MXPA97008701A - Vacuna de neospora viva atenu - Google Patents

Abstract

The present invention relates to live attenuated cultures of the pathogenic protozoan Neospora parasite and live vaccines against neosporosis prepared therewith, which are useful in the prevention of clinical disease and abortion in mammals.

Description

VACCINE OF NEOSPORA VIVA ATENUATED FIELD OF THE INVENTION The present invention relates to attenuated strains of the pathogenic protozoan Neospora, and to live vaccines against neosporosis prepared from attenuated strains axis which are useful in the prevention of clinical diseases and abortion in mammals BACKGROUND OF THE INVENTION Neospora is a pathogenic protozoan parasite of animals that has recently been recognized as an important cause of abortion, neonatal death, congenital infection and encephalitic disease in mammals. Dubey and Lindsay, 1993, Parasitology Today, 9: 452-458. N. caninurn infects dogs and congenitally infects puppies, often leading to paralysis. N. caninorm tachyzoites have been isolated from naturally infected puppies. Lindsay and Dubey, 1989, 3. Parasitol. 75: 163-165. Neospora spp. they are an important cause of abortion in dairy cattle. Cases of Neospora-related disease, that is, neosporosis, in goats, sheep and horses have also been cited. Although N. caninum is superficially similar to the pathogen, Toxoplasma gondii, N. caninum and T. gondii have distinguished themselves from one another from the antigen and structural point of view. Dubey and Lindsay, 1993, supra. In addition, the protozoan parasites similar to Neospora isolated from the brains of aborted bovine fetuses and grown continuously in vit-ro showed that they were different from the antigen and ultrastructural axis view of T. gondii and Hamrnondia ha mondi and more similar to N caninum. Conrad et al., 1993, Parasitology 106: 239-249. In addition, the analysis of ribosomal RNA genes from small subunits of the nucleus did not reveal nucleotide differences between Neospora spp. isolated from cattle and dogs, but did show consistent differences with respect to T. gondii. Marsh et al., 1995, 3. Parasitol. 81: 530-535. The etiological role of a Neospora bovine isolate in bovine abortion and congenital disease has been confirmed. Barr et al., 1994, 3. Vet. Diag. Invest. 6: 207-215. A model has been developed in the neosporosis rodent of the central nervous system using inbred BALB / c mice infected with N. caninum. Lindsay et al., 1995, 3. Parasitol. 81: 313-315. In addition, they have been described by Cole et al., 1995, 3. Parasitol, 81: 730-732 and by Long et al., 1996, 3. Parasitol. 82: 608-611, models for the study of placental transmission of N. caninum in exogamous and inbred pregnant mice, respectively. Additionally, a model of an experimental N. caninum pygmy goat has been demonstrated that closely resembles abortion in cows induced by Neospora naturally acquired. Lindsay et al., 1995, Am. 3. Vet. Res. 56: 1176-1180. UO 9525541 describes a biologically pure Neospora axis culture, methods for detecting anti-Neospora antibodies and Neospora-specific nucleic acids, and a composition containing a bovine Neospora antigen and a vehicle for use as a vaccine. UO 9525541 does not disclose, however, live attenuated cultures of Neospora or live vaccines prepared therefrom which are capable of eliciting a protective immune response in a vaccine animal.

BRIEF DESCRIPTION OF THE INVENTION In a first aspect, the present invention provides cell cultures of a strain derived from a pathogenic progenitor strain of a Neospora species, cells that show attenuated pathogenicity compared to those of the parent strain, but which are capable of shaking an immune response that protect a mammal against neosporosis when administered as a live vaccine. In a second aspect, the present invention provides vaccines that protect a mammal against neosporosis, composed of an immunologically effective amount of living cells of a strain derived from a pathogenic progenitor strain, a Neospora species, cells that show attenuated pathogenicity compared with those of the parent strain, but which are capable of triggering an immunological response that protects the mammal against neosporosis when administered as a live vaccine, and a veterinarily acceptable vehicle. The invention vaccines may also contain one or more additional components, including, for example, an adjuvant. The vaccines of the present invention can be administered to any animal species susceptible to being infected and becoming ill due to Neospora, including, but not limited to, dogs, cows, goats, sheep and horses. In a third aspect, the present invention provides methods for the preparation of cultures of cells attenuaejas axis a pathogenic strain of Neospora for use in a vaccine that protects a mammal against neosporosis, which comprises modifying cells of a pathogenic progenitor strain of a species from Neospora; select and propagate by cloning one or more modified cells showing attenuated pathogenicity compared to those of the progenitor strain cells; and selecting and propagating by cloning one or more immune cells that protect the mammal against neosporosis when administered in a live vaccine. In a fourth aspect, the present invention provides methods for the preparation of a vaccine that protects a mammal against neosporosis, which comprises modifying cells of a pathogenic progenitor strain of an axis species Neospora; select and propagate by cloning those modified cells that show attenuated pathogenicity compared to cells of the parent strain but that are capable of eliciting an immune response in the mammal that protects against neosporosis when administered in a live vaccine; and combining an effective immunologically effective amount of the attenuated cells with an acceptable veterinary vehicle in a form suitable for administration in a live vaccine to a mammal. In a fifth aspect, the present invention provides methods for vaccinating a mammal against neosporosis, comprising administering to a mammal an immunologically effective amount of a vaccine composed of living cells of a strain derived from a pathogenic parent strain of a Neospora species, cells that show attenuated pathogenicity compared to those of the progenitor strain, but that are capable of triggering an immunological response that protects the mammal against neosporoeis when they are administered as a live vaccine, and a veterinary vehicle is acceptable. In a sixth aspect, the present invention provides combination vaccines, comprising an immunologically effective amount of living cells of a strain derived from a pathogenic progenitor strain, a Neospora species, cells that show attenuated pathogenicity compared to those of the parent strain, but which are capable of triggering an immunological response that protects the mammal against neosporosis when administered as a live vaccine; one or more different antigens that trigger an immune response that protects the animal against a pathological disease or disorder; and a veterinarily acceptable vehicle. The combination axis vaccines may additionally comprise one or more additional components including, for example, an adjuvant.

DETAILED DESCRIPTION OF THE INVENTION Applicants have discovered that the cells of a pathogenic strain of a Neospora axis species can be attenuated, and that the resulting attenuated cells are capable of eliciting an immune response that protects mammals against neosporosis when they are co-administered or a live vaccine . The present invention thus provides cell cultures of a strain derived from a pathogenic progenitor strain of a Neospora species, cells that exhibit attenuational pathogenicity compared to those of the parent strain, but which can trigger an immunological response that protects a mammal against to neosporosis when administered as a live vaccine. The present invention further provides methods for the preparation of attenuated cell cultures of a Neospora species for use in a vaccine that protects a mammal against neosporosis, which comprises modifying cells of a pathogenic progenitor strain of a Neospora species, for example, by a large number of serial passages, or by exposure to a nonatagen agent, or by genetic engineering using recombinant DNA techniques; selecting and propagating - by cloning one or more modified cells showing attenuated pathogenicity compared to the stem cells the progenitor strain; and selecting and propagating by cloning one or more attenuated cells that are capable of causing an immune response that protects the mammal against neosporosis when administered in a live vaccine. As used herein, the term "neosporosis" refers to the infection of a mammal by a Neospora species or strain, or to any symptom, disorder, event or clinical condition associated with infection of the mammal by Neospora. The term "attenuated" as used herein, describes a Neospora cell, culture or strain strain that shows a detectable reduction in infectivity or smallpox capacity in vitro or in vivo when compared to that of the Neospora progenitor strain. which said cell, culture or attenuated strain is derived. The reduction of virulence includes any detectable decrease in any attribute axis virulence, including the ability of infection in vitro and in vivo, or any decrease in the intensity or speed progression of any symptom or clinical disorder associated with the infection. The term "parent strain" refers to a Neospora strain that exhibits a relatively greater degree of pathogenicity when administered to an animal than an attenuated strain derived therefrom by one or more passage in vivo or in vitro and / or a or more stages of attenuation. The present invention further includes the preparation and use is a cell vaccine of a strain of Neospora derived from a strain or species that is non-pathogenic in a particular amelia species, but whose cells have been modified by chemical or genetic means so that they can trigger an immunological response in members of said mammalian species. The live attenuated cells of the invention can deduce an immune response that protects a mammal against neosporosis, then one or more administrations as a live vaccine. A "protective immune response" is defined as any immunological response, either antibody-induced or cell-mediated immunity, or both, that originates in the mammal that avoids or measurably reduces subsequent infection, or eliminates or reduces detectably the intensity, or detectably decrease the rate of progression, of one or more symptoms or clinical disorders associated with neosporosis. The term "immunologically effective amount" refers to the amount or dose of vaccine or antigen that triggers a protective immune response when administered to an animal.

Preparation of attenuated Neospora strains Since the invention is based on the axes axis hub that cells of a pathogenic Neospora strain can be attenuated, and that the resulting attenuated cells can elicit an immune response that protects a mammal against neosporosis when co-administered or a live vaccine, the practical embodiment of the invention is not limited to any particular attenuation procedure. Rather, the attenuation of cells from a pathogenic Neospora axis strain can be carried out by any technique or procedure well known in the art including, but not limited to, high number of serial passages, or exposure to a rnutágeno agent, or by genetic engineering using recombinant DNA technology or some combination thereof. Serial passage a large number of times can be carried out by in vitro passage repeated axis cells of a Neospora pathogenic strain in susceptible host cells until sufficient attenuation occurs. The passage can be carried out under conditions in a specific environment to select the attenuated cells. For example, the passage can be carried out at a temperature below the body temperature of the mammal to be vaccinated to select the Neospora-axis-sensitive strains that will not develop, or will only develop at a reduced rate, when administer the mammal in a vaccine.

The rnutagenesis can be carried out by exposure of Neospor-a cells to chemical mutagen or radiation, as described in the art. A non-limiting example of a chemical mutant useful in the practice of the invention is N-methyl-N'-nitro-N-mtrosoguanidine (MNNG) (Sigma), the use of which is described later in Example 1. Radiation can be Select between ultraviolet light and ionizing radiation. The exposure graft to the reactor is to perform the chemical concentration, or the level of radiation, as well as the duration of the exposure, preferably reaching a level sufficient to produce one or more viable Neospora cells that show a positive result. attenuated by protrusion but that may trigger an immune response that protects against neosporosis when administered as a live vaccine to a mammal. Suitable parameters for use with rnutagenic agents can be determined empirically using conventional techniques. Pathogenic Neospora strains can also be attenuated using recornbinant DNA technology according to procedures known in the art, and the present invention is intended to include such modified strains and vaccines prepared therefrom. Non-limiting examples of such recombinant DNA techniques that can be used to carry out the invention include gene replacement or gene knockout to neutralize one or more genes, resulting in a strain having attenuated pathogenicity.

Genes that can be neutralized include, for example, an essential metabolic gene, or a gene encoding a virulence factor, or a gene encoding a surface antigen that plays a role in the modulation of the immune response in the mammalian host. . A non-limiting example of an essential metabolic gene axis that can be usefully designated for disruption in the Neospor-a genome is the dihydrofolate reductasatirnidylate synthetase (DHRF-TS) gene. Tilus et al., 1995, Proc. Nati Acad. Sci. USA, 92: 10267-10271 describe the progressive elimination of the DHFR-TS gene to produce a safe, live Leishmania vaccine, a publication which is incorporated herein by reference. Breaking the DHFT-TS gene in Neospora will create auxotrophic mutants that require thymidine to continue development, that exhibit attenuated pathogenicity and that are capable of triggering an immune response in a mammal that protects against neosporosis when administered as a live vaccine. The techniques of recombinant DNA for gene replacement or progressive elimination axis genes are known in the art and include, but are not limited to, those that take advantage of homologous recombination. For example, axis cells a pathogenic Neospora axis strain can be transformed or transfected with a vector, such as a plasmid, composed of homologous nucleotide sequences that are normally located next to or, for example, in an essential metabolic gene, preferably a gene of a single copy, in a pathogenic strain of Neospora. Within or within the homologous nucleotide sequences, the vector may further comprise a nucleotide sequence which corresponds to the nucleotide sequence of the pathogenic strain but which is defective as a result of, for example, a non-silent change or a deletion in one. or more nucleotides compared to the sequence of the pathogenic strain. The transformation of a cell of the pathogenic strain with the vector is followed by the integration of the defective gene sequence in the Neospora genome, which also serves to replace the original or "wild-type" sequence. In this way, the selected gene is neutralized in the transformed cell. Transformed cells can be screened for those cells that show attenuated pathogenicity. Transformed cells that show attenuated pathogenicity can then be traced back to the new axis for those cells that are capable of eliciting an immune response in a mammal that protects against neosporosis when administered as a live vaccine. To help in the selection of transformants, the vector can be engineered to additionally comprise a coding sequence for a reporter gene product or other selectable marker. Reporter genes that may be utility axis in the invention are well known in the art and include, for example, the gene encoding chloramphenicol acetyl transferase (CAT) or the gene encoding luciferase. A further non-limiting example of a reporter gene is a sequence encoding | 3-galactosidase in E. coli, which can insert into the vector and use to confirm the transformants by detecting the enzymatic activity by converting a substrate such as, for example, red-ß-D-galactopyranoside, in a colored product. Seeber and Boothroyd, 1996, Gene, 169: 39-45, used this reporter enzyme to detect transformants in Toxoplasrna gondii, the publication of which is incorporated herein by reference. Coding sequences encoding selectable markers that may be useful in the invention are also known in the art, and include those that encode gene products that confer resistance to antibiotics or antimetabolites, or that provide an auxotrophic requirement. Examples of such axes include those that confer resistance to hygromycin, or to neomycin or to fleromycin. An example of the use of a marker of resistance to an antibiotic in a different pathogen is presented by Messina et al., 1995, Gene, 165: 213-217, which describes the use of a marker of resistance to phleomycin to construct stable transformants. in T. gondii, and whose publication is incorporated in the present reference. Any coding sequence for a reporter gene product or selectable marker will preferably be inserted into a vector in operative association with a sequence encoding the regulatory element. As used herein, a "regulatory element" includes, but is not limited to, inducible and non-inducible promoters, enhancers, operators and other elements known in the art that serve to drive and / or regulate expression. In addition, as used herein, a DNA coding sequence is in "operative association" with one or more regulatory elements when the regulatory elements regulate axis form effective and allow transcription of the coding sequence eg DNA and / or DNA. translation of the corresponding mRNA. For example, expression vectors of the selectable marker in Neospora axis cells can be constructed bordering the open reading frame (ORF) with regulatory regions of Neospor-a or Apicomplexa, Toxoplasma genes very similar. The regions eg the 5 'side of the Neospora or Toxoplasma single copy gene may be used to express ble. Examples of single-copy Toxoplasma genes are: (i) SAG1, which encodes the main tachyzoite surface antigen, p30 (Burg et al., 1988, 3. Immunol.141: 3584-3591); (ii) GRAl encoding a secretory protein, p23 (Cesbron-Delauw et al., 1989, Proc.Nat.Aca.Sci.86: 7537-7541); and (iii) GRA2, which encodes a secretory protein, p28 (Mercier et al., 1993, Mol. Biochem. Parasitol, 58: 71-82). Examples of single copy Neospora genes include: (i) Toxoplasma gene homologs SAG1, GRA1 and GRA2, identified using conventional PCR methods, based, for example, on the published Toxoplasrna sequences; (ii) the gene encoding the major surface protein, NC-p43, of the tachyzoites of N. caninum (Hemphill, 1996, Infect, Irnmunon, 64: 4279-4287); and (iii) "genes encoding immunodominant proteins axes 17, 29, 30 and 37 kDa excretory / secretory (Bjerkas et al., 1994, Clin, Diag.Lab. Immun.1: 214-221). The 3 'side region of the SAG1 gene can be used to provide a polyadenylation sequence. The vector structure for the insertion of the sequences of the 5 'promoter, gen ble and 3' -polyadenylation can be any commercially available conventional plasmid, such as pBLUESCRIPT ™ (Stratagene). Once the appropriate vector has been constructed, it is used to transform or transfect one or more cells from a progenitor strain of Neospora. The vector can be introduced into the cells according to known techniques, including, but not limited to, electroporation, icroinjection, vial transfection, liposome-induced transfection, microprojectile-borne, etc. Once the vector has been introduced into the Neospora cells, the presence, integration and maintenance of the coding sequence introduced into the genome of the host cell can be confirmed and controlled, or episode by conventional techniques including, but not limited to, Southern hybridization analysis, PCR analysis, including reverse transcriptase PCR (RT-PCR); immunological or colori-etric assay for the expected product protein; detection of the presence or absence of a marker gene function, such as the appearance of a new auxotroph; or by detection of an attenuation in pathogenicity. Examples of vector construction, transformation, axis selection, transformations, expression of host cells, etc., as applied specifically to pathogenic protozoa are described in the following publications, which are incorporated herein by reference: Seeber and Boothroyd, 1996, supra; Titus et al., 1995, supra; Messina et al., 1995, Gene, 165: 213-217; ? ibley et al., 1994, Proc. Nati Acad. Sci. USA, 91: 5508-5512; Donald and Roos, 1994, Mol. Biochem. Parasitol., 63: 243-253; Kirn et al., 1993, Science, 262: 911-914; Ryan and others, 1993, Proc. Nati Acad. ? ci. USA, 90: 8609-8613; Soldati and Boothroyd, 1993, Science, 260: 349-352; Eid and Sollner-ebb, 1991, Proc. Nati Acad. Sci. USA, 88: 2118-2121; LeBowitz et al., 1990, Proc. Nati Acad. Sci. USA, 87: 9736-9740; Lee and Van der Ploeg, 1990, Science, 250: 1583-1586; Asbroek, et al., 1990, Nature, 348: 174-175; Cruz and Beverley, 1990, Nature, 348: 171-173; and Laban et al., Nature, 343: 572-574. General techniques of genetic recombination, including vector construction, transformation, selection of transformants, expression of host cells, etc., are further described in Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates »Uiley Interscience, N.Y .; Sanbrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2Q Ed, Cold Spring Harbor-Laboratory Press, Cold Spring Harbor, N.Y .; Innis et al., (Eds), 1995, PCR Strategies, Academic Press, Inc., San Diego, Ca .; and Erlich (ed), 1992, PCR Technology, Oxford University Press, N.Y., which are incorporated herein by reference. Then, from the attenuation step, cells showing one or more attenuated pathogenicity axis indicators are selected from the culture and propagated by cloning axes after axis limiting the dilution. Examples of such indicators include, but are not limited to, emergence of a new temperature sensitivity or a new in vitro auxotroph, or a reduction of an attribute of virulence such as the infectious capacity or the intensity or speed of the progression of one or more symptoms or disorders in a mammal after the administration of cells of the strain compared with the infection with the progenitor strain, among others. A particular non-limiting example of a temperature sensitivity that is useful in the practice of the invention is that in which the cells of the attenuated strain will develop at 32 ° C but not at 37 ° C. Said temperature-sensitive strain will cause lysis of infected host cells at 32 ° C, resulting in the appearance of axis lesions or plaques in a monolayer of host cells. When developed at 37 ° C, the attenuated strain is not 1.8 it will replicate sufficiently and be unable to produce plaques in the monolayers of host cells. An attenuated Neospora strain can be derived from any pathogenic strain of any species of the genus, including, but not limited to, N. Caninurn. A non-limiting example of a particular pathogenic strain N.e. caninurn from which an attenuated strain can be usefully derived is strain NC-1, which is present in MARC145 monkey kidney cells infected from the American Type Culture Collection (ATCC ), 12301 Parklawn Drive, Rockville, MD 20852 USA (ATCC Access Hub, CRL-12231). Strain NC-1 is also described in Dubey et al., 1988, 3. Am. Vet. Med. Assoc., 193: 1259-63, the publication of which is incorporated herein by reference. Alternatively, the pathogenic Neospora axis strains can be obtained from tissues, organs or body fluids of infected animals that present clinical symptoms of neosporosis using conventional isolating techniques axioms, for example, in the publications reviewed above. A non-limiting example of an attenuated and live strain derived from the NC-1 strain of N. caninum is NCTS-8, which is present in MARC145 monkey kidney cells infected with the ATCC axis (access ns of the ATCC CRL- 12230). Both the parental and attenuated Neospora strains can be cultured in vitro by infecting any receptive cell line, preferably a mammalian cell line, with tachyzoite axis strain according to procedures .1.9 known ones described in the art. Mammalian cell lines in which Neospora tachyzoites can be cultured include, for example, human foreskin fibroblasts (Lindsay et al., 1993, A. 3. Vet. Res., 54: 103-106); bovine cardiopulmonary aortic endothelial cells (March et al., 1995, supra); and bovine rhonocytes (Lindsay and Dubey, 1989 supra), among others. For example, N. caninum tachyzoites can be cultured in monolayers of human foreskin fibroblast cells Hs68 (ATCC Accession No. CRL-1635) (Lindsay et al., 1993, supra). The bradizoitos can be cultivated and manipulated in the same way. Cultures of mammalian cells can be grown, and cell cultures infected with Neospora can be maintained in any one of several culture media described in the art. For example, stationary rnonolane cultures of bovine cardiopulmonary aortic endothelial cells infected with N. caninum tachyzoites can be grown in Dulbecco's Minimum Essential Medium (DMEM: Gibco Laboratories, NY), supplemented with thermally inactivated fetal bovine serum (FBS). 10% (v / v) or adult equine serum (ES), 2 mM L-glutamine, 50 U / ml penicillin and 50 μg / ml streptomycin axis (Conrad et al., 1993 supra). The monolayers of human foreskin fibroblast Hs68 cells can be maintained in RPMI 1640 medium (Gibco), containing 2% fetal bovine serum (v / v), 1.0 M sodium pyruvate, 1 x 10 * U / ml of penicillin, 1 x 10 * g / ml streptomycin, 2-mercaptoethanol 5 x 10 -2 rnM and 0.3 mg / rnl L-glutamine axis (maintenance medium axis). Monolayer cultures of human foreskin fibroblast Hs68 cells infected with Neospora can be maintained in identical media, but in which fetal bovine serum is increased to 10% (v / v) (growth medium). Attenuated strains of Neospora that have new auxotrophs will require an adequate modification to the culture medium to support the development, as is known in the art. Neospora-infected monolayer cultures of mammalian cells are normally maintained under conventional tissue culture conditions such as at 37 ° C and 5% C02. The tachyzoites are generally passed on to uninfected rnonolapa cultures when 70-90% of mammalian cells have been infected in the culture, which can be determined microscopically using conventional techniques. Tachyzoites can be harvested from cultures of infected mammalian cells using host cells using any conventional technique that allows tachyzoites to maintain their viability and by collecting the tachyzoites from filtration or by centrifugation., for example. Preparation and Use of Vaccines The present invention provides vaccines against neosporosis, comprising an immunologically effective amount of living cells of a strain derived from a pathogenic progenitor strain of a Neospora species, cells that show attenuated pathogenicity compared to those of the strain progenitor, but which are capable of triggering an immune response that protects the mammal against neosporosis when they are administered as a live vaccine, and an aminally acceptable veterinary vehicle. The present invention further provides methods for the preparation of a vaccine that protects a mammal against neosporosis, which comprises modifying cells of a pathogenic progenitor strain, a species of Neospora.; select- and propagate by cloning those modified cells that show attenuated pathogenicity compared to the axis cells of the progenitor strain but that are capable of triggering an immunological response in the mammal that protects against neosporosis when administered in a live vaccine; and combining an immunologically effective amount of the attenuated cells with a veterinarily acceptable carrier in a manner suitable for administration as a live vaccine to a mammal. The present invention further provides methods of vaccinating a mammal against neosporosis, which comprises administering to the mammal an effective immunologically effective amount of a vaccine composed of living cells of a strain derived from a pathogenic progenitor layer of a Neospora species, cells that show attenuated pathogenicity compared to those of the parental strain but that are capable of triggering an immunological response that protects the mammal against neoporosis when it? ? they administer it as a live vaccine, and a veterinarily acceptable vehicle. The vaccine of the invention comprises living cells of an attenuated Neospora strain, either as the only antigenic component or in combination with one or more different antigens which axes trigger a mmunological response that protects the mammal against a pathological disease or disorder that may or may not be related to neosporosis. Thus, the present invention further provides combination vaccines, composed of an immunologically effective amount of living cells of a strain derived from a pathogenic progeny strain, a Neospora species, cells that show attenuated pathogenicity compared to those of the parent strain but which are capable of triggering an immunological response that protects the mammal against neosporosis when administered as a live vaccine; one or more different antigens that trigger an inrnunological response that protects the mammal against a pathological disease or disorder; and a veterinarily acceptable vehicle. The combination vaccines may further comprise one or more additional components including, for example, an adjuvant. The vaccine is conveniently administered parenterally, for example, by subcutaneous or intramuscular injection. However, the vaccine may also be administered by intraperitoneal or intravenous injection, or by other routes, including oral, intranasal, rectal or vaginal, and, when the vaccine is administered in this way, a veterinarily acceptable vehicle is selected. The vaccine may comprise only cells attenuated in a culture fluid, which are administered directly to the mammal. Alternatively, the vaccine may comprise attenuated cells combined with a veterinary or pharmaceutically acceptable carrier, selected from the knowledge of those skilled in the art depending on the route of administration and its ability to maintain cell viability. Non-limiting examples of such vehicles include water, saline, tarninated vehicles and the like. Other vehicles for suitable vaccines and additives are known, or will be apparent to those skilled in the art. See for example, Re ington's Pharmaceutical Science, 18th ed., 1990, Mack Publisching, which is incorporated herein by reference. The vaccine may further comprise one or more other components such as an immunomodulatory agent including, for example, interleukin-1, or another substance that enhances immunity as a veterinarily acceptable adjuvant. Non-limiting examples of adjuvants include complete and incomplete Freund axis adjuvants, mineral gels including aluminum hydroxide and formulations water in oil and oil in water. The immunornodulatory agents are selected on the basis of their ability to maintain both the viability of the attenuated Neospora cells and the ability of the cells to elicit a protective immune response in the vaccinated mammal. Non-limiting examples of oil-in-water formulations that are useful as adjuvants in the vaccines of the invention include the following emulsions 1.3. Emulsion 1 is composed of: (a) ME6201 (5% v / v squalene, 0.1% v / v vitamin E, and 0.8% v / v Tween ™ 80 dispersant); (b) Preparation of Saponin Quil A (OA) (Superfos) (200 μg / rnl); and (c) cholesterol (col) (100 μg / ml). The emulsion 2 is composed of: (a) ME6201; and (b) Avridine lipoidal amine (1 rng / rnl). Emulsion 3 is composed of ME6201 (5% v / v squalene, 1.0% v / v vitamin E and 0.8% Tween ™ 80 dispersant). An effective dose can be exemplified by conventional means, starting with a low dose of attenuated cells and then increasing the dose while controlling the effects, and also systematically varying the dose. Numerous factors can be taken into account when determining the optimal dose per animal. Mainly among these is the species, the size of the animal, the age of the animal, the general condition of the animal, the presence of other drugs in the animal, the virulence of a particular Neospora strain against which the animal is vaccinated and the like. The actual dose will preferably be chosen after the axis to consider the results axis other animal studies. The vaccination guidelines are also selected based on the factors described above. Animals can be vaccinated at any time, including just before, or at the time of reproduction. Supplementary administrations, or reinforcements, may be necessary for full protection. A procedure to detect whether adequate immunological protection has been achieved is to determine seroconversion and antibody concentrations in the animal after vaccination. In this way, the vaccine of the invention can be administered at any time during the life of a particular animal to be vaccinated, depending on several factors including, for example, the duration of an outbreak of neosporosis among other animals, etc. The vaccine can be administered to animals at the weaning age or younger, or more mature animals, for example, with a pre-breeding vaccine to protect the animal against congenital disease or abortion related to Neospora. Effective vaccination may only require a first vaccination, or a first vaccination with one or more booster vaccinations. Booster vaccinations can be administered at any time after the first vaccination, depending, for example, on the immunological response after the first vaccination, the intensity of the outbreak, the virulence of the Neospora strain causing the outbreak, the health of the vaccinated animals, etc. The vaccination program and the number of reinforcements, if any, will preferably be determined by a veterinarian on the basis of an analysis of all relevant factors, some of which have been described above.

The Neospora axis cells used in the vaccine are preferably tachyzoites, although they may be bradyzoites, or oocytes, or some combination thereof. The concentration of the attenuated cells in the vaccine preferably ranges from about 1 x 10 3 / ml to about 1 x 10 8 / rnl, and more preferably from about 2 x 10 6 / ml to about 2 x 10 7 ml. A suitable dosage size ranges from about 0.5 ml to about 1.0 rnl. In general, depending on the dose, the number of attenuated cells administered to an animal preferably ranges from about 2 x 10 * to about 2 x 108; more preferably axis about 2 x 10 * to about 2 x 107; The most preferable axis is about 1 x 106 to about 1 x 107. The vaccine of the invention protects a mammal against infection or disease caused by Neospora. The vaccine is useful to protect both pregnant and non-pregnant mammals including, but not limited to, bovine, ovine, caprine, canine and equine species, against infection, clinical disease or abortion resulting from neosporoeis. The term "protection" is widely used and is not limited to the absolute prevention of Neospora infection, but includes a reduction in the infectiousness, or in the intensity of a disease or disorder resulting from the infection, including a reduction detectable in one or more of the pathological effects or symptoms resulting from the infection, or a detectable reduction in the rate of progression of one or more of such effects or pathological symptoms. The vaccine of the invention is also innocuous, that is, it does not cause disease or significant side effects in the vaccinated animal. The following examples are offered to further illustrate, but not to limit, the compositions and methods of the invention.

EXAMPLE 1 Determination of temperature sensitive strains of N. caninum and analysis of pathogenicity in BALB / c mice The objective of this study was to determine strains sensitive to the temperature of N. caninum (NCTS) and to test the pathogenicity of these strains by analyzing the response to serum antibodies, tissue cysts and production of brain injury, and the development of clinical symptoms in BALB mice. / c, which are known to be highly susceptible to neosporosis. Materials and procedures Tachyzoites of N. caninum strain NC-1 were cloned.

(Dubey et al., 1988, supra) twice limiting dilution and maintaining at 37 ° C, as described (Lindsay and Dubey, 1989, supra). The tachyzoites were propagated in 25 cpv flasks * and cloned into 96-well plates containing monolayers of human foreskin fibroblast Hs68 cells (ATCC accession no. CRL-1635) (Lindsay et al., 1993, supra). The Hs68 cells were previously rotated in RPMI 1640 medium containing 2% fetal bovine serum (v / v), 1.0 mM sodium pyruvate, 1 x 10 * u / ml penicillin axis, 1 x 10 * g / ml streptomycin. , 2-mercaptoethanol 5 x 10-2 rnM and 0.3 rng / rnl of L-glutarnine (maintenance medium axis). The rnonolaps of cultured axis infected cells were maintained in identical medium, but in which the fetal bovine serum was increased up to 10% (v / v) (means of "jesarrollo"). A clone was isolated and designated in the laboratory as the NC-1-2C line, hereafter referred to simply as strain NC-1. The tachyzoites of strain NC-1 were mutagenized by exposure to 0.5 μM N-rnethyl-N'-nitro-N-nitrosoguanidine (Sigma) in growth medium for 24 hours and then grown at 32.5 ° C for 3 months in cells Hs68 in maintenance medium, after which the tachyzoites were cloned by limiting the dilution axis. Twelve clones were initially isolated. Three clones, named NCTS-4, NCTS-8 and NCTS-12 (NCTS = N. caninurn temperature sensitive) were selected for further study after having been maintained in Hs6B cells in continuous culture at 32.5 ° C for more than 8 days. months in the middle of maintenance. The serological test of the mice was carried out as follows. An indirect innunofluorescent antibody (IFAT) assay (Dubey et al., 1988, supra) to analyze the sera of mice and determine the presence of antibodies directed against N, caninum. The sera of the mice were obtained immediately before the inoculation by stimulation induced in the axis vaccination studies. Serum was also obtained from all the mice that survived the pathogenicity experiments. The sera were examined at double dilutions at 1:50 and at the end of the titraejo. The tachyzoites were used as an antigen. The positive samples showed a complete fluorescence axis axis the tachyzoites. Negative samples showed no fluorescence or only a fluorescence at the anterior end. The presence of lesions in the axis brains of the mice inoculated by stimulation caused with different strains of N. caninum was determined as follows. The brain of each mouse was excised at autopsy, and a first half was fixed in 10% (v / v) neutral tarnponaeja forrnaline solution for histopathological examination. Tissue sections were prepared using routine root histological techniques and stained with hematoxylin and eosin to detect the presence of lesions under a microscope. The evaluation of the lesions of the brain tissue sections stained with hematoxylin and eosin was carried out according to the criteria described in Lindsay et al., 1995, 3. Parasitol. 81: 313-315.

No. of inflamed / necrotic foci Value without foci 1 1-5 foci 2 6-10 foci 3 > 10 foci 4 Average size of the foci Value None 1 100-200 μrn 2 200-500 μrn 3 > 500 μrn 4 Intensity of injuries Value No injuries 1 1 light 2 moderate 3 moderate 4 pointed 5 A mean value of the lesion was obtained using the three values presented above. A normal uninfected mouse brain has a mean axis value of the 3.0 axis lesion. The mean values of the lesion were evaluated using a non-parametric Kruskal-Uallis test, and multiple comparison procedures of free distribution. The number axis mice in each group with lesions was examined using Fisher's exact test. The statistical significance was determined at a value of p < 0.05.

The brain sections of each mouse were further examined using a mouse non-clonal antibody, 6G7, specific for N. caninum, in combination with an avidin-biotin peroxidase complex (ABC) inrnunohistoquírnico assay to detect the stages of N. caninum ( Cole and others, 1993, 3. Vet. Diag. Invest. 5: 574-589). The second half of each mouse axis brain was digested in acid-pepsin and used to inoculate mammalian cell cultures and detect the presence of N. caninurn tissue cysts. For digestion, the second half of the mouse brain was placed in 3 ml of Hank's balanced salt solution (HBS?) And passed twice through a syringe with a 23 gauge needle. Three my solution axis were added of acid-pepsin (0.52 g of pepsin, 0.50 g of NaCl, 98.6 rnl axis H2O d, 1.4 rnl of HCl concentrate, pH 0.8) to the homogenate and incubated for 10 minutes at 37 ° C in a water bath. The acid-pepsin solution was separated by centrifugation and the pellet, representing the entire second half of the brain, was inoculated into a 25 cm2 tissue culture flask containing a monolayer of human foreskin fibroblast Hs68 cells cultured as described above. described above. After 30 minutes, the inoculum was removed and the monolayer of Hs68 cells was washed and incubated in fresh maintenance medium as above. The cell cultures were then examined for 30 days to detect the presence of N. caninurn (Lindsay and Dubey, 1989, supra).

The pathogenicity of NC-1 and the three selected NCTS strains, ie NCTS-4, NCT-8 and NCTS-12, of N. caninum was determined as follows. BALB / c mice (8 weeks, female) (Harían Sprague Dawley (HSD)) were inoculated subcutaneously with HBSS (control) or with 5 x 105 tachyzoites of the NC-1 strains, NCTS-4, NCTS-8 or NCTS-12 of N. caninum in HBSS (0.5 to 1.2 ml of total volume). The surviving mice were examined microscopically at autopsy 42 or 56 days after inoculation (Pl) (see Table 1). The serum of the surviving mice was collected at autopsy. The brains of these mice were examined to determine the value of the lesions V '.a inrnunohistologia, and one half of each brain was used for digestion in acid-pepsin to determine the presence of tissue cysts as described above.

TABLE 1 Results of the inoculation of BflLB / c mice with tachyzoites of the strains NCTS-4, NCTS-8, NCTS-12 and NC-1 of N. caninum TABLE 2 Mean value of lesions of BflLB / c mice inoculated with different strains of N. caninum * = significant difference of control (HBSS) (p <0.05) * > = significant difference of NCTS-4, NCTS-8 and NCTS-12 (p <0.05) TABLE 3 Reverse of antibody concentrations in mouse serum Results Clinical neosporosis and mortality occurred only in BALB / c mice inoculated with strain NC-1 (Table 1). Only three of 10 BALB / c mice survived infection with strain NC-1. Strains NCTS-4, NCTS-8 and NCTS-12 did not cause mortality in BALB / c mice. The mean lesion values are presented in Table 2. Lesions were found in the brains of some mice inoculated with the NCTS strains., but the mean lesion values were not statistically significant when compared with the control (HBSS). Significant difference was found in mean lesion values and number of mice with lesions when mice inoculated with strain NC-1 were compared with mice inoculated with HBSS (control) or with any axis the NCTS strains of N. caninurn. The concentrations of antibodies in serum are presented in Table 3. Significant IFAT concentrations were detected (_ >; 400) in mice inoculated with stimulated stimulation (30/30) with NCTS strains. This indicates that NCTS strains can stimulate a B-cell axis response in an immunologically intact but genetically susceptible animal. IFAT concentrations in mice stimulated with NCTS were equal to, or in some cases greater than, that of mice stimulated with NC-1. No tissue cysts were detected in any of the brain regions examined using the digestion technique with aciejo-pepsma centroscpta previously. This indicates that the tissue cysts, if present, are few in mice inoculated with the NCTS or NC-1 strains of N. camnum.

EXAMPLE 2 Analysis of the pathogenicity of N. caninum NCTS strains in HSD exogamic mice: ICR The objective of this study was to determine the pathogenicity of NCTS strains of N. canmum in outbred mice HSD: ICR, which are immunocompetent and more resistant to Neospora than BALB / c inbred mice.

Materials and methods In this experiment, HSD: ICR mice (4 weeks, female) were used. All the HSD: ICR mice, except the controls, were inoculated with 5 x 105 axis tachyzoites the appropriate strain of N. caninurn in HB? S (total volume 0.5 to 1.2 ml).

Control mice were inoculated with HBSS only. All the surviving mice were sacrificed at 56 μL. The serum was collected for the IFAT assay. The brains of these mice were harvested and a first half was used to assess the lesions and immunohistology as before. The second half axis each brain was used in digestion with acid-pepsin to detect tissue cysts as previously described. Results None of the N. caninurn strains tested, including NC-1 caused mortality in HSD: ICR mice (Table 4) and no significant differences were observed in the number of mice with lesions or in the mean values of the lesion compared with the controls not inoculated by induced stimulation (Table 5). No tissue cysts were observed in the brain in sections stained with ABC or histologically. No parasites were isolated in cell cultures.

TABLE 4 Results of the inoculation of HSD.ICR mice with tachyzoites of the strains NC-1, NCTS-4, NCTS-8 or NCTS-12 of N. caninum TABLE 5 Mean value of lesions of HSD mice: ICR inoculated with different strains of N. caninum a = No significant differences were observed between the treatment groups.

TABLE 6 Reverse of antibody concentrations in mouse serum » a = All concentrations were determined on day 56 Pl The antibody concentrations of surviving mice are presented in table 6. Significant IFAT concentrations (_> 400) were screened in the majority of the mice (13/15) inoculated by stimulation caused with the NCTS strains, which indicates that these strains are capable of inducing a response in B cells in immunologically intact but genetically resistant animals. No tissue cysts were detected in the brain protection examined histologically, immunohistologically or using the axis-digestion procedure with acid-pepsin. These results confirm those presented in Example 1, above, using BflLB / c mice.

EXAMPLE 3 Analysis of the pathogenicity of N. caninum NCTS strains in HSD mice: immunodepressed ICR A first objective of this study was to determine the pathogenicity of NCTS strains of N. caninurn in H? D mice: ICR inrnunodeprirnidos. A second objective of this study was to determine if the reversal of pathogenicity occurred after an in vitro passage of NCTS strains of N. caninum at 37 ° C. Materials and procedures HSD: ICR mice were inoculated (4 weeks, female) by intramuscular administration of 2 mg of methylprednisolone acetate (UPA) (Upjohn-Phar acia) on days -7, 0 and 7 Pl. See Lindsay and Dubey, 1989, 3. Parasitology 75: 772-779. HSD: ICR mice in uno deprirnidos were inoculated on day 0 with 2 x 10 * tachyzoites of strains NC-1, NCTS-4, NCTS-8 or NCTS-12 or one of the axis reversal potential controls, denominated NCTS-4- 37, NCTS-8-37, or NCTS-12-37, in HBSS (total volume of 0.5 to 1.2 mi). The mice were subsequently examined serologically, histologically and clinically, as described above. Clones NCTS-4, NCTS-8 and NCTS-12 were examined for pathogenicity reversion to development at 37 ° C for 88 days (25 cell culture passages), followed by inoculation in HSD: immunosuppressed ICR mice (strains) of potential reversal are called NCT? -4-37, 4-37, NCTS-8-37 or NCTS-12-37). Results Strains NC-1, NCTS-4-37 and NCTS-12-37 of N. caninum caused a 100% mortality axis in HSD mice: immunosuppressed ICR (table 7). The strains NCTS-4, NCTS-8, NCTS-12 and NCTS-8-37 were pathogenic to the HSD: ICR inrnunodeprirnicjos mice and caused only 0 to 20% mortality. The mean values of the lesion are presented in Table 8. No tachyzoites were detected in digests acid-pepsin axis in any of the mice examined at autopsy 56 Pl. Antibody concentrations of surviving mice are presented in Table 9. significant IFflT concentrations (>; _ 800) were detected in NCTS-4, -8, and 12, and NCTS-8-37 mice at 56 days after the induced inoculation, indicating that these strains are capable of stimulating a response of B cells in a animal inmonodeprirnido but genetically resistant.

TABLE 7 Results of inoculation of HSD mice: immunosuppressed ICR with tachyzoites from strains NC-1, NCTS-4, NCTS-B, or NCTS-12 or potential reversal strains of N. caninum * = Mice that were moribund due to clinical encephalitic neosporosis were euthanized for human reasons.

TABLE 8 Mean value of lesions of HSD mice: immunosuppressed ICR inoculated with different strains of N. caninum a = Significant difference of control (Group 16) (p < 0.05) TABLE 9 Reverse of antibody concentrations in mouse serum * a = All concentrations were determined on day 56. Pl Lae NCTS strains were pathogenic in HSD mice: immunosuppressed ICR than strains NC-1, NCTS-4-37 and NCTS-12-37. The relatively high survival rate (4/5) of mice inoculated with the NCTS-8-37 axis reversal strain cornparaeja with the 100% axis-axis mortality rate in the mice inoculated with the reversal strains NCTS-4-37 or NCTS- 12-37 indicates that the reversal axis strain NCTS-8-37 maintains attenuated pathogenicity after a serial pass at 37 ° C. Based on this demonstrated retention of the attenuated pathogenicity, the NCTS-8 N. caninum strain was selected as a potential vaccine candidate and was used in subsequent studies as described below.

EXAMPLE 4 Vaccination of BflLB / c mice against N. caninum-induced encephalitis A first objective of this study was to determine whether vaccination with a live strain sensitive to the N. caninum axis temperature can provide protection against disease caused by inoculation by subsequent induced stimulation with a pathogenic strain, eg, NC-1 N. caninum. A second objective of this study was to determine the level of protection provided by the vaccination of BALB / c mice with dead (frozen) tachyzoites of NCTS-8 that were subsequently inoculated by stimulation caused with the NC-1 strain of N. caninum. lIFlTERIflLES AND PROCEDURES BALB / c mice (9 weeks old, female) were vaccinated by subcutaneous injection with HBSS (control) (0.5 ml) or 5 x 10 * live tachyzoites of the strain NCTS-8 in HBSS (0.5 ml) or 2 x 105 dead tachyzoites ( frozen) in HBSS (0.5 ml) (table 10). The vaccinates were reinforced 21 days Pl with the same material as in the first injection. Mice were inoculated by stimulation elicited by subcutaneous administration of 1 x 10 * tachyzoites of N. caninurn strain NC-1 in HB? S, or only with HB? S (control) (total volume 0.5 ml), 14 days after the reinforcement. IFAT was used to test the sera of the mice that survived the experiment to determine the presence of antibodies against N. caninum, as described above. The brain axis each mouse was excised at autopsy. The first half was used for histopathology and in unohistology, and the second half was used for digestion with acid-pepsin as described above. The viability of the tachyzoites was determined after freezing by inoculating cultures in monolayer human Hp68 foreskin fibroblast cells, as described above.

RESULTS None of the mice in any of the test groups died after the first or booster vaccination. Two out of 10 mice vaccinated with HBSS (control) and 3 to 10 mice vaccinated with dead NCTS-B tachyzoites died after stimulation with tachyzoites NC-1. The mean lesion axis values of the mice that survived are presented in Table 11. The inverse antibody concentrations of the surviving mice are presented in Table 12. Significant IFAT concentrations (> 800) were detected in mice vaccinated with Live NCTS-B tachyzoites, after reinforcement, confirming the previous results described above. In contrast, there were no significant IFAT concentrations present in any mice vaccinated with NCTS-8 tachyzoites killed before the stimulation evoked, demonstrating that dead tachyzoites are unable to induce a significant antibody response in a genetically susceptible host. N. caninum tachyzoites were isolated from cell cultures inoculated with brain-axis tissue digested with acid-pepsin from two mice stimulated and vaccinated in simulated form (group 24, mice numbers 1 and 2) and from a mouse vaccinated with tachyzoites NCTS-B dead (group 31, mouse number 5). Tachyzoites were not isolated from cultures inoculated with brain tissue from any other mouse.

TABLE 10 Protocol for vaccination and stimulation of BALB / c mice.

TABLE 11 Mean value of lesions of BflLB / c mice stimulated and vaccinated a = Significant difference of groups 24 + 25 (p < 0.05) b = Significant difference of groups 30 + 31 (p < 0.05) caapeo 12 Reverse of antibody concentrations in mouse serum before and after stimulation. •faith Mice vaccinated with live tachyzoites from N. caninurn strain NCTS-8 did not die or develop clinical disease symptoms. Mice vaccinated with live tachyzoites of the strain NCTS-8 and subsequently inoculaejos by stimulation caused with tachyzoites of the strain NC-1 (table 11, groups 28 + 29) had lesion values that were almost identical to those of the mice vaccinated with live tachyzoites of strain NCTS-8 followed by administration of HBSS (Table 11, groups 26 + 27). This indicates that vaccination with live tachyzoites of the strain NCTS-8 provides substantial protection against the disease caused by infection with the strain NC-1 axis N. caninum. Vaccination of mice with dead tachyzoites from the NCTS-8 strain offered little protection against neosporosis (Table 11, groups 30 + 31).

EXAMPLE 5 MOUSE VACCINATION BflLB / c WITH A SMALL DOSE OF Lfl CEPA NCTS-8 FROM N. CRNINUM The objective of this study was to determine if a small dose, that is, 5 x 10 * tachyzoites, of the NCTS-8 strain of N. caninum can provide protection against subsequent infection caused by stimulation.

MATERIALS AND PROCEDURES BALB / c mice (7 weeks, female) were vaccinated subcutaneously with HBSS, or with 5 x 10 * tachyzoites axis NCTS-8 strain of N. caninum in HBSS (0.5 ml) (Table 13). This dose of tachyzoites is one tenth the amount used in previous examples. The mice were boosted 21 days Pl with the same material as in the first injection. The mice were inoculated by stimulation caused 14 days after the reinforcement with 1 x 106 tachyzoites of the NC-1 strain of N. caninum. IFAT was used to test the serum of the mice that survived the experiment for the antibodies against N. caninum as described above. The brain of each mouse was excised at autopsy. A first half of the brain was used for histopathology and in onehistology, and the remaining second half was used for digestion in acid-pepsin, as previously described.

RESULTS A control mouse (administered only with HBSS) died after stimulation caused by tachyzoites of N. caninum strain NC-1. None of the mice vaccinated with the small dose of tachyzoites of the NCT? -8 strain of N. caninurn died after stimulation with tachyzoites of strain NC-1. The mean lesion values and the number of mice with lesions were significantly higher in the control mice vaccinated in simulated form with HBSS only than in the mice vaccinated with a small dose of the NCTS-B strain (Table 14). Table 15 shows the inverse axis concentration of antibodies.

TABLE 13 Protocol for small dose vaccination and induced stimulation of BflLB / c mice TABLE 14 Mean value of lesions in mice a = Significant difference of group 36 (p < 0.05) TABLE 15 Reverse of antibody concentrations in mouse serum before inoculation by induced stimulation EXAMPLE 6 Efficacy of vaccines with and without adjuvant The objective of this study was to determine the effect of the addition of an adjuvant to a modified live Neospora vaccine and the degree of protection obtained with it against neosporoeie.

Materials and procedures Previous in vitro results (data not shown) indicated that tachyzoites of at least one modified live N. caninum strain, ie, NCTS-8, maintained viability and ability to partially infect in vitro when incubated together with a of several different formulations of oil in water. On the basis of these reults in vitro, three specific formulations were selected for an in vivo evaluation as adjuvants. Groups of ten 15-week BALB / c mouse females were subcutaneously subcutaneously (0.2 rnl) on days 0 (first vaccination) and 21 (booster) PI, with HBSS alone (control or with 5 x lQis tachyzoites of the NCTS strain -8 of N. caninurn in HBSS, or with 5 x 105 tachyzoites of N. caninum strain NCTS-8 in one of the following three oil-in-water emulsions: Emulsion 1 was composed of: (a) P1E6201 (squalene at 5% v / v, vitamin E at 0.1% v / v, and TweenTM at or 0.8% v / v as dispersant); (b) Preparation of Saponin Quil (0A) (Superfos) (200 μg / ml); (c) cholesterol (col) (100 μg / ml). The emulsion 2 is composed of: (a) ME6201; and (b) Avridine lipoidal amine (1 mg / ml). Emulsion 3 is composed of: ME6201 (5% v / v squalene, 1.0% v / v vitamin E and 0.8% Tween ™ 80 dispersant).

TABLE 16 Protocol for the testing of vaccine formulations with and without adjuvant On day 35 Pl, the stimulation caused by all the mice was carried out by subcutaneous administration of the axis 1 x 10 * tachyzoites of the strain NC-1 of N. caninum in HBSS (0.2 ml). Groups of 3 to 5 mice were sacrificed on days 49 and 63 Pl to evaluate the efficacy of the vaccine. Beginning on day 0, the disease was assessed on the basis of mortality, as well as on the basis of the appearance of disheveled hair cover, irregular movements, pelvic limb paralysis and generalized weakness. Histopathological analyzes were carried out as follows. Pulmonary samples were obtained on day 49, fixed in 10% neutral buffered formalin (v / v) and the tissue was cut and stained using routine histological techniq Lung sections stained with hematoxylin and eosin were coded and blind shape axis lesions were assessed without knowledge of the treatment groups. The pneumonia lesions were assessed using the following system: 0 = none; 1 = mild; 2 = moderate; 3 = stressed; 4 = intense.

Results Mice vaccinated with a formulation composed of tachyzoite axis, the NCTS-8 strain of N. caninurn and an adjuvant had a significantly lower incidence of mild pneumonia (33%) after stimulation with the NC-1 strain of N. caninum. than control mice vaccinated only with HBSS (56%) (? <0.01). None of the mice vaccinated with NCTS-8 and unstimulated showed any sign encephalitic disease axis or parasites when examined 9 weeks after vaccination (data not shown), confirming that the administration of NCTS-B does not produce clinical disease. The results indicate that a formulation composed of live attenuated Neospora tachyzoites and an adjuvant is at least as effective and safe to use as a vaccine against neosporosis than the same formulation without the adjuvant (table 17).

TABLE 17 Histopathological analysis of mouse lung tissue after administration of vaccine, with or without adjuvant and stimulation caused with the NC-1 strain of N. caninum EXAMPLE 7 Protection of dwarf goats from neosporosis The objective of this study was to determine whether vaccination of dwarf goats with a live attenuated strain of N. caninum can protect goats against neosporosis. More specifically, the ability of a vaccine composed of live tachyzoites in the N. caninum strain NCTS-8 was tested to protect female dwarf goats against Neospora-induced abortion.

Materials and procedures Randomly assigned axis to the groups A-E (table 10) Female dwarf goats from approximately 2 to 5 years old. The dose in each administration of non-simulated vaccine (groups A-C) was composed of 4 x 10 * tachyzoites axis the indicated strain. After subcutaneous vaccination (1.0 ml / dose) on day 0 (first) and day 21 (booster), the females were synchronized using LUTALYSE ™ prostaglandin preparation (Upjohn-Pharrnacia) (10 mg / go intramuscularly) on days 28 and 39. The pregnant females were determined by ultrasound and between gestation day 41 and 55 on the day of the stimulation caused. The axis females groups A-D were inoculated by stimulation caused by 4 x 106 tachyzoites of N. caninum strain NC-1 in the serum-free maintenance medium (0.45 ml) administered by i.v. jugular. The females were controlled by ultrasound, taking the temperature daily during the 7 days after the stimulation and visual observation twice a day, and blood sample was taken once a week after the stimulation caused.

TABLE 18 Female dwarf goat treatment groups Results All goats vaccinated with live N. caninum strain NC (N.) (group A) or N. caninurn attenuated NCT? -8 strain (group B and C) seroconverted and had measurable IFAT concentrations 10 days after reinforcement. (table 19). These data show that NC-1 and NCTS-B are immunogenic in pregnant goats. The GMT for group A was numerically greater than for groups B and C, promoting improved replication in the host of strain NC-1 compared to that of the attenuated NCTS-8 strain. Table 20 shows the capacity of a vaccine composed of live attenuated tachyzoites from Neospora to protect female dwarf goats against Neospora-induced abortion. The four female goats vaccinated with the live strain in NC-1 (A) experienced abortion after stimulation pr-ovocada with NC-1 (protection elel 0%). Five of 6 female goats that were vaccinated in a simulated manner (D) experienced abortion after stimulation with NC-1 (17% protection). In contrast, only 2 out of 5 female goats vaccines with NCTS-8 (B) aborted after stimulation with NC-1 (60% protection) and only 2 out of 4 female goats vaccinated with NCIS-8 with adjuvant (C) aborted after the stimulation provoked with NC-1 (50% protection). The results show that pregnant female dwarf goats are substantially protected against abortion induced by Neospora by vaccination with live attenuated tachyzoic from the NCT5-8 strain of N. caninurn. This is the first explanation of the protection of a pregnant mammal against abortion induced by Neospora by vaccination with attenuated live tachyzoites exerted from a pathogenic neospore strain.

TABLE 19 Reverse of the mediageometric antibody (GMT) antibody concentration of goats 10 days after reinforcement TABLE 20 Protection provided by the live attenuated vaccine against fetal abortion induced by N. caninum in dwarf goats h2 Deposit of biological materials The following materials were deposited in the America Type Culture Collection (ATCC) at 12301 Parklawn Drive, Roc ville, MD. 20852, USA on November 6, 1996 and the following access numbers were assigned: 1.- Neospora caninurn strain NC-1 in MARC145 axis kidney cells, ATCC accession number CRL-12231 2.- NCTS-8 strain of Neospora caninurn in monkey kidney axle cells MARC145, ATCC Accession NRL CRL-12230 All patents, patent applications and publications cited above are incorporated in the presence as a reference in their entirety. The scope of the present invention is not limited to the specific embodiments described, which are intended to be simple illustrations of individual aspects of the invention, functionally equivalent compositions and methods are within the scope of the invention, in fact, various modifications of the invention , in addition to those shown and described herein, will be apparent to experts in the field of microbiology, parasitology, molecular biology, veterinary medicine and fields related to the foregoing description. It is intended that such modifications be within the scope of the appended claims. Having described the invention as above, the contents of the following are declared as property

Claims (31)

NOVELTY OF THE INVENTION CLAIMS

1. - A culture of strains of a strain derived from a pathogenic progenitor strain of a Neospora species, cells with attenuated pathogenicity purchased with the axis of the parent strain but which are capable of triggering a mmunological response that protects a mammal against neosporosis when administered as a live vaccine.

2. The culture according to claim 1, whose cells are sensitive to temperature.

3. The culture according to claim 1, wherein the species of the progemtora strain is N. caninurn.

4. The culture according to claim 3, wherein the progenitor strain of N. caninum is NC-1, which is present in kidney cells of rnono MARC145 with accession number ATCC CRL-12231.

The culture according to claim 4, wherein the attenuated axis of the cell strain is NCTS-8, which is present in axis cells kidney nono MARC145 with the access axis number ATCC CRL-12230.

6. A vaccine to protect a mammal against neosporos, which comprises an immunologically effective cantilev of living cells axis a strain derived from a pathogenic progenitor strain of a Neospora axis species, cells that show attenuated pathogenicity compared to the ee strain progenitor but that are capable of triggering an immunological response that protects the mammal against neosporosi when administered as a live vaccine, and a veterinarily acceptable vehicle.

7. The vaccine according to claim 6, wherein the attenuated cells are sensitive to temperature.

8. The vaccine according to claim 6, wherein the axis species of the progenitor strain is N. caninurn.

9. The vaccine according to claim 8, wherein the N. caninum axis progenitor strain is NC-1, which is present in MARC145 monkey kidney cells with accession number ATCC CRL-12231.

10. The vaccine according to claim 9, wherein the attenuated cell strain is NCTS-8, which is present in MARC145 nono kidney cells with accession number ATCC CRL-12230.

11. The vaccine according to claim 6, further comprising an adjuvant.

12. The vaccine according to claim 11, wherein the adjuvant is an oil-in-water emulsion.

13. A method for the preparation of a culture of attenuated cells of a Neospora species for use in a vaccine that protects a mammal against neosporosis, which comprises modifying cells of a pathogenic progenitor strain of a Neospora species; select and propagate by cloning one or more attenuated cells that are capable of eliciting an immune response that protects the mammal against neosporosis when administered in a live vaccine.

14. The method according to claim 13, wherein the cells of attenuated culture are sensitive to temperature.

15. The method according to claim 13, wherein the species of the parent strain is N. caninum.

16. The method according to claim 15, wherein the progenitor strain of N. caninurn is NC-1, which is present in monkey kidney cells MARC145 with accession number ATCC CRL-12231.

17. A method for the preparation of a vaccine to protect a mammal against neosporosis, which comprises modifying cells of a pathogenic progenitor strain of a Neospora species; selecting and propagating by cloning those modified cells that show attenuated pathogenicity compared to cells of the parent strain but that are capable of eliciting an immune response in the mammal that protects against neosporosis when administered in a live vaccine; and combining an immunologically effective amount of the attenuated cells with a veterinarily acceptable carrier in a form suitable for administration as a live vaccine to the mammal.

18. The method according to claim 17, wherein the attenuated cells are sensitive to temperature.

19. - The method according to claim 17, in which the species of the progenitor strain is N. caninum.

20. The method according to claim 19, wherein the progenitor strain of N. camnurn is NC-1, which is present in kidney cells of rnono MARC145 with accession number ATCC CRL-12231.

21. The method according to claim 20, wherein the strain of attenuated cells is NCTS-8, which is present in kidney cells of rnono MARC145 with accession number ATCC CRL -12230.

22. The method according to claim 17, further comprising the addition of an adjuvant to the vaccine.

23. The process according to claim 22, wherein the adjuvant is an oil-in-water emulsion.

24. A method for vaccinating a mammal against neosporosis, which comprises administering to the mammal a uniquely effective amount of a vaccine composed of living cells of a strain derived from a pathogenic progenitor strain of a Neospora species, cells that show Attenuated pathogenesis compared to those of the progenitor strain but which are capable of triggering an immunological response that protects the mammal against neosporosis when administered as a vaccine, and an acceptable veterinary vehicle.

25. The method according to claim 24, wherein the attenuated cells are sensitive to temperature.

26. - The method according to claim 24, wherein the species of the progeny strain is N. caninurn.

27. The method according to claim 26, wherein the parent strain of N. canmurn is NC-1, which is present in kidney cells of rnono MARC145 with accession number ATCC CRL-12231.

28. The method according to claim 27, wherein the strain of attenuated cells is NCTS-8, which is present in kidney cells of rnono MARC145 with accession number ATCC CRL-L2230.

29. The method according to claim 24, wherein the vaccine further comprises an adjuvant.

30. The process according to claim 29, wherein the adjuvant is an oil-in-water emulsion.

31. The method according to claim 24, wherein the animal is selected from the group consisting of dogs, cows, goats, sheep and horses. 32.- A combination vaccine, comprising an immunologically effective amount of living cells of a strain derived from a pathogenic progenitor strain, a Neospora species, cells that show attenuated pathogenicity compared to those of the progenitor strain but that are capable of triggering a unique immune response that protects the mammal against neosporosis when administered as a live vaccine; one or more different antigens that trigger an unrunological response that protects the mammal against a disease or pathological disorder; and a veterinarily acceptable vehicle.