MXPA99008507A - Isolated viable parasite intestinal cells - Google Patents

Abstract

The present invention provides a homogenous population of parasite cells, wherein the cells are not mosquito cells, capable of prolonged culture in vitro. Fractions from the cells, both cellular and non-cellular are also provided. Further provided is a method of treating or preventing parasite involvement with an animal comprising administering immunogenic amounts of the parasite cells of the invention or fractions derived from the cells to the animal, thereby treating or preventing the parasite involvement with the animal. Further provided is a method of detecting the presence of a parasite in an animal, comprising contacting either the parasite cells or cell fractions of the invention or antibodies of the invention with either an antibody containing sample or an antigen containing sample from the animal and detecting the presence of binding of either the antibodies in the sample with the cells or fractions of the invention or the binding of the antigens in the sample with the antibodies of the invention, the presence of binding indicating the presence of a parasite in the animal. In addition, the invention provides a method of culturing parasite cell populations in vitro comprising culturing a parasite in parasite culture medium under conditions which allow for decomposition and/or degradation of the cuticle layer of the parasite such that cellular buds are produced;disrupting the culture to cause the cellular buds to shear from the cuticle layer;and culturing the parasite cellular buds in cell culture medium. Finally provided is a population of differentiated nematode cells capable of prolonged culture in vitro.

Description

PARASITE INTESTINAL CELLS, VIABLE AND ISOLATED Field of the Invention The present invention relates to viable and isolated parasite cells, as well as to the use thereof. In particular, the present invention relates to vaccines for the prevention or treatment of parasitic infection or infestation by the use of immunogenic material derived from viable and isolated parasite cells.

Background of the Invention Parasite infections are widespread in the livestock and equine production industries. The losses attributed to parasitism are estimated in hundreds of millions of dollars (Gibbs and Herd, 1986). Parasitism can manifest itself as either a clinical or subclinical condition. Although clinical parasitism may appear in a more dramatic form, subclinical parasitism is more pervasive, causing decreases in feeding efficiency, reproductive function and susceptibility to disease. These effects are complicated by the age of the animal, types of parasites present, nutritional and environmental stresses, administration systems, the presence of other disease conditions, genetic histories and numerous other factors (Gibbs and Herd, 1986, Hawkins, 1993). . The control of parasitism in the United States of America has focused on the use of anthelmintics, literally with millions of dollars spent annually on the administration of such products (Lanusse and Prichard, 1993). Traditionally, control has been therapeutic and curative in nature; animals are treated to prevent death rather than infection. Mortality and serious diseases have decreased, but subclinical losses between treatments persist as a result of re-infection from pastures or contaminated feedlot areas. More recently, a more preventative approach to nematode control has become popular, depending either on strategic treatment with anthelmintics alone, or in combination with pasture / pasture management practices (Williams, 1986, Miller, 1993, Stromberg and Corwin). , 1993). Rather than concentrating on adult parasites, the intention of these programs has been to reduce the contamination of pastures with infective larvae, thus reducing the risk of exposure to parasites. This in turn reduces the effects of subclinical parasitism within a herd of cattle. Although anthelmintics provide many economic advantages, their use entails different disadvantages; the remarkable development of resistance and the potential dangers of persistent residues and ecotoxicity (Waller, 1993). The complication of anthelminthic efficacy is the development of resistance, which has been well documented in ruminants. It was first reported in 1957 when it was found that Haemonchus contortus was resistant to phenothiazine (Drudge, 1957). Resistance continues to be a problem even with the new classes of anthelmintics. Although it is mainly a problem in horses and small ruminants, some resistance has also been reported in cattle parasites. The resistance of Ostertagia to levamisole (Lyons, 1981; Geerts, 1987; Williams, 1991; Williams, 1991) and the sustained release of large morantel pills (Borgsteele 1988), with lateral resistance to levamisole (Borgsteele 1991) has been documented. , and the resistance of Trichostrongylus axei and Coopería oncophora to oxfenbendasol (Eagleson &Bowie, 1986, Jackson, 1987). Resistance to anthelmintics occurs with all classes of medications used to control nematodes. Cross resistance, multiple resistance and lateral resistance has been reported (Craig, 1993). It is believed that the development of resistance is promoted by rapid "rotation" between different preparations. Reversal or selection away from the resistance, once the selection pressure has been eliminated, is low (Kelly &; Hall, 1979). The development of vaccines against gastrointestinal parasites of cattle has generally produced less than optimal results. Due to the fact that the gastrointestinal nematodes of ruminants thrive without taking into account the immune system, a vaccine that mimics this immune balance is unlikely to be highly efficient. Vaccines capable of inducing protection through a mechanism different from that of imitation of natural immunity would theoretically be more successful (Willadsen, 1993). Due to their physical location, "hidden" antigens from intestinal tissue are not exposed or "visible" to the host immune system and therefore do not normally elicit an immune response. Vaccination of the host with preparations isolated from "hidden" antigens from several parasites has shown some potential in the induction of lethal immune response. The intestinal tissue from the Anopheles mosquito was used for the first time as a source of antigen for the production of a vaccine. Mosquitoes that had been fed with blood from rabbits injected with homogenates of heterologous cell fractions from the midgut of mosquito had a greater proportion of mortality than those fed with control rabbits (Alger &Cabrera, 1972). Cattle and guinea pigs were immunized with homogenates of heterologous cell fractions containing antigens extracted from the intestine of partially-fed Dermacentor andersoni ticks. Egg production and bloating were significantly reduced in ticks that were fed with vaccinated animals (Alien &Humhreys, 1979). A similar success was achieved with calves vaccinated with Amblyomma americanum (McGowan, 1981). These successes and the emergence of strains of ticks with acaricide resistance promoted the work on the specific tick of cattle, Boophilus microplus. Immunization of cattle with crude extracts of partially fed ticks decreased tick populations (Johnston, 1986). This protection was different from the resistance acquired naturally and involved a hypersensitivity reaction at the tick-binding site, which is a reaction that is not present in the response to immunization (Kemp, 1986). Histopathology of intestinal tissue from ticks fed immunized cattle showed no apparent damage in ticks fed cattle with natural tick infestation (Agbede &Kemp, 1986). In addition, cattle injected with membrane and raw tick intestine adjuvant had significantly higher antibody levels than naturally infested cattle. Cattle vaccinated with crude intestinal membrane antigen and subsequently challenged with parasites did not present any obvious anamnestic response, although the challenge dose was sufficient to produce a low but significant antibody response in unaffected animals (Opdebeeck & Dlay, 1990). These observations support the argument that vaccination with the intestinal membrane and the natural infestation of ticks do not invoke the same immune response.

Experiments with tick extract, crude and purified, showed that the immunoprotective antigen was associated with the parasite gut membrane (Opdebeeck, 1988, Willadsen, 1988, Willadsen, 1989). Additional characterization of the antigen revealed that it consisted of a membrane-bound glycoprotein referred to as Bm86. The immunization of host animals with this antigen decreased the survival of ticks, the weights by overcrowding with blood and fecundity. Antibodies to this antigen rapidly inhibited the endocytotic activity of parasite digestive cells (large lumen lateral gut cells separated from the basement membrane) in the intestine of ticks. This antigen was cloned and expressed as inclusion bodies in E. coli. Ticks fed cattle vaccinated with these inclusion bodies were significantly damaged, but not exterminated (Rand, 1989). Monoclonal antibodies, produced against precipitous antigens of the midgut membrane of Boophilus microplus, were protectors greater than 99% in challenge studies. These antigens separated in a larger band and five minor bands in the conventional application of SDS-PAGE, indicate that the epitope recognized by the monoclonal antibody is repeated in several antigens. It is believed that these antigens are different from Bm86 because vaccination with these antigens results in the extermination of ticks (Lee &Opdebeeck, 1991). Antigens may be common to more than one stage of the life cycle of a parasite and shared reactive epitopes may occur in different proteins at different stages (Maizels, 1987). Because antibody levels have been correlated with the level of protection afforded by immunization with tick-intestine antigen (Opdebeeck, 1988; Lee &Opdebeeck, 1991), larval and adult anthogenic extracts were purified using antigen antibodies. anti-intestine. The protection provided by purified antigens of both larvae and adult was greater than 80%, in this way protective antigens can be common to both stages. Tissue egg membrane extracts were found to be immunogenic, but not protective, to menacing infections. Antibody membrane and anti-gut membrane antibodies were cross-reactive, recognizing common antigens for the tick egg and intestine (Kimaro, 1993).

Because anti-tick antibodies in the serum of cattle vaccinated with tick-bodied membrane and naturally infested cattle with ticks reacted with salivary gland and adult tick gut antigens, as well as with antigens of larva, it was thought that Boophilus microplus intestine antigens were not truly "hidden" antigens (Opdebeeck &Daly, 1990). It was determined, however, that when antisera from naturally infested cattle reacted with Bm86, it was through a cross-reactive carbohydrate epitope that had no deleterious effect on ticks (Willadsen &McKema, 1991). Thus, the Bm86 intestine antigen is "hidden" and its polypeptide epitopes are responsible for providing immunoprotection. The "hidden" antigens have been proposed as a means to control flea infestation in cattle that involve the species Ctenocephalides feli. Immunoglobulins produced in rabbits immunized with homogenates of cell fractions containing antigens from the midgut of fleas were fed to fleas of cattle and showed to have harmful effects. Dogs immunized with crude antigens and challenged with fleas had fewer surviving fleas than the control animals and the surviving females laid fewer eggs (Heath, 1994). The species Haemonchus contortus, an economically important nematode that feeds on blood in sheep, has also been the target of vaccine development. Non-specific immune responses induced by injections of Freund's complete adjuvant provided some protection against Haemonchus contortus (Bautista-Garfias, 1991). The vaccination with cuticular collagen was not protective, although it was immunogenic (Boisvenue, 1991). In contrast, soluble antigens from adults and larvae of the third stage proved to be poor immunogens (Cuquerella, 1991). Contortin is an extracellular polymeric protein that is loosely associated with the luminal surface of the plasma membrane of the intestinal epithelium of nematodes. Vaccination with an extract rich in contortin prepared from total helminth homogenates is protective in young sheep. The population of nematodes in vaccinated animals is smaller in numbers than those found in control animals (Munn, 1987). Whey antibodies precipitated several components of the extract rich in contortin.

Vaccination with crude intestinal tissue extracts from adult nematodes and third stage larvae (L3) provided similar protection in goats (Jasmer &McGuire, 1991). Reductions in the numbers of both worm and egg production were achieved in the immunized group. The antibodies from immune serum recognized seven intestine proteins, some of which were integral membrane proteins. This antigen preparation may contain a significant amount of contortin (Munn, 1993b). Immunohistochemistry provided confirmation that the antigen originated from parasite intestinal cell populations and demonstrated cross-reactivity with microvillar proteins in Ostertagia ostertagi and several small equine strongyles. The reduction in the number of nematodes recovered after immunization with intestine extract from Haemonchus contortus was confirmed by Smith (1993) in young Suffolk sheep. Serum from sheep that exhibit natural immunity to Haemonchus contortus did not react with intestinal membrane proteins, confirming the "hidden" nature of these proteins. Passive transfer with immune serum from vaccinated sheep decreased egg production in the animals that received the vaccine. The presence of host antibodies coating the microvilli can neutralize the necessary proteins (that is, enzymes) that result in the death of 1 worm, or the coating can mechanically block the absorption of nutrients, practically starving the nematode. The Hl 1 antigen, present in the larva of the fourth stage (L4) and the fifth stage (L5) of the Haemonchus contortus is the largest protein of the integral microvilar membrane of the Haemonchus contortus. The vaccination of young Merino sheep with Hl l and with a preparation enriched with Hl l (containing a small amount of peripheral membrane Pl protein) resulted in a reduction in the mean number of nematodes and the production of nematode eggs. The late onset of egg production was observed, suggesting that the effector mechanism may act on pre-adult stages of the parasite (Munn, 1993a). The reduction in the number of helminths and in the production of eggs correlated with the titration of serum antibodies to Hl l (Smith, 1993; Tavernor, 1992a, b). The enzymatic nature of Hl 1 has been deduced from DNA sequencing and confirmed by assay and specific inhibitor studies (Munn, 1993). The activity of Hl 1 is inhibited by serum antibodies from vaccinated animals. Most of the antibodies produced are targeted in Hl 1 (Munn, 1993) and levels of inhibition correlated with protection levels (Munn, 1993). As with contortin, host immunoglobulin appears to be the effector mechanism. Host antibodies bind to the parasite's intestine as early as seven days after infection, with mortality observed in nematodes between days 7 and 14. Younger larvae 7 days after infection are apparently not susceptible to infection. immune response. Lambs immunized with Hl 1 antigens and challenged with drip inoculations were extensively protected against anemia and egg production was observed in the inoculated controls. They grew as efficiently as the controls without infection and acquired natural immunity during the course of the drip infection. Animals challenged with either strains of Haemonchus contortus resistant or susceptible to benzimidasol were similarly protected by vaccination of Hl 1 (Smith &Smith, 1993). The female parasites were lost more quickly than the males, counting for the reduction of egg production. Vaccination with fractions of the enriched extract of Hl 1 of complete helminth showed that the protective activity was mainly associated with Hl 1. Another fraction, Pl or H45 was also protective but in much greater quantities than Hl l. Immunization with Hl l-enriched extract (containing Pl) conferred protection in Dorset lambs and Clun Forest sheep (Tavernor, 1992aMunn, 1993b), but a greater reduction of nematodes was observed in the Clun Forest sheep. This difference in protection could be due to the feeding, quantity of antigen, or age of the lambs. In a direct comparison of the protection conferred by vaccination with contortin-free antigen enriched with Hl l (Munn, 1993b) and by vaccination with antigen enriched with contortin (Munn, 1987), mean protection (ie, number of helminths) and egg production declines) achieved with the contortin-free preparations and enriched with Hll was equal to the best protection achieved with contortin-enriched preparations, even when smaller amounts of Hll protein were used. Thus, Hl l is more effective than contortin. Sufficient protection was achieved with immunization using 100 mg of Hl l antigen and no further protection was demonstrated with higher doses of antigens (Travernor, 1992a). Vaccination with 95% pure Hl l reduced the number of nematodes up to 93% with a 94.6% reduction in egg production. No cross-protection was demonstrated in challenge studies with Ostertagia circumcincta and Nematodirus battus. This may be a reflection of the antigenic difference or due to ingestion of the host immunoglobulin nematode was in insufficient quantities to promote lethal damage (Smith, 1993). Monoclonal antibodies made against surface epitopes of the intestine of Haemonchus contortus identified epitopes also located in the body wall, the cuticle region and in internal organs of third stage larvae (L3), as well as in the intestine and tissues of Ostertagia ostertagi, Trichostrongylus colubriformis, small strongyles of equine and Caenorhabditis elegans (Jasmer, 1992). Another protective component was isolated from the integral membrane fraction of intestinal cells using lectins as ligands to purify the microvilar glycoproteins from complete helminth extracts. This fraction, Haemonchus galactose containing glycoprotein complex or H-gal-GP is easily separated from Hl l or Pl by SDS-PAGE, its lectin binding specificity and its low isoelectric point. In a side-by-side comparative study, H-gal-GP was less protective than Hl 1 and reductions in the number of nematodes were not as large as for Hl 1, although reductions in egg production were similar. As for Hl 1, H-gal-GP is more effective against female helminths than with male helminths. Comparisons in the literature show that H-gal-GP is more effective than the H45 complex. The differences in protection induced by H-gal-GP and H45 may be due to the protocol used for specific immunization. ' - The establishment of a heterogeneous cell line from a plant parasite (caterpillar stage) has been described (Manousis &Ellar, 1990). These authors established that this was the first time that this technique had been successfully performed with a nematode. Populations of heterogeneous (non-specific) cells survived for no more than three months. Supplementation of the growth medium with fetal bovine serum (FBS, 10% v / v) supported the propagation of cell populations during a period of just over five months, (Kurti et al., 1988) describes the spread of heterogeneous cell lines derived from tick embryos Dermacentor variabilis, Rhipicephalus appendiculatus, Rhipicephalus sanguineus and Boophillus microplus \ pn growth medium containing 10% FBS, although there was no attempt to propagate a specific cell line through selective laboratory techniques. In another description of the propagation of a parasite cell line, whole parasites were homogenized as starting material, so that no attempt was made to selectively culture a specific cell line (Hobbs et al, 1993). Homogenato The parasite was transferred to wells of a culture plate with tissue containing a "feeder" cell layer of buffalo rat liver and "free" serum culture medium was used. The modification of a growth medium similar to DMEM to contain less KCl and glucose allowed maintenance of viable cell lines for four weeks or more. Culturing juvenile helminth cells on an irradiated buffalo rat liver (BRL) feeder layer lengthened the viability of cell clusters from a few weeks to as long as six months. The feeder layers of bovine endothelial cells or mouse embryo (3T3) were less effective. Kurtti and Munderloh (1984) described the production of mosquito cell cultures from larval tissues, embryonic and adult ovarian tissues, resulting in the cultivation of heterogeneous mixtures of mosquito cells for several years. Munderloh et al. (1994) described the spread of heterogeneous cell populations from embryonated tick eggs. These researchers described the difficulty in keeping the cells in liquid nitrogen for long periods of time. The cells were propagated in tissue culture growth medium containing fetal bovine serum (FBS 20% v / v). The time interval between the initiation of the main culture and the first subculture varied from 6 to 12 months. As can be seen from the studies described above, antigens expressed by parasite cells have been shown to be potential in providing protective immunity in sheep and cattle. Unfortunately, these fractions were derived from intestinal parasite tracts that had been harvested manually by microdissection. Thus, it is expensive and labor-intensive to obtain sufficient amounts of immunogenic proteins. In addition, it is difficult to obtain sufficient purity of antigens and identify antigens that may be useful in vaccines. The cell lines that have been established from parasites have been heterogeneous and undifferentiated populations difficult to sustain in an environment of culture medium for long periods of time. Thus, there is a need for homogeneous populations of parasite cells that can be sustained in culture for extended periods of time. The present invention satisfies this need by providing homogeneous populations of parasite cells that are susceptible to sustain in culture for extended periods of time, as well as methods to produce such homogeneous populations.

OBJECTIVES OF THE INVENTION The present invention provides a homogenous population of parasite cells, wherein the cells are not mosquito cells, capable of prolonged in vitro culture. Fractions from cells, both cellular and non-cellular, are also provided. Antibodies that specifically bind to cells or cell subfractions and anti-idiotype antibodies are also provided. Also provided is a method for the treatment or prevention of the wrapping of an animal, with parasites, which comprises the administration of immunogenic amounts of the cells of the parasite of the invention or fractions derived from the cells to the animal, treating or preventing in this way the wrapping with parasites of the animal. The invention also provides a screening method for a compound for determining anthelmintic activity, which comprises contacting the compound with the cells of the invention and determining whether the compound has a damaging effect on the cells. Furthermore, a method for detecting the presence of a parasite in an animal is provided, which comprises contacting the cells of the parasite of the invention or fractions derived from the cells with an antibody containing sample from the animal and detecting the presence of binding of the antibodies that are in the sample with the cells or fractions, the presence of binding indicating the presence of a parasite in the animal. A method for detecting the presence of a parasite in an animal is also provided, which comprises contacting antibodies of the invention with a sample from the animal potentially containing parasite antigens, and detecting the presence of binding of the antibodies with an antigen. antigen, the presence of binding indicating the presence of a parasite in the animal. In addition, the invention provides a method for culturing populations of parasite cells in vitro, which comprises culturing a parasite in parasite culture medium under conditions that allow the decomposition and / or degradation of the parasite cuticle layer, such as so that cell outbreaks occur; dissolving the culture to cause the cell shoots to detach from the cuticle layer; and culturing the parasite cell buds in cell culture medium. The present invention also provides a population of parasite cells, not including mosquito cells, capable of prolonged in vitro culture, produced by the method of culturing populations of parasite cells in vitro comprising culturing a parasite in low parasite culture medium. conditions that allow the decomposition and / or degradation of the cuticle layer of the parasite, in such a way that cellular outbreaks occur; dissolving the culture to cause the cell shoots to detach from the cuticle layer; and culturing the parasite cell buds in cell culture medium. Finally, the present invention provides a population of differentiated nematode cells capable of prolonged in vitro culture. Various other objects and advantages of the present invention will become clearer from the following description.

Brief Description of the Drawings Figure 1 shows parasite cells propagated in a tissue culture environment (in vitro): A: the clusters represent cellular outbreaks of parasites that have been expelled from the cuticular envelope of the larvae. B: the cell clusters are of a diameter larger than that of the individual larvae, indicating the in vitro proliferation of the cell shoots after the expulsion of the larva.

Detailed Description of the Invention The present invention is described more particularly in the following examples, which are intended to be illustrative only since numerous modifications and variants will become clear to those skilled in the art. As used in the claims, "a / a" may include multiples. This invention provides a homogenous population of non-mosquito parasite cells capable of prolonged in vitro culture. By "homogeneous" it is meant that the cells are substantially only of one type. For example, the homogeneous population can consist of any number in the range of between 100 and 80% of cells of a type, such as 100%, >95%, > 90%, > 85%, > 80%, 79% and in the preferred range, especially 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%. The percentage of cells of a cell type in the homogeneous population can be determined by standard methods in the art, such as, for example, fluorescence-activated cell sorting (Harlow and Lane, 1988). By "capable of prolonged culture" is meant that the cells can be passaged and frozen and reconstituted in such a way that the cells can be maintained as a homogeneous population for an indefinite period of time. For example, the cells of the present invention have been maintained as a homogenous population of cells for up to three years. Because these cells are maintained as a suspension culture, these cells are passed in the best form when it can be seen that the bottom of the tissue culture flask contains a significant amount of cell pellet. The cells of this invention can be passed either by "cleaving" the cells in a culture flask into • additional tissue culture flasks or by adding additional cell culture medium to the existing vial. Additionally, the cells of the present invention can be frozen for a long storage as viable cells according to the standard protocols that are known in the art for the cryopreservation of cultured cells. For example, medium containing cells can be centrifuged for granulation of the cells and the supernatant medium can be discarded. The cell pellets can then be resuspended in frozen medium (for example, IPL-41 or SF-900 medium or a 50/50 mixture of IPL-41 and SF-900) containing, for example, 10% (v / v) of DMSO and supplemented with amikacin (2.5 mg / ml) and oxacillin 2.5 mg / ml). The medium and the cells are then transferred to cryophrases and placed at -96 ° C for one hour. The cryophrases are then placed inside and continuously maintained in liquid nitrogen until they are reconstituted. To reconstitute the cells, for example, the cryophrases containing the cells are removed from the liquid nitrogen and the outside of the bottle is sterilized with alcohol. The cells are thawed by placing the bottle in a water bath at room temperature (RT, 25 ° C). Two ml of cell culture medium are added to the bottle to dilute the DMSO and the cells are granulated at low speed (200-300 x g). The supernatant is decanted and fresh medium is added to the cells and they are subjected to granulation again. The supernatant is decanted again and the cells are resuspended in tissue culture medium which is approximately 90% fresh medium and 10% half spent, and transferred to a tissue culture flask. Populations of cells cultured from larvae or reconstituted after cryopreservation can be passed an unlimited number of times and can be maintained as viable cells indefinitely (ie, for a period of two years). The parasite cells derived or used in this invention may be of any type of cells found in the adult or larval parasite. For example, such cells may include, but are not limited to, intestinal, secretory, esophageal, muscular, neurological and reproductive cells, such as uterine cells. The intestinal cells are especially useful for the treatment and prevention of involvement with parasites. As described in more detail below, the cells can be used, for example, for treatment, prevention and diagnostic purposes. For either of these purposes, the cells can be used either intact, partially or fully used, as well as with or without the culture medium in which the cells are being cultured. Thus, different cell fractions can be derived from the parasite cells. For example, membrane-associated antigens can be separated from the rest of the culture and cellular components as membrane fractions and used for various purposes. Other cell fractions can include, but are not limited to, total cell fractions, subcellular organelle fractions, enzyme fractions, genetic material (eg, RNA, DNA), etc. In addition, the medium in which the cells are cultured can be used without the cells, since certain cellular components of the cells will have been released into the medium that can be harvested and analyzed as non-cellular fractions. The medium with and without the cells and / or the cells, used or without lysate, can be used in therapeutic, prophylactic or diagnostic assays to optimize as best as possible the ratio of cells to medium and the ratio of cells used to un-lysed. Optimization would involve conducting in vitro and / or in vivo efficacy tests according to standard protocols in the art, to determine or identify those proportions of medium: cell and lysed cell: unlysed cell proportions capable of producing therapeutic, prophylactic results or optimal diagnosis (for example, Smith, 1993). The cells of the invention can be used by any standard means in the art, such as detergent solubilization and mechanical disruption (Travenor et al., 1992). Various cell fractions can be separated from cell lysate by standard cell fractionation techniques, such as, for example, gel filtration chromatography, ion exchange chromatography, affinity chromatography, high pressure liquid chromatography (HPLC) and the like. , as are well known in the art (eg, Travenor et al., 1992; Munn et al., 1993; McKerrow et al., 1990; Gambel et al., 1990). Alternatively, a protein fraction of parasite cells can be obtained by treating the cells with an ionic detergent such as sodium dodecyl sulfate or a non-ionic detergent such as Triton X-100 (C34H6O11, average) or ethylphenyl-polyethylene glycol (NP) -40, Shell Oil Company). The protein fractions thus obtained can be tested to determine the immunogenicity, specificity and biochemical enzyme activity as described above. Other immunogenically specific determinants of the parasite cells can be obtained by standard methods, for example, as described above. Proteins and protein fragments produced by cells of this invention can be isolated and purified, and the amino acid sequence and nucleic acid sequence of these proteins and protein fragments can be determined according to standard methods in the art. The nucleic acids encoding proteins and protein fragments can be cloned into vectors and expressed in transgenic cells and / or animals according to molecular genetic protocol well known to those skilled in the art (see, for example, Sambrook et al, 1989).

Other components can be added to the used or unlysed cells, cell fractions derived from cells or medium removed from the cell cultures. Thus, the invention provides compositions comprising these components and may include an effective amount of the cells, fractions thereof or non-cellular fractions, in combination with a pharmaceutically acceptable carrier and, in addition, may include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc. By "pharmaceutically acceptable" is meant a material that is not biologically undesirable or in some other way, that is, the material can be administered to an individual in conjunction with the selected component without causing any undesirable biological effects and which are important, or interact in a pernicious way with any of the other components of the pharmaceutical composition in which it is contained. Current methods for the preparation of the dosage form are known, or are clear, to those skilled in the art (see, for example, Martin, latest edition, Arnon, 1987). The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject. In another embodiment the composition could include an adjuvant to improve the therapeutic or prophylactic effect of the active ingredient. The adjuvant can be selected by standard criteria based on the particular antigen used, the mode of administration and the subject (Arno, 1987). For example, the composition may include Freund's complete adjuvant, incomplete Freund's adjuvant, aluminum hydroxide or any other adjuvant known to improve the immunogenicity of an antigen. Examples of parasites that can be used to generate the homogenous populations of parasite cells of the present invention may include, but are not limited to, nematodes, trematodes, annelid helminths and cestodes, as well as arthropod and arachnid species (e.g. ticks, mites, lice and fleas). A particularly useful parasite homogenous cell line is derived from a nematode. Examples of nematodes useful in the present invention may include, but are not limited to Cooperia, Oesophagostomum, Ostertagia, Haemonchus, Dirofilaria immitis and Dictyocaulus. Economically important parasites that may be employed in the present invention may include, for example, Cooperia bisonis, Cooperia cuticei, Cooperatia mcmasteri, Cooperatia oncophora, Cooperia pectinata, Cooperia punctata, Cooperia spatulata, Cooperia surnabada, Dictyocaulus viviparus, Haemonchus contortus, Haemonchus placei, Haemonchus similis, Oesophogostomum radiatum, Ostertagia bisonis, Ostertagia orloffi, Ostertagia ostertagi, Trichostrongylus axei, Trichostrongylus, Trichostrongylus longispicularis, magna Fasciola, hepatic Fasciola, Amblyomma americanum, Amblyomma cajennense, Amblyomma maculatum, Boophilus annulatus, Dermacentor albopictus, Dermacentor andersoni, Dermacentor occidentalis , Dermacentor variabilis, Ixodes cookei, Ixodes pacificus, Ixodes scapularis, Chorioptes bovis, Psorergates bos, Psoroptes ovis, Sarcoptes scabiei, Oesophogostomum columbianum, Oesophogostomum venulosum, Ostertagia circumcincta, Ostertagia occidentalis, Ostertagia trifurcata, capricola Trichostrongylus, Nematodirella longispiculata, Nematodirus abnormalis, Nematodirus davtiani, Nematodirusfilicollis, Nematodirus helvetianus, Nematodirus lanceolatus, Nematodirus spathiger, Ascaris suum, Hyostrongylus rubidus, Oesophagostomum brevicaudum, Oesophagostomum dentatum, Oesophagostomum georgianum, Oesophagostomum quadrispinulatum, Strongyloides ransomi, Strongyloides westeri, Trichuris suis, Strongylus edentatus, Strongylus equinus, Strongylus vulgaris, Strongylus westeri, Dirofilaria immitus and Ascaris canis. The invention also provides an antibody or ligand that specifically binds to the parasite cells or fractions of the cells of the present invention. As used herein, the antibodies can include immunoreactive antibody fragments. These antibodies can be made by standard techniques well known in the art (see for example, Harlow &; Lane, 1988). Monoclonal or polyclonal antibodies elicited against antigens (e.g., derivatives of the present intact cells or from purified cell fractions from cell lysates) can be used as diagnostic reagents to detect antigens in tissue or body fluids of a animal, as well as to purify parasite antigens through the use of affinity capture and other antigen purification techniques. The antibodies of this invention can also be used in therapeutic applications to treat or prevent the wrapping of an animal with parasites. The antibodies can be either purified directly from an immunized animal, or spleen cells producing antibodies can be obtained from the animal by producing hybridoma. Spleen cells are fused with an immortal cell line and are maintained as hybridomas for antibody secretion. Similarly, purified polyclonal antibodies, specifically reactive with the antigen, are within the scope of the present invention. Polyclonal antibodies can be obtained by standard protocols of immunization and purification (Harlow and Lane, 1988). Detection of the reaction of the ligand or antibody with antigen can be facilitated by the use of a ligand or antibody that is bound to a detectable moiety. Said detectable fraction will allow visual detection of a precipitate or a color change, visual detection by microscopy, or automated detection by spectrophotometry, radiometric measurements or the like. Examples of detectable fractions include fluorescein and rhodamine (for fluorescence microscopy), horseradish peroxidase (either for light microscopy or electron microscopy and biochemical detection), biotin-streptavidin (for light or electron microscopy) and phosphatase alkaline (for biochemical detection by color change). The detection method and the detectable fraction used can be selected from the above list or other appropriate examples according to standard criteria applied to said selections (Harlow and Lane, 1988). An anti-idiotype antibody that binds specifically to antibodies is also provided. Said anti-idiotype antibody could be used naturally as an immunogen to provide therapeutic or prophylactic effect against a parasite. The anti-idiotype antibodies represent the image of the original antigen and can function in a vaccine preparation to induce an immune response to a pathogenic antigen, thus preventing immunization with the pathogen or the pathogenic antigen itself (Harlow &Lane, 1988). The invention also provides a method for the treatment or prevention of the wrapping of parasites with an animal, including humans, which comprises the administration of immunogenic amounts of the parasite cells or fractions derived from parasite cells, to the animal, treating or preventing in this way the parasite envelopment with the animal. By the phrase "encirclement of the animal with parasites" is meant any interaction or relationship between a parasite and an animal, whereby the parasite infects or infests an animal, attaches to the animal or takes fluid feed from the blood, other tissue or of the animal's body, whether the animal is alive or dead. Parasite cells, cell fractions or non-cellular fractions can be tested for immunogenicity by methods known in the art (Harlow &Lane, 1988, Arnon, 1987). Briefly, several concentrations of potentially immunogenic cells or specific cell fractions are prepared and administered to the animal in various concentrations and the immunological response (e.g., antibody production or cell-mediated immunity) of the animal at each concentration is determined by standard protocols. The amount and type of immunogen administered will depend on the species, size and condition of the animal. Thereafter, an animal thus inoculated with the immunogen can be exposed to the parasite to test the effect of the potential vaccine of the specific immunogen. The specificity of the potential immunogen can be ascertained by testing sera and other fluids, as well as lymphocytes from the inoculated animal, to determine cross-reactivity with other closely related parasites. Once immunogenicity is established, the amount of the immunogen to be administered to a particular animal can be optimized according to standard procedures and as is known in the art (Harlow & amp; amp;; Lane, 1988, Arnon, 1987). In a preferred embodiment, the immunogen may comprise a "hidden antigen" which is an antigen produced by the parasite which is located in or within the parasite in such an anatomical location that, under typical circumstances of animal wrapping with parasites, including humans , the animal's immune system does not have direct access to the antigen (for example, antigens expressed in intestinal parasite cells). If the animal is effectively challenged with an immunogen that is a "hidden antigen" of a parasite, it is possible to induce a cellular and / or humoral immune response in an animal that is therapeutic or protective against the immunogen and, thus, against the parasite. . For example, if an antigen from an intestinal parasite cell is properly presented to the animal and a humoral and / or cellular immune response is induced, subsequent ingestion of parasites having the antigen may promote a fatal event for the parasite, through either a direct cytotoxic effect on the parasite or an interference with the nutrient absorption properties of the intestinal tract of the parasite due to antibody binding, or a combination of both mechanisms. Also in a preferred embodiment, the immunogens or antigens of the present invention are cross-reactive with immunogens or antigens from different species of parasites, ie, these antigens or immunogens are "shared" between different species of parasites. Such shared antigens or immunogens can be identified as being immunologically reactive cross-linked with antigens or immunogens of other species according to standard serological protocols in the art to identify cross-reactive antigens. The antibodies used in these cross-reactive studies can be produced according to the methods described herein. The antigens can be selected for the determination of their immunological cross-reactivity capacities based on the similarities in molecular weight, binding affinity With-A, enzymatic activity, etc. according to the methods provided here. The mode of administration of the immunogen may vary depending on the species, size and condition of the animal. The therapeutic or prophylactic immunogen of the invention is typically administered parenterally, either subcutaneously or by intramuscular injection. Of course, the immunogenic amount can be given in divided doses or administered at multiple sites in the animal. For example, the immunogen can be administered in a single dose or in two doses at several intervals (e.g., two or four weeks). Booster immunizations may also be given at various intervals (eg, at biweekly intervals) as required to maintain the desired therapeutic or prophylactic effect. Immunizations may also be administered to subjects as "drip inoculations", such as, for example, by administering about 50-100 μg of immunogen to a subject either subcutaneously or intramuscularly every other day for a period of 14-30 days. . Parenteral administration is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. A recently revised approach for parenteral administration involves the use of a slow release or sustained release system, such that a constant level of immunogen is maintained. See, for example, U.S. Patent No. 3,710,795. The animal can be any animal in which there is a real or potential need for prevention of involvement with parasites. Typical animals can be selected from the group consisting of, for example, humans, cattle, equines, pigs, goats, sheep, canines, felines and poultry species. Similarly, the animal could be a kind of wild animal in which there is a desire to control parasites, for example, in zoological facilities where wild animals are kept and / or in situations where the parasites could be passed from one animal to another. wild animal to a pet or to the human being. The invention also provides a method for the treatment or prevention of the wrapping of an animal with parasites, which comprises the administration of antibodies of the invention to the animal. In a related manner, the invention provides a method for the treatment or prevention of the wrapping of an animal with a parasite, which comprises the administration to the animal of anti-idiotype antibodies for the antibodies of the invention. Specifically, whole cells or antigenic fractions can be harvested from populations of parasite cells of the present invention and subsequently purified according to the methods provided herein. These antigens can be used in the production of monoclonal antibodies according to well-known protocols for the production of antibody-secreting hybridomas, as well as for the generation of polyclonal antibodies by the immunization of animals (for example, mice, rabbits, etc.). with the antigens and purifying the antibodies resulting from the animal's serum by well-known protocols such as affinity chromatography. These antibodies can then be used as a source of secondary antigen to produce anti-idiotype antibodies according to the same protocols described herein. The invention further provides a screening method for a compound for determining anthelmintic activity, which comprises contacting the compound with the cells of the invention and determining whether the compound has a detrimental effect on the cells. The only requirement for a successful screening of the compounds is that a quantifiable number of cells from the culture of homogeneous parasite cells is viable so that the number of cells that are affected by the compound can be determined.

Thus, the composition may further comprise cells that do not come from the homogenous population of parasite cells. By "detrimental effect", as used herein, can include any effect that can be observed and that can be recognized as atypical of physiologically normal cells in tissue culture, such as, for example, a change in the condition or appearance of the cells of parasite that is pathological (eg, cytopathology, syncytial formation, altered biochemical function, failure in the production or expression of antigens, adherence or non-adherence to tissue culture substratum, rounding or crushing of cells, breakage of a monolayer, abnormal clumping of cells, growth in multiple layers, abnormal cell inclusions, etc.) or death of parasite cells. In the screening method, the compound can be an antibody or another molecule, including synthetic, organic or normal-occurring molecules (Baron et al., 1989; DeClercq, 1989). Such organic molecules may have active site-directed properties that inhibit parasites in vitro or in vivo. Additionally, any molecule that interferes with any phase of the parasite's life cycle can be identified and sifted according to the methods present. The compound can be screened by contacting various concentrations of the compound of interest with the cells of the present invention. The concentrations can be selected empirically or can be extrapolated from the teachings of the art in relation to the use of the compound for other applications. If the compound is added to cells in a tissue culture environment, variables such as pH, temperature and adjunct compounds can also be evaluated according to standard protocols in the art, to determine their influence on the efficacy of the compound under study. produce a detrimental effect on the cells. After an appropriate period of time following the contact of the compound with the cells, the cells can be examined to determine such detrimental effects as cytopathology, cell death, etc. Those compounds that demonstrate a detrimental effect on the cells of the present invention can then be tested on complete parasites to determine detrimental effects on the whole organism, or administered to animals to test the ability of the compound to treat or prevent the wrapping of the animal with parasites. .

The invention also provides a method for detecting the presence of a parasite in an animal, which comprises contacting the parasite cells of the invention, or fractions derived from the cells, which contain parasite antigens, with an antibody containing sample from the animal, and detect the presence of binding of the antibodies in the sample with the antigens in the cells or fractions, the presence of binding indicating the presence of a parasite in the animal. Well-known detection methods, such as immunofluorescence assays (IFA), enzyme-linked immunosorbent assays (ELISA) and Wester blotting can quickly be adapted to carry out the detection of either parasite antigen or antibodies specifically reactive with them . Specific reagents and protocols for use in the detection methods described herein and similar assays can be selected from those available in the art based on standard criteria (Harlow and Lane, 1988). An example of the method for detecting antibodies specifically reactive with parasite cells and cell fractions can be performed by contacting an antibody containing sample from the subject with an amount of parasite cells or cell fractions of the present invention, and detecting the reaction of the antibody with a parasite antigen. A specific embodiment of the antibody detection method of the present invention may be one of ELISA. Briefly, purified parasite cells or cell lysates are attached to a substrate (eg, membrane, bead, plate); Non-specific proteins are blocked with a suitable blocking agent and subsequently contacted with a sample of the subject for the capture of antibody by parasite antigen. Subsequently, a secondary antibody is added, which binds to the antibody captured by the antigen. The secondary antibody can include a portion of enzyme that can produce a colored reaction product which can be detected by the addition of the appropriate enzyme substrate and by observing and / or measuring the colored reaction product. The invention also provides a method for the detection of the presence of a parasite in an animal, which comprises contacting antibodies of the invention with a sample from the animal potentially containing parasite antigen, and detecting the presence of binding of the parasite. antibodies with a parasite antigen, the presence of binding indicating the presence of a parasite in the animal. An example of the method of detecting parasite antigen is by contacting a sample of tissue or fluid from the subject with an amount of a purified antibody of the present invention, and detecting the reaction of the antibody with a parasite antigen. . A specific embodiment of the antigen detection method of the present invention may be one of ELISA. Briefly, antibodies are attached to a substrate (e.g., membrane, count, plate); Non-specific proteins are blocked with a suitable blocking agent and subsequently contacted with a sample of the subject for the capture of parasite antigen by antibody. Subsequently, a secondary antibody is added, which binds to the antigen captured by the antibody. The second antibody can include an enzyme portion that can produce a colored reaction product which can be detected by the addition of the suitable enzyme substrate and by observing and / or measuring the colored reaction product. As contemplated herein, the antibody can include any ligand that binds to a parasite antigen, for example, an intact antibody, a fragment of an antibody or any other reagent or compound that is responsive to a parasite antigen. The subject sample of this method can comprise any body tissue or fluid that may contain a parasite antigen or a cell containing a parasite antigen, such as a biopsy material, blood, plasma, serum, saliva or urine. Other possible examples of bodily fluids include sputum, mucus, semen, gastric fluids, joint fluids, cavity fluids and the like. In addition, the invention provides a method of culturing parasite cell populations in vitro, which comprises culturing a parasite in a parasite culture medium (eg, IPL-41 medium with antibiotics) under conditions that allow for decomposition and / or degradation of the cuticle layer of the parasite, such that cell shoots occur, breaking the culture to cause the cell shoots to detach from the cuticle layer of the parasite; and culturing outbreaks of parasite cells in cell culture medium (eg, a mixture of IPL-41 / SF900 media with antibiotics or only SF900 medium with antibiotics). This method may further comprise purification of the cell shoots in a Percol density gradient, rinsing the cells in PBS and inoculating the cells in the cell culture medium. Cell sprouts can be purified in a Percol density gradient shortly after cell buds have begun to form inside and / or outside the parasite's cuticle layer and cell sprouts can be subsequently removed from the gradient and placed in cell culture medium. Cell sprouts can also be purified in a gradient with Percol density after having been transferred from the parasite medium to the cell culture medium. One or both stages of purification can be carried out by density gradient. A layer may also be formed in the Percol density gradient consisting of parasites from which only cell shoots and parasites have only been partially formed from which cell shoots have not yet formed. These parasites can be removed from the gradient and placed back into the parasite culture medium and allow cell buds that can be harvested for culture to form. This stage can be repeated until the parasites that are able to form cell shoots have done so. The method of culturing parasite cells of this invention can also comprise transferring the cell shoots from the cell culture medium to fresh cell culture medium. In this method of cultivation, it is preferred that an average of at least two outer cell shoots per parasite be produced before the breaking step. It is also preferred that the degradation be smooth, for example, with low speed centrifugation (200 to 400 x g) in conical tubes, followed by serial aspiration with manual pipette. It is further preferred that the degradation be gradual, for example, the degradation or decomposition of the cuticle layer of the parasite may occur in two to three weeks as monitored by direct microscopy examination. However, some parasite species require shorter or longer degradation periods and can be optimized by evaluating the degradation process by direct microscopic examination. The medium used to grow parasites, particularly larvae, can be IPL-41 medium or another medium of similar composition to which antibiotics are added at concentrations of at least 50 to 100 mg / ml. For example, the parasite medium can be IPL-41 medium to which an aminoglycoside antibiotic (eg, amikacin) has been added at a concentration of at least about 50 to 100 mg / ml and a β-lactam and / or antibiotic of Cephalosporin (for example oxacillin) has been added at a concentration of at least about 50 to 100 mg / ml. Antifungal agents (e.g., fungizone at about 50 μg / ml) can also be added to the medium. The parasite medium may also contain yeast extract as a non-whey protein source. The cell culture medium can be IPL-41, SF900, a mixture of IPL-41 / SF900 in any proportion, or other medium of similar composition to which antibiotics have been added at a concentration of at least 2.5 mg / ml. For example, the cell culture medium can be SF900 medium to which amikacin has been added at a concentration of approximately 2.5 mg / ml and oxacillin has been added to a concentration of approximately 5.0 mg / ml. Antifungal agents (e.g., fungizone at about 50 μg / ml) can also be added to the medium. The cell culture medium can also contain yeast extract as a non-whey protein source. Tissue culture flasks containing the cells of this invention can be filled with media to about 33 to 80% of the total volumetric capacity. The bottles can be manually sealed and agitated (aerated) at the time of media supplementation (this is at approximately one week intervals). The method of the present invention may also comprise dissecting an organ from a larva at any stage, L1-L5, according to artery dissection protocols known in the art and placing the cut organ in the culture medium (e.g. , medium IPL-41 with antibiotics) under conditions that allow cell growths to occur; fractionating the crop to cause the cell shoots to detach from the cut organ; and by growing the cell shoots in cell culture medium (eg, mixture of IPL-41 / SF900 medium with antibiotics or SF900 medium with antibiotics). The same considerations, modifications and additional steps described herein as applicable to the culture of a population of homogenous parasite cells from parasites are also applied to sectioned organs. The viability of the cells of the homogeneous population of the present invention can be determined with life-dyeing protocols well known in the art, such as, for example, the MTT staining protocol (3- [4,5-dimethylthiazole-] bromide. 2-yl] -2,5-diphenyl tetrazolium; Sigma) Coyne et al., 1993 (a); Coyne et al., 1993 (b)) described in the examples presented here. The types of homogeneous parasite cell populations can be identified by a variety of methods well known in the art. For example, the cells in the culture can be examined microscopically to determine morphologically that all the cells in the population are of the same type. Cell types can be further characterized by measuring or detecting the expression of cell-specific markers, such as enzymes or membrane antigens. Expression of cell surface antigens can be detected by several immunocytochemical protocols well known in the art, such as, for example, immunofluorescence, flow cytometry (fluorescence activated cell sorting), immunostaining, immunoblotting, etc. Cell types can also be characterized by biochemical assays of cell-specific enzymes employing specific substrates of enzyme and electron microscopy. Polymerase chain reaction (PCR) protocols can also be used to characterize cell types by the presence of cell-specific nucleic acids. Specific examples of how the populations of parasite cells of the present invention can be characterized as a cell type can be found in the Examples provided herein. The present invention also contemplates methods for improving the adhesion of parasite cell populations to the surface of tissue culture flasks through a variety of mechanisms including, but not limited to, preventing the breakdown of cellular contents within the cells. flasks, sealing of the lid on the tissue culture flasks, supplementation of tissue culture growth medium with lipid extracts, continued incubation at 37 ° C in a tissue culture environment and / or continued supplementation with growth medium in a weekly basis. The potential application of these methods would include the use of adherent parasite cell populations as a biological tool for screening the efficacy of new and conventional pharmaceutical agents (e.g., anthelmintic preparations). The present invention further provides a population of parasite cells, not including mosquito cells, capable of prolonged in vitro culture, produced by the method of culturing a parasite in a parasite culture medium (eg, IPL-41 medium with antibiotics) under conditions that allow the decomposition and / or degradation of the cuticle layer of the parasite, such that cell shoots occur, rupture of the culture to cause the cell shoots to detach from the cuticle layer of the parasite; and culture of the outbreaks of parasite cells in cell culture medium (eg, a mixture of IPL-41 / SF900 media with antibiotics or only SF900 medium with antibiotics). The population of parasite cells produced by this method can be either a homogenous population of parasite cells wherein the parasite cells are substantially of a cell type as defined herein, or a homogenous population of parasite cells wherein the parasite cells are not substantially of a single type. The population of cells produced by this method can be of any type of parasite cells and preferably are intestinal parasite cells. These cells can be used, fractionated and / or provided in a pharmaceutically acceptable carrier under all the conditions described herein. In addition, antigens from these cells can be isolated and purified as described herein and used as immunogens in vaccine preparations and for the production of antibodies and anti-idiotype antibodies as described herein. The amino acid and nucleic acid sequences of the proteins of these cells can be determined as described herein and the genes for the proteins of these cells can be cloned and expressed in appropriate expression systems. The parasite from which the population of cells is obtained by the method described herein can be of any parasite as described above and is preferably a nematode, and more preferably is a nematode selected from the group consisting of Cooperia species, Oessophagostomum, Ostertagia, Haemonchus, Dirofilaria and Dictyocaulus. Finally, in a particular embodiment, the present invention provides a population of differentiated nematode cells capable of prolonged in vitro culture. As used herein, "differentiated" means that the cells originated from an organ / tissue system derived from a developed parasite (eg, larva of stage L? -L5 and / or adult parasites) as determined by the expression of antigens and / or enzymatic activities that are known to be expressed only by adult larvae and parasites. The expression of such antigens and / or enzymatic activities can be detected according to the protocols provided in the Examples herein.

The population of nematode cells can be either a homogenous population of nematode cells wherein the nematode cells are substantially of a cell type as defined herein, or a heterogeneous population of nematode cells where the nematode cells do not they are substantially of a single cell type. The population of nematode cells produced by this method can be of any type of nematode cells and preferably are intestinal nematode cells. These cells can be used, fractionated and / or provided in a pharmaceutically acceptable carrier under all of the same conditions as described herein. In addition, antigens from these cells can be isolated and purified as described herein and used as immunogens in vaccine preparations and for the production of antibodies and anti-idiotype antibodies as described herein. In addition, the amino acid and nucleic acid sequences of the proteins of these cells can be determined as described herein and the genes for the proteins of these cells can be cloned and expressed in appropriate expression systems. The nematode from which the population of nematode cells is obtained can be of any nematode and more preferably is a nematode selected from the group consisting of Cooperia, Oesophagostomum, Ostertagia, Haemonchus, Dirofilaria and Dictyocaulus species. The present invention is described more particularly in the following examples which are intended to be illustrative only since numerous modifications and variants to these will be clear to those skilled in the art.

EXAMPLES Preparation, purification and propagation of parasite cell populations: L3 stage parasite larvae from the nematode species Cooperia, Haemonchus for example, Haemonchus contortus), Oesophagostomum, Ostertagia for example, Ostertagia ostertagi) Dictyocaulus for example, Dictyocaulus viviparous ) and Dirofilaria for example, Dirofilaria immitus) were used as a source for the preparation of homogenous parasite cell populations in vitro. L3 stage larvae were selected on the basis that, at this stage of development, the cells of the gastrointestinal tract of the parasite predominated over other types of cells because other organ systems of the organism are still very immature. Therefore, the probability that the cells obtained from the cell shoots of the L3 stage larvae are identical cells is substantially increased. However, any stage of parasite larvae (eg, L ?, L2, L, L5) of parasite larvae can be used as starting material in the present invention. Initial larval preparations: bacterial decontamination and debris removal from faecal plants: viable larval populations ingested plant debris and bacterial flora as a source of nutrients from the lumen of the host's intestinal tract. For this reason, variable larval preparations were suspended in physiological brine supplemented with antibiotics and anti-fungal formulations (ampicillin, oxacillin, fungizone) and incubated at 4 ° C for 24 to 48 hours to decontaminate the preparations prior to tissue culture procedures. The larvae treated with antibiotics were rinsed in sterile phosphate-buffered brine (PBS) (pH 7.4) and separated from fecal waste in Percol density gradients (S.G. 1025, 1050, 1075, 1100). The larval populations that were concentrated in stratified bands were harvested by pipette and their identity was verified by light microscopy. The harvested larvae were then suspended in PBS and centrifuged at low speed (200 x g). The resulting supernatant containing residual Percol density gradient medium was removed by pipette and discarded. The rinsing of the larvae was necessary to remove the entire medium of the Percol density gradient by virtue of adversely affecting the tissue culture medium formulations. Percol density gradient separation procedures were repeated as necessary to obtain a rich larval preparation with increasing purity free from contamination by fecal plant material. Frequently, there is a minimum amount of waste that can not be separated from the larvae by pure purification by Percol density gradient. The larval preparations can be further purified by differential centrifugation using low speed centrifugation (200-300 x g) and / or gravity sedimentation. Alternative procedures that are effective for the separation and harvesting of viable larval populations from faecal plant debris may include the application of a fine wire mesh division or multiple layers of cloth. In this procedure, the larvae that migrated actively to the other site of a fine wire mesh or cloth layers are harvested for culture (Baerman technique).; Ivens et al., 1978). Propagation of parasite cell populations in a tissue culture environment (in vitro): Larvae that have been decontaminated from material of bacterial flora and fecal plant, in addition to the elimination of residual medium of Percol density gradient, are inoculated in tissue culture flasks containing serum-free growth medium such as Grace tissue culture medium, IPL-41 (Cooperia, Haemonchus, Oesophagostomum), or SF-900 (Ostertagia) supplemented with amikacin (50-100 mg / ml), oxacillin (50-100 mg / ml) and fungizone (50 μg / ml). Following the antibiotic and antifungal supplementation, the larval medium has a final pH of approximately 4.5 in conditions of 5.9% CO2 and humidified air. Vials with tissue culture are aerated periodically by gentle agitation. The larval cultures are incubated at 37 ° C for a period of between 14 to 21 days, during which time the cuticle layer of the larva is gradually degraded and the cells can be observed that protrude through defects in the cuticle layer of individual larvae ("blooming" cell). The concentration of antibiotics and the pH level of the medium seem to directly affect the field of this process and the speed at which this process advances. Optimal cell "blooming" has been demonstrated with IPL-41 tissue culture medium, in contrast to other formulations evaluated. The relatively low pH of this modified tissue culture medium can stimulate the abomasal environment to which many of the larval populations have adapted. Bacterial flora can potentially improve the spread of parasite cell populations (eg, intestinal cells) in a tissue culture environment, based on the ingestion of such organisms by intact parasites as a source of nutrients. If tissue culture bottles become excessively "contaminated" with bacterial growth, additional antibiotics can be added to the culture flasks. The growth medium is then screened for contaminating bacterial growth by inoculating the medium onto MacConkey agar plates, Mueller-Hinton plates and blood agar.

The flasks were inspected on a uniform basis under an inverted microscope to detect cellular "flowering" through defects in the cuticle layer of the larvae. When the "flowering" has been reached, the larval culture is subjected to soft breaking by serial pipetting in conjunction with low speed centrifugation. This procedure seems to gently "detach" the cellular "buds" and slightly collapses the cuticle layer. As a result, the parasite cells are "crushed" or squeezed out from within the lumen of the cuticle layer cylindrically. This approach allows the soft harvesting of parasite cells that have been allowed to proliferate in situ without excessive breaking. Further separation of viable cells from cuticle and other non-viable wastes can be achieved by layering the larval culture material on a Percol density gradient, centrifuging the gradient to separate the cell shoots from the other materials, separating the cell shoots from the gradient, by rinsing the cell shoots in PBS and re-inoculating the cell shoots into tissue culture flasks containing cell culture medium (for example a mixture of IPL-41 / SF900 media or only SF900 medium) supplemented with amikacin (2.5 mg / ml), oxacillin (5.0 mg / ml) and fungizone (amphotericin B) (50 μg / ml). Parasite cells appear to propagate optimally in vitro when fresh cell culture medium is periodically emptied into the tissue culture flasks at regular intervals. The addition of medium is therefore performed on a basis of what is required, usually at intervals of about one week, depending on the existing level of proliferation, as assessed by observation and direct visual examination under an inverted microscope. For example, cell shoots are initially transferred to tissue culture flasks of 75 cm 2 and approximately 15 ml of cell culture medium is added. At approximately a week interval, a small volume of fresh medium is added and the bottle is aerated by shaking. This is repeated until the volume of medium reaches approximately 50 ml, at which time the cells and medium are transferred to a tissue culture flask of 250 cm 2 and fresh medium is added for an initial volume of approximately 60 ml. The medium is added at intervals of approximately one week with agitation, until the volume of medium reaches approximately 225 ml. The cells and medium are then transferred to a tissue culture flask of 500 cm.sup.2 and fresh media is added at approximately one week intervals with shaking until the volume of the medium reaches approximately 450 ml. At this time, the cells are divided into multiple bottles as described here. Once the capacity of a tissue culture bottle has been exceeded as determined by the volume of medium and the microscopic observation of a substantial amount of cell pellet at the bottom of the tissue culture bottle, the cells are "scraped" of the bottle by a sterile technique and are either transferred (passed) to a jar of larval tissue culture or "divided" 50/50 or 33/33/33 between two or three bottles. In any situation of "division". Additional fresh cell culture medium is added to each tissue culture flask. It has been observed that, if the entire medium of the tissue culture vial is replaced with fresh cell culture medium when the parasite cells are being passed (which is typically done in mammalian tissue culture), the Parasite cells demonstrate a delay or termination of antigen expression and proliferation. Therefore, when the cells are passed, fresh cell culture medium is typically added to the tissue culture flask in a proportion of between 33 and 50% of the "exhausted" cell culture medium and 67 and 50% of the cell culture medium. fresh cell culture medium, although any proportion of spent-to-fresh cell culture medium can be used as long as the percentage of spent medium is at least 10-15%. Stimulation of the rate of delayed proliferation: Populations of parasite cells will occasionally exhibit patterns of slowed growth and proliferation rates. In such cases, several procedures can be applied to stimulate cell division and propagation. Examples of such procedures that have been effective include (a) low speed centrifugation and resuspension of the granule in the same "spent" growth medium.; (b) cryopreservation in liquid nitrogen for a short period of time (for example, at least 48 hours) and subsequent recultivation in fresh cell culture medium; (c) sterile "scraping" of cells from the surfaces of tissue culture flasks; (d) preventing the exposure of the tissue culture flasks to atmospheric air for several days; (e) placing the tissue culture flasks on a diagonal inclination; and (f) inoculation of loose cell granules in "spent" medium followed by centrifugation at low speed (200-300 x g).

Cell lysis, fractionation and preparation of antigen sample: Parasite cells were centrifuged at 200 xg to form a cell pellet, the medium was discarded and the cell pellet was resuspended in Trtiton X-100 (1-5%) supplemented with EDTA (2-5 mM) and aprotinin (3 mg / ml) at 0 ° C to 4 ° C (ice bath). The cells were incubated at 25 ° C for one hour with periodic gentle shaking and subsequently centrifuged at 500 x g. The resulting supernatant extract was harvested. Excessive processing of parasite cell granules can break the integrity and / or alter the harvest of important antigenic and / or enzymatic fractions. The granulation of parasite cell populations should ideally be by single centrifugation at low speed (eg, 200 x g) for a relatively short period of time at 4 ° C. The centrifugation procedure for harvesting populations of parasite cells should be performed only once. If a second centrifugation procedure is necessary to harvest parasite cells in a single collective granule, then the resulting "second" supernatant must be harvested for analysis and not discarded. The basis for this precaution was found on the observation that membrane-associated antigens (eg, aminopeptidase-M) are apparently "leached" easily away from the outer surface of the parasite cells. Only "spent" or fresh cell culture medium should be used during centrifugation procedures to suspend and harvest parasite cells in vitro. The application of regulatory systems such as PBS and Tris-HCl, which are traditionally considered to be physiologically mild with populations of mammalian cells, appear to break excessively and substantially reduce the cell mass harvested. Verification of cell viability and estimation of in vitro proliferation rate: Parasite cells were transferred in aliquots (300 μl) into individual compartments of a 48-well microtiter plate. A 60 μl aliquot of MTT staining reagent (Sigma) was added to each well and the plates were incubated in a humidified incubator at 37 ° C for 12 hours. The microtiter plates were then centrifuged, the resulting supernatant was removed by pipette and the parasite cells were destained with isopropyl alcohol for 20 minutes at 25 ° C. The resulting supernatant was transferred by pipette to a 96-well microtiter plate and the adsorbance of each well was read at 450 nm using a microtiter plate reader integrated with a computer. The tests were repeated at various times to estimate the approximate proliferation rate (double time) of the parasite cells propagated in a tissue culture environment. The MTT reagent is reduced to dark blue formazole crystals within the cytosol of viable cells, which is subsequently solubilized with isopropyl alcohol before the measurement of the spectrophotometric absorbance at 450 nm. Mammalian cells and bacteria reduce the MTT reagent to formazon crystals in approximately 3-4 hours and 15 minutes, respectively. The parasite cells reduced the MTT reagent to intracellular formazone crystals in a period of 8 to 12 hours. Verification of cell population homogeneity in vitro: Populations of parasite cells were examined under an inverted microscope and morphologically evaluated to determine the homogeneity of the cell population. The cells were examined for uniformity in shape and size, the appearance of fine granules within the cells and the presence of aggregates or clusters of varying size of cells in the culture flask. It was observed that all of the parasite cells of the present invention were of a uniform size and shape and that all contained fine granules., of similar appearance in the totality of the observed cells. Thus, on the basis of these observations of the formological characteristics of the cells of this invention, it appears that all of the cells were of a single cell type, demonstrating that the parasite cells in culture consisted of a homogeneous population of cells . Verification of viability and estimation of the proliferation rate: The proliferation speed of Ostertagia and Uaemonchus cells propagated in vitro was determined as described here, with the application of MTT vitality dye reagent, as follows: Ostertagia: Flask 1: 8.9 times increase / 6days. Bottle 2: 3.5 times increase / 14 days. Haemonchus: Bottle 1: 1.5 times increase / 6 days. Bottle 2: 1.49 times increase. Bottle 3: 1.90 times increase. These data demonstrate that the parasite cells of this invention were viable and proliferated under the culture conditions described herein. Analysis of surface membrane antigens by SDS-PAGE: Samples of "spent" growth medium and extracts of parasite cells solubilized with Triton X-100 detergent (1-5%) were analyzed by non-denaturing SDS-PAGE (10% acrylamide, 20 constant voltage, 4 ° C) according to standard techniques (Laemmli, 1970). The gels were stained in silver according to standard methods to identify membrane-associated antigens produced by the parasite cells of the homogenous population of cells. SDS-PAGE Analysis: The following are the estimated molecular weights observed for membrane-associated antigens (in kDa) expressed by populations of parasite cells propagated in vitro.

MW (kDA) 12 14 18 20 29 32 40 50 60 70 80 Parasite Ostertagia M H L L L H M H M - - Haemonchus M M M M - H M H M M H Oesophagostomum H - M - - - - H - L - Cooperia M M L L H L L L H M H H = high level of expression relative to other protein fractions identified within the same track (sample).

M = moderate level of expression relative to other protein fractions identified within the same track (sample).

L = low level of expression relative to other protein fractions identified within the same track (sample).

Additional membrane-associated antigens and expressed by parasite cells of the present invention include the following: Haemonchus, Cooperia 120 kDa Haemonchus, Cooperia, Oesophagostomum 180 kDa These experimental findings serve to: (a) partially characterize the identity of cell lines being currently propagated in vitro; (b) demonstrating a relatively minimal loss of expression of membrane-associated antigens in a population of parasite cells propagated for long periods in vitro; and (c) identifying membrane-associated proteins / glycoproteins that may be "shared" antigens expressed by each of the four genera of bovine parasites described herein. Such "shared" membrane-associated antigens may provide a means to induce "cross-reactive" protective immunity to these parasites in challenged hosts. Western blot analysis of membrane-associated microfilarial antigens: Membrane-associated antigens were harvested from the microfilaria cell population by detergent extraction (e.g., Triton X-100, Thesit). The proteins in the fraction were separated according to the molecular weight with SDS-PAGE. The proteins were transferred to nitrocellulose membranes according to standard blotting protocols (Harlow and Lane, 1988, catalog and BioRad manual). A monoclonal antibody (catalog No. DFI 023-40470; Capricorn Products, Inc., Scarborough, ME), directed against an antigen expressed by heart helminths (Dirofilaria immitus) of adult canine was added to the nitrocellulose membranes. A secondary anti-murine antibody conjugated to horseradish peroxidase (HRPO) (Pierce Chemicals) was then added to the membranes and H2O2 was added to the membrane for the development of a detectable color reaction. Wester Blot Analysis of SDS-PAGE: A monoclonal antibody directed against an antigen expressed by heart helminths (Dirofilaria immitus) of adult canine showed binding avidity by fractions containing membrane-associated antigens solubilized from populations of microfilaria cells. These experimental findings indicate that cells cultured from microfilaria larvae have been successfully propagated in a tissue culture environment and that the cultured cells are microfilarial cells that continue to express the antigen bound by the monoclonal antibody. Detection of proteolytic enzyme fractions by SD-PAGE in gelatin: Samples of "spent" growth medium and Triton X-100 detergent extracts from parasite cells were analyzed for proteolytic enzymatic activity by SDS-PAGE in undenatured gelatin (no reducer) (0.1% gelatin, 10% acrylamide, constant voltage of 20.4 ° C) (McKerrow et al., 1990, Gambel et al., 1996). The gels were rinsed (20 minutes x 3) in Triton X-100 (2.5%) and incubated 24-48 hours at 37 ° C in Tris-HCl (0.1 M, pH 7.0) supplemented with CaC12 (1 mM). Gelatin SDS-PAGE gels were stained with Coomassie Brilliant Blue 450 (0.1%) (Sigma) for three hours at 25 ° C, followed by staining in methane-1-acetic acid in water (35:10 v / v). The proteolytic enzymes were detected as clear zones against the background of gelatin SDS-PAGE positively stained with Coomassie Brilliant Blue 450, signifying the enzymatic degradation of the gelatin matrix. In Ostertagia ostertagi cells, the proteolytic enzymatic activity was detected in protein fractions that had the approximate molecular weights of >200 kDa, 116-150 kDa, 63-75 kDa and 45 kDa. The proteolytic fractions located at 116-150 kDa and 63-75 kDa were very faint in appearance. In Haemonchus contortus cells, the proteolytic enzyme activity was detected in a protein fraction having the approximate molecular weight of 30 kDa. These results match the known gelatin SDS-PAGE profiles of whole parasite larvae, demonstrating that the Ostertagia and Haemonchus cells of the present invention are larval cells and that the cells in culture are producers of proteolytic enzymes. Assays for aminopeptidase-M proteolytic activity: M-aminopeptidase is an enzyme produced in intestinal parasite cells (McMicahel-Phillips et al., 1995), therefore, an assay to determine aminopeptidase activity M was carried out to characterize additionally the homogenous population of parasite cells with respect to the cell type. Precautions were taken in the processing of parasite cell populations for the assay of aminopeptidase-M activity because this enzyme appeared to "leach" out of the outer membrane during processing.

Experimental samples (50 μl) of whole cells of spent medium, rinsed and mechanically fractured complete cells or extracts of Triton X-100 were combined with MOPS buffer (50 mM, pH 7.0, 100 μl) and incubated at 25 ° C for 15 minutes in a 96-well microtiter plate to allow the enzyme to equilibrate in this regulator. At the end of the incubation period, enzyme-specific substrate reagents, leucine-paranitroanalide (pNA) and methionine-pNA (2 mM, 100 μl) were added into individual wells. The plates were incubated for variable times (0 to 48 hours) in a humid incubator at 37 ° C and the presence of M-aminopeptidase was determined by measurements of the proteolytic release of pNA as detected by spectrophotometric absorbance at 405 nm. In addition, samples of spent medium and Triton X-100 were fractionated by molecular weight in a microfiltration device (Amicon, Inc., Beverly, MA) and prepared as described above. Negative controls included the application of metal chelating reagent (zinc), 1,10 phenanthroline (10 mM, 4 μl), to wells containing experimental sample, regulator and enzyme substrate, because the aminopeptidase enzymes are classified as metalloproteases of zinc. Positive controls included porcine aminopeptidase-M (Sigma, St Louis, MO), regulator and enzyme substrate.

Expression of Aminopeptidase-M: The results of the spectrophotometric assay for aminopeptidase-M demonstrated that this enzymatic activity was present in the whole-cell preparations, "spent" medium samples and membrane-associated antigen fractions solubilized with detergent from the populations of Parasite cells of Ostertagia ostertagi and Haemonchus contortus, Oesophagostomum, Dirofilaria immitus and Cooperia of the present invention. The results of the experiments in which the samples were fractionated by molecular weight before the test showed that the aminopeptidase activity was present in fractions of approximately 45-50 kDa molecular weights and > 100 kDa. In the presence of the metal chelating agent, 1,10 phenanthroline, no aminopeptidase-M activity was detected in any of the experimental samples. These data demonstrate that the Ostertagia and Haemonchus cells of the present invention are intestinal cells on the basis that these cells express proteins having aminopeptidase-M activity and that the proteins expressing this activity are of molecular weights similar to those of proteins. extracted from intestinal parasite cells that are known to express M-aminopeptidase activity (McMichael-Phillips et al., 1995). Assays for phosphorylase activity: In addition to the M-aminopeptidase assays, assays were carried out for phosphorylase (phosphorylhydrolase), another enzyme known to be produced in intestinal parasite cells (Gambel et al., 1980; Knowles and Oakes, 1979; Gambel & Mansfield, 1996; Gambel et al., 1996; Barrett, 1981) to further characterize cells from homogeneous populations of cells such as intestinal parasite cells. To detect phosphorylase activity in the parasite cells of this invention the protocol described above for the detection of aminopeptidase-M activity was carried out with the exception that the phosphorylase-specific substrate, paranitrophenyl phosphate, was replaced by aminopeptidase-substrate. M. Experimental samples possessing phosphorylase activity were identified by the development of a yellow color representing the enzymatic release of the chromogenic substrate fraction as detected by absorbance measurements at 405 nm. The negative controls contained tartaric acid (lmM) as a phosphorylase inhibitor. Expression of phosphorylase: The activity of phosphorylase was detected in experimental samples from Ostertagia ostertagi, Dictyocaulus vivaparus, Haemonchus contortus, Cooperia, Oesophagostomum and Dirofilaria immitus in culture. Because it is known that this enzyme is expressed by intestinal parasite cells, these data provide additional support that the parasite cells of the present invention are intestinal cells. Assays for other enzyme markers: Tests were also carried out for enzymes known to be produced in parasite cells of other types than * that of intestinal cells, according to the protocol described above to further characterize cultured parasite cells of the present invention. For example, phospholipase-C, chymotrypsin, cathepsin C, dipeptidylpeptidase and N-acetylglycosamidase have all been detected in secretory-excretory products in fourth-stage larvae (Gamble and Mansfield, 1996) and have not been detected in intestinal parasite cells. The other enzymes for which the screening was carried out and the specific substrates that were used are the following: Enzyme marker Phospholipase-C enzyme specific para-nitrophenylphosphorylcholine chymotrypsin succinyl-phenylalanine paranitroanilide cathepsin C glycine-phenylalanine dipeptidylpeptidase paranitroanilide IV glycine-proline para-nitroanilide N-acetyl-β-D-glucosamidase paranitrophenyl-N-acetyl-β- D-glucosamide Detection of the expression of other parasite cell enzyme markers: The assays of the Ostertagia and Haemonchus cells of the present invention for the expression of phospholipase-C, chymotrypsin, cathepsin C, dipeptidylpeptidase IV and N-acetylglycosamidase, with the respective specific substrates of enzyme from the above list, all produced negative results. These results provide further support that the Ostertagia and Haemonchus parasite cells of the present invention are intestinal cells on the basis that none of the cells demonstrated any activity of phospholipase-C, chymotrypsin, cathepsin C, dipeptidylpeptidase IV, and N-acetylglycosamidase , which is consistent with what would be expected in intestinal cells and that the activity of aminopeptidase-M and phosphorylase detected in these cells is not associated with excretory-secretory products. These data also show that the cell populations of the present invention do not contain cells of the types expressing these enzymes, further demonstrating that the cell populations of the present invention are homogeneous. Ligand binding assay with concavalin A lectin: Experimental samples of "spent" tissue culture medium and extracts solubilized with Triton X-100 detergent were applied to nitrocellulose membranes using a 96 well Dot Blot apparatus in combination with negative pressure (BioRad). Non-specific binding was minimized by incubating the nitrocellulose membranes in bovine serum albumin (BSA) or skimmed milk as blocking buffer (100 mM Tris, pH 7.4), for two hours at 25 ° C. The nitrocellulose membranes were rinsed (3 x 20 minutes) in Tris-HCl (50 mM pH 7.0). Biotinylated concavalin-A (Con-A, 10 μg / ml) (EY labs) was added to the nitrocellulose membranes for 90 minutes at 25 ° C. The residual biotinylated Con-A was removed by rinsing (3 x 20 minutes) in Tris-HCL (50 mM, pH 7.0). Streptavidin-horseradish peroxidase (streptavidin-HRPO; 2 μl / ml of a 0.5 mg / ml standard) (Pierce Chemical Co.) in Tris-HCl (50 nM, pH 7.0) was added to the nitrocellulose membranes for 90 minutes at 25 ° C. The membranes were rinsed again (3 x 20 minutes) in Tris-HCl and the development of a detectable reaction was achieved by the addition of H2O2 (2 μl / ml of a 30% solution) which functions as a catalyst for enzymatic activity of horseradish peroxidase. Dot Blot Analysis of lectin (Biotinylated Con-A): The binding of Con-A to experimental samples from spent medium and fractions of Triton X-100 was detected by the development of a visible reaction product on the nitrocellulose membranes. The results of these experiments showed that the proteins contained in both the "spent" growth medium and the Triton X-100 detergent extracts of in vitro propagated parasite cells showed positive binding avidity for biotinylated Con-A reagent. Gel extracts with ligand affinity, "spent" growth medium and membrane-associated antigens: experimental samples of "spent" growth medium and membrane-associated antigens were applied to Con-A conjugated to sepharose in regulator A (pH 5.2 ) consisting of sodium acetate (5 mM), manganese chloride (1 mM), calcium chloride (1 mM), sodium chloride (0.1 mM) and sodium azide (0.02%). Next, extensive rinsings of Con-A gel with sepharose with regulator A bound protein fractions were displaced from the gel using regulator B (pH 5.2), consisting of methyl-alpha-D-glucopyranoside (0.5 M) and methyl-mannoside ( 0.2 M), and subsequently were harvested in the resulting supernatant. Portions of these samples were then analyzed by SDS-PAGE under reducing conditions to determine the approximate molecular weights of the Con-A-bound proteins in the parasite cells. In addition, protein fractions possessing A-binding Avidity were further tested for aminopeptidase-M activity according to the protocol described above. SDS-PAGE of proteins with binding to Con-A: The proteins of Ostertagia ostertagi and Haemonchus contortus containing Con-A binding activity had approximate molecular weights of >; 200 kDa, 100-116 kDa, 50-55 kDa, 40-45 kDa and 29-33 kDa. In addition, these fractions with Con-A binding exhibited M-aminopeptidase activity. The importance of these data is that similar proteins of similar molecular weights harvested from intestinal parasite cells possess both aminopeptidase-M activity and Con-A binding activity (McMichael-Phillips et al., 1995). Propagation of Fasciola hepatica and Fasciola magna cell populations. The methods of the present invention have also been used to produce cell populations capable of prolonged in vitro culture originated from Fasciola hepatica and Fasciola magna metacercaria. These cell populations are grown and maintained in growth medium RPMI-1640 supplemented with either bovine calf serum (10% to 20% v / v) or yeast protein extract. Thus, the present invention provides a homogeneous population of Fasciola hepatica cells capable of prolonged culture in vitro and a homogeneous population of Fasciola magna capable of prolonged culture in vitro. While the present process has been described with reference to specific details of certain embodiments thereof, it is not intended that such details be referred to as limitations on the scope of the invention, except as and to the extent that these are included in the claims. that are accompanied. Throughout this application several publications have been referred. The descriptions of these publications, in their entirety, by means of the present are incorporated by reference in this application for the purpose of describing in greater detail the state of the art with which this invention has to do.

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Claims (58)

1. Novelty of the Invention 1. A homogenous population of parasite cells, not including mosquito cells, capable of prolonged in vitro culture.
2. 2. The cells of claim 1, wherein the parasite cells are intestinal cells.
3. 3. The cells of claim 1, wherein at least a portion of the cells have been used.
4. 4. A composition comprising the cells of any of claims 1 to 3.
5. The composition of claim 4, further comprising a pharmaceutically acceptable carrier.
6. 6. A substantially non-cellular fraction derived from the composition of claim 4.
7. 7. A cellular fraction derived from the cells of claim 1.
8. 8. A composition comprising the fraction of claim 7 and a pharmaceutically acceptable carrier.
9. 9. The cellular fraction of claim 7, wherein the fraction is substantially membrane associated antigens.
10. 10. The cells of claim 1, wherein the parasite is a nematode.
11. 11. The cells of claim 10, wherein the nematode is selected from the group consisting of the species of Cooperia, Oesophagostomum, Ostertagia, Haemonchus, Dirofilaria and Dictyocaulus.
12. 12. The cells of claim 1, wherein the parasite is selected from the group consisting of Fasciola hepatica and Fasciola magna.
13. 13. An antibody that specifically binds to the cells of claim 1.
14. 14. An anti-idiotype antibody that specifically binds to the antibody of claim 13.
15. 15. An antibody that specifically binds to the fraction of claim 7. .
16. 16. An anti-idiotype antibody that specifically binds to the antibody of claim 15.
17. 17. A method for the treatment or prevention of wrapping an animal with parasites, which comprises the administration of immunogenic amounts of the parasite cells of claim 1 or fractions derived from the cells to the animal, thus treating or preventing the wrapping of the animal. animal with parasites.
18. 18. The method of claim 17, wherein the parasite cells are intestinal cells.
19. 19. The method of claim 17, wherein the wrapping of the animal with the parasites is the infestation or infection of the animal.
20. The method of claim 17, wherein the wrapping of the animal with parasites is the taking of a body fluid or blood from the animal.
21. 21. The method of claim 17, wherein the parasite is a nematode.
22. 22. The method of claim 21, wherein the nematode is selected from the group consisting of the species of Cooperia, Oesophagostomum, Ostertagia, Haemonchus, Dirofilaria and Dictyocaulus.
23. 23. The method of claim 17, wherein the parasite is selected from the group consisting of Fasciola hepatica and Fasciola magna.
24. The method of claim 17, wherein the animal is selected from the group consisting of human, bovine, equine, porcine, caprine, ovine, canine, feline and poultry animals.
25. 25. A method for treating or preventing the wrapping of an animal with a parasite, which comprises administering to the animal the antibodies of claim 13 or 15.
26. 26. A method for the treatment or prevention of the wrapping of an animal with a parasite. , which comprise the administration to the animal of the anti-idiotype antibodies of claim 14 or 16.
27. 27. A method of screening a compound for anthelmintic activity, which comprises contacting the compound with the cells of the claim 1, and determine if the compound has a detrimental effect on the cells.
28. 28. The method of claim 27, wherein the cells are in a composition.
29. 29. The method of claim 28, wherein the composition comprises cells that do not originate from a homogeneous population.
30. 30. The method of claim 27, wherein the deleterious effect is cell death.
31. 31. A method to detect the presence of a parasite in an animal, which comprises contacting the parasite cells of claim 1 or fractions derived from the cells with an antibody containing sample from the animal and detecting the presence of binding of the antibodies in the sample with the cells or fractions, the presence of binding indicating the presence of a parasite in the animal.
32. 32. A method for detecting the presence of a parasite in an animal, which comprises contacting antibodies of any of claims 13 or 15 with a sample from the animal containing potentially antigens of the parasite and detecting the presence of binding of the parasites. antibodies with an antigen, the presence of binding indicating the presence of a parasite in the animal.
33. 33. A method for culturing parasite cell populations in vitro, which comprises: (a) culturing a parasite in parasite culture medium under conditions that allow the decomposition and / or degradation of the parasite cuticle layer, such way cell breakouts occur; (b) breaking the culture to cause the cell shoots to detach from the cuticle layer; and (c) culturing the parasite cell buds in cell culture medium.
34. 34. The method of claim 33, further comprising purifying the parasite cells on a Percol density gradient, rinsing the cells in phosphate-regulated brine and inoculating the cells in cell culture medium.
35. 35. The method of claim 33, wherein the parasite cells are intestinal cells.
36. 36. The method of claim 33, further comprising, after step (c), transferring the cell shoots from the cell culture medium to fresh cell culture medium.
37. 37. A population of parasite cells, not including mosquito cells, capable of prolonged in vitro culture, produced by the method of claim 33.
38. 38. The cells of claim 37, wherein the parasite cells are intestinal cells.
39. 39. The cells of claim 37, wherein at least a portion of the cells have been used.
40. 40. A composition comprising the cells of any of claims 37 to 39.
41. 41. The composition of claim 40 further comprising a pharmaceutically acceptable carrier.
42. 42. A substantially non-cellular fraction derived from the composition of claim 40.
43. 43. A cellular fraction derived from the cells of claim 37.
44. 44. A composition comprising the fraction of claim 43 and a pharmaceutically acceptable carrier.
45. 45. The cellular fraction of claim 43, wherein the fraction consists substantially of membrane-associated antigens.
46. 46. The cells of claim 37, wherein the parasite is a nematode.
47. 47. The cells of claim 46, wherein the nematode is selected from the group consisting of the species of Cooperia, Oesophagostomum, Ostertagia, Haemonchus, Dirofilaria and Dictyocaulus.
48. 48. The cells of claim 37, wherein the parasite is selected from the group consisting of Fasciola hepatica and Fasciola magna.
49. 49. A population of differentiated nematode cells capable of prolonged culture in vitro.
50. 50. The cells of claim 49, wherein the nematode cells are intestinal cells.
51. 51. The cells of claim 49, wherein at least a portion of the cells have been used.
52. 52. A composition comprising the cells of any of claims 49 to 51.
53. 53. The composition of claim 52 further comprising a pharmaceutically acceptable carrier.
54. 54. A substantially non-cellular fraction derived from the composition of claim 52.
55. 55. A cell fraction derived from the cells of claim 49.
56. 56. A composition comprising the fraction of claim 55 and a pharmaceutically acceptable carrier.
57. 57. The cellular fraction of claim 55, wherein the fraction consists substantially of membrane associated antigens.
58. 58. The cells of claim 49, wherein the nematode is selected from the group consisting of the species of Cooperia, Oesophagostomum, Ostertagia, Haemonchus, Dirofilaria and Dictyocaulus.