WO2005084704A1

WO2005084704A1 - Digestive bypass composition for arthropod and uses thereof

Info

Publication number: WO2005084704A1
Application number: PCT/CA2005/000351
Authority: WO
Inventors: Grant W. Vandenberg
Original assignee: Peros Systems Technologies Inc.
Priority date: 2004-03-02
Filing date: 2005-03-02
Publication date: 2005-09-15

Abstract

The present invention relates to a composition and uses thereof to induce physiological reaction in arthropods. More particularly, the composition comprises a physiological reaction inducing agent diluted into a buffer comprising compounds allowing to improve the pH in the digestive tract. The composition also comprises a vegetable extract under form of oil or crude homogenized legumes, and a choleretic or intestinal uptake-increasing agent.

Description

DIGESTIVE BYPASS COMPOSITION FOR ARTHROPOD AND USES THEREOF

BACKGROUND OF THE INVENTION a) Field of the invention The present invention relates to a composition useful in per us delivery of prophylactic and therapeutic agent for the management of production yield or microbial diseases in aquatic animals. More particularly, the composition of the present invention allows to improve the metabolic performance of shrimp at the level of the growth and induced resistance to microbial pathogens. b) Description of the prior art The earliest shrimp farms consisted of near shore impoundments to which small numbers (1-3 shrimp/m²) of wild caught shrimp post-larvae (wild seed) were stocked. Several hundred kilograms/hectare of adult shrimp were harvested periodically. These farms in essence practiced natural balanced ecosystem grow out; no feed was added to the growth medium, no water was exchanged, no aeration or mixing was performed, and no water treatment was provided. Most shrimp farm operations have retained dependence upon wild seed, but have raised stocking densities. Yields of 1,000-3,000 g/m² became standard, and some farm operations reported yields of 10,000 g/m². Some prerequisites to these higher yields were substantial modifications in farm management practices including but not limited to use and dependence upon supplemental feeds such as trash fish, manufactured feed or both; water exchange; and aeration/mixing. Generally the higher the stocking density, the more the fanning system depended upon quality high protein and high energy feeds, water exchange, and aeration/mixing. Currently, the impact and spread of shrimp diseases and mortality rates of shrimp are the dominant concerns of shrimp farmers around the world. Pathogenic viruses and other disease agents for shrimp are commonly found in wild shrimp populations and in river and coastal waters where shrimp or other crustaceans are or may be farmed. These pathogens are also carried by workers, birds, and wind, and may contaminate the shrimp population, with devastating results on crop growth and yield. . Few solutions have been rendered available in the world to favorably impact the different microbial diseases affecting shrimp production plans. Good farming practices allow to reach a certain level of bio-security in these types of production plans, but a lot of production batches are still lost because of the incapacity to avoid microbial contamination. The innate immune response of arthropods also relies on the production of antimicrobial peptides which possess activity against a large range of pathogens. In the horseshoe crab (Chelicerata), endotoxin activation of the hemocytes results in the release by exocytosis of the contents of two types of granules, such as, on the one hand, the clotting factors, essential for hemolymph coagulation, proteases, tachylectins, and on the other hand, antimicrobial peptides, tachyplesins, big defensin, tachycitin and tachystatins which possess agglutinating activity. In insects, the synthesis of antimicrobial peptides induced upon injury is the hallmark of the immune response of higher insect orders. A septic injury induces the rapid and transient transcription of several genes encoding potent antibacterial and antifungal peptides that are released into the blood where they act to destroy the invading microorganisms. The activation cascades that control the expression of the antimicrobial peptide genes in higher insects show striking structural and functional similarities with activation cascades involved in cytokine-induced expression of acute phase proteins in mammals. The production of antimicrobial peptides is in fact widespread in the living kingdom, from bacteria to plants and from vertebrates to invertebrates. Surprisingly, in crustaceans, until now the innate defense reaction involving the synthesis of antimicrobial peptides has been poorly studied. To date, constitutive hemocytic proteins have been isolated in the crab Carcinus maenas, and a 6.5 kDa antimicrobial peptide has been partially characterized (Schnapp et al. 1996, Eur. J. Biotech. 240:532-539). In the penaeid shrimp, P. vannamei, three antimicrobial peptides have been purified from the hemocytes and the plasma. They have been fully characterized and their cDNA cloned. According to their biochemical and structural features, the three peptides cannot be associated to other peptide families hitherto described, and they were named penaeidins, after the genus Penaeus. Research is being continued on with the role of the penaeidins in the immune response against pathogens as well as with the characterization of other peptides in shrimp. The consideration of production yield is also very important concern for shrimp producers. The way to improve the production yield, through improvement of the growth of shrimp has been studied under different angles in the past decades. Food additives, nutriments, or nutraceutical products have proved their value in keeping shrimp healthy, and have an certain impact, on the growth of the animals, but remains quite limited. Few approaches were proposed to provide alternative solutions or methods or products to make production cycles faster. The only one criteria that is admitted to shorter the production cycles is a faster growth of shrimp, which allow to have animals with commercial standards more rapidly. Thus, there is a need for an economical food composition for intensive production of highly growing and highly resistant shrimp which minimizes environmental side effects.

SUMMARY OF THE INVENTION One aim of the present invention is to provide a composition for modulating a physiological reaction or inducing an immune reaction in arthropod comprising: a) at least one physiological reaction inducing agent in a physiologically acceptable buffer; b) at least one vegetable extract selected from the group consisting of homogenized legumes, oilseed or pulse grains; and c) at least one of choleretic or intestinal uptake-increasing agent. The physiological reaction is growth improvement or production of recognition binding protein. The recognition binding protein can be an immunoglobulin or an antibody. The arthropod can be a crustacean, but preferentially a shrimp. The physiological reaction inducing agent is preferably a growth factor, an antigen or an antimicrobial compound, and is mixed to a buffer that comprises at least one compound allowing to increase digestive tract pH above 4, but preferentially above 6. The compound is generally at concentration between 1% to 60% w/w in the composition of the present invention. For example, but not limited to, the compound can be selected from the group consisting of anti-acids, sodium bicarbonate, sodium carbonate, sodium citrate, sodium hydrogencarbonate, calcium phosphate, calcium carbonate, magnesium salts, magnesium carbonate, magnesium trisilicate, magnesium .hydroxide, magnesium phosphate, magnesium oxide, bismuth subcarbonate, and combinations thereof. The concentration of the vegetable extract is between 1% to 50%, and the choleretic or uptake increasing agent is at concentration between 0.1% to 50% w/w. It will be recognized by someone skilled in the art that the vegetable extract, for example but not limited to, can be selected from the group consisting of anti-protease, egg albumin, plant-derived inhibitors from oilseed, soybean, kidney bean, faba bean, rice bran, wheat bran, ethylenediamine tetraacetate, alpha- 1-antitrypsin, albumin, ovalbumin, and proteosomes. Also, the choleretic or intestinal uptake increasing agent is preferentially selected from the group consisting of bile salt, (3α,5β,12α)-3,12-dihydroxy-5-cholan-24- oic acid, 17β-(l-methyl-3-carboxypropyl)etio-cholane-3α,12α-diol, deoxycholic acid, or salts or derivatives thereof, and are generally found in the composition invention at concentration between 1% to 5%. According to one aim of the invention, the composition is orally administered to said arthropod. In accordance with another aim of the present invention, there is provided a composition for delivery of a physiological reaction inducing agent in arthropod hemolymph comprising: a) at least one physiological reaction inducing agent in a physiologically acceptable buffer; b) at least one vegetable extract selected from the group consisting of homogenized legumes, oilseed or pulse grains; and c) at least one of choleretic or intestinal uptake-increasing agent. Another aim of the present invention is to provide a method for modulating a physiological reaction or inducing an immune response in crustacean comprising orally administrating composition as defined herein to crustaceans. The physiological reaction can be, preferentially, at least one of body growth, immune reaction, fat metabolism, or muscle synthesis. Also is provided with the present invention a method for enhancing hemolymph concentration of a physiological reaction inducing agent to an arthropod comprising orally administering a physiologically effective amount of a composition as defined herein to crustaceans. The composition of the present invention can also be used in the manufacture of a drug or a food that will allow to induce, through the physiological reaction inducer agent, a physiological reaction such as, but not limited to, growth improvement or induction of synthesis of recognition binding proteins.. Another object of the present invention is to provide a method for modulating a physiological reaction or inducing an immune response in a shrimp comprising orally administrating a sufficient amount of a composition of the present invention defined herein. For the purpose of the present invention the following terms are defined below. The term "therapeutic agent " is used in a generic sense and includes treating agents, prophylactic agents, and replacement agents, antimicrobial agents. The term "common mucosal immune system" refers to the fact that immunization at any mucosal site can elicit an immune response at all other mucosal sites._. The terms "protein", "peptide" and "polypeptide" refer to both the naturally occurring chemical entities and the structurally similar bioactive equivalents derived from either endogenous, exogenous, or synthetic sources and is used to mean polymers of amino acids linked together by an amide type linkage known as a peptide bond. The term "structurally similar bioactive equivalent" is meant a polypeptide with an amino acid sequence which, although not identical to that of the naturally occurring peptide, is sufficiently similar in structure to produce substantially equivalent therapeutic effects on the subject to that produced by the natural peptide itself. The term "therapeutically effect amount" of a medicament is meant a sufficient amount of the compound to obtain the intended therapeutic benefit, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the medicaments and compositions of the present invention will be decided by the attending physician with the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start at doses lower than required to achieve the desired therapeutic effect and to gradually increase the. dosage until the desired effect is achieved.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates the concentration of HRP in shrimp hemolymph at different time periods following feeding of shrimp with the composition according to one embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which preferred embodiments of the invention are shown. This invention, may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The effectiveness of using the vaccine composition to deliver vaccines orally in order to overcome current production constraints facing the aquaculture industry is hereby clearly demonstrated. The present invention relates to the administration of therapeutic proteins and polypeptides in oral dosage form. The invention provides increased absorption through the GI tract and greatly improved bioavailability of the proteins/peptides as compared to that of the prior art formulations. The invention is useful in shrimp nutrition, therapy and treatment. As used herein and in the appended claims, the term "polypeptide" encompasses proteins and peptides as well as polypeptides within its scope. The compounds and compositions of the subject invention are useful for administering biologically or chemically active agents to shrimps. The system is particularly advantageous for delivering physiologically, biologically or chemically active agents which would otherwise be degraded or rendered less effective by conditions encountered before the active agent reaches its target zone (i.e. the area in which the active agent of the delivery composition is to be released) within the body of the animal to which they are administered. Particularly, the compounds and compositions of the present invention are useful for orally administering active agents, especially those which are not ordinarily orally deliverable. The present invention is particularly useful for the administration of polypeptides, including proteins, such as, but not limited to, therapeutical agents, nutritional products, mucopolysaccharides, lipids, carbohydrates, steroids, hormones, growth hormone (GH), growth hormone releasing hormone (GHRH), epithelial growth factor, vascular endothelial growth and permeability factor (VEGPF), nerve growth factor, cytokines, interleukins, interferons, GMCSF, hormone-like product, neurological factor, neurotropic factor, neurotransmitter, neuro-modulator, enzyme, antibody, peptide, protein fragment, vaccine, adjuvant, an antigen, immune stimulating or inhibiting factor, heomatopoietic factor, anti-cancer product, anti-inflammatory agent, anti-parasitic compound, anti-microbial agent, nucleic acid fragment, plasmid DNA vector, cell proliferation inhibitor or activator, cell differentiating factor, blood coagulation factor, immunoglobulin, anti-angiogenic product, negative selective markers or "suicide" agent, toxic compound, anti-angiogenic agent, polypeptide, and anti-cancer agent nucleotides, and the like, and structurally similar bioactive equivalents thereof. In accordance with one embodiment of the present invention, there is provided a composition for oral administration and intestinal delivery of a nutritional compound or a therapeutic polypeptide that can be formulated, but without limitation to products described herein, with deoxycholate and saponins in a ratio to provide a substantially increased absorption and systemic bioavailability of the peptide by the intestine of the host. The composition also comprises a pH neutralizing agent, such as but not limited to sodium carbonate and calcium carbonate, and at least one inhibitor of digestive enzymes, such as but not limited to egg albumin. This composition is preferably solid so as to be easy to manipulate in formulating oral composition forms. Neutralization of pH is intended to mean increasing the pH into the digestive tract to acid-base equilibrium compatible with most of known active biological products in nature or synthesized. The digestive tract's pH may be, but not limited to, between about 5 and 9, and preferably between about 6.5 and 7.5. It is to be understood that the above list of drugs is for illustration purposes only and is not provided as an all inclusive list of all the drugs which may be beneficially formulated or reformulated using the oral delivery compositions of the present invention. Other physiologically-active compounds that can be encapsulated in the compositions of the present invention include biologically-active compounds, such as proteins, enzymes, anti-enzymes, peptides, catecholamines, anti-histamines, analgesics, and the like. For the purposes of the present invention "biological" is defined to mean any nutritionally or medically useful composition derived from a biological source and/or a synthetic pharmacological equivalent thereof such as insulin, heme, hemoglobin, and hormones; "enzyme" or "enzyme system" is defined to mean any protein or conjugated protein produced biologically or synthetically and which functions as a biocatalyst. Other medically useful compositions known to those skilled in the art, for example, globulin, one or more glycoproteins, such as erythropoeitin, may also be incorporated in the composition of the present invention. The amount of therapeutic polypeptide will vary widely, depending on various factors such as the particular peptide to be delivered, the indication to be treated, the individual patient, and the like. The amount will be a therapeutically effective amount, that is, an amount that will provide a therapeutic effect, to be determined in accordance with well-established medical practice. Another embodiment of the present invention is the use of enteric coatings, which are available for tablets and capsules. Enteric coatings will remain intact in the stomach but will rapidly dissolve once they arrive at the small intestine, thereafter releasing the drug at sites downstream in the intestine (e.g., the ileum and colon). Enteric coatings are well known in the art. Alternatively, a controlled release oral delivery vessel designed to release a drug after a predetermined period of time, and thus after the vessel has passed into the ileum or colon, can be used to deliver the formulation of the present invention. Such vessels include the CHRONSETTM delivery device (ALZA Corporation, Palo Alto, Calif.) and the PulsincapTM delivery device (R.P. Scherer Co.). The composition may further comprises an ion-pair forming reagent wherein the mole ratio of ion-pair forming reagent to drug is from about 2:1 to about 10:1. The ion- pair-forming reagent is added to increase the lipophihcity of the dissolved physiologically active agent or drug and thereby increase its membrane permeability. Increasing the drug's lipophihcity may also provide some protection of the drug from enzymatic deactivation as much of the peptide degradation that occurs in vivo does so in the aqueous environment of the gastrointestinal tract. Representative ion-pair forming reagents include sodium decanesulfonate, sodium lauryl sulfate, and sodium benzoate. In one embodiment of the present invention is that the composition may optionally comprise from about 1% to about 5% based on the total volume of the composition of an intestinal mucosal membrane transport enhancing agent, deoxycholate. Such agents facilitate the absorption of the therapeutic agent across the mucosal tissues in the intestinal mucosa and directly into the bloodstream of the subject. Also tissue transport enhancing agents suitable for use in the present compositions are selected from essential or volatile oils or from non-toxic, pharmaceutically acceptable organic and inorganic acids or salts and esters thereof. Essential or volatile oils which may be employed in the composition are selected from soybean oil, faba oil, rice oil, fish oil. The preferred essential oil is fish oil. In another embodiment of the present invention, the composition may contain additional agents such as preservatives and antioxidants. Typical preservatives include sodium benzoate, sorbic acid, and the methyl and propyl esters of p-hydroxy-benzoic acid (parabens). Representative antioxidants include butylated hydroxy anisole, butylated hydroxy toluene, nordihydroguaiaretic acid, the gallates such as propyl gallate, hydroquinone, propenyl methyl guaethol and alkyl thiopropionates, or water soluble agents such as alkanolamines, alcohols, and propylene glycol. The most preferred antioxidant is Tenox™ GTl (1:1 vitamin E-soybean oil), present in a concentration of between about 5% to about 25% based on the total volume of the droplet. To prepare the pharmaceutical formulation of the present invention, the ingredients are dry blended together, after which the small amount of oil is added. These materials are mixed together until a homogeneous mixture of ingredient results. The resulting solid formulation can be pressed into tablets that can then be coated with a suitable enteric coating. Alternatively, the solid formulation can be placed into a capsule formed of gelatin or the like and coated with an enteric compound, or placed into a controlled release delivery device such as the CHRONSET™. The solid formulation provides a mean for easily and conveniently fabricating a dosage form. In one embodiment of the present invention, the composition may comprise, for example, but not limited to, Egg albumin, Sodium carbonate, Calcium carbonate, EDTA, Soybeans, Faba beans, Rice huul, Deoxycholate, Fish oil, Brewers yeast. The products can be previously processed, such as being homogenized or crushed, or dissorlve, or be kept crude for the preparation of the composition of the invention. One embodiment of the invention is to provide a method for delivering hormones and pharmaceuticals or vaccines to shrimp in a very simple manner through feeding. Among the agricultural production field, the production of different species of shrimps is importantly pointing out. The control of the reproduction physiology is of particular importance. The first indication of the invention is for manipulating growth and microbial resistance by feeding bioactive materials with the composition of the present invention. A particular embodiment of the present invention is to provide a composition and a method allowing the use of the oral route for vaccination that offers significant advantage in that it reduces labor costs, is time-saving, decreases the possibilities for cross-contamination with needles and does not involve inventory handling or require disposal of treatment waters. According to the present invention, a host can be immunized by oral administration of bacterial protein immunogens, preferably mixed with an adjuvant, such as cholera toxin (CT). Of course, as an adjuvant, the amount of cholera toxin used is non- toxic to the host. The ability of a vaccine to protect against microbial colonization, as provided herein, means that the active component may protect against disease not only in the immunized host but, by eliminating carriage among immunized individuals, the pathogen and hence any disease it causes may be eliminated from the population as a whole. Oral administration may also prevent sepsis resulting from administration of microbials, so that the vaccine can protect against both microbial colonization and sepsis (systemic infection). Immunostimulatory agents or adjuvants have been used for many years to improve the host immune response to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccharides, normally are the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non- covalently linked to antigens and are formulated to enhance the host immune response. Aluminum hydroxide and aluminum phosphates (collectively commonly referred to as alum) are routinely used as adjuvants veterinary vaccines. The efficacy of alum in increasing antibody responses to diphtheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum. A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund's complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes. To efficiently induce humoral immune response (HIR) and cell-mediated immunity (CMI), immunogens are preferably emulsified in adjuvants. Compositions of the invention, especially for oral administration may be conveniently provided as liquid preparations, e.g., isotonic aqueous solutions, suspensions, emulsions or viscous compositions which may be buffered to a selected pH. However, since delivery to the digestive tract is preferred, compositions of the invention may be in a "solid" form of pills, tablets, capsules, caplets and the like, including "solid" preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach and/or small intestine for delivery to the gut and/or digestive system. The composition of the invention may also contain pharmaceutically acceptable flavoring and/or coloring agents for rendering them more appealing. The viscous compositions may be in the form of gels, lotions, ointments, creams and the like and will typically contain a sufficient amount of a thickening agent so that the viscosity is from about 2500 to 6500 cps, although more viscous compositions, even up to 10,000 cps may be employed. Viscous compositions have a viscosity preferably of 2500 to 5000 cps, since above that range they become more difficult to administer. However, above that range, the compositions can approach solid or gelatin forms that are then easily administered as a swallowed pill for oral ingestion. Liquid preparations are normally easier to prepare than gels and other viscous compositions, and solid compositions. Additionally, liquid compositions are somewhat more convenient to administer, especially to animals, children, particularly small children, and others who may have difficulty swallowing a pill, tablet, capsule or the like, or in multi-dose situations. Niscous compositions, on the other hand can be formulated within the appropriate viscosity range to provide longer contact periods with mucosa, such as the lining of the stomach or intestine. Suitable nontoxic pharmaceutically acceptable carriers, and especially oral carriers, will be apparent to those skilled in the art of pharmaceutical and especially oral or peroral pharmaceutical formations. Obviously, the choice of suitable carriers will depend on the exact nature of the particular dosage form, e.g., liquid dosage form (e.g., whether the composition is to be formulated into a solution, a suspension, a gel or another liquid form, or a solid dosage form, or e.g., whether the composition is to be formulated into a pill, tablet, capsule, caplet, time release form or liquid-filled form). Solutions, suspensions and gels, normally contain a major amount of water (preferably purified water) in addition to the antigen. Minor amounts of other ingredients such as pH adjusters (e.g., a base such as ΝaOH), emulsifiers or dispersing agents, buffering agents, preservatives, wetting agents, jelling agents, (e.g., methylcellulose), coloring and/or flavoring agents may also be present. The compositions can be isotonic, i.e., it can have the same osmotic pressure as blood and lachrymal fluid. Those skilled in the art will recognize that the components of the composition must be selected to be chemically inert with respect to microbial antigens. This will present no problem to those skilled in chemical and pharmaceutical principles, or problems can be readily avoided by reference to standard tests or by simple experiments (not involving undue experimentation), from this disclosure. The immunological effective compositions of this invention are prepared by mixing the ingredients following generally accepted procedures. For example the selected components may be simply mixed in a blender, or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity. Generally the pH may be from about 3 to 7.5. The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I Oral delivery of HRP in shrimp

Preliminary research has provided evidence that by using proprietary composition of the present invention described herein, it is possible to deliver functional bioactive compounds past the stomach of gastric fish whereby the compound is absorbed and elicits a biological response. The following research plan proposes to investigate the utility of the Oralject™ to orally deliver a model protein to shrimp.

Long-Term Objective Investigate the potential feasibility to apply the Oralject™ formulation to marine shrimp.

Short-Term Objectives

1) Conduct short-term palatability studies to determine the ability of shrimp to ingest the Oralject™ formulation. If required, alter the formulation to assure adequate palatability 2) Using the formulation developed above, delivery orally a model protein, namely horseradish peroxidase (HRP) to shrimp.

METHODOLOGY Peneaus vannamei is used as the model, as it is an important farmed species and has been the predominant model for research. Animals are maintained at standard laboratory conditions with temperature, pH, ammonia, nitrite, nitrate and salinity monitored at regular intervals to ensure optimal environmental conditions. For these experiments temperature is 30°C, salinity held at 31 ppt, and other parameters measured every 48 hours.

Palatability Study Short-term studies will determine an Oralject™ formulation that is palatable for marine shrimp.

Basal Oralject Formulation Initially, the current Oralject™- vaccine formulation will be pelleted using a cold-pelleted process (2 mm dia pellets, 35 mm in length). These pellets will be fed uncoated or coated with a standard lipid mixture.

Feeding Study Shrimp (n=20 /tank; initial weight= 0.25 grams) are stocked into 9 liter tanks in an experimental system 4 days prior to the start of the experiment. Water temperature is held at 30°C (photoperiod is regulated by regular room lighting, roughly lOhrs light per day). Animals are fed a standard feed high protein feed (Zeigler Brothers SI-35) during the acclimation period and during the non-treatment periods. Shrimp are fasted for 24 hours prior to feeding Oralject™. Tanks are cleaned of all fecal matter prior to the start of the experimental feeding. Using the experimental Oralject™ formulation and a commercial feed control diet, shrimp are fed manually for 60 minutes until active feeding ceases. Observations relating to feeding behavior are noted. At the end of the study, total feed consumption is noted and expressed as a percentage of animal tank weight. Should the above experimental formulations prove to promote reduced intake, a number of formulation modifications are attempted in order to increase palatability. A number of feeding attractants (squid meal/oil, others?) are added to the formulation. Furthermore, ingredients having a probability to affect intake are reduced/removed, and the experiments repeated as above.

Oral HRP delivery Using a palatable Oralject™ formulation for shrimp HRP is delivered into the hemolymph following oral administration.

Oral HRP delivery HRP is mixed into the formulation derived above at a given dose (1 and 10 mg/g) and pelleted as described previously. Each dose of Oralject-HRP will be fed to (3 tanks of shrimp (n=10 4.0 gm shrimp/tank) previously fasted for 24 h, and at predetermined time periods following feeding (0, 5, 10, 15, 30 60, 120 minutes), 100 μl hemolymph will be collected using standard methods. Samples are frozen and shipped to the analysis laboratory on dry ice for analysis.

HRP quantification HRP will be quantified using a capture ELISA with goat-anti-HRP antibody (1 : 1000). Prior to HRP analysis, a preliminary study will investigate the recovery of HRP from shrimp hemolymph to ensure a lack of interference with this fluid. Intact enzyme is measured in the hemolymph of individuals treated with Oralject™ containing HRP. HRP was mixed into the Oralject™ formulation at a given dose (1 and 5 mg/g). Each dose of Oralject™-HRP was fed to shrimp (initial weight = 4 grams) previously fasted for 24 h._At predetermined time periods following feeding (0, 0.25, 0.5, 1, 4, 8 and 36 h postfeeding), 100 μl of hemolymph was collected from 4 shrimp using standard methods. Intact HRP in shrimp hemolymph was quantified using a capture ELISA with goat-anti-HRP antibody. RESULTS Results showed that using the Oralject™ system, a model bioactive protein can be delivered orally to marine shrimp, resulting in significant accumulation in hemolymph. While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come, within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.

Claims

WE CLAIM:

1. A composition for modulating a physiological reaction or inducing an immune reaction in arthropod comprising: . a) at least one physiological reaction inducing agent in a physiologically acceptable buffer; b) at least one vegetable extract selected from the group consisting of homogenized legumes, oilseed or pulse grains; and c) at least one of choleretic or intestinal uptake-increasing agent.

2. The composition of claim 1, wherein physiological reaction is growth improvement or production of recognition binding protein. .

3. The composition of claim 2, wherein said recognition binding protein is an immunoglobulin.

4. The composition of claim 3, wherein said immunoglobulin is an antibody.

5. The composition of claim 1, wherein said arthropod is a crustacean.

6. The composition of claim 5, wherein said crustacean is shrimp.

7. The composition of claim 1, wherein said physiological reaction inducing agent is a growth factor, an antigen or an antimicrobial compound.

8. The composition of claim 1, physiologically acceptable buffer comprises a compound allowing to increase digestive tract pH above 6.

9. The composition of claim 8, wherein said compound is at concentration between 1% to 60% w/w.

10. The composition of claim 8, wherein said compound is selected from the group consisting of anti-acids, sodium bicarbonate, sodium carbonate, sodium citrate, sodium hydrogencarbonate, calcium phosphate, calcium carbonate, magnesium salts, magnesium carbonate, magnesium trisilicate, magnesium hydroxide, magnesium phosphate, magnesium oxide, bismuth subcarbonate, and combinations thereof.

11. The composition of claim 1, wherein said vegetable extract is at concentration between 1% to 50%,

12. The composition of claim 1, wherein said choleretic or uptake increasing agent is at concentration between 0.1% to 50% w/w.

13. The composition of claim 1, wherein said vegetable extract is selected from the group consisting of anti-protease, egg albumin, plant-derived inhibitors from oilseed, soybean, kidney bean, faba bean, rice bran, wheat bran, ethylenediamine tetraacetate, alpha- 1 -antitrypsin, albumin, ovalbumin, and proteosomes.

14. The composition of claim 1, wherein said choleretic or intestinal uptake increasing agent is selected from the group consisting of bile salt, (3α,5β,12 )-3,12- dihydroxy-5 -cholan-24-oic acid, 17β-( 1 -methyl-3 -carboxypropyl)etio-cholane-3α, 12α- diol, deoxycholic acid, or salts or derivatives thereof.

15. The composition of claim 14, wherein said choleretic or intestinal uptake- increasing agent is at concentration between 1% to 5%.

16. The composition of claim 1 which is orally administered to said arthropod.

17. A composition for delivery of a physiological reaction inducing agent in arthropod hemolymph comprising: a) at least one physiological reaction inducing agent in a physiologically acceptable buffer; b) at least one vegetable extract selected from the group consisting of homogenized legumes, oilseed or pulse grains; and c) at least one of choleretic or intestinal uptake-increasing agent.

18. A method for modulating a physiological reaction or inducing an immune response in crustacean comprising orally administrating composition as defined in claim 1 to said crustacean.

19. The method of claim 18, wherein said physiological reaction is at least one of body growth, immune reaction,, fat metabolism, or muscle synthesis.

20. A method for enhancing hemolymph concentration of a physiological reaction inducing agent to an arthropod comprising orally administering a physiologically effective amount of a composition as defined in claim 1 to said crustacean.

21. Use of a composition according to claim 1 in the manufacture of a drug or a food.