EP0717629A1 - Novel eimeria antibodies and antigens and methods of using the same - Google Patents

Abstract

Novel stage-specific and intracellular protective Eimeria antigens are disclosed. The antigens are identifiable using antibody preparations produced locally, at the site of parasitic infection. The antibodies and antigens are useful as diagnostic reagents to detect coccidiosis as well as to determine the state of immunity of a particular avian subject. The antibodies and antigens are also useful in vaccine compositions to provide protection against Eimeria infection.

Description

NOVEL EIMERIA ANTIBODIES AND ANTIGENS AND METHODS OF

USING THE SAME

Technical Field The present invention relates generally to the field of parasitology. More particularly, the invention relates to novel antibodies and antigens from Eimeria and methods of using and detecting the same.

Background of the Invention Avian coccidiosis is an enteric, parasitic disease of domestic and wild bird species caused by members of the protozoan genus Eimeria. The disease is spread by ingestion of sporulated oocysts from feces of an infected host. The infectious process is rapid and characterized by parasite replication in host cells, causing extensive damage to the intestinal mucosa. The avian eimerian life cycle is complex and occurs in two stages - the exogenous phase which takes place in the litter, and the endogenous phase, which occurs in the intestinal tract of the host. In typical field operations, newly hatched chicks ingest infectious, sporulated oocysts from contaminated litter shortly after arrival in the broiler house. Narious stimuli interact to release infectious sporozoites within the intestinal tract which rapidly penetrate, in a species-specific manner, villous or surface epithelial cells of the gut mucosa. Intracellular sporozoites then undergo genetically programmed asexual reproduction, or schizogony, to form merozoites bounded by a defined membrane or parasitophorous vacuole. The merozoites erupt to invade adjacent intestinal cells and reinitiate merogony (asexual development). At each point during the endogenous eimerian life cycle, different stage-specific antigens become the potential targets of host protective B and T-cell immune responses. Thus, active infection is able to generate a natural immune response in some animals. However, there is no practical method for determining whether a particular animal is indeed immune to infection, which can only be confirmed by visually inspecting the intestinal tract of a sacrificed bird.

In the absence of a protective host immune response, a few infectious events and exponential amplification of the number of parasites rapidly leads to clinical coccidiosis, manifested by surface and crypt cell loss, malsorption, diarrhea, and dehydration. Sexual gametogony then occurs, resulting in the continual shedding of oocysts in the fecal material and the spread of further infection. The disease, therefore, causes acute morbidity and results in economic losses to the poultry industry through decreased growth and feed utilization. Previous methods of immunization against coccidiosis have involved the use of inactivated, attenuated and precocious forms of parasite material. For example, both viable and attenuated oocysts from Eimeria species have been used as a method of inducing an immune response. See, e.g., Joyner, L.P. and Norton, C.C. Parasitology (1973) 62:333-340; Galmes, M.M. et al. Ann. Parasitol. Hum. Comp. (1991) £6:144X48; Jenkins et al. Infect. Immun. (1991) 52:4042-4048. However, effective immunization with such material is tentative and the production and delivery of these preparations can be problematic. Side effects, such as reversion to virulent forms, have also been encountered. Anύ-Eimeήa polyclonal and monoclonal antibodies have been produced, both for immunization purposes and in an effort to elucidate events occurring during the various developmental stages of the parasite.

For example, immunization using crude globulin fractions prepared from chickens infected with oocysts of Eimeria species has been described (see, e.g., Rose, M.E. Parasitology (1974) 6&285-292; Long, P.L. and Rose, M.E. Parasitology (1972) 65:437-445; Rose, M.E. Parasitology (1971) 62:11-25; Rose, M.E. and Long, P.L. Parasitology (1971) 62:299-313) and passive immunization using monoclonal antibodies produced against E. tenella merozoites and sporozoites has been suggested (see, e.g., EPA Publication Nos. 135,073 and 135,712, published 27 March 1984 and 3 April 1985, respectively). Danforth, H.D. Am. J. Vet. Res.

(1983) 44:1722-1727 produced monoclonal antibodies to E. tenella in order to study developmental stages of the organism and effects on sporozoite penetration and intracellular development. All of the above-described antibody preparations were prepared using conventional techniques. However, the production of antibodies at the local site of Eimeria infection has not heretofore been described and it now appears that a local response with involvement of the secretory immune system and/or cell mediated immunity, is involved in protective immunity to the parasite.

Subunit vaccine preparations have been developed in an attempt to alleviate the problems inherent in the use of the above-described preparations. For example, EPA Publication No. 453,055 (published 23 October 1991 ) describes multicomponent vaccine compositions including mixtures of a 25 kDa E. tenella antigen, a 26 kDa E. necatrix antigen or a 55 kDa E. maxima antigen, derived from Eimeria oocysts. EPA Publication No. 256,536 (published 24 February 1988) describes the isolation of E. maxima macrogametocytes and microgametocytes and vaccines comprising heterogenous protein extracts derived from the gametocytes. Similarly, EPA Publication No. 167,443 (published 8 January 1986) describes the use of heterogenous extracts, containing at least 15 polypeptides, from sporozoites and sporulated oocysts of E. tenella for immunizing chickens. Finally, ΕPA Publication No. 164,176 (published 11 December 1985) and Australian Patent Publication No. 87/199,027 (published 4 June 1987) describe the isolation of a 25 kDa protein composed of two polypeptide chains having molecular masses of 17 kDa and 8 kDa, respectively, from E. tenella sporocysts and use of the same in a vaccine composition. The gene encoding the protein has also been cloned and sequenced.

All of these subunit vaccines have been derived from the extracellular parasitic forms of Eimeria, i.e., the extracellular sporozoite and merozoite forms of the organism. Similarly, previously identified Eimeria genes have been isolated and characterized based on antibody probes raised against the extracellular parasitic forms. However, it appears that acquired protective immunity is associated with the developing asexual stages and that intracellular sporozoite metabolism and/or early asexual development is mandatory for the transcription and translation of parasite- specific gene products recognized by the immune system. Accordingly, the identification, isolation, and biochemical characterization of these intracellular parasite metabolic products is desirable for the design of effective anticoccidial vaccines.

Disclosure of the Invention

The present invention is based on the development of novel Eimeria antibody preparations, produced locally or which traffic to, the site of parasitic infection. The antibodies so produced provide for the discovery of protective extracellular and intracellular Eimeria antigens in biological samples, including in culture systems which support high levels of parasite growth and development.

Intracellular antigens have not previously been identifiable using conventional cell culture techniques and polyclonal or monoclonal antibody preparations. The antigens and antibodies can be used in protective Eimeria vaccines. Diagnostic tests as well as bioassays to measure B- and T-cell dependent immune responses at the local site of parasite entry are also made possible by the present discoveries.

Accordingly, in one embodiment, the invention is directed to an isolated, locally generated, Eimeria antibody preparation. In particularly preferred embodiments, the antibody preparation comprises cecal lymphocyte immune products (CLIP), splenic lymphocyte immune products (SLIP), rectal antibody test (RAT) or cage dropping antibody test (CD AT). In another embodiment, the invention is directed to a method for detecting the presence or absence of an Eimeria antigen in a biological sample, the method comprising:

(a) contacting the biological sample with a locally generated, Eimeria antibody preparation under conditions whereby a complex is capable of being formed between an antigen present in the biological sample and an antibody present in the antibody preparation; and

(b) detecting any complexes formed using a revealing label.

In yet another embodiment, the subject invention is directed to a method for detecting the presence or absence of an Eimeria tenella antigen in a biological sample, the method comprising:

(a) contacting the biological sample with at least one locally generated, Eimeria tenella antibody preparation selected from the group consisting of CLIP, SLIP, RAT and CD AT, under conditions whereby a complex is capable of being formed between an antigen present in the biological sample and an antibody present in the antibody preparation; and

(b) detecting any complexes formed using a revealing label.

In another embodiment, the invention is directed to a method for diagnosing coccidiosis infection in an avian subject, the method comprising: (a) providing a biological sample from the avian subject;

(b) contacting the biological sample with a locally generated, Eimeria antibody preparation under conditions whereby a complex is capable of being formed between an antigen present in the biological sample and an antibody present in the antibody preparation; and (c) detecting any complexes formed using a revealing label.

In still a further embodiment, the invention is directed to an intracellular Eimeria antigen, identifiable using a locally generated, Eimeria antibody preparation. In particularly preferred embodiments, the antigen has a molecular weight of approximately 28 kDa, 35 kDa, 38 kDa, 40 kDa, 43 kDa, 55 kDa, 70 kDa, 100 kDa or 110 kDa, as determined by Western immunoblot analysis.

In another embodiment, the invention is directed to a method for detecting the presence or absence of an Eimeria antibody in a biological sample, the method comprising:

(a) contacting the biological sample with an intracellular Eimeria antigen under conditions whereby a complex is capable of being formed between the antigen and an antibody present in the biological sample; and

(c) detecting any complexes formed using a revealing label. In still further embodiments, the invention is directed to a kit for diagnosing coccidiosis in an avian subject, the kit comprising a locally generated, Eimeria antibody preparation, packaged in a suitable container.

In another embodiment, the invention is directed to a kit for diagnosing coccidiosis in an avian subject, the kit comprising an Eimeria tenella monoclonal antibody reactive with an intracellular Eimeria antigen, packaged in a suitable container.

In another embodiment, the invention is directed to a kit for detecting the presence or absence of antibodies to Eimeria in a biological sample, the kit comprising an intracellular Eimeria antigen, packaged in a suitable container.

These and other embodiments of the subject invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

Detailed Description The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (MJ. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984); Animal Cell Culture (R.K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series, Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); and Handbook of Experimental Immunology, Vols. I-IV

(D.M. Weir and C.C. Blackwell eds., 1986, Blackwell Scientific Publications).

All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.

A. Definitions

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. An "Eimeria antigen" refers to a molecule derived from an Eimeria species which contains one or more epitopes that will stimulate a host's immune system to make a secretory, humoral and/or cellular antigen-specific response. For purposes of the present invention, antigens can be derived from any of the known Eimeria species, the choice of species being dependent on the host and coccidial disorder to be treated. By way of example, domestic fowl (Gallus domesticus) can be infected by any of E. tenella, E. necatrix, E. brunetti, E. maxima, E. acervulina and E. praecox. Turkeys (Meleagris) are susceptible to infection by E. melagrimitis, E. dispersa, E. meleagήdis, E. gallopavonis, E. adenoides, E. innocua and E. subrotunda. Domestic and wild ducks (Anas) suffer from infections caused by E. anatis and geese (Anser) can be infected by E. anseris, E. nocens, E. parvula, E. hermani, E. striata and E.fulva. Antigens of the present invention can therefore be identified and derived from any of the above species.

Furthermore, for purposes of the present invention, an "Eimeria antigen" includes antigens substantially homologous and functionally equivalent to the corresponding native Eimeria antigen. Thus, the term "Eimeria antigen" encompasses modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequences. Such modifications of the primary amino acid sequence may result in proteins which have enhanced or decreased activity as compared to the native sequence. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens. All of these modifications are included, so long as the molecule remains capable of eliciting an immunological response, as defined below, and activity is not destroyed.

Additionally, an "Eimeria antigen" denotes a protein which may be modified by combination with other biological materials, such as lipids and saccharides, or by side chain modification, such as acetylation of amino groups, phosphorylation of hydroxyl side chains including phosphorylation of tyrosine, serine, threonine or any other side chains, whether or not these residues are normally phosphorylated in the native molecule, or oxidation of sulfhydryl groups, as well as other modifications of the encoded primary sequence. Thus, included within the definition are glycosylated and unglycosylated forms, the amino acid sequences with or without associated phosphates, and amino acid sequences substantially homologous to the native sequence which retain immunological activity.

By "intracellular antigen" is meant an Eimeria antigen, as described above, which is expressed during intracellular stages of parasite development, i.e., during asexual development within the villous or epithelial cells of the intestinal mucosa. The intracellular stage includes intracellular sporozoite metabolism, trophozoites and asexual development into multinucleate schizonts or meronts. Accordingly, intracellular antigens produced during these events are covered by the definition. The production of such intracellular antigens can be seen in cell lines adapted to support the growth of intracellular Eimeria spp. forms, including both primary and continuous cell lines. Representative cell lines are described further below. Thus, intracellular antigens are also referred to herein as "tissue culture derived antigens." The term "intracellular antigen" encompasses proteins which are both secreted into the cell culture media in which the parasite is developing, as well as those antigens which are retained within the cell, such as an antigen associated with the cell membrane, endoplasmic reticulum and so forth. An intracellular antigen can be either soluble or insoluble. An "extracellular antigen" is an Eimeria antigen which is produced during the extracellular stages of parasite development, i.e., the exogenous phase of the Eimeria lifecycle which occurs in the litter and the endogenous phases where the organism has not yet invaded and entered host cells. Such antigens include the extracellular sporozoite and merozoite forms of the organism. For purposes of the present invention, antigens of Eimeria spp., are sometimes identified below with reference to their molecular mass in kilodaltons (kDa). For example, an antigen having a molecular mass of about 35 kDa is identified herein as P35; an antigen of about 40 kDa in molecular mass is identified as P40, and so on. A "locally generated" antibody preparation is a composition containing one or more antibodies which are produced at, or traffic to, the local site of Eimeria infection. Examples of locally generated antibody preparations include antibody preparations derived from immune lymphocyte populations found within the intestinal tract of a previously or currently infected avian subject, such as those derived from any portion of the cecum or intestine and termed cecal lymphocyte immune products ("CLIP") herein; splenic lymphocyte immune products ("SLIP"), produced by trafficking memory splenic lymphocytes in infected avian subjects; coprantibody preparations (i.e., antibodies derived from fecal material) isolated from fecal material either directly from the intestinal tract or externally, of a previously infected or currently infected avian subject, such as rectal antibody test

("RAT") which is isolated from the intestinal digesta of the rectum of infected avian subjects, and cage dropping antibody test ("CDAT") which is derived from bird cage droppings of infected avian subjects.

An "isolated" antigen or antibody an antigen or antibody which is separate and discrete from a whole organism (live or killed) with which the protein sequence is normally associated in nature. Thus, an antigen contained in a cell free extract would constitute an "isolated" antigen, as would an antigen synthetically or recombinantly produced. Similarly, an "isolated" antibody preparation includes antibodies present in crude mixtures, blood, serum, etc., so long as the mixture is separate and discrete from the organism with which the antibodies are normally found. The term "isolated" encompasses both polyclonal and monoclonal antibody preparations. Additionally, the term "isolated" with respect to both antigens and antibodies, is not meant to imply a particular degree of purity. Thus, a crude extract is encompassed by the term, as is a highly purified preparation.

The terms "polypeptide" and "protein" are used interchangeably and refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the terms "polypeptide" and "protein" include oligopeptides, protein fragments, analogs, muteins, fusion proteins and the like.

The term "epitope" refers to the site on an antigen or hapten to which specific B cells and T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." An "immunological response" to a composition or vaccine is the development in the host of a secretory, cellular and/ or antibody-mediated immune response to the composition or vaccine of interest. Usually, such a response includes but is not limited to one or more of the following effects; the production of antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G or M; B cells; helper T cells; suppressor T cells; and or cytotoxic T cells and/or δ T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest.

Of particular interest is an immunological response wherein the production of immunoglobulin A (IgA) is stimulated, since this is the principle immunoglobulin produced by the secretory system of warm-blooded animals. In particular, avian species have a mucosal immune network consisting of gut-associated lymphoid tissue (termed GALT or Peyer's patches), bronchial-associated lymphoid tissue (BALT), and the harder gland, located ventrally and posteriomedially to the eyeball. Presentation of antigen to these tissues triggers proliferation and dissemination of committed B cells to the secretory tissues and glands in the body, with the ultimate production of secretory IgA (slgA). SIgA serves to block the colonization and invasion of specific surface antigens that colonize on, and pass through, a mucosal surface.

The terms "immunogenic" antigen or protein refer to an antigen or protein having an amino acid sequence which elicits an immunological response as described above. An "immunogenic" antigen or protein, as used herein, includes the full length sequence of the desired Eimeria protein or an immunogenic fragment thereof. By "immunogenic fragment" is meant a fragment of a polypeptide which includes one or more epitopes and thus elicits an immunological response, as defined above. Such fragments can be identified by, e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the Eimeria protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Patent No. 4,708,871; Geysen, H.M. et al. (1984) Proc. Natl. Acad. Sci. USA £1:3998-4002; Geysen, H.M. et al. (1986) Molec. Immunol. 22:709- 715, all incorporated herein by reference in their entireties. Such fragments will usually be at least about 2 amino acids in length, more preferably about 5 amino acids in length, and most preferably at least about 10 to 15 amino acids in length. There is no critical upper limit to the length of the fragment, which could comprise nearly the full length of the protein sequence, or even a fusion protein comprising fragments of two or more of the Eimeria antigens or one or more of the Eimeria antigens fused to, e.g., a bacterial, fungal, viral or protozoal protein.

Two polypeptide sequences are "substantially homologous" when at least about 65% (preferably at least about 80% to 90%, and most preferably at least about 95%) of the amino acids match over a defined length of the molecule. As used herein, substantially homologous also refers to sequences showing identity to the specified polypeptide sequence.

By "avian subject" is meant domestic, wild and game birds, including animals belonging to the order Galliformes, such as chickens, turkeys, pheasants, partridges, quail, grouse, guinea fowl and peacocks, as well as birds of the order Anseriformes, such as ducks and geese. The definition encompasses birds of all ages, including subjects in ovo.

As used herein, a "biological sample" refers to a sample of tissue or fluid isolated from an avian subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, respiratory, intestinal, and genitourinary tracts, blood cells, organs, egg constituents and also samples of in vitro cell culture constituents

(including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, recombinant cells, and cell components). A "biological sample" also refers to a sample taken from any of the various developmental stages of Eimeria spp., including samples of sporulated oocysts, infectious sporozoites, intracellular sporozoites, merozoites, gametocytes, and the like. The term "treatment" as used herein refers to both (i) the prevention of infection or reinfection (prophylaxis), and (ii) the reduction or elimination of symptoms (therapy) of coccidiosis.

B. General Methods

Central to the present invention is the development of novel antibody preparations, produced locally at the site of parasitic infection, and methods for the identification of parasite antigens using these preparations. The antibodies can be used in a variety of assays, including assays to identify antigens produced during the intracellular and extracellular phases of the eimerian life cycle. The antibodies also allow the detection and characterization of antigens appearing at particular time periods during the infective process as well as at particular sites of infection. The antibodies can be used as diagnostic reagents, to detect coccidiosis infection, as well as to determine host immunity levels; to immunopurify Eimeria antigens; and as screening agents to detect the presence of homologous genes in other medically important species.

Localized antibodies, produced in response to Eimeria infection, have not heretofore been reported. Furthermore, the identification of intracellular antigens has not previously been possible and such antigens are believed to be responsible for providing protective immunity against coccidiosis. The antigens so identified can be isolated, characterized and used in subunit vaccine compositions, thus avoiding problems inherent in prior attenuated and killed vaccine preparations. The identified antigens can also be used as diagnostic tools, for detecting Eimeria infection in biological samples and for determining the level of host immunity to the Eimeria species of interest.

Several classes of antibody preparations are described herein which are produced at, or traffic to, the local site of parasitic infection. In particular, described herein are cecal lymphocyte immune products ("CLIP"), produced by intestinal immune lymphocytes; and splenic lymphocyte immune products ("SLIP"), produced by trafficking memory splenic lymphocytes. Also disclosed are slg A containing coprantibody preparations derived from the intestinal digesta of Eimeria spp - immune birds. These antibody preparations are termed rectal antibody test ("RAT"), isolated from the intestinal digesta of the rectum; and cage dropping antibody test ("CDAT"), derived from bird cage droppings. More specifically, CLIP and SLIP antibody preparations can be derived from splenic and cecal lymphocytes isolated from an appropriate immune bird subject. Particularly useful subjects are inbred bird lines which have been treated to simulate natural immunity to coccidiosis infection, as described in the examples. Splenic and cecal lymphocytes can be isolated from the spleens and cecal pouches, respectively, of the bird subject and cultured in vitro in single cell suspension, using cell isolation and cultivation techniques known to those of skill in the art. In general, lymphocytes are cultivated for approximately five days after which time culture supernatants containing secreted Eimeria spp.-specific antibodies are removed, clarified by centrifugation, filtered, and stored at -20°C or lower. Antibody-containing supernatants can also be generated from in vitro cultivation of defined immune T- and B-cell lymphocyte subsets, or combinations thereof, using known techniques.

RAT and CD AT slgA-containing coprantibodies are isolated from the rectum digesta and from bird fecal droppings, respectively, of Eimeria spp.- immune birds. Exemplified herein is the isolation of RAT and CD AT from an inbred natural avian immune model. Avian slgA coprantibody samples may also be obtained from naturally exposed Eimeria spp. outbred broilers raised in wire batteries, floor pens, or broiler houses. To isolate the antibodies, wet fecal material is resuspended in any suitable physiological balance solution or media, such as phosphate-buffered saline (PBS), followed by agitation, the addition of protease inhibitors, and two or more successive centrifugations at low and moderate speeds. The antibody-containing solution is adjusted to physiological pH, filtered and stored at at least -20°C. Further purification of slgA can be achieved using ammonium sulfate precipitation, size and affinity chromatography or other biophysical methods readily known to those of skill in the art.

The antibody preparations produced above can be further characterized and used for a variety of purposes. For example, eimerian SLIP, CLIP, RAT, and CD AT reagents can be characterized in anti-parasite assays, such as a parasite neutralization assay (PN) and in vitro parasite inhibition assays (PI). Total, isotype- specific and Eimeria spp.-spcci c antibodies, present in SLIP, CLIP, RAT, and CD AT, can be quantified using conventional ELISAs, known to those of skill in the art and described further in the examples.

Of particular interest is the use of SLIP, CLIP, RAT, and CDAT for the identification of Eimeria spp. antigens produced during the intracellular stage of parasitic development. Detection of Eimeria antigens will find utility not only for the identification of vaccine candidates, but also for diagnostic purposes, to determine the presence or absence of coccidiosis in an avian subject and to assess the level of host immunity to the Eimeria species of interest. Eimeria antigens can be identified in a variety of biological samples, including in samples containing sporulated oocysts, extracellular sporozoites and merozoites, intracellular sporozoites and merozoites, and the like. Furthermore, tissue and fecal samples, e.g., samples from the spleen, cecum, rectum and from bird droppings, can be taken directly from infected avian subjects or subjects suspected of having coccidiosis and antigens detected using the antibodies of the present invention. Intracellular antigens can be identified in media from cell culture systems that support the growth of the intracellular stages of the parasitic life cycle. Thus, the present invention makes it possible to discriminate between antigens having similar molecular weights but produced during different stages of parasitic infection and/or at different sites of infection. For example, by use of the invention, intracellular and extracellular antigens that specifically appear in the spleen, ceca, intestinal digesta and feces, after challenge in immune versus naive birds, can be identified.

A number of known extracellular antigens have been identified in Eimeria sporozoites and merozoites in Western immunoblots, using the novel antibody preparations described herein, confirming that these antibodies are indeed able to selectively identify immunogenic, extracellular proteins. More importantly, the present antibody preparations for the first time allow the identification of stage- specific and/or intracellular antigens of Eimeria. These antigens are of particular interest since they are believed to be primarily responsible for a protective immune response to the organism. Intracellular antigens have not heretofore been recognized by conventional antibody probes such as by sera produced in Eimeria immune chickens, nor have they been identified using polyclonal antibodies produced in common animal species, such as rabbits, or by monoclonal antibodies from mice immunized with Eimeria sporozoites and merozoites. The identification of Eimeria intracellular antigens is now possible using the antibody preparations of the invention to assay culture media from avian Eimeria spp. grown in continuous cell lines able to support growth oi Eimeria during its entire life cycle, from sporozoite to oocyst, particularly during the intracellular phases of development. Such cell lines are described in International Publication No. WO 93/01276 (published 21 January 1993) and include cell clones SB-CEV- 1/P (ATCC Accession

No. CRL10497), SB-CEV- 1/F7 (ATCC Accession No. CRL10495) and SB-CEV- 1/G7 (ATCC Accession No. CRL10496). However, any cell line which will support the growth of Eimeria during the intracellular stages of asexual development will also find use for the identification of these stage-specific antigens. Representative intracellular Eimeria antigens have been identified from

Eimeria spp. grown in cell line SB-CEV-1/F7 (ATCC Accession No. CRL10495), using the above antibodies. This cell line is optimally cultured in Medium 199 (Gibco Laboratories, Grand Island, NY) under incubation conditions of 5% CO₂ and 40.5°C. Fetal bovine serum, antibiotics and antifungal agents can also be added at proportions readily determined by one of skill in the art. Culturing conditions for SB-CEV- 1/F7 are detailed further in International Publication No. WO 93/01276 (published 21 January 1993).

In particular, a number of intracellular antigens, including antigens having molecular masses of 28 kDa, 35 kDa, 38 kDa, 40 kDa, 43 kDa, 55 kDa, 70 kDa, 100 kDa and 110 kDa, respectively, have been identified in E. tenella-deήvcd ammonium sulfate-treated tissue culture supernatants from SB-CEV- 1/F7 using the antibodies of the present invention, as well as using hyperimmune sera prepared from naturally exposed, inbred chickens. The 35, 38, 43, 55 and 70 kDa antigens are recognized by SLIP, CLIP, RAT and immune sera; the 40 kDa antigen by CLIP, RAT and immune sera; the 100 kDa antigen by SLIP, RAT and immune sera; the 100 kDa antigen by SLIP and RAT; the 28 kDa antigen by CLIP and RAT; and the 55 and 70 kDa antigens by SLIP, CLIP and RAT. Furthermore, as can be seen in the examples that follow, the antigens appear at different times during the infective process. Additionally, the 35, 38, 40, 43 and 70 kDa antigens are present in both E. tenella and E. maxima strains tested and the 38 and 43 kDa antigens can be identified in both E. tenella and E. acervulina. Finally, the 38, 40 and 43 kDa antigens are present in several E. tenella strains tested and appear to be the immunodominant reactive species using the above antibodies. Accordingly, these three antigens are particularly important for providing protection against a variety of coccidiosis-causing agents, as well as for use as diagnostic reagents in immunoassays for the detection of Eimeria antibodies, thereby indicating the presence of Eimeria infection.

These and other important Eimeria antigens can be identified in a biological sample using the above-described antibodies and any of several standard identification techniques. For example, the presence of proteins reactive with the antibodies can be detected using standard electrophoretic and immunodiagnostic techniques. The antibodies can be used in immunoassays, such as competition, direct reaction, or sandwich type assays, for identifying the presence or absence of the proteins by forming complexes therewith. Such assays include, but are not limited to, Western blots, agglutination tests, enzyme-labeled and mediated immunoassays, such as ΕLISAs, biotin/avidin type assays, radioimmunoassays, immunoelectrophoresis, immunoprecipitation, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, or enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen present in the biological sample and the antibody or antibodies reacted therewith.

Typically, an immunoassay for detecting one or more of the Eimeria proteins will involve selecting and preparing the test sample and then reacting it with one or more of SLIP, CLIP, RAT, and CD AT, under conditions that allow protein-antibody conjugates to form. Solid supports can be used such as nitrocellulose, in membrane or microtiter well form; polyvinylchloride, in sheets or microtiter wells; polystyrene latex, in beads or microtiter plates; polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, and the like. Typically, the solid support is first reacted with the biological sample, washed and then the antibodies applied. If a sandwich type format is desired, such as a sandwich ELISA assay, a commercially available anti-immunoglobulin (i.e. anti- rabbit immunoglobulin) conjugated to a detectable label, such as horseradish peroxidase, alkaline phosphatase or urease, can be added. An appropriate substrate is then used to develop a color reaction.

A particularly convenient method for identifying and characterizing antigens using the antibodies of the invention involves immunoblot analysis. Briefly, parasite antigens are prepared and separated on SDS polyacrylamide preparative minigels. Separated proteins are electroblotted onto membranes, cut into strips, and residual protein-binding sites on the membrane are blocked with an appropriate agent, such as non-fat milk, bovine serum albumin (BSA), or heat-inactivated normal bovine serum (NBS). The test sample can be applied neat, or more often, it can be diluted, usually in a buffered solution which contains a small amount of protein, such as milk, BSA, or NBS. After incubating for a sufficient length of time to allow specific binding to occur, the membrane is washed to remove unbound sample and then incubated with a combination of conjugated anti-chicken immunoglobulins (total antibody)(ie., IgA + IgG + IgM) or a single labeled anti- chicken immunoglobulin (isotype antibody)(ie., IgA). Sufficient time is allowed for specific binding to occur again, the membrane is washed to remove unbound conjugate, and the substrate for the enzyme is added. Color is allowed to develop and the reaction stopped by rinsing in appropriate solution.

Alternatively, a "two antibody sandwich" assay can be used to detect the proteins of the present invention. In this technique, the solid support is reacted first with one or more of the antibodies of the present invention, washed and then exposed to the test sample. Antibodies are again added and the reaction visualized using either a direct color reaction or using a labeled second antibody, such as an anti-immunoglobulin labeled with horseradish peroxidase, alkaline phosphatase or urease.

Assays can also be conducted in solution, such that the eimerian proteins and antibodies thereto form complexes under precipitating conditions. The precipitated complexes can then be separated from the test sample, for example, by centrifugation.

Once identified and isolated, the antigens can be further purified using any of a variety of conventional methods including liquid chromatography, both normal or reverse phase, HPLC, FPLC and the like; affinity chromatography; size exclusion chromatography; immobilized metal chelate chromatography; gel electorphoresis; etc. The amino acid sequences of the purified antigens can be determined using techniques well known in the art such as repetitive cycles of Edman degradation, followed by amino acid analysis.

The purified antigens can be immunologically characterized using standard techniques such as MHC-restricted response profiles in genetically inbred animals. These measurements include, without limitation, Western blot, molecular weight determinations using standard techniques such as SDS-PAGE/staining, T-cell recognition assays, and assays to infer immune protection or immune pathology by adoptive transfer of cells, proteins or antibodies. Genes encoding the subject antigens can be identified by constructing gene libraries, using the resulting clones to transform a suitable host cell and pooling and screening individual colonies using the antibodies of the present invention, polyclonal serum or monoclonal antibodies to the desired antigen.

Alternatively, once the amino acid sequences of the subject antigens are determined, oligonucleotide probes which contain codons for a portion of the determined amino acid sequences can be prepared and used to screen DNA libraries for genes encoding the subject proteins. See, e.g., DNA Cloning: Vol. I, supra; Nucleic Acid Hybridization, supra; Oligonucleotide Synthesis, supra; T. Maniatis et al., supra. Synthetic DNA sequences, encoding the proteins of interest, can also be prepared, based on the determined sequence, using known techniques. See, e.g., Edge (1981) Nature 222:756; Nambair et al. (1984) Science 222:1299; Jay et al. (1984) /. Biol. Chem. 252:6311.

The coding sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. See, generally, DNA Cloning: Vols. I & II, supra; T. Maniatis et al, supra; B. Perbal, supra. The gene can be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired protein is transcribed into RNA in the host cell transformed by a vector containing this construct. The coding sequence may or may not contain a signal peptide or leader sequence. If so, the proteins can be expressed with or without the native sequences. Alternatively, heterologous signal sequences can be used. Leader sequences can be removed by the bacterial host in post-translational processing. See, e.g., U.S. Patent Nos. 4,431,739; 4,425,437; 4,338,397.

Other regulatory sequences may also be desirable, which allow for regulation of the expression of the protein sequences relative to the growth of the host cell. Regulatory sequences are known to those of skill in the art, and exarnples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.

The control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector, such as the cloning vectors described above. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

In some cases it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the appropriate orientation; i.e., to maintain the proper reading frame. It may also be desirable to produce mutants or analogs of the Eimeria antigen of interest. Mutants or analogs may be prepared by the deletion of a portion of the sequence encoding the antigen, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are described in, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra. The expression vector is then used to transform an appropriate host cell. The transformed host cells are cultured under conditions providing for expression of the antigen of interest. The antigen is then isolated from the host cells and purified. If the expression system secretes the protein into growth media, the protein can be purified directly from the media. If the protein is not secreted, it is isolated from cell lysates. The selection of the appropriate growth conditions and recovery methods are within the skill of the art. The antigens of the present invention may also be produced by chemical synthesis, such as solid phase peptide synthesis, using known amino acid sequences or amino acid sequences derived from the DNA sequence of the genes of interest. Such methods are known to those skilled in the art. Chemical synthesis of peptides may be preferable if a small fragment of the antigen in question is capable of raising an immunological response in the subject of interest.

The isolated, recombinantly or synthetically produced Eimeria antigens can be used in immunoassays, such as the competition, direct reaction or sandwich-type assays described above, to detect the presence of Eimeria antibodies in biological samples. In this way, not only can the diagnosis of coccidiosis be made, but the host level of immunity can be determined. For example, naturally occurring nnύ-Eimeria spp. antibodies are produced by the infected chicken in its fecal material. The presence of these antibodies can be determined by reacting a sample of fecal material with one or more of the Eimeria antigens of the present invention. Antibodies present in the fecal sample will form an antibody- antigen complex with the antigen. The reaction mixture can be analyzed to determine the presence or absence of these antibody-antigen complexes using any of a number of standard methods, such as those immunodiagnostic methods described above. For example, the isolated antigens can be conjugated to a solid support, such as any of the above-described supports, a fecal sample is then incubated with the conjugate, and the reaction mixture analyzed to determine the presence of the antibodies.

Other useful assay formats include the filter cup and dipstick. In the former assay, the antigen of this invention is fixed to a sinter glass filter to the opening of a small cap. The fecal sample is resuspended in diluent and then passed through the filter. If the antibody is present, it will bind to the filter which is then visualized through a second antibody detector. The dipstick assays involves fixing an antigen to a filter, which is then dipped in the resuspended fecal sample, dried and screened with a detector molecule. The Eimeria proteins of the present invention or their fragments can also be used to produce antibodies, both polyclonal and monoclonal. If polyclonal antibodies are desired, a selected mammal, (e.g., mouse, rabbit, goat, horse, etc.) is immunized with an antigen of the present invention, or its fragment, or a mutated antigen. Serum from the immunized animal is collected and treated according to known procedures. If serum containing polyclonal antibodies is used, the polyclonal antibodies can be purified by immunoaffinity chromatography, using known procedures. Monoclonal antibodies to the Eimeria antigens of the present invention, and to fragments thereof, can also be readily produced by one skilled in the art. The production of several monoclonal antibodies raised against E. te«e//α-infected SB- CΕV/F7 tissue culture supernatants is described in the examples. Of particular interest is MAb 1-2-6, which reacts with the 40 kDa intracellular protein which has been identified as being protective herein. The Eimeria monoclonal antibodies are produced by using hybridoma technology. In particular, immortal antibody-producing cell lines can be created by cell fusion, as well as by other techniques such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., M. Schreier et al., Hybridoma Techniques (1980); Hammerling et al., Monoclonal Antibodies and T-cell Hybridomas (1981); Kennett et al., Monoclonal Antibodies (1980); see, also, U.S. Patent Nos. 4,341,761; 4,399,121; 4,427,783; 4,444,887; 4,452,570; 4,466,917; 4,472,500, 4,491,632; and 4,493,890. Panels of monoclonal antibodies produced against the Eimeria antigen of interest, or fragment thereof, can be screened for various properties; i.e., for isotype, epitope, affinity, etc.

Monoclonal antibodies are useful in purification, using immunoaffinity techniques, of the individual antigens against which they are directed. The antibodies are also useful in diagnosis of coccidiosis infection, and can be used e.g., in immunoassays such as those described above, as well as in therapeutic compositions for the passive immunization of avian subjects.

The above-described antigens and antibodies, including the various intracellular antigens, polyclonal and monoclonal antibodies raised against the antigens, CLIP, SLIP, RAT and CD AT, can be provided in kits, with suitable instructions and other necessary reagents, in order to conduct immunoassays as described above. For example, the antibodies, eimerian antigens, or both, can be provided in a diagnostic immunoassay test kit to provide for the detection of coccidiosis infection or to test the state of immunity to coccidiosis of an avian subject. The kit can also contain, depending on the particular immunoassay used, suitable labels and other packaged reagents and materials (i.e. wash buffers and the like). Standard immunoassays, such as those described above, can be conducted using these kits.

The antigens (either purified, partially purified, or crude mixtures thereof), immunogenic fragments of the antigens, chimeric proteins comprising the same, and antibodies described above, can also be formulated into subunit vaccine compositions to provide immunity to coccidiosis. The antigens and antibodies of the present invention can be used either alone or in combination with other antigens and antibodies, from the same or different species of Eimeria. For example, intracellular Eimeria antigens may be combined with extracellular Eimeria proteins, such as those present in extracellular sporozoites and merozoites. In such combinations, the antigens may be provided in the form of a fusion protein or a larger, multimeric protein. These fusion proteins or multimeric proteins may be produced recombinantly, as described in, e.g., U.S. Patent No. 4,366,246, or may be synthesized chemically. Further, antigens of this invention may be employed in combination with antigens from other avian pathogens, to provide broad spectrum protection against a variety of avian diseases. Additionally, crude mixtures of the antigens, such as partially purified mixtures of intracellular antigens derived from ammonium sulfate precipitation of culture media which supports the growth of intracellular forms of Eimeria spp., can be used in vaccine compositions without further purification. See, e.g., the examples, where such crude extracts are shown to be protective against E. tenella challenge. The vaccine compositions are generally formulated with a pharmaceutically acceptable vehicle or excipient. Suitable vehicles are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the vehicle may contain minor amounts of auxiliary substances such as wetting or emulsifying agents and pH buffering agents. Preservatives known in the art, such as thimerosal, phenol and other phenolic compounds, as well as antibiotics, can also be added to the vaccine compositions of the present invention. Suitable vaccine vehicles and additives are known, or will be apparent, to those skilled in the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Εaston, Pennsylvania, 18th edition, 1990. Adjuvants which enhance the effectiveness of the vaccine, may also be added to the formulation. Adjuvants may include for example, muramyl dipeptides, avridine, aluminum hydroxide, oils, oil in water emulsions, saponins, cytokines, and other substances known in the art.

The protein may be linked to a carrier in order to increase the immunogenicity thereof. Suitable carriers include large, slowly metabolized macro- molecules such as proteins, including serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art; polysaccharides, such as sepharose, agarose, cellulose, cellulose beads and the like; polymeric amino acids such as polyglutamic acid, polylysine, and the like; amino acid copolymers; and inactive virus particles. The Eimeria antigens may be used in their native forms or functional groups modified for attachment to these carriers. As explained above, avian species have a mucosal immune network consisting of gut-associated lymphoid tissue termed GALT or Peyer's patches), bronchial-associated lymphoid tissue (BALT), and the Harder gland, located ventrally and posteriomedially to the eyeball. Presentation of an antigen to these tissues triggers proliferation and dissemination of committed B- cells to the secretory tissues and glands in the body, with the ultimate production of secretory IgA (slgA). SlgA serves to block the colonization and invasion of specific surface antigens that colonize on, and pass through, a mucosal surface. It appears that the intestinal slgA system plays an essential role in the protective immune response to Eimeria. Davis, PJ. et al. Immunology (1978) 24:879-888. Accordingly, the

Eimeria antigens of the present invention may also be administered using avirulent carrier microbes, able to invade and proliferate in the cells of the GALT and BALT. Such delivery allows for a generalized secretory immune response as well as humoral and cellular immune responses. For example, recombinant plasmids containing genes for the Eimeria antigens can be introduced into one of several avirulent strains of bacteria, designed for delivering antigens to avian subjects. Such avirulent organisms generally contain mutations in genes necessary for long- term survival and include mutant derivatives oi Salmonella, E. coli and E. coli- Salmonella hybrids. Such mutants are described in e.g., Curtiss, R. Ill, et al. Infect. Immun. (1987) 55:3035-3043 and U.S. Patent Nos. 4,968,619; 4,888,170 and

4,190,495, incorporated herein by reference in their entirety.

Furthermore, the proteins may be formulated into vaccine compositions in either neutral or salt forms. Pharmaceutically acceptable salts include the acid addi¬ tion salts (formed with the free amino groups of the active polypeptides) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

Vaccine formulations are prepared by combining an effective amount of one or more antigens described above, the exact amount being readily determined by one skilled in the art. For purposes of the present invention, an "effective amount" of an antigenic vaccine component will be that amount required to generate an amount of circulating antibody sufficient to prevent or reduce coccidiosis disease symptoms. An effective amount of an Eimeria antigen will vary, depending on the mode of administration, the particular species oi Eimeria targeted, the degree of protection desired and the age and health of the subject to be treated. Such amounts are readily determinable by the skilled artisan however, by way of example, for compositions to be delivered parenterally, generally between about 10 μg to about 1 mg, more preferably about 25 μg to about 200 μg, and most preferably about 50 μg to about 100 μg, in about 0.5 to about 10 ml, preferably about 1 ml to 3 ml, will constitute an effective amount of antigen.

The vaccine compositions of the present invention can be administered parenterally, e.g., by intramuscular, subcutaneous, intravenous or intraperitoneal injection. It may also be desirable to introduce the vaccine composition directly into the gut or bronchus, to stimulate a preferred response of the GALT or BALT, such as by oral administration, intranasal administration, gastric intubation or aerosol administration, as well as air sac and intratracheal inoculation. For example, for oral administration, the vaccines can be conveniently placed directly into water given to the avian subjects. Other suitable methods of administering the vaccines of the invention are, e.g., via the conjunctiva to reach the Harder gland or in ovo administration, by inoculating avian eggs before they hatch. A combination of these routes of administration can also be used. For example, the initial inoculation might involve parenteral administration while subsequent boosters might be given orally. The avian subject is immunized by administration of the vaccine formulation, in at least one dose, and preferably two or more doses. However, the animal may be administered as many doses as is required to maintain a state of immunity against coccidiosis.

For example, boosters can be given at regular intervals, i.e., at six months or yearly, in order to sustain immunity at an effective level.

C. Experimental

Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used

(e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

The antigens of Eimeria spp., are sometimes identified in the examples with reference to their molecular mass in kilodaltons (kDa). Thus, an antigen having a molecular mass of about 35 kDa is identified below as P35; an antigen of about 40 kDa in molecular mass is identified as P40, and so on. The following acronyms are used in the examples and are defined as follows: UI/UC, unimmunized, unchallenged (naive) UI/C, unimmunized, challenged

NE/UC, naturally exposed, unchallenged (also referred to as "tricklefed")

NE/C, naturally exposed, challenged SLIP, splenic lymphocyte immune product CLIP, cecal lymphocyte immune product RAT, rectal antibody test CD AT, cage dropping antibody test

MAb, monoclonal antibody spz, extracellular sporozoite mrz, extracellular merozoite

Example 1

Establishment and Testing of Inbred Natural Exposure (NE) Model Chickens (B¹⁹B¹⁹, B^MB²⁴, and B³⁰B³⁰, New Hampshire Poultry Research Center), serologically typed and previously determined to differ at the MHC-locus (B-complex), were housed in wire cages. Chicks were fed a nonmedicated starter/grower diet and water ad libitum. Groups (NE/C) of one-day chicks were immunized with 500 E. tenella oocysts from a strain designated L.S. 65 (provided by D. Strout, Univ. of New Hampshire) per os for five consecutive days. Unless specifically noted, all trickle immunizations and challenge infections were performed with E. tenella L.S. 65. Control groups (UI/C) were similarly immunized with distilled water. At either 6, 8, 11 or 15 days following the last day of parasite exposure, birds were weighed and challenged with 3.5 x IO⁴ E. tenella oocysts. Mean body weight gain and cecal lesions were determined at 6 days post- challenge. The duration of immunity to homologous challenge in trickle- immunized chicks was found to be at least 2 weeks. NE/C birds from all three haplotypes showed significantly higher weight gains and lower lesions than their

UI/C counterparts at the timepoints examined, supporting the concept that this method of trickle immunization affords protection against homologous parasite challenge. Therefore, the reagents obtained from NE/UC, NE/C and UI C birds at various timepoints post-challenge are representative of the different levels of immunity which exist following parasite challenge. Example 2 Preparation of SLIP and CLIP Samples from Inbred NE Model For preparation of SLIP samples, spleens were removed (typically 4- 6/group) at the desired timepoint and mononuclear cells isolated using standard Histopaque 1077 centrifugation (Sigma, St. Louis, MO.). Viable cells were counted using trypan blue and a hemacytometer and cultivated at a density of 5 x IO⁶ cells/ml in serum-free modified LM-Hahn medium (LMH) (Calnek, et. al. Infect. Immun. (1981) 24:483-491.) for 5 days (40.5°C, 5% CO₂). Supernatant was harvested, spun (800 x g, 10 min., 4°C), and cell-free supernatant collected. Sodium azide was added to 0.1% (v/v) final concentration and samples filtered (0.2 μM). SLIP samples were stored at 4°C for up to 2 weeks or aliquots prepared and frozen at -20°C until use.

For preparation of CLIP samples, both cecal pouches per bird were removed (typically 4-6 birds/group) at the desired timepoint and intestinal lymphocytes isolated according to standard procedures. Briefly, cecal pouches were cut at the ileocecal junction, the distal end excised, and cecal contents expressed out. Tissue was placed into a tube containing cHBSS (Ca²⁺ and Mg²⁺-free HBSS containing 2X antibiotic/antimycotic, 25 mM HEPES, pH 7.4) and placed on ice until further manipulations. Tubes were shaken vigorously for 10 sec. to remove additional gut contents, and S/N discarded following gravity sedimentation of tissue. Cecal tissue was placed into a petri dish and each cecum opened longitudinally. The mucosal surface was gently scraped to remove any residual clumps of fecal material and tissue minced into small 1-2 cm fragments. Tissue fragments were placed in a 50 ml tube containing 10-20 ml cHBSS, and samples hand shaken vigorously for 10-20 sec, followed by a low speed spin (70 x g, 1 min. R.T.). S/N was discarded and 10- 20 fresh cHBSS added to tissue. The shake/spin procedure was repeated two additional times, followed by the addition of 10-20 ml cHBSS/DTT (lOmM DTT, IX genticin and polymyxin B sulfate) per sample. Tubes were briefly shaken to disrupt tissue pellet, incubated in a horizontal position on a platform shaker for 20 min. (150 rpm, R.T.), and spun at low speed as above. S/N was discarded and tissue washed two additional times at low speed to remove residual DTT. Approximately 10 ml of freshly prepared cHBSS/ES (cHBSS containing 1 mg/ml collagenase (Clostridiopeptidase A, Type VII, Sigma) was added and minced tissue solution transferred to a glass flask containing a stir bar. Samples were incubated with moderate stirring (400-500 rpm) on a multiple magnetic stir plate for 60 min (37°C) and solution transferred into a 50 ml tube and spun at low speed to pellet any .transferred tissue fragments. S/N containing desired cells was carefully poured off and kept on ice until further manipulations. The cHBSS ES treatment was repeated one additional time and desired cells from the second incubation pooled with cells from initial enzymatic treatment. Cells were pelleted (450 x g, 10 min. 4°C) and resuspended to homogeneity in 40% isotonic Percoll/cHBSS. Volume for resupsension was dependent on cell pellet size. Typically, 9 ml of 40% Percoll was used per 10⁸ cells. Approx. 3 ml of 40% cell suspension was gently layered onto a equal volume of 70% isotonic Percoll in 15 ml tubes, spun (600 x g, 20 min. 4°C), and the 40/70 cell interface collected. Using this procedure, both intrepithelial and lamina propria cecal intestinal lymphocytes were isolated together in the 40/70 cell interface. Cells were washed 3-4 times in cHBSS (450 x g, 10 min.4°C), viable cell count determined, and cultured for 5 days as described above for splenic lymphocytes. CLIP samples were prepared and stored as previously described for SLIP samples.

Example 3 Preparation of RAT from Intestinal Digesta and CD AT from Fresh Cage droppings

For RAT, fecal material (typically 4-6 birds/group) was collected from the ileocecaljunction down to the cloaca and placed in a preweighed 50 ml conical tube. For CD AT, fresh cage droppings were collected from litter pans and placed in a preweighed 50 ml conical tube. The net wet weight of samples were determined, and 3 mis of Dulbecco's phosphate buffered saline (DPBS) were added per gram feces. Samples were vortexed at moderate speed for 15-20 sec. to resuspend fecal material. Samples were spun (2750 x g,4o^sC, 15 min.) and supernatant above fecal pellet transferred to 30 ml Sorvall GSA-600 rotor tubes. Samples were spun

(32,571 x g, 4°C, 20 min.) and supernatant above pellet transferred to a clean tube. The pH of the sample was determined and adjusted to pH 6.5 to 7.0 if necessary. Protease inhibitors (1 mM 1, 10-phenanthroline, 1 mM benzamidine HCL hydrate, 10 μg/ml pepstatin, 0.5 mM PMSF (all final concentrations) and sodium azide (0J% final concentration) were then added. Samples were stored at -20°C until ready for use.

Example 4 In Vitro Parasite Neutralization Assay In order to measure B-cell derived antibodies to extracellular sporozoites present in CLIP, control and trickle-immunized B²⁴B²⁴ and B³⁰B³⁰ birds were prepared as described in Example 1. Thirteen days following the last parasite exposure, birds were challenged with 3.5 x IO⁴ oocysts and CLIP samples prepared at days 1, 3 and 5 post-challenge as described in Example 2. One ml samples were incubated with an equal volume of freshly excysted L.S. 65 E. tenella sporozoites (5 x lOVml) on a rocker platform (1 hr, 40.5°C). The mixture was then directly added in quadruplicate to microtiter wells containing SB-CEV/F7 cells (ATCC Accession No. CRL10495) plated (in medium 199/5% FBS) at 1 x 10⁵ cells/well 24 hrs previously. After 2 hrs (40.5°C, 5% CO₂) extracellular sporozoites were removed by washing, and one μCi per well of tritiated uracil (Amersham, 5 mCi/mmol) added. After 24 hr incubation (40.5°C,5% CO₂), cells were harvested onto glass fiber mats (MACH III Harvester, TomTech, Orange, Conn.) and incorporation of radioactivity determined (Matrix 96, Packard Instrument Co., Sterling, VA.). Percentage inhibition was calculated as the mean counts per minute (cpm) (mean cpm equals mean of quadruplicates using Dicksons Outlier Test to exclude extreme values (Dickson, WJ. Biometrics (1953) 2:74-89)) of sample treated wells divided by the cpm of untreated wells times 100%. The kinetics of CLIP sporozoite neutralizing activity differed between naive and immune challenged birds. At day 1 post-challenge, CLIP from both NE/C haplotypes contained significantly higher parasite neutralization activity compared to CLIP from UI/C groups. At day 3 post- challenge, only NE/C CLIP from the B^B²⁴ haplotype contained higher parasite neutralization activity. At day 5 post-challenge, no measurable activity differences between naive and immune groups in either haplotype were observed. These results indicate that the anti-parasitic activity of CLIP from NE/C birds appears more quickly in immune versus naive birds following parasite challenge.

In order to determine whether particular molecular weight extracellular sporozoite antigens were recognized by these antibody-containing CLIP samples, samples exhibiting varying degrees of neutralizing activity were screened by Western blot against E. tenella spz antigen (see Example 7). Results indicated that CLIP samples with strong IgG reactivity to sporozoite antigens having molecular weights of 43 kDa, 40 kDa, 38 kDa and 26 kDa, possessed the highest levels of anti- sporozoite neutralizing activity. These results indicate that these antigens are recognized by IgG produced by cecal lymphocytes at the site of parasite replication. Finally, these results clearly indicate that the antigen-specific IgG in immune CLIP was produced through T-cell dependent immune mechanisms. Example 5 In Vitro Parasite Inhibition Assay In order to measure soluble, T-cell derived anti-parasitic factors present in CLIP, the same CLIP samples obtained from Ul/C and NE/C B²⁴B²⁴ and B³⁰B³⁰ birds in Example 4 were used for the assay. The duck embryo (DE) cell Hne

(ATCC Accession No. CCL 141) was seeded in 96 well flat microtiter plates at 1 x IO⁴ cells/well (in medium 199/5% FBS) overnight (40.5°C, 5% CO₂). Cells were then pretreated with 0.2 mis/well of duplicate log₂ dilutions of CLIP samples. One row of cells was treated with media alone (control). After 24 hrs incubation (40.5°C, 5% C0₂), fresh dilutions of test CLIP, 1 x 10⁵/well freshly excysted extracellular E. tenella sporozoites (obtained as described in Example 7), and one μ Ci per well of tritiated uracil was added. After 24 hr incubation (40.5°C, 5% CO₂), cells were harvested onto glass fiber mats and incorporation of radioactivity determined. Percentage inhibition was calculated in a similar fashion as that described in Example 4, i.e., as the mean cpm of sample treated wells divided by the cpm of media control wells times 100%. The kinetics of CLIP parasite inhibition differed between naive and immune challenged birds. At day 1 post-challenge, CLIP from both NE/C haplotypes contained significantly higher parasite neutralization activity as compared to CLIP from UI/C groups. At day 3 post- challenge, only NE/C CLIP from the B² B²⁴ haplotype contained higher parasite inhibition activity. At day 5 post-challenge, no significant differences in the inhibitory activity between naive and immune groups in either haplotype was observed. These results indicate that the anti-parasitic activity in CLIP, directed against the intracellular parasite forms, appears more quickly in immune versus naive birds following parasite challenge.

The peak inhibitory activity of these NE/C T-cell derived cytokines parallels the peak inhibitory activity of the NE/C B-cell derived antibodies obtained in Example 4. By 24 hrs post-challenge in NE/C birds, antigen-specific T_H2cells located in the cecal lining elaborate specific cytokines, most likely IL5 and IL6, which enhance antibody production and extracellular sporozoite neutralization. But perhaps more importantly, there is a simultaneous induction of antigen specific T_H1 cells and secretion of anti-parasitic cytokines, probably interferon-gamma and tumor necrosis factor beta, which exert their effects directly on the intracellular replicating parasite. Therefore, the same CLIP sample can be used to measure both antibody and cell-mediated immune effector molecules. Example 6 Ouantitation of total and sporozoite-specific IgA in RAT For quantitation of total IgA in RAT samples, a murine anti-chicken IgA MAb (MAb 6.2.3-1 purchased as ascites from Dr. S. Naqi, Cornell University, Ithaca, NY) or MAb Jl 26.189.96 (Janssen Biochemica) was diluted 1 :500 in 50 mM sodium borate, pH 9.5. One hundred microliters per wells was added to ELISA multiwell plates (Nunc ImmunoPlate Maxisorb F96, Nunc, Denmark) and incubated at 40°C (2hrs) or at 4°C (overnight). Plates were washed 3X with PBST (phosphate buffered saline/0.05% Tween) and then blocked for 2hrs (40°C) at overnight (4°C) PBST/5% skimmed milk (Difco 0032-01-1). Plates were then washed 3X with

PBST. Test RAT samples were initially diluted 1:100 in PBST/5% milk and serial two-fold dilutions added in duplicate to wells (100 μl/well). Serial two-fold dilutions of reference serum containing IgA (Bethyl Labs, RS10-102-l)(initial concentration 4.0 μg/ml) were similarly prepared for each plate. Plates were incubated at 4°C overnight. Primary RAT antibody incubation was performed at 4°C to decrease endogenous protease activity in the samples. Plates were washed 3X with PBST and then 100 μl/well of a 1:500 dilution (PBST/5% milk) of horseradish peroxidase conjugated goat anti-chicken IgA (Bethyl Labs, A30-103P- 3) added for lhr (40°C). Plates were washed 3X with PBST and then developed by the addition of 100 μl/well TMB peroxidase substrate/peroxidase solution

(Kirkegarrd & Perry). After 5-30 min. the reaction was stopped by the addition of lOμl/well IN NaOH. Optical density at 450 nm was determined on a VMAX ELISA plate reader (Molecular Devices). The VMAX program was used to calculate unknown concentrations based on the IgA reference standard curve. For quantitation of sporozoite-specific IgA RAT samples, a similar procedure was followed except the plates were coated with 100 μl/well of sonicated E. tenella antigen (3 μg/ml) for 2 hrs (40°C) or overnight (4°C). Two rows were coated with anti-chicken IgA MAb as above for capture of reference serum. The remainder of the assay was performed as described above for the total IgA ELISA, except the RAT samples were tested at an initial 1 : 1 dilution.

Example 7 Immunoblots The following procedure was used for several of the examples to follow. Immunoblots were modified from previously published procedures (J.T. Roehrig et al. Virology (1985) 142:347-356 and H. Towbin et al. Proc. Natl. Acad. Sci. USA (1979) 76:4350-4354). E. tenella (LS65) oocysts were produced and maintained by passage in chickens. Pure oocysts and sporozoites were obtained essentially as previously described by Schultz, D.M. et al. J. Protozol. (1984) 3-1: 181-183. Sporozoite and merozoite antigens were obtained by resuspending sporozoites and in vitro merozoites in PBS containing 0.5 mM phenylmethyl sulfonyl fluoride (Calbiochem-Behring, La Jolla, CA). The solution was freeze-thawed three times on dry ice and sonicated (Heat Systems Ultrasonics, model W-380) on ice for one min using a one second pulse, 80% duty cycle. After five cycles, each one min long, samples were transferred to microcentrifuge tubes and spun at 10,000 x g, 10 min at 4°C. Soluble material above the pellet was collected and protein concentrations determined using standard procedures. Sonicated parasite preparations were adjusted to 1 mg/ml in serum-free media 199, aliquoted and stored at -20°C for further use.

E. tenella sonicated sporozoites or merzoites, and uninfected or E. tenella SB-CΕN/F7 30% and 45%(ΝH₄)₂SO₄44-72 h tissue culture proteins (10 μg/lane) were separated on 10%, 12.5% or 4-20% polyacrylamide preparative minigels (U.K. Laemmli Nature (1970) 27_7_:680-685)(Daiichi gels, Integrated Separation Systems, Hyde Park, MA) using high and low molecular prestained BioRad markers as standards (BioRad, Richmond, VA). The gels were stopped when the bromophenol marker began to migrate off the bottom of the resolving gel. Molecular weight determinations were based on average gel mobilities (n=5).

Proteins were immunoblotted (overnight, 40 mA, 4°C) onto Immobilon-P membranes (0.45 μM, Millipore Corp., Bedford, MA.) If necessary, membranes were cut into desired size strips, prior to subsequent manipulations. Membranes or membrane strips were washed three times in TTBS (wash buffer, Tris-buffered saline/0.01% Tween 20). Membranes were rinsed in wash buffer between all subsequent incubation steps. Blots to be used for SLIP, CLIP, and sera antibody incubations were blocked in TTBS/2% skim milk/1% gelatin for a minimum of 2 hrs (R.T.); blots to be used for RAT incubations were blocked in TTBS/3% BSA for a minimum of 6 hrs. In some instances, blots were blocked overnight (R.T.). For primary SLIP, CLIP, and sera antibody incubations, samples were diluted 1/2, 1/2, and 1/500 respectively, in TTBS/1% gelatin/0J% NaN₃; for primary RAT incubations, samples were diluted 1/5 in TTBS/3% BSA/10% FBS. All primary antibody incubations were carried out overnight (R.T.). For detection of bound chicken IgG from SLIP, CLIP, and sera samples, goat phosphatase-labeled anti- chicken IgG (Kirkegaard & Perry) was used at a 1/1000 dilution (TTBS/0J% BSA). For detection of bound rabbit IgG from rabbit sera, mouse phosphatase- labeled anti-rabbit IgG (Kirkegaard & Perry) was used at a 1/1000 dilution (TTBS/0J% BSA). For detection of bound chicken IgA from CLIP and RAT samples, goat anti-chicken IgA (Bethyl Labs, Montgomery TX) was used at a 1/500 dilution (TTBS/3% BSA/10% FBS), incubated for a minimum of 2 hrs. (R.T.), followed by incubation in phosphastase-labeled rabbit anti-goat IgG (Kirkegaard & Perry) at al/1000 dilution (TTBS/3% BSA/10% FBS). All phosphatase incubations were carried out for 1 hr (R.T.). For immunodetection, the BCIP/NBT 1 component substrate system (Kirkegaard & Perry) was used at the recommended concentration. Blots were allowed to develop for 5 to 15 minutes and the reaction was stopped by rinsing in water.

Example 8 Determination of Total and Sporozoite-specific IgA Levels in RAT Samples Prepared from Inbred NE Model and Correlation to Protection Against Disease RAT samples were prepared from NE/UC and NE/C B¹⁹B¹⁹ birds following different periods of rest. Day-old chicks were trickle immunized with 500 E. tenella oocysts for 5 consecutive days. Then 10, 17 and 24 days after the last parasite exposure, 10 birds/group were weighed and either mock challenged or challenged with 3.5 x 10" homologous oocysts. Groups of age-matched naive birds were also weighed and challenged (UI/C). At day 2 post-challenge, RAT samples from 5 birds per group were prepared. At day 6 post-challenge, the remaining birds were weighed and mean weight gains of NE/C birds compared to UI/C birds. RAT samples were assayed for both total and spz-specific IgA concentrations. Substantial increases in total IgA levels were observed following parasite challenge. The increases were dependent on the rest period examined. At day 2 post-challenge following either a 10 or 17-day rest period, total IgA levels were elevated in NE/C birds compared to NE/UC birds, indicating that parasite challenge resulted in stimulation of an IgA memory response. After 24 days post-parasite exposure, no significant increase in total IgA was detected, indicating that the memory response had begun to wane. The spz-specific IgA results are similar in that IgA concentrations increased following parasite challenge after 10 and 17, but not 24, days rest. The increases in total and spz-specific IgA levels following parasite challenge after 10 or 17 days rest, but lack of antibody increase after 24 days rest, is consistent with the weight performance data obtained. Significant protection against weight loss in NE/C groups was observed after 10 and 17 days rest, but not after 24 days rest. Using this or a similarly derived ELISA, the concentrations of both total and spz-specific IgA in RAT in birds can be determined. The results can be used to determine the minimum total and spz-specific IgA titers required for protection against homologous parasite challenge. Results can also be used to better evaluate flock immunity.

Example 9

Determination of Total and Sporozoite-specific IgA Levels in RAT Samples Prepared from Outbred Broilers Chickens from a commercial poultry operation broiler line, raised on wire, were used for this study. 16-day old birds were trickle immunized with 500 E. tenella L.S. 65 or L.S. 80 oocysts for four consecutive days, rested for two days and then trickle immunized once more with 500 oocysts. Birds were subsequently rested for 14 days, challenged orally with 50,000 oocysts of the homologous immunizing strain, rested again for 15 days, and then a portion of each group challenged orally with either the homologous or heterologous immunizing strain. At days 2 and 6 post-challenge, RAT samples were prepared (2-3 birds/group) from all groups and assayed for total and sporozoite-specific IgA concentrations. The data indicate that it is possible to measure total and spz-specific IgA levels in outbred broilers using a RAT ELISA, and that the levels of RAT may correlate to the immune status. For example, the highest levels of spz-specific IgA were detected in immune birds at day 6 post-challenge (groups 8 and 9) and these values were considerably higher than values obtained from naive birds at day 6 post- challenge (groups 2 and 3). results also indicated that birds immune to one strain of E. tenella (group 4) increased spz-specific IgA levels following exposure to a second E. tenella strain (group 8). The results obtained can be used to determine the minimum total and spz-specific IgA titers required for protection against homologous or heterologous species challenge. Results can also be used to better evaluate flock immunity.

Example 10 Protection Against E. tenella Challenge in Naive Inbred Birds Vaccinated with

30%rNH_AXSO₁ Antigen Prepared from SB-CEV/F7 E. tenella Infected

44-72 hr Tissue Culture Supernatant In vivo efficacy of the 30%(NH₄)₂SO₄ antigen prepared from SB-CΕV/F7 44-72 hr tissue culture supernatant was tested in three independent vaccine trials. In all 3 battery trials, 30%(NH₄)₂SO₄44-72 hr E. tenella antigen was used to immunize 4-day old inbred B¹⁹B¹⁹ chicks. Chicks were vaccinated subcutaneously at the base of the neck (1.0 ml total volume) with antigen adjuvanted in an oil in water emulsion containing Amphigen. At 7 days of age, birds were orally boosted with the same amount of vaccine. Age-matched control birds (U C) were immunized and boosted with adjuvanted tissue culture media obtained from uninfected F7 cells. At 10 days of age, all birds were weighed and challenged with 3.5 x IO⁴ sporulated E. tenella oocysts per os. At 16 days of age, final bird weights were measured. In trial 1, birds were vaccinated with approximately 50 μg total protein per dose and in trials 2 and 3, birds were vaccinated with approximately 100 μg total protein per dose. A group of unimmunized, unchallenged birds (UI/UC) was also used in all 3 trials. Statistical comparisons of weight gains were performed using least square analysis and values compared to UI/C controls.

Results of the 3 inbred efficacy studies are summarized in Table 1. In all 3 vaccination trials, 44-72 30% immunized, challenged birds showed statistically higher weight gains compared to UI/C controls. In trial 1, the vaccinated group also showed numerically higher weight gains compared to the UI/UC group. These results demonstrate that protection against weight loss associated with E. tenella infection in young birds is afforded by vaccination with a 30%(NH₄)₂SO₄ soluble parasite antigen fraction prepared from an E. tene/Zα-infected F7 cell line. This particular antigen fraction was therefore selected for extensive Western analysis and characterization using a panel of NΕ/C immune reagents including SLIP, CLIP, and RAT, in order to identify lead protective antigen (s) candidates.

Example 11 Identification of E. tenella Antigens in NΕ Model Using SLIP Prepared from 3

MHC Haplotypes SLIP samples were obtained from UI/C, NΕ/UC, and NΕ/C B¹⁹B¹⁹, B^B²⁴, and B³⁰B³⁰ groups. Birds were trickle-immunized with 500 E. tenella oocysts/bird for 5 days, rested for 13 days, and then challenged orally with 3.5 x IO⁴ homologous oocysts. Day 1 post-challenge, SLIP samples were prepared as described above and assayed for Western reactivity against E. tenella sonicated sporozoites and 30%(NH₄)₂SO₄44-72 hr supernatants from E. tenella SB-CΕV/F7 infected cells.

The IgG Western reactivity profiles of the SLIP samples are summarized in Table 2.

Results indicate that the spleens of all 3 UI/C haplotypes contain B cell populations capable of producing IgG reactive with the spz 40 kDa antigen (termed "P40" herein). This antigen induces an immunodominant response, since the spleens of all 3 NE/UC haplotypes contain B cell reactivity to P40 15 days after the last 500 oocyst parasite exposure. Interestingly, this same splenic B cell population is not present at day 1 post-challenge in the B¹⁹B¹⁹ and B² B²⁴ haplotypes, suggesting that this population has emigrated from the spleen.

Results also indicate that the SB-CEV/F7 30%(NH₄)₂SO₄ antigens having molecular masses of 110 kDa and 70 kDa (termed "Pl 10 and P70," respectively) induce immunodominant responses, since NE/UC groups contain B-cell reactivity to these antigens 15 days following the last trickle immunization.

In summary, these results show that the spz P40 and SB-CEV/F7 30%(NH₄)₂SO₄ P110 and P70 are non-MHC restricted E. tenella antigens important for protection.

Example 12 Identification of E. tenella Antigens in NE Model Using CLIP Prepared from 3

MHC Haplotypes CLIP samples were obtained from UI/C, NE/UC, and NE/C B¹⁹B¹⁹, B²⁴B²⁴, and B³⁰B³⁰ groups day 1 and 3 post-challenge, as described in Example 10. These samples were prepared and assayed for Western reactivity against E. tenella sonicated sporozoites and 30%(NH₄)₂SO₄44-72 hr supernatants from E. tenella SB- CΕV/F7 infected cells. The IgG Western reactivity profiles of the day 1 and 3 post- challenge CLIP samples are summarized in Tables 3 and 4, respectively. Results show the CLIP reactivity profile within a single NE/C haplotype and group is similar but clearly different, between day 1 and day 3 post-challenge. Thus, while some non-MHC restricted NE/C intestinal B-cells are present at both days 1 and 3 post-challenge (e.g., P70 and P40), other non-MHC restricted B-cells are not present until day 3 post-challenge (e.g., P43). Moreover, the differential presence of these antigen-specific B-cells is a direct result of parasite challenge, since almost identical day 1 and day 3 CLIP reactivity profiles were observed in the 3 NE/UC haplotypes. Results further indicate that the SB-CEV/F7 30%(NH₄)₂SO₄ P70 and P38 antigens induce non-MHC restricted immunodominant responses, since groups from all 3 NE/UC MHC haplotypes contain intestinal B-cell IgG reactivity to these two antigens 15 days following the last trickle immunization.

Results also clearly demonstrate that NE/C CLIP from all 3 haplotypes recognize a P40 protein present in 30%(NH₄)₂SO₄ material, but not in the sonicated spz preparation. These results suggest that the 30%(NH₄)₂SO₄ P40 is distinct from the spz P40 previously described in example 11. Further analysis of the spz vs. 30%(NH₄)₂SO₄ profiles on days 1 and 3 post-challenge reveals several immunodominant extracellular-specific antigens (e.g., P110, P28, P26) and several immunodominant intracellular-specific 30%(NH₄)₂SO₄ antigens (e.g., P70, P55, P38).

Finally, comparison of the day 1 NE/C SLIP (Example 11, Table 2) profile to the day 1 NE/C CLIP profile shows that the reactivity pattern between the two biological compartments is different indicating unique, localized immune responses occur simultaneously within the same immune host.

Example 13 Identification of E. tenella Antigens in NE Model Using RAT Prepared from 2 MHC Haplotypes Following a Low or High Oocvst Challenge

RAT samples were prepared from UI/C and NE/C B¹⁹B¹⁹, B²⁴B²⁴, and B³⁰B³⁰ groups. Birds were trickle-immunized with 500 E. tenella oocysts/bird for 5 days, rested for 16 days, and then challenged orally with either a predetermined low or high oocyst dose. At day 2 post-challenge, RAT samples were prepared (3 birds/group) and assayed for Western reactivity against E. tenella sonicated sporozoites and 30%(NH₄)₂SO₄44-72hr supernatants from E. tenella SB-CEV/F7 infected cells.

Day 6 performance results show that protection against challenge in the NE/C groups was dose and MHC haplotype dependent. Both NE/C haplotypes showed significant protection against weight loss at the low challenge dose, but only the B³⁰B³⁰ NE/C group were significantly protected against weight loss at the high challenge dose. However, the B¹⁹B¹⁹ NE/C group appeared to be partially protected against high dose challenge based on a significant reduction in lesions. The immunological basis for the dichotomy in performance between the B¹⁹B¹⁹, and B³⁰B³⁰ lines was investigated by examination of the IgA Western reactivity profiles of the RAT samples.

Western results (Tables 5 and 6) indicate that the reactivity profiles were dependent on a combination of parameters: challenge dose, immune status and MHC haplotype. For the low dose challenge, specific antigen reactivity appeared to be correlative to protection in both NE/C haplotypes. B¹⁹B¹⁹ NE/C, but not UI/C birds, responded to several antigens: 30%(NH₄)₂SO₄ P120, P55 and P28, 45%(NH₄)₂SO₄ P120, P55 and P28. B³⁰B³⁰ NE/C, but not UI/C birds, responded to 45%(NH₄)₂SO₄ P28. For the high dose challenge, specific antigen reactivity was more difficult to correlate to protection. However, results clearly indicate that the RAT profiles identified several MW antigens previously identified by CLIP and SLIP. Example 14 Identification of E. tenella Antigens Using Sera Prepared from NE Model Sera samples were obtained from UI/C and NE/C B¹⁹B¹⁹ groups. Birds were trickle-immunized with 500 E. tenella oocysts/bird for 5 days, rested for 14 days, and then challenged orally with 3.5 x IO⁴ homologous oocysts. At day 1, 3 and 5 post-challenge, serum samples were collected (5/group) by cardiac puncture, pooled and assayed for Western reactivity against E. tenella sonicated sporozoites, merozoites, and 30%(NH₄)₂SO₄44-72 hr supernatants from E. tenella SB-CEV/F7 infected cells. The IgG Western reactivity profiles of the sera samples are summarized in Table 7.

As expected from the early post-challenge timepoints examined, sera from the UI/C group did not recognize any proteins. The lack of detectable IgG sera immunoreactivity in UI/C birds at days 1 through 5 post-challenge, but immunoreactivity to Eimeria antigens in other biological compartments at the same timepoints (as shown in several of the above examples), highlights one of the critical aspects of the present invention - namely, that analysis of the antibody response and identification of immunodominant antigens using sera alone, does not reflect the local intestinal B and T-cell immune responses and cell trafficking events which occur at the site of parasite replication. At day 1 post-challenge, sera from NE/C identified P35 present in both spz and 30%(NH₄)₂SO₄44-72. At day 3 post- challenge, sera from NE/C identified P38 present in mrz. At day 5 post-challenge, a broader number of antigens were identified, such as P70, P55, P43, P40, P38 and P35. similar molecular weight proteins have been described in previous examples herein using SLIP, CLIP and RAT. However, these proteins were generally identified earlier, days 1 through 3 post-challenge. These data suggest that E. tenella antigens are initially recognized by local immune responses (CLIP, RAT) and then appear later as measured by systemic immune responses (sera). Therefore, kinetic measurements and specificity determinations of the earlier, localized memory immune responses provides a more comprehensive and meaningful analysis of the critical E. tenella antigens important for induction of a protective immune response.

Example 15 Purification of slgA from NE/C CLIP Fourteen B¹⁹B¹⁹ birds were infected with 5 x IO⁴ E. tenella oocysts at 10 days of age, and then challenged with the same dose at 22 and 36 days of age. Two days following the last challenge, CLIP reagent was prepared as described in Example 2. Approximately 40 mL CLIP was treated with ammonium sulfate to a concentration of 35%. After stirring one hour at 4°C, the solution was centrifuged (16,800 x g), precipitate collected, dissolved in PBS and dialyzed against PBS. Western blot results show that all reactivity using a murine MAb specific for chicken alpha chain (Cornell, 6:3-2) was in the 35%NH₄)₂SO₄ precipitate, with no detectable reactivity in the 35%NH₄)₂SO₄ supernatant. The Western positive sample was then applied to a 5 mL Jacalin column (Pierce) at a flow rate of 0.5 mL/min and absorbance monitored (280 nm). the column was washed with PBS (30 mL) to remove unbound protein and fractions collected, pooled and concentrated. Bound material was eluted using 100 mM melibiose/PBS and fractions with highest absorbance pooled. Unbound and bound pooled fractions were subjected to SDS- PAGE (reducing) and analyzed by silver staining and Western blot. Silver stain revealed a predominant P70 species with a few minor contaminating bands. The molecular weight of the reduced alpha heavy chain is 70 kDa. A portion of the positive staining fraction was applied to a Superose 6 column (1.6 x 53 cm) using a 0.5 mL/min. flow rate. Analysis of the collected fractions by SDS-PAGE silver stain identified a major P70 species. Based on the Superose 6 molecular weight standard profile, an approximate molecular weight of 170,000 daltons was assigned, indicating the presence of monomeric IgA.

Example 16 Purification of slgA from NE/C RAT RAT reagent was collected and prepared from the same group of birds used in Example 15 and purified in a similar manner. The eluate from the Jacalin column tested positive in the spz-specific IgA ELISA and was applied to a Superose 6 column, individual fractions collected and analyzed by SDS-PAGE silver stain. Results showed a P70 species present in both the early and late fractions. Analysis of the individual fractions staining positive for P70 using the SMART system (Pharmacia, Superose 6 column) suggested that tetrameric slgA (700,000 kDa) was present in the early fractions and that dimeric slgA (350,000 kDa) was present in the late fractions. The anti-chicken IgA probe only reacted with tetrameric IgA. These results indicate that both the tetrameric and dimeric forms of slgA are found in immune RAT and can be purified using the procedures described herein. Example 17 Identification of E. tenella Antigens Using Purified slgA Obtained from NE/C

Model The fraction containing CLIP dimeric slgA (Example 15) was used as a probe to identify antigens present in E. tenella-infected SB-CEV 30% and 45%(NIi,)₂SO₄44-72 hr supernatant. IgA Western results identified immunodominant 30%(NH₄)₂SO₄ P100 and weaker reactivity to P140 and P30. Using 45%(NH₄)₂SO₄, P140, P120, P70 and P35, were identified. In addition, numerous spz antigens were detected. These E. tenella antigens, identified using the partially purified IgA isolated from the local site of protective immunity in immune birds, are candidates for incorporation into Coccidiosis vaccines.

Example 18 Comparison of Western Reactivity of Anti-E. tenella Rabbit Polyclonal Sera to RAT Prepared from Immune Inbred NΕ/C Model

A standard NΕ/C RAT sample lot was prepared from B¹⁹B¹⁹ birds. Chicks were trickle-immunized with 500 E. tenella oocysts/bird for 5 days, rested for 15 days, challenged with 5 x IO⁴ oocysts and then rechallenged 14 days later. Seven days later, RAT reagent was prepared. Polyclonal rabbit anti-E. tenella sera, designated Rb 15/16, was obtained by immunizing rabbits with freshly excysted and adjuvanted E. tenella sporozoites three times. Rb 15/16 and RAT samples were assayed for IgG and IgA reactivity, respectively, against E. tenella spz, mrz, and E. te/ιe//α-mfected SB-CΕV/F7 30% and 45%(NH₄)₂SO₄0-44 and 44-72 supernatants. The Western reactivity profiles of the Rb 15/16 and RAT samples are summarized in Table 8. Results show a clear difference in the antigen reactivity between the two sources of anti-E. tenella antibodies. Most striking is an immunodominant P26 species recognized by Rb 15/16 that is not seen by immune RAT. In addition, Rb 15/16 identified at least 6 different merozoite antigens, whereas RAT reacts with a restricted number of merozoite proteins, namely P40, P35 and P29. These results support the contention that anti-E. tenella IgA antibodies present in RAT identified several, unique extracellular and SB-CΕV/F7 intracellular E. tenella antigens not recognized by conventional rabbit antisera raised against E. tenella sporozoites. Antigens recognized by NE/C RAT represent novel vaccine candidate targets. Example 19 Comparison of Anti-Eimeria Antibody Responses in Different Biological Compartments of NE/C B^B^ Birds Sera, SLIP and CLIP samples were obtained from NE/C B¹⁹B¹⁹ birds. Day- old chicks were trickle-immunized with 500 E. tenella oocysts/birds for 5 days, rested for 15 days, boosted with 5 x IO⁴ homologous oocysts, rested an additional 14 days, challenged with 5 x IO⁴ oocysts, and samples prepared at day 2 post-challenge as outlined above. Samples were assayed for Western reactivity against E. tenella spz, 30% and 45%(NH₄)₂SO₄44-72 hr supernatants from SB-CEV F7 infected and uninfected cells, and 44-72 hr infected and uninfected SB-CEV/F7 whole cell- associated antigens. Whole cell-associated antigens were prepared using a cell soluble lysis buffer (0.5% Brij-35, 300 mM NaCl, 50 mM Tris-Cl, pH 7.6 containing protease inhibitors (100 mM 1, 10 phenanthroline, 100 mM benzamidine HCL hydrate, 1 mg/ml pepstatin, 50 mM PMSF, 2 mg/ml leupeptin, 5 mg/ml soybean trypsin inhibitor, and 4 mg ml aprotinin)) for soluble membrane and cytosolic proteins, and a cell insoluble lysis buffer (0.2% sodium deoxycholate, 0.2% SDS) for insoluble material. The soluble and insoluble preparations were pooled for Western analysis. Results of the Western reactivity profile as summarized in Table 9. Only immunodominant proteins unique to infected preparations are listed. The antigen reactivity was specific to the immune compartment examined. For example, in 30%(NH₄)₂SO₄, CLIP recognized only P55, SLIP identified P120, Pl 10, P100 and P70. In contrast, immune chicken sera failed to identify any strong reactive 30%(NH₄)₂SO₄ species. In the infected cell- associated material, immune sera recognized P55, SLIP identified P38 and P35, and CLIP only identified P29. These data support the general claim that the immunodominant antigens identified are dependent on the type of antibody probe used. Antibodies produced at the local site of infection in an immune host, i.e., CLIP and/or RAT, typically recognize a more restricted set of antigens as compared to SLIP and sera. Moreover, the antigens recognized by immune CLIP and/or RAT are different than those recognized by SLIP or sera at a given timepoint, particularly early post-challenge. As stated previously, antigens in this invention are specified by MHC haplotype recognition, biological compartment, response time and immune status of the bird. Example 20 Identification of Cross-Reactive E. tenella Strain Antigens Using RAT Prepared from L.S. 65 E. tenella NE/C Birds A standard RAT sample lot was obtained from NE/C B¹⁹B¹⁹ birds. Chicks were trickle-immunized with 500 E. tenella L.S. 65 oocysts/bird for 5 days, rested for 15 days, boosted with 5 x IO⁴ homologous oocysts, rested an additional 14 days, challenged with 5 x 10⁴ oocysts, and samples prepared at day 7 post-challenge as outlined above. In order to identify important Eimeria antigens conserved between different E. tenella strains, immune RAT raised against E. tenella L.S. 65 was assayed for Western reactivity against antigens prepared from two heterologous E. tenella field strains isolated from two different geographic areas. The first field strain, designated GP5, was isolated from a poultry farm in Mississippi in 1992 (Dr. Linda Pote, Mississippi State) and the second field stain was isolated from an poultry farm in Arkansas in 1992 (Dr. Phil Davis, Univ. of Arkansas). Oocysts from both field strains were purified and amplified in Peterson Arbor Acres broilers using standard techniques, and used to infect SB-CEV/F7 cells. The following panel of E. tenella L.S. 65, GP1 and PD1 antigens were prepared as previously described: sporozoites, 30% and 45%(NH₄)₂SO₄44-72 hr supernatants from SB- CEV/F7 infected and uninfected cells, and infected and uninfected SB-CEV/F7 whole cell-associated antigens. Results of the Western reactivity profile are summarized in Table 10 and only immunodominant proteins specific to infected preparations are listed. Results indicate that the P43, P40, P38 triplet previously identified, is conserved among the 3 E. tenella strains examined. These results demonstrate that E. tenella RAT IgA antibodies raised against one E. tenella strain are capable of recognizing antigens from other E. tenella strains. Although strain cross-reactive serum antibodies have been previously described, this is the first example in which antibodies produced at the local site of infection, i.e., immune RAT, have been shown to contain IgA antibodies which are strain cross-reactive. This strategy can be used to confirm the conservation of P43, P40 and P38 in other E. tenella and heterologous Eimeria spp. field isolates (see below).

Example 21 Identification of Cross-Reactive Heterologous Eimeria spp. Antigens Using RAT Prepared from E. tenella L.S. 65 NE/C Birds In order to identify important antigens conserved between different Eimeria spp., RAT raised against E. tenella L.S. 65 was used to identify extracellular and SB-CEV/F7 intracellular antigens obtained from different Eimeria spp.: E. acervulina and E. maxima. Sporozoites obtained from pure oocyst cultures of each species were used to infect SB-CΕV/F7 cells. Sporozoites, 30% and 45%(NH₄)₂SO₄ 44-72 hr supernatants from SB-CEV/F7 infected and uninfected cells, and infected and uninfected SB-CEV/F7 whole cell-associated antigens, were prepared. For some species, limited SB-CEV/F7 intracellular replication was achieved, thus in these cases several of the standard antigen preparations were not available for screening. A standard RAT sample lot was obtained from NE/C B¹⁹B¹⁹ birds. Results of the Western IgA reactivity profile showed that 35, 38, 40, 43 and 70 kDa antigens were present in both E. tenella and E. maxima strains and 38 and 43 kDa antigens were identified in both E. tenella and E. acervulina.

Example 22 Identification of E. tenella Antigens Using E. tenella UI/C. NΕ/UC and NΕ/C RAT Prepared from Four Different Outbred Commercial Broiler Lines and Correlation to Protection

Groups of UI/C, NΕ/UC and NΕ/C outbred commercial broilers arbitrarily designated lines 1 through 4 were raised on wire and used as a source of RAT. Ten- day old birds were infected with 5 x IO⁴ E. tenella oocysts, rested for 11 days, then weighed and challenged with the same strain and dose. A group of age-matched naive birds was also weighed and challenged. A group of age-matched uninfected naive birds from each line was used as controls. At 39 hrs post-challenge, cecal and rectal feces from 2-3 birds/group were collected and pooled. Remaining birds were weighed at day 6 post-challenge and weight gains of the UI/C group from each line compared to their UI/UC counterparts. Weight performance was expressed as the percentage weight loss of the UI/C group compared to UI/UC controls. Pooled

RAT from each group of each line was used to screen E. tenella L.S. 65 sporozoites and 30%(NH₄)₂SO₄44-72 hr supernatants from SB-CΕV/F7 infected cells. The IgA Western reactivity profiles of the RAT samples are summarized in Table 11. The spz and 30% antigen reactivity profiles were dependent on both the bird line and the immune status. Results confirm several previously identified antigens, including

P35, P38, P40, P55 and P70. Additional antigens identified include P28 and P21. Importantly, these results show a direct correlation between weight performance and the ability to respond to P55, P40 and P35 spz antigens and P40 and P28 30%(NH₄)₂SO₄ antigens. UI/C birds from lines 3 and 4 demonstrated the lowest reduction in weights (more resistant), and these two lines produced IgA to several of the antigens described above. Birds from line 1 were partially susceptible to acute challenge and showed intermediate IgA antigen- specific responses. Birds from line 2 were most susceptible to challenge and showed very little IgA reactivity. These observations are also supported by the IgA antigen-specific responses of the NE/C groups from each line. NE/C birds from line 2 showed no detectable antigen- specific reactivity. Based on the RAT profiles, results suggest that lines 3 and 4 are high responders to acute infection (more resistant), whereas line 2 is a poor responder (more susceptible). Finally, these results confirm that several of the E. tenella antigens described in the NE/C model using the 3 MHC haplotype lines, i.e., P35, P38, P40 and P70, are also recognized by outbred broilers from several commercial production lines. These results also demonstrate the feasibility of using outbred RAT S/N to identify Eimeria antigens as well as to provide a better indication of the speed and level of immune protection afforded by initial parasite infection.

Example 23 Identification of E. tenella Antigens Using CD AT Prepared from Four Different E. tenella UI/C Outbred Broiler Lines and Correlation to Protection The same outbred commercial broiler lines used in the previous example were raised on wire and used as a source of CD AT. Birds were weighed at 14 days of age, and challenged with 5 x IO⁴ oocysts (UI/C). Age-matched naive birds from each line were weighed and mock-infected (UVUC). At 36-40 hrs post-challenge, fresh cage droppings from all the groups were collected, CD AT prepared and screened by Western blot analysis using anti-IgA against E. tenella spz antigen. UI/C bird weights at day 6 post-challenge were determined and compared to their UI/UC counterparts. Weight performance was expressed as the percentage weight loss of the UI/C group compared to the UI/UC controls. Only antigens identified in UI/C and not their UVUC counterparts are shown in Table 12. CD AT from line 1 identified Pl 10 and CD AT from line 3 recognized P55 and P35. These results indicate that in 14-day old birds acutely infected with E. tenella, RAT IgA spz- specific responses appear earliest in line 3. This result is consistent with the RAT results obtained in Example 21. In addition, CD AT responses to P55 and P35 E. tenella spz is likely indicative of a protective response, since line 3 showed the lowest drop in weight (-10%) among the 4 UI/C lines tested. Finally, this example shows that fresh CD AT can be used in a non-damaging, non-invasive diagnostic test to better determine the level of flock immunity to the Eimeria species of interest. Example 24 Identification of E. tenella Antigens Using RAT Prepared from Commercial Poultry Field Operation Farms Infected with Eimeria spp. RAT samples were collected from 15 different commercial poultry farms (4 birds/farm, pooled) during periodic, conventional coccidiosis diagnostic screening procedures. Samples were from either broiler or roaster production lines and birds ranged in age from 2-6 weeks. RAT samples were subjected to preliminary Western blot against L.S. 65 E. tenella sporozoite antigen. Two of fifteen farms showed very strong reactivity. These same two farms were subsequently confirmed by an independent laboratory to have the highest incidences of E. tenella cecal lesions in the birds examined. Five of fifteen farms tested positive for reactivity against 3 E. tenella spz antigens, P92, P40 and P38. These five RAT were then screened against 30%(NH₄)₂SO₄44-72 hr supernatant antigen from E. tenella SB-CΕV/F7 infected cells. A summary of the Western reactivity is shown in Table 13, and includes the type of bird, age, and most prevalent Eimeria spp. confirmed to be present by an independent laboratory. Results show that the local humoral immune response, recognized by RAT, identified 3 antigens common to all five farms. These five farms differed in the most common Eimeria spp. found. Therefore, these results show that the P43, P40, and P38 antigens are cross-reactive and most likely conserved among field isolates of E. tenella, E. maxima and E. acervulina. These 3 antigens are obvious targets for inclusion in a multivalent coccidiosis vaccine designed to be protective against the most economically important Eimeria species.

Example 25 Identification of E. tenella Antigens Using RAT Prepared from Commercial Poultry

Field Operation Farms Infected with E. maxima RAT samples were collected from 8 different commercial poultry farms (4 birds pooled/farm) during periodic, conventional coccidiosis diagnostic screening procedures. Samples were from broiler poultry houses and birds ranged in age from 2-5 weeks. RAT was prepared and used to screen E. tenella 30%(NH₄)₂SO₄44-72 hr supernatant antigen from E. tenella SB-CEV/F7 infected cells. The IgA Western reactivity profiles of the RAT samples are summarized in Table 14. Of the 8 pooled samples tested, 3 showed strong reactivity to both spz and 30%(NH₄)₂SO₄ antigens. A positive correlation between high clinical incidence of E. maxima and reactivity to E. tenella L.S. 65 spz antigens P43, P28 and P26 and E. tenella L.S. 65 SB- CEV/F7 30%(NH₄)₂SO₄ P70 was seen. As previously shown in Example 24, the strongest degree of Western reactivity was directly correlative to the highest incidence of E. maxima coccidiosis. No clinical incidence of E. tenella coccidiosis was found in any of the farms examined. Therefore, the P70, P43, P28 and P26 E. tenella antigens are cross-reactive with field RAT samples raised against E. maxima and represent important antigens for the induction of cross-protective immune responses.

Example 26

Characterization of Monoclonal Antibodies Raised Against E. tenella-lniected SB-

CEV F7 44-72 hr Tissue Culture Antigens or Merozoites Monoclonal antibodies were derived from five independent fusion experiments. In the first series (fusions #3 and 4), mice were immunized i.p., at two-week intervals, with 5 x IO⁶ SB-CEV/F7 culture-derived E. tenella merozoites, adjuvanted 1:1 with complete (primary immunization) or incomplete (boost) Freund's adjuvant. Three days prior to fusion, mice were immunized both IP and IV with IO⁶ unadjuvanted merozoites. Spleen cells were fused with mouse myeloma cell line SP2/0 and hybridoma supernatants initially screened by ELISA against E. tenella sporozoite and merozoite antigens. Reactive colonies were expanded and ^• screened against uninfected and 44-72 hr infected SB-CΕV/F7 supernatant. A total of nine MAbs were cloned by limiting dilution and characterized by Western blot and immunofluorescence assays. One MAb, designated 3-1, reacted with a single P21 protein. MAbs 4-1, 4-4 and 4-7 reacted with a series of proteins, suggesting reactivity to a common carbohydrate moiety present in all proteins. In the second series (fusions # 1, 2 and 5), mice were immunized and boosted as described above, with 25-30 μg of partially purified protein obtained by the biochemical separation of E. tenella infected 44-72 hr SB-CEV/F7 tissue culture supernatant. Hybridoma colonies were screened against the immunogen, and a total of three cloned MAbs were further characterized as above. Although MAb 1-2-6 recognized at least four different molecular weight proteins, it did show reactivity to P40, one of the protective proteins identified herein. The fact that MAb 1-2-6 reacted with segmenting schizonts by immunofluorescence suggests that P40 is expressed during the intracellular life cycle, and subsequently appears in the 44-72 hr tissue culture supernatant. A portion of P40 also appears to be associated with the large molecular weight (>200 kDa) 44-72 hr tissue culture aggregate. P40 is a component of the 30%(NH₄)₂SO₄ tissue culture vaccine shown to be protective in birds (Example 10), based on the SLIP, CLIP and RAT reactivity described in the present invention. It should be possible to obtain the cDNA clone for P40 using MAb 1-2-6 and conventional cDNA library screening techniques. Example 27 Partial N-terminal Amino Acid Sequence Determination of 45%fNH₁ ^')₂SO_J 110/100 Doublet Reactive with Rb 15/16. MAb 2-3 and SLIP. CLIP and RAT The 45%(NH₄)₂SO₄44-72 hr SB-CEV/F7 infected supernatant was applied to a Superose 6 column equilibrated with 4 M GdSCN in PBS. Western blot results using MAb 2-3 identified a strongly reactive 92 kDa species. Fractions containing MAb 2-3 reactivity were combined, dialyzed against PBS and the dialysate applied to a MAb 2-3 immunoaffinity column. Silver stain and Western blot results using MAb 2-3 and RB 15/16 identified a strongly reactive 110/100 doublet in the flow- through fractions. No reactivity with MAb 2-3 was detected under nonreducing SDS-PAGE. The doublet was strongly reactive with Rb 15/16 and associated with a 92 kDa species present in both the MAb 2-3 immunoaffinity flow-through and eluted fractions. Immunoreactive flow-through and eluate fractions were pooled and concentrated in an Amicon microconcentrator (MWCO 10,000). An

SDS-PAGE was run and submitted for microsequencing. Following electroblotting onto Problott membranes, seven lanes of the upper band of the 110/100 doublet were loaded for sequencing. The sequence obtained for the doublet is as follows: 110: 'X-P-L-P-Y-T-Y-I-P-Q¹⁰ 100: 'X-P-L-E-A-V-A-G-X-L-E¹¹.

The sequence shows no matches to the GenEMBL database.

Thus, novel Eimeria antibody preparation and antigens, as well as methods for producing and using the same, are disclosed. Although preferred embodiments of the subject invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and the scope of the invention as defined by the appended claims.

TABLE 1

E. tenella 30% Ammonium Sulfate SB-CEV/F7 Antigens Protect Inbred Birds Against Homologous Parasite Challenge

Footnotes:

N.T. = not tested

¹ UVUC = unimmunized.unchallenged; UVC = mock immunized/challenged;

30%/C = 30%(NH₄)₂SO₄ immunized/challenged

^•p≤O.02

^bp≤0.001

TABLE 2

Day 1 Post-Challenge IgG SLIP Reactivity From 3 Inbred MHC Haplotypes

Footnotes:

prepared from E. tenella infected SB-CEV/F7 44-72 hr. tissue culture supernatant

TABLE 3

Day 1 Post-Challenge IgG CLIP Reactivity in 3 MHC Haplotypes

Footnotes:

* strongest reactive species(s)

^b E. tenella infected SB-CEV/F7 44-72 hr. supernatant

TABLE 4

Day 3 Post-Challenge IgG CLIP Reactivity in 3 MHC Haplotypes

Footnotes:

^* strongest reactive species(s) ^b E. tenella infected SB-CΕV/F7 44-72 hr. supernatant

TABLE 5

Day 2 Post-Low Dose Challenge IgA RAT Reactivity in 2 MHC Haplotypes

Footnotes:

^* E. tenella infected SB-CΕV/F7 44-72 hr. supernatant

TABLE 6

Day 2 Post-High Dose Challenge IgA RAT Reactivity in 2 MHC Haplotypes

Footnotes:

^* E. tenella infected SB-CEV/F7 44-72 hr. supernatant

TABLE 7

Reactivity of Immune Sera from Inbred NE Model at Days 1, 3, and 5 Post- Challenge

Footnotes:

* E. tenella infected SB-CEV IF! 44-72 hr. supernatant

TABLE 8

IgG Polyclonal Rabbit Anti-E. tennella sporozoite and IgA NΕ/C RAT Western

Reactivity

Footnotes:

* denotes strongest immunoreactivity

TABLE 9

Comparison of Anti-Eimeria Antibody Responses in Different Biological Compartments of NE/C B¹⁹B¹⁹ Birds

Footnotes:

^* E. tenella infected SB-CΕV/F7 44-72 hr. supernatant

E. tenella infected SB-CEV/F7 cell-associated 44-72 hr.

TABLE 10

IgA Western Cross-Reacitvity of Immune NE/C RAT Raised Against E. tenella L.S. 65 to Heterologous E. tenella Field Isolates

Footnotes:

¹ CA = cell-associated 44-72 E. tenella-infected SB- CEB/F7

TABLE 11

Comparison of Weight Performance and RAT IgA Western Reactivity to E. tenella Antigens in Four Commercial Broiler Lines

TABLE 12

Comparison of Weight Performance and CD AT IgA Western Reactivity to E. tenlla SPZ Antigen in Four Commercial Broiler Lines

TABLE 13

Identification of E. tenella Antigens Using RAT Obtained From Commercial Poultry Houses Infected with Eimeria spp.

Footnotes:

' R = Roaster; B = Broiler

age in weeks

TABLE 14

Identification of E. tenella Antigens Using RAT Obtained From Commercial Poultry Houses Infected with E. maxima

Footnotes:

' age in weeks

based on lesions and oocyst counts

relative intensity

Deposits of Strains Useful in Practicing the Invention

A deposit of biologically pure cultures of the following strains was made with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland. The accession number indicated was assigned after successful viability testing, and the requisite fees were paid. Access to said cultures will be available during pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 USC 122. All restriction on availability of said cultures to the public will be irrevocably removed upon the granting of a patent based upon the application. Moreover, the designated deposits will be maintained for a period of thirty (30) years from the date of deposit, or for five (5) years after the last request for the deposit; or for the enforceable life of the U.S. patent, whichever is longer. Should a culture become nonviable or be inadvertently destroyed, or, in the case of plasmid-containing strains, lose its plasmid, it will be replaced with a viable culture(s) of the same taxonomic description.

These deposits are provided merely as convenience to those of skill in the art, and are not an admission that a deposit is required under 35 USC §112. A license may be required to make, use, or sell the deposited materials, and no such license is hereby granted.

Cell Line Deposit Date ATCC No.

SB-CEV- 1\F7 3 July 1990 CRL10495

Claims

Vhat is Claimed is:

1. An isolated, locally generated, Eimeria antibody preparation.

2. The antibody preparation of claim 1 wherein said preparation is derived from an avian subject which has been exposed to Eimeria tenella.

3. The antibody preparation of claim 2 wherein said preparation is derived from a lymphocyte population found within the intestinal tract of said avian subject.

4. The antibody preparation of claim 3 wherein said preparation comprises cecal lymphocyte immune products (CLIP).

5. The antibody preparation of claim 2 wherein said preparation comprises splenic lymphocyte immune products (SLIP).

6. The antibody preparation of claim 2 wherein said preparation comprises a coprantibody preparation.

7. The antibody preparation of claim 6 wherein said preparation is derived from the intestinal digesta of said avian subject.

8. The antibody preparation of claim 7 wherein said preparation comprises rectal antibody test (RAT).

9. The antibody preparation of claim 7 wherein said preparation comprises cage dropping antibody test (CDAT).

10. A method for detecting the presence or absence of an Eimeria antigen in a biological sample, said method comprising:

(a) contacting said biological sample with a locally generated, Eimeria antibody preparation under conditions whereby a complex is capable of being formed between an antigen present in said biological sample and an antibody present in said antibody preparation; and (b) detecting any complexes formed using a revealing label.

11. The method of claim 10 wherein said biological sample comprises an extract from a continuous cell line capable of supporting the propagation of Eimeria during the intracellular stages of development.

12. The method of claim 11 wherein said continuous cell line supports the propagation of Eimeria tenella.

13. The method of claim 10 wherein said biological sample is derived from extracellular sporozoites or merozoites.

14. The method of claim 10 wherein said antibody preparation is derived from an avian subject which has been exposed to Eimeria tenella.

15. The method of claim 10 wherein said antibody preparation comprises cecal lymphocyte immune products (CLIP).

16. The method of claim 10 wherein said antibody preparation comprises splenic lymphocyte immune products (SLIP).

17. The method of claim 10 wherein said antibody preparation comprises a coprantibody preparation.

18. The method of claim 17 wherein said antibody preparation is derived from the intestinal digesta of said avian subject.

19. The method of claim 18 wherein said antibody preparation comprises rectal antibody test (RAT).

20. The method of claim 18 wherein said antibody preparation comprises cage dropping antibody test (CD AT).

21. A method for detecting the presence or absence of an Eimeria tenella antigen in a biological sample, said method comprising:

(a) contacting said biological sample with at least one locally generated, Eimeria tenella antibody preparation selected from the group consisting of cecal lymphocyte immune products (CLIP), splenic lymphocyte immune products (SLIP), rectal antibody test (RAT) and cage dropping antibody test (CDAT), under conditions whereby a complex is capable of being formed between an antigen present in said biological sample and an antibody present in said antibody preparation; and

(b) detecting any complexes formed using a revealing label.

22. A method for diagnosing coccidiosis infection in an avian subject, said method comprising:

(a) providing a biological sample from said avian subject;

(b) contacting said biological sample with a locally generated, Eimeria antibody preparation under conditions whereby a complex is capable of being formed between an antigen present in said biological sample and an antibody present in said antibody preparation; and

(c) detecting any complexes formed using a revealing label.

23. An intracellular Eimeria antigen, identifiable using a locally generated,

Eimeria antibody preparation.

24. The antigen of claim 23, equivalent to an antigen derived from an extract of a continuous cell line capable of supporting the propagation of Eimeria tenella during the intracellular stages of development.

25. The antigen of claim 23 having a molecular mass of approximately 28 kDa, as determined using Western immunoblot analysis.

,

26. The antigen of claim 23 having a molecular mass of approximately 35 kDa, as determined using Western immunoblot analysis.

27. The antigen of claim 23 having a molecular mass of approximately 38 kDa, as determined using Western immunoblot analysis.

28. The antigen of claim 23 having a molecular mass of approximately 40 kDa, as determined using Western immunoblot analysis.

29. The antigen of claim 23 having a molecular mass of approximately 43 kDa, as determined using Western immunoblot analysis.

30. The antigen of claim 23 having a molecular mass of approximately 55 kDa, as determined using Western immunoblot analysis.

31. The antigen of claim 23 having a molecular mass of approximately 70 kDa, as determined using Western immunoblot analysis.

32. The antigen of claim 23 having a molecular mass of approximately 100 kDa, as determined using Western immunoblot analysis.

33. The antigen of claim 23 having a molecular mass of approximately 110 kDa, as determined using Western immunoblot analysis.

34. Monoclonal antibodies reactive with the antigen of claim 23.

35. A method for detecting the presence or absence of an Eimeria antibody in a biological sample, said method comprising:

(a) contacting said biological sample with an intracellular Eimeria antigen under conditions whereby a complex is capable of being formed between said antigen and an antibody present in said biological sample; and (c) detecting any complexes formed using a revealing label.

36. The method of claim 35 wherein said antigen is an Eimeria tenella antigen.

37. The method of claim 36 wherein said Eimeria tenella antigen is an intracellular antigen selected from the group of Eimeria tenella intracellular antigens having molecular weights of approximately 28 kDa, 35 kDa, 38 kDa, 40 kDa, 43 kDa, 55 kDa, 70 kDa, 100 kDa and 110 kDa, respectively, as determined using Western immunoblot analysis.

38. The method of claim 35 wherein said biological sample is derived from the intestinal digesta of said avian subject.

39. The method of claim 38 wherein said biological sample comprises intestinal digesta of the rectum of said avian subject.

40. The method of claim 38 wherein said biological sample comprises cage droppings from said avian subject.

41. A kit for diagnosing coccidiosis in an avian subject, said kit comprising a locally generated, Eimeria antibody preparation, packaged in a suitable container.

42. The kit of claim 41 wherein said Eimeria antibody preparation is derived from Eimeria tenella and is selected from the group consisting of cecal lymphocyte immune products (CLIP), splenic lymphocyte immune products (SLIP), rectal antibody test (RAT) and cage dropping antibody test (CD AT).

43. A kit for diagnosing coccidiosis in an avian subject, said kit comprising an Eimeria tenella monoclonal antibody reactive with an intracellular Eimeria antigen, packaged in a suitable container.

44. A kit for detecting the presence or absence of antibodies to Eimeria in a biological sample, said kit comprising an intracellular Eimeria antigen, packaged in a suitable container.

45. The kit of claim 44 wherein said Eimeria antigen is derived from an

Eimeria tenella intracellular antigen selected from the group of Eimeria tenella intracellular antigens having molecular weights of approximately 28 kDa, 35 kDa, 38 kDa, 40 kDa, 43 kDa, 55 kDa, 70 kDa, 100 kDa and 110 kDa, respectively, as determined using Western immunoblot analysis.