CA2251547A1 - Recombinant neospora antigens and their uses - Google Patents

Abstract

The present invention provides isolated bovine Neospora cultures. The cultures are used to develop diagnostic assays for the detection of Neospora infections in cattle and other animals. Also provided are pharmaceutical compositions for the treatment and prevention of Neospora infections.

Description

CA 022~1~47 1998-10-14 Recombinant Neospora Antigens and Their Uses BACKGROUND OF THE INVENTION
5This invention relates to the diagnosis and prevention of bovine diseases caused by the protozoan parasite, Neospora. The invention specifically relates to isolated cultures of the p~l~sile and nucleic acids and ploteills isolated from them.
A distinct pattern of infl~mm~tory lesions, consisting of focal 10 non~ ulali~e necrotizing encephalitis, non-~up~ulàli~/e myocarditis and myositis have been observed in many aborted bovine fetuses submitted for diagnosis. The pattern of lesions, particularly in the brain, is similar to those seen with Toxoplasma gondii infections in sheep. However, cattle have been reported to be resistant to T. gondii infection Dubey, Vet. Parasit. 22:177-202 (1986). In 1988, 15 a cyst-forming protozoal parasite was first itlPntffl~l by histopathological ex~min~tinn in fetuses (Barr et al., Vet. Parasit. 27:354-61 (1990)). This p~làSil~
was morphologically similar to Toxoplasma, except that some of the cysts had thick walls, which was more similar to the Neospora caninum-like protozoan observed by Thilsted & Dubey (J. Vet. Diagnos. Invest. 1:205-9(1989)) in abortedfetuses from a dairy in New Mexico.
Further studies showed that the protozoal l~alasiles associated with infl~mm~tory lesions in aborted fetuses and neonatal calves in California had ultrastructural and antigenic features that were most similar to N. caninum parasites which were originally isolated from dogs (Dubey et al., JA17l~A
25193:1259-63 (1988)). However, dirf~lcnces in the antigenic reactivity of the bovine protozoan and N. caninum when tested with a panel of antisera indicated that they may not be the same species (Barr et al., Vet. Pathol. 28:110-16 (1991)).
A more complete underst~n~ing of the identity and biology of these bovine protozoa requires estab1ishing continuous in ~itro cultures of the parasites.
30Such cultures would also be valuable in the development of diagnostic assays and ph~ e~1tic~1 compositions for the treatment and prevention of Neospora infections. The present invention addresses these and other needs.

.. . ..

CA 022~1~47 1998-10-14 SUMMARY OF THE INVENTION
The present invention provides biologically pure cultures of bovine Neospora. Two such cultures have been deposited with the ATCC and given ATCC Accession No. (BPA1), and ATCC Accession No.
5 (BPA6).
The invention also provides methods of detecting the presence of antibodies specifically immnnnreactive with a bovine Neospora antigen in a biological sample (e.g., bovine serum). The method comprises cont~cting the sample with the Neospora antigen, thereby forming a antigen/antibody complex, 10 and ~letecting the plesel1ce or absence of the complex. The Neospora antigen is typically an isolated recombinantly produced immunodominant Neospora antigen.
In some embo~im~nt~, the antigen is immobilized on a solid surface and the complex is ~let~ctecl using a fluorescently labeled anti-bovine antibody.
The invention further provides methods of detecting the presence of 15 Neospora in a biological sample. These mPtho~s comprise cont~cting the samplewith an antibody specifically immllnf)reactive with a Neospora antigen, thereby forming a antigen/antibody complex, and ~et~cting the presence or absence of thecomplex. The antibody (e.g., a monoclonal antibody) may be immobilized on a solid surface and the complex (letect~l using a second labeled antibody. Typically, 20 the biological sample is bovine fetal neurological tissue.
The methods of the invention also include detecting the presence of Neospora-specific nucleic acids in a biological sample by contacting the sample with a oligonucleotide probe which specifically hybridizes with a target Neospora-specific polynucleotide sequence and ~etecting the presence or absence of 25 hybridization complexes. The methods may further comprise amplifying the target Neospora-specific polynucleotide sequenre.
The invention further provides ph~ ceutic~l composition comprising a ph~rm~e~-tir~lly acceptable carrier and an immlln~genically effective amount of a bovine Neospora antigen, such as a recombinantly produced bovine 30 Neospora polypeptide.
The ph~rm~relltic~l compositions are used in proteclillg a bovine animal from infection by bovine Neospora. The compositions are preferably CA 022~1~47 1998-10-14 a(lmini~tered to a cow or heifer when the animal is breeding. The ph~ ceutic~l composition is usually ~mini.ctered parenlel~lly.
Definitions "Antibody" refers to an immllnoglobulin molecule able to bind to ~ 5 a specific epitope on an ~ntigen Antibodies can be a polyclonal mixture or monoclonal. Antibodies can be intact immllnoglobulins derived from natural sources or from recombinant sources and can be immllnc)reactive portions of intact immlln~globulins. Antibodies may exist in a variety of forms including, for example, Fv, Fab, and F(ab)2, as well as in single chains. Single-chain antibodies, in which genes for a heavy chain and a light chain are combined into a single coding sequence, may also be used.
"Biological sample" refers to any sample obtained from a living or dead organism. Examples of biological samples include biological fluids and tissue specimens. Examples of tissue specimens include fetal brain tissue, spinal cord,and placenta. Examples of biological fluids include blood, serum, plasma, urine,ascites ~luid, cerebrospinal fluid and fetal fluid.
A "biologically pure bovine Neospora culture" refers to a continuous in vitro culture of bovine Neospora org~ni~m~ (e. g. tachyzoites) which is substantially free of other org~ni~m.c other than the host cells in which Neospora 20 tachyzoites are grown. A culture is subst~nti~lly free of other org~ni~m~ if standard harvesting procedures (as described below) result in a pl~l)aldtion which coln~lises at least about 95%, preferably 99% or more of the org~ni~m, e.g., Neospora tachyzoites.
"Bovine Neospora" refers to Neospora or "Neospora-like"
25 protozoans i-lentif1ed in or isolated from bovine tissues and fluids. Typically, the protozoal ~al~siles can be isolated from neurological tissue of aborted bovine fetuses or congenitally infected calves. Exemplary isolates have been deposited with the American Type Culture Collection, as described below.
A bovine Neospora "protein" or "polypeptide" includes allelic 30 variations normally found in the natural population and changes introduced byrecombinant t~r.hni(lues. Those of skill recognize that proteins can be modified in a variety of ways including the addition, deletion and substitution of amino acids.

CA 022~1~47 1998-10-14 WO 97/42g71 PCT/US97107500 A "recombinantly produced immllnndominant Neospora antigen" is recombinantly produced polypeptide CO~ lisillg one or more immlmodominant epitopes. Exemplary recombinant antigens of the invention include SEQ ID NO:
5 10 and SEQ ID NO:12. Such antigen are encoded by which are encoded by SEQ
ID No:9 and SEQ ID NO:11. Terms used to describe the recombinant antigens and the nucleic acids which encode them will be understood by those of skill in the art to include sequences which are subst~nti~lly identical to the exemplified sequences. Substantial identity can be determined as described below.
"Nucleic acids" and "polynucleotides", as used herein, may be DNA
or RNA. One of skill will recognize that for use in the expression of Neospora ~rotehls or as diagnostic probes, polynucleotide sequences need not be i~lçnti~
and may be subst~nti~lly identical to sequences disclosed here. In particular, where the inserted polynucleotide sequence is transcribed and tr~n.~l~tçd to produce 15 a functional polypeptide, one of skill will recognize that because of codon degeneracy a number of polynucleotide sequenres will encode the same polypeptide.
"Percentage of sequen~e identity" for polynucleotides and polypeptides is ~leterminp~l by co~ )alillg two optimally aligned sequences over a 20 comparison window, wllcleill the portion of the polynucleotide or polypeptidesequence in the colll~alison window may comprise additions or deletions (i.e., gaps) as com~ared to the ,~,fe~ ce sequetl~e (which does not colll~lise additions or deletions) for optimal ~lignm~nt of the two sequences. The pel~el,~ge is c~lr~ te~1 by det~-",in;-~g the number of positions at which the identical nucleic 25 acid base or amino acid residue occurs in both sequen~es to yield the number of matched positions, dividing the number of m~tcll~cl positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the peicelltage of sequence identity. Optimal ~lignment of sequences for comparison may be con~luctçd by computerized implemP-nt~tions of known 30 algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Colll~ul~l Group (GCG), 575 Science Dr., Madison, WI), or by in.cpection.

CA 022~l~47 l998- l0- l4 Substantial identity of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 75% sequence identity, preferably at least 80%, more preferably at least 90% and most preferably at least 95%, Typically, two polypeptides are considered to be subst~nti~lly identical if5 at least 40%, preferably at least 60%, more preferably at least 90%, and most preferably at least 95 % are identical or conservative substitutions. Sequences are preferably compared to a reference sequence using GAP using default parameters.

Another indication that polynucleotide sequences are substantially 10 identical is if two molecules hybridize to each other under stringent conditions.
Sl~hlgelll conditions are sequenre dependent and will be dirrelcnl in different cirC~lm~t~nres. Generally, stringent conditions are selected to be about 5~ C lower than the thPrrn~l melting point (Tm) for the specific sequenre at a defined ionic ~llenglh and pH. The Tm is the temperature (under defined ionic ~llen~lll and pH) 15 at which 50% of the target sequence hybridizes to a perfectly m~tcl~Pcl probe.
Typically stringent conditions for a Southern blot protocol involve washing at room temperature with a SXSSC, 0.1% SDS wash.
The phrase "selectively hybridizing to", refers to a hybridization bclwcen a probe and a target seqUçnre in which the probe binds subst~nti~lly only 20 to the target seq~lence when the target is in a heterogeneous l~ lu~e of polynucleotides and other compounds. Such hybridization is dete""hlalive of the presence of the target seqUçnre. Although the probe may bind other unrelated sequences, at least 90%, preferably 95% or more of the hybridization complexes formed are with the target sequence.
The phrase "specifically immllnoreactive with", when Icfe~hlg to a protein or peptide, refers to a binding reaction between the protein and an antibody which is determinative of the presence of the protein in the presence of a heterogeneous population of p,oleills and other compounds. Thus, under de~ign~te~l immnno~s~y conditions, the specified antibodies bind to a particularprotein and do not bind in a signific~nt amount to other pruteills present in the sample. Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of CA 022~1~47 1998-10-14 WO 97/42971 PCT/US97tO7500 immnno~s~y formats may be used to select antibodies specifically immnnoreactive with a particular protein and are described in detail below.
The phrase "substantially pure" or "isolated" when referring to a Neospora peptide or protein, means a chemical composition which is free of other5 subcellular components of the Neospora olg~l~slll. Typically, a monomeric protein is subst~nti~lly pure when at least about 85% or more of a sample exhibits a single polypeptide backbone. Minor variants or chemical modifications may typically share the same polypeptide sequence. Depending on the purification procedure, purities of 85 %, and preferably over 95 % pure are possible. Protein10 purity or homogeneity may be inrlic~te~l by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed byvi~ li7ing a single polypeptide band on a polyacrylamide gel upon silver staining.
For certain purposes high resolution will be needed and HPLC or a similar means for purification utili7P~.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows indirect fluorescent antibody (IFA) titers of serial samples from four cows that aborted Neospora-infected fetuses.
Figure 2 shows IFA test titers of serial samples from two cows that 20 aborted Neospora-infected fetuses and subsequently delivered congenitally infected calves.
Figure 3 shows seroconversion by heifers ~pc.;~ lly infected with Neospora. (Figure 5A, SEQ ID NO:10; SPPQS(S/Y)PPEP = SEQ ID
NO:13; HP(H/Y)P = SEQ ID NO:14; SPP(E/Q) = SEQ ID NO:15;
25 SY(A/P)P(D/E)PS - SEQ ID NO:17; Figure 5B, SEQ ID NO:16) Figure 4 shows ~FA test titers: co~ )a~ g cattle with no evidence of infection to dams with Neospora-infected fetuses or calves.
Figures 5A and 5B show sequence motifs in two immlmodominant cDNA clones isolated from Neospora.

CA 022~1~47 1998-10-14 DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention provides Neospora cultures isolated from cattle. The cultures are useful in a variety of applications including the production of nucleic acids or proteins for diagnostic assays and the preparation of immllnogenic proteins for use in vaccine compositions.
Neospora tachyzoite cultures of the invention have been deposited with the American Type Culture Collection, Rockville, Maryland on March 17, 1994 and given Accession Numbers (BPA1) and (BPA6).
These isolates were obtained from tissue homogenates of brain and/or spinal cord of an aborted bovine fetus and congenitally infected calves.
Tmmllnohi.ctoch-omi.~try was used to identify the tachyzoite and/or cysts associated with lesions in the tissues of t'nese fetuses and calves as Neospora parasites prior to isolation. Tachyzoite stages of the isolates were grown in stationary monolayer cultures of bovine fetal trophoblasts, aortic endothelial cells and/or macrophages.
Electron microscopic studies were used to characterize the ultrastructural features of the BPA1 isolate. Antigenically, tachyzoites of 5 separate isolates react strongly with antisera to Neospora and show little or no reactivity with a,-lis~,d to Toxoplasma gondii or Hammondia hammondi. Based on these ultrastructural and ~nti~eniC cha~ .lics, t'nese p~ siles can be distinguished from the most closelyrelated and morphologically similar genera of protozoa, Toxoplasma, Hammondia and Sarcocystis.
In addition, partial sequences (500-550 base pairs) of the 5 end of the nuclear small subunit (nss)-rRNA gene for three of the bovine Neospora isolates (BPA1, BPA3 and BPA5) have been obtained and shown to be i~1entir~l.
The more complete 1.8 kilobase sequen~e of the nss-rRNA gene of the BPA1 isolate was obtained and compared to the sequences for this gene in other coccidial parasites. ~lignmPnt of these sequences with published sequences of Neospora caninum, Cryptosporidium parvum, Sarcocystis muris and Toxoplasma gondii showed that the bovine Neospora isolate is genotypically unique.
As explained in detail below, the isolates are used to develop a variety of diagnostic assays as well as ph~rm~elltic~l compositions for treatment and prevention of infection.

CA 022~1~47 1998-10-14 Preparation of Neospora polypeptides and nucleic acids Standard protein purification techniques can be used to isolate proteins from the tachyzoites or bradyzoites derived from the cultures provided here. Such techniques include selective precipitation with such substances as 5 ammonium sulfate, column chromatography, immllnopurification methods, and the like. See, for instance, R. Scopes, Protein Purification: Principles and Practice, Sp~ gel-Verlag: New York (1982).
Using standard immllnoblot techniques 11 proteins with molecular weights of approximately 106, 49.5, 48, 33, 32.5, 30, 28, 26, 19, 18 and 17 10 kilodaltons (kd) have been identified. All of these proteins are specificallyrecognized by antibodies from Neospora infected cattle. Standard protein purification methods can be used to purify these proteins and produce polyclonalor monoclonal antibodies for use in diagnostic methods described below. Two of these antigens (approximately 106 and 19 kd) have been shown to be useful in 15 enzyme-linked iîr~nunoassays (ELISA) for the detection of Neospora-specific antibodies in infected cattle.
Rather than extract the ploleills directly from cultured tachyzoites, nucleic acids derived from the cultures can be used for recombinant ~ ession of il.-..-...~odo~llina~l antigens of the invention. In these methods, the nucleic acids 20 encoding the ~rot~ins of interest are introduced into suitable host cells, followed by induction of the cells to produce large amounts of the protein. The isolationof two exemplary nucleic acids is described in Example 6, below. The invention relies on routine techniques in the field of recombinant genetics, well known tothose of or.lhlaly skill in the art. A basic text disclosing the general methods of 25 use in this invention is Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Publish., Cold Spring Harbor, NY 2nd ed. (1989).
Nucleic acids for use as diagnostic oligonucleotide probes or for the recombinant expression of protei..s can be isolated using a number of techniquesFor in~t~nre, portions of ~ tt;illS isolated from the cultures discussed above can 30 be seqllenrecl and used to design degel~ldt~ oligonucleotide probes to screen a cDNA library. Amino acid sequencing is pclrol.lled and oligonucleotide probes are synthrsi7ecl according to standard techniques as described, for in~t~nre, in CA 022~1~47 1998-10-14 wo 97/42971 PCT/US97/07500 Sambrook et al., supra. Alternatively, oligonucleotide probes useful for identification of desired genes can also be prepared from conserved regions of related genes in other species.
Alternatively, polymerase chain reaction technology (PCR) can be 5 used to amplify nucleic acid sequences of the desired gene directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. Polymerase chain reaction (PCR) or other in vitro amplification methods may also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed,to make nucleic acids to use as probes for cletecting the presence of the mRNA in 10 physiological samples, for nucleic acid sequencing, or for other purposes. For a general overview of PCR see PCR Protocols: A Guide to Methods and Applications. (Innis, M, Gelfand, D., Sninsky, J. and White, T., eds.), AcademicPress, San Diego (1990).
Standard transfection methods are used to produce prokaryotic, 15 ~ n, yeast or insect cell lines which express large ql-~ntitirs of the desired polypeptide, which is then purified using standard techni-lues. See, e.g., Colley et al., J. Biol. Chem. 264:17619-17622,1989; and Guide to Protein Purification, supra.
The nucleotide seq-lenres used to lld~r~;l ehe host cells can be 20 modified to yield Neospora polypeptides with a variety of desired plopel lies. For example, the polypeptides can vary from the naturally-occurring sequence at the plillldl~ structure level by amino acid, insertions, substitutions, deletions, and the like. These moriifir~tions can be used in a number of combinations to produce the final modified protein chain.
The amino acid seq~lenre variants can be pl~pdl~ d with various objectives in mind, including facilitating purification and prel)aldtion of the recombinant polypeptide. The modified polypeptides are also useful for modifyingplasma half life, improving lh~ldl~tulic efficacy, and lessening the severity oroccurrence of side effects during therapeutic use. The amino acid sequence variants are usually predetermined variants not found in nature but exhibit the same immlln~ genic activity as naturally occurring protein. In general, modifications of the sequences encoding the polypeptides may be readily CA 022~1~47 1998-10-14 WO 97/42971 rCT/US97/07500 accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see, Gillman and Smith, Gene 8:81-97, 1979) and Roberts, S. et al.,Nature 328:731-734, 1987). One of ordinary skill will appreciate that the effectof many mutations is difficult to predict. Thus, most modifications are evaluated S by routine scl~e~ g in a suitable assay for the desired characteristic. For in~t~nre, the effect of various modifications on the ability of the polypeptide to elicit a protective immlmf~ response can be easily determined using in vitro assays.
For in.ct~nre, the polypeptides can be tested for their ability to induce Iymphoproliferation, T cell cytotoxicity, or cytokine production using standard 10 techniques.
The particular procedure used to introduce the genetic material into the host cell for expression of the polypeptide is not particularly critical. Any of the well known procedures for introducing foreign nucleotide sequences into hostcells may be used. These include the use of calcium phosphate transfection, 15 spheroplasts, electropol~tion, liposomes, microinjection, plasmid vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (seeSambrook et al., supra). It is only nf~ces~ry that the particular procedure utilized be capable of successfully introducing at least one gene into the host cell which is 20 capable of e~ressillg the gene.
Any of a number of well known cells and cell lines can be used to express the polypeptides of the invention. For in~n~e, prokaryotic cells such asE. coli can be used. Eukaryotic cells include, yeast, Chinese hamster ovary (CHO) cells, COS cells, and insect cells.
The particular vector used to transport the genetic information into the cell is also not particularly critical. Any of the conventional vectors used for expression of recombinant pl'OIeillS in prokaryotic and eukaryotic cells may be used. Expression vectors for m~mm~ n cells typically contain regulatory elements from eukaryotic viruses.
The e~leSSiOn vector typically contains a transcription unit or e~pression c~sette that contains all the elements required for the expression of the polypeptide DNA in the host cells. A typical e~ SSiOn c~sette contains a CA 022~l~47 l998- l0- l4 WO 97/42971 PCT/US97l07500 promoter operably linked to the DNA sequence encoding a polypeptide and signals required for efficient polyadenylation of the transcript. The term "operably linked" as used herein refers to linkage of a promoter ul~sll~all~ from a DNA
sequence such that the promoter mf~ t~S transcription of the DNA sequence. The 5 promoter is preferably positioned about the same ~i~t~nre from the heterologous Lldl~sclip~ion start site as it is from the tldllsclil,tion start site in its natural setting.
As is known in the art, however, some variation in this ~ t~n~e can be accommodated without loss of promoter function.
Following the growth of the recombinant cells and expression of the 10 polypeptide, the culture m~dillm is harvested for purification of the secreted protein. The media are typically clarified by centrifugation or filtration to remove cells and cell debris and the proteins are concell~r~ted by adsorption to any suitable resin or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration. Other routine means known in the art may be 15 equally suitable. Further purification of the polypeptide can be accomplished by standard techni~lues, for example, affinity chromatography, ion exchange chromatography, sizing chromatography, His6 tagging and Ni-agarose chromatography (as described in Dobeli et al. Mol. and Biochem. Parasit. 41:259-268 (1990)), or other protein purification teçhn~ es to obtain homogeneity. The 20 puriffed pl'O~illS are then used to produce ph~rrn~ceuti-~l compositions, as described below.
An ~ltçrn~ive method of l~lel,alillg recombinant polypeptides useful as vaccines involves the use of recombinant viruses (e.g., vaccinia). Vaccinia virus is grown in suitable cultured ~.. -.. ~li~n cells such as the HeLA S3 spinner cells, as described by Mackett, Smith and Moss, "The construction and characterization of Vaccinia Virus Recombinants Expressing Foreign Genes" in "DNA cloning Vol. II. A practical approach", Ed. D.M. Glover, IRL Press, Oxford, pp 191-211.
Antibody Production The isolated l!loteil s or cultures of the present invention can be used to produce antibodies specifically reactive with Neospora antigens. If isolated CA 022~1~47 1998-10-14 cins are used, they may be recombinantly produced or isolated from Neospora cultures. Synthetic peptides made using the protein sequences may also be used.
Methods of production of polyclonal antibodies are known to those of skill in the art. In brief, an immllnogen, preferably a purified protein, is mixed 5 with an adjuvant and ~nim~l~ are imml~ni7ed. When appropriately high titers ofantibody to the immnnogen are obtained, blood is collected from the animal and antisera is plcpalcd. Further fractionation of the antisera to enrich for antibodies reactive to Neospora proteins can be done if desired. (See Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Pubs., N.Y. (1988)).
Polyclonal antisera to the BPA1 and BPA3 isolates have been produced and evaluated. The polyclonal antisera are used to identify and characterize Neospora tachyzoite and bradyzoite stages in the tissues of infected anim~ using, for i~ e, imml~noperoxidase test procedures described in Anderson et al. JAVMA 198:241 (1991) and Barr et al. Vet. Pathol. 28:110-15 116(1991).
Monoclonal antibodies may be obtained by various techni~ es famili~r to those skilled in the art. Briefly, spleen cells from an animal i..l....li~i7~d with a desired antigen are immortalized, commonly by fusion with a myeloma cell (See, Kohler and Milstein, Eur. J. Immunol. 6:511-519 (1976)). Alternative 20 methods of immortalization include transformation with Epstein Barr Virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells may be enh~n~e-l by various techniques, 25 including injection into the peritoneal cavity of a vertebrate host.
For in~t~n~e, the BPA1 isolate has been used to ;---.-~ e mice to obtain sensitized B cells for hybridoma production. Using these cells, monoclonal antibodies to the 48 kd and 70 kd Neospora proteins have been obtained. The monoclonal antibodies produced are used, for in~t~n~e, in ELISA diagnostic tests, 30 immunohistoch~mic~l tests, for the in vitro evaluation of parasite invasion, to select c~n~ te antigens for vaccine development, protein isolation, and for sclce~ g genomic and cDNA libraries to select al)l)lopliate gene sequences.

CA 022~1~47 1998-10-14 Diagnosis of Neospora infections The present invention also provides methods for detecting the presence or absence of Neospora in a biological sample. For in~t~nr.e, antibodies specifically reactive with Neospora can be ~letecte-l using either proteins or the 5 isolates described here. The proteins and isolates can also be used to raise specific antibodies (either monoclonal or polyclonal) to detect the antigen in a sample. In addition, the nucleic acids disclosed and claimed here can be used to detect Neospora-specific seq~ enreS using standard hybridization techniques. Each of these assays is described below.
A. Immunoassays For a review of immlm-logical and immllm)assay procedures in general, see Basic and Clinical Immunology 7th Edition (D. Stites and A. Terr ed.) 1991. The immllnoassays of the present invention can be performed in any of several configurations, which are reviewed extensively in En~:yme Immlmoassay, 15 E.T. Maggio, ed., CRC Press, Boca Raton, Florida (1980); "Practice and Theoryof Enzyme Tmmllnl~cs~ys, " P. Tijssen, Laboratory Techniques in Biochemistry andMolecular Biology, Elsevier Science Publishers B.V. Amsterdam (1985). For in~t~nre, the pl~o~eills and antibodies disclosed here are conveniently used in ELISA, imml-noblot analysis and ag~ tion assays. Particularly preferred assay 20 formats include the indirec~ fluo~sce~ll antibody assay as described in Example 2.
In brief, immllnoa~s?ys to measure anti-Neospora antibodies or antigens can be either competitive or noncompetitive binding assays. In co"lpe~ e binding assays, the sample analyte (e.g., anti-Neospora antibodies) c~ .e~es with a labeled analyte (e.g., anti-Neospora monoclonal antibody) for 25 specific binding sites on a capture agent (e.g., isolated Neospora protein) bound to a solid surface. The concell~ation of labeled analyte bound to the capture agent is inversely ~fopolLional to the amount of free analyte present in the sample.
Noncompetitive assays are typically sandwich assays, in which the sample analyte is bound between two analyte-specific binding reagents. One of the 30 binding agents is used as a capture agent and is bound to a solid surface. The second binding agent is labelled and is used to measure or detect the resultant complex by visual or instrument means.

CA 022~1~47 1998-10-14 A number of combinations of capture agent and labelled binding agent can be used. For in~t~nl~e, an isolated Neospora protein or culture can beused as the capture agent and labelled anti-bovine antibodies specific for the constant region of bovine antibodies can be used as the labelled binding agent.
S Goat, sheep and other non-bovine antibodies specific for bovine imm11noglobulin constant regions (eg. ~ or ~1) are well known in the art. Alternatively, the anti-bovine antibodies can be the capture agent and the antigen can be labelled.
Various components of the assay, including the antigen, anti-Neospora antibody, or anti-bovine antibody, may be bound to a solid surface.
lO Many methods for immobilizing biomolecules to a variety of solid surfaces are known in the art. For in~ e, the solid surface may be a membrane (e.g., nitrocellulose), a microtiter dish (e.g., PVC or polystyrene) or a bead. The desired component may be covalently bound or noncovalently ~tt:~h~d through nonspecific bonding.
Alternatively, the imm-m-as~y may be carried out in liquid phase and a variety of separation methods may be employed to separate the bound labeled component from the unbound labelled components. These methods are known to those of skill in the art and include immllno~lccipil~tion~ column chlonlatography, adsoll,lioll, addition of magn~ti7~1~1e particles coated with a20 binding agent and other similar procedu1es.
An immnno~ y may also be carried out in liquid phase without a sel)~alion procedure. Various homogeneous i""-"~-oa~y methods are now being applied to i,-,---.-.-oassays for protein analytes. In these methods, the binding of the binding agent to the analyte causes a change in the signal emitted by the label, 25 so that bi1ldillg may be measured without sepalalillg the bound from the unbound labelled component.
Western blot (imm11noblot) analysis can also be used to detect the presence of antibodies to Neospora in the sample. This technique is a reliable method for collr1ll11i1lg the presence of antibodies against a particular protein in the 30 sample. The technique generally comprises separating p1Ot~i11S by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, CA 022~1~47 1998-10-14 or derivatized nylon filter), and inrubating the sample with the separated proteins.
This causes specific target antibodies present in the sample to bind their respective plo~eills. Target antibodies are then cletecte~l using labeled anti-bovine antibodies.
The immnnnassay formats described above employ labelled assay 5 components. The label can be in a variety of forms. The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. A wide variety of labels may be used. The component may be labelled by any one of several methods. Traditionally a radioactive labelincorporating 3H, 125I, 35S, 14C, or 32p was used. Non-radioactive labels include 10 ligands which bind to labelled antibodies, fluorophores, chemil-~min~scent agents, en_ymes, and antibodies which can serve as specific binding pair members for a labelled ligand. The choice of label depends on sensitivity required, ease of conjugation with the compound, stability requirements, and available instr~m~nt~tion.
Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidored~lct~ces, particularly peroxidases. Fluorescelll compounds include fluorescein and its derivatives, rho~l~min~ and its derivatives, dansyl, umbelliferone, etc.
Ch~mill-minPscent compounds include luciferin, and 2,3-dihydrophth~1~7.in~-1iones, 20 e.g., luminol. For a review of various labelling or signal producing systems which may be used, see U.S. Patent No. 4,391,904, which is incorporated herein by reference.
Non-radioactive labels are often ~tt~ l by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule.25 The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently ~letectable or covalently bound to a signal system, such as a ~etPct~ble en_yme, a fluorescent compound, or a ch~mih-min~scent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in colljull~lion with the 30 labelled, nat~urally occurring anti-ligands. Alternatively, any haptenic or antigenic compound can be used in combination with an antibody.

CA 022~l~47 l998- l0- l4 Some assay formats do not require the use of labelled components.
For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutin~t~d by samples comprising the target antibodies. In this format, none of the components need beS labelled and the presence of the target antibody is ~letecte~l by simple visual inspection.
B. Detection of Neospora nucleic acids As noted above, this invention also embraces methods for ~letecting the presence of Neospora DNA or RNA in biological samples. These sequences 10 can be used to detect all stages of the Neospora life cycle (e.g., tachyzoites, bradyzoites, and oocysts) in biological samples from both the bovine host and the definitive host. A variety of methods of specific DNA and RNA measurement using nucleic acid hybridization techniqlles are known to those of skill in the art.
See Sambrook et al., supra.
One method for dete, .. li.. i.~g the presence or absence of Neospora DNA in a sample involves a Southern tl~l~r~l. Briefly, the digested DNA is run on agarose slab gels in buffer and transferred to membranes. In a similar manner, a Northern ~ rer may be used for the detection of Neospora mRNA in samples of RNA. Hybridization is carried out using labelled oligonucleotide probes whichspecifically hybridize to Neospora nucleic acids. Labels used for this purpose are generally as described for imml-noa~s~ys. Vi~ li7~tion of the hybridized portions allows the qualitative determination of the presence or absence of Neospora genes.
A variety of other nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in "Nucleic Acid Hybridization, A Practical Approach, " Ed. Hades, B.Dand Higgins, S.J., IRL Press, 1985; Gall and Pardue (1969), Proc. Natl. Acad Sci., U.S.A., 63:378-383; and John, Burnsteil and Jones (1969) Nature, 223:582 587.
Sandwich assays are commercially useful hybridization assays for ~let~cting or isolating nucleic acid sequences. Such assays utilize a "capture"
nucleic acid covalently immobilized to a solid support and labelled "signal" nucleic CA 022~1~47 1998-10-14 acid in solution. The biological sample will provide the target nucleic acid. The "capture" nucleic acid and "signal" nucleic acid probe hybridize with the targetnucleic acid to form a "sandwich" hybridization complex. To be effective, the signal nucleic acid cannot hybridize with the capture nucleic acid.
The sensitivity of the hybridization assays may be enh~nre~ through use of a nucleic acid amplification system which multiplies the target nucleic acid being detrctPd. Exarnples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBATN,Cangene, Missi~s~ , Ontario) and Q Beta Replicase systems.
An alternative means for detecting Neospora nucleic acids is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer, et al., Methods Enymol., 152:649-660 (1987). In situ hybridization assays use cells or tissue fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are dendlurl_d with heat or alkali. Thecells are then contacted with a hybridization solution at a moderate temperature to perrnit ~nnP~ling of labelled Neospora specific probes. The probes are preferably labelled with radioisotopes or fluolescellt reporters.
Exemplary nucleic acid sequenres for use in the assays described 20 above include sequences from the nss-rRNA sequ~nres disclosed here. For in~t~nre, the primer and probe sequences disclosed in Example 4 can be used to amplify and identify nucleic acids of bovine Neospora in blood, cerebrospinal fluid and fetal fluids, as well as in frozen or formalin-fixed tissue. These primers are particularly useful for the diagnosis of neosporosis and i(l~ntifir~tion of the source 25 of Neospora parasite stages (tachyzoites, bradyzoites and oocysts) in various animal hosts.
Phartn~reutir~l Compositions comprisin~ Neospora A pharrn~reutir~l composition prepared using anti-Neospora monoclonal antibodies or fr~gmrnt~ thereof as well as Neospora l)lOleil-S or their i.. ~.. ogenic equivalents can be used in a variety of ph~rtn~relltic~l preparations for the Llc,~ l and/or prevention of Neospora infections.

~ .. . . .

CA 022~1~47 1998-10-14 The ph~ relltic:~l compositions are typically used to vaccinate cattle, sheep, goats and other ~nim~l~ infected by Neospora. The compositions ofthe invention can also be used to treat the definitive host to prevent the ~h~dtling of oocysts and subsequent transfer to cattle. The compositions for ~ lion 5 to either cattle or the definitive host can comprise tachyzoite and/or bradyzoite antigens.
An attenuated Neospora vaccine can only be used in the absence of a risk of human infection should the milk or tissues of immllni7~d ~nim~l.c be con.cnm~cl. Thus, plefe.,cd vaccines are subunit vaccines that elicit antibody and 10 cell-m~ t~d illll~ y (CMI) to antigens of bovine Neospora. Experimental evidence in-lir~t~s that CMI is an important component of the protective immnn-~response in cattle. A variety of methods for ev~ ting the specificity of the helper and cytotoxic T cell response to selected antigens in vitro can be used. In addition, as demoJ-~llated below, cows infected using culture-derived tachyzoites 15 mount a ~ lecli~e immlln~ onsc and prevent transp]nce~t~l infection of the fetus.
The vaccines of the invention are typically ~ 1cd orally or parcllteldlly, usually h-l-~ lccl-l~rly or subcutaneously. For palc,-lc-dl ~lmini~tration, the antigen may be combined with a suitable carrier. For example, 20 it may be a~lmini~tered in water, saline or buffered vehicles with or withoutvarious adjuva-lls or i..l..l~ n-~locllll~ting agents such as ~l--,--i----,-l hydroxide, al-~,-lill...-- phosphate, alllminllm potassium sulfate (alum), beryllium sulfate, silica, kaolin, carbon, water-in-oilemulsions, oil-in-wateremulsions, lllul~-lyldipeptide, bacterial endotoxin, lipid, Bordetella pertussis, and the like. Such adjuvants are 25 available co.---.-clcially from various sources, for example, Merck Adjuvant 6 (Merck and Company, Inc., Rahway, N.J.). Other suitable adjuvants are MPL+TDM Emulsion (RIBBI Tmmlln~chem Research Inc. U.S.A.). Other immuno-stimlll~nt~ include interleukin 1, interleukin 2 and in~elrelol1-gamma These proteil~s can be provided with the vaccine or their corresponding genetic 30 sequence provided as a functional operon with a recombinant vaccine system such as vaccinia virus. The proportion of antigen and adjuvant can be varied over a broad range so long as both are present in effective amounts.

CA 022~1~47 1998-10-14 Vaccine compositions of the invention are a~lmini~tered to a cattle, sheep, or goats susceptible to or otherwise at risk of infection to elicit an immllnP
response against the antigen and thus enhance the patient's own immlln~ responsecapabilities. Such an amount is defined to be an "imml-nogenically effective 5 amount." In this use, the precise amounts depend on the judgement of the prescribing veterinarian and would include consideration of the patient's state of health and weight, the mode of ~-imini~tration, the nature of the formulation, and the like. Generally, on a per-dose basis, the concentration of the Neospora antigen can range from about 1 ,ug to about 100 mg per bovine host. For a~lmini~tration 10 to cattle, a preferable range is from about 100 ~g to about 1 mg per unit dose.
A suitable dose size is about 1-10 ml, preferably about 1.0 ml. Accordingly, a typical dose for subcutaneous injection, for example, would comprise 1 to 2 ml cont~ining 0.1 to 10 mg of antigen.
A variety of vaccination l~;gillRIIS may be effective in illllllllt~ g 15 cattle and other :~nim~l~. Preferably, female cattle (heifers and cows) are vaccinated just prior to or at the time of breeding so as to prevent abortion and reduce the possibility of congenital infections. A second immlmi7~tion will be given 2-4 weeks after initial i,,,,,.~ on. Calves and adult males may also be vaccinated, if desired. Animals that have been previously exposed to Neospora 20 or have received colostral antibodies from the mother may require booster in~ections. The booster injection is preferably timed to coincide with times of m~xim~l challenge and/or risk of abortion. Dirrelenl i.. ,-i~i7~tion regimes may be adopted depellding on the judgement of the v~lelillalian.
Vaccines of the invention may comprise a crude extract of Neospora tachyzoites, brady;coiles or other stages. Ch~mic~lly fixed parasites or cells can also be used. As noted above, plel~l~cd vaccines comprise partially orcompletely purified Neospora protein preparations. The antigen produced by recombinant DNA technology is pferell~d because it is more economical than the other sources and is more readily purified in large q~ntiti~s.
In addition to use in recombinant expression systems, the isolated Neospora gene seq len~es can also be used to llal~rolm viruses that transfect host cells in :lnim~l~. Live atten~-ated viruses, such as vaccinia or adenovirus, are . .

CA 022~1~47 1998-10-14 convenient alternatives for vaccines because they are h~e,~ellsi~re to produce and are easily transported and ~lmini~ered.
Suitable viruses for use in the present invention include, but are not limited to, pox viruses, such as capripox and cowpox viruses, and vaccinia viruses, alpha viruses, adenoviruses, and other animal viruses. The recombinant viruses can be produced by methods well known in the art, for example, using homologous recombination or ligating two plasmids. A recombinant canarypox or cowpox virus can be made, for example, by inserting the DNA's encoding the Neospora protein or fragments thereof into plasmids so that they are flanked by viral sequences on both sides. The DNA's encoding Neospora polypeptides are then inserted into the virus genome through homologous recombination.
Ple~l~l,Lially, a viral vaccine using recombinant vaccinia virus is used. A vaccine prepared lltiti7:ing the gene encoding the Neospora protein incorporated into vaccinia virus would c~Jml,lise stocks of recombinant virus where the gene encoding the Neospora protein is integrated into the genome of the virus in a form suitable for ~ ression of the gene.

EXAMPLES
Example 1 This example desclibes the isolation and in vitro cultivation of Neospora from aborted bovine fetuses. The isolation of these 2 cultures (BPA1 and BPA2) is described in Conrad et al. Parasitol. 106:239-249 (1993) Additional cultures (BPA3-6) were also isolated using the same technique, exceptthat 1 ml (instead of 2 ml) of brain or spinal cord homogenate was Llyl~sil~ized and then in~llh~t~d for 2-4 hours, rather than overnight, on the cell monolayer. In addition, the bovine aortic endothelial cell line (CPAE: American Tissue CultureCollection #CCL209) was found to be the best cell monolayer for the cultivation of bovine Neospora. One of these cultures (BPA6) has been shown to induce bradyzoite cysts in mice.

CA 022~1~47 1998-10-14 WO 97t42971 PCT/US97/07500 MATERIALS AND METHODS
Pathological ex:lmin~tion and immlmohistochemistry of fetal tissues Aborted bovine fetuses submitted to the California Vele~ a, y Diagnostic Laboratory System were neclol)sied using standard techni~ues. The 5 brains from fetuses ~uspect~d of having protozoal infections were removed aseptically from the cranium. One half of the brain was placed in sterile saline(0-85% w/v) cont~ining 1000 U/ml penicillin G and 100 ~g/ml streptomycin (antibiotic saline) and stored at 4 ~C until a diagnosis of protozoal infection was confirm~1, at which time the brain could be processed for in vitro cultivation.
10 Multiple tissues, including portions of the brain, liver, kidney, heart, lung, spleen, ga:i~roi"le~l ;"~l tract, skeletal muscle, adrenal, trachea and thymus, were collected from each fetus and fixed in 10% neutral buffered formalin for 24 h. Fixed tissues were trirnmed, embedded in paraffin, sectioned, stained with h~m~toxylinand eosin, and ~ d by light microscopy for the presence of lesions and 15 parasites, as previously described (Barr et al. 1990 Vet. Pathol. 27:354-61).Fetuses with multifocal microgliosis and/or necrosis in the brain, suggesting protozoal infection, were further ex~minP(l by immlmt~histoch.orni~try for the presence of parasites in brain tissue sections using an avidin-biotin peroxidase complex procedure (Vector Laboratories, Burling~m~, CA, USA) with 20 anti-rabbit serum to detect tissue-binding of rabbit polyclonal anti-N. caninum serum. The i""~ pel~xidase method employed was basically as described previously (Barr et al. 1991 J. Vet. Diag. Invest. 3:39-46) except that tissue sections were processed using a microprobe system (FisherBiotech, Pil~bu~
PA, USA) and Probe-On glass slides (FisherBiotech). ~minoethylcarbazole 25 (A.E.C. Substrate System, Dako, Santa Barbara, CA, USA) was the chromogen.
Parasites in the tissue sections of brains from the 66th and 93rd fetus (hereafter referred to as fetus 66 and 93) were further characterized by the same i.,~ ohistoch~mic~l procedure to test their reactivity with alllisel~ to additional apicomplexan protozoal parasites. Tissue sections were incubated at 30 room temperature for 1 h with optimal dilutions of the following antisera: 1:1000 dilution of antiserum to N. caninum tachyzoites (Lindsay & Dubey, 1989 Am. J.
Vet. Res. 50:1981-3), 1:50 dilution of antiserum to Hammondia hammondi tissue CA 022~1~47 1998-10-14 cysts and 4 different alllisel~ to T. gondii (Tgl-4). Antiserum Tgl was producedby the infection of a rabbit with live sporulated oocysts of the ME-49 strain (Lindsay & Dubey, supra) of T. gondii and used at a 1 :400 dilution. Toxoplasma gondii antiserum Tg2 (Dr J. C. Boothroyd, Stanford University) was produced by 5 i,."".l.~ ion of a rabbit with a tachyzoite Iysate of the RH strain of T. gondii and was used at a dilution of 1:300. Antisera Tg3 (BioGenex Laboratories, Dublin, CA, USA) and Tg4 (I.C.N. Tmml~nobiologicals, Lisle, IL, USA) were developed by il"---"~ -g rabbits with tachyzoites of the RH and H44 strains, respectively.Antiserum Tg3 was applied as supplied by the manufacturer and Tg4 was used at 10 a 1:80 dilution. The optimal dilution chosen for each anlisel-llll produced astrongly positive reaction against the l.,sl.e.;li~e positive control parasite with no appreciable non-specific, background st~inin~. Control tissues consisted of paraffin-embedded sections of murine brain with N. caninum tachyzoites, murine brain with T. gondii cysts, murine spleen with T. gondii tachyzoites, murine 15 skeletal muscle with H. hammondi cysts and bovine tongue with Sarcocystis cruzi cysts (Barr et al. 1991 Vet. Path. 28: 110-116).
Parasite cultures Stationary monolayer cultures of bovine cardiopulmonary aortic endothelial cells (CPAE: ATCC iYCCL209) and M617 bovine macrophages (Speer 20 et al. 1985 Infect. and Immun. 50:566-71) were m~int~in~d in Dulbecco's i",.-,., Essential Medium (DMEM:GIBCO Laboratories, Grand Island, NY, USA) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS) or heat-inactivated adult equine serum (HS), 2 mM L-glnt~min~, 50 U/ml penicillin and 50 ~g/ml sl,~,pto"-ycin (DMEM-FBS or DMEM-HS). Bovine fetal trophoblast 25 cells (87-3) were grown is previously described by Munson et al. 1988 J. Tissue Cult. Methods 11:123-8) in DMEM supplemented with 10% (v/v) FBS, 5 ~g/ml transferrin, 5 ~g/ml insulin, 5 ng/ml selenium, 10 ng/ml epidermal growth factor, 100 ,ug/ml ~ ,ptolllycin, 100 U/mi penicillin and 0.25 ~glmi amphotericin B
(DMEM-FBS*). Control cultures of T. gondii (~ strain provided by Dr J.
30 Boothroyd) and N. caninum (NCI; Dubey et al. 1988 J Am Vet Med Assoc 193:1259-1263) were m~int~inP~l in the CPAE and M617 monolayer cultures.
Parasite-infected and uninfected monolayer cultures were m~int~inP~ in 25 or 75 CA 022~1~47 1998-10-14 WO 97/42971 rCT/US97/07500 cm2 flasks inrubatPd at 37 ~C with 5% C02. Culture medium was changed 3 times weekly. Established parasite cultures were passaged to uninfected monolayers when 70-90% of cells were infected. To passage parasites, the infected monolayerwas removed from the flask by scraping into the media and passed 3 times through5 a 25-gauge needle to disrupt the cells. The cell suspension was then diluted from 1:2 to 1:8 in fresh complete media and added to a conflllent, uninfected monolayer.
After protozoal infection was confirmP~ by histology, fetal brain tissue that was stored at 4 ~C for a variable period of time in antibiotic saline was 10 processed for in vitro cultivation. In all cases where isolates were obtained in vitro, tissue cysts were seen in tissue sections of bovine fetal brain. Half of the fetal brain in approximately 25 ml of antibiotic saline was ground with a pestle and mortar and filtered through sterile gauze. Aliquots of 2 ml of brain homogenate were placed in 10 ml of 0.05% (v/v) trypsin and inr~b~ted at 37 ~C for 1 h.
15 After trypsin digestion, the material was pelleted by ce~ irugation at 600 g for 10 min. The supe, .l~t~l~l was discarded and the pellet was resuspended in 1-3 ml of either DMEM-HS or DMEM-FBS. Brain from fetus 66 was p~paled for culture 48 h after submission and 1 ml of ~ly~sini7Pd brain suspension was dispensed into a 25 cm2 flask of bovine 87-3 trophoblast cells. Brain from fetus 93 was 20 processed 10 days after submission when half of the lly~si~ ed brain was placed in a 25 cm2 flask of 87-3 trophoblast cells and the rem~in~1Pr in a 75 cm2 flask of endothelial cells. After inr~lb~tion overnight, the brain suspension from both fetuses was removed from the flask and the monolayers were washed 3 times with the ~L,l.~pliate media before adding 5-10 ml of fresh media. Cultures were 25 m~int~inPd as described above and ex~minPd with an inverted microscope for the presence of parasites.
Tmml-nnhi~tochemistry of tachyzoites in vitro Antigenic reactivity of the two in vitro isolates from aborted bovine fetuses was compared to that of tachyzoites from control cultures of T. gondii and 30 N. caninum. Tachyzoites of each isolate were harvested during logarithmic growth by scraping the infected CPAE monolayer from a 25 cm2 tissue culture flask.
Monolayer cells were disrupted by repeated passage through a 25-gauge needle.

CA 022~1~47 1998-10-14 The suspension was passed through a 5 ~m filter to remove cellular debris and pelleted by centrifugation at 1500 g for 10 min. After removing the supernatant fraction, the pelleted tachyzoites of each isolate were resuspended in DMEM-HS
and inoc~ ted into each of the wells on two 4-chambered tissue culture slides 5 (Lab-Tek, Nunc, Naperville, IL, USA). Each of the 4 chambers on these slides were seeded 24-48 h prior to parasite inoculation with CPAE cells so that the monolayers were 60-80% confluent at the time of infection. The applol,liate slides were inoculated with the slower growing bovine fetal isolates first to allow theparasites to grow for 48 h, whereas the isolates of T. gondii and N. caninum were 10 cultivated on slides for 24 h before being processed for immllnohistoch,~n~iç~l evaluation.
To pret)ale the parasite cultures for immlm~histochPn~ try, culture supellldtallls were removed with monolayers rem~ining adherent to glass slides.
These slides were fixed in 100% methanol (4 ~C) for 10 min and allowed to air 15 dry completely before being washed 3 times for 5 min each in physiologically burr~led saline (PBS:pH 7-2), inrub~t~d for 10 min in 3 % (v/v) hydrogen peroxide in methanol, washed again 3 times for 5 min each in PBS and inrllb~te-l for 30 min with 20 % goat serum to block non-specific antibody binding sites. Each slide was then incubated for 1 h with 3 wells cont~ining dir~e.c.ll primary antiserum and 20 1 well serving as a negative control with a pre-infection rabbit serum. The optimal antisera dilutions were selected to produce a strongly positive reactionagainst the homologous culture-derived antigen with no appreciable non-specific,background staining. The dilutions of antisera used for st~ining pal~iles in vitro were 1:3000 for N. caninum, 1: 800 for Tgl, 1:40 for Tg2, 1:1 for Tg3, 1:2000 25 for Tg4 and 1:50 for H. hammondi. Slides were washed 3 times for 5 min each in PBS and the secondary antibody and conjugate were applied as described above for the tissue sections except that the slides were processed m~n--~lly and the chromogen was applied for only 2 min.

The first parasite isolate (BPA1) was obtained from fetus 66 which was e~ d to be approximately 4 months gestational age and in relatively good CA 022~1~47 1998-10-14 Wo 97/4297l PCT/USg7/07500 postmortem condition at the time of necropsy. Significant gross lesions were restricted to focal epicardial petechi~e. On histological e~r~min~tion there were infrequent, random, small foci of gliosis and 5 protozoal cysts were seen in sections of the fetal brain. The tissue cysts ranged from 8 to 10 ~m in ~ 3m~ter5 and had distinct, thin walls ( < 1 ,~4m) surrounding at least 25-40 closely packed bradyzoites. In addition there were scattered mononuclear infl~mm~tory cell infiltrates in the heart, portal tracts of the liver and throughout the renal cortex.
In the lung, macrophages and neullu~hils were present within alveolar septa, adjacent to bronchioles and free in the lumen of bronchioles and alveoli.
0 Escherichia coli and Proteus spp. were isolated from the lung, liver, spleen and abomasal contents of this fetus.
The second isolate (BPA2) was obtained from fetus 93 which had an e~ n-t~d gestational age of 6 months and was mildly autolyzed. Histological e~r~min~tion revealed infrequent random foci of gliosis in the brain with adjacent 15 capillaries that had ~lolllinelll hypertrophied endothelium. There was also a mild diffuse meningeal infiltrate of mononuclear cells, con~i~ting predomin~ntly of Iymphocytes with occasional plasma cells. Four randomly located protozoal tissuecysts were found in the brain; one being located adjacent to a focus of gliosis.The cysts were 8-13 ~m in r1i~m~t~r with at least 25-50 bradyzoites. Two of the 20 cysts had thick (1-2 ~m) walls. Focal, mixed mononuclear infl~mm~tory cell infiltrates were also present in skeletal muscle and in the renal cortex.
Table 1 ~-"~ es the i"~"~ olcactivity of protozoal tissue cysts in the brains of fetus 66 and fetus 93 with dirr~ltlll polyclonal antisera and compares these antigenic reactions to those of N. caninum, T. gondii, H.
25 hammondi and S. cruzi. The piotozoal cysts in fetuses 66 and 93 reacted most strongly to N. caninum antiserum and had weaker reactions to H. hammondi antiserum. With both antisera, st~ining was predomin~ntly to the cyst wall with some st~ining of zoites within the cysts. Overall, the reactivity of cysts in the two bovine fetuses was more similar to the reactivity of N. caninum tachyzoites than30 to that of T. gondii tachyzoites or cysts, H. hammondia cysts or S. cruzi cysts.

Table 1 Reactivity of tissue cysts and tachyzoites with rabbit polyclonal antisera against different parasites in an immlln- peroxidase test Antisera lhV~7A----- g(~diii S ~?~ F~ A
Parasite antigen Tissue canLn~n h~ 1~ 3 Tg4 Fetus 66 cysts Bc~vine brain ++ ~ + - - -Eetus 93 cysts Ba~i~e brain ++ +
L ~ ll~I~ ~se brain ~ _ ~+
tactyzoites 0 Ihr~)7A.'-q g~i ~use spleen - ~+ +++ +++ + +
t~chyzoites 1~7~ gandii r~use brain + ~+ +++ + ++ ++
cy~;ts y ''A ~ use nuscle + f++ +++ - + ++
cysts cysts nuscle Prin~rily cyst wall st:ltnin~.

In approximately 14 months, over 100 fetuses were submitted specifically as protozoal abortion sl~spect~ from dairies in California. Parasite isolations were attempted with brain from the 49 fetuses which had Neospora-like25 protozoa identified by immlmohi~toç~ . y. The first evidence of parasite growth in these cultures was detectecl in the 87-3 cell line 34 days after inoculation of brain tissue from fetus 66 (isolate BAPl). The next sllcce~sful in vitro isolation wasapl,alclll when tachyzoites were first observed in 87-3 and CPAE cultures on day15 after inoculation with brain tissue from fetus 93 (isolate BPA2). In cultures of 30 both isolates, parasites first ap~ealcd as small clusters of intracellular tachyzoites in pairs or random groups. Extracellular tachyzoites were seen escaping from thebovine cell monolayers and moving by gliding and twisting in the culture m-o-limn.
On Giemsa-stained smears of infected monolayers, extracellular tachyzoites were 1.5-2.5 ~m wide at the nucleus and 6-8 ,um long. The number of tachyzoite clusters 35 and number of tachyzoites in each cluster increased gradually in the cultures as they became established with continuous parasite growth. Generally, parasite clusterscontained approximately 10-100 tachyzoites. Growth of both isolates was CA 022~1~47 1998-10-14 WO 97/42971 PCT/US97tO7500 m~int~in~d in cultures of 87-3,CPAE, or M617. However, the best growth was observed in the 87-3 and CPAE cultures. Within 2-3 months of establishment, the cultures were passaged weekly whenever approximately 80-95~Zo of the bovine monolayer cells were infected with tachyzoites. Routinely, the established BPAl 5 and BPA2 cultures were passaged by adding a 1:8 dilution of needle-passaged monolayer in fresh media to cultures of uninfected bovine monolayers. By comparison, in our laboMtory, cultures of T. gondii (R~I isolate) are routinely passaged at a 1:200 dilution and N. caninum (NC-1 isolate) cultures are passagedat a 1:10 dilution every 2-3 days. As of mid-May 1992, cultures of the BPA1 and BPA2 isolates had been m~int~in~d with continuous growth for 10 and 6 months, cs~ecli~/ely~
The results of an antigenic comparison of in vitro cultivated tachyzoites of BPAl and BPA2 to those of cultivated N. caninum and T. gondii tachyzoites are shown in Table 2. The reactions of the bovine fetal isolates to the 15 dirr.,lcn~ al,lisel~. were sirnilar to that clerno~ .dl~d by N. caninum, and (li~tinrtly dirr~lelll from the pattern of reactivity observed with T. gondii tachyzoites (Table 2).

Table 2. Reactivity of in vitro cultivated tachyzoites with rabbit polyclonal antisera 20 against dirrelent parasites in an immlmoperoxidase test Antisera Toxoplasma gondii Parasite Neospora u. ~n~i a antigen caninum hammondi Tgl Tg2 Tg3 Tg4 25 Foetus 66 (BPAl) +++ - - +
Foetus 93 (BPA2) +++ - + +
Neospora c~ni n~m I ++ _ _ +
Toxoplasma gondii - +++ +++ +++ +++ +++

By tr~n~mi~sion electron microscopy, the in vitro tachyzoites of isolates BPAl and BPA2 were morphologically similar. Therefore, the following CA 022~1~47 1998-10-14 ultrastructural description applies to both isolates. Individual tachyzoites or clusters of multiple tachyzoites were usually located within a parasitophorous vacuole in the cytoplasm of bovine monolayer cells. Tachyzoites had a pellicle consisting of a complex of 2 inner membranes beneath a plasmalemmal membrane, a prominent nucleus in the central or posterior portion of the tachyzoite, 1 to 3 profiles of long tubular cristate mitochondria, a Golgi complex, rough and smooth endoplasmic reticulum, single- or multiple-membraned vesicles, and numerous free ribosomes.
Ultrastructural apical features characteristic of apicomplexan parasites were present in tachyzoites of both isolates, including a polar ring which gave rise to 22 10 longi~din~l subpellicular microtubules, a cylindrical or cone-shaped conoid within the polar ring and numerous electron dense rhoptries. The number of rhoptries visible in individual tachyzoites varied greatly and was dependent to some extent on the plane of section. A maximum of 24 rhoptries was counted in the anterior end of transversely sectioned tachyzoites. Rhoptries were not seen posterior to 15 tachyzoite nuclei. In longitll~lin~l sections, rhoptries were elongated, club-shaped structures with narrow, dense necks that extended into the conoid. As many as 14-32 electron-dense bodies were observed primarily posterior to the nucleus.
Smaller numbers of these dense bodies were found anterior to the nucleus. Unlilce rhoptries, dense bodies were generally round or oval in longi~ in~l sections. Large 20 numbers of micronemes were seen in the anterior end of tachyzoites and only rarely observed posterior to the nucleus. The micronemes were most often arranged in olg~ l arrays or sheets that were olienlated parallel to the pellicular membraneor longi~-~lin~l axis of the tachyzoite. The number varied greatly in individualtachyzoites but as many as 60-100 micronemes were counted in longitll~lin~l or 25 oblique sections of selected tachyzoites. In addition, a single micropore, located anterior to the nucleus was seen in some tachyzoites. Parasites multiplied by endodyogeny and many were observed in the process of forming 2 progeny zoites within a single tachyzoite. Rarely, as many as 4 zoites with intact nuclei were seen in division but still ~tt~rh~d to each other at the posterior end.
A concerted effort has been focused on the in vitro isolation of Neospora- like protozoal parasites from cattle after studies showed that they were the major diagnosed cause of abortion in California dairy cattle. Histologically, the CA 022~1~47 1998-10-14 wo 97/42971 PCT/US97tO7500 two bovine fetuses from which isolates were first obtained in 1991 had compatible lesions, including multifocal non-supportive encephalitis, and protozoal tissue cysts like those seen in other natural infections of Neospora-like protozoa in bovine fetuses. The imm~lnological reactivity of tissue cysts in the brains of fetus 66 and 5 fetus 93 was also similar to that seen with many of the cysts in naturally infected fetuses which had strong reactions with N. caninum and H. hammondi antisera and occasional reactions to some of the T. gondii antisera.
Isolation of these Neospora-like protozoal parasites from aborted bovine fetuses was difficult because fetuses are generally moderately to severely 10 autolyzed at the time of abortion and protozoal tissue cysts are present in arelatively small proportion of the infected fetuses. Previous ultrastructural studies suggested that most of the protozoal cysts in these fetal tissues were affected by autolysisandwereprobablynon-viable(Barretal. 1991 Vet. Path.28:110-16). The two fetuses from which isolates were obtained were in comparatively good post-15 mortem condition. This fact, plus the relatively large number of cysts in thesefetuses may have been critical factors in the successful isolation of the protozoal parasites. In addition, the isolation methods were modified. In particular, a longer period of trypsinization in pl e~,a- h~g the brain material for cultivation, the overnight inr~lb~tion of the brain homogenate on the monolayer, and use of the 87-3 bovine20 trophoblastic cell line for the initial ~al~sil~ isolation were modifications in these procedures which appeared to be particularly helpful in obtaining the BPA1 and BPA2 isolates. Parasite growth was best m~int~in~-l in 87-3 and CPAE monolayer cells. This contrasts with N. caninum and Hammondia heydorni which are reported to grow better in bovine monocyte cells (Speer et al. 1988 Infect. and Immun.
25 50:566-71).
In colllpa,illg these bovine protozoal isolates to those of T. gondii and N. caninum that have been re-isolated from mouse brains, it was found that the bovine protozoal isolates grew more slowly during isolation and after establishment of continuous growth. Whether this reflects a dirrerelue in the virulence of the30 org~ni~m~ or a dirr~ ce in adaptation to culture remains to be determined.
By light microscopy, tachyzoites of the bovine isolates were morphologically similar to in vitro tachyzoites of T. gondii and N. caninum.

... .. . .

CA 022~1~47 1998-10-14 Cultivated tachyzoites of the bovine isolates had similar immllnl~histochptnic~lreactions to tachyzoites of N. caninum, reacting strongly with N. caninum anti-serum and weakly to serum Tgl which was produced by i,~ "l~ tion of a rabbit with tachyzoite Iysates of T. gondii. These antigenic reactions were distinctly 5 difrelelll from those seen with culture-derived tachyzoites of T. gondii. Dirrelcnces in antigenic reactivity of the cultivated BPA1 and BPA2 tachyzoites, as comparedto those of tissue cysts in the source fetuses, could be explained by the stage-specific antigen expression of the different parasites and variations in the methods used to produce the antisera (i.e. imml~ni7~tion with cysts, oocysts or 10 tachyzoite Iysates). For example, tissue cyst wall antigens that reacted withantiserum to H. hammondi al-l c~red to be lacking on tachyzoites of the bovine isolates in vitro. Ullrol ~unately, a direct comparison of dirrel ~ parasite stages was not always possible since tachyzoites were not identified in the brains of the two bovine fetuses and true cysts have not been observed in the BPA1 or BPA2 cultures.
15 Similarly, N. caninum tissue cysts and culture-derived H. hammondi tachyzoites were not available for colll~alison. Differences in antigenic expression may also be affected by host-specific factors. To evaluate this possibility, efforts are under way to obtain material from cattle, dogs, rats, cats and mice that have been experimentally infected with N. caninum or the bovine isolates so as to make a 20 direct collll)alison of antigenic leaclivily of the parasites in the same host species.
Thus far, chal~cleli~lion of the in vitro isolates from the two aborted bovine fetuses has shown that these parasites are antigenically and/or ultra-structurally distinct from T. gondii, H. hammondi, S. cruzi, Besnoitia spp. and Frenkelia spp. These isolates most closely resemble N. caninum parasites which 25 have been most extensively studied in the USA and Sc~n~in~via. The similaritybetween these parasites in~ at~s that the BPA1 and BPA2 isolates belong to the genus Neospora. At present little is known about the life-cycle, including the definitive host, of these Neospora parasites in dogs or cattle. A better underst~n-ling of the biology of these parasites is essential to determine their30 taxonomic relationship to each other and to other apicolllplexan parasites.

CA 022~1~47 1998-10-14 Example 2 This example describes an indirect fluorescent antibody (IFA) test for the detection of parasite-specific antibody responses in cattle that were naturally or experimentally infected with Neospora parasites. The methods used here are 5 generally as described in Conrad et al. 1993 J. Vet. Diagn. Invest. 5:572-578 (1993) MATER~ALS AND METHODS
Parasites and antigen slide ~lepalaLion Antigen slides were prepared using tachyzoites of the BPA1 described 10 above. Culture media consisted of Dulbecco's Mi~ Essential Medium (DMEM) supplemented with 10% (v/v) heat-inactivated adult equine serum, 2 mM
L-gl~-t~min~, 50 U/ml penicillin and 50 ug/ml streptomycin (DMEM-HS).
Tachyzoites of Toxoplasma gondii (RH isolate; kindly provided by Dr. J.
Boothroyd) were obtained from CPAE monolayer cultures grown in the same 15 medium except that 10% (v/v) heat-inactivated fetal bovine serum was used instead of equine serum. Parasite-infected cultures were m~int~in~d in 25 or 75 cm2 flasks incubated at 37~C in an atmosphere of 5% CO2.
Parasites were harvested for antigen ~ alaLion when 2 80% of the CPAE
cells in the culture flask were infected with clusters of tachyzoites. The infected 20 monolayer was removed from the flask by scraping into the mPdil-m and then passed 3x through a 25 ga needle to disrupt the cells. The suspension was passedthrough a 5~m filter to remove cellular debris and tachy~oil~s were pelleted by centrifugation at 1300 xg for 10 min. After removing the ~upe~ ..l, the pellet was washed twice in sterile phosphate l ul~d saline pH 7.2 (PBS) and then 25 resuspended in a modified PBS saline (137mM NaCI, 3mM KCI, 3mM
Na3C6H5O7.2H2O, 0.4mM NaH2PO4.H2O, 12mM NaHCO3, 6mM glucose) to a final concentration of approximately 2,000/,ul. Aliquots of 10,ul of tachyzoite suspension were dispensed into each 4 mm well on 12-well heavy teflon coated (HTC) antigen slides. Slides were air-dried at room l~ p~làture and stored at -70~ C.
30 Cattle Test sera was obtained from naturally infected cows that aborted Neospora-infected fetuses, as well as col1ge~ lly infected calves. In addition, sera was CA 022~1~47 1998-10-14 obtained from two pregnant heifers that were experimentally infected at approxirnately 120 days gestation with tachyzoites of the BPA1 isolate derived from CPAE cultures. Tachyzoites were obtained from cultures using the procedure described for harvesting tachyzoites for antigen preparation except that the parasites 5 were not washed in PBS and only the inoculum given to each heifer intravenously was filtered to remove cellular debris. After centrifugation, tachyzoites were resuspended in DMEM and allministPred by inoculation to each heifer so that 3X106 tachyzoites were given IV and 5X106 were given IM. A control heifer from the same herd and at the same stage of gestation was inoc~ ted with an equivalent 10 amount of uninfected CPAE cell culture material which was p~ al~d and admini~tPred using the same procedures as for the infected heifers. Natural or experimental infections were co--ri. ~I-Pd by identification of Neospora tachyzoites and/or tissue cysts in fetal or calf tissues using an i, ~ . . - -ol)eroxidase test procedure (Andersonetal. (1991)JAm VetMedAssoc 198:241-244andBarretal. (1991) Vet 15 Path 28:110-116).
For serological comparison with samples from Neospora-infected cattle, sera were obtained from the following additional sources: 1) cows that aborted fetuses which did not have lesions or pa~siL~s typical of Neospora infections, 2) weak calves that were ~ pe-;L~d of having Neospora infections, but showed no lesions or 20 parasites on post-mortem histopathologic e~min~tion, 3) 20 heifers that were purchased as weanling~ from a closed beef herd in Todd County, Nebraska and m~int~inecl under strict isolation, on range conditions at the Agricultural Research Development Center, University of Nebraska-Lincoln in Mead, Nebraska, 4) 20 pregnant heifers that were m~int~inP~I on pasture in California and 5) 21 adult beef 25 bulls or cows that were originally on pasture and then m~int~inPd in the same feedlot as the ~elilllentally infected heifers.
Serum collection and testin~
Test and control sera were obtained from blood samples that were collected by venipul~Lul~ into v~c~lt~inPr tubes without anticoagulant. After storage at 4~ C
30 for 2-12 hr, the blood was centrifuged at 500 xg for 10 min and the serum wasremoved. Sera was stored either at 4~ C for <48 hr or frozen at -70~ C until tested.

CA 022~1~47 1998-10-14 Antigen slides were thawed at room lelllpelalu~e immP~ tely prior to use.
Sera were initially titrated in 2-fold dilutions from 1:40 to 1:40,960 to determine the end-point titer. Ten ul of diluted test or control sera were placed in separate wells on the antigen slides. Slides were incubated at 37~ C for 1 hr in a moist 5 cha~"bel, washed 3x for 5 min each in PBS, and then tapped gently to remove excess PBS. Fluorescein-labelled affinity-purified rabbit anti-bovine IgG diluted 1:500 in PBS was added in 10 ul aliquots to each well. Slides were in~1b~t~ d at37~ C for 30 min, washed 3 times with PBS for 5 min each wash, tapped to remove excess PBS, cover-slipped with buffered glycerol (25 % [w/v] glycerine in TRIS-10 HCL:pH 9.0), and ex~min~d at 200 magnification using a fluorescence microscope.The end-point titer was the last serum dilutions showing distinct, whole parasite fluorescence.
RESULTS
Natural infections Sera collected at the time of abortion from 64 cows were tested for serologic reactivity to Neospora antigen (isolate BPA-l) using the IFA test procedure.
Aborted fetuses from 55 of these cows had nol~u~pul~live encephalitis and/or myocarditis which was con~i.ct~nt with a protozoal infection. In addition tachyzoite and/or cyst stages of Neospora were identified by immllnnhi.~toch~ try in the 20 tissues of these 55 fetuses (Table 3). In the r~m~ining 9 fetuses there was no indication of encephalitis and/or myocarditis and no detect~ble protozoal parasites.
All of the cows that aborted Neospora-infected fetuses had titers of 320 to 5,120 to Neospora antigens (Table 3). Eight of the cows that aborted fetuses with no detect~ble Neospora parasites had titers ' 160 and one had a titer of 320.

CA 022~l~47 l998-l0-l4 W O 97/42971 PCTAUS97tO7SOO

TABLE 3: Titers of cow sera collected after abortion of Neospora-infected fetuses to bovine Neospora (BPA1 isolate) antigens.
Neospora Number Neospora tlssue staqes in fetus 5 titer of cows cysts tachys cysts & tachys 320 1 o 1 o 1280 12 2 10 o Six of the cows that aborted Neospora-infected fetuses were m~int~in~ on their 4 dairies of origin so that these cows could be tested repeatedly over a 6 to 12 month period to determine changes in the Neospora titer. Peak titers of 640 to 2,560 were appar~llt within the first 20 days after abortion in all of the cows (Figs.
1 & 2). Subsequently, the titers of 4 of the cows (Fig. 1, cows 9, 970 & 522; Fig.
2, cow 578) decreased to 640, whereas the titers of cow 3 (Fig. 1) and cow 1328 (Fig. 2) dropped to 160 within 150 days post-abortion. C~ows 578 and 1328 were rebred and became pregnant again within approximately 50 to 70 days of aborting Neospora-infected fetuses. When these cows were approximately 4 to 5 months pregnant their Neospora titers increased to their original peak levels of 1,280 and rem~in~l at this level until the cows gave birth to full-terrn calves (Fig. 2). The calf born to cow 1328 had a pre-colostral titer of 20,480 and twin calves born to COw 578 both had precolostral titers of 10,240 to the bovine Neospora isolate.
Upon neclopsy at 2 to 6 days of age, these calves showed mild nol.sup~uldli~le encephalomyelitis or focal mononuclear cell infiltrates the brain parenchyma.
Neospora tissue cysts were seen in association with infl~mm~tory lesions in all 3 calves. The post-colostral titers of sera taken from each calf prior to ~ cia were the same as their precolostral titers.
Serologic titers were determined for four additional calves that were diagnosed as having congellilal Neospora infections based on the presence of characteristic cyst stages in the brain and/or spinal cord which reacted 40 immllnohistochemically with antisera to the BPA-1 bovine Neospora isolate.
Neospora was isolated from the brains and/or spinal cords of calves 1-3 and the . , . ~

CA 022~1~47 1998-10-14 parasites were grown continuously in vitro, using a previously described method for isolation of Neospora from aborted bovine fetuses. At necropsy, calves 1 and 2 had Neospora titers of 20,480, calf 3 had a titer of 10,240 and calf 4 had a titer of 5,120. Sera collected from the dam of calf 4 at calving had a Neospora titer of 5 2,560. Precolostral calf sera and sera from the dams of calves 1-3 were not available for testing.
The titers observed in the 7 congenitally infected calves with confirmed Neospora infections were m~rkP~lly greater than those obtained with sera from 4 weak 1-5 day old calves which were suspected of having Neospora infections, but 10 showed no histopathologic evidence of characteristic lesions or parasites on post-mortem ex~min~tion. One of these unillfecled calves had a titer of 160, while the others had titers < 80 to bovine Neospora antigens. Whether or not these calves had received colostrum was not known.
Expelillle~ l infections Repeated sera samples taken from the 3 pregnant heifers prior to experimental innc~ tion on day 43 had titers of ~ 80 to Neospora BPA1 antigens.
The 2 heifers that were infected with culture-derived tachyzoites of the BPA1 bovine isolate developed Neospora titers of 640 by day 9 and 1,280 by day 18 after parasite inoculation (Fig. 3). The heifer that received unillr~cled cell culture20 material had titers < 80 to Neospora antigens throughout the experiment. She was euth~niti7P~l 32 days after inoculation to remove her fetus which was viable, histologically normal and uninfected, with no cletçct~ble titer to Neospora. Peak titers for both infected heifers were ~letçcted 32 days after parasite inoculation, at which time the fetus of heifer 413 was removed by caesarian section.
25 Histologically, the fetus had infl~mm~tory lesions and numerous Neospora tachyzoites present in its central nervous system. In addition, Neospora tachyzoites were isolated from fetal tissues and grown continuously in cell culture. Sera collected from the fetus had a titer of 640 to Neospora antigens. After her fetus was removed, the Neospora titer of heifer 413 fl~ctu~te~l between 1,280 and 5,120, until 30 day 193 post-infection when it dropped to 640 (Fig. 3). Heifer 416 calved 158 days after pal~sile inoculation at which time she had a Neospora titer of 1,280 (Fig. 3).
The calf had a precolostral Neospora titer of 10,240 which was the same as the .

CA 022~1~47 1998-10-14 sample which was collected 2 days later, after ingestion of the dam's colostrum.Clinically the calf appeared normal except that it had decreased conscious proprioception in all 4 limbs when e~r~min~l prior to el~th~n~ at 2 days of age.There were minim~l histological lesions, consisting of focal gliosis in the central S nervous system, but no parasites were ~etecte~l in fetal tissues.
Uninfected cattle Fifty three of the 61 (87~c) adult cattle tested which had no history of Neospora infection had titers < 80, and all but one animal had titers 5 160 to both Neospora and Toxoplasma antigens (Table 4). The pastured cattle that were moved 10 and subseq~ently m~int~inP-1 under feedlot conditions did not have higher serologic titers to tachyzoites of bovine Neospora or Toxoplasma gondii than those kept onpasture. End-point titer determinations of all samples from infected or uninfected cattle were always based on whole tachyzoite fluulcscel~ce. However, in testing the appalell~ly umllrecled ~nim~l~, sera samples from 3 of the cows and 7 of the bulls 15 that were housed in the UCD feedlot had parasite fluorescence which was restricted to the apical end of the parasite. This reaction was particularly marked with the 7 sera from bulls which had apical fluorescence titers of 160 to 320 to both Neospora and Toxoplasma, while the whole parasite fluorescence titer was C 80.

CA 022~l~47 l998-l0-l4 W O 97/42971 PCTrUS97/07S00 TABLE 4: Titers of sera from cattle with no evidence of Neospora infection to bovine Neospora (BPA1 isolate) and Toxoplasma ~ondii.
Cattle Location Neo Number Toxo 5Number titer positive titer positive 0 20 heifers Nebraska <80 16 <80 14 pasture 160 3 160 6 20 heifers California <80 17 <80 13 pasture 160 3 160 3 9 cows UCD feedlot <80 9 ~80 7 12 bulls UCD feedlot ~80 ll ~80 10 Figure 4 shows the serologic titers of the 61 uninfected adult cattle plus the 9 cows that aborted fetuses without evidence of Neospora infections compared to the titers of Neospora-infected cows at the time of abortion or calving.
Although a majority of the infected cattle had titers 2 1280 to Neospora and most 30 of the cattle that had no evidence of infection had titers ~ 80, there was some overlap between these groups in the 160 to 640 titer range (Fig. 4).

Example 3 This example describes isolation of DNA encoding nss-rRNA (SEQ
35 ID NO:1).
Parasites Bovine Neospora isolates BPA-1 BPA-2, BPA-3, BPA-4, BPA-5 were used for DNA isolation. Parasites were harvested for DNA preparation when > 80% of the CPAE cells were infected with large clusters of tachyzoites. The 40 infected monolayer was removed from the flask by sc,~L,pillg. The tachyzoites in tissue culture media were pelleted by centrifugation at room tel~lpe.dl~lre, 1300Xg for 10 minutes. The ~upelllal~lll was removed and the pellet was r~u~ended in 10mls sterile physiologically buffered saline (PBS: pH 7.4), passed through a 25 gauge needle three times to disrupt the CPAE cells, and then filtered through a 5 .. . ........

CA 022~1~47 1998-10-14 WO 97t42971 PCT/US97/07500 um disc filter (Gelman Sciences, Acrodisc) to remove cellular debris. The filtered material was pelleted at 1300Xg for 10 mimlt~s and washed in PBS (pH 7.4). The supernatant was removed and the tachyzoite pellet was stored at -70~C until used.
Uninfected CPAE monolayer cells were processed by the same procedure and used as controls.
Two methods were used to prepare the DNA from the tachyzoites and control CPAE cells. Initially, DNA was prepared as follows. Briefly, the parasite or control cell pellets were suspended in 1.0 ml STE with 0.5% SDS
treated with proteinase K (100~g/ml) and RNAase (100~4g/ml) then extracted twicewith phenol, once with phenol-chloroform-isoamyl alcohol, and once with chloroform-isoamyl alcohol. DNA was subsequently precipitated with ethanol, dried and resuspended in TE buffer. Other DNA samples were ~l~pal~d with the Isoquick DNA Extraction kit (Microprobe, Corp., Garden Grove, Calif.) following manufacturer's directions.
DNA plepaldLions were electrophoresed in 0.8% (w/v) agarose (FMC) Bioproducts) in 0. 5 M Tris/borate/EDTA (TBE) buffer (89 rnM Tris, 89mM
boric acid, 2mM EDTA) gels stained with ethi~ m-bromide (0.5 ug/ml) and e~min~-~ under by ultraviolet (UV) light.
Amplification of rRNA gene sequences:
DNA sequences were amplified by the polymerase chain reaction (PCR) using a prog~all~ able thermal cycler (Perkin-Elmer). Reactions were performed in 50 to 100 ul volume samples cont~inin~ approximately 50-100 ng of DNA template, 50 mM Tris buffer (pH 8.3), 1.0 mM MgC12, 200 mM of each of the four deoxynucleoside triphosphates, 0.5 U of Taq polymerase (Promega) and 100 pM of universal primer A (5'CCG AAT TCG TCG ACA CCT GGT TGA TCC
CCG ACG ACC GTG GTC TGA ACG GGA G' (SEQ ID NO:2)) and primer C
(5'GGG CCC TAG GTG GCG CCG ACG ACC GTG GTC TGA ACG GGA G 3' (SEQ ID NO:3). The PCR cycling parameters consisted of a single step at 94~C
for 3 mimltes followed by 30 cycles dend~ dlion at 94~C for 1 minute, 1 minute of Ann~ling at 55, and 2 minutes of extension at 72 with a final extension step of 7 minutes. The PCR amplification product was an approximately 550-bp sequence from the 5' end of the nss-rRNA gene. A more extensive 1.8 kb sequence of the CA 022~1~47 1998-10-14 nss-rRNA gene was amplified from BPA1 DNA using universal primer A and primer B (5' CCC GGG ATC CAA GCT TGA TCC TTC TGC AGG TTC ACC
TAC 3' (SEQ ID NO:4)).
These reactions were performed using 50ul reaction samples that 5 contained lOOpM of each primer, 1 mM MgC12, 50-100 ng of template DNA, 50 mM Tris buffer (pH 8.3), 1.0 mM MgC12, a 200 mM of each of the four deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP), and 0.5 U of Taq polymerase (Promega). The amplification cycles were performed by an initial denaturation step at 94~C for 3 minlltes followed by 45 cycles of denaturation at 10 94~C for 1 minute, 1 minute of ~nnP~Iing at 55~C, and 4 minlltl~c of extension at 72~C with the final extension step for 7 minl~t~.s. For both amplification reactions, the stationary CPAE cell DNA was used as the negative template control. Sterile water was used for the PCR reaction condition controls. Aliquots of each PCR
product were sized by comparison with DNA standards (Bioventures, Inc. TN) after15 electrophoresis through a 3% (w/v) NuSieve, 1% (w/v) Se~Kem agarose gel (FMC
BioProducts, Rockland ME) stained with ethitlium bromide and vi~ l~li7ed under UV light.
Amplification products from 3 to 5 reactions were pooled prior to yuliricalion to reduce the possibility of any nucleotide misincorporation errors by 20 the Taq polymerase during the elongation step of the newly synthesi7~d complement strain. The PCR amplification products were purified either by gel electroelution or by spin-columns. Two dirrelcll~ spin-columns were used at dirrelcnl times.
First, Magic PCR Prep DNA purification System (Promega Corp.) was used following m~mlf~ctllrer's directions. Briefly, the products were electrophoresed25 through a low telll~el~lul~ melting agarose (Low Melt Agarose, FMC
BioPorducts,). The DNA was vi~-~1i7Pd in the gel by ethidium bromide st~ining and the DNA band was excised into an eppendorf tube. The agarose and DNA
were heated (70~C) to melt the agarose. The DNA was sepalaled from the agarose using columns and reagents provided in the kit. Later simpler and less time 30 intensive methods were used by purifying the PCR products using the PCR Select II (5 Primer-3 Prime, Inc) columns which do not require electrophoresis and excision of the product in low melt agarose.

CA 022~l~47 l998- l0- l4 WO 97/42971 PCTtUS97/07500 DNA sequencing of the purified PCR products were perforrned following m~m-f~ctl-rer's instructions for the PCR Cycle Sequencing System (BRL
ds DNA Cycle Sequencing System). The cycling parameters consisted of a two step program after complete denaLula~ion at 95~C for 3 minutes. The first program step amplified DNA using 20 cycles that included 30 seconds for denaturation, 30 seconds for ~nne~ling, and 1 minute of extension. The second program step alternated between denaturation (95~C) and extension (72~C) only. Initially, theuniversal primers (A, C or B) were used to obtain the first nucleotide sequence data from which internal primers could be constructed and used to amplify internal 10 sequences. All sequencing primers were 5' labelled with adenosine 5'[y32p]
triphosphate (Amersham). Reactions were heated to 95~C for 5 minutes prior to loading onto either a 6% (w/v) or 8% (w/v) polyacrylamide, 8 M urea (0.4 mm thick) non-gradient gel using a Model S2 Sequencing Gel Apparatus (GIBCO BRL, Gaith~l~lJulg, Md). The seql~en~ing gels were fixed in 10% acetic acid and 10%
15 meth~nol to remove the urea prior to transfer of the sequencing products in the gel onto filter paper. The gels were dried using a gel drying apparatus (Biorad Gel Drier, X) for 1 to 2 hours at 70-80~C. The membrane filters were autoradiographed using Kodak X-OMAT X-ray film.
DNA Sequence analysis:
DNA seq~en~es were constructed from at least 3 sel)aldt~ reactions to ensure the accuracy of the nucleotide sequenre data obtained. Autoradiographsof the sequencing products were read using Hibio MacDNASIS DNA and Protein Sequence Analysis System (Hitachi Software Fngineering Co,). This program and the GCG programs (SEQED, Fragment Assembly, ~ineup, and Pretty) (University 25 of Wisconsin Genetics Coll~ul~r Group) on a VMS system facilitated the construction of the DNA sequences.

Example 4 The DNA prepared in Example 3 was used to design ~ e.s and 30 probes for the detection of Neospora. The protocol used was as follows.

CA 022~1~47 1998-10-14 Oligonucleotide PCR primers:
1) Bovine Neospora Forward Primer (5'-AAGTATAAGCTTTTATACGGCT-3' (SEQ ID NO:5)) 2) Bovine Neospora Reverse Primer (5'-CACTGCCACGGTAGTCCAATAC-3' (SEQ ID NO:6)) DNA amplification was carried out in a total volume of 50 ,ul. The reaction mixture contained 10 mM Tris-HCl (pH 9.0), 50 mM potassium chloride, 0.1% Triton X-100, 1.0 mM magnesium chloride, 200 mM of each 10 deoxynucleoside triphosphates, 0.42 ,~4M Bovine Neospora Forward primer and 0.384 ~M Bovine Neospora Reverse primer. After precycle denaturation at 94~C
for 4 min to reduce nonspecific amplification, 2.5 U of Taq DNA polymerase (P~ ega Corp., Madison, WI) was added and the mixture was overlaid with 50 ,ul of mineral oil. Amplification was performed in a DNA Thermal Cycler (Perkin 15 Elmer Cetus Corp., Norwalk, CT) for 31 cyc}es as follows: denaturation at 94~C
for 1 min, ~nn~ling at 54~C for 1 min, and extension at 72~C for 2 min. The lastcycle was given a prolonged extension period of 7 min. After amplification, 5 Ill of each sample or a BioMarker Low (BioVentures, Inc., Murfreeboro, TN) DNA
size standard were mixed with 1 ~l of 6X loading dye and electrophoresed on a 3 %
20 Nusieve 3:1 agarose gel (FMC Bioproducts). The gel was stained in a 0.5 ~g/ml ethi(~ m bromide solution for 30 min and observed for the presence of amplification products under ultraviolet ill~ tion.

Oligonucleotide DNA probes:
25 3) BPA/Neospora Internal Probe Seq~enre (5'-AGTCAAACGCG-3'(SEQ ID NO:7)) 4) Toxoplasma Internal Probe Seq~lenre (5'-AAGTCAACGCG-3'(SEQ ID NO:8)) Amplification products were denatured in the gel and transferred to nylon membranes (Hybond-N; Amersham Corp., Arlington ~eight~, Il) by Southern blotting method. DNA was cross-linked to nylon membrane using a Strat~lin'-~r CA 022~1~47 1998-10-14 UV crosslinker (Stratagene, La Jolla, CA). Prehybridization, preparation of the labeled internal probe, and hybridization were performed as recommended by the manufacturer for the F.nh~nred Chemill-minPscence 3'-oligolabeling and DetectionSystems (Amersham). Labeled internal probe was added to a final concentration 5 of 10 ng/ml of hybridization solution and incubated overnight at 30~C with gentle agitation. After hybridization, the membranes were washed twice for 5 min each at room telllpe~ re in 5X SSC and 0.1 % (w/v) SDS, and then washed twice for 5 min each at room t~ pelatule in 0.5X SSC and 0.1% (w/v) SDS. Membrane blocking, antibody incubations, signal generation and detection were performed as 10 described by the m~m-f~cturer. Membranes were exposed to Kodak X-Omat film for 3-10 min.
Results Using the Neospora-specific primers 294 bp PCR products were amplified from DNAs of BPA-1 and Toxoplasma (RH and BT isolates). In 15 addition, a 350 bp product was amplified from Sarcoc ystis cruzi DNA. No products were produced with DNAs from various bacteria, CPAE cells, and bovine thymocytes. Only the Neospora-specific probe hybridized to the Neospora amplification product. Similarly, the Toxoplasma-specific hybridized only to theToxoplasma amplification product.
Example 5 This example describes experimental infections of ple~nallt cows with culture-derived Neospora tachyzoites.
Three cows were inoculated with 8 x 106 tachyzoites of the BPA1 25 Neospora isolate (3 x 106 tachyzoites IV, and 5 x 106 tachyzoites IM). These cows were inoculated at 95 days gestation (Cow #412), 100 days gestation (Cow #416), and 105 days gestation (Cow #413). In each case, a Neospora fetal infection was confirm~d (Cow #412 expelled an infected mllmmifi~(l fetus; Cow #416 gave birth to a calf that was in utero exposed; and an infected fetus was removed surgically 30 from Cow #416). Two control cows were inoc~ ted with uninfected cell culture and gave birth to uninfected live calves.

CA 022~l~47 l998- lO- l4 WO 97/42971 PCTrUS97/07500 These cows were kept and rebred without any illlel ~nlion. All three experirnental cows gave birth to seronegative, clinically normal calves (not all post mortem tissues e~minPd to date).
The cows were kept and rebred once again. The previously infected - 5 cows (#S 412,416,413 were then rechallenged by giving them the same inoculum (8 x 106 tachyzoites, divided and given IV and IM) at 89, 83, and 83 days gestation, respectively. Control cows were rebred and observed. Two infected cows (#s 413 and 416) gave birth to live calves which were clinically normal and seronegative to Neospora antigens. The third cow (#412) aborted 27 days post inoculation. The fetus was recovered. Although mild lesions suggestive of Neospora infection werefound, Neospora infection, to date, has not been confirmPd (formalin-fixed paraffin embedded tissues negative by immunnhi~tQchemistry). The cow was rebred and resorbed its fetus. She was rebred again and she aborted once again at 97 days gestation. This second fetus was not recovered. Thus far histopathologic e~min~tion or the tissues from the two clinically normal calves in(lic~tes that they were not tranpl~eçntAlly infected with Neospora parasites.
This is the first experiment to show that cattle can be protected against Neospora abortion by ;~ ni7~tion with culture-derived tachyzoites of theBPA-1 Neospora isolate.
Example 6 This example describes identifi~tion of two clones from Neospora cDNA library. These cDNAs can be used to produce recombinant immllnndo---;..~ proteins useful for vaccines and immnnndiagnostics.

25 Methods and Methods:
In vitro cultivation Neospora tachyzoites (BPA-1 isolate) and Toxoplasma gondii (R~
strain) tachyzoites were cultivated in tissue culture as described in Conrad, Parasitology, 106:239-249 (1993). Briefly, BPA-1 tachzoites were grown in 30 confluent layers of BAE (bovine aortic endothelial) cells, harvested and filtered through 5 ~m disc filters to remove cellular debris, washed twice in phosphate buffered saline (PBS), and pelleted for use. After rnRNA for cDNA library CA 022~l~47 l998- l0- l4 construction, BPA-1 and Toxoplasma tachyzoites were subsequently grown in Vero cells.

Isolation of nucleic acids Total RNA was isolated from pelleted tissue culture cells with TRISOLV~ reagent (Biofecx Laboratories, Houston, Texas). Poly A+ RNA was selected by oligo (dT)-cellulose chromatography (Avis, et al., Proc. Natl. Acad.Sci. USA 69, 1408-1412 (1972)). Integrity of RNA was monitored by electrophoresis on a form~ Pllyde gel and vi~ li7ed with ethi~ m bromide using 10 standard methods.
DNA was p,~aled with the IsoQuick NDA Extraction Kit (Microprobe Corp., Garden Grove, California).

Construction of a bovine Neospora cDNA library in ~gtl 1 Construction of a Neospora cDNA e~ ession library in the vector )~gtll was l)elÇc,ll,led essenti~lly according to the m~nl-f~ctllrer's protocol in the cDNA Sy"~,esis Kit (Stratagene, La Jolla, California). In short, first strand cDNA
was ~yll~tlpsi~d from S ~u of tachyzoitic poly(A) RNA using StataScript RNasc H-7 reverse transcriptase and the second strand was synthP~i7p~l with E. coli DNA
20 polymerase. Subsequently, the double stranded cDNA was blunt-ended with the Klenow fr~gmPnt, ligated to Eco R1 adaptors on both ends, kin~ed with T4 polynucleotide, and size-fractionated on a Sephacryl S-400 spin column. The final cDNA product was then ligated into the Eco R1 digested and dephosphorylated ~gtl 1 vector and packaged using the Gig~rarl~ III Gold P~c~ging Extract 25 (Strategene, La Jolla, California). The library contained 7 x 106 phages withapproximately 97% of these being recombinants. Subseq~ently, one round of library amplification was completed in E. coli Y1088.

Immunoscreening of the cDNA library The library was plated on E. coli Y109Or- and duplicate nitrocellulose plate lifts were screened with high titer sera from naturally infected cow D91-4696 and e~ Pnt~lly infected cow #416 described in Conrad, et al., .. . . . . . . . . . . .

CA 022~1~47 1998-10-14 J. Vet. Diegn. Invest., 5:572-578 (1993). Sera were first pre-absorbed with Y109Or-bacterial Iysates and then diluted 1:300 in TBS-T (10 mM Tris-HCI pH 8, 150 mM NaCl, 0.05% Tween 20) cons~inin~ 5% horse serum and used for screening. Bound antibodies were vi~ li7.ed with ~Ik~lin~ phosphatase conjugated5 goat anti-bovine IgG diluted 1:5000 in TBS-T, 5% HS. Tmml-noscreening procedure was essentially as described for imm1-noblot assays below except 5%
nonfat dry milk was replaced with 5 % horse serum.

DNA Sequencing PCR products gemldted with universal ~gtll primers fl~nking the vector's Eco Rl restriction site (Promega, Madison, WI) were used as templates for sequencing. Cycling conditions were as described by Obar, et al., Methods in Cell Biology, 37, 361-406 (1993). Two sets of templates for sequencing were made for each clone and both were sequenced in the forward and reverse directions.
Automated DNA sequencing was accomplished with the ABI 373 DNA Seq~len~er and the DNA Sequencing Kit with AmphiTaq Polymerase FS
(Perkin-Elmer Corp., Foster City, CA). Dye t~ nllator c~mi~try in conjunction with cycle sequencing was used.

20 Southern and Northern blot analysis For Southern blotting, 4 ug of genomic DNA was digested with re;,~li.;Lion enzyme and S~al~l~d on a 0.8% agarose gel according to standard procedures. After the gel was de~ a~ed with 250 mM HCI for l0 ...;....~es, de~ ul~,d with I.5 M NaCl, 0.5 M Tris-HCI, pH 7.5 for 30 Ini..~ s, the nucleic 25 acids were transferred overnight to Hybond-Nnylon membranes (Amersham Corp., Allil*~on Heights, II) and cro~link~d to the membrane via a UV crosslinker (Stratagene, La Jolla, CA). For probes, Eco Rl inserts from the recombinant clones were labeled with the ECI Direct Nucleic Acid Labeling and Detection Systems (Amersham Corp.). Blots were prehybridized with the ECL Gold 30 Hybridization buffer cont~ining 5% blocking agent and 0.5 M NaCl (.Am~rh~m Corp.) for I hour and hybridized with the probes overnight at 42~C. The blots CA 022~1~47 1998-10-14 were washed at 42~C (2 x 20 min.) in 6M Urea, 0.4% SDS, 0.5 x SSC and rinsed with 20x SSC prior to autoradiography.
For Northern blotting, the above Southern blot procedure was essenti~lly followed except that 0.4 ug mRNA (clone N54) or S ug of total RNA
5 (clone N57) were run on a 1 % formaldehyde gel and no extensive treatment of the gel was required prior to transfer. Also, when the Eco R1 insert from clone N54 was used as a probe, the wash protocol was adjusted to 0.4% SDS, O.Sx SSC at 45~C (2 x 20 min.).

10 Recombinant Protein Expression and Purification To construct the e~l,rcssion vector, PCR products of the clone inserts were digested with Eco R1 and gel purified. This DNA was ligated into the Eco R1digested and dephosphorylated pRSET B vector (Kroll, et al., DNA and Cell Biology, 12 441-453 (1993)) and the res-llting plasmid was transformed into BL2115 DE3 pLysS E. coli (Studier, et al., Methods En~:ymol. 185, 60-89 (1990)). Fusion proteins expressed from this plasmid contained a h~mer of hi~titlin~s which allowed purification through a Ni2+ affinity column under denaturing conditions.Single colonies with highexpression levels were inoc~ t~-l into Luria Broth cont~inin~ 100 ug/mL ampicillin and grown at 37~C to an O.D.600 of 0.6.
20 O~iele~ression of fusion pl~ ls was initi~te~ with the addition of isopro~yl-D-thiogalaclopyldlloside (0.4 mM final) and ~h~kin~ for 3 hours. Affinity purification with a nickel-NTA-agarose affinity (Qiagen, Chetsworth, CA) under denaLulillg conditions was completed as described by Kroll, et al., DNA and Cell Biology, 12441-453 (1993). Samples were concentrated with a Centricon-10 (Amicon, 25 Beverly, MA) and further purified by (size chromatography) with Sephadex G-150 ~u~c~rine (Pharmacia, Uppsala, Sweden) equilibrated in 8 M urea, 0.1 M
Napholsphate, 0.01 M Tris, pH 4.5 using standard methods. Denalul~ in the final proteins were removed by a slow 1 M urea stepwise dialysis to a final PBS,pH 7.4 buffer. Samples were concentrated again with Centricon-10 (Amicon, 30 Beverly, MA) and q~ ilt~d with the BCA Protein Assay (Pierce, Rockford, II).

.. . ..

CA 022~1~47 1998-10-14 Wo 97/42971 PcTluss7lo75oo Polyclonal Antibody Production Female New 7e~l~n~l White rabbits were i~ ed subcutaneously with 400 ug of recombinant plOltil~S in a 50% emulsion of Freund's complete adjuvant and thereafter boosted twice at 4 weeks intervals with 200 ug and then 100 5 ug of protein in incomplete Freund's adjuvant. Sera were obtained two weeks after the third immnni7~tion and used for immllnnblots.

Immunoblots Proteins were analyzed on a 12% polyacrylamide gel or a 4-20%
10 gradient slab gel (Anderson, et al. J. Am. Vet. Med. Assoc., 207:1206-1210 (1995)). Parasite antigens were harvested from tissue culture as described above, washed 2x in PBS, lysed with water, freeze thawed 3x, and sonic~te~l. Both recombinant antigens and parasite antigens were q~ ed with the BCA Protein Assay (Pierce), denatured in T ~,ommli's sample buffer and boiled for 5 mimlt~s 15 prior to electrophoresis. SDS-PAGE and Western blots were pelrolnled under standard conditions.
Rabbit antisera to the recombinant antigens, Neospora (BPA1) (Conrad, Parasitology, 106:239-249 (1993)), and Toxoplasma gondii (Conrad, Parasitology, 106:239-249 (1993)) were diluted 1:300 in TBS-Tween, 5% Blotto 20 and inrllbate~l for 5 hours at room lelll~el~lul~. The secondary antibody, HRP-goat anti-rabbit (Jackson Laboratories) was diluted 1:1000 in the same buffer and inr~b~t~cl for 2 hours at room tt~llpcf~luf~. Blots were developed with 4-chloron~rhthol and H2O2.

25 Results After one round of amplification, a bovine Neospora )~gtll cDNA
library was ge~ led cont~ining 5.3 x 10'~ pfu/mL. Sera from cow D91-4696 and cow 416 which had high titers to Neospora antigens were chosen to screen the library. No signifir~nt banding pattern dirr~,ellces were seen between Western 30 blots which were inr~lbat~d with these sera (data not shown). Both naturally infected cow D91 -4696 and experim~nt~lly infected cow 416 was tested negative for Toxoplasma gondii. ExperimPnt~lly infected cow 416 was a subject of a previous CA 022~l~47 l998- l0- l4 Neospora abortion study (Conrad, et al., J. Vet. Diegn. Invest., 5:572-578 (1993)).
Primary screening of 200,000 cDNA clones with the above sera identified 61 double positive clones. Two were further characterized and designated N54 and N57. Based on agarose gel electrophoresis, the ~gtl 1 Eco R1 inserts were 5 430 and 630 based pairs respectively.
Sequence analysis of clone N54 revealed that the cDNA insert was 407 bases within an open reading frame. The clone was not full length as indicated by the absence of a 5'-methione start site and the absence of a 3' stop codon and poly A tail (SEQ ID NO:9). Translation of the sequences into its corresponding 10 amino acids revealed that the protein sequence was highly proline rich (32%) and composed of several repeat units (SEQ ID NO:10). Of particular interest is the sequence, SPPQS(S/Y)PPEP (SEQ ID NO: 13) shown in bold in Figure 5A, which occurs twice and has a high surface probability (Emini ave. index = 3.025) and moderate antigenicity index (Jameson-Wolf ave. index = 1.24). Other unique 15 repeats within the region are the L~Lldye~lides~ HP(H/P)P (SEQ ID NO:14) and SPP(E/Q) (SEQ ID NO: 15), which occur four times each within the 135 amino acid sequence and the sequence, SY(A/P)P(D/E)PSP (SEQ ID NO: 17), which contains conserved substitutions.
Unlike clone N54, clone N57 contained a 3' stop codon and a long 20 poly A tail (SEQ ID NO:11); however, because of difficulties in sequencing the l~,peLili~e noncoding region, only the coding sequences are shown (SEQ ID NO: 12).
While this partial clone did not have any discernible peptide repeats within its 76 amino acid sequence, upon hlsye~;lion at the DNA level, multiple units of nucleotide repeats were identifie~l. In particular, a long tandem repeat with 74% homology 25 was present at the 3' end (Figure SB). While these repeats were similar at the nucleotide level, the multiple deletions within the repeats resulted in frame shifts that tr~n~l~ted into peptide sequences with only 2 out of 10 amino acids being similar. By Emini analysis, these two regions appeared to be likely surface exposed and a correlation between surface exposure and antigenicity was seen. The second30 repeat also al,~ealed to have a potential glycosylation at the position 50 asparagine.
In addition, there was a high frequency of nine GGA(A/G) repeats clustered within a 105 base region at the 3' end of the gene seql-~nre.

., CA 022~1~47 1998-10-14 Clones N54 and N57 hybridized to bands of dirrerellL mo}ecular weights on Southern blots of Neospora (BPA-1) DNA, inl1ic.~ting that the clones were derived from separate Neospora genes. Clone N54 showed some hybridization to a higher molecular weight band of Toxoplasma gondii DNA, - 5 whereas clone N7 did not bind to Toxoplasma DNA or to Vero DNA.
Northern blots with the same probes also showed that both clones recognized different molecular weight RNA transcripts and thus, encoded distinctNeospora proleills. Clone N54 bound to a 4.2 kb Neospora tldl]SC~ipt, while clone N57 bound to a shorter 1.4 kb Ll~nsclil)t. Neither clones hybridized to Toxoplasma gondii RNA or Vero RNA.
When clones N54 and N57 were expressed as hi~ti-lin~ fusion plol~ins from the pRSET vectors, the resl~lting protein product of clone N54 was29.3 kD whereas clone N57 was 20.1 kD. By Western blot analysis, both plol~ s were recognized by rabbit antisera to BPA1 and not by rabbit Toxoplasma gondii antisera (data not shown). This suggests that the recombinant antigens are more diagnostically useful than the whole lysate which did have some reactivity to rabbit Toxoplasma gondii antisera.
In addition, polyclonal monospecific antisera to clones N54 and N57 only bound to Neospora antigens on reducing and nonreducing Western blots.
Rabbit anti-N54 recognized Neospora bands of molecular weights 97.2 kD, 87.9 kD, 77.1 kD, 67.4 kD, 64.3 kD, 60.1 kD, 55.3 kD, and 28.3-28 (reducing) and 126.7 kD, 89.4 kD, 68.7 kD, 58.4 kD, 55.2 kD, 54.5 kD, 52.7 kD, 49.7 kD, 46.7 kD and 26.5-27.9 (~ lucing). Rabbit anti-N57 recognized Neospora bands to molecular weights 33.6 kD, 31.4 kD, 27.5 kD, and 22.4 kD (reducing) and 32.8 kD, 30.6 kD, 28.4 kD, 26.3 kD, and 21.0 kD (nonreducing). Neither polyclonal antisera bound to Toxoplasma gondii antigens nor to Vero cell antigens, making these two recombinant antigens prc ~llising c~n~ t~s for a highly specific ELISA.

Conclusion:
Current ELISA protocols require the use of in vitro cultivated of Neospora tachyzoites for coating antigens. (Pare, et al., J. Vet. Diag. Invest.,7:352-9 (1995); Bjorkman, et al., Parasite ~mmunology, 16:643-8). When using , . . , .. .. . ,, . . . .... . . . . . . , . .. .. ~ . , CA 022~l~47 l998- l0- l4 such a crude mixture of antigens, one runs the risk of generating false positives due to crossreactivity between proteins from closely related parasites. By using one or two Neospora specific recombinant antigens in an ELISA, potentially crossreactive antigens are removed. In addition, use of whole parasites requires time con~l-ming 5 and expensive tissue culture methods.
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily al)palell~ to one of ordinary skill in the art and are encompa~sed by the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated 10 by reference.

CA 022~1547 1998-10-14 SEQUENCE LISTING

(1) GENERAL INFORMATION:
(i) APPLICANT: THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

(ii) TITLE OF INVENTION: Bovine Neospora Isolates and Their Uses (iii) NUMBER OF SEQUENCES: 17 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Robbins, Berliner & Carson LLP
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(A) NAME: Berliner, Robert (B) REGISTRATION NUMBER: 20,121 (C) REFERENCE/DOCKET NUMBER: 5555-437 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (213) 977-1001 (B) TELEFAX: (213) 977-1003 CA 022~1~47 1998-10-14 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1747 base pairs (C) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TAACCGTGGT AATTCTATGG CTAATACATG CGCACATGCC lLlIL~ILTG CAA~ T 180 GTTTATTAGA TAC~r~Arrq ACCCACCTTC CGGTGGTCCT CGGGTGATTC ATAGTAACCG 240 SUBSTITUTE SHEET (RULE 26) . .

CA 0225l547 l998-l0-l4 WO97/42971 PCT~US97/07500 GTGAATTCTA GCAlC~l-l~l GGA~ C ACACTTCATT GTGTGGAGTT TTTTCCAGGA 720 CTTTTACTTT GAGAAAATTA GA~lG~ A AGCAGGCTTG TCGCCTTGAA TACTGCAGCA 780 TGGAATAATA AGATAGGATT TCGGCCCTAT l-l-l~l-lGGTT TCTAGGACTG AAGTAATGAT 840 AGATAGGAAA ACGTCATGCT TGACTTCTCC TGCACCTTAT GAGAAATCAA A~l~l-l-lGGG 1080 TTCTGGGGGG AGTAlG~lCG CAAGGCTGAA ACTTAAAGGA ATTGACGGAA GGGCACCACC 1140 AGGAAGGATT GACAGATTGA TAG~ -lC TTGATTCTAT GG~l~GlGGT GCATGGCCGT l260 TCTTAGTTGG TGGAGTGATT l~l~lGGTTA Al-lCC~l-lAA CGAACGAGAC CTTAACCTGC 1320 TAAATAGGAT CAGGAACTTC Gl~l-l~l-l~l ATCACTTCTT AGAGGGACTT TGC~lGl~lA 1380 CCGTCGCTCC TACCGATTGA ~l~l-lCCG~l GAATTATTCG GACC~l-ll-lG TGGCGCGTTC 1680 G~l-l-lCC 1747 ~2) INFORMATION FOR SEQ ID NO:2:
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Gln His Pro His Pro His Pro Pro Arg Pro Asn Pro Pro Glu Ala l 5 l0 15 Ser Pro Pro Gln Ser Ser Pro Pro Glu Pro Gln Arg Pro Phe Ser Gln Trp Pro His Thr Pro His Phe Phe His Tyr His Pro Tyr Pro Gly Tyr AAT CTT CCT TAT TTT ACT TAC CAT CAG TCT CCT CTT CCT TAT GGT CCC l90 Asn Leu Pro Tyr Phe Thr Tyr His Gln Ser Pro Leu Pro Tyr Gly Pro ... .

CA 022~l~47 l998-l0-l4 W O97/42971 PCT~US97/07500 Tyr Gly Arg Asp Pro Cys Pro Cys Ala Ser His Pro Tyr Pro Ala Asp Asp Ser Pro Leu Gly Ser Tyr Ala Pro Asp Pro Ser Pro Pro Gln Ser Tyr Pro Pro Glu Pro Ser Pro Ser Lys Pro Ser Pro Pro Glu Gly Ser Ser Pro Arg Val Pro Ser Pro His Arg His Pro Ser Arg Ser Arg Leu Pro Ser Ala Val Glu Pro Ser Pro (2) INFORMATION FOR SEQ ID NO:10:
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~ln His Pro His Pro His Pro Pro Arg Pro Asn Pro Pro Glu Ala Ser ~ro Pro Gln Ser Ser Pro Pro Glu Pro Gln Arg Pro Phe Ser Gln Trp Pro His Thr Pro His Phe Phe His Tyr His Pro Tyr Pro Gly Tyr Asn Leu Pro Tyr Phe Thr Tyr His Gln Ser Pro Leu Pro Tyr Gly Pro Tyr Gly Arg Asp Pro Cys Pro Cy5 Ala Ser His Pro Tyr Pro Ala Asp Asp ~er Pro Leu Gly Ser Tyr Ala Pro Asp Pro Ser Pro Pro Gln Ser Tyr ~ro Pro Glu Pro Ser Pro Ser Lys Pro Ser Pro Pro Glu Gly Ser Ser ~ro Arg Val Pro Ser Pro His Arg His Pro Ser Arg Ser Arg Leu Pro Ser Ala Val Glu Pro Ser Pro (2) INFORMATION FOR SEQ ID NO:ll:
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Ile Ala Ala Leu Val Ala Ala Ala Ala Phe Ala Gly Leu Gly Leu Ala Arg Thr Phe Arg His Phe Val Pro Lys Lys Ser Lys Thr Val Ala Ser Glu Asp Ser Ala Leu Gly Asn Ser Glu Glu Gln Tyr Val Glu Gly Thr Val Asn Gly Ser Ser Asp Pro Glu Gln Glu Arg Ala Gly Gly Pro Leu Ile Pro Glu Gly Asp Glu Gln Glu Val Asp Thr Glu ~2) INFORMATION FOR SEQ ID NO:12:
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Ile Ala Ala Leu Val Ala Ala Ala Ala Phe Ala Gly Leu Gly Leu Ala ~rg Thr Phe Arg His Phe Val Pro Lys Lys Ser Lys Thr Val Ala Ser Glu Asp Ser Ala Leu Gly Asn Ser Glu Glu Gln Tyr Val Glu Gly Thr Val Asn Gly Ser Ser Asp Pro Glu Gln Glu Arg Ala Gly Gly Pro Leu Ile Pro Glu Gly Asp Glu Gln Glu Val Asp Thr Glu (2) INFORMATION FOR SEQ ID NO:13:
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(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
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(A) NAME/KEY: Modified-site (B) LOCATION: 6 (D) OTHER INFORMATION: /product= ~'OTHER"
/note= nXaa = Ser or Tyr"

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Ser Pro Pro Gln Ser Xaa Pro Pro Glu Pro (2) INFORMATION FOR SEQ ID NO:14:
(i) S~uu~N~: CHARACTERISTICS:
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(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LOCATION: 3 (D) OTHER INFORMATION: /product= "OTHER"
/note= ~Xaa = His or Pro"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
His Pro Xaa Pro (2) INFORMATION FOR SEQ ID ND:15:
~:N~ CHARACTERISTICS:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

CA 0225l547 l998-l0-l4 WO97/42971 PCT~US97/07500 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS:
~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide tix) FEATURE:
~A) NAME/REY: Modified- 8 ite (B~ LO Q TION: 3 (D) OTHER INFORMATION: /product= nOTHER"
/note= ~Xaa = Ala or Pro"
(ix) FEATURE:
(A) NAME/KEY: Modified-site (B) LO QTION: 5 (D) OTHER INFORMATION: /product~ "OTHER~
/note= ~Xaa = Asp or Glu"

(xi) SEQUENCE ~ESCRIPTION: SEQ ID NO:17:
Ser Tyr Xaa Pro Xaa Pro Ser

Claims (22)

WHAT IS CLAIMED IS:

1. An isolated recombinant nucleic acid construct comprising a nucleic acid encoding an immunodominant Neospora antigen.

2. The construct of claim 1, wherein the nucleic acid encodes a polypeptide having a sequence as shown in SEQ ID NO:10 or SEQ ID NO:12.

3. The construct of claim 1, wherein the nucleic acid has a sequence as shown in SEQ ID NO:9 or SEQ ID NO:11.

4. A method of detecting the presence of antibodies specifically immunoreactive with a bovine Neospora antigen in a biological sample, the methodcomprising contacting the sample with an isolated recombinant immunodominant Neospora antigen, thereby forming an antigen/antibody complex, and detecting thepresence or absence of the complex.

5. The method of claim 4, wherein the Neospora antigen is SEQ
ID NO:9.

6. The method of claim 4, wherein the biological sample is bovine serum.

7. The method of claim 4, wherein the antigen is immobilized on a solid surface.

8. The method of claim 4, wherein the complex is detected using a labeled anti-bovine antibody.

9. The method of claim 8, wherein the anti-bovine antibody is fluorescently labeled.

10. A method of detecting the presence of Neospora-specific nucleic acids in a biological sample, the method comprising contacting the sample with a oligonucleotide probe which specifically hybridizes with a target Neospora-specific polynucleotide sequence, thereby forming a hybridization complex, and detecting the presence or absence of the complex.

11. The method of claim 10, further comprising amplifying the target Neospora-specific polynucleotide sequence.

12. The method of claim 10, wherein the target Neospora-specific polynucleotide sequence is immobilized on a solid surface.

13. The method of claim 10, wherein the oligonucleotide probe is labelled.

14. The method of claim 10, wherein the target nucleic acid is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:9 or SEQ ID NO:11.

15. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an immunogenically effective amount of a bovine Neospora antigen.

16. The composition of claim 15, wherein the bovine Neospora antigen is an isolated bovine Neospora polypeptide.

17. The composition of claim 16, wherein the polypeptide is recombinantly produced.

18. The composition of claim 15, wherein the bovine Neospora antigen is expressed by a recombinant virus.

19. A method for protecting a bovine animal from infection by bovine Neospora, the method comprising the administration of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an immunogenically effective amount of a bovine Neospora antigen.

20. The method of claim 19, wherein the bovine animal is a cow or heifer.

21. The method of claim 19, wherein the pharmaceutical composition is administered when the bovine animal is breeding.

22. The method of claim 19, wherein the pharmaceutical composition is administered parenterally.