WO2010094011A1 - Compositions and methods for improved livestock and aquaculture performance - Google Patents

Abstract

Methods and compositions for improved livestock and aquaculture productivity are disclosed. The methods include administering an agent, such as an antibody raised against phospholipase A₂ or immunogenic fragments or variants thereof. Animal feed and fish or shellfish food comprising antibodies as described herein are also disclosed.

Description

COMPOSITIONS AND METHODS FOR IMPROVED LIVESTOCK AND AQUACULTURE PERFORMANCE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 61/152,795, filed February 16, 2009, the entire contents of which are hereby incorporated by reference in its entirety.

TECHNICAL FILED

[0002] The present disclosure relates generally to the field of livestock and aquaculture feeds and compositions for improving livestock and aquaculture productivity.

SUMMARY

[0003] The present disclosure relates to antibodies which may be used to speed animal growth and survival and improve productivity in animals. The present invention further relates to animal feed compositions and supplements comprising such as anti-phospholipase A₂ (PLA₂) antibodies and antibodies to fragments and variants of PLA₂.

[0004] In one aspect, the present disclosure provides isolated peptide immunogens and antibodies which bind to this sequence in intact proteins. The antibodies are useful in the preparation of supplements for animal feed. In one embodiment, the antibodies are raised against an isolated PLA₂ peptides or a variant thereof, wherein the PLA₂ peptide comprises a fragment of 6 to 20 contiguous amino acids of a PLA₂ protein, wherein the variant has from one to five amino acid substitutions, additions, or deletions. The fragment may be from a PLA₂ protein, such as porcine, bovine, ovine, avian, or fish PLA₂ protein. In a particular embodiment, the PLA₂ protein has a sequence according to any one of SEQ ID NOs: 29-48. In some embodiments, the variant has from one to five amino acid substitutions, additions, or deletions in the sequences selected from the group consisting of: SEQ ID NOs: 1-28. In specific embodiments, the PLA₂ peptide or variant is selected from the group consisting of: SEQ ID NOs: 1-28. Antibodies raised against such PLA₂ peptides or variants may bind to the same epitope present in other peptides or intact proteins. [0005] In one embodiment, immunogenic compositions comprise an isolated PLA₂ peptide or a variant thereof, wherein the PLA₂ peptide comprises a fragment of 6 to 20 contiguous amino acids of a PLA₂ protein, and wherein the variant has from one to five amino acid substitutions, additions, or deletions. The fragment may be from a PLA₂ protein, such as porcine, bovine, ovine, or avian PLA₂ protein. In a particular embodiment, the fragment is derived from a PLA₂ protein having a sequence according to any one of according to any one of SEQ ID NOs: 29-48. In some embodiments, the variant has from one to five amino acid substitutions, additions, or deletions in the sequences selected from the group consisting of: SEQ ID NOs: 1-28. In specific embodiments, the PLA₂ peptide or variant is selected from the group consisting of: SEQ ID NOs: 1-28. In some embodiments, the peptide immunogen is conjugated to a high molecular weight carrier protein, e.g., keyhole limpet hemocyanin (KLH), ovalbumin, or bovine serum albumin.

[0006] In one aspect, the present disclosure provides a composition comprising an animal feed and a supplement, wherein the supplement comprises an antibody which binds specifically to an isolated PLA₂ peptide or a variant thereof, wherein the PLA₂ peptide comprises a fragment of 6 to 20 contiguous amino acids of a PLA₂ protein, wherein the variant has from one to five amino acid substitutions, additions, or deletions. The fragment may be from a PLA₂ protein, such as porcine, bovine, ovine, or avian PLA₂ protein. In a some embodiments, the PLA₂ protein has a sequence according to SEQ ID NO: 29-48. In some embodiments, the variant has from one to five amino acid substitutions, additions, or deletions in the sequences selected from the group consisting of: SEQ ID NOs: 1-28. In specific embodiments, the PLA₂ peptide or variant is selected from the group consisting of: SEQ ID NOs: 1-28.

[0007] In one aspect, the present disclosure provides a method for improving mineral utilization in animals, including, but not limited to, calcium and phosphorus utilization. In particular embodiments, the phosphorus content of animal feces is reduced by administering to the animal an effective amount of a composition comprising an animal feed and a supplement, wherein the supplement comprises an antibody which binds specifically to an isolated PLA₂ peptide or a variant thereof, wherein the PLA₂ peptide comprises a fragment of 6 to 20 contiguous amino acids of a PLA₂ protein, wherein the variant has from one to five amino acid substitutions, additions, or deletions, and wherein the antibody is present in an effective amount to decrease the phosphorus content of feces from animals administered the composition compared to the phosphorus content of feces from animals not administered the composition.

[0008] In one aspect, the present disclosure provides a method for increasing the specific gravity of eggs comprising administering to poultry an effective amount of a composition comprising an animal feed and a supplement, wherein the supplement comprises an antibody which binds specifically to an isolated PLA₂ peptide or a variant thereof, wherein the PLA₂ peptide comprises a fragment of 6 to 20 contiguous amino acids of a PLA₂ protein, wherein the variant has from one to five amino acid substitutions, additions, or deletions, and wherein the antibody is present in an effective amount for increasing the specific gravity of eggs laid by poultry administered the composition compared to eggs laid by poultry not administered the composition. In some embodiments, the antibody is present in an effective amount for increasing the specific gravity of eggs laid by poultry administered the composition compared to eggs laid by poultry not administered the composition, and wherein the poultry feed comprises less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% calcium. The antibodies may increase the specific gravity of the egg by at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, or at least 1% in comparison to eggs laid by chickens not administered the antibodies of the present invention. The antibodies will typically not increase the specific gravity of the egg by more than about 2%, more than about 5%, more than about 10%, more than about 20%, or more than about 30%.

[0009] In some embodiments, the antibody is present in an effective amount for increasing the specific gravity of eggs laid by poultry administered the composition compared to eggs laid by poultry not administered the composition. Typically, the poultry feed comprises less than 5% calcium. In some embodiments, the number of eggs having a specific gravity of at least 1.081, at least 1.082, at least 1.083, at least 1.084, or at least 1.085 laid by poultry administered the composition is increased relative to the number of eggs laid by poultry not administered the composition. In other embodiments, from about 25% to 100% of the eggs laid by poultry administered the composition will have a specific gravity of at least 1.081, at least 1.082, at least 1.083, at least 1.084, or at least 1.085.

[0010] In one aspect, the present disclosure provides a method for increasing the growth rate, the feed conversion ratio, or the survivability of an animal comprising administering to the animal an effective amount of a composition comprising an animal feed and a supplement, wherein the supplement comprises an antibody which binds specifically to an isolated PLA₂ peptide or a variant thereof, wherein the PLA₂ peptide comprises a fragment of 6 to 20 contiguous amino acids of a PLA₂ protein, wherein the variant has from one to five amino acid substitutions, additions, or deletions, wherein the antibody is present in an effective amount for increasing the growth rate of the animal administered the composition compared to the growth rate of the animal not administered the composition. In some embodiments, the growth rate, feed conversion ratio, and/or the survivability of the animal is increased at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30% or at least about 50% compared to the growth rate, feed conversion ratio, and/or survivability of an animal not administered the composition.

[0011] Also disclosed herein are compositions and methods to reduce the amount of phosphorus in manure. Compositions and methods for significantly reducing the phosphorus content of animal feces have been discovered. In one aspect, the present disclosure provides a composition comprising an animal feed and a supplement comprising an antibody, wherein the antibody is present in an effective amount for reducing the phosphorus content of feces from animals administered the composition compared to feces from animals not administered the composition. The antibodies may decrease the phosphorus content of the feces by at least at least 1%, at least 5%, at least 10%, at least 15% or at least 20% in comparison to feces from animals not administered the antibodies of the present invention. The antibodies will typically not reduce the fecal phosphorus by more than about 25%, more than about 50%, or more than about 75%.

[0012] The antibody may be administered by any methods known to those skilled in the art and may be conveniently administered by feeding it to the animals in food. In some embodiments, the antibody supplement comprises an egg preparation, e.g. a powdered egg preparation, which comprises the antibody. Antibodies described herein can be administered by injection or by oral delivery, and may be administered in combination with a suitable carrier of the type commonly used in delivery of pharmaceuticals or nutritional supplements. Injection methods include, but are not limited to, subcutaneous, intraperitoneal, intramuscular, or intravenous injection. Oral administration, can include, but is not limited to, administration in tablet or powder form. Most preferably, the agent is fed directly by mixing with feed or by coating feed particles as described in U.S. Pat. No. 5,725,873.

[0013] In one method, antibodies are prepared as follows. A producer animal is immunized with a peptide or protein, such as any of the peptides of SEQ ID NOS: 1-28 or variants thereof or full-length recombinant PLA₂, against which antibodies are desired so that the producer animal produces an antibody to the peptide or protein. A substance containing the antibody is obtained from said producer animal. The antibody can be subject to further purification if desired or can be used without further preparation in an animal feed.

BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 presents data showing the improved eggshell quality (as indicated by specific gravity) of eggs laid by chickens administered the compositions of the present invention. The data compare the percentage of eggs having a particular specific gravity before (0 weeks) and after (4 weeks) administration of the composition.

DETAILED DESCRIPTION

[0015] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the invention are described below in various levels of detail in order to provide a substantial understanding of the present invention.

[0016] In practicing the present invention, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. These techniques are well-known and are explained in, e.g. , Current Protocols in Molecular Biology, VoIs. I-III, Ansubel, Ed. (1997); Sambrook et ah, Molecular Cloning: A Laboratory Manual, Second Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989); DNA Cloning: A Practical Approach, VoIs. I and II, Glover, Ed. (1985); Oligonucleotide Synthesis, Gait, Ed. (1984); Nucleic Acid Hybridization, Hames & Higgins, Eds. (1985); Transcription and Translation, Hames & Higgins, Eds. (1984); Animal Cell Culture, Freshney, Ed. (1986); Immobilized Cells and Enzymes (IRL Press, 1986); Perbal, A Practical Guide to Molecular Cloning; the series, Meth. EnzymoL, (Academic Press, Inc., 1984); Gene Transfer Vectors for Mammalian Cells, Miller & Calos, Eds. (Cold Spring Harbor Laboratory, NY, 1987); and Meth. EnzymoL, VoIs. 154 and 155, Wu & Grossman, and Wu, Eds., respectively.

[0017] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. For example, reference to "an antibody" includes a combination of two or more antibodies, and the like.

[0018] As used herein, the "administration" of an agent or drug to a subject or subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including orally, intranasally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, or topically. Administration includes self-administration and the administration by another. In particular embodiments, the antibody supplements disclosed herein are administered orally to animals, e.g., in animal feed, or to fish and shellfish, e.g., in fish or shellfish food.

[0019] As used herein, the term "amino acid" includes naturally-occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally-occurring amino acids. Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally-occurring amino acid, i.e., an α-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally-occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally- occurring amino acid. Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0020] As used herein, the term "animal" refers to a vertebrate or invertebrate animal, including, but not limited to, mammals, reptiles, birds, and fish. In some embodiments, the animal is a mammal, including, without limitation, bovine, ovine, and porcine. In some embodiments, the animal is a bird, including any of the species of poultry. In other embodiments, the animal is a fish, e.g., trout, tilapia, perch, catfish, salmon, bass, sunfish, and baitfish. In some embodiments, the animal is a shellfish, e.g., shrimp, prawns, mussels, clams, oysters, lobster, crab, and crayfish. [0021] As used herein, the term "antibody" means a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen, e.g., PLA₂ or a PLA₂ peptide. Use of the term antibody is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding fragments thereof. The term "antibody" includes bispecifϊc antibodies and multispecifϊc antibodies so long as they exhibit the desired biological activity or function.

[0022] As used herein, the term "effective amount" or "pharmaceutically effective amount" or "therapeutically effective amount" of a composition, is a quantity sufficient to achieve a desired biological effect, e.g., an increase in an animal's growth rate or survival, a decrease in the animal's feces phosphorus content, or an increase in eggshell specific gravity. Typically, the biological effect is measured in comparison to an animal not administered the composition. The amount of a composition of the invention administered to the animal will depend on such factors as general health, age, sex, body weight and tolerance to the composition. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions of the present invention can also be administered in combination with one or more additional supplements known in the art, e.g., antibiotics.

[0023] As used herein, the terms "identical" or "percent identity", when used in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region when compared and aligned for maximum correspondence over a comparison window or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site). Such sequences are then said to be "substantially identical." This term also refers to, or can be applied to, the complement of a test sequence. The term also includes sequences that have deletions and/or additions, as well as those that have substitutions.

[0024] As used herein, the term "immune response" refers to the concerted action of lymphocytes, antigen presenting cells, phagocytic cells, granulocytes, and soluble macromolecules produced by the above cells or the liver (including antibodies, cytokines, and complement) that results in selective damage to, destruction of, or elimination from the animal body of invading pathogens, cells or tissues infected with pathogens.

[0025] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. In a particular embodiment, the polypeptide is a PLA₂ polypeptide or a subsequence or variant thereof.

[0026] As used herein, the term "substitution" refers to variants that have at least one amino acid residue in the PLA₂ polypeptide or a fragment thereof replaced by a different residue. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as hydrophobic, polar, acidic or basic side chains. Conservative substitution tables providing functionally similar amino acids are well known in the art. For example, the following six groups each contain amino acids that are conservative substitutions for one another: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur- containing: Methionine (M), Cysteine (C); Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E); Polar: Serine (S), Threonine (T), Asparagine (N), Glutamine (Q).

[0027] As used herein, the term "survivability" refers to the survival rate of the animals {i.e., fish or livestock) which remain alive throughout the period of interest. In some embodiments, the "survivability" of fish may refer to fish which remain alive from the juvenile period until adulthood, or until they are harvested for food purposes.

[0028] As used herein, the term "poultry" refers to any domesticated birds raise primarily for meat, egg, and/or feather production, including, but not limited to chickens, turkeys, ducks, geese, guinea fowl, pigeons, pheasants, quail, other game birds, ostriches, emus, swans, and peafowl.

Peptide Immunogens

[0029] In one aspect, present disclosure provides immunogens that are PLA₂ peptide fragments or variants thereof. As used herein, the term "PLA₂ peptide" refers to an immunologically active fragment of a full length PLA₂ protein and includes PLA₂, homo logs (such as mammalian orthologs) and isoforms, unless otherwise noted. A peptide fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 or 39 amino acids in length.

In some embodiments, the PLA₂ peptide fragments are derived from animal PLA₂ polypeptide sequences, such as fish or livestock PLA₂ polypeptides. In an illustrative embodiment, the PLA₂ peptide fragments are derived from porcine PLA₂. The full-length sequence porcine PLA₂ is available at GenBank Accession No. NP 001004037 (SEQ ID NO: 29). In some embodiments, PLA₂ polypeptides useful in the methods and compositions of the present invention include, but are not limited to: chicken (GenBank Accession No. NP OOl 138961, SEQ ID NO: 30), bovine (GenBank Accession No. Q56JZ2, SEQ ID NO: 31); rabbit (Gen Bank Accession No. P14422, , SEQ ID NO: 32), human (GenBank Accession No. NP 000919, SEQ ID NO: 33); hamster (GenBank Accession No. CAB62564, SEQ ID NO: 34), monkey (GenBank Accession No. XP 001088684, SEQ ID NO: 35); dog (GenBank Accession No. NP 001003320, SEQ ID NO: 36); chimpanzee (GenBank Accession No. XP OOl 160305, SEQ ID NO: 37); horse (GenBank Accession No. XP 001075312, SEQ ID NO: 38); sheep (GenBank Accession No. P14419, SEQ ID NO: 39); rat (GenBank Accession No. NP l 13773, SEQ ID NO: 40); mouse (GenBank Accession No. NP 035237, SEQ ID NO: 41); sea snake (GenBank Accession No. BAC03245, SEQ ID NO: 42); and opossum (GenBank Accession No. XP 001376870, SEQ ID NO: 43). Fish and shellfish PLA₂ polypeptides useful in the methods and compositions of the present invention include, but are not limited to: zebra finch (GenBank Accession No. XP 002199575, SEQ ID NO: 44); seabass (GenBank Accession No. CAA10765, SEQ ID NO: 45); red seabream (GenBank Accession No. BAB47142, SEQ ID NO: 46); starfish (GenBank Accession No. BAA37115, SEQ ID NO: 47); and mummichog (GenBank Accession No. AAU50527, SEQ ID NO: 48). [0030] As used herein, the term "variants" includes PLA₂ peptides where residues have been altered with respect to the native sequence. For example, it may be desirable to improve the biological properties of the peptides, such as immunogenicity. In some embodiments, variants PLA₂ peptides differ from the native sequence by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, or 20 amino acids. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of, residues within the amino acid sequences of the PLA₂ peptides. Any combination of deletion, insertion, and substitution is made to obtain the PLA₂ peptide of interest, as long as the obtained peptide possesses the desired properties, i.e., immunogenicity. In some embodiments, the PLA₂ peptide has at least 75%, 80%, at least 85%, at least 90%, at least 95%, at least 97% sequence identity to the corresponding fragment from the native PLA₂ protein. Amino acid sequence variants of PLA₂ peptides may be prepared by introducing appropriate nucleotide changes into the nucleic acid encoding the peptide, or by peptide synthesis.

[0031] In some embodiments, the sequence modifications of the PLA₂ peptides include addition of one or more cysteine or lysine residues may be added to the N- and/or C-terminus of the PLA₂ peptides. Such residues provide side chains for linkages to other peptides or chemical groups, via e.g., disulfide bonds. In other embodiments, the sequence modifications of the PLA₂ peptides include the addition of a CSGS or SGSC sequence to either the N- or C- terminus, respectively, of the PLA₂ fragment, as in SEQ ID NOS: 2 and 3.

[0032] In particular embodiments, the PLA₂ peptide immunogens have a sequence according to any one of SEQ ID NOS: 1-28 shown in Table 1, or variants thereof having 1, 2, or 3 amino acid substitutions, deletions, or insertions.

TABLE 1. PLA₂ Peptide Immunogens

[0033] A peptide segment can be chemically synthesized which corresponds precisely with the amino acid sequence of the region of interest of the PLA₂ protein being studied and the peptide may be coupled to a carrier protein and injected into a suitable host to obtain antibodies.

[0034] Peptides may be synthesized according to any methods known in the art. For example, Fmoc synthesis technology may be used for the synthesis. Wang Fmoc resins are added into the synthesis well. After de-protection (removing the Fmoc protection group), an Fmoc amino acid is coupled onto the resins. The resins are then de-protected again before adding the next Fmoc amino acid. The process is repeated according to the desired peptide sequence until the last amino acid is coupled. After all amino acids are coupled onto the resins, the peptides are de-protected to remove Fmoc and any other protected side group. The peptides are then cleaved off from the resins, precipitated with ether, washed, and dried. The purification of peptides may be performed using a preparative HPLC and the MW of the peptide may be measured on a mass spectrometer.

Methods of Preparing Antibodies

[0035] Initially, a target peptide is chosen to which the antibodies of the invention can be raised (see previous section). Techniques for generating antibodies directed to target polypeptides are well known to those skilled in the art. Target polypeptides within the scope of the present invention include any polypeptide or polypeptide derivative which is capable of exhibiting antigenicity. Examples include, but are not limited to PLA₂, peptides, polypeptides, and fragments thereof.

[0036] It should be understood that not only are naturally-occurring antibodies suitable as antibodies for use in accordance with the present disclosure, but recombinantly engineered antibodies and antibody fragments, e.g., antibody-related polypeptides, which are directed to PLA₂ polypeptide and fragments thereof are also suitable. Antibodies that can be subjected to the techniques set forth herein include monoclonal and polyclonal antibodies, and antibody fragments such as Fab, Fab', F(ab')₂, Fd, scFv, diabodies, antibody light chains, antibody heavy chains and/or antibody fragments. Methods useful for the high yield production of antibody Fv-containing polypeptides, e.g., Fab' and F(ab')₂ antibody fragments have been described. See U.S. Pat. No. 5,648,237.

[0037] Methods of generating antibodies typically include immunizing a subject (generally a non-human subject such as a chicken, mouse or rabbit) with a purified PLA₂ polypeptide or fragment thereof. Any immunogenic portion of the PLA₂ polypeptide can be employed as the immunogen. An appropriate immunogenic preparation can contain, e.g., a recombinantly-expressed PLA₂ peptide or a chemically-synthesized PLA₂ polypeptide. An isolated PLA₂ polypeptide, or a portion or fragment thereof, can be used as an immunogen to generate a PLA₂ antibody using standard techniques for polyclonal and monoclonal antibody preparation. The full-length PLA₂ polypeptide can be used or, alternatively, the invention provides for the use of the PLA₂ polypeptide fragments as immunogens. For example, the PLA₂ polypeptide comprises at least four amino acid residues of the amino acid sequence shown in SEQ ID NO.: 29-48, and encompasses an epitope of the PLA₂ polypeptide such that an antibody raised against the peptide forms a specific immune complex with the PLA₂ polypeptide. Preferably, the antigenic peptide comprises at least 5, 8, 10, 15, 20, or 30 amino acid residues. Longer antigenic peptides are sometimes preferable over shorter antigenic peptides, depending on use and according to methods well known to those skilled in the art. Typically, the immunogen will be at least about 8 amino acyl residues in length, and preferably at least about 10 acyl residues in length. Multimers of a given epitope are sometimes more effective than a monomer. In some embodiments, the peptide is selected from the group consisting of SEQ ID NOs: 1-28.

[0038] If needed, the immunogenicity of the PLA₂ polypeptide (or fragment thereof) can be increased by fusion or conjugation to a hapten such as keyhole limpet hemocyanin (KLH) or ovalbumin (OVA). Many such haptens are known in the art. One can also combine the PLA₂ polypeptide with a conventional adjuvant such as Freund's complete or incomplete adjuvant to increase the subject's immune reaction to the polypeptide. Various adjuvants used to increase the immunological response include, but are not limited to, Freund's (complete and incomplete), mineral gels (e.g., aluminum hydroxide), surface active substances (e.g., lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, etc.), human adjuvants such as Bacille Calmette-Guerin and Corynebacterium parvum, or similar immunostimulatory compounds. These techniques are standard in the art.

[0039] Following appropriate immunization, the PLA₂ binding agent, e.g., anti- PLA₂ polyclonal antibody can be prepared from the subject's serum, or for chickens, from the egg. If desired, the antibody molecules directed against the PLA₂ polypeptide can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.

[0040] In one embodiment, the antibody is an anti-PLA₂ monoclonal antibody. For preparation of monoclonal antibodies directed towards a particular PLA₂ polypeptide, or derivatives, fragments, analogs or homologs thereof, any technique that provides for the production of antibody molecules by continuous cell line culture can be utilized. Such techniques include, but are not limited to, the hybridoma technique (see, e.g., Kohler & Milstein, 1975. Nature 256: 495-497); the trioma technique; the human B-cell hybridoma technique (see, e.g., Kozbor, et ah, 1983. Immunol. Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see, e.g., Cole, et ah, 1985. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).

[0041] The method of Tokoro (U.S. Pat. No. 5,080,895), as exemplified in Cook (U.S. Pat. No. 6,383,485), can be used to produce a preparation of egg-yolk antibodies. Laying hens can be inoculated with an immunogen, e.g., full-length PLA₂ or a PLA₂ peptide or variant thereof. Typically, a suitable adjuvant is administered in conjunction with the immunogen to enhance the immunization. An adjuvant useful for this purpose is a water-in-oil emulsion adjuvant such as complete Freund's adjuvant. In some embodiments, the immunogen causes the hens to produce anti-PLA₂ antibodies which are transferred into the egg yolk of eggs laid by the hens. [0042] An egg preparation, e.g., egg yolks or whole eggs, containing the antibody can be collected and homogenized to form an emulsion. The resulting emulsion can be dried to form a powder containing the antibody. This powder can then be formulated in a manner appropriate to the administration route and then administered to the desired animals using methods known in the art. The preparation is typically administered orally, e.g., as a supplement to the animal's diet. It is well within the skill in the art to adjust the amount of antibody administered to the animal based on particular application, the results desired, and the guidance provided herein.

[0043] In certain embodiments the antibody supplements are added to animal or fish feed. The main ingredients used in commercially prepared feed are the feed grains, which include corn, soybeans, sorghum, oats, and barley. Micro-ingredients such as vitamins, minerals, chemical preservatives, antibiotics, fermentation products, and other essential ingredients may be added to the feed preparation.

Methods for Improving Growth Rate, Feed Conversion Ratio and/or Survivability of Fish and Shellfish

[0044] In accordance with one aspect of the present invention, antibodies are added to feed to improve the growth rate, feed conversion ration and/or survivability of fish and shellfish. Antibodies described herein can be administered by injection or by oral delivery, and may be administered in combination with a suitable carrier of the type commonly used in delivery of pharmaceuticals or nutritional supplements. Injection methods include, but are not limited to, subcutaneous, intraperitoneal, intramuscular, or intravenous injection. Oral administration, can include, but is not limited to, administration in tablet or powder form. Most preferably, the agent is fed directly by mixing with feed or by coating feed particles as described in U.S. Pat. No. 5,725,873, incorporated herein by reference in its entirety.

[0045] In one embodiment, the antibodies may be administered in fish or shellfish feed. Where the feed is provided to fish or shellfish having an aggressive feeding behavior, an egg powder comprising the anti-PLA₂ peptide antibody may be mixed directly with the fish feed. Because the fish consume the feed quickly, the antibody is unlikely to leech from the feed. However, in some other aquaculture species, e.g., yellow perch, shrimp, etc., feeding normally takes up to 30 minutes between the time feed is provided into the tanks and when the feed is completely consumed. This creates a potential problem, as the antibody may leech from the feed before it is consumed. Accordingly, in one embodiment, vegetable oil may be sprayed on the feed preparation. For example, a 2% solution of vegetable oil in water may be sprayed on the feed after the antibody powder has been mixed with the feed.

[0046] The amount of antibody administered is effective to increase feed efficiency and/or weight gain compared to fish or shellfish which have not been administered the antibody. Suitable antibodies include, for example, anti-PLA₂ peptide antibody and the like. The weight gain of the fish or shellfish to which the antibody is administered may range, e.g., from about 1% to about 30%, to about 50% or even to about 100%. For example weight gain can be at least 5%, at least 10%, at least 15%, at least 20%, or at least 25% compared to fish or shellfish that have not been administered an antibody as described herein. Likewise, the increase in feed efficiency in fish or shellfish administered the antibody may range from about 1% to about 5%, to about 10%, to about 15%, or even to about 20%, and can be at least 5%, at least 6%, at least 7%, at least 8%, at least 9% or even at least 10% in comparison to the untreated fish or shellfish. Likewise, the increase in survivability in fish and shellfish administered the antibody may range from about 1%, to about 5%, to about 10%, to about 15%, to about 20%, and can be at least 5%, at least 10%, or at least 30% in comparison to untreated fish or shellfish.

[0047] In another aspect of the invention, the methods include administering to a fish or a shellfish an antibody that enhances weight gain by at least 5%, improves feeding efficiency by at least 1%, or both, compared to untreated fish. In some embodiments the weight gain is enhanced by at least 10% and/or the increase in feed efficiency is at least 2%, at least 3%, at least 4% or at least 5%. Suitable antibodies include anti-PLA₂ petide antibodies and the like. In some embodiments, the antibody used is an anti-PLA₂ peptide antibody.

[0048] A wide variety of fish are suitable for use in methods described herein, including, but not limited to trout, tilapia, perch, catfish, salmon, bass, sunfish, and baitfish. Likewise, various shellfish may be used in methods described herein. Exemplary shellfish include, but are not limited to shrimp, prawns, mussels, clams, oysters, lobster, crab, and crayfish. The antibody may be administered by any methods known to those skilled in the art and may be conveniently administered by feeding it to the fish or shellfish in food. In some embodiments, the food comprises an egg preparation that comprises the antibody. [0049] In another aspect, the invention provides compositions containing an antibody of the invention. Thus, for example, there are provided compositions that include fish or shellfish food and an effective amount of an antibody that enhances weight gain by fish or shellfish by at least 10% or improve feeding efficiency of the fish or shellfish by at least 5% compared to fish or shellfish which have not been administered the antibody. Typically, the fish and shellfish food includes protein and fat. In some embodiments, the food includes by weight from 30% to 65% protein and from 5% to 25% fat. In other embodiments, the fish food includes by weight from 40% to 50% protein and from 10% to 20% fat. In certain embodiments, the fish food comprises an egg preparation that comprises the antibody, e.g., a powdered egg preparation is added in an amount from about 0.01% to about 10% by weight, from about 0.1 to about 5% by weight, from about 0.1% to about 3% by weight, from about 0.1% to about 2% by weight, from about 0.1% to about 1% by weight, or from about 0.15% to about 1% by weight. The amount of added egg preparation in the feed will vary according to the concentration of antibody in the egg preparation and the particular application. It is well within the skill in the art to select the appropriate amount for a particular application in view of the guidance provided herein. In some embodiments, the fish food composition may be sprayed with an oil to reduce product separation such as leeching. For example, the oil may be a vegetable oil that is added to a level of from about 0.5 wt % to about 5 wt % such as about 0.5 wt %, about 1 wt %, about 2 wt %, or about 5 wt % of the fish food composition. In some embodiments, the oil is suitably added at about 2 wt % in the final ration.

[0050] In some embodiments, synthetic or plant-derived lipids and protein may be substituted for fish oil and fish-meal protein in fish and shellfish food. Therefore, in some embodiments, the compositions do not contain any fish oil or fish-derived proteins. Canola oil and soybean meal are the most familiar plant-derived substitutions used in commercial fish feed, although flaxseed, hempseed, and soy oils, and wheat, corn, barley, oats, canola, and sunflower meals are also employed (K. S. Betts, Environmental Science and Technology, Feb. 12, 2004). For example, fish food containing soybean meal can trigger stress in many species of fish resulting in suppressed appetite, weakened immune function, intestinal damage, and physical alterations which damage the market value (Overturf et ah, Aquaculture America, p. 220 (2006)). Thus, fish food is typically formulated with large amounts of fish meal as the primary protein source, with fish oil as the nutritional lipid source. In the present compositions, the addition of antibodies allows the fish to better tolerate plant-derived food sources. Thus, compositions provided herein may include plant- derived proteins such as soybean-meal. In some embodiments, the amount of plant-derived protein ranges from about 1% to about 100% from about 1% to about 75%, and preferably from about 1% to about 50%. The compositions provided herein may include plant-derived lipids, such as rapeseed oil, in addition to or as a replacement for fish oil (see Bell et ah, Aquaculture 218: 515-528 (2003)). In some embodiments. The amount of plant-derived lipids ranges from about 1% to about 100% from about 1% to about 75%, and suitably from about 50% to about 100%.

[0051] One or more additional fish food supplements may be combined with an anti-PLA₂ antibody to generate a fish food composition. In one embodiment, the fish food composition may further comprise probiotics {e.g., Bacillus, Lactobacillus, Enterococcus, Carnobacteriam, Saccharomyces, or Candida). The fish food composition may also comprise prebiotics (e.g., fructooligosaccharides, lipopolysaccharides, and glucans). In addition, the fish food composition may include supplements such as nucleic acids, nucleotides, metabolites, enzymes, antibiotics, or other egg antibody products. In other embodiments, taurine, methionine, beta-glucans, hormones, or immunostimulants may be added to the fish food composition. Taurine, methionine, and beta-glucans have been used to boost growth in fish grown with diets high in plant materials (Gaylord et ah, 2007. Aquaculture 269: 514-525; Sealey et ah, 2006. Book of Abstracts Aquaculture America). Hormones have also been studied to increase growth and muscle mass in fish, although possibly this can only be effectively administered by injection (Simpson, et ah, 2004. General and Comparative Endocrinology 135: 324-333). Additionally, various immunostimulants have been proposed as feed supplements (Galindop-Villegas, J. & H. Hosokawa. 2004. Immunostimulants: Towards Temporary Prevention of Diseases in Marine Fish In: Advances en Nutricion Acuicola VII Memorias del VII Simposium Internacional de Nutricion Acuicola, 16-19 November 2004. Hermosillo, Sonora, Mexico) which could possibly have auxiliary benefits of increased growth.

Methods for Improving Livestock Performance

[0052] In accordance with one aspect of the present invention, antibodies are added to feed to improve the growth rate, and/or feed conversion ratio of livestock. In particular embodiments, the antibodies are added to feed to improve the growth rate and/or feed conversion ratio of chickens. In one embodiment, the antibodies may be administered in chicken feed. [0053] In accordance with one aspect of the present invention, antibodies are added to feed to improve the dietary mineral utilization of animals — including, but not limited to, utilization of phosphorus, calcium, magnesium, sodium, chloride, zinc, and selenium. For example, it was surprisingly discovered that the antibody compositions of the present invention improve the animal's utilization of dietary calcium and phosphorus. Increased mineral utilization by the animal may allow for reduction of mineral supplementation in animal feed. Improved mineral utilization may also help to protect the animal from mineral deficiency problems, e.g., rickets disease.

[0054] As a result of increased calcium utilization, the eggshell quality of eggs laid by the poultry is improved. One measure of eggshell quality is the specific gravity of the egg. Specific gravity is the unitless ratio of the density of an object to the density of water. As the specific gravity of an egg goes down, the number of cracks generally increase. Specific gravity gives the producer an idea of the probability of the eggs being cracked during handling. The specific gravity of an egg may be determined by weighing the egg and then dividing the weight by the volume of the egg. Specific gravity of an egg indicates the quantity of shell relative to other components of the egg. Therefore, differences in specific gravity among eggs of similar weights are mainly due to variations in the amount of shell. In another method to measure eggshell quality, one could break the egg, wash away excess albumin from the shell, let the shell dry and then weigh the eggshell. Also, when the eggshell is dry, thickness can be determined and used as a measure of eggshell quality.

[0055] In some embodiments, antibodies are added to poultry feed and the amount of calcium typically in the feed is concomitantly reduced, without any reduction in the specific gravity of the eggs laid by the poultry administered the feed. Thus, feed containing the antibodies of the present invention may comprise less calcium, yet still result in eggs that have a specific gravity equal to or better than eggs laid by poultry fed conventional feed mixtures with calcium. In some embodiments, the amount of calcium in feed compositions comprising the antibodies of the present invention is reduced by at least about 5%, at least about 10%, at least about 25%, or at least about 50%, but not more than about 90% or about 95%.

[0056] In some embodiments, the antibody replaces a growth promoting antibiotic (GPA) as a supplement in the animal feed, thus eliminating a major source of GPA contamination. GPA contamination is believed to result in antibiotic-resistant microbial agents which endanger public health.

Methods for Reducing Phosphorous Content of Manure

[0057] In accordance with one aspect, antibodies are added to feed to reduce the phosphorus content of feces excreted by animals, e.g., livestock. Excess phosphorus and antibiotics in the soil that result from manure spreading is a serious threat to the environment and even to public health. It has unexpectedly been discovered that the composition and methods described herein result in a remarkable decrease in the phosphorus content of manure from animals to which certain antibodies are administered. Although not wishing to be bound by theory, it is believed that the decreased phosphorus content of manure results from increased phosphorus absorption in the animal by lowering gastric inflammation with antibodies.

EXAMPLES

[0058] The present invention is further illustrated by the following examples, which should not be construed as limiting in any way.

Example 1 - aPLA? Feed Compositions Increase Specific Gravity of Chicken Eggs

[0059] Eggs (n=100) were collected randomly from a layer house and were moved to aOvaTech laboratory for specific gravity analysis. The layer house was then supplemented with aPLA₂ egg antibody product at 2.5 lb/ton. After 4 weeks, eggs (n=100) were collected randomly from the layer house and moved to the same lab for analyzing specific gravity. Three specific gravity solutions (1.075, 1.08, and 1.085) were prepared by dissolving salt in the same amount of water. Eggs were tested to determine if they floated or sank. Eggs were placed in the lowest specific gravity solution. If the egg floated, it was then moved up to the next higher specific gravity solution. The process was repeated until the egg was placed in a specific gravity solution in which it sank.

[0060] The results are shown in FIG. 1 and indicate that feeding aPLA₂ supplement for 4 weeks shifted the specific gravity distribution curve higher. In particular, the percentage of eggs having specific gravity >1.085 represents 34% at week 4 versus 11% at week 0. As such, the antibody compositions of the present invention are useful in methods for increasing the thickness of eggshells.

Example 2 - Preparation of PLA? Peptide Antibody Compositions

[0061] Chickens (laying hens) were injected intramuscularly with 0.6 mg PLA₂ peptide KC 17 (Alpha 1) (SEQ ID NO: 1) in complete Freund's adjuvant. Chickens received two subsequent boosts of the same amount of KC17 in Freund's adjuvant at two week intervals. Three weeks after the last boost, liquid whole eggs containing a KC 17 antibody (aKC17) were collected. Liquid whole eggs were dried to a powder and added to animal feed. The peptides were synthesized using conventional solid phase synthesis.

Example 3 - aPLA? and aKC17 Antibody Compositions Reduce Phosphorus Content of Chicken Feces

[0062] The effects of aPLA₂ and aKC17 antibodies on the phosphorus content of chicken feces were investigated. A chick trial was conducted in a battery cage of 16 pens with 4 pens per treatment group. Treatment groups were: (1) control egg powder; (2) T300 (ratio of 29% aPLA₂ egg powder/71% control egg powder); (3) T900 (ratio of 87% aPLA₂ egg powder/13% control egg powder); and (4) aKC17 (ratio of 13% aKC17 egg powder/87% control egg powder). The feed is formulated by adding 0.1% egg powder to chick mash diet comprising: corn (63%), soybean meal (30.2%), salt (0.5%), fat (2.8%), dicalcium phosphate (1.59%), calcium carbonate (1.48%), Dl- methionine (0.18%), vitamins and trace mineral (0.25%). After 3 weeks, the fecal samples were sent to the Soil and Plant Analysis Lab at UW-Madison for analysis of total phosphorus analysis in manure samples.

[0063] The results are shown in Table 2. All treatment groups showed a decreased phosphorus content of manure relative to the control group. The T300, T900, and aKC17 treatment groups showed 92%, 90%, and 87% of the total phosphorus relative to the control, respectively. The aKC17 treatment group showed the greatest reduction in phosphorus content. These results suggest that antibodies raised against the KC 17 peptide are more effective at reducing phosphorus content than antibodies raised against the full length PLA₂ protein.

Example 4 - Effects of Peptide Antibodies on Growth Rate and Feed Conversion Ratio in Chickens

[0064] The effects of aPLA₂ and peptide antibodies on the growth rate and feed conversion ratio (FCR) of chickens were investigated. A chicken trial was conducted with 120 birds (3 birds per cage, 5 cages per treatment). Treatment groups were: (1) control egg powder; (2) aPLA₂ egg powder; (3) KC 17 egg powder; and (4) DS 14 egg powder; (5) alpha-3 egg powder; (6) alpha-4 egg powder; (7) alpha-5 egg powder and (8) gamma- 1 egg powder. The feed is formulated by adding 0.1% egg powder to chick mash diet comprising: corn (63%), soybean meal (30.2%), salt (0.5%), fat (2.8%), dicalcium phosphate (1.59%), calcium carbonate (1.48%), Dl- methionine (0.18%), vitamins and trace mineral (0.25%). The amount of feed given to the chickens was recorded. The results are shown in Table 3. In this trial, anti-gamma 1 antibody has 6.2% improvement in weight gain and is significant at p<0.08. Improvements by aKC17 were 2.2% on gain and 3.1 % on feed efficiency. Improvements on alpha5 was 2.4% on gain and 2.3% on feed efficiency.

[0065] In a second trial, the effects of aPLA₂ and peptide antibodies on the growth rate and feed conversion ratio (FCR) of chickens were investigated. A chicken trial was conducted with 120 birds (3 birds per cage, 5 cages per treatment). Treatment groups were: (1) control egg powder; (2) aPLA₂ egg powder; (3) KC17 egg powder; and (4) DS14 egg powder; (5) alpha-3 egg powder; (6) alpha-4 egg powder; (7) alpha-5 egg powder and (8) gamma- 1 egg powder. The feed is formulated by adding 0.1% egg powder to chick mash diet comprising: corn (63%), soybean meal (30.2%), salt (0.5%), fat (2.8%), dicalcium phosphate (1.59%), calcium carbonate (1.48%), Dl- methionine (0.18%), vitamins and trace mineral (0.25%). The amount of feed given to the chickens was recorded. The results are shown in Table 4. In this trial, anti-gamma 1 antibody has 11.3% improvement in weight gain (p<0.12) and significant improved feed/gain at 4% at p<0.02. Improvements by alpha 5 were 5.9% on gain and 2.1% on feed efficiency. aPLA2 gave a boost of 7.6% gain increase and improved feed/gain (p<0.07).

Example 5 - Effects of Peptide Antibodies on Growth Rate and Feed Conversion Ratio in Fish and Shellfish

[0066] A blind study is conducted to test the effects of anti-PLA₂ peptide antibodies on juvenile rainbow trout growth. The study includes three treatment groups: control, 0.15% and 0.30% anti-PLA₂ antibody powder produced as in U.S. Patent No. 6,383,485. Each antibody is prepared from a fragment or variant of a PLA₂ polypeptide, e.g., any of the fragments of SEQ ID NOs: 1-28. Each treatment group involves six 115-L tanks, each stocked with 12 fish (~25 g initial weight) for a total of 18 tanks. The feed is extruded Silver Cup steelhead diet (45% protein, 16% fat). The fish are carefully fed to apparent satiation once daily, and total feed input to each tank was recorded.

[0067] It is predicted that the anti-PLA₂ peptide antibodies are a very effective stimulator of growth in rainbow trout. The antibodies are also predicted to improve the FCR and surviviability of the trout. As such, feed comprising the anti-PLA₂ antibody is useful in methods to improve feeding efficiency and/or weight gain in fish or shellfish administered the composition.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All nucleotide sequences provided herein are presented in the 5' to 3' direction.

[0069] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed.

[0070] Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification, improvement and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications, improvements and variations are considered to be within the scope of this invention. The materials, methods, and examples provided here are representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention.

[0071] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0072] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0073] All publications, patent applications, issued patents, and other documents referred to in the present disclosure are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Claims

CLAIMSWhat is claimed is:

1 . An isolated antibody that binds specifically to a PLA₂ fragment or a variant thereof, wherein the PLA₂ fragment is from 6 to 20 contiguous amino acids of a PLA₂ protein, and the variant has one or two conservative or non-conservative amino acid substitutions.

2. The isolated antibody of claim 1 , wherein the PLA₂ protein is selected from the group consisting of: SEQ ID NOs: 29-48.

3. The isolated antibody of claim 1 , wherein the PLA₂ fragment or a variant thereof is selected from the group consisting of: SEQ ID NOs: 1-28.

4. An isolated peptide having the amino acid sequence selected from the group consisting of: SEQ ID NOs: 1-28.

5. A peptide immunogen comprising the peptide of claim 4 conjugated to a high molecular weight carrier protein.

6. The peptide immunogen of claim 5 wherein the high molecular weight carrier protein is selected from the group consisting of: keyhole limpet hemocyanin (KLH), ovalbumin, and bovine serum albumin.

7. A method of producing antibodies comprising immunizing an animal with the peptide immunogen of claim 4 combined with an acceptable adjuvant.

8. The method of claim 7, wherein the animal is a chicken.

9. A method for enhancing the growth rate, feed conversion ratio, or survivability of an animal, the method comprising administering to the animal an effective amount of an antibody that binds specifically to a PLA₂ fragment or variant selected from the group consisting of SEQ ID NOs: 1-28.

10. The method of claim 9, wherein the animal is a vertebrate or invertebrate animal.

11. The method of claim 10 ,wherein the animal is mammal.

12. The method of claim 11 , wherein the animal is a bovine, ovine, or porcine animal.

13. The method of claim 10, wherein the animal is a bird.

14. The method of claim 10, wherein the animal is a fish or shellfish.

15. The method of claim 14, wherein the fish or shellfish is selected from the group consisting of: trout, tilapia, perch, catfish, salmon, bass, sunfish, baitfish, shrimp, prawns, mussels, clams, oysters, lobster, crab, and crayfish