E.coli DnaK specific Mabs .

A single polypeptide of approximately 70 kDa was present in all of the bacterial strains (LVS3, LVS8, GR8 and GRlO) tested and increased in staining intensity in heat shocked samples

Description of the Invention

In a first objective, the present invention provides a composition supplemented with heat shock proteins for controlling and/or preventing diseases in aquatic organisms, in particular a feed composition.

In a composition supplemented with heat shock proteins, the total amount of heat shock proteins present, is increased when compared to the non-supplemented composition. For example in case of feed compositions consisting essentially of or comprising life food organism, the heat shock supplemented composition would comprise life food organisms enriched in heat shock proteins, such as for example by; • exposure to a non-lethal heat shock (i.e. the feed composition accordingly comprises or essentially consists of life food organisms, in particular Artemia, previously exposed to a non-lethal heat shock) ; • submersion in a heat shock protein containing emulsion (solution) , wherein said heat shock protein

is optionally in a formulated form (infra) (i.e. the feed composition accordingly comprises or essentially consists of life food organisms submersed in a heat shock containing emulsion) ; • or by submersion in an emulsion (solution) of nonpathogenic micro-organisms previously exposed to a non-lethal heat shock (i.e. the feed composition accordingly comprises or essentially consists of life food organisms submersed in an emulsion (solution) of non-pathogenic micro-organisms (supra) previously exposed to a non-lethal heat shock) .

A composition supplemented with heat shock proteins, comprises at least an amount of heat shock proteins sufficient to enhance the resistance of aquatic organisms against opportunistic or obligate pathogenic microorganisms .

As used herein, the heat shock proteins are a group of proteins whose expression is increased when the cells are exposed to elevated temperatures or other stress. This increase in expression is transcriptionally regulated. This dramatic upregulation of the heat shock proteins induced mostly by Heat Shock Factor (HSF) is a key part of the heat shock response.

The function of heat-shock proteins is similar in virtually all living organisms, from bacteria to humans.

The HSPs are named according to their molecular weights, for example Hsp70 and Hsp90 each define families of

chaperones. The major classes of heat shock proteins are tabulated below.

Given the knowledge on the immunomodulating properties of certain fragments within heat-shock proteins, see for example Wang Y et al. (I.Immunol. 2005, 174; 3306-3316), it is also an object of the present invention, to provide the use of such fragments in the different embodiments of the present application. For HSP70, for example, said fragments include but are not limited to the peptide binding domain (p359-494), the C-terminal fragment (p359- 610) and the peptide binding base between loops 3,4 and 4,5 of HSP70.

Table 1

Although the most important members of each family are tabulated here, it should be noted that some species may express additional chaperones, co-chaperones, and heat

shock proteins not listed. Additionally, many of these proteins may have multiple splice variants (Hsp90α and Hsp90β, for instance) or conflicts of nomenclature (Hsp72 is sometimes called Hsp70) .

In a particular embodiment of the present invention, the heat-shock proteins are selected from the group classes enlisted in Table 1; more in particular from the bacterial (prokaryotic) HSP60 or HSP70 family; even more in particular from the bacterial (prokaryotic) HSP70 family.

In one embodiment, the HSPs are purified HSPs and include recombinantly produced HSPs, e.g. DnaK overproduced in E.coli. The purified proteins can be either mixed with the feed and fed directly to the larvae or invertebrates. In another formulation the HSP homologs can be protected from the hydrolysing activity of the stomach or the front gut by co-feeding with protective agents such a bicarbonate, aluminiumhydroxide, protease inhibitors which are compounds that are known to enhance the delivery of peptids to target organisms [Gastrointestinal delivery of peptide and protein drugs to aquacultural teleosts. McLean, E., Ronscholdt, B., Sien, C, Najamuddin, Aquaculture 177: 231-247 (1999)]. To enhance the delivery of HSP homolog, the protein can also be offered encapsulated in coated particles that allow for colon specific hydrolysis. As an examples of such colon specific delivery, HSP70 homologs could be incorporated in coated particles according to the COLAL techniques [Biorelevant dissolution testing of colon-specified delivery systems activated by colonic microflora. Yang L., Journal of Controlled release 125 : 77-86 (2008 ) ]

In a further embodiment, the composition comprises heat- shocked non-pathogenic organisms. In particular, nonpathogenic organisms selected from the group of microorganisms that have the GRAS (GRAS: generally regarded as safe) status, such as for example Saccharomyces cerevisiae, Lactobacillus acidophilus, Lactobacillus lactis, and Pediococcus acidilactici.

Heat-shocked non-pathogenic organisms as used herein, refers to non-pathogenic organisms, in particular any of the aforementioned micro-organisms, who have been exposed to a non-lethal heat shock. A non-lethal heat shock (NLHS) generally refers to exposure of the organisms to a temperature shock that induces thermotolerance, i.e. after exposure to a significantly increased temperature when compared to the ambient temperature for said organism (the non-lethal heat shock) , the organism is protected to an otherwise lethal temperature exposure. For example in case of Artemia a NLHS would consist of an exposure to a temperature ranging from 28 ⁰ C to 37 ⁰ C for a period ranging from 15 to 60 min., with a recovery ranging from 2 to 12 h.

In one embodiment, compositions comprising heat-shocked non-pathogenic organisms include, live food organisms enriched with heat-shocked micro-organisms such as for example Rotifers or Artemia submerged into a suspension of yeast cells that were previously exposed to a non-lethal heat shock.

In a particular embodiment, the composition consists essentially of live food organisms enriched with heat- shocked micro-organisms such as for example Rotifers or

Artemia submerged into a suspension of heat-shocked microorganisms that have the GRAS (GRAS: generally regarded as safe) status, such as for example Saccharomyces cerevisiae, Lactobacillus acidophilus, Lactobacillus lactis, and Pediococcus acidilactici; and any possible combinations thereof. As used herein, a composition consisting essentially of a particular component, is meant to refer to any composition containing said component as the main component. Containing for example about 30% to about 35%; about 40% to about 50%; about 50% to about 60%; about 60% to about 70%; about 70% to about 80%; about 80% to about 90%; about 90% to about 95%; about 98%; or about 99% by weight of said component.

In a second objective, the present invention provides the use of a composition as defined hereinbefore, in controlling and/or preventing diseases in aquatic organisms. In particular the use of a composition according to the invention in enhancing the resistance of aquatic organisms against opportunistic or obligate pathogenic micro-organisms. In a particular embodiment it provides the use of a composition according to the invention in enhancing the resistance of ANA' s against opportunistic or obligate pathogenic micro-organisms.

As used herein, the term ^aquatic organism' is to be understood in its broadest sense and it includes any aquatic species such as fish species, including as examples salmon, trout, carp, bass, bream, turbot, sole, milkfish, grey mullet, grouper, bream, halibut; crustaceans such as for example shrimp, lobster, crayfish and crabs; ornamental fish and shellfish; and molluscs such as bivalves, like clams and oysters.

The methods and compositions are particularly useful in controlling and/or preventing diseases in aquatic Antibody Negative Animals (ANA) organisms, such as for example selected from crustaceans and bivalves or larvae of aquatic organisms; more in particular in enhancing the resistance of fish larvae and of crustaceans and bivalves against opportunistic or obligate pathogenic microorganisms .

As shown in the examples hereinafter, the methods and compositions of the present invention are used in enhancing the resistance against opportunistic or obligate pathogenic micro-organisms selected from the group consisting of Gram negative bacteria, Vibrio, Aeromonas, Photobacterium, Yersinia, Edwarsiella, Renibacterium, Flavobacterium; Gram positive bacteria, including but not limited to species of Streptococcus, Lactococcus; viruses including but not limited to nodaviridae/ and parasites including but not limited to Ceratomyxa shasta, Ichthyophthirius multifillius, Cryptobia salmositica, Lepeophtheirus salmonis, Tetrahymena species, Trichodina species and Epistylus species. In a further embodiment of the present invention, the methods and compositions are useful in controlling Gram negative pathogens, including but not limited to Vibrio harveyi , V. paraheamolytocus, V. splendidus, V. mimicus, V. alginolyticus, V. anguillarum.

In a particular embodiment the present invention provides the use of the methods and compositions in enhancing the resistance of the aquatic organisms, in particular larvae of aquatic organisms, against a challenge with pathogens selected from any one of the above.

In the methods for controlling and/or preventing diseases

in aquacultures of aquatic organisms, the compositions as defined herein, are administered to the aquatic organisms. In particular fed to the aquatic organisms. In said embodiment the heat shock proteins, fragments thereof or heat shocked non-pathogenic micro-organisms are incorporated into an animal feed which is added to the water containing the aquatic animal, i.e. the food compositions enriched in heat shock proteins is added directly to the containment systems of the aquatic animals such as aquaria, tanks, cages, raceways, ponds or other enclosures .

In particular embodiments of the invention the methods and animal feed include: - feeding feed such as Brachionus and Artemia enriched with HSP (infra) to marine fish larvae, or crustacean the enrichment of feed with heat shock proteins, including purified heat shock proteins; or with microorganisms comprising heat-shocked proteins - the enrichment of feed by including heat-shocked microorganisms that have the GRAS status, such as yeast

Optionally, the methods and feed further comprise or include the co-feeding with agents (protective agents) known to enhance the delivery of peptides to the target organisms. Such protective agents include; a bicarbonate, aluminiumhydroxide, and protease inhibitors [Gastrointestinal delivery of peptide and protein drugs to aquacultural teleosts. McLean, E., Ronscholdt, B., Sien, C, Najamuddin, Aquaculture 177: 231-247 (1999)].

This invention will be better understood by reference to the Experimental Details that follow, but those skilled in the art will readily appreciate that these are only illustrative of the invention as described more fully in the claims that follow thereafter. Additionally, throughout this application, various publications are cited. The disclosure of these publications is hereby incorporated by reference into this application to describe more fully the state of the art to which this invention pertains.

EXAMPLES

The following examples illustrate the invention. Other embodiments will occur to the person skilled in the art in light of these examples.

Example 1: Heat shocked bacteria

Artemia instar II nauplii were grown in a germ free

(gnotobiotic) environment and fed once with bacteria subjected to a non-lethal heat shock by increasing the temperature of the culture medium from 28 ⁰ C to 37 ⁰ C for 30 min. Subsequently these heat shock bacteria were fed to 50 Artemia nauplii in 30 ml of sterile seawater. After 6 hour feeding, Artemia was challenged with Vibrio campbellii at 10 ⁷ CFU/ml. The table below show the results of the challenge test after feeding heat shocked, Bacillus (LVS2) , Aeromonas (LVS3), Vibrio sp (LVS8) , Cytophaga (GR8) or Roseobacter (GRlO) .

Bacteria control HS strains Survival Survival

I (% )

LVS 2 42 + 6 ^a 63 ± r ^*

LVS 3 29 + 8 ^b 52 ± gab*

LVS 8 22 ± 5 ^b 45 ± 6 ^b*

GR 8 43 ± ₂ a 63 ± 3 ^a*

GR 10 46 ± 4 ^a 65 ± 3 ^a*

Protein extracts of the heat-shocked bacteria were resolved in 10% SDS polyacrylamide gels, transferred to polyvinylidene fluoride membranes (BioRad Immun-Blot™ PVDF, USA) before incubation with blocking buffer (50 ml of phosphate buffered saline containing 0.2% (v/v) Tween-20 and 5% (w/v) bovine serum albumin) for 60 min . Membranes were incubated for 1 h at room temperature (RT) with rabbit polyclonal antibody raised against E. coli DnaK, i.e. HSP70 (a generous gift from Dr. Bernd Bukau, ZMBH, Germany) (Bucca et al . , 2000-bukau folder). Subsequent to membrane washing, horseradish peroxidase conjugated goat anti-rabbit IgG (Gentaur BVBA, Belgium) was added. After washing, antibody reactive proteins were detected by use of 0.7 mM diaminobenzidine tetrahydrochloride dihydrate and 0.01 % (v/v) H ₂ O ₂ in 0.1 M Tris-HCl (pH 7.6).

A single polypeptide of approximately 70 kDa was present in all of the bacterial strains (LVS3, LVS8, GR8 and GRlO) tested and increased in staining intensity in heat shocked samples (Fig 1) . Increased DnaK (heat shock protein) correlated with enhanced larval survival of Artemia.

Further evidence to the protective effect of heat shock proteins came from an experiment wherein a purposely build E. coli strain overproducing DnaK-DnaJ-grpE under the control of a arabinose-inducible promoter (strain P3) was used. Again, similar results were obtained. Namely feeding E. coli cells not overproducing these proteins resulted in 43 ± 3* survival in Artemia after challenge, while using E. coli cells overproducing these proteins (heat shock proteins) as feed resulted in 71± 6 after challenge (significant different at the 5% level)

Example 2 : Feeding Seabream larvae with HSP-protein enriched Artemia or Brachionus .

Seabream larvae are fed directly with purified HSP s part of inert diets, or are fed with live feed (rotifers or Artemia) that is enriched with purified HSP. For the latter DnaK, the bacterial HSP70 homolog, is overproduced in E. coli as a hybrid protein, containing a tag (6x HIS tag) that allows for easy isolation and purification. The purified proteins are either mixed with the live feed in that it is taken up by the filter-feeders (for instance rotifers or Artemia) or fed directly to the larvae as part of inert diets .

Such an inert diet could for example consist of the "IFREMER-INRA diet" (patent WO 0064273) conprising about 58.4% protein, 21.3% lipid (7.8% neutral lipid and 11.6% phospholipid), 12.2% ash related to dry matter and 9.8% moisture. It is prepared by mixing the dietary ingredients with water, pelletising the resultant mixture, and drying at 45 ⁰ C for 20 min . The pellets are then sieved to obtain

particles of two size ranges, 120-200 μm for larvae from 25 to 29 dph and 200-400 μm for larvae from 29 dph onwards.

Example 3: Feeding P. monodon with heat-shocked yeast- enriched Artemia

Penaeus monodon larvae are fed with Artemia or Brachionus that were enriched with HSP' s by submerging them in a suspension of yeast cells that were previously exposed to a non-lethal heat shock.

Example 4 : Feeding Sea bass larvae with Artemia enriched with HSP-containing lipid emulsion

Artemia nauplii are for 24 hours enriched with a commercially available lipid emulsion containing additionally added heat shock proteins.

Afterwards, the Artemia nauplii are rinsed and concentrated and fed to Sea bass larvae.

Example 5: Feeding Seabass larvae with encapsulated heat- shocked bacteria

Bacillus or a micro-organisms with the GRAS status are exposed to a non-lethal heat shock by increasing the temperature from 28 ⁰ C to 37 ⁰ C for 30 minutes. Subsequently these bacteria are encapsulated in coated particles according to the COLAL technique . The encapsulated bacteria are fed directly to Seabass or to Artemia nauplii, which are enriched for 24 hours on a feed

containing such encapsulated bacteria. In the latter case, the Artemia nauplii are rinsed and concentrated and fed to seabass lavae.

References

1. Graham R Stewart_ and Douglas B Young 2004 Heat-shock proteins and the host-pathogen interaction during bacterial infection. Current Opinion in Immunology 2004, 16:506-510

2. Singh, V. and Aballay, A. 2006 Heat-shock transcription factor (HSF)-I pathway required for Caenorhabditis elegans immunity. Proceeding National Academy of Science 103: 13093

3. Yeong Y.S, Pineda, C, MacRae, T.H. , Sorgeloos, P. and Bossier, P. 2007 Exposure of gnotobiotic Artemia franciscana larvae to abiotic stress promotes heat shock protein 70 synthesis and enhances resistance to pathogenic Vibrio campbellii. Cell Stress and Chaperones; in press

4. Yeong, Y.S., Van Damme, E.J.M., Sorgeloos, P., Bossier, P. (2007). Non-lethal heat shock protects gnotobiotic Artemia franciscana larvae against virulent Vibrios. Fish & Shellfish Immunology 22: 318-326