US20150165008A1 - Use of attenuated strains of parasites for the prevention or treatment of pathologies associated with an apicomplexan - Google Patents

Use of attenuated strains of parasites for the prevention or treatment of pathologies associated with an apicomplexan Download PDF

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US20150165008A1
US20150165008A1 US14/418,984 US201314418984A US2015165008A1 US 20150165008 A1 US20150165008 A1 US 20150165008A1 US 201314418984 A US201314418984 A US 201314418984A US 2015165008 A1 US2015165008 A1 US 2015165008A1
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neospora
toxoplasma
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Audrey Gnahoui-David
Fabrice Laurent
Marie-Noelle Mevelec
Edouard Seche
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VITAMFERO
Institut National de la Recherche Agronomique INRA
Universite de Tours
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VITAMFERO
Institut National de la Recherche Agronomique INRA
Universite Francois Rabelais de Tours
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/012—Coccidia antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/522—Bacterial cells; Fungal cells; Protozoal cells avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the use of attenuated strains of parasites for preventing or treating pathologies associated with an apicomplexan.
  • the Apicomplexa are predominantly obligate intracellular parasites that have a life cycle that may involve several hosts.
  • the phylum of these parasites is subdivided into several families.
  • Toxoplasma gondii ( T. gondii ) belongs to the Sarcocystidae family. This protozoon exists in three infectious forms which vary depending on the host and the infectious stage:
  • the strains of T. gondii are classified according to their degree of virulence in vivo: the type I strains (i.e. the strain RH) are highly virulent whereas the type II strains (i.e. the strains ME49, 76K or Pru) and type III strains (i.e. the strains CEP or M7741) are less virulent and generally establish chronic infections.
  • type I strains i.e. the strain RH
  • type II strains i.e. the strains ME49, 76K or Pru
  • type III strains i.e. the strains CEP or M7741
  • numerous atypical strains not assignable to the first three types have been identified, in particular in Africa and in South America (Howe D K et al., 1995 , J. Infect. Dis ., 171, 1561-1566; Rajendran C et al., 2011 , Infect. Genet. Evol ., 12, 359-368; Mercier A et al.
  • Toxoplasma gondii the parasite responsible for toxoplasmosis
  • This strain designated Toxo mic1-3 KO, generated a strong and specific immune response against Toxoplasma gondii and makes it possible to prevent the effects of subsequent infection in the mouse (Isma ⁇ l et al., 2005 , J. Infect.
  • Neospora caninum is an intracellular parasite, responsible for neosporosis. It also belongs to the Sarcocystidae family. The life cycle of Neospora caninum is very similar to that of T. gondii with two distinct phases: a sexual phase in the final host (i.e. the canids and the dog in particular) which leads to the production of oocysts, containing sporozoites, which are eliminated in the faeces, and an asexual phase in an intermediate host (i.e. ovines, caprins, bovines, equines, etc.), which leads to the production of tachyzoites and then cysts containing the bradyzoites.
  • a sexual phase in the final host
  • oocysts containing sporozoites, which are eliminated in the faeces
  • an asexual phase in an intermediate host i.e. ovines, caprins, bovines, equi
  • Neospora caninum an attenuated live strain of Neospora caninum was obtained, the strain Neo ncmic1-3 KO, in which the ncmic1 and ncmic3 genes were knocked out by homologous recombination.
  • the ncmic3 gene is replaced by a DHFR cassette, which confers resistance to pyrimethamine
  • the ncmic1 gene is replaced by a CAT-GFP cassette, which endows the parasite with resistance to chloramphenicol and makes the parasite fluorescent.
  • the parasite no longer expresses the NcMIC1 and NcMIC3 proteins. It has been shown that this mutant strain has infectious and immunogenic properties, endowing mammals with vaccine protection against the harmful effects of neosporosis.
  • Toxoplasma gondii and Neospora caninum have in common a specific process of invasion of the host cells in several steps leading to the formation of a parasitophorous vacuole in which the parasite multiplies and develops.
  • the Cryptosporidiidae constitute another family belonging to the phylum of the apicomplexans and are responsible for cryptosporidiosis, an extremely common disease which in particular affects humans and many animal species including farm animals (ovin, caprin, bovine, etc.) by the ingestion of parasites present in their food.
  • the parasite then multiplies in the intestines, firstly asexually and then, secondly, sexually.
  • the contaminated individuals excrete and disseminate new parasites, thus contaminating their environment (i.e. pastureland, water, etc.).
  • Cryptosporidiidae Several species of Cryptosporidiidae have been identified, among which Cryptosporidium parvum is one of the commonest and most virulent.
  • cryptosporidiosis is generally a benign disease.
  • the consequences of this disease the incidence of which is increasing every year, in particular in the United States, may be extremely serious in young children and immunodepressed persons, especially patients infected with the HIV virus.
  • cryptosporidiosis is responsible for severe diarrhoea that may cause significant dehydration or, without suitable treatment, even death of the individual.
  • Cryptosporidiosis In immunocompetent adult animals, cryptosporidiosis is also benign. Conversely, cryptosporidiosis generally has grave consequences in very young animals, a few days to a few weeks old, and whose immune system is immature. Thus, Cryptosporidium spp, and in particular C. parvum , has proved to be one of the commonest etiological agents of neonatal diarrhoea, which causes severe growth retardation and without suitable treatment may be fatal to the animal.
  • Neonatal diarrhoea constitutes a constant threat for breeders of bovines, ovines and caprins, and represents a considerable economic loss due to the loss of income resulting from retardation of growth, mortality of neonates, intervention of veterinarians and the costs of therapeutic treatment and rehydration.
  • the etiology of neonatal diarrhoea is often multiple, several infectious agents in fact being responsible for these symptoms, in particular rotaviruses, coronaviruses, BVDV (Bovine Viral Diarrhoea Virus), Escherichia coli and, of course, C. parvum .
  • One of the aims of the invention is to provide an immunostimulant capable of inducing a non-specific immune response in the neonate, with the production of IL-12 and IFN ⁇ .
  • Another aim of the invention is to provide an immunostimulant capable of limiting the effects of infection of mammals, human or animal, by Cryptosporidium parvum.
  • Yet another aim of the invention is to provide a prophylactic treatment, and in particular a vaccine, against cryptosporidiosis.
  • the present invention relates to strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp, isolated from their natural environment and having an immunostimulant effect, for the use thereof in the prevention or the treatment, in a mammal, of a disorder associated with an apicomplexan of the family Cryptosporidiidae.
  • the present invention relates to attenuated strains of Sarcocystidae selected from attenuated Toxoplasma spp or attenuated Neospora spp, isolated from their natural environment and having an immunostimulant effect, for the use thereof in the prevention or the treatment, in a mammal, of a pathology associated with an apicomplexan of the family Cryptosporidiidae.
  • the present invention relates to strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect, for the use thereof in the prevention or the treatment, in a mammal, of a pathology associated with an apicomplexan of the family Cryptosporidiidae, said strains having attenuated virulence relative to the wild strains that induce a pathology associated with Toxoplasma spp or Neospora spp.
  • such wild strains of apicomplexans may be illustrated by a virulent strain of T. gondii of type RH for toxoplasmosis, or a virulent strain of N. caninum of type NC1 for neosporosis.
  • the present invention relates to strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect, for the use thereof in the prevention or the treatment, in a mammal, of a pathology associated with an apicomplexan of the family Cryptosporidiidae, said strains having attenuated virulence relative to a virulent strain, i.e. a strain having virulence substantially identical to the virulence of the strain from which the strain with attenuated virulence was obtained.
  • immunostimulation is meant the ability of a strain of Toxoplasma spp or Neospora spp to induce early activation of the immune system of the host. This activation involves components of the immune system such as interferons, cytokines, phagocytic cells, NK (Natural Killer) cells, dendritic cells and the complement system, which will act in a non-specific fashion on the targeted pathogen. This immunostimulation is not based on the establishment of adaptive immunity.
  • prevention is meant prophylaxis with the aim of preventing the appearance or spreading of a disease. It is in particular a question of protecting an individual with predisposition to contracting and developing a disorder associated with an apicomplexan of the family Cryptosporidiidae. Such individuals are in particular neonates that have an immature immune system or individuals with an immune system dysfunction. It is also a question of protecting a mammal exposed to a risk of contamination from its environment.
  • treatment is meant not only inhibition of the progression of the pathology but also attenuation of the symptoms associated with this pathology.
  • the treatment has the aim of reducing the extent of the symptoms until they disappear completely, allowing the individual to recover a normal physiological state.
  • mammal human beings, certain commercial or farm animals and certain pet animals.
  • Attenuated strains of Toxoplasma spp or Neospora spp is meant strains of Toxoplasma spp or Neospora spp having an attenuated virulence, less than the virulence of the wild strains of T. gondii or of N. caninum capable of inducing a pathology, but which nevertheless conserve immunogenicity so as to be able to be used in the prevention or the treatment of a disorder associated with an apicomplexan of the family Cryptosporidiidae.
  • the attenuation of virulence may result either from a natural process of evolution of the species or may be induced in particular by techniques of molecular biology familiar to a person skilled in the art.
  • the attenuation of virulence is due to the absence of expression of virulence factors or to the expression of one or more virulence factors that are non-functional or have an altered function.
  • the in vitro modification of the genetic heritage of Toxoplasma spp or Neospora spp confers a mutant character on the strain, relative to the wild-type strain from which it is derived.
  • the wild strains of parasites not only have an immunogenic potential but are also virulent, i.e. they are capable of inducing a pathology associated with Toxoplasma spp or Neospora spp (i.e.
  • toxoplasmosis and neosporosis respectively
  • the virulence of the strains of Toxoplasma spp or of Neospora spp may in particular be evaluated by in vitro cellular infectivity tests or by infectivity tests in animals.
  • the cellular infectivity tests are carried out by depositing tachyzoites on confluent cells, for example HFF (Human Foreskin Fibroblast) cells or Vero cells, cell lines frequently used for the production of tachyzoites of T. gondii or of N. caninum .
  • HFF Human Foreskin Fibroblast
  • Vero cells cell lines frequently used for the production of tachyzoites of T. gondii or of N. caninum .
  • the number of vacuoles formed and the number of parasites in each vacuole is determined by microscopic observation.
  • the various strains are injected into the animals and the survival of these animals is monitored over time (Cérborne et al., 2005).
  • pathologies associated with an apicomplexan of the family Cryptosporidiidae is meant the diseases resulting from an infection by a protozoon belonging to the phylum of the apicomplexans, and in particular parasites belonging to the family Cryptosporidiidae, which comprises the genus Cryptosporidium .
  • Several tens of species of Cryptosporidium are referenced (Fayer R, 2010 , Exp. Parasitol ., 124, 90-7). These parasites are capable of invading the mucosal epithelia.
  • said mammal in the use according to the present invention of the strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect, said mammal is a neonate.
  • the present invention relates to strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp, isolated from their natural environment and having an immunostimulant effect, for use in the prevention or the treatment, in a neonate mammal, of a pathology associated with an apicomplexan of the family Cryptosporidiidae.
  • Neospora spp a mammal from the time of its birth until it is weaned, i.e. until the moment when the mammal becomes capable of feeding itself and is no longer dependent on its mother's milk.
  • the main advantage of using the mutant strain of Toxoplasma spp or Neospora spp for the prevention and/or the treatment of pathologies associated with an apicomplexan of the family Cryptosporidiidae in the neonate is stimulation of its immature immune system in order to induce a non-specific immune response by synthesizing molecules that inhibit the growth of the Apicomplexa.
  • said mammal in the use according to the present invention of the strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect, said mammal is a human being or an animal.
  • the animals to which the present invention relates are mainly commercial or farm animals, which are of interest to the agricultural and food industries, but also certain pet animals.
  • said animal belongs to the group comprising or constituted by of ovines, caprids, porcines, bovines, equines, camelids, canids or felids (Fayer, 2004 , Vet. Parasitol ., 126, 37-59).
  • said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect said strains of Toxoplasma spp or of Neospora spp have at least an adhesin MIC1 and/or an adhesin MIC3 inactivated by a genetic modification relating to at least one of the genes mid and/or mic3.
  • an adhesin MIC1 and/or an adhesin MIC3 is meant the proteins of the micronemes, also called adhesins, MIC1 and/or MIC3 which play a role in the mobility, the migration or the invasion of parasites of the phylum Apicomplexa in its host. These proteins have linkage modules that allow them to bind to the target cells of the host.
  • an inactivated adhesin is meant an adhesin the function of which can no longer be ensured within the cell.
  • An adhesin is inactivated when it is not produced or when it is produced but does not have functional activity or has reduced functional activity.
  • the inactivation also relates to an adhesin that can no longer bind to other proteins in order to form a complex.
  • genetic modification is meant any mutation made in the nucleic acid sequence of a gene leading to the absence of expression of the protein encoded by that gene or leading to expression of a non-functional or less functional form of the protein encoded by that gene. This operation requires human intervention when it is carried out in vitro.
  • This mutation may consist of the deletion of all or part of the gene, or of its coding region, or of its promoter region, and of the insertion or the substitution of nucleotides in the nucleotide sequence of the gene.
  • mic1 gene is meant the gene coding for the protein of the micronemes MIC1, also called adhesin MIC1. This protein contains several modules, including binding domains that bind lactose specifically. The protein MIC1 is also capable of binding to the surface of the host cells.
  • mic3 gene is meant the gene coding for the protein of the micronemes MIC-3, also called adhesin MIC3. This protein homodimerizes in order to form a complex of 90 kDa.
  • MIC3 comprises domains of the EGF type and a domain of the lectin type. The protein MIC3 is also capable of binding to the surface of the host cells.
  • said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect said strains of Toxoplasma spp or of Neospora spp have the two adhesins MIC1 and MIC3 inactivated by a genetic modification relating to the two genes mid and mic3.
  • mutant strains of Toxoplasma spp or of Neospora spp are called Toxo mic1-3 KO or Neo ncmic1-3 KO respectively and have a very attenuated virulence in comparison with the wild strains of T. gondii of type RH or of N. caninum of type NC1 from which they are derived.
  • Toxo mic1-3 KO or Neo ncmic1-3 KO strains retain a strong immunogenicity.
  • the detailed construction of the Toxo mic1-3 KO strain is described in documents U.S. Pat. No. 7,946,185 B2 and EP 1 703 914 B1.
  • the detailed construction of the Neo ncmic1-3 KO strain is described in the present application.
  • the Toxo mic1-3 KO and Neo ncmic1-3 KO strains retained their capacity for colonizing the target tissues without the development of a pathogenic effect following administration of said strains to a mammal.
  • the knock out of the genes mid and mic3 has little or no effect on the immunogenic potential of these strains, but reduces their virulence considerably relative to a virulent strain, i.e. a strain having a virulence substantially identical to the virulence of the strain from which the strain with attenuated virulence was obtained.
  • the inoculation of the Toxo mic1-3 KO strain in neonate mice leads to the production of cytokines IL-12 and IFN- ⁇ and effectively protects the mouse pups from subsequent infection with Cryptosporidium parvum.
  • the mutant Toxo mic1-3 KO strain effectively stimulates the production of IL-12 and IFN- ⁇ from cells of the mesenteric lymph nodes and splenocytes of lambs, confirming the possibility of using this mutant strain as an immunostimulant in this target animal species.
  • said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect are respectively Toxoplasma gondii or Neospora caninum.
  • said immunostimulant effect of said strains leads to the secretion of interleukin-12 (IL-12) and then of interferon- ⁇ (IFN- ⁇ ).
  • IL-12 interleukin-12
  • IFN- ⁇ interferon- ⁇
  • IL-12 interleukin-12
  • monocytes the monocytes
  • dendritic cells the dendritic cells
  • macrophages the cytokine synthesized by immune system cells such as the monocytes, the dendritic cells and the macrophages.
  • the cytokine is secreted early and will thus activate the target cells (T cells and Natural Killer cells) so that the latter secrete IFN- ⁇ in their turn.
  • IFN- ⁇ interferon- ⁇
  • NK Natural Killer
  • the secretion of IFN- ⁇ by the cells of the organism will allow production of an innate response protective against C. parvum .
  • This cytokine will have a pleiotropic activity owing to the diversity of the targeted cell lines.
  • cytokines is meant all the molecules involved in the development and the regulation of the immune system.
  • the cytokines are glycosylated or non-glycosylated proteins, which may be classified according to their biological activity:
  • said secretion of interleukin-12 (IL-12) and of interferon- ⁇ (IFN- ⁇ ) begins between 3 and 9 days after using said strains of Toxoplasma spp or of Neospora spp as immunostimulant.
  • said pathology associated with an apicomplexan of the family Cryptosporidiidae is cryptosporidiosis.
  • cryptosporidiosis is meant the pathology characterized by symptoms such as cramps, fever, fatigue, nausea and especially diarrhoea, which may lead to the dehydration of the mammal infected by Cryptosporidium parvum or by another Cryptosporidium species.
  • Cryptosporidiosis mainly affects the intestines of mammals but may also infect the biliary and pancreatic tracts and the respiratory tract in an immunodepressed individual.
  • the infestation occurs by ingestion of oocysts present in the excrement of parasitized animals with which the environment is contaminated (water, earth or raw food). The symptoms appear two to ten days after the infection occurred and persist for more than two weeks.
  • This pathology varies depending on the age of the infected host but more particularly on the state of its immune system.
  • cryptosporidiosis has no effect on the health of the host.
  • a host that is very young, very old or has an immature or deficient immune system may develop very severe forms of cryptosporidiosis.
  • immaturity or deficient immune system is meant the immune system for which one or more cell lines are either absent, or deficient.
  • the immaturity of the immune system is a common trait in neonate mammals, which makes them particularly vulnerable to parasitic infections.
  • the absorption of antibodies from the mother via the colostrum endows the neonate with a certain degree of protection while its immune system is being established.
  • the immaturity or the deficiency of the immune system may also result from pathologies of a genetic origin, in particular in humans, such as Wiskott-Aldrich syndrome, an X-linked lymphoproliferative syndrome.
  • This deficiency may also arise after an infection by the human immunodeficiency virus (HIV) or following a therapeutic treatment associated with an organ graft or the medical management of certain cancers by chemo- or radiotherapy.
  • HIV human immunodeficiency virus
  • the mammals that have an immature or deficient immune system are particularly vulnerable to infections with apicomplexans.
  • said apicomplexan of the family Cryptosporidiidae responsible for cryptosporidiosis is at least one apicomplexan selected from the group constituted by Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium andersoni, Cryptosporidium ryanae, Cryptosporidium muris, Cryptosporidium ubiquitum, Cryptosporidium hominis, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium baileyi, Cryptosporidium meleagridis or Cryptosporidium xiaoi.
  • Cryptosporidium parvum Cryptosporidium bovis, Cryptosporidium andersoni, Cryptosporidium ryanae, Cryptosporidium muris, Cryptosporidium ubiquitum, Cryptosporidium hominis, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium baileyi, Cryptosporidium meleagridis or Cryptosporidium xiaoi ” is meant the protozoa of the phylum Apicomplexa capable of causing intestinal pathologies of a hydric nature in the majority of mammals.
  • These parasites are in particular capable of causing cryptosporidiosis in ovines, caprins, porcines, bovines, equines, camels, camelids, canids, felids or humans (Payer, 2004 , Vet. Parasitol ., 126, 37-59) that have an immature or deficient immune system.
  • the present invention also relates to the strains of Toxoplasma gondii or of Neospora caninum that have the two adhesins MIC1 and MIC3 inactivated by a genetic modification relating to the two mid and mica genes for the use thereof in the prevention or the treatment, in a mammal, of a pathology associated with an apicomplexan of the family Cryptosporidiidae, said pathology associated with an apicomplexan of the family Cryptosporidiidae being cryptosporidiosis.
  • the present invention also relates to the strains of Toxoplasma gondii or of Neospora caninum isolated from their natural environment and having an immunostimulant effect, for use in the prevention or the treatment, in a mammal, of a pathology associated with an apicomplexan of the family Cryptosporidiidae, said strains of Toxoplasma gondii or of Neospora caninum having the two adhesins MIC-1 and MIC-3 inactivated by a genetic modification relating to the two mic-1 and mic-3 genes, and said pathology associated with an apicomplexan of the family Cryptosporidiidae is cryptosporidiosis.
  • the invention also relates to the strains of Toxoplasma gondii or of Neospora caninum isolated from their natural environment and having an immunostimulant effect, said strains of Toxoplasma gondii or of Neospora caninum having the two adhesins MIC1 and MIC3 inactivated by a genetic modification relating to the two mic1 and mic3 genes for the use thereof in the prevention or the treatment, in a mammal, of a pathology associated with an apicomplexan of the family Cryptosporidiidae, said strains of Toxoplasma gondii or of Neospora caninum being administered to said mammal at a rate from 20 to 10 9 tachyzoites.
  • tachyzoite is meant the rapidly multiplying form of Toxoplasma gondii or of Neospora caninum .
  • the tachyzoite has a crescent shape and a variable size of 5-8 ⁇ 2-3 ⁇ m.
  • the apical part of the parasite comprises conoids which participate in the penetration of the parasite into the host cell.
  • the micronemes, the rhoptries and the dense granules constitute the three major organelles of the tachyzoite, which also comprises a nucleus, an apicoplast, a Golgi apparatus, an endoplasmic reticulum and an organite similar to the mitochondrion.
  • an effective dose of tachyzoites for the prophylactic treatment of mammals makes it possible to limit the infection or the transmission of the pathogenic agent responsible for cryptosporidiosis.
  • Such a treatment may be adapted and/or repeated as many times as necessary by a person skilled in the art, depending on the age and immunological status of the mammal.
  • the tachyzoites may be brought into contact with the mammal not only on a mammal presenting the symptoms characteristic of cryptosporidiosis but also on a mammal without any of the symptoms of cryptosporidiosis but which is in contact with other mammals infected by Cryptosporidium parvum or by another species of Cryptosporidium able to induce cryptosporidiosis.
  • the strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect for the use thereof according to the present invention are in a galenic form selected from the group comprising or constituted by liquid suspensions, solid or liquid dispersions, powders, pastes or lyophilizates.
  • the galenic form is adapted by a person skilled in the art depending on the method of administration selected. All the conventional methods of administration may be envisaged: by parenteral route (intravenous, subcutaneous, intradermal, intramuscular, intraperitoneal, and intranasal) or by enteral route.
  • the strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp isolated from their natural environment and having an immunostimulant effect for the use thereof according to the present invention are associated with at least one other antigen, or at least one adjuvant, or at least one stabilizer, or at least one preservative or a mixture of at least two of said products for increasing the immune response of said mammal.
  • antigen any natural or recombinant protein, in its native or mutated form, originating from a parasite or from a pathogen other than Toxoplasma spp or Neospora spp capable of inducing a cellular or humoral immune response in a mammal.
  • the aim of combining the mutant strain of Toxoplasma spp or Neospora spp with such an antigen is to amplify the mammal's immune response and thus endow it with better protection against an apicomplexan infection.
  • adjuvant any substance capable of reinforcing and prolonging the immune response directed against the targeted antigen.
  • the mechanism involved in order to make the immune response more effective is dependent on the adjuvant used.
  • the adjuvants are substances which are well known to a person skilled in the art and in particular include aluminium salts, squalene, saponins, the bacterial constituents or toxins, or also certain proteins (peptone, albumin, casein).
  • stabilizers or preservatives is meant the compounds allowing perfect preservation of the strains of Toxoplasma spp or Neospora spp in their packaging.
  • Stabilizers or preservatives are substances which are well known to a person skilled in the art and in particular include the carbohydrates (sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose), the polar organic solvents, such as DMSO (dimethylsulphoxide), and the polysorbates.
  • carbohydrates sorbitol, mannitol, lactose, sucrose, glucose, dextran, trehalose
  • polar organic solvents such as DMSO (dimethylsulphoxide)
  • DMSO dimethylsulphoxide
  • IL-12 interleukin-12
  • IFN- ⁇ interferon ⁇
  • the present invention also relates to a method of inducing an immune response in a mammal comprising a step of administration, to said mammal, of tachyzoites of strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp, isolated from their natural environment and having an immunostimulant effect, allowing an immune response to be induced.
  • the present invention also relates to a method of inducing an immune response in a neonate mammal comprising a step of administration, to said neonate mammal, of tachyzoites of strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp, isolated from their natural environment and having an immunostimulant effect, allowing an immune response to be induced.
  • immune response is meant the two essential phases constituted by the recognition of the antigen and the reaction intended to eliminate said antigen.
  • an antigen enters the body for the first time, it is the non-specific immune response that is activated. It uses non-clonal mechanisms, since it does not require specific cellular clones.
  • the inflammatory reaction created by this non-specific response will lead to adaptive immunity. This response involves cells specifically recognizing the antigen and will then lead to clonal expansion. This clonal expansion will result in a very effective immune response and a memory response (specific immune response following the second entry of the antigen into the organism).
  • said mammal is a neonate.
  • said mammal is a human being or an animal.
  • the animal belongs to the group comprising or constituted by ovines, caprins, porcines, bovines, equines, camelids, canids or felids.
  • said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp have at least one adhesin MIC1 and/or one adhesin MIC3 inactivated by a genetic modification which relates to at least one of the mic1 and/or mic3 genes.
  • said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp have the two adhesins MIC1 and MIC3 inactivated by a genetic modification which relates to the two mid and mic3 genes.
  • said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp are respectively Toxoplasma gondii or Neospora caninum.
  • the strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp induce a non-specific immune response in particular characterized by the secretion of IL-12 and/or IFN- ⁇ .
  • the present invention also relates to a method for protecting a mammal against a parasitosis associated with an apicomplexan of the family Cryptosporidiidae comprising a step of administration, to said mammal, tachyzoites of strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp, isolated from their natural environment and having an immunostimulant effect, for inducing protection against said parasitosis.
  • the mammal is a neonate.
  • the mammal in the method according to the present invention, is a human being or an animal.
  • the animal belongs to the group comprising or constituted by ovines, caprins, porcines, bovines, equines, camelids, canids or felids.
  • said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp have at least one adhesin MIC1 and/or one adhesin MIC3 inactivated by a genetic modification which relates to at least one of the mic1 and/or mic3 genes.
  • said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp have the two adhesins MIC1 and MIC3 inactivated by a genetic modification which relates to the two mid and mic3 genes.
  • said parasitosis associated with an apicomplexan of the family Cryptosporidiidae is cryptosporidiosis.
  • the apicomplexan of the family Cryptosporidiidae responsible for the cryptosporidiosis is at least one apicomplexan selected from the group constituted by Cryptosporidium parvum, Cryptosporidium bovis, Cryptosporidium andersoni, Cryptosporidium ryanae, Cryptosporidium muris, Cryptosporidium ubiquitum, Cryptosporidium hominis, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium baileyi, Cryptosporidium meleagridis or Cryptosporidium xiaoi.
  • the administration, to said mammal, of said tachyzoites of said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp is carried out by enteral route or by parenteral route.
  • the administration, to said mammal, of said tachyzoites of said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp is carried out before exposure of said mammal to the apicomplexan of the family Cryptosporidiidae responsible for cryptosporidiosis.
  • the administration, to said mammal, of said tachyzoites of said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp is carried out during exposure of said mammal to the apicomplexan of the family Cryptosporidiidae responsible for cryptosporidiosis.
  • the administration, to said mammal, of said tachyzoites of said strains of Sarcocystidae selected from Toxoplasma spp or Neospora spp is carried out after exposure of said mammal to the apicomplexan of the family Cryptosporidiidae responsible for cryptosporidiosis.
  • said mammal may be a neonate.
  • FIG. 1 this figure illustrates the 2 steps of homologous recombination in order to obtain the strain Neo ncmic1-3 KO.
  • the first step of homologous recombination allows integration of the gene coding for the enzyme dihydrofolate reductase (DHFR) at the locus of the ncmic3 gene. Selection with pyrimethamine makes it possible to amplify the mutant single strain Neo ncmic3 KO.
  • DHFR dihydrofolate reductase
  • Neo ncmic3 KO strain thus obtained serves for the second step of homologous recombination, which allows the integration of the gene coding for the chimeric protein chloramphenicol-acetyl-transferase/green fluorescent protein (CAT-GFP) at the locus of the ncmic1 gene. Selection with chloramphenicol then makes it possible to amplify the double mutant strain Neo ncmic1-3 KO.
  • CAT-GFP chloramphenicol-acetyl-transferase/green fluorescent protein
  • FIG. 2-A this figure is a schematic representation of the pNcMic3KO-DHFR plasmid.
  • This plasmid with 11,312 base pairs comprises the DHFR selection gene flanked by the homologous regions (5HR-NcMic3 and 3HR-NcMic3) of the sequences flanking the ncmic3 gene, the ampicillin resistance gene (Amp) as well as the Not I restriction site which permits its linearization.
  • FIG. 2-B this figure is a schematic representation of the pNcMic1KO-CAT-GFP plasmid.
  • This plasmid with 10,069 base pairs comprises the CAT-GFP selection gene flanked by the homologous regions (3HR-NcMic1 and 5HR-NcMic1) of the sequences flanking the ncmic1 gene, the ampicillin resistance gene (Amp) as well as the Kpn I restriction site which permits its linearization.
  • FIG. 3-A shows the electrophoretic profiles of the PCR products obtained respectively in the wild-type strain NC1 of N. caninum and in the mutant strain Neo ncmic3 KO, using the sets of PCR primers No. 1, No. 2 or No. 3 in Table II which correspond to SEQ ID NO: 5 to SEQ ID NO: 10.
  • FIG. 3-B shows the electrophoretic profiles of the PCR products obtained respectively in the wild-type strain NC1 of N. caninum and in the mutant strain Neo ncmic3 KO, using the sets of PCR primers No. 4, No. 5, No. 6 or No. 7 in Table II which correspond to SEQ ID NO: 11 to SEQ ID NO: 16.
  • FIG. 4-A this figure illustrates the analysis for detecting the NcMIC3 protein in the wild-type strain NC1 of N. caninum by immunofluorescence, using an antibody specifically recognizing the NcMIC3 protein.
  • One and the same microscopic field is visualized in direct light (image A) or in fluorescence (image B).
  • FIG. 4-B this figure illustrates the analysis for detecting the NcMIC3 protein in the mutant strain of N. caninum Neo ncmic3 KO by immunofluorescence, using an antibody specifically directed against the NcMIC3 protein.
  • One and the same microscopic field is visualized in direct light (image A) or in fluorescence (image B).
  • FIG. 5 shows the electrophoretic profiles of the PCR products obtained respectively in the wild-type strain NC1 of N. caninum , in the mutant strain Neo ncmic3 KO and in the mutant strain Neo ncmic1-3 KO using the sets of PCR primers No. 1 to No. 12 in Table VII which correspond to SEQ ID NO: 7 to 16 and to SEQ ID NO: 21 to 30.
  • FIG. 6 this figure illustrates the analysis for detecting the GFP protein in the mutant strains Neo ncmic3 KO (images A and B) and Neo ncmic1-3 KO (image C and D) by immunofluorescence, using the fluorescent properties of the CAT-GFP protein.
  • One and the same microscopic field is visualized in direct light (top images A and C) or in fluorescence (bottom images B and D).
  • FIG. 7 illustrates the assay of antibodies of type IgM anti- Toxoplasma gondii in mouse pup serum.
  • the assay of antibodies of type IgM anti- Toxoplasma gondii in the serum was carried out 3 days (empty grey squares) or 9 days (filled black squares) after the mouse pups had received 20 tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route.
  • the mouse pups that had not received any tachyzoite of the strain Toxo mic1-3 KO served as controls.
  • FIG. 8-A illustrates the measurement of the expression of interferon-gamma (IFN- ⁇ ) in the ileum of the mouse pup.
  • IFN- ⁇ interferon-gamma
  • the expression level of the gene coding for IFN- ⁇ was measured by quantitative PCR on the total RNAs extracted from a fragment of ileum using specific primers (SEQ ID NO: 33 to 34), 9 days after the mouse pups had received 20 tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route (filled black squares). The mouse pups that had not received any tachyzoites of the strain Toxo mic1-3 KO served as controls (empty grey squares).
  • FIG. 8-B illustrates the measurement of the expression of interleukin-12 (p40 subunit) (IL-12p40) in the mouse pup ileum.
  • the expression level of the gene coding for IL-12p40 was measured by quantitative PCR on the total RNAs extracted from a fragment of ileum using specific primers (SEQ ID NO: 31 to 32), 9 days after the mouse pups received 20 tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route (filled black squares).
  • the mouse pups that had not received any tachyzoites of the strain Toxo mic1-3 KO served as controls (empty grey squares):
  • FIG. 9 illustrates the detection of the presence of tachyzoites of Toxo mic1-3 KO in the intestine of the mouse pup.
  • the detection of the presence of tachyzoites of Toxo mic1-3 KO was carried out by immunohistology on sections of the intestine 9 days after the mouse pups received 20 tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route Immunolabelling is carried out using a rabbit anti-SAG-1 polyclonal antibody and an anti-rabbit secondary antibody coupled to fluorescein isothiocyanate.
  • the detection of tachyzoites of Toxoplasma gondii mic1-3 KO is shown for the intestines of the mouse pups No. 4 (B) and No. 6 (D).
  • the tachyzoites appear in the form of white dots in the intestinal muscles of the mouse pup No. 4 (B).
  • nuclei of the cells of the intestinal villi and intestinal muscles of the mouse pups No. 4 (C) and No. 6 (E) are labelled with Hoechst and appear in the form of white dots.
  • FIG. 10 illustrates the presence of Toxoplasma gondii via the expression of the surface antigen SAG-1 in the ileum of the mouse pup.
  • the cDNAs obtained from the total RNAs extracted from a fragment of ileum served as a matrix for the amplification of SAG-1 by PCR using specific primers (SEQ ID NO: 37 to 38).
  • the mouse pups were sacrificed three days (T1) or 9 days (T2) after receiving 20 tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route (V).
  • the mouse pups that had not received any tachyzoite of the strain Toxo mic1-3 KO served as controls (NV).
  • the PCR products were deposited on agarose gel.
  • SAG-1 was also amplified from the genomic DNA of the wild-type strain of Toxoplasma gondii (RH) and from the genomic DNA of the mutated strain of Toxo mic1-3 KO (KO).
  • the amplification products serve as controls of the size of the amplification product of SAG-1.
  • the mouse pups are shown by their number (1, 2, 3, 4, 5, 6) in FIG. 10 .
  • FIG. 11 illustrates the count of the oocysts of Cryptosporidium parvum in the intestine of the mouse pup.
  • the number of oocysts of Cryptosporidium parvum was counted in a Thoma cell from an extract of ground material of the intestine placed in a sugar solution.
  • the intestines originate from:
  • mice pups are sacrificed 6 days after the infection by Cryptosporidium parvum.
  • FIG. 12 illustrates the detection of the presence of oocysts of Cryptosporidium parvum in the intestine of the mouse pup.
  • the number of oocysts of Cryptosporidium parvum was counted in a Thoma cell from an extract of ground material of the intestine placed in a sugar solution.
  • the intestines originate from:
  • mice pups are sacrificed 7 days after the infection by Cryptosporidium parvum.
  • FIG. 13-A illustrates the assay of interleukin-12 (IL-12) in the mononuclear cells from the spleen of lambs and of adult sheep.
  • Samples of mononuclear cells from the spleen of lambs (grey triangles) and of adult sheep (black squares) were infected in vitro by three different strains of Toxoplasma gondii : type I wild-type strain (RH), type I mutant strain Toxo mic1-3 KO (KO) and type II wild-type strain (Pru).
  • RH type I wild-type strain
  • KO type I mutant strain Toxo mic1-3 KO
  • Pru type II wild-type strain
  • Mononuclear cells from the spleen of lambs and of adult sheep not infected in vitro serve as controls (M).
  • FIG. 13-B illustrates the assay of interleukin-12 (IL-12) in the mononuclear cells originating from the mesenteric lymph nodes of lambs and of adult sheep.
  • Samples of mononuclear cells originating from the mesenteric lymph nodes of lambs (grey triangles) and of adult sheep (black squares) were infected in vitro by three different strains of Toxoplasma gondii : type I wild-type strain (RH), type I mutant strain Toxo mic1-3 KO (KO) and type II wild-type strain (Pru).
  • RH type I wild-type strain
  • KO type I mutant strain Toxo mic1-3 KO
  • Pru type II wild-type strain
  • Mononuclear cells from the mesenteric lymph nodes of lambs and of adult sheep not infected in vitro serve as controls (M).
  • FIG. 13-C illustrates the assay of interferon gamma (IFN ⁇ ) in the mononuclear cells from the spleen of lambs and of adult sheep.
  • IFN ⁇ interferon gamma
  • FIG. 14 illustrates the change in body weight of the lambs immunostimulated with 10 6 tachyzoites Toxo mic1-3 KO, one day after their birth (batch A-black square) in comparison with control lambs not immunostimulated (batch B-grey circle).
  • FIG. 15 illustrates the production of IFN- ⁇ after restimulation with the total extract of the strain Toxo mic1-3 KO of mononuclear cells of the spleen of lambs immunostimulated with 10 6 tachyzoites Toxo mic1-3 KO, one day after their birth (lambs No. 1416-1418-1421 and 1424) or of control lambs (lambs 1428 and 1423), sacrificed 15 days after the immuno stimulation,
  • FIG. 16 illustrates the production of IFN- ⁇ produced from the ex vivo culture of cells of subiliac and popliteal lymph nodes of lambs immunostimulated with 10 6 tachyzoites Toxo mic1-3 KO, one day after their birth (lambs No. 1416-1418-1421 and 1424) or of control lambs (lambs 1428 and 1423), sacrificed 15 days after the immunostimulation.
  • FIG. 17 illustrates the survival of lambs immunostimulated with 10 6 tachyzoites Toxo mic1-3 KO, one day after their birth and challenged with 5.10 6 oocysts of C. parvum (black square) and the control lambs only challenged (grey circle).
  • the survival curves are of the Kaplan-Meier type.
  • FIG. 18 illustrates the daily weight gain of lambs immunostimulated with 10 6 tachyzoites Toxo mic1-3 KO, one day after their birth and challenged with 5.10 6 oocysts of C. parvum (black square) and the control lambs only challenged (grey diamond).
  • FIG. 19 illustrates the mean excretion of oocysts C. parvum per gram of excrement of lambs immuno stimulated with 10 6 tachyzoites Toxo mic1-3 KO, one day after their birth and challenged with 5.10 6 oocysts of C. parvum (black square) and the control lambs only challenged (grey diamond).
  • the first step of homologous recombination makes it possible to obtain a simple mutant KO (strain Neo ncmic3 KO).
  • the second step of homologous recombination is carried out in the strain Neo ncmic3 KO in order to obtain a doubly deleted strain (Neo ncmic1-3 KO) ( FIG. 1 ).
  • Neospora caninum The haploidy of the genome of Neospora caninum during the proliferative phase allows inactivation of a gene in a single homologous recombination.
  • HFF human fibroblasts
  • FCS foetal calf serum
  • FCS foetal calf serum
  • the plasmid pNcMic3KO-DHFR contains the DHFR (dihydrofolate reductase) selection gene, which confers resistance to pyrimethamine.
  • the DHFR selection gene is placed under the control of the ⁇ -tubulin promoter of Toxoplasma gondii (aTUB5 promoter) to allow expression of the gene in the parasite. The efficacy of this heterologous promoter had been demonstrated beforehand in N. caninum .
  • This cassette is framed by the homologous regions (5HR-NcMic3 and 3HR-NcMic3) of the sequences flanking the ncmic3 gene.
  • the DHFR selection cassette makes it possible to carry out selection for pyrimethamine.
  • the 5′UTR region of the ncmic3 gene was amplified by PCR from the genomic DNA of the strain NC1 of Neospora caninum .
  • the primers 5 HR NCmic3 F KpnI and 5 HR NCmic3 R ClaI allow amplification of the 5′UTR region of the ncmic3 gene and creation of two restriction sites, which were used for cloning the 5HR fragment upstream of the DHFR selection cassette into the plasmid pT230 DHFR (KpnI at 5′ and ClaI at 3′ of the PCR fragment).
  • the 3′UTR region of the ncmic3 gene was amplified by PCR from the genomic DNA of the strain NC1 of Neospora caninum .
  • the primers 3 HR NCmic3 F XbaI and 3 HR NCmic3 R NotI allow amplification of the 3′UTR region of the ncmic3 gene and creation of two restriction sites, which were used for cloning the 3HR fragment downstream of the DHFR selection cassette in the plasmid pT230 5HR-NcMic3-DHFR (XbaI at 5′ and NotI at 3′ of the PCR fragment).
  • the sequences of the primers are shown in Table I below.
  • plasmid pNcMic3KO-DHFR 50 ⁇ g of the plasmid pNcMic3KO-DHFR, purified and then linearized by NotI, was added to 5 ⁇ 10 7 NC1 tachyzoites of Neospora caninum suspended in the CYTOMIX electroporation medium containing ATP (3 mM) and glutathione (3 mM) (Van den Hoff et al., Nucleic Acid Research , June 11; 20(11): 2902), and electroporation was carried out in a cuvette with a 4 mm gap, in a volume of 800 ⁇ L on a BioRad apparatus (parameters: 2000 V, 50 ohms, 25 ⁇ F, with two electric shocks).
  • the tachyzoites were deposited on a monolayer of HFF cells in culture.
  • the culture medium is replaced and supplemented with the selection agent (2 ⁇ M pyrimethamine), 24 h after electroporation. Three culture passages are carried out in this medium.
  • the resistant parasites are cloned by limit dilution in the wells of 96-well plates of HFF cells. After amplification, the lysis plaques caused by the parasite are investigated. The parasites are subcultured and their genomic DNA is extracted for PCR analyses. These PCR analyses should confirm integration of the transgene but should also allow differentiation of the parasites that have randomly integrated the transgene from the parasites of interest the ncmic3 gene of which has been effectively suppressed by homologous recombination.
  • Neospora PCR Neo ncmic3 KO caninum NC1 1 3824 2163 2 — 850 3 504 — 4 — 3127 5 — 3374 6 2890 — 7 3258 —
  • FIG. 3-A The electrophoretic profiles of the PCR products are presented in FIG. 3-A .
  • certain clones display a specific band of DHFR (PCR 3) but no specific band of ncmic3 (PCR 2).
  • PCR No. 1 which was carried out on these clones, revealed a band of 3824 bp specific for a Neo ncmic3 KO clone.
  • PCRs New PCR analyses were carried out on these clones of interest with new sets of primers. These PCRs, called integration PCRs, allow validation of the genetic KO using a primer present on the genome upstream or downstream of the sequences flanking the ncmic3 gene and a second primer present in the selection cassette (dhfr gene) or in the gene of interest (ncmic3) ( FIG. 3-B ).
  • PCRs No. 4 and No. 5 make it possible to show the presence of ncmic3 at the locus of ncmic3.
  • PCR No. 4 is carried out with the primer set Integ NCmic3 F (SEQ ID NO: 11) and ORF NCmic3 R2 (SEQ ID NO: 12).
  • PCR No. 5 is carried out with the primer set Integ NCmic3 R (SEQ ID NO: 14) and ORF NCmic3 F2 (SEQ ID NO: 15).
  • the presence of bands for the wild-type strain NC1 of Neospora caninum and the absence of these bands for the mutant strain Neo ncmic3 KO are observed.
  • FIG. 3-B PCRs No. 6 and No.
  • PCR No. 6 is carried out with the primer set Integ NCmic3 F (SEQ ID NO: 11) and ORF DHFR R2 (SEQ ID NO: 13).
  • PCR No. 7 is carried out with the primer set Integ NCmic3 R (SEQ ID NO: 14) and ORF DHFR F2 (SEQ ID NO: 16).
  • the absence of bands for the wild-type strain NC1 of Neospora caninum and the presence of bands for the strain Neo ncmic3 KO are noted.
  • the presence of a non-specific band for PCR No. 6 at approximately 1000 bp should be noted.
  • the cells infected by the parasites are washed twice with 1 ⁇ PBS and then fixed with paraformaldehyde (3.7% in 1 ⁇ PBS) for 30 min After 3 washings with 1 ⁇ PBS, the cells are permeabilized with Triton solution (0.1% in 1 ⁇ PBS) for 5 minutes. After 3 washings with 1 ⁇ PBS, a saturation step is carried out with a solution of 1 ⁇ PBS/10% FCS (foetal calf serum) for 30 min.
  • the cells are then incubated with the primary antibody diluted in a solution of PBS/2% FCS (foetal calf serum) for 1 hour, washed 3 times and then incubated with the secondary antibody diluted in a solution of PBS/2% FCS (foetal calf serum) for 1 hour. After 2 washings with 1 ⁇ PBS, the coverslips are mounted on a slide with Immu-Mount and are observed with a fluorescence microscope.
  • the primary antibody used is an antibody that allows detection of expression of the protein NcMIC3 in the parasite (primary antibody: rabbit anti-mic3 antibody and commercial secondary antibody: Alexa fluor® 594 goat anti-rabbit, Life Technologies ref. A-11012).
  • the plasmid pNcMic1KO-CAT-GFP ( FIG. 2-B ) contains a CAT-GFP selection cassette coding for a fusion protein giving both resistance to chloramphenicol (CAT) and green fluorescence (GFP: Green Fluorescent Protein).
  • CAT chloramphenicol
  • GFP Green Fluorescent Protein
  • the 3′ UTR region of the ncmic1 gene was amplified by PCR from the genomic DNA of the strain NC1 of Neospora caninum .
  • the primers 3 HR NCmic1 F KpnI and 3 HR NCmic1 R HindIII allow amplification of the 3′UTR region of the ncmic1 gene and creation of two restriction sites, which were used for cloning the 3HR fragment upstream of the CAT-GFP selection cassette into the plasmid pT230 CAT-GFP (KpnI at 5′ and HindIII at 3′ of the PCR fragment).
  • the 5′ UTR region of the ncmic1 gene was amplified by PCR from the genomic DNA of the strain NC1 of Neospora caninum .
  • the primers 5 HR NCmic1 F BamHI and 5 HR NCmic1 R NotI allow amplification of the 5′ UTR region of the ncmic1 gene and creation of two restriction sites, which were used for cloning the 5HR fragment downstream of the CAT-GFP selection cassette into the plasmid pT230 3HRNcMic1CAT-GFP (BamHI at 5′ and NotI at 3′ of the PCR fragment).
  • the sequences of the primers are shown in Table IV below.
  • plasmid pNcMic1KO-CAT-GFP 50 ⁇ g of the plasmid pNcMic1KO-CAT-GFP, purified and then linearized by KpnI, must be added to 5 ⁇ 10 7 NC1 tachyzoites suspended in CYTOMIX electroporation medium containing ATP (3 mM) and glutathione (3 mM) (Van den Hoff et al., Nucleic Acid Research , June 11; 20(11): 2902), and electroporation must be carried out in a cuvette with a 4 mm gap, in a volume of 800 ⁇ L on a BioRad apparatus (parameters: 2000 V, 50 ohms, 25 ⁇ F, with two electric shocks).
  • the tachyzoites will be deposited on a monolayer of HFF cells in culture.
  • the culture medium will be replaced and supplemented with the selection agent (50 ⁇ M chloramphenicol), 24 h after electroporation. Three culture passages must be carried out in this medium.
  • the resistant parasites will be cloned by limiting dilution in the wells of 96-well plates of HFF cells. After amplification, the lysis plaques caused by the parasite will be investigated. The parasites will be subcultured and their genomic DNA will be extracted for PCR analyses.
  • Neospora PCR Neo ncmic1 KO caninum NC1 1 3359 — 2 3421 — 3 — 3746 4 — 3046 5 — 449 6 472 —
  • Neo ncmic3 KO tachyzoites suspended in the CYTOMIX electroporation medium containing ATP (3 mM) and glutathione (3 mM) (Van den Hoff et al., Nucleic Acid Research, June 11; 20(11): 2902), and electroporation was carried out in a cuvette with a 4 mm gap, in a volume of 800 ⁇ L on a BioRad apparatus (parameters: 2000 V, 50 ohms, 25 ⁇ F, with two electric shocks).
  • the tachyzoites were deposited on a monolayer of HFF cells in culture.
  • the culture medium is replaced and supplemented with the selection agent (chloramphenicol 50 ⁇ M), 24 h after electroporation. Three culture passages are carried out in this medium.
  • the resistant parasites are cloned by limiting dilution in the wells of 96-well plates of HFF cells. After amplification, the lysis plaques caused by the parasite are investigated. The parasites are subcultured and their genomic DNA is extracted for PCR analyses.
  • Neospora PCR Neo ncmic1-3 KO caninum (NC1) Neo ncmic3 KO 1 3359 — — 2 3421 — — 3 — 3746 3746 4 — 3046 3046 5 — 3127 — 6 — 3374 — 7 2890 — 2890 8 3258 — 3258 9 — 449 449 10 472 — — 11 — 850 — 12 504 — 504
  • PCR No. 1 is carried out with the set of primers Integ NCmic1 F (SEQ ID NO: 21) and ORF CATGFP R (SEQ ID NO: 22).
  • PCR 2 is carried out with the set of primers ORF CATGFP F (SEQ ID NO: 23) and Integ NCmic1 R (SEQ ID NO: 24).
  • PCR No. 3 is carried out with the set of primers Integ NCmic1 F (SEQ ID NO: 21) and ORF NCmic1 R (SEQ ID NO: 25).
  • PCR No. 4 is carried out with the set of primers Integ NCmic1 R (SEQ ID NO: 24) and ORF NCmic1 F (SEQ ID NO: 26).
  • PCR No. 5 is carried out with the set of primers Integ NCmic3 F (SEQ ID NO: 11) and ORF NCmic3 R2 (SEQ ID NO: 12).
  • PCR No. 6 is carried out with the set of primers Integ NCmic3 R (SEQ ID NO: 14) and ORF NCmic3 F2 (SEQ ID NO: 15).
  • PCR No. 7 is carried out with the set of primers Integ NCmic3 F (SEQ ID NO: 11) and ORF DHFR R2 (SEQ ID NO: 13).
  • PCR No. 8 is carried out with the set of primers Integ NCmic3 R (SEQ ID NO: 14) and ORF DHFR F2 (SEQ ID NO: 16).
  • PCR No. 9 is carried out with the set of primers ORF NCmic1 F2 (SEQ ID NO: 27) and ORF NCmic1 R2 (SEQ ID NO: 28).
  • PCR No. 10 is carried out with the set of primers ORF CATGFP F2 (SEQ ID NO: 29) and ORF CATGFP R2 (SEQ ID NO: 30).
  • PCR No. 11 is carried out with the set of primers ORF NCmic3 F (SEQ ID NO: 7) and ORF NCmic3 R (SEQ ID NO: 8).
  • PCR No. 12 is carried out with the set of primers ORF DHFR F (SEQ ID NO: 9) and ORF DHFR R (SEQ ID NO: 10).
  • the electrophoretic analyses of the PCR products show that the strain Neo ncmic1-3 KO no longer has the ncmic1 and ncmic3 genes (wells 3, 4, 5, 6, 9 and 11, FIG. 5 ) and does have the dhfr and cat-gfp genes (wells 1, 2, 7, 8, 10 and 12, FIG. 5 ), thus validating production of the strain Neo ncmic1-3 KO.
  • All of the PCR results demonstrate that homologous recombination has indeed taken place and the strain Neo ncmic1-3 KO has indeed been deleted from the ncmic1 and ncmic3 genes.
  • Immunofluorescence analysis was carried out solely by direct observation of the fluorescence of the parasite ( FIG. 6 ).
  • the parasites of the two mutant strains are visualized in direct light (images A and C).
  • One and the same microscopic field is visualized in fluorescence.
  • Green fluorescence due to expression of the recombinant chimeric protein CAT-GFP, is only detected in the mutant strain Neo ncmic1-3 KO (image D) following insertion of the CAT-GFP cassette.
  • the strain Neo ncmic3 KO which does not have a CAT-GFP cassette, does not express the CAT-GFP protein and consequently does not display fluorescence (image B).
  • Immunostimulation is carried out on the C57BL/6 mouse pups aged 3 days. These mouse pups were obtained and bred in the INRA Centre in Nouzilly (Indre et Loire, France). The mouse pups are kept throughout the experiments in an animal house of containment level 2 in order to minimize the risk of external contamination.
  • strain Toxo mic1-3 KO The mutant strain of Toxoplasma gondii , in which the genes coding for the proteins MIC1 and MIC3 were knocked out (called strain Toxo mic1-3 KO) is maintained by successive passages on a human foreskin fibroblast (HFF) line cultured in DMEM medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin.
  • HFF human foreskin fibroblast
  • the wild-type strain RH of Toxoplasma gondii is also maintained by successive passages on a human foreskin fibroblast (HFF) line cultured in DMEM medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin.
  • HFF human foreskin fibroblast
  • the tachyzoites of the strain RH are washed, sonicated at 60 W/s, three times for 10 min and centrifuged at 2000 g for 30 min at 4° C. The supernatant is concentrated and divided into aliquots. The concentration is determined by BCA assay, using BSA (Bovine Serum Albumin) as standard. The aliquots are stored at ⁇ 20° C.
  • mice pups in batch A received 20 tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route.
  • the humoral immune response was studied by evaluating, by ELISA, the kinetics of appearance of the IgM specific anti- Toxoplasma gondii antibodies in the serum.
  • the sera are taken at the moment of sacrifice of the mouse pups on D3 and on D9 post-infection.
  • the sample is left for 10 mM at room temperature to allow clotting.
  • the serum is recovered by centrifuging the samples at 2000 rpm for 10 min at +20° C.
  • the supernatant is divided into aliquots in a clean tube and stored at ⁇ 20° C.
  • the non-specific sites are saturated by incubation of the plates for 1.5 h at 37° C. under humid atmosphere with PBS with 4% BSA.
  • the assumed positivity threshold was determined as a function of the values of absorbance (OD) of the mouse pups in the control batch: it is fixed at 0.23 of OD for a 1/50 serum dilution.
  • IL-12 is a cytokine produced in response to intrusion of a pathogen that stimulates secretion of IFN- ⁇ , a cytokine produced by the immune system cells in response to inflammation on the site of infection by the pathogen.
  • the intestine is removed and the ileum (1 cm above the caecum) is used for analysis by qPCR.
  • the tissue is incubated in 1 mL of Trizol® (Invitrogen) and then is ground in a Thurax, incubated at room temperature for 5 min and centrifuged for 10 minutes at 12,000 g. The supernatant is then recovered and mixed by pipetting up and down approximately ten times.
  • RNA is isolated from the DNA and from the proteins with chloroform.
  • chloroform One millilitre of chloroform is added to the supernatant and the Trizol®/chloroform mixture is stirred vigorously for 15 seconds, then incubated at room temperature and finally centrifuged at 12,000 g for 15 minutes at 4° C. After centrifugation, the mixture is separated into an organic phase (phenol/chloroform, pink, lower phase), an interphase (white film, DNA and cell debris) and an aqueous phase (upper phase) containing the total RNAs.
  • RNA is taken up in approximately 20 ⁇ L of 0.1% DEPC water.
  • RNA extraction yield is quantified using a spectrophotometer.
  • RNA solution is incubated with 2 ⁇ L of dNTP (dATP, dTTP, dGTP, dCTP each at 20 mM), 4 ⁇ L of reverse transcriptase buffer (5 ⁇ Eurogentec; 250 mM Tris-HCl (pH 8.3); 375 mM KCl; 50 mM DTT; 15 mM MgCl 2 ) and 0.4 ⁇ L of MuMLV (25 U/ ⁇ L; 50 mM Tric-HCL (pH 8.3); 1 mM EDTA, 0.1% Triton X-100, 0.1 M NaCl; 5 mM DTT; 50% (v/v)
  • dNTP dATP, dTTP, dGTP, dCTP each at 20 mM
  • reverse transcriptase buffer 5 ⁇ Eurogentec; 250 mM Tris-HCl (pH 8.3); 375 mM KCl; 50 mM DTT; 15 mM MgCl
  • the primer pair SEQ ID NO: 31 (5′-CTCACATCTGCTGCTCCACAA-3′) and SEQ ID NO: 32 (5′-GACGCCATTCCACATGTCACT-3′) was used for IL-12
  • the primer pair SEQ ID NO: 5′-TCTTCTTGGATATCTGGAGGAA-3′) and SEQ ID NO: 34 (5′-AGCTCATTGAATGCTTGGCGCTG-3′) was used for assaying IFN ⁇
  • the primer pair SEQ ID NO: 35 (5′-GGATACAGGCCAGACTTTGTTG-3′) and SEQ ID NO: 36 (5′-GAGGGTAGGCTGGCCTATAG-3′) was used for assaying the murine HPRT reference gene.
  • cDNA diluted to 1/10 from the reverse transcription reaction is incubated with 0.3 ⁇ L of the 5′ primer (25 ⁇ M); 0.3 ⁇ L of the 3′ primer (25 ⁇ M); 7.5 ⁇ L of Mix PCR (BioRad) in a final volume of 15 ⁇ L.
  • the conditions selected for the PCR reaction are as follows: 1) denaturation at 95° C. for 5 minutes, 2) denaturation at 95° C. for 10 seconds, 3) pairing and elongation at 62° C. for IL-12, IFN′ and HPRT for 15 seconds, 4) repeating the cycle starting from step 2: 39 times, 5) melting curve from 55° C. to 95° C. to verify the presence or absence of the dimers.
  • the level of infection of the mouse pups was analysed by three different techniques: 1) tissue dissemination of the tachyzoites on HFF cells, 2) immunohistology on sections of intestine from the infected mouse pups and 3) PCR from the ileum of the mouse pups in batches A and B.
  • the HFF cells are deposited in a 24-well plate one week before depositing the organs at a rate of 1 ⁇ 10 4 cells/well.
  • the cells are cultured in 1 mL of DMEM cell culture medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin.
  • FCS foetal calf serum
  • the spleen of the mouse pups of batches 1 and 2 is removed and ground in 2 mL of 1 ⁇ PBS. Ten microlitres of ground material is deposited per well containing the HFF cells. Twenty-four hours after depositing the ground materials from the spleen, the cells are washed with DMEM and then 1 mL of clean medium is deposited in each well. The cells are incubated at 37° C., 5% CO 2 until the lysis plaques revealing the presence of Toxo mic1-3 KO tachyzoites are detected.
  • the intestine of the sacrificed mouse pups is rolled up like a Swiss roll and then kept in this form using paper wrapped around the Swiss roll and stapled.
  • the tissues are then washed in 1 ⁇ PBS and then incubated at 4° C. for 8 h in clean 1 ⁇ PBS. This last step is repeated a second time.
  • the tissues are incubated for 8 hours at 4° C., in 30% sucrose solution diluted in 1 ⁇ PBS and filtered. Finally the tissues are transferred to moulds of a suitable size filled with OCT embedding medium. After 5 minutes of incubation in the OCT, the samples are frozen using dry ice and stored at ⁇ 80° C.
  • the samples of intestine are then cut into sections using a cryostat which maintains the sample at ⁇ 20° C. Histological sections with a thickness of 7 ⁇ m are prepared and then deposited on a slide by electrostatic force. The slides are stored at ⁇ 80° C.
  • the slides of histological sections are thawed and then left to dry at room temperature for 1 h.
  • the zone of the sample on the slide is delimited with a pen with hydrophobic ink (Dakocitamation pen).
  • the histological sections of intestine are permeabilized with 50 ⁇ L of a solution of 1 ⁇ PBS, Mg 2+ , Ca 2+ free, Triton X-100 and 1% BSA at room temperature and in a humid chamber for 10 min.
  • the permeabilizing solution is removed by aspiration and the samples are saturated in 50 ⁇ L of a solution of 1 ⁇ PBS, Mg 2+ , Ca 2+ free, Triton X-100 and 10% BSA at room temperature and in a humid chamber for 1 h.
  • a PCR was carried out from cDNAs obtained by reverse transcription reaction (cf. paragraph 1.5).
  • the SAG-1 gene specific to the parasite Toxoplasma gondii was amplified by PCR with the primers SEQ ID NO: 37 (5′-CTGCACCACTTCATTATTTCTTCTG-3′) and SEQ ID NO: 38 (5′-ACTCACGCGACACAAGCTG-3′).
  • 2 ⁇ L of cDNA is incubated with 1 ⁇ L of 5′ primer (10 ⁇ M); 1 ⁇ L of 3′ primer (10 ⁇ M); 25 ⁇ L of GoTaq® Green Master Mix (2 ⁇ , Promega) in a final volume of 50 ⁇ L.
  • the conditions selected for the PCR are as follows: 1) denaturation at 94° C. for 5 min, 2) denaturation at 94° C. for 30 s, 3) pairing at 60° C. for 30 s, 4) elongation at 72° C. for 1 min, 5) repeating the cycle starting from step 2: 34 times, 6) elongation at 72° C. for 5 min.
  • the presence of the parasite Toxoplasma gondii in the intestine is verified by an amplified fragment of 1001 base pairs.
  • mice pups in the control batch (A) have not developed a humoral response on D3 and on D9 post-infection in contrast to 2 mouse pups out of 3 of the batch immunostimulated with the strain Toxo mic1-3 KO (B), which produced anti-toxoplasmic IgM antibodies at 9 days post-infection; these are the mouse pups No. 4 and No. 6.
  • FIGS. 8-A and 8 -B The results of the quantitative PCR tests for expression of the gene coding for IFN- ⁇ and the gene coding for IL-12 carried out on D9 post-immunostimulation are shown in FIGS. 8-A and 8 -B respectively.
  • the mouse pups in the control batch (A) have not developed an inflammatory response (secretion of IL-12 and IFN- ⁇ ) on D9 post-infection.
  • 9 days after immunostimulation the mouse pups No. 4, 5 and 6 display significantly greater expression of IL-12 than that of the mouse pups in the control batches ( FIG. 8-B ).
  • mice pups out of 3 in the batch immunostimulated with the strain Toxo mic1-3 KO display expression of IFN- ⁇ significantly greater than that of the mouse pups in the control batches ( FIG. 8-A ); these are the mouse pups No. 4 and No. 6.
  • HFF cells The dissemination of the spleens on HFF cells is illustrated in Table IX. No lysis plaque is detected in the HFF cells infected with the spleens from the mouse pups of the control batch. In contrast, the HFF cells infected with the ground material from the spleen originating from the mouse pups immunostimulated with the strain Toxo mic1-3 KO have lysis plaques, demonstrating that tachyzoites are present in the spleen.
  • the technique of tissue dissemination of the tachyzoites on HFF cells is a semiquantitative technique which shows that the mouse pups in the batch immunostimulated with the strain Toxo mic1-3 KO have different states of infection.
  • the mouse pups No. 4 and No. 6 seem be the most infected as they have the largest number of parasites in the spleen, followed by mouse pup No. 5, which has far fewer parasites in the spleen (Table IX).
  • the immunohistology sections of the intestines from the mouse pups are illustrated in FIG. 9 .
  • PCR results are presented in FIG. 10 .
  • Amplification of the SAG1 gene of Toxoplasma gondii from a DNA sample originating from ground material from the ileum is representative of the presence of Toxo mic1-3 KO tachyzoites (amplified fragment at 1001 bp).
  • This semiquantitative technique shows absence of a band for the mouse pups in the control batch (NV) sacrificed on D3 (T1) or on D9 (T2).
  • Immunostimulation is carried out on C57BL/6 mouse pups aged 3 days. These mouse pups were obtained and bred in the INRA Centre in Nouzilly (Indre et Loire). The mouse pups are kept throughout the experiments in an animal house of containment level 2 in order to limit the risk of external contamination as far as possible.
  • the oocysts of Cryptosporidium parvum are obtained from excrement of calves infected with 10 7 C. parvum oocysts.
  • the stool undergoes various treatments until a suspension of purified, sterile parasites is obtained, suitable for use in cell culture. Throughout the treatment, the oocysts are manipulated at 4° C., to prevent excystation of the oocysts.
  • the latter is diluted in fresh water and then passed through a 100- ⁇ m filter and centrifuged at 1900 g for 10 minutes at 4° C.
  • the pellets obtained, containing the oocysts, are taken up in 2% potassium dichromate solution (Prolabo, ref. 26 776 290, CAS 7778-50-9), then washed twice with cold water by centrifugation at 1900 g for 10 minutes at 4° C. to remove the potassium dichromate.
  • the pellet of coccidia is taken up in a mixture of water and ether (Ethyl Ether, Carlo Erba, CAS No.
  • the purified oocysts are then sterilized. After centrifugation at 1900 g for 10 minutes at 4° C., the pellet of oocysts is incubated for 15 minutes in a solution of bleach (sodium hypochlorite (Sigma 239305-500ML Titre: 4.5% of active chlorine) diluted to 10% in demineralized water, then washed 3 times in sterile 1 ⁇ PBS (diluted from a solution of 10 ⁇ PBS: sodium chloride, NaCl 80 g/litre water; potassium chloride, KCl 2 g/litre; potassium dihydrogen phosphate, KH 2 PO 4 : 2 g/litre; disodium hydrogen phosphate, Na 2 HPO 4 , 12 H 2 O: 29 g/litre).
  • bleach sodium hypochlorite (Sigma 239305-500ML Titre: 4.5% of active chlorine) diluted to 10% in demineralized water
  • sterile 1 ⁇ PBS diluted from a solution of 10 ⁇ PBS
  • the purified and sterilized oocysts are counted on a slide (5 ⁇ L of solution of oocysts and 495 ⁇ L of malachite green), adjusted to a concentration of 2 ⁇ 10 8 oocysts/mL, divided into aliquots in 1.5-mL tubes and stored at 4° C.
  • strain Toxo mic1-3 KO The mutant strain of Toxoplasma gondii , in which the genes coding for the proteins MIC1 and MIC3 have been knocked out (called strain Toxo mic1-3 KO) is maintained by successive passages on a human foreskin fibroblast (HFF) line cultured in DMEM medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin.
  • HFF human foreskin fibroblast
  • the mouse pups in batch 1 received 20 tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route.
  • mice pups in batch 1 and batch 2 were challenged with 500,000 parasites of Cryptosporidium parvum.
  • the state of infection of the mouse pups was analysed by the technique of tissue dissemination of the tachyzoites on HFF cells described above.
  • the HFF cells are deposited in a 24-well plate, one week before depositing the organs at a rate of 10 4 cells per well.
  • the cells are cultured in 1 mL of DMEM cell culture medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin. After sacrifice of the mouse pups, the spleen of each of the mouse pups is taken and ground in 2 mL of 1 ⁇ PBS.
  • FCS foetal calf serum
  • the protection of the mouse pups against Cryptosporidium parvum is analysed by counting the oocysts in the intestine. After the mouse pups are sacrificed, the intestines are recovered, weighed, placed in 1 mL of water (4° C.) and ground for 20 seconds. 100 microlitres of ground material is added to 400 ⁇ L of sugar solution at 4° C. (500 g of powdered sugar, 320 mL of distilled water, Na azide at 0.02%). After homogenizing the solution by pipetting, 20 ⁇ L is deposited on a Thoma cell.
  • the state of infection of the mouse pups is evaluated from parasitaemia in the case of Cryptosporidium parvum ( C. parvum ) and Toxoplasma gondii ( T. gondii ).
  • Parasitaemia in the case of C. parvum is determined by the total number of oocysts of C. parvum counted in the mouse pup intestine.
  • Parasitaemia in the case of T. gondii is evaluated by dissemination of the spleens of the mouse pups on HFF cells.
  • the parasitaemias for C. parvum and T. gondii are presented in Table X.
  • the number associated with parasitaemia in the case of T. gondii corresponds to the number of tachyzoites counted in 20 ⁇ L of supernatant of the HFF cells infected after dissemination of the spleen. Infected C.
  • the mouse pups infected with C. parvum only have a total number of oocysts of C. parvum in the intestine in the range from 5 ⁇ 10 5 to 1 ⁇ 10 6 oocysts.
  • T. gondii parasitaemia For mouse pups No. 2, 3 and 7, which have the highest T. gondii parasitaemia, the total number of oocysts of C. parvum in the intestine varies from 5 ⁇ 10 4 to 3.3 ⁇ 10 5 oocysts.
  • the state of protection of the mouse pups is shown in FIG. 11 .
  • mice pups out of 7 have a total number of oocysts of C. parvum in the intestine that is significantly reduced, by approximately 62% to 94%.
  • These three mouse pups are the mouse pups No. 2, No. 3 and No. 7, which displayed the highest parasitaemia with T. gondii of all the mouse pups in this group.
  • Immunostimulation is carried out on C57BL/6 mouse pups aged 3 days. These mouse pups were obtained and bred in the INRA Centre in Nouzilly (Indre et Loire). The mouse pups are kept throughout the experiments in an animal house of containment level 2 in order to limit the risk of external contamination as far as possible.
  • Oocysts of Cryptosporidium parvum are obtained from excrement of calves infected with 10 7 C. parvum oocysts.
  • the stool undergoes various treatments until a suspension of sterile, purified parasites is obtained, suitable for use in cell culture. Throughout the treatment, the oocysts are manipulated at 4° C. to prevent excystation of the oocysts.
  • the latter is diluted in fresh water and then passed through a 100- ⁇ m filter and centrifuged at 1900 g for 10 minutes at 4° C.
  • the pellets obtained, containing the oocysts, are taken up in 2% potassium dichromate solution (Prolabo ref. 26 776 290, CAS 7778-50-9), then washed twice with cold water by centrifugation at 1900 g for 10 minutes at 4° C. to remove the potassium dichromate.
  • the pellet of coccidia is taken up in a mixture of water and ether (ethyl ether, Carlo Erba CAS No.
  • the purified oocysts are then sterilized. After centrifugation at 1900 g for 10 minutes at 4° C., the pellet of oocysts is incubated for 15 minutes in a solution of bleach (sodium hypochlorite, Sigma 239305-500ML Titre: 4.5% of active chlorine) diluted to 10% in demineralized water and then washed 3 times in sterile 1 ⁇ PBS (diluted from a solution of 10 ⁇ PBS: sodium chloride, NaCl 80 g/litre water; potassium chloride, KCl 2 g/litre; potassium dihydrogen phosphate, KH 2 PO 4 : 2 g/litre; disodium hydrogen phosphate, Na 2 HPO 4 , 12 H 2 O: 29 g/litre).
  • bleach sodium hypochlorite, Sigma 239305-500ML Titre: 4.5% of active chlorine
  • the purified and sterilized oocysts are counted on a slide (5 ⁇ L of solution of oocysts and 495 ⁇ L of malachite green), adjusted to a concentration of 2 ⁇ 10 8 oocysts/mL, divided into aliquots in 1.5-mL tubes and stored at 4° C.
  • strain Toxo mic1-3 KO The mutant strain of Toxoplasma gondii , in which the genes coding for the proteins MIC1 and MIC3 have been suppressed (called strain Toxo mic1-3 KO) is maintained by successive passages on a human foreskin fibroblast (HFF) line cultured in DMEM medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin.
  • HFF human foreskin fibroblast
  • the mouse pups in batch 1 received 20,000 tachyzoites of the strain Toxo mic1-3 KO by oral route.
  • the state of infection of the mouse pups was analysed by the technique of tissue dissemination of the tachyzoites on HFF cells described above.
  • the HFF cells are deposited in a 24-well plate, one week before depositing the organs at a rate of 10 4 cells per well.
  • the cells are cultured in 1 mL of DMEM cell culture medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin. After sacrifice of the mouse pups, the spleen of each of the mouse pups is taken and ground in 2 mL of 1 ⁇ PBS.
  • FCS foetal calf serum
  • the protection of the mouse pups against Cryptosporidium parvum is analysed by counting the oocysts in the intestine. Once the mouse pups have been sacrificed, the intestines are recovered, weighed, placed in 1 mL of water (4° C.) and ground for 20 seconds. 100 microlitres of ground material is added to 400 ⁇ l of sugar solution at 4° C. (500 g of sucrose, 320 mL of distilled water, Na azide at 0.02%). After homogenizing the solution by pipetting, 20 ⁇ L is deposited on a Thoma slide.
  • the mouse pups infected only with C. parvum have a total number of oocysts of C. parvum in the intestine in the range from 1.4 ⁇ 10 6 to 2.4 ⁇ 10 6 oocysts.
  • T. gondii parasitaemia For the mouse pups No. 1 and 2, which have severe T. gondii parasitaemia, the total number of oocysts of C. parvum in the intestine varies from 2.6 ⁇ 10 5 to 4.8 ⁇ 10 5 oocysts.
  • the state of protection of the mouse pups is shown in FIG. 12 .
  • mice pups out of 4 have a total number of oocysts of C. parvum in the intestine that is significantly reduced by approximately 75% and 87%. These are the mouse pups No. 1 and No. 2.
  • the mesenteric lymph nodes and the spleens of neonates used in this experiment originate from lambs of the Ile de France breed aged 6-12 days. Until sacrifice, the lambs were kept with their mothers in a sealed sheep house (INRA-Nouzilly) in order to limit the risks of natural contamination. They were anaesthetized by electronarcosis and then euthanased to collect the different organs. Only the animal keepers and the experimenters, equipped with clothing for use inside the sheep house, may enter the buildings, in order to avoid contamination of the environment, and they only leave the sealed zone after showering. The utensils used and the biological material are only taken out after passing through a disinfectant bath, and the waste is incinerated.
  • the mesenteric lymph nodes and the spleens of adult subjects used in this experiment originate from adult sheep of the Ile de France breed aged from 1 to 3 years.
  • the wild-type RH and Pru strains and the mutant strain Toxo mic1-3 KO are maintained by successive passages on a human foreskin fibroblast (HFF) line cultured in DMEM medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin.
  • HFF human foreskin fibroblast
  • the mesenteric lymph nodes are removed as quickly as possible after the animals are euthanased, and transferred to a sterile sample tube containing culture medium (HBSS supplemented with 2% foetal calf serum (FCS) and 1% penicillin/streptomycin (P/S)).
  • HBSS 2% foetal calf serum
  • P/S penicillin/streptomycin
  • Each lymph node is defatted using previously sterilized forceps, scissors or scalpel.
  • the lymph node is then deposited on a sterile (autoclaved) nylon mesh measuring 3 cm ⁇ 3 cm placed at the bottom of a sterile Petri dish containing 10 mL of medium (HBSS; 2% FCS; 1% P/S).
  • the lymph node is crushed and comminuted using a piston of a 5-mL syringe to release the cells contained in the lymph node.
  • the medium thus enriched with cells is filtered using a 60- ⁇ m sterile nylon cloth positioned above a 50-mL tube for recovering the medium enriched with filtered cells.
  • the 90 mL of cell suspension is divided into 3 to be purified in a Ficoll-Hypaque gradient. 30 mL is carefully deposited per tube (3 tubes in total) containing 15 mL of Ficoll-Hypaque.
  • the 3 tubes of Ficoll-Hypaque enriched with cell suspension are centrifuged at 1500 g without braking (deceleration 2, acceleration 5), for 30 minutes at room temperature.
  • an upper phase is obtained composed of cell debris, a ring composed of mononuclear cells, positioned as an interphase between the cell debris and the Ficoll phase, and finally a lower phase composed of red blood cells.
  • the 3 rings (20 mL/tubes) are recovered and divided into two 50-mL tubes and washed with a final volume of 50 mL of medium (HBSS; 2% FCS; 1% P/S) at 700 g for 10 min.
  • the pellets of mononuclear cells obtained are pooled.
  • the final pellet is washed with 50 mL of medium (HBSS; 2% FCS; 1% P/S) by centrifugation at 400 g for 10 min.
  • the pellet is taken up in 5 mL of RPMI, 10% FCS, 1% P/S, 5.10 ⁇ 5 M of beta-mercaptoethanol.
  • the mononuclear cells are stored in ice; a proportion of the cells is used for counting and observation of viability with Trypan blue (sample diluted to 1/100). Once counted, the cells are distributed in a 96-well plate at a rate of 3.10 5 cells/well.
  • the spleen is removed as quickly as possible after euthanasia and transferred to a sterile sample tube containing culture medium (HBSS; 2% FCS; 1% P/S).
  • HBSS fetal bovine serum
  • FCS fetal calf serum
  • P/S fetal calf serum
  • Each spleen is defatted using previously sterilized forceps, scissors or scalpel.
  • the spleen is then deposited on a metal grid (autoclaved) placed at the bottom of a sterile Petri dish containing 10 mL of medium (HBSS; 2% FCS; 1% P/S).
  • the spleen is crushed and comminuted using a piston of a 5-mL syringe to release the cells contained in the spleen.
  • the medium thus enriched with cells is deposited in a 50-mL tube.
  • the 50-mL tube for recovery of the medium enriched with cells is left to settle for 5 min at 4° C.
  • the supernatant is filtered on a 60 ⁇ m nylon mesh above a 50-mL tube.
  • Ten millilitres of medium is added to the remaining sediments and the second supernatant is recovered in the same way.
  • the 50-mL tube for recovery of the two supernatants is centrifuged for 30 s at 400 g at 4° C.
  • the supernatant is recovered and filtered on a 60- ⁇ m sterile nylon mesh above a 50-mL tube. This last tube is centrifuged for 10 min at 400 g at 4° C.
  • the cell pellet thus obtained is resuspended in a final volume of 50 mL of medium (HBSS; 2% FCS; 1% P/S). Washing is carried out by centrifugation for 10 min at 400 g at 4° C. The pellet is then taken up in a final volume of 120 mL in three 50-mL tubes of medium (HBSS; 2% FCS; 1% P/S) at room temperature.
  • medium HBSS; 2% FCS; 1% P/S
  • the 120 mL of cell suspension is divided into 3 to be purified in a Ficoll-Hypaque gradient. 30 mL is carefully deposited per tube (4 tubes in total) containing 15 mL of Ficoll-Hypaque.
  • the 4 tubes of Ficoll-Hypaque enriched with cell suspension are centrifuged at 1500 g without braking (deceleration 2, acceleration 5), for 30 minutes at room temperature.
  • an upper phase is obtained composed of cell debris, a ring composed of mononuclear cells positioned as an interphase between the cell debris and the Ficoll phase, and finally a lower phase composed of red blood cells.
  • the 4 rings (20 mL/tubes) are recovered and divided into three 50-mL tubes and washed with a final volume of 50 mL of medium (HBSS; 2% FCS; 1% P/S) at 700 g for 10 min.
  • the pellets of mononuclear cells obtained are pooled.
  • the final pellet is washed with 50 mL of medium (HBSS; 2% FCS; 1% P/S) by centrifugation at 400 g for 10 min.
  • the pellet is taken up in 5 mL of RPMI, 10% FCS, 1% P/S, 5.10 ⁇ 5 M of beta-mercaptoethanol.
  • the mononuclear cells are stored in ice; a proportion of the cells is used for counting and observation of viability with trypan blue (sample diluted to 1/100). Once counted, the cells are distributed in a 96-well plate at a rate of 3.10 5 cells/well.
  • the cells are distributed in a 96-well plate at a rate of 3.10 5 cells/well.
  • the tachyzoites are also counted on a Malassey slide; pellets are obtained and are taken up in RPMI medium, 10% FCS, 1% P/S.
  • the solutions of the tachyzoites (RH or KO) are adjusted to obtain a concentration of 3.10 6 tachyzoites/mL.
  • the conditions for stimulation are as follows: 3 mononuclear cells are stimulated per tachyzoite. Parasites, lymph node mononuclear cells and spleen mononuclear cells are cultured at 37° C. in a stove at 5% CO 2 and 95% humidity.
  • the supernatants of the lymph node mononuclear cells and the supernatants of the spleen mononuclear cells are used for assay of interleukin-12 and IFN- ⁇ by the ELISA technique.
  • the technique used for assay of interleukin-12 is an ELISA of the sandwich type on a 96-well plate.
  • the antibody (anti-IL-12) immobilized on a plate reacts specifically with the IL-12 present in the test sample.
  • the quantity of antigen-antibody is measured for a second time after it has reacted with an antibody of identical specificity coupled to an enzyme.
  • the range is prepared as follows: 50 ⁇ L of the standard range of rov IL-12 is deposited at different concentrations: 16 U/mL, 8 U/mL, 4 U/mL, 2 U/mL, 1 U/mL, 0.5 U/mL, 0.25 U/mL, 0.125 U/mL, 0.0625 U/mL, 0.03125 U/mL.
  • the samples (supernatant of stimulated mononuclear cells, diluted 1/2) are deposited at a rate of 50 ⁇ L per well.
  • the samples and the standard range are incubated for 1 h at room temperature. 4 washings are carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • biotinylated detection antibodies Mae anti-bovine interleukin-12: biotin clone CC 326 (Serotec MCA2173B) Initial concentration: 500 ⁇ g/ml) diluted to 1/500 is deposited per well, for an incubation time of one hour at room temperature. A series of 4 washings is carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • IL-12 is detected by a biotin-ExtrAvidine® affinity reaction followed by a colorimetric reaction between peroxidase and its substrate: 50 ⁇ L of modified avidin coupled to a peroxidase (ExtrAvidine-Peroxidase conjugate® (Sigma E2886)) diluted to 1/2000th is deposited per well and incubated for 20 minutes at room temperature. A series of 4 washings is carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • Detection is carried out using 50 ⁇ L/well of peroxidase substrate originating from a solution A and B mixed volume for volume (TMB Peroxidase substrate Eurobio KPL solution A 50-76-01 and solution B 50-65-00). The substrate solution is incubated for 15 minutes at room temperature. 50 ⁇ L of stopping solution (phosphoric acid 1M Sigma 43,080-1, CAS 7664-38-2) is added per well under a fume hood.
  • stopping solution phosphoric acid 1M Sigma 43,080-1, CAS 7664-38-2
  • the standard curve is determined by the following function: OD value as a function of the concentration of recombinant IL-12.
  • the OD values of the samples tested are converted to concentration using the standard curve. To be taken into account, the samples must give ODs located on the linear portion of the standard curve.
  • the technique used for assay of interferon is an ELISA, comparable to the assay of interleukin-12 described in the preceding paragraph, of the sandwich type on a 96-well plate.
  • the antibody (anti-IFN ⁇ ) immobilized on a plate reacts specifically with the IFN ⁇ present in the test sample.
  • the quantity of antigen-antibody is measured for a second time after it has reacted with an antibody of identical specificity to an enzyme.
  • All the dilutions of the different reagents are prepared in 1 ⁇ PBS, 0.05% Tween 20 and 1% BSA except for the capture antibody, which is diluted in 1 ⁇ PBS.
  • the range is prepared as follows: 50 ⁇ L of the standard range of Recombinant bovine IFN ⁇ (Perbio Endogen robIFNG ⁇ , initial concentration: 30 ⁇ g/ml) is deposited at different concentrations: 4 ng/mL, 2 ng/mL, 1 ng/mL, 0.5 ng/mL, 0.25 ng/mL, 0.125 ng/mL, 0.0625 ng/mL, 0.03125 ng/mL, 0.015 ng/ml).
  • the samples (supernatant of stimulated mononuclear cells, diluted 1/2) are deposited at a rate of 50 ⁇ L per well.
  • the samples and the standard range are incubated for 1 h at room temperature. 4 washings are carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • biotinylated detection antibodies Mae anti-bovine interferon gamma: biotin clone CC 302 (Serotec MCA1783B) Initial concentration: 500 ⁇ g/ml) diluted to 1/500 is deposited per well, for an incubation time of one hour at room temperature. A series of 4 washings is carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • IFN ⁇ is detected by a biotin-ExtrAvidine® affinity reaction followed by a colorimetric reaction between peroxidase and its substrate: 50 ⁇ L of modified avidin coupled to a peroxidase (ExtrAvidine-Peroxidase conjugate® (Sigma E2886)) diluted to 1/2000th is deposited per well and incubated for 20 minutes at room temperature. A series of 4 washings is carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • Detection is carried out using 50 ⁇ L per well of peroxidase substrate originating from a solution A and B mixed volume for volume (TMB Peroxidase substrate Eurobio KPL solution A 50-76-01 and solution B 50-65-00). The substrate solution is incubated for 15 minutes at room temperature. 50 ⁇ L of stopping solution (phosphoric acid 1M Sigma 43,080-1, CAS 7664-38-2) is added per well under a fume hood.
  • stopping solution phosphoric acid 1M Sigma 43,080-1, CAS 7664-38-2
  • the standard curve is determined by the following function: OD value as a function of the concentration of recombinant IFN ⁇ .
  • the OD values of the samples tested are converted to concentration using the standard curve. To be taken into account, the samples must give ODs located on the linear portion of the standard curve.
  • the level of IL-12 and of IFN- ⁇ secreted by the mononuclear cells of spleens and the level of IL-12 secreted by the mononuclear cells of mesenteric lymph nodes (MLN) isolated from lambs aged from 6 to 12 days and from adult sheep aged from 1 to 3 years and stimulated with different strains of the parasite Toxoplasma gondii are shown in FIGS. 13-A , 13 -B and 13 -C.
  • Immunostimulation is carried out on 1-day-old neonate lambs. After ingestion of colostrum, the lambs were isolated from their mothers in a sealed sheep house (INRA-Nouzilly) in order to limit the risks of natural contamination. They were anaesthetized by electronarcosis and then euthanased to collect the various organs. Only the animal keepers and the experimenters, equipped with clothing for use inside the animal house, may enter the buildings, to prevent contamination of the environment, and they only leave the sealed zone after showering.
  • strain Toxo mic1-3 KO The mutant strain of Toxoplasma gondii , in which the genes coding for the proteins MIC1 and MIC3 have been suppressed (called strain Toxo mic1-3 KO) is maintained by successive passages on a human foreskin fibroblast (HFF) line cultured in DMEM medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin.
  • HFF human foreskin fibroblast
  • the lambs in batch A received 10 6 tachyzoites of the strain Toxo mic1-3 KO subcutaneously. After immunostimulation, the temperatures and the weights of the lambs are recorded daily.
  • the spleen is removed as quickly as possible after euthanasia and transferred to a sterile sample tube containing culture medium (HBSS; 2% FCS; 1% P/S).
  • HBSS fetal bovine serum
  • FCS fetal calf serum
  • P/S fetal calf serum
  • Each spleen is defatted using previously sterilized forceps, scissors or scalpel.
  • the spleen is then deposited on a metal grid (autoclaved) placed at the bottom of a sterile Petri dish containing 10 mL of medium (HBSS; 2% FCS; 1% P/S).
  • the spleen is crushed and comminuted using a piston of a 5-mL syringe to release the cells contained in the spleen.
  • the medium thus enriched with cells is deposited in a 50-mL tube.
  • the 50-mL tube for recovery of the cell-enriched medium is left to settle for 5 min at 4° C.
  • the supernatant is filtered on a 60 ⁇ m nylon mesh above a 50-mL tube.
  • Ten millilitres of medium is added to the remaining sediments and the second supernatant is recovered in the same way.
  • the 50-mL tube for recovery of the two supernatants is centrifuged for 30 s at 400 g at 4° C.
  • the supernatant is recovered and filtered on a 60- ⁇ m sterile nylon mesh above a 50-mL tube. This last tube is centrifuged for 10 min at 400 g at 4° C.
  • the cell pellet thus obtained is resuspended in a final volume of 50 mL of medium (HBSS; 2% FCS; 1% P/S). Washing is carried out by centrifugation for 10 min at 400 g at 4° C. The pellet is then taken up in a final volume of 120 mL in three 50-mL tubes of medium (HBSS; 2% FCS; 1% P/S) at room temperature.
  • medium HBSS; 2% FCS; 1% P/S
  • the 120 mL of cell suspension is divided into 3 to be purified in a Ficoll-Hypaque gradient. 30 mL is carefully deposited per tube (4 tubes in total) containing 15 mL of Ficoll-Hypaque.
  • the 4 tubes of Ficoll-Hypaque enriched with cell suspension are centrifuged at 1500 g without braking (deceleration 2, acceleration 5), for 30 minutes at room temperature.
  • an upper phase is obtained composed of cell debris, a ring composed of mononuclear cells positioned as an interphase between the cell debris and the Ficoll phase, and finally a lower phase composed of red blood cells.
  • the 4 rings (20 mL/tubes) are recovered and divided into three 50-mL tubes and washed with a final volume of 50 mL of medium (HBSS; 2% FCS; 1% P/S) at 700 g for 10 min.
  • the pellets of mononuclear cells obtained are pooled.
  • the final pellet is washed with 50 mL of medium (HBSS; 2% FCS; 1% P/S) by centrifugation at 400 g for 10 min.
  • the pellet is taken up in 5 mL of RPMI, 10% FCS, 1% P/S, 5.10 ⁇ 5 M of beta-mercaptoethanol.
  • the mononuclear cells are stored in ice; a proportion of the cells is used for counting and observation of viability with trypan blue (sample diluted to 1/100). Once counted, the cells are distributed in a 96-well plate at a rate of 3.10 5 cells/well.
  • the popliteal and subiliac lymph nodes, situated near the injection site, are removed as quickly as possible after euthanasia of the animals and are transferred to a sterile sample tube containing culture medium (HBSS supplemented with 2% foetal calf serum (FCS) and 1% penicillin/streptomycin (P/S)).
  • HBSS 2% foetal calf serum
  • P/S penicillin/streptomycin
  • Each lymph node is defatted using previously sterilized forceps, scissors or scalpel.
  • the lymph node is then deposited on a sterile (autoclaved) nylon mesh measuring 3 cm ⁇ 3 cm placed at the bottom of a sterile Petri dish containing 10 ml of medium (HBSS; 2% FCS; 1% P/S).
  • the lymph node is crushed and comminuted using a piston of a 5-mL syringe to release the cells contained in the lymph node.
  • the medium thus enriched with cells is filtered using a 60- ⁇ m sterile nylon mesh positioned above a 50-mL tube for recovering the medium enriched with filtered cells.
  • the pellet is stored in ice; a proportion of the cells is used for counting and observation of viability with trypan blue (sample diluted to 1/100). Once counted, the cells are distributed in a 96-well plate at a rate of 3.10 5 cells/well.
  • the mononuclear cells of the spleen have been isolated by Ficoll Histopaque gradient and distributed in a 96-well plate, at a rate of 3.10 5 cells per well, the latter are stimulated in vitro with 30 ⁇ g/ml of total parasite extract of the strain Toxo mic1-3 KO.
  • the cells are stimulated with concanavalin A.
  • the cells are cultured in a medium without stimulant. The supernatants are taken after 24 hours post-stimulation in vitro.
  • the technique used for assay of IFN ⁇ is an ELISA of the sandwich type on a 96-well plate.
  • the antibody (anti-IFN ⁇ ) immobilized on a plate reacts specifically with the IFN ⁇ present in the test sample.
  • the quantity of antigen-antibody is measured in a second phase after it has reacted with an antibody of identical specificity coupled to an enzyme.
  • All the dilutions of the various reagents are prepared in 1 ⁇ PBS, 0.05% Tween 20 and 1% BSA, except for the capture antibody, which is diluted in 1 ⁇ PBS.
  • the range is prepared as follows: 50 ⁇ L of the standard range of rBoIFN ⁇ is deposited at different concentrations: 4 ng/mL, 2 ng/mL, 1 ng/mL, 0.5 ng/mL, 0.25 ng/mL, 0.125 ng/mL, 0.0625 ng/mL, 0.03125 ng/mL, 0.015 ng/mL.
  • the samples (supernatant of stimulated mononuclear cells) are deposited at a rate of 50 ⁇ L per well.
  • the samples and the standard range are incubated for 1 h at room temperature. 4 washings are carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • biotinylated detection antibodies Mae anti-bovine IFNg: biotin clone CC 302 (Serotec MCA1783B) Initial concentration: 500 ⁇ g/ml) diluted to 1/500 is deposited per well, for an incubation time of one hour at room temperature. A series of 4 washings is carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • IFN ⁇ is detected by a biotin-ExtrAvidine® affinity reaction followed by a colorimetric reaction between peroxidase and its substrate: 50 ⁇ L of a solution of modified avidin coupled to a peroxidase (ExtrAvidine-Peroxidase conjugate® (Sigma E2886)) diluted to 1/2000th is deposited per well and incubated for 20 minutes at room temperature. A series of 4 washings is carried out in 1 ⁇ PBS buffer with the addition of 0.05% Tween 20 (PBS-T).
  • Detection is carried out using 50 ⁇ L/well of peroxidase substrate originating from a solution A and B mixed volume for volume (TMB Peroxidase substrate Eurobio KPL solution A 50-76-01 and solution B 50-65-00). The substrate solution is incubated for 15 minutes at room temperature. 50 ⁇ L of stopping solution (phosphoric acid 1M Sigma 43,080-1, CAS 7664-38-2) is added per well under a fume hood.
  • stopping solution phosphoric acid 1M Sigma 43,080-1, CAS 7664-38-2
  • the standard curve is determined by the following function: OD value as a function of the concentration of the recombinant IFN ⁇ .
  • the OD values of the samples tested are converted to concentration using the standard curve. To be taken into account, the samples must give ODs located on the linear portion of the standard curve.
  • lymph node cells distributed in a 96-well plate at a rate of 3.10 5 cells per well are cultured without any stimulant in order to detect expression of the cytokines ex-vivo.
  • the supernatants of the lymph node cells are taken after 24 hours. IFN ⁇ is assayed by the ELISA technique as described above.
  • the lambs After ingestion of colostrum, the lambs are separated from their mothers and are fed with a lamb feeder bucket.
  • the 4 lambs in batch A were immunostimulated with 10 6 tachyzoites of the strain Toxo mic1-3 KO, freshly produced. After inoculation with the strain Toxo mic1-3 KO, no severe clinical sign was found and the lambs immunostimulated with the strain Toxo mic1-3 KO have a weight curve similar to the weight gain of the control lambs ( FIG. 14 ).
  • the lambs from batch A and from batch B were euthanased 15 days after immunostimulation.
  • the lambs of the control batch did not develop an inflammatory response (IFN- ⁇ secretion), either in the spleen, or in the subiliac and popliteal lymph nodes.
  • the cells of the subiliac lymph nodes of 3 lambs out of 4 produce IFN- ⁇ . These lymph nodes are situated downstream of the injection site, in contrast to the popliteal lymph nodes, situated upstream and in which no production of IFN- ⁇ is detected.
  • the inflammatory response of the lambs immunostimulated with the strain Toxo mic1-3 KO is confirmed after restimulation of the splenocytes with total parasite extract of the strain Toxo mic1-3 KO, since production of IFN- ⁇ is induced for the 4 lambs.
  • the splenocytes are also stimulated with concanavalin A, a protein of the lectin family, known to be a polyclonal activator of the T lymphocytes, which serves as positive control of stimulation of the immune system cells.
  • Immunostimulation is carried out on 1-day-old neonate lambs. After ingestion of colostrum, the lambs were isolated from their mothers in a sealed sheep house (INRA-Nouzilly) in order to limit the risks of natural contamination.
  • the lambs were anaesthetized by electronarcosis and then euthanased to collect the various organs. Only the animal keepers and the experimenters, equipped with clothing for use inside the animal house, may enter the buildings, in order to prevent contamination of the environment, and they only leave the sealed zone after showering.
  • Oocysts of Cryptosporidium parvum are obtained from excrement of calves infected with 10 7 C. parvum oocysts.
  • the stool undergoes various treatments until a suspension of sterile, purified parasites is obtained, suitable for use in cell culture. Throughout the treatment, the oocysts are manipulated at 4° C. to prevent excystation of the oocysts.
  • the latter is diluted in fresh water and then passed through a 100- ⁇ m filter and centrifuged at 1900 g for 10 minutes at 4° C.
  • the pellets obtained, containing the oocysts, are taken up in 2% potassium dichromate solution (Prolabo, ref. 26 776 290, CAS 7778-50-9), and are then washed twice with cold water by centrifugation at 1900 g for 10 minutes at 4° C. to remove the potassium dichromate.
  • the pellet of coccidia is taken up in a mixture of water and ether (ethyl ether, Carlo Erba, CAS No.
  • the purified oocysts are then sterilized. After centrifugation at 1900 g for 10 minutes at 4° C., the pellet of oocysts is incubated for 15 minutes in a solution of bleach (sodium hypochlorite (Sigma 239305-500ML Titre: 4.5% of active chlorine)) diluted to 10% in demineralized water, then washed 3 times in sterile 1 ⁇ PBS (diluted from a solution of PBS 10 ⁇ : sodium chloride, NaCl 80 g/litre water; potassium chloride, KCl 2 g/litre; potassium dihydrogen phosphate, KH 2 PO 4 : 2 g/litre; disodium hydrogen phosphate, Na 2 HPO 4 , 12 H 2 O: 29 g/litre).
  • bleach sodium hypochlorite (Sigma 239305-500ML Titre: 4.5% of active chlorine)
  • the purified and sterilized oocysts are counted on a slide (5 ⁇ L of solution of oocysts and 495 ⁇ L of malachite green), adjusted to a concentration of 2 ⁇ 10 8 oocysts/mL, divided into aliquots in 1.5-mL tubes and stored at 4° C.
  • strain Toxo mic1-3 KO The mutant strain of Toxoplasma gondii , in which the genes coding for the proteins TgMIC1 and TgMIC3 have been knocked out (called strain Toxo mic1-3 KO) is maintained by successive passages on a human foreskin fibroblast (HFF) line cultured in DMEM medium supplemented with 10% of foetal calf serum (FCS), 2 mM of glutamine, 50 U/mL of penicillin and 50 ⁇ g/mL of streptomycin.
  • HFF human foreskin fibroblast
  • the lambs in batch B received 10 6 tachyzoites of the strain Toxo mic1-3 KO by intraperitoneal route.
  • the lambs in batch A and in batch B were weighed every 2-3 days to evaluate their weight gain.
  • the lambs After ingestion of colostrum, the lambs are separated from their mother and are fed with a lamb feeder bucket. 3 lambs from batch B and 1 lamb from batch A were unable to feed properly and were euthanased. Since euthanasia took place before the infectious challenge, these animals were statistically removed from the protocol.
  • the daily weight gain (DAG, daily average gain) of the lambs in batch A and in batch B is presented in FIG. 18 .
  • a significantly lower DAG is found for batch A challenged only with C. parvum than for batch B immunostimulated with the strain Toxo mic1-3 KO and then challenged with C. parvum.

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