MXPA99002705A - Method of identification of animals resistant or susceptible to diseases such as ruminant brucellosis, tuberculosis, paratuberculosis and salmonellosis - Google Patents

Abstract

The present invention relates to materials and methods for identifying animals that are resistant or susceptible to diseases associated with intracellular parasites such as brucellosis, tuberculosis, paratuberculosis and salmonellosis. More particularly, the present invention relates to the identification of a gene, called NRAMP1, which is associated with the susceptibility or resistance of an animal, such as an artiodactyla to diseases such as brucellosis, tuberculosis, paratuberculosis and salmonellosis. Still more particularly, the present invention relates to the identification of specific sequences of bovine NRAMP1 which associate with resistance or susceptibility to ruminant brucellosis, tuberculosis, paratuberculosis and salmonellosis, and to the method of identifying said sequences to identify animals who are susceptible or resistant to disease.

Description

METHOD OF IDENTIFICATION OF RESISTANT ANIMALS OR SUSCEPTIBLE TO SUCH DISEASES AS BRUCELOSIS OF CATTLE, TUBERCULOSIS, PARATUBERCULOSIS AND SALMONELOSIS BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for identifying animals that are resistant or susceptible to diseases associated with intracellular parasites. More particularly, the present invention relates to the identification of a gene, called NRAMP1, associated with the susceptibility or resistance of an animal, such as artiodactyls, or diseases such as brucellosis, tuberculosis, paratuberculosis and salmonellosis. Even more particularly, the present invention relates to the identification of specific sequences in the 3 'untranslated region (3' UTR, for its acronym in English) NRAMP1 of bovine, which is associated with resistance or susceptibility to brucellosis, tuberculosis, paratuberculosis and salmonellosis of bovines and the use of general sequence patterns to identify artiodactyl animals that contain those sequences in situ, thus allowing the identification of animals provided for being resistant or susceptible to diseases associated with intracellular parasites. 2. General Background Intracelular zoonotic bacterial diseases similar to brucellosis and tuberculosis, cause significant losses in the livestock industry despite the widespread application of antimicrobials, vaccination, isolation and quarantine, testing and killing, or a combination of these. The lack of success in eradicating infectious animal diseases using these approaches indicates the need for a different strategy, such as the development of a means to identify genetic sequences associated with resistance and / or susceptibility, wherein said means could allow the identification of animals that are resistant or susceptible to the disease. This could then allow treatment, either prophylactic or therapeutic, or the elimination of susceptible animals and the selective use and / or breeding of resistant animals (See for example, Tempieton et al., 1988). Diseases such as brucellosis, tuberculosis, paratuberculosis and salmonellosis in ruminants cause a calculated loss of $ 250,000,000 annually to the meat and dairy industry of the U.S.A. In addition, tuberculosis especially is a threat to the health of all ungulates including rare and endangered mammals. These are diseases for which the usual eradication programs have been delayed, expensive and somewhat inaccurate. For example, tuberculosis in cattle began to become a disease of old in 1970 but re-emerged as an endemic disease in dairy herds in El Paso, Texas. In the past five years, bovine tuberculosis outbreaks have been reported in California, Idaho, Indiana, Louisiana, Missouri, Montana, Nebraska, New Mexico, New York, North Carolina, Pennsylvania, South Carolina, Texas, Wisconsin and Virginia (Essey and Koller 1994; and Essey MA 1991). In addition, each of these specific diseases are zoonotic diseases that continually threaten the population of the United States. The benefits of livestock naturally resistant to these and other diseases could be a key component of pre-harvest pathogen reduction programs such as the proposed National Hazard Analysis Critical Control Point (HACCP) Program. for use on farms (Pierson, MD and Corlett, DA, 1992, and Vanderzant, C, 1995). Furthermore, it is convenient that said approach used to control these diseases use natural resistance since it is environmentally compatible. The only method currently available for the detection of artiodactyles resistant to brucellosis or tuberculosis is by a potent in vivo comparison with Brucella abortus, Salmonella dublin, Mycobacterium paratuberculosis, or virulent Mycobacterium bovis (Templeton and Adams 1996). Unfortunately for this analysis, tested ungulates have to be euthanized in order to cultivate the specific pathogen. Males confronted with B. abortus or M. bovis should be necropsied and cultured to determine if the bacterium has been eliminated (resistant) or persists (susceptible). Nonpregnant females confronted with M. bovis should be necropsied and cultured for resistance or susceptibility. Although gametes, both males and females, can be stored frozen and used in a breeding program to produce naturally resistant progenies with some success, this is both extremely expensive and inefficient. The viability of frozen gametes and embryos is variable and a much lower birth rate and natural mating occurs. Additionally, the breeding program could be based on phenotypic selection (so-called mass selection) that is not as efficient in determining genotypes and selecting the resistance associated with genetic sequences directly. (See, for example, Martin and others 1994; and Dietrich and others 1986). The present invention solves these problems of the prior art by providing an efficient and reliable method for determining whether an animal, such as an artiodactyl, is susceptible or resistant to diseases such as brucellosis, tuberculosis, paratuberculosis and salmonellosis. SUMMARY OF THE INVENTION In this invention, we identify murine NRAMP1 homologs from bovine, bison and other artiodactyls and show that the particular 3 'UTR sequences of these NRAMP1 homologs have a highly significant association with resistance or susceptibility to diseases associated with bacterial pathogens. More specifically, this invention relates to the discovery of different sequences present in the nature of NRAMP1 of bovine, where the presence of a particular sequence is strongly correlated (P = 0.0089) with resistance or susceptibility, among other things, to brucellosis, tuberculosis , paratuberculosis and salmonellosis in unrelated cattle. The genetic sequences associated with NRAMP1 of artiodactyls that is statistically associated with susceptibility or resistance, imply a transversion at position 1782 of the complementary DNA (c) of NRAMP1 and a polysatellite DNA microsatellite sequence difference, both of which are located in 3 'UTR. The sequence associated with resistance contains a thymine at position 1782 and a polymorphic microsatellite sequence starting at a position 1782 characterized by: SEQ ID NO. 31 5 '(GT) 10AT (GT) 3 (N) 61 (GT) 5 (N) 24 (GT) 133' where "N" symbolizes any of the four nucleotide bases A, C, G or T. In In contrast, sequences associated with continuous susceptibility contain a guanine at position 1782 and a microsatellite region of polymorphic DNA characterized by: 5 '(GT) < 1oAT (GT) 3 (N) > 61 (GT) 5 (N) < 24 (GT) > 133 'where "N" again symbolizes any of the four nucleotide bases A, C, G or T These sequence differences in 3' UTR of the NRAMP1 gene can be used to detect whether the animals are susceptible or resistant to the disease . For example, by screening animals for the presence of sequences associated with susceptibility or resistance, one can easily and accurately predict the susceptibility or resistance of an animal to diseases such as brucellosis, tuberculosis, paratuberculosis, salmonellosis and other diseases associated with macrophage infections. Once identified, susceptible animals can be segregated, treated, prophylactically or therapeutically or sacrificed. Resistant animals, on the other hand, can be managed safely, used to produce food products and / or procreated to produce disease-resistant animals. BRIEF DESCRIPTION OF THE DRAWINGS: For a better understanding of the nature and objectives of the present invention, reference should be made to the following detailed description, taken together with the accompanying drawings, wherein: FIGURE 1A, shows the sequence of initiators of PCR useful in the detection of NRAMP1 sequences of bovines associated with susceptibility and resistance to disease (SEQ ID NO.1 and SEQ ID NO.2); FIGURE 1B shows the sequences of primers used to clone bovine NRAMP1 (SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO 5 and SEQ ID NO.6); FIGURE 2 shows the predicted amino acid sequence of Nrampl of bovine (SEQ ID NO.7) and Nrampl homologs of human (SEQ ID NO.8) and murine (SEQ ID NO.9) and their alignment with each other; FIGURE 3A shows the analysis of ACSH of 22 heads of unrelated cattle, determined phenotypically (by in vivo confrontation and / or by an analysis of death of macrophages in vitro) to be resistant or naturally susceptible to the disease; FIGURE 3B shows the tabular results of the experiment carried out in FIGURE 3A; FIGURE 3C shows the analysis of ACSH and genealogy of naturally resistant bulls conferred to a generally susceptible cattle and its progeny; FIGURE 4 shows the nucleotide sequence (SEQ ID NO.10) and the predicted amino acid sequence (SEQ ID NO.9) of NRAMP1 of bovines; FIGURE 5 shows the hydrophobicity profile of the predicted amino acid sequence of NRAMP1 of bovine; FIGURE 6 shows a schematic representation of the putative structure of the Nrampl protein of cattle; FIGURE 7A shows the NRAMP1 sequences of bovines associated with susceptibility and disease resistance (SEQ ID NO.11, 12, 13 and 14): FIGURE 7B shows the generalized sequence of bovine NRAMP1 associated with resistance (SEQ ID NO 15), FIGURE 7C shows the generalized sequence of bovine NRAMP1 associated with susceptibility, FIGURES 8A1 and 8A2 show the sequence alignment of amino acids conserved in 3 'UTR of several ungulates, FIGURE 8B, shows an alignment of the sequence of amino acids encoded by NRAMP1 of bovine (BovNrampl) and bison (BisNrampl), FIGURE 8C, shows the length and pattern of microsatellites for several Mammalian Species, FIGURE 8D, shows the 3 'untranslated Sequence of NRAMP1 of Bison in Resistant Bison (SEQ ID NO 29) and Susceptible (SEQ ID NO 30), FIGURE 9, shows the genetic mapping of NRAMP1 of bovine in BTA2, FIGURE 10, shows a representation of conserved chromosomal segments among other species around the NRAMP1 site, FIGURE 11 shows a Northern blot analysis of RNA isolated from bovine tissues and cells DETAILED DESCRIPTION OF THE PREFERRED MODALITY: Genetic studies in mice have shown that the innate susceptibility to Mycobacterium bovis (BCG), Leishmama donovani Salmonella typhimupm and atypical mycobacterium vanes is controlled by a single gene in the Autosome 1 of Altered Muscle (MMU) called Bcg, Lsh or Ity (Mock and others, 1990, Plant and others, 1982, Schurr and others 1991, Goto and others, 1989, Skamene and others, 1984, de Chastellier and others, 1993; and Frelier et al., 1990). Bcg mediates the antimicrobial activity of macrophages against intracellular parasites in the early stage of infection (Gros et al., 1983, Blackwell et al., 1991, Roach et al., 1994, Roach et al., 1991). Bison cattle naturally resistant (R) or susceptible (S) to brucellosis was identified by confrontation experiments against Brucella abortus, in vivo (Harmon et al., 1985). Studies showed that resistant cattle macrophages were better able to control the intracellular replication of B. abortus in an in vitro analysis (Harmon et al., 1989, Price et al., 1990, Campbell et al., 1992). These observations were compared to differences in macrophage function between resistant and susceptible mice of M. bovis-BCG, Salmonella typhimurium and L. donovani controlled by the Bcg / Lsh / lty genes (Radzioch et al., 1991; Kramnik and others 1994). Blackwell et al., 1994; Gros et al., 1983; Blackwell et al., 1991; Blackwell et al., 1994; Roach et al., 1991). In mice, it was reported that a segment of approximately 30 cM MMUl (Mock et al., 1990; Skow et al. 1987; Malo et al. 1993) including Bcg. it is conserved in autosomes of Homo sapiens (HSA, for its acronym in English) 2q (Cellier and others 1994, White and others 1994) and autosoma of Bos taurus (BTA) 2 (Womack et al. 1986; Fries et al. 1993; Adkinson and others 1988; Beever and others 1994). Vidal et al. (Vidal et al. 1993) isolated a candidate gene from murine Bcg, designated macrophage protein associated with natural resistance (NRAMP1), which apparently encodes a polytopic integral membrane protein having structural aspects similar to prokaryotic and eukaryotic transporters. Recent studies using unconscious mice have shown that NRAMP1 is the gene of Bcg / Lsh / lty. It is suggested that murine Nrampl protein can function in phagolysosoma membranes as a concentrator of nitric oxide oxidation products, mediating cytocidal activity against ingested parasites of infected macrophages (Vidal et al., 1993; Malo et al., 1994a; Cellier and others, 1994; Malo et al., 1994b). Recently, it has been indicated that the mammalian Nramp protein (at least Nramp 2) functions as a specifically broad divalent cation transporter (Gunshin et al., Nature 388: 482, 1997; Fleming et al., Nature Genet., 16: 383 , 1997). In the present invention, a study was carried out to determine if a bovine homologue in the NRAMP1 gene was expressed from murine in bovine macrophages and was involved in the susceptibility of cattle to, for example, B. abortus. Human, murine and bovine homologs of the bovine NRAMP1 gene product indicate a notorious degree of homology (see Figure 2) The NRAMP1 cDNA of bovine encodes a protein with an overall predicted amino acid sequence homology of 86.9% and 88.6% for the gene products of human NRAMP1 and murine NRAMP1, respectively. Northern blot analysis and RCP-RT analysis indicate that, similar to human and murine gene products, bovine NRAMP1 is mainly expressed in reticuloendothelium (ER) and macrophage organs (Vidal et al 1993; Cellier et al. 1994; Gruenheid and others 1995). All three homologues contain 12 helical potential membrane expansion domains and several functional sequence motifs including an N-terminal SH3-binding PNNP motif, a 20 amino acid transport motif, also known as the "membrane component subject" motif. of the transport system that depends on the binding proteins "within segment 8-9 of the transmembrane (TM) (Vidal et al., 1993; Malo et al., 1994a; Cellier et al., 1994; Malo et al., 1994b) four phosphorylation sites of Protein Kinase C (PKC) and an N-linked glycosylation site (Figure 2). Additionally, very few substitutions in the Nrampl protein appear to be tolerated in the membrane expansion regions. Bovine NRAMP1 has been mapped to BTA 2 within a group of syntenic sites conserved in HSA 2q and murine chromosome 1 overlapping the Lsh / lty / Bcg site (Adkinson et al., 1988 Beever et al., 1994 Cellier et al. 1994; White et al., 1994). Additionally, the interleukin-8 receptor is linked to bovine NRAMP1. The data presented here also extend a large conserved large synthety of bovine, human and murine genes on these chromosomes. All together, these findings indicate that the observed collective properties have important structural and mechanical roles to mediate Nrampl's function. The analyzes of ACSH (single-strand conformational analysis) and PCSH (single-strand conformational polymorphism) are two very similar techniques commonly used to detect differences in DNA sequences. ACSH tends to be slightly more sensitive, it can be used to detect nucleotide differences alone, between two sequences. Frequently, ACSH is used when multiple sequence differences are detected such as those that occur in the microsatellite DNA sequence regions. In the present invention, both analyzes of ACSH and PCSH, together with direct DNA sequencing, were used to show the differences in NRAMP1 sequences of bovines associated with susceptibility or resistance to infection. The significant association of bovine NRAMP1 conformational polymorphisms (i.e., sequence variations) associated with natural resistance or natural susceptibility to bovine brucellosis, among other things, strongly suggest that, although the inventors do not wish to be linked to a In theory, the NRAMP1 of cattle is the homologue of cattle Bcg or it is equally important that Bcg regulate natural resistance to intracellular parasites. In fact, the finding that variants of NRAMP1 sequences of bovines associated with resistance or susceptibility reinforces the case for the proposed role of NRAMP1 to control the natural resistance in brucellosis, salmonellosis and tuberculosis in all artiodactyls. Potential mechanisms to control NRAMP1 of bovines, or associating it with resistance / susceptibility, have been made by others, which are incorporated herein by reference (Vidal et al. 1993, Celher et al., 1994, Blackwell et al., 1994, Ivanyi et al., 1994, Vidal et al. others, 1995, Blackwell et al., 1995) Given the conservation of NRAMP1 genes in at least three species, it is very likely that the fundamental function of NRAMP1 homologs against different intracellular pathogens, such as, but not limited to, Mycobactepae, Brucellae, Salmonellae and Leishmama are conserved and can be related to the level of death of macrophages. The exact mechanism can vary with different pathogens and may include transport and production of nitrogen oxide, production of reactive nitrogen and oxygen intermediates, respiratory spasms and the derivation of hexose monophosphate, SH3 and tyrosma cmase signal transduction, superior regulation of MHC Class II expression and production of interleukin-1 Given the complex structure and conservation of the Nrampl protein predicted in three species, it will not be surprising if the proposed signaling and bactericidal mechanisms are implicated in the antimicrobial activity / parasites of macrophages While it is possible that the regulation of the Nrampl activity may be different in several species, with the high degree of similarity between species, it is more likely that the fundamental function of Nrampl is conserved against different intracellular pathogens, namely Mycobacteriae, Brucellae, Salmonellae and Leishmania. Therefore, the present invention which relates to the use of genetic variation discovered in the NRAMP1 gene to select and breed domestic and free-ranging artiodactyls that are naturally resistant to these important diseases, could have a key role to previously produce the reduction of pathogens in the National Hazardous Critical Control Point Program (HACCP) for use on farms (Pierson, MD Corlett, DA, 1992; and Vanderzant, C, 1985). The mechanisms by which the sequence variations in 3 'UTR of NRAMP1 contribute to the susceptibility or resistance of the disease caused by infection by intracellular parasites are not precisely known. However, while it is not intended to be linked to a particular theory, the applicants suggest that variations in the 3'UTR sequence could affect the translation of the NRAMP1 message of bovine, a sequence being transcribed more or less than the other. A possible mechanism by which this could occur could be the selective instability of ribosomes either on the mRNA associated with resistance or associated with susceptibility. This instability can result in a complex translation that is most likely out of the message of a sequence type that of the other. One embodiment of the present invention, therefore, involves the identifion, cloning and use of an artiodactyl gene associated with resistance and susceptibility to disease (s) involving (n) intracellular parasites, such as brucellosis, tuberculosis, paratuberculosis and salmonellosis. . More particularly, the present invention relates to the discovery that the artiodactyls and especially ungulates and more specifically le, have a homologue (NRAMP1 of le) of the human and murine NRAMP1 gene. The present invention describes that this NRAMP1 gene has at least two different sequences in the 3 'untranslated region of the gene that is significantly associated (P = 0.0089) with any resistance or susceptibility of an animal that contains the sequence of less the diseases brucellosis, tuberculosis, paratuberculosis and salmonellosis. Even more particularly, the present invention shows that at least these two different NRAMP1 sequences can be easily differentiated by analysis of ACSH and PCSH or any other suitable technique for detecting a particular genetic sequence, for example, but not limited to, direct sequencing. so that one can easily sift animals for the presence of any sequence associated with resistance or sequence associated with NRAMP1 susceptibility.

This information as to whether the animal contains a sequence associated with resistance or a sequence associated with susceptibility, can be used to predict whether the sifted animal is likely susceptible or resistant to diseases caused by intracellular parasites such as brucellosis, tuberculosis, paratuberculosis and salmonellosis. According to the screening results of the present invention, the susceptible animals can be segregated, prophylactically or therapeutically treated or sacrificed. Resistant animals, on the other hand, can be safely bred, recovered and / or procreated to form animals resistant to the disease. In addition, according to the present invention, breeding of animals for resistance to disease can be easily monitored by practicing the genetic screening methods of the invention in order to analyze the transmission of resistance to the disease. In addition, the method of the present invention allows the selective breeding of disease-resistant animals based on the selective screening of only a single genetic trait and the analysis of that trait via genetic analysis, rather than phenotypic selection. This allows the favorable trait to segregate and be tracked independently, allowing selective screening of the favorable genetic sequence, which can avoid the unnecessary selection of unwanted traits and allow the simultaneous tracing of other favorable traits.

The invention further relates to the use of the discovered sequences of NRAMP1 of bovine as indicated, or "genetic markers", of susceptibility or disease resistance in artiodactyles. Specifically, the invention includes the detection and identification of these specific gene sequences via conventional molecular biological techniques such as, but not limited to, ACSH and PCSH. Even more specifically, the present invention illustrates specific methods and materials (such as, for example, specific PCR primers) to identify and distinguish these sequences via ACSH; PCSH or direct sequencing. This allows an animal to be sifted and to detect which NRAMP1 sequences the animal has, which then allows the accurate prediction of the animal's susceptibility or resistance to the disease. In addition, the present invention relates to the use of these predictive genetic markers in animal economics including the production of food and the selective breeding of disease-resistant animals including cattle. The conception of this invention is based, in part, on a series of reports published on the genetic selection of pigs naturally resistant to brucellosis in the 30s and 40s. These publications reported that pigs that do not produce antibodies for an oral confrontation of Brucella Viruses, produced offspring that do not produce an antibody to a similar challenge of approximately 70% of the time compared to a frequency of approximately 20% of the progeny of the unselected control groups. Id. The observation that approximately 20% of unvaccinated control cattle confronted with a virulent strain of Brucella abortus S2308 did not show signs of brucellosis (infection with Brucella abortus) and the lack of antibody production after the confrontation led to inventors to hypothesize that this was a natural resistance to brucellosis of bovines. The inventors then began breeding studies to determine if this natural resistance was inherited and to investigate genes that could control this natural resistance, if it was inheritable. Natural resistance was shown to be inheritable given that they responded to the selection; a higher percentage of offspring were naturally resistant to brucellosis (57% compared to 37%) when naturally resistant progeny were formed for naturally susceptible mothers. Genetic studies in other animals indicated that a major gene called Bcg can control natural resistance to Mycobacterium bovis-BCG. A candidate gene was reported in mice (Vidal, and others 1993). However, unlike the field of the present invention, this report was not in an animal artiodactyl. The inventors then proposed that cattle could have a conserved homologue of the murine gene and that this conserved homologue could have a major control effect on the main resistance to brucellosis in the cattle that the inventors had been breeding. The inventors tested then the previous hypothesis and cloned, sequenced and genetically mapped NRAMP1 from bovine in bovine NRAMP1 from bovine was mapped to BTA 2, within the conserved synthetic site in HSA 2q and MMUI NRAMP1 from bovine was expressed mainly in macrophages and tissues of the reticuloendotehal system and it is predicted that it encodes a 548 amino acid protein having 12 transmembrane segments with the hydrophilic N-terminal region containing a Src binding motif with homology 3 (SH3) located on the cytoplasmic surface and a conserved conserved transport motif The gene is designated as NRAMP1 of bovines given to genetic ligation with servada, expression of tissues and homology of amino acid sequence with NRAMP1 of mupnos The inventors discovered that the macrophage restricted the expression of the NRAMP1 gene of bovine, more importantly, they discovered the sequence and conformational differences in the NRAMP1 gene of bovine which was significantly associated with natural resistance or natural susceptibility to brucellosis in cattle The test was also carried out to determine if NRAMP1 of cattle was conserved in other artiodactyls Significantly, pigs, goats, sheep, bison (American buffalo), llamas elk (Wapiti), red deer, Japanese deer, water buffalo, fallow deer and white-tailed deer, in addition all artiodactyls (for a definition of artiodactyls, see Nowak, R. M, and others 1983) analyzed more fully, have a conserved NRAMP1 gene. The present invention has also shown that cattle whose phenotype has been evaluated as being resistant to a confrontation of virulent B. abortus are significantly different in their ability to control the intracellular replication of Brucella abortus, Mycobacterium bovis-BCG and Salmonella dublin and in the in vitro macrophage death analysis that cattle whose phenotypes have been evaluated for being susceptible (85% correlation with the confrontation phenotype) (Qureshi, T., Templeton, JW, and Adams, LG 1996). This cattle was phenotyped both for in vivo concentration with Strain 2308 of Brucella abortus and for an in vitro macrophage death analysis of Strain 2308 of Brucella abortus, Mycobacterium bovis strain - BCG and Salmonella dublin to determine its resistance or susceptibility to brucellosis and tuberculosis of bovines. Using ACSH or PCSH, a genetic polymorphism was discovered in 3 'UTR of the gene. This polymorphism has two different forms which are significantly associated (p = 0.0089) with the naturally resistant and naturally susceptible phenotypes of brucellosis, tuberculosis, salmonellosis and paratuberculosis of bovines in unrelated cattle. By screening the particular polymorphism and / or sequence that a given animal has, one can accurately and efficiently predict the susceptibility or resistance of that animal to brucellosis, tuberculosis, paratuberculosis and salmonellosis of ruminants and other diseases involving intracellular macrophage parasites. The bovine NRAMP1 gene was conserved in Bison bison Bos spp., Odocoileus virginianus, Capra hirus, Moose elk, Cervus canadensis, Cervus alaphus, Dama dama, Elaphurus davidianus, Ursus spp. Sus scrofa and Oreamnos americanus (SEQ ID Nos. 16-27, respectively) and most probably all domestic and wild artiodactyls (see Figures 8A1 and 8A2 SEQ ID Nos. 16-27 and Figure 8C). In addition, the Nrampl protein was also highly conserved (see, for example, Figure 8B, SEQ ID Nos. 9 and 28). The genetic variation discovered in 3 'UTR of the NRAMP1 gene of artiodactyls, can be used, among other things, to select and breed free-ranging domestic artiodactyls, which are resistant, among other things, to brucellosis, tuberculosis, paratuberculosis and salmonellosis . The NRAMP1 polymorphism of bovines results from a transversion at position 1782 of the NRAMP1 cDNA of cattle, to thymine in the resistant sequence, to guanine in the susceptible sequence. Additionally, there is a microsatellite sequence difference of polymorphic DNA between resistant and susceptible cattle implying a number of dinucleotide repeats (GT) and separation in the 3'UTR of cattle Nrampl. This sequence in resistant animals that begins at position 1779 is: SEQ ID NO 15: GGGTGT (GT) 10AT (GT) 3 (N) 6? (GT) 5 (N) 24 (GT) 13 where "N" symbolizes any of the four nucleotide bases A, C, G or T. In contrast, the DNA sequences associated with susceptible cattle follows the form: ( GT) < 10AT (GT) 3 (N) > 61 (GT) 5 (N) < 24 (GT) > 13 where "N" again symbolizes any of the four nucleotide bases A, C, G or T. The detection of the sequence associated with resistance or the sequence associated with susceptibility can be performed by ACSH, PCSH, polymerase chain reaction (PCR) followed by direct DNA sequencing or any other technique known to those skilled in the art capable of detecting genetic sequence differences. The sequence of PCR primers used to detect the genomic DNA sequence of NRAMP1 from bovine containing the polymorphic DNA sequences associated with resistance or susceptibility is indicated in Figure 1A, SEQ ID Nos. 1 and 2. These PCR primers will amplify the resistant and susceptible allelic sequences in genomic DNA (g) or cDNA. However, it should be established that any of the PCR primers that will amplify the polymorphic region can also be used in this invention. In a screening analysis (see Figure 3, for example), the NRAMP1 sequences of cattle correctly identified animals as resistant or susceptible in 18 of 22 heads of cattle naturally resistant or susceptible to brucellosis (Figure 3A and Figure 3B) ( Significant association, p = 0.0089, Fisher's exact analysis). Most importantly, these 22 heads of cattle were unrelated animals. Bovine NRAMP1 sequences can be detected in gDNA isolated from any tissue including gDNA isolated from, but not limited to, peripheral blood samples, semen, mucosal scrapings, etc., using PCR amplification. As shown in Example 9, for example, approximately 82% of naturally resistant or susceptible cattle to brucellosis and tuberculosis can be identified by typing it for the resistant or susceptible polymorphism to NRAMP1 of cattle using ACSH (or PCSH). The zygosity of cattle for the associated polymorphism resistant (heterozygous or homozygous genotype) can be determined and a breeding program can be practiced to efficiently produce cattle naturally resistant to brucellosis and tuberculosis. Additionally, the bovine NRAMP1 gene is a good candidate gene for the production of transgenic animals that have genes of outstanding production traits and by the action of transgenes are naturally resistant to, among other things, brucellosis, salmonellosis, paratuberculosis and tuberculosis . The genetic selection of the breeding animals for a single site is not detrimental to the overall production of animals, that is, production of meat, muscles, grains or milk while a breeding plan is constructed to regulate this effect. The other chromosomes will segregate by independent classification and perpetuate heterozygosity. Additionally, with the current availability of microsatellite markers separated across the bovine genome, the selection of disease-resistant genotypes can be achieved without compromising other convenient production traits while heterozygosity is maximized at approximately 100 microsatellite sites . With the development of bovine gene maps and the identification of major genes that control economically important traits in cattle and other livestock, the ability to identify a genotype evaluated for disease resistance and high quality production will be possible in the near future . A surprising process in raising cattle will be done when it is possible to select superior genotypes directly identifying important genes. Detection based on ACSH and PCSH of polymorphic sequences of NRAMP1 can be carried out on gDNA isolated from antemortem or postmortem tissues, since postmortem tissue has been reasonably protected from an environment that degrades DNA where tissue autolysis could occur. In a preferred embodiment, one mode for the detection of NRAMP1 sequences of artiodactyls is in a laboratory with a routine RNA isolation, DNA PCR amplification, electrophoresis technique, ACSH analysis, PCSH, direct DNA sequencing or any another technique suitable to detect differences in the genetic sequences. In a preferred embodiment, a mode for the identification of the sequences associated with NRAMP1 of resistant and susceptible artiodactyls is by the amplification of specific PCR of gDNA isolated from the peripheral blood of an individual animal collected in an anticoagulant. The PCR amplification can be carried out in a common laboratory with the capacity to carry out the polymerase chain reaction and with ordinary experience in molecular biology. With manual thermal cyclists, it is also possible to carry out the PCR amplification of the alleles outside the laboratory in the so-called "water channel lateral" analysis in a short time that the blood is collected. In a preferred embodiment, one way to utilize the present invention for the purpose of detecting resistant and susceptible NRAMP1 sequences is by PCR amplification of gDNA isolated from the peripheral blood of artiodactyl animals followed by the analysis of ACSH or PCSH of the product of RCP The isolation of gDNA from blood cells can be performed by normal methods suitable for the subsequent amplification of PCR. As shown in Example 8 and Figure 1A, the PCR primer sequences can be used to amplify the polymorphic DNA region of resistant and susceptible animals, namely, cattle. The PCR products can be specifically labeled, either by using radioactive nucleotides in the PCR reaction (as is the case of ACSH) or by using an initiator labeled at one end specifically (as is the case with PCSH) in the PCR reaction. It could be established that other radioactive (i.e., 32P, 33P, etc.) or non-reactive alternatives (eg, but not limited to, DIG marking) can be used to specifically mark the PCR products. These amplicons can be run on a polyacrylamide gel and the migration of the amplicons can be visualized by normal autoradiographic techniques. It should be noted that if, for example, non-radioactive marking techniques are used, alternative detection methods may be employed. The associated susceptible and associated resistant DNA sequences can be easily distinguished (Figure 3A). The banding patterns of the DNA amplicons associated with resistance and associated with susceptibility are very different. The amplicons of the DNA sequences associated with resistance show faster migration through the gel than is expected from their smaller size amplicons (175 bp for amplicons associated with resistance against more than 175 bp for amplicons associated with susceptibility). A major advantage of a diagnostic test using an ACSH or PCSH analysis is that this technology is readily available. It is relatively easy to compare many techniques used to identify DNA sequences; It is relatively inexpensive to equip a laboratory with the necessary equipment; This is conductive technology for the massive passage of large numbers of samples: and relatively simple technology occurs in minimal false test results (positive or negative) when properly controlled. In addition, because the sequences associated with resistance or susceptibility are genetic, they are transferable, meaning, for example, that resistance may be an inherited trait. Because the transmission of these diseases depends on a susceptible host, resistant animals offer an excellent opportunity to break the disease dissemination cycle and begin eradication. There are no particular unique disadvantages for the proposed analyzes of ACSH or PCSH based on the analysis purchased with other molecular biological diagnostic tests. All these tests require some specialized equipment, a laboratory that uses good basic laboratory practices and at least currently, the recovery of tissue (blood) and transportation to a laboratory. Occasional tensions associated with an animal's restriction for blood recovery will not affect these results. A main purpose of this invention is to identify sequences of NRAMP1 associated with resistance or susceptibility to disease, using techniques based on ACSH or PCSH which results in the correct identification of artiodactyls that are naturally resistant to brucellosis, tuberculosis, salmonellosis and paratuberculosis with a high degree of precision, for example 82%.

There are several possibilities that could be taken into account for the lack of the association from ACSH to 100% with resistant phenotypes. It is possible that Bcg is not just a gene, but that it is a gene complex and NRAMP1 is one of the genes of Bcg, or that there are two or more genes that control the natural resistance to brucellosis of bovines. Alternatively, NRAMP1 may be in linkage disequilibrium with the Bcg gene and is a marker gene for the Bcgr ° X alleles. The lack of 100% association of ACSH of NRAMP1 from bovine with resistant or naturally susceptible phenotypes could also be due to the incorrect assurance that some of the R or S phenotypes of cattle resistance or susceptibility caused by genetic heterogeneity, phenocopies, lack of penetration or error in the confrontation procedure. We tried to minimize the effects of phenocopies by a uniform powerful confrontation (conjunctive instillation of 107 colony formation units of B. abortus). An indication of lack of penetration is not a major problem but the fact that the phenotypes of the parents respond to genetic selection in the breeding studies (Templeton et al., 1990a, Templeton et al., 1990b). The fact that the cattle used in these studies is derived from four different progenies implies that genetic heterogeneity does not confuse phenotypes and provides strong direct evidence that NRAMP1 is a likely candidate for a major gene that controls natural resistance to, among Other things, brucellosis and in particular bovine NRAMP1 is probably a. candidate for a major gene that controls natural resistance to, among other things, bovine brucellosis. The following examples are intended to illustrate the embodiments of the present invention and are in no way intended to limit the scope of the invention. Example 1: Cloning of Bovine NRAMP1 and Isolation of Bovine NRAMP1 cDNA The bovine NRAMP1 gene was cloned in the following manner.

Based on the genomic sequence of murine NRAMP-1, the oligonucleotide primers SEQ ID NO. 3 and SEQ ID NO.4 (also designated as 1F and 1R) were used to apply a segment of 155 bp in bovine genomic DNA (Figure 1B). From this 155 bp bovine sequence, RCP-RT is performed on the mRNA of bovine macrophages using a bovine-specific forward primer designated SEQ ID NO.5 (PE2): 5 'CGTGGTGACAGGCAAGGACT 3' and a reverse primer SEQ ID NO.6 (MUT2): 5 'CCAAGAAGAGGAAGAAGAAGG-TGTC 3' of the murine NRAMP1 cDNA sequence (Vidal et al. 1993). The reverse transcription was carried out in the following manner. Total RNA was extracted from cattle macrophages, spleen, lung or heart as described (Chirgwin et al., 1979). 0.5 μg of the total RNA was transcribed in a 25 μl ration at 37 ° C for 60 minutes with MMLV reverse transcriptase (Gibco-BRL). Amplification of cDNA at 95 ° C (5 min.) Followed by 32 cycles of 94 ° C (1 min.) Was carried out. 58 ° C (1 min.) And 72 ° C (1 min.) With 1 mM MgCl2, 2 μL 10X CPR regulator solution, 2 Units of Taq polymerase (Perkin-Elmer) and 4 μL of RT standard in a final volume of 25 μl. A 222 bp product was amplified from the total RNA of bovine macrophages transcribed in reverse. Sequence analysis showed that this PCR product contained 90% nucleotide identity with the third exon (nucleotide positions 338-458) of the murine homologue. The 222 bp product was generated to screen a cDNA library of splenic bovine gt11 (Clontech). A total of 1 X 106 clones were screened by in situ plate hybridization radiolabelled with [32 P] -a-dCTP (3000 Ci / mmoles) (Dupont, NEN Research Products) by the hexamer primer (1-3 X 109 cpm / μg ) (Feinberg et al., 1983). The filters were washed under conditions of increasing restriction up to 1 X SSC, 0.1% SDS at 65 ° C for 30 minutes. Positive clones were checked using PCR with the primers SEQ ID NO.5 (PE2) and SEQ ID NO.6 (MUT-2) and subsequently amplified by PCR to obtain a 2.3 kb insert with the insert screening sieve amplimers? gt11. This PCR product was gel purified and ligated into pT7BlueT-Vector (Novagen). Both strands of plasmid DNA were sequenced with the dideoxy method of Sanger et al. (Sanger et al., 1977) using modified T7 DNA polymerase (USB) and [35 S] - dATP (3000 Ci / mmole) (NEN Research Products, Boston, MA). All sequence data were pooled and analyzed using MacVector 4.1 software (Eastman Kodak Comp.New Haven, CT). Twenty NRAMP1 clones of potentially complete length were obtained ("2.3 kb), eight of which were sequenced and used to construct the complete sequence. As shown in Figure 4, the ATG frame start codon was located at position 73 of nucleotides at the 5 'end and was followed by a segment of 1644 nucleotides, forming a single open reading frame (MLA) encoding a 548 residue protein with a calculated molecular weight of 59.6 KDa. A TGA stop codon located immediately downstream of glycine 548 (nucleotide pst 1717) was followed by an intact AATAAA polyadenylation signal, position 2257. Example 2: Analysis of the NRAMP1 structure of bovine envisaged. As used herein, it is to be understood that the term "NRAMP1" is understood to include the coding sequence and at least the 3 'UTR of the gene. The first 64 N-terminal amino acids of NRAMP1 from bovine are rich in proline (11/64), glycine (10/64), serine (8/64) and loaded amino acids (10/64) and include two phosphorylation sites of PKC putatives at amino acid positions 37 and 51 (Figure 2). Because the SH3 domains interact specifically with proline-rich peptides, we compared the proline-rich coding fragment PPSPEP (positions 21-26) to several identified SH3-binding sequences. The analysis revealed that the binding motif of "PNNP" (Musacchio et al., 1994) was retained in cattle Nrampl, which indicates that Nrampl of bovine contains an N-terminal SH3 binding domain. The Kyte-Doolittle hydrophilicity analysis (Figure 5) described that the surface probability of PPSPEP of peptides is from 50.3% to 67.6%, which indicates that the SH3 Nrampl binding motif of bovines is more likely localized on the surface of membrane. Analysis of bovine NRAMP1 indicates that the predicted protein is highly hydrophobic with 12 putative transmembrane domains (Figure 6) according to the putative structure of murine and human Nrampl (Vidal et al., 1993; Barton et al., 1994; Cellier et al., 1994). The bovine NRAMP1 gene product contains a potential N-linked glycosylation site at position 335, within a highly hydrophilic region between domains 7 and 8 of the transmembrane (TM) provided and the three phosphorylation sites of PKC in serine (positions 37, 51 and 269, respectively). A transport motif of 20 amino acids is located between domains 8 and 9 of the TM provided and are retained in murine Nrampl (Figure 2). This conserved motif is known as the "internal membrane component assignment of transport system that depends on the binding proteins". Based on the hydropathic analysis and the reverse transport motif, we propose, but not in a limiting sense, that the topography associated with Nrampl bovine membranes is as follows: the NH2 terminus is located in the cytoplasm and the following 12 TM residues result in the 5 consecutive transmembrane cycles. This arrangement could place the SH3 binding motif on the cytoplasmic membrane surface; the SH3 domain with two potential phosphorylation sites and the transmembrane (TM) 2 and 3 cycles and the transmembrane 6 and transmembrane 7 cycles that each contain the phosphorylation site, projecting into the cytoplasm; the TM7 and TM8 cycle containing an expected N-linked extracellular glycosylation site and the carboxyl terminus in the cytoplasm. Example 3: Homology between human, murine and bovine Nramp proteins Comparison of predicted human, murine and bovine Nramp protein sequences (Figure 2) indicates a notorious degree of homology (86.9% amino acid sequence identity between murines and cattle, 88.6% sequence identity between humans and cattle). The TM 1-8 segments provided are domains associated with highly conserved hydrophobic membranes in the three species with a 99% identity between human and bovine and 96% identity between murine and bovine. The most conserved consecutive region is TM 8-9 with 100% identity from position 346 to 456 between humans and cattle; 98.2% identity between murine and bovine. Within segment 8-9 of TM, the "subject of internal membrane component of the transport system that depends on bovine binding proteins" was identical to the human and human Nramp with one exception (substitution of lysine to arginine to the position 392 in humans (Figure 2) Also among these three species, a predicted N-linked glycosylation site was conserved within the fourth putative extracellular cycle between TM 7 and 8 and a consensual PKC phosphorylation site was retained in the cycle intracytoplasmic predicted between TM 6 and 7 at position 37 (Figure 2) (Vidal et al., 1993; Cellier et al. 1994; Gruenheid et al., 1995) .Amino acid substitutions were not randomly distributed throughout the sequence of the protein, but were grouped significantly within certain regions.The most outstanding differences were located at the ends of the proteins, the NH2 terminus (identity of 57.4% of positions 1-47). among murine and bovine; 66% identity of positions 1-50 between humans and cattle) and COOH termination (57.6% identity of position 516-548 between murine and bovine, 69.6% identity between humans and cattle). The third and fourth extracellular cycles predicted at positions 215-237 and positions 307-346 were less conserved in amino acid sequences than the TM domains. The identity was 78.2% between murine and bovine and 82.0% between humans and cattle for the third extracellular cycle, respectively, and 75.0% identity between murine and bovine and 85% identity between humans and cattle, respectively, for the fourth extracellular cycle planned. Example 4: Genetic Mapping of Bovine NRAMP1 Genetic mapping is carried out in the following manner. The spots of hybrid somatic cell panels of cattle-hamsters (Womack and others 1986; Adkinson and others 1988; Beever et al. 1994), were hybridized with the RCP 1F / 1R-generated probe (4-8 x 108 cpm / μg) (Feinberg et al. 1983). Hybridization was carried out at 43 ° C for 18 hours in 20 ml of 50% formamide, 5X SSC, 1X Denhardt's solution and 20 mM NaPO4 (pH-6.8), followed by washing once in 2X SSC , 0.5% SDS at room temperature for 15 minutes, two successive washes in 1X SSC, 0.1% SDS at 65 ° C for 30 minutes (Adkinso.ny others, 1988). All gene probes were labeled in the randomized initiated DNA labeling method which is α- [32 P] dCTP (3000 Ci / mmoles) (NEN Research Products, Boston, MA (Feinberg et al., 1983)). Synthesis was assessed by concordance analysis of the probe with known marker genes as described (Womack et al., 1986; Adkinson et al., 1988; Womack et al., 1994). The syngeneic arrangement of bovine NRAMP1 was determined using the segregation analysis of somatic cell hybrids (Womack et al., 1986, White et al., 1994). DNA from 87 bovine / rodent somatic hybrid cells was digested with Hind III and hybridized to a generated PCR generated probe using primers SEQ ID NO. 3 and SEQ ID NO.4 (1F and 1R) (Figure 1B). The 4.7 kb bovine-specific HindIII restriction fragment was easily discriminated from the fragments representing the hamster and mouse homologues, allowing detection of specific bovine fragments in each cell line. A concordance analysis in the form of pairs indicated that NRAMP1 of cattle segregated 100% concordantly with Cry- ?, which was assigned to BTA 2 (Figure 9). An analysis of 87 somatic hybrids revealed that 28 were positive and 59 were negative for Cry-? and NRAMP1 of bovines. A group of syngenic sites of bovines, villin, Cry-? (Adkinson et al., 1988; Beever et al., 1994) and interleukin-8 receptor was mapped to a region of BTA 2 and retained in HSA 2q (White et al., 1994) and proximal MMU 1 (Cerretti et al. 1993), which were tightly linked to the Lsh / lty / Bcg site in the mouse (Figure 10). These results also support the homology between NRAMP of human, bovine and murine. Example 5: Single-strand conformational analysis (ACSH) We identified phenotypically resistant cattle susceptible to brucellosis by in vivo challenge (Harmon et al., 1985). Screening 22 offspring, individuals not related by ACSH, revealed the existence of two general single-strand polymorphic forms of NRAMP1 from cattle (Figure 3A). Sequencing analysis of fragments amplified by PCR showed a microsatellite length polymorphism that split at position 1785 of the two types; one being SEQ ID NO. 31: 5 '(GT), oAT (GT) 3 (N) 61 (GT) 5 (N) 24 (GT) 133, i the other being (GT) < .oAT (GT) 3 (N) -. 6. (GT) 5 (N) &. 24 (GT) > 133 \ where "N" symbolizes any of the four nucleotide bases A, C, G or T (Figure 7A). These polymorphisms correlate with both their distinctive patterns analyzed by ACSH or PCSH and with their respective in vivo phenotypes [p = 0.0089, Fisher Exact Analysis]. We will designate the initiator DNA sequence as ACSHr and the last sequence as ACSHX. The relative risk (RR) of susceptibility, if an animal has ACSHS is 4.5. Example 6: Specific expression of bovine Nrampl mRNA cells To test whether bovine NRAMP1 was expressed mainly in macrophage populations, we analyzed total RNA prepared from 15 different bovine tissues (peripheral blood lymphocytes, liver, lymph node, spleen , amygdala, lung, kidney, thymus, heart, skeletal muscle, jejunum, colon, ovary, uterus, brain and cultured macrophages) by Northern blot analysis using a bovine DNA probe generated by PCR of SEQ ID NO.3 and SEQ ID NO.4 (Figure 11). Northern blot analysis was carried out in the following manner. Monocyte-derived macrophages were recovered and cultured as described (Campbell and Adams, 1993). Total RNA was isolated from these macrophages and lymphocytes using normal techniques (Chirgwin et al., 1979). 10 μg of total RNA from macrophages and lymphocytes were separated on 1% formaldehyde agarose gels transferred to "Nytran" plus membranes (Schleicher &Schuell). The spots were prehybridized in 20 ml of 50% formamide, 10% dextran sulfate, 4.7X ACSH (1X ACSH is 10 mM sodium phosphate, 1 mM EDTA, 150 mM NaCl), 0.47X Denhardt's solution. 0.1% SDS, 0.18 mg / ml heat-denatured salmon sperm DNA and 0.34% fat-free milk for 4 hours at 42 ° C. Hybridization at 42 ° C for 18 hours was carried out in the same solution containing 2 X 108 cpm / m radiolabeled with [32 P] of the fragment SEQ ID NO.3 and SEQ ID NO.4. the final wash conditions were 0.2 X SSC, 0.1% SDS at 68 ° C for 30 minutes. A band of approximately 2.3 kb was detected in macrophage, spleen and lung RNA, but was absent in RNA analyzed from other tissues. These results indicate that Nrampl of bovines is mainly expressed in macrophages and in the reticuloendothelial system (RE). The following examples are intended to illustrate the embodiments of the present invention and are not intended to limit the scope of the invention in any way. Example 7: Identification of naturally resistant or disease-susceptible animals (traditional method) Cattle were phenotyped for resistance or susceptibility by in vivo challenge experiments with Brucella abortus strain 2308 and by in vitro macrophage death analysis . In vivo analysis: Unvaccinated control cattle were challenged with a virulent strain of S2308 of Brucella abortus. Those who did not exhibit signs of brucellosis, who were cultured bacteriologically negative for B. abortus, and who lack production of anti-lipopolysaccharide antibodies of B. abortus after challenge, were considered naturally resistant. Those who exhibited signs of brucellosis, were bacteriologically positive for culture of B. abortus or who produced antibodies of B. abortus anti-poiisacarid after confrontation, were considered naturally susceptible (Harmon et al 1985). In vitro analysis: The macrophages of unvaccinated control cattle were isolated according to normal techniques and compared, subsequently with B. abortus in an in vitro analysis (Harmon and others 1989, Price and others 1990, Campbell and others 1992) . Cattle macrophages that were able to control the intracellular replication of B. abortus in vitro were considered resistant, whereas cattle macrophages that were not as good at controlling the intracellular replication of B. abortus in vitro were considered susceptible. Example 8: Single Thread Conformational Analysis (ACSH): In the 15 μl total PCR reaction used for ACSH, the inventors mixed 0.2 mM each of dGTP, dGTP, dCTP, 0.07 mM dATP, 1 mM MgCl2m 10X M RCP buffer, 0.2 Units of Taq DNA polymerase (Cetus-Perkin Eimer) and 8 μCi of [35S ] -dATP with 50 ng of genomic DNA. The parameters for PCR amplification were a single step of denaturation of 5 minutes at 94 ° C followed by 32 cycles of denaturation (1 minute at 94 ° C), annealing (40 seconds at 60 ° C) and extension (1 minute at 72 ° C). ° C) and a final extension step of 7 minutes at 72 ° C. Oligonucleotide primers designed from 3'-UT were SEQ ID NO.1 (Fmicrol) = 5 'AAGGCAGCAAGACAGACAGG 3', nucleotide positions 1814-1833 and SEQ ID NO.2 (3 and 3) = 5 'ATGGAACTCACGTTGGCTG 3 ', nucleotide positions 1970-198). The ACSH polymorphisms of 232 unrelated cattle heads were detected by separating these PCR fragments from 175 bp in 6% poiiacrylamide gels with or without urea at room temperature at 90W for 2.5 hours. Analysis of ACSH ACSH was carried out essentially the same as PCSH with the following two exceptions. First, for ACSH analysis, SEQ ID NO.1 or SEQ ID NO.2 was marked at one end before the PCR reaction instead of having [35S] -dATP in the PCR reaction mixture. Second, for PCSH analysis, polyacrylamide gels always contained urea. Example 9: Specific Genetic Sequences Associated with NRAMP1 in situ Significantly Associated with Resistance or Susceptibility to Brucellosis, Tuberculosis, Paratuberculosis and Salmonellosis of Ruminants The cattle used in these experiments were unrelated. One was purebred Angus (Bos taurus) and the other twenty-two were cross-breed cattle produced by a three-way crossbreed - Fi (Bos taurus) X American Brahmin (Bos indicus)] (Harmon et al., 1985). All animals used in the experiments were housed in USDA approved facilities with daily supplemental feed. All experimental protocols are reviewed and approved by the University. DNA PCR amplification isolated from susceptible or resistant cattle was carried out in the following manner. In a PCR reaction of 15 μl used for ACSH, 0.2 mM of dGTP, dTTP and dCTP, 0.07 mM of dATP, 1 mM of MgCl2, 1.5 μL of 10X of PCR solution, 0.2 units of Taq DNA polymerase (Cetus -Perkin Elmer) and 8 μCi of [35S] -dATP were added to 50 ng of genomic DNA. The parameters for PCR amplification were a single step of denaturation of 5 minutes at 94 ° C followed by 32 cycles of denaturation (1 minute at 94 ° C), annealing (40 seconds at 60 ° C) and extension (1 minute at 72 ° C). ° C) and a final step of final extension of 7 minutes at 72 ° C. Oligonucleotide primers designated from 3 'UTR were SEQ ID NO.1 and SEQ ID NO.2 (Figure 1A). This amplified DNA was then subjected to electrophoresis under conditions of ACSH or PCSH. Namely, the PCR fragments were run on a 6% polyacrylamide gel (National Diagnostics, G.A.) at room temperature at 90W for 2.5 hours. The different migration patterns of the amplified DNA under these gel conditions were correlated with the cattle phenotype previously determined by the in vivo or in vitro challenge experiments with B. abortus (Harmon et al., 1985). Amplification of DNA PCR (using SEQ ID NO.1 and SEQ ID NO.2 as primers) of cattle previously determined to be resistant produced an amplicon that migrated faster than the isolated and amplified DNA of cattle determined to be sensitive when the amplicons were run on a 6% polyacrylamide gel under said conditions (Figure 3). Subsequent sequencing of the amplicons of some of these cattle revealed two significant sequence differences (Figure 7A). These sequence differences were correlated with resistance / susceptibility determined in vivo and can be categorized into two general groups. The amplified DNA of the resistant cattle corresponds to DNA sequences of the type: GGGTGT (GT) 10AT (GT) 3 (N) 6. (GT) 5 (N) 24 (GT) 13 While the amplified DNA of susceptible cattle corresponds to DNA sequences of the type: GGGGGT (GT) < 10AT (GT) 3 (N) < 61 (GT) 5 (N) < 24 (GT) > 13 Although ACSH and PCSH can not determine the exact DNA sequence of cattle in this region, the difference in the migration patterns of the amplicons associated with susceptibility and resistance allows us to define these amplicons because they are ACSHr or PCSHS for the conformation polymorphism of a single thread of the resistant or susceptible type, respectively. In addition, due to the nucleotide sequence conserved among pigs, goats, sheep, bison (American buffalo), llamas, moose (Wapiti), red deer, Japanese deer, water buffalo, fallow deer and white-tailed deer, and most probably all domestic and wild artiodactyls in this region, we can expect all these sequence differences in their relationship to susceptibility to disease are applicable to all artiodactyls (see, Figures 8A1, 8A2 and 8C). With respect to this, the intracellular survival of Brucella abortus and Mycobacterium bovis BCG was determined in an in vitro monocyte death analysis of macrophages using bison macrophages genotypically determined to be naturally resistant and susceptible to Brucella abortus infection, see Table I Table 1: Intracellular survival of Brucella abortus and Mycobacterium bovis BCG in a death analysis of monocyte-derived macrophages in vitro using bison macrophages phenotypically determined to be naturally resistant or susceptible to Brucella abortus infection. 1 R and S designate phenotypically the bison determined to be naturally resistant (R) to, or susceptibly (S) to, Brucella abortus. This was determined by a confrontation of pregnant bison not previously exposed, either by natural exposure, vaccination, at mid-gestation with 1 x 107 organisms of virulent B. abortus. 2 Percent survival refers to the number of B. abortus organisms that survive after being phagocytized by macrophages compared to the numbers of bacteria at Time 0 after 3 days of culture for B. abortus and 14 days of culture for M bovis BCG. In addition, these resistance / susceptibility profiles phenotypically determined in vitro of bison were compared with the resistance / susceptibility profiles genotypically determined from 3'UTR of the bison NRAMP1 gene. Similar to bovine studies, there is a correlation between the 3'UTR sequence of Bison NRAMP1 and the resistance / susceptibility phenotype. The bison phenotypically determined to be resistant, had a gene sequence associated with resistance in 3'UTR while the bison phenotypically determined to be susceptible, had a gene sequence associated with susceptibility in 3'UTR (Figure 8D). Figure 8D shows the 3'UTR NRAMP1 cDNA sequences in naturally susceptible and resistant bison. The nucleotides were positively enumerated in the 5 'to 3' orientation to the right of each line, starting from the coding nucleotide G in 1676 and ending with the last nucleotide. The ATG stop codon is indicated by (@). Three repetitions of TG including (TG) 13, (TG) 8 and (TG) 16 are marked in bold separately. The differences between the nucleotide sequences R and S are that the sequence of S has a TG less in the first repetition (TG) 12 against (TG) 13 and therefore it is two bases shorter in general (2259) versus (2261) ) for this area of the 3'UT sequence of NRAMP1. Polymorphisms containing the first (TG) 13 were detected by ACSH using Fmicro and Bmicrol 'primers as indicated.

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Because many variable and different modalities can be made within the scope of the inventive concept taught herein and because many modifications can be made to the detailed embodiments herein in accordance with the descriptive requirement of the law, understand that the details in the present will be interpreted as illustrative and not in a limiting sense.

LIST OF SEQUENCES (1) GENERAL INFORMATION (I) APPLICANT: Joe W. Templeton, Jianwei Feng, L. Garry Adams, Erwin Schurr, Philippe Gros, Donald S. Davis and Roger Smith (ii) TITLE OF THE INVENTION: IDENTIFICATION METHOD OF ANIMALS RESISTANT OR SUSCEPTIBLE TO DISEASES SUCH AS BRUCELLOSIS OF CATTLE, TUBERCULOSIS, PARATUBERCULOSIS AND SALMONELOSIS (ii) NUMBER OF SEQUENCES: 31 (iv) ADDRESS OF CORRESPONDENCE (TO) RECIPIENT: Pravel, Hewitt, Kimball & Krieger (B) STREET: 1177 West Loop South, 10th Floor (C) CITY: Houston (D) STATE: TX (E) COUNTRY: E.U.A. (F) ZP: 77027-9095 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIUM: Flexible Disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reeléase # 1.0, Version # 1.25 (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: (B) SUBMISSION DATE: (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) NUMBER OF APPLICATION: PROVISIONAL APPLICATION NUMBER 60 / 031,443 (B) SUBMISSION DATE: September 20, 1996 (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: Krieger, Paul E. (B) NUMBER. REGISTRATION: 25,886 (C) REFERENCE / CASE NUMBER: 00162-3 / V96171 US (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 713-850-0909 (B) TELEFAX: 713-850-0165 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: AAGGCAGCAA GACAGACAGG (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: ATGGAACTCA CGTTGGCTG (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TCTCTGGCTG AAGGCTCTCC (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (¡) i) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: CCAAGCTCAC CTTAGGGTAG (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: CGTGGTGACA GGCAAGGAC (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 6: CCAAGAAGAG GAAGAAGAAG GTGTC (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 547 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 7: Mee Thr Gly Asp Lys Gly Pro Gln Arg Leu Ser Gly 5 10 Ser Ser Tyr Gly Ser Be Ser Pro Thr Ser Pro 15 20 Gl? Pro Gln Qln Wing Pro Pro Arg Glu Thr Tyr Leu 25 3 ° 35 Ser Glu Lys He Pro Pro Pro Asp Thr Lys Pro Gly 40 45 Thr Phe Ser Leu Arg Lys Leu Trp Wing Phe Thr Gly Pro Gly Phe Leu Met Be He Wing Phe Leu Asp Pro 65 70 Gly Asn He Glu Be Asp Leu Gln Wing Gly Wing Val 75 80 Wing Gly Phe Lys Leu Leu Trp Val Leu Leu Trp Wing 85 90 95 Thr Val Leu Gly Leu Leu Cys Gln Arg Leu Ala Ala 100 105 Arg Leu Gly Val Val Thr Gly L s Asp Leu Gly Glu 110 115 120 Val Cys His Cys Tyr Tyr Pro Lys Val Pro Arg Thr 125 130 Val Leu Trp Leu Thr He Glu Leu Ala lie Val Gly 135 140 As Asp Met Gln Glu Val He Gly Thr Ala lie Wing 145 150 155 Phe Asn Leu Leu Be Wing Gly Arg He Pro Leu Trp 160 165 Gly Gly Val Leu He Thr He Val Asp Thr Phe Phe 170 175 180 Phe Leu Phe Leu Asp Asn Tyr Gly Leu Arg Lys Leu 185 190 Glu Wing Phe Phe Gly Leu He Thr He Met Wing 195 '200 Leu Thr Phe Gly Tyr Glu Tyr Val Val Wing Arg Pro 205 210 215 Glu Gln Gly Ala Leu Leu Arg Gly Leu Phe Leu Pro 220 225 Ser Cys Pro Gly Cys Gly His Pro Glu Leu Leu Gln 230 235 240 Wing Val Gly He Val Val Gly Wing He Met Met Pro His 245 250 Asn He Tyr Leu His Ser Wing Leu Val Lys Ser Arg 255 260 Glu He Asp Arg Wing Arg Arg Wing Asp He Arg Glu 265 270 275 Wing Asn Met Tyr Phe Leu He Glu Wing Thr He Ala 280 285 Leu Ser Val Ser Phe He He Asn Leu Phe Val Met 290 295 300 Wing Val Phe Gly Gln Wing Phe Tyr Gln Lye Thr Asn 305 310 Gln Wing Wing Phe Asn He Cys Wing Asn Ser Ser Leu 315 320 His Asp Tyr Wing Lys He Phe Pro Met Asn Asn Wing 325 330 335 Thr Val Wing Val Asp He Tyr Gln Gly Gly Val He 340 345 Leu Gly Cys Leu Phe Gly Pro Wing Ala Leu Tyr lie 350 355 360 Trp Wing He Gly Leu Leu Wing Wing Gly Gln Ser Ser 365 370 Thr Met Thr Gly Thr Tyr Wing Gly Gln Phe Val Met 375 380 Glu Gly Phe Leu Arg Leu Arg Trp Ser Arg Phe Wing 385 390 395 Arg Val Leu Leu Thr Arg Ser Cys Wing He Leu Pro 4Q0 405 Thr Val Leu Val Wing Val Phe Arg Asp Leu Arg Asp 410 415 420 Leu Ser Gly Leu Asn Asp Leu Leu Asn Val Leu Gln 425 430 Ser Leu Leu Pro Phe Ala Val Leu Pro He Leu 435 440 Thr Phe Thr Ser Met Pro Thr Leu Met Gln Glu Phe 445 450 455 Wing Asn Gly Leu Leu Asn Lys Val Val Thr Ser Ser 460 465 He Met Val Leu Val Cys Ala He Asn Leu Tyr Phe 470 475 480 Val Val Ser Tyr Leu Pro Be Leu Pro His Pro Wing 485 490 Tyr Phe Gly Leu Wing Wing Leu Leu Wing Wing Wing Tyr 495 500 Leu Gly Leu Ser Thr Tyr Leu Val Trp Thr Cys Cys 505 510 515 Leu Ala His Gly Ala Thr Pro Leu Ala His Ser Ser 520 525 His His His Phe Leu Tyr Gly Leu Leu Glu Glu Aap 530 535 540 Gln Lys Gly Glu Thr Ser Gly 545 (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 548 amino acids (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE: SEQ ID N0: 8: Met He Ser Asp Lys Ser Pro Pro Arg Leu Ser Arg 5 10 Pro Ser Tyr Gly Ser He Be Ser Leu Pro Gly Pro 15 20 Wing Pro Gln Pro Pro Wing Pro Cys Arg Glu Thr Tyr Leu 25 30 35 Ser Glu Lys He Pro Pro Pro Being Wing Asp Gln Gly 40 45 Thr Phe Ser Leu Arg Lys Leu Trp Wing Phe Thr Gly 50 55 60 Pro Gly Phe Leu Met Be lie Wing Phe Leu Asp Pro 65 70 Gly Asn He Glu Be Asp Leu Gln Wing Gly Wing Val 75 80 Wing Gly Phe Lys Leu Leu Trp Val Leu Leu Trp Wing 85 90 95 Thr Val Leu Gly Leu Leu Cys Gln Arg Leu Ala Ala 100 105 Arg Leu Gly Val Val Thr Gly Lys Asp Leu Gly Glu 110 115 120 Val Cys His Leu Tyr Tyr Pro Lys Val Pro Arg He 125 125 Leu Leu Trp Leu Thr He Glu Leu Wing He Val Gly 135 140 Being Asp Met Gln Glu Val He Gly Thr Wing He Ser 145 150 155 Phe Asn Leu Leu Be Wing Gly Arg He Pro Leu Trp 160 165 Gly Gly Val Leu He Thr He Val Asp Thr Phe Phe 170 175 180 Phe Leu Phe Leu Asp Asn Tyr Gly Leu Arg Lys Leu 185 190 Glu Wing Phe Phe Gly Leu Leu He Thr He Met Wing 195 200 Leu Thr Phe Gly Tyr Glu Tyr Val Val Wing His Pro 205 _10 215 Be Gln Gly Wing Leu Leu Lys Gly Leu Val Leu Pro 220 225 Thr Cys Pro Gly Cys Gly Gln Pro Glu Leu Gluc 230 235 240 Wing Val Gly He Val Gly Wing He Met Met Pro His 245 250 Asn He Tyr Leu His Ser Wing Leu Val Lys Ser Arg 255 260 Glu Val Asp Arg Thr Arg Arg Val Asp Val Arg Glu 265 270 275 Wing Asn Met Tyr Phe Leu He Glu Wing Thr He Ala 280 285 Leu Ser Val Ser Phe He He Asn Leu Phe Val M 290 295 300 Wing Val Phe Gly Gln Wing Phe Tyr Gln Oln Thr Asn 305 310 Glu Glu Wing Phe Asn He Cys Wing Asn Ser Ser Leu 315 320 Gln Asn Tyr Wing Lys He Phe Pro Arg Asp Asn Asn 325 330 335 Thr Val Ser Val Asp He Tyr Gln Gly Gly Val He 340 345 Leu Gly Cys Leu Phe Gly Pro Wing Ala Leu Tyr He 350 355 360 Trp Wing Val Gly Leu Leu Wing Wing Gly Gln Ser Ser 365 370 Thr Met Thr Gly Thr Tyr Wing Gly Gln Phe Val Me 375 380 Glu Gly Phe Leu Lys Leu Arg Trp Ser Arg Phe Wing 385 390 395 Arg Val Leu Leu Thr Arg Ser Cys Wing He Leu Pro 400 405 Thr Val Leu Val Wing Val Phe Arg Asp Leu Lys Asp 410 415 420 Leu Ser Gly Leu Asn Asp Leu Leu Asn Val Leu Gln 425 430 Ser Leu Leu Leu Pro Phe Wing Val Leu Pro He Leu 435 440 Thr Phe Thr Ser Met Pro Wing Val Met Gln Glu Phe 445 450 455 Wing Asn Gly Arg Met Ser Lys Wing He Thr Ser Cys 460 465 He Met Wing Wing Leu Val Cys Wing He Asn Leu Tyr Phe 470 475 480 Val He Ser Tyr Leu Pro Be Leu Pro His Pro Wing 485 490 Tyr Phe Gly Leu Val Wing Leu Phe Wing He Gly Tyr 495 - 500 Leu Gly Leu Thr Wing Tyr Leu Wing Trp Thr Cys Cys 505 510 515 He Ala His Gly Ala Thr Phe Leu Thr Has Ser Ser 520 525 His Lys His Phe Leu Tyr Gly Leu Pro Asn Glu Glu 530 535 540 Gln Gly Gly Val Gln Gly Ser Gly 545 (2) INFORMATION FOR SEQ ID N0: 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 548 amino acids (B) TYPE amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF SEQUENCE : SEQ ID NO: 9: Met Ser Gly Asp Thr Gly Pro Pro Lys Gln Gly Gly 5 10 Thr Arg Tyr Gly Ser Be Ser Ser Pro Pro Ser Pro 20 Glu Pro Gln Gln Wing Pro Pro Gly Gly Thr Tyr Leu 25 30 35 Ser Glu Lys He Pro He Pro Asp Thr Glu Ser Gly 40 45 Thr Phe Ser Leu Arg Lys Leu Trp Wing Phe Thr Gly 50 55 60 Pro Gly Phe Leu Met Be He Wing Phe Leu Asp Pro 65 70 Gly Asn He Glu Be Asp Leu Gln Wing Gly Wing Val 75 80 Wing Gly Phe Lys Leu Leu Trp Val Leu Leu Trp Wing 85 90 95 Thr Val Leu Gly Leu Leu Cys Gln Arg Leu Ala Ala 100 105 Arg Leu Gly Val Val Thr Gly Lys Asp Leu Gly Glu 110 115 120 Val Cys His Leu Tyr Tyr Pro Lys Val Px-o Arg He 125 130 Leu Leu Trp Leu Thr He Glu Leu Wing He Val Gly 135 140 As Asp Met Gln Glu Val He Gly Thr Ala He Wing 145 150 155 Phe Ser Leu Leu Ser Wing Gly Arg He Pro Leu Trp 160 165 Gly Gly Val Leu He Thr Val Val Asp Thr Phe Phe 17Q 175 180 Phe Leu Phe Leu Asp Asn Tyr Gly Leu Arg Lys Leu 185 190 Glu Wing Phe Phe Gly Phe Leu He Thr He Met Wing 195 200 Leu Thr Phe Gly Tyr Glu Tyr Val Val Wing Gln Pro 205 210 215 Wing Gln Gly Wing Leu Leu Gln Gly Leu Phe Leu Pro 220 225 Ser Cys Pro Gly Cys Gly Gln Pro Glu Leu Leu Gln 230 235 240 Wing Val Gly He He Gly Wing He He Met Met Pro His 245 250 Asn He Tyr Leu His Ser Ser Leu Val Lys Ser Arg 255 260 Glu Val Asp Arg Ser Arg Arg Wing Asp He Arg Glu 265 270 275 Wing Asn Met Tyr Phe Leu He Glu Wing Thr He Ala 280 285 Leu Ser Val Ser Phe Leu He Asn Leu Phe Val Met 290 295 300 Wing Val Phe Gly Gln Wing Phe Tyr Lys Gln Thr Asn 305 310 Gln Wing Wing Phe Asn He Cys Wing Asp Being Ser Leu 315 320 His Asp Tyr Ala Pro He Phe Pro Arg Asn Asn Leu 325 330 335 Thr Val Wing Val Asp He Tyr Gln Gly Gly Val He 340 345 Leu Gly Cys Leu Phe Gly Pro Pro Wing Leu Tyr He 350 355 360 Trp Wing Val Gly Leu Leu Wing Wing Gly Gln Ser Ser 365 370 Thr Met Thr Gly Thr Tyr Wing Gly Gln Phe Val Met 375 380 Glu Gly Phe Leu Lys Leu Arg Trp Ser Arg Phe Wing 385 390 395 Arg Val Leu Leu Thr Arg Ser Cys Wing He Leu Pro 400 405 Thr Val Leu Leu Wing Val Phe Arg Asp Leu Arg Asp 410 415 420 Leu Ser Gly Leu Asn Asu Leu Leu Asn Val Leu Gln 425 430 Ser Leu Leu Leu Pro Phe Ala Val Leu Pro He Leu 435 440 Thr Phe Thr Ser Met Pro Ala Leu Met Gln Glu Phe 445 4SO 455 Wing Asn Gly Leu Val Ser Lys Val He Thr Ser Ser 460 465 He Met Val Leu Val Cys Wing Val Asn Leu Tyr Phe 470 475 480 Val He Ser Tyr Leu Pro Ser Leu Pro His Pro Wing 485 490 Tyr Phe Ser Leu Val Wing Leu Leu Wing Wing Wing Tyr 495 500 Leu Gly Leu Thr Thr Tyr Leu Val Trp Thr Cys Leu 505 510 515 He Thr Gln Gly Ala Thr Leu Leu Ala His Ser Ser 520 525 His Gln Arg Phe Leu Tyr Gly Leu Pro Glu Glu Asp 530 535 540 Gln Glu Lys Gly Arg Thr Ser Gly 545 (2) INFORMATION FOR SEQ ID NO: 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH 2271 base pairs (B) TYPE, nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GCTTGCCATG CCCGTGAGGG GCTGCCCGGC ACGCCAGCCA CTCGCACAGA 50 GAGTGCCCGA GCCTGCGGTC CTCATGTCAG GTGACACGGG CCCCCCAAAG 100 CAGGGAGGGA CCAGATATGG CTCCATCTCC AGCCCACCCA GTCCAGAGCC 150 ACAGCAAGCA CCTCCCGGAG GGACCTACCT AAGTGAGAAG ATCCCCATTC 200 CGGATACAGA ATCGGGTACA TTCAGCCTGA GGAAGCTGTG GGCCTTCACG 250 GGGCCTGGAT TCCTCATGAG CATCGCATTC CTGGACCCAG GAAACATTGA 300 GTCGGATCTT CAGGCTGGGG CTGTGGCTGG ATTCAAACTG CTCTGGGTGC 350 TGCTGTGGGC CACAGTGTTG GGCTTGCTTT GCCAGCGACT GGCTGCCCGG 400 CTGGGCGTGG TGACAGGCAA GGACTTGGGC GAGGTCTGCC ATCTCTACTA 450 CCCTAAGGTG CCCCGCATTC TCCTCTGGCT GACCATCGAG CTAGCCATCG 500 TGGGCTCAGA CATGCAGGAA GTCATTGGCA CAGCTATTGC ATTCAGTCTG 550 CTCTCCGCCG GACGAATCCC ACTCTGGGGT GGTGTCCTCA TCACCGTCGT 600 GGACACTTTC TTCTTCCTCT TCCTCGATAA CTACGGGTTG CGGAAGCTGG 650 AAGCCTTTTT TGGATTTCTT ATTACCATAA TGGCCTTGAC CTTCGGCTAT 700 GAGTACGTGG TGGCTCAGCC TGCTCAGGGA GCATTGCTTC AGGGCCTGTT 750 CCTGCCCTCG TGCCCAGGCT GTGGCCAGCC CGAGCTGCTG CAAGCCGTGG 800 GCATCATTGG CGCCATCATC ATGCCCCACA ACATCTACCT GCATTCCTCC 850 CTGGTCAAGT CTCGAGAGGT AGACCGGTCC CGGCGGGCGG ACATCCGAGA 900 GGCCAACATG TACTTCCTGA TTGAAGCCAC CATCGCCCTG TCTGTCTCCT 950 TCCTCATCAA CCTGTTTGTC ATGGCTGTCT TTGGGCAAGC CTTCTACAAG 1000 CAAACCAACC AGGCTGCGTT CAACATCTGT GCCGACAGCA GCCTCCACGA 1050 CTACGCGCCG ATCTTTCCCA GGAACAACCT GACCGTGGCA GTGGACATTT 1100 ACCAAGGAGG CGTGATCCTG GGCTGCCTCT TTGGTCCTCC AGCCCTGTAC 1150 ATCTGGGCCG TGGGTCTCCT GGCTGCTGGG CAGAGCTCCA CCATGACCGG 1200 CACCTACGCG GGACAGTTTG TGATGGAGGG CTTCCTGAAG CTGCGGTGGT 1250 CACGCTTCGC CCGAGTCCTG CTCACTCGCT CCTGCGCCAT CCTGCCCACT 1300 GTGCTCCTGG CTGTCTTCAG GGACTTGCGG GACCTGTCAG GCCTCAACGA 1350 CCTGCTCAAT GTGCTGCAGA GCCTGCTGCT TCCCTTCGCT GTGCTGCCCA 1400 TCCTCACCTT CACCAGCATG CCCGCCCTGA TGCAGGAGTT TGCCAATGGC 1450 CTGGTGAGCA AAGTTATCAC TTCCTCCATC ATGGTGCTGG TCTGCGCCGT 1500 CAACCTTTAC TTCGTGATCA GCTACTTGCC CAGCCTCCCC CACCCTGCCT 1550 ACTTCAGCCT TGTAGCACTG CTGGCCGCAG CCTACCTGGG CCTCACCACT 1600 TACCTGGTCT GGACCTGTCT CATCACCCAG GGAGCCACTC TTCTGGCCCA 1650 CAGTTCCCAC CAACGCTTCC TGTATGGGCT TCCTGAAGAG GATCAGGAGA 1700 AGGGGAGGAC CTCGGGATGA GCTCCCACCA GGGCCTGGCC ACGGGTGGAA 1750 TGAGTGGGCA CAGTGGCCTG TCAGACAAGG GTGTGTGTGT GTGTGTGTGT 1800 GTGTATGTGT GTGAAGGCAG CAAGACAGAC AGGGAGTTCT GGAAGCTGGC 1850 CAACGTGAGT TCCAGAGGGA CCTGTGTGTG TGTGACACAC TGGCCTGCCA 1900 GACAAGGGTG TGTGTGTGTG TGTGTGTGTG TGTGCATGCA CAGCAAGACG 1950 GAGAGGGAGT TCTGGAAGGC AGCCAACGTG AGTTCCATAG GGACCTGCTA 2000 TTTCCTAGCT CAGATCTCAG TGTTCTTGAC TATAAAATGG GGACACCTAC 2050 CTTGGAGTGG TTGTAAATAA GACACTTGAA CGCAGAGCCT AGCACTTCAG 2100 ATTTAAAAAC AAAAGAATCA TAATTCCAAA AGTTACTGAG CACTATCACA 2150 GGAGTGACCT GACAGACCCA CCCAGTCTAG GGTGGGACCC AGGCTCCAAA 2200 CTGATTTAAA ATAAGAGTCT GAAAATGCTA AATAAATGCT GTTGTGCTTA 2250 GTCCCCGAGA AAAAAAAAAA A 2271 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 155 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) ) SEQUENCE DESCRIPTION: SEQ ID N0: 11: GGGTGTGTGT GTGTGTGTGT GTGTGTATGT GTGTGAAGGC AGCAAGACAG 50 ACAGGGAGTT CTGGAAGCTG GCCAACGTGA GTTCCAGAGG GACCTGTGTG 100 TGTGTGACAC ACTGGCCTGC CAGACAAGGG TGTGTGTGTG TGTGTGTGTG 150 TGTGT 155 (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 155 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGGGGTGTGT GTGTGTGTGT ATGTGTGTGA AGGCAGCAAG ACAGACAGGG 50 AGTTCTGGAA GCTGGCCAAC GTGAGTTCCA GAGGGACCTG TGTGTGTGTG 100 ACACACTGGC CTGCCAGACA AGGGTGTGTG TGTGTGTGTG TGTGTGTGTG 150 TGTGT 155 (2) INFORMATION FOR SEQ ID NO: 13: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 155 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 13: GGGGGTGTGT GTGTGTGTGT GTATGTGTGT GAAGGCAGCA AGACAGACAG 50 GGAGTTCTGG AAGCTGGCCA ACGTGAGTTC CAGAGGGACC TGTGTGTGTG 100 TGACACACTG GCCTGCCAGA CAAGGGTGTG TGTGTGTGTG TGTGTGTGTG 150 TGTGT 155TION FOR SEQ ID N0: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 155 base pairs (B) TYPE: nucleic acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GGGGGTGTGT GTGTGTGTGT GTGTATGTGT GTGAAGGCAG CAAGACAGAC 50 AGGGAGTTCT GGAAGCTGGC CAACGTGAGT TCCAGAGGGA CCTGTGTGTG 100 TGTGACACAC TGGCCTGCCA GACAAGGGTG TGTGTGTGTG TGTGTGTGTG 150 TGTGT ) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 155 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 15: GGGTGTGTGT GTGTGTGTGT GTGTGTATGT GTGTNN NNN t-ps-TONNNlSI 50 p ^ INNpmNNNN NNOT? S? SGN ^ isrN NNirepsrNW N S ?? ITNNNN NNNNGTGTG 100 TGTGT NN NN ^^ prNJNNN ^ NNNN! Psp. { NG TGTGTGTGTG TGTGTGTGTG 150 TGTGT (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 499 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 16: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAAGGG 50 GAGGACCTCG GGATGAGCTC CCACCAGGGC CTGGCCACGG GTGGAATGAG 100 TGGGCACAGT GGCCTGTCAG ACAAGGGTGT GTGTGTGTGT GTGTGTGTGT 150 ATGTGTGTGA AGGCAGCAAG ACAGACAGGG AGTTCTGGAA GCTGGCCAAC 200 GTGAGTTCCA GAGGGACCTG TGTGTGTGTG ACACACTGGC CTGCCAGACA 250 AGGGTGTGTG TGTGTGTGTG TGTGTGTGTG CATGCACAGC AAGACGGAGA 3O0 GGGAGTTCTG GAAGGCAGCC AACGTGAGTT CCATAGGGAC CTGCTATTTC 350 CTAGCTCAGA TCTCAGTGTT CTTGACTATA AAATGGGGAC ACCTACCTTG 400 GAGTGGTTGT AAATAAGACA CTTGAACGCA GAGCCTAGCA CTTCAGATT 449 (2) INFORMATION FOR SEQ ID N0: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 433 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple, (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 17: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAAGGG 50 GAGGACCTCG GGATGAGCTC CCACCAGGGC CTGGCCACGG GTGGAATGAG 100 TGGGCACAGT GGCCTGTCAG ACAAGGGTGT GTGTGTGTGT GTGTGTGTGT 150 GTGAAGGCAG CAAGACAGAC AGGGAGTTCT GGAAGCTGGC CAACGTGAGT 200 TCCAGAGGGA CCTGTGTGTG TGTGTGTGTC TGGCCTGCCA GACAAGGGTG 250 TGTGTGTGTG TGTGTGTGTG TGTGTGTGTA CAGCAAGACG GAGAGGG GT 300 TCTGGAAGGC AGCCAACGTG AGTTCCATAG GGACCTGCTA TTTCCTAGCT 350 CAGATCTCAG TGTTCTTGAC TATAAAATGG GGACACCTAC CTTGGAGTGG 400 TTGTAAATAA GACACTTGAA CGCAGAGCCT AGCACTTCAG ATT 443 ) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 445 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID N0: 18: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAAGGG 50 GAGGACCTCG GGATGAGCTC CCACCAGGGC CTGGCCACGG GTGGGATGAG 100 TGGGCACAGT GGCCTGTCAG ACAAAGGGGT GTGTGTGTGT GTGTGTGTAT 150 GTGTGCGAAG GCAGCAAGAC AGACAGGGAG TTCTGGAAGC TGGCCAACGT 200 GAGTTCCAGA GGGACCTGTG TGTGTGTGAC ACACTGGCCT GCCAGACAAA 250 GGTGTGTGTG TGTGTGTGTG TGTGTGCATG CACAGCAAGA CGGAGAGGGA 300 GTTCTGGAAG GCAGCCAACG TGAGTTCCAT AGGGACCTGC TATTTCCTAG_350_CTCAGATCTC AGTGTTCTTG ACTATAAAAT GGGGACACCC ACCTTGGAGT 400 GGTTGTTAAT AAGACACTTG AACGCAGAAC CTAGCACCTC AGATT 445 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 401 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 19: CACCAACGCT TCCTGTATGG GCTTCCTGGA GAGGATCAGG AGGAGGGGAG 50 GACCTCGGGA TGAACTCCCA CCAGGGCCTG GCCACGGGTG GGATGAGTGA 100 CCACAGTGGC CTGCCAGACA AGGGTGTGTG TGTGTGTGTG TGTGTGTGTG 150 TGTGTGCATG CACAGCAAGA TGGAGAGGGA GTTCACGGGT GGGATGAGTG 200 GGCACAGTGG CCTGCCAGAC AAGGGTGTGT GTGTGTGTGC ACGCACAGCA 250 AGATGGACAG GGAATTTTGG AAGCCGGCCA AGCCATAGGG ACCTGCTATT 300 TCCTAGCTCA GATCTCGGTA TTCTTGAGTA TTAAATGGGG ACACCTACCT 350 TGCAATGGTT GTAAATAAGA CACTTGAACG CAGAGCCTAG CACTTCAGAT 400 T (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 344 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 20: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAAGGG 50 GAGGACCTCA GGATGAGCTC CCACCAGGGC CTGGCCACGG GTGGAATGAG 100 TGGGCACAGT GGCCTGCCAG ACAAGGGTGT GTGTGTGTAT GTGTGTGTGT 150 GTGTGTGTGT GTGTGTGCGC GCTCACCCAC AACAAGACGG AGAGGGAGTT 200 CTGGAAGCCG GACAACGTOA GTTCCATAGG GACCTGCTGT TTCCTAGCTC 250 AGATCTCAGT GTTCTTGATT ATAAAATGGG GACACCTACC TTGCAACGGT 300 TGTAAATAAG ACACATTGGA ACGCAGAGGC TAGCACTTCA GATT 344 (2) INFORMATION FOR SEQ ID NO : 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 349 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 21 TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGGAGAATG 5O GGAGGACCTC AGGATGAGCT CCCACCAGGA CCCTGCCACG GGTGGGATGA 100 GTGGGCACAG TGGCCTGCCA GACAAGGGTG TGTGTGTGTG TGTGTGTGTG 150 TGTGTGTGTG CGCGCGCGCG CGCGAGCGCT CACACACAGC AAGACAGAGA 200 GGGAGTTCTG GAAGCCGGAC GACGTGAGTT CCATAGGGAC CTGCTGTTTC 250 CTAGCTCATT CTTCACTATA AAATGGGGAC ACCTACCTTG CAATGGTTGT 300 AAATAAGAGT AAATAAGACA CTTGAATGCA GAGCCTAGCA CTTCAGATT 349 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 348 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 22: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAATGG 50 GAGGACCTCG GGATGAGCTC CCACCAGGAC CCGGCCACGG GTGGGATGAG 100 GTGTGTGTGT GTGTGTGTGT TGGGCACAGT GGCCTGCCAG ACAAGGGTGT 150 GTGTGTGTGT GTGTGCGCGC GCGCGCGCTC ACACACAGCA AGACAGAGAG 200 GGAGTTCTGG AAGCAGGACG ACGTGAGTTC CATAGGGACC TGCTGTTTCC 250 TAGCTCAGAT CTCAGTGTTC TTCACTATAA AATGGGGACA CCTACCTTGC 300 AATGGTTGTA AATAAGACAC TTGAATGCAG AGCCTAGCAC TTCAGATT 348 (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 344 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 23: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAATGG 50 GAGGACCTCG GGATGAGCTC CCACCAGGAC CCGGCCACGG GTGGGATGAG 100 GTGTGTGTGT GTGTGTGTGT TGGGCACAGT GGCCTGCCAG ACAAGGGTGT 150 GTGTGTGTGT GCGCGCGCGC GCGCTCACAC ACAGCAAGAC AGAGAGGGAG 200 TTCCGGAAGC CGGACGACGT GAGTTCCATA GGGACCTGCT GTTTCCTAGC 250 TCAGATCTCA GTGTTCTTCA CTATAAAATG GGGACACCTA CCTTGCAATG 300 GTTGTAAATA AGACACTTGA ATGCAGAGCC TAGCACTTCA GATT 344 (2) INFORMATION FOR SEQ ID NO: 24: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 326 base pairs (B) TYPE: nucleic acid '(C) THREAD FORM: simple (D) TOPOLOGY: linear ( ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 24: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAATGG 50 GAGGACCTCG GGATGAGCTC CCACCAGGGC CCGGCCACGG GTGGGATGAG 100 TGGGCACAGT GGCCTGCCAG ACAAGGGGGT GTGTGCACGC GTGTGTGTGT 150 GCGCGCTCAC ACACAGCAAG ACAGAGAGGG AGTTCTGGAA GCAGGACGAC 200 GTGAGTTCCA TAGGGACCTG CTGTTTCCTA GCTCAGATCT CAGTGTTCTT 250 CACTATAAAA TGGGGACACC TACCTTGCAA TGGTTGTAAA TAAGACACTT 300 GAACGCAGAG CCTAGCACTT CAGATT 326 (2) INFORMATION FOR SEQ ID NO: 25: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 308 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 25: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAGGGG 50 GAGGACCTCA GGATGAGCTC CCACCAGGGC CTGGCCACGG GTGGGATGAG 100 TGGGCACAGT GGCCTGCCAG ACAAGGGTGT GTGTGTGGTC ACCCACAGCA 150 AGACGGAGAG GGAGTTCTGG AAGCCGGACA ACGTGAGTTC CATAGGGACC 200 TGCTGTTTCC TAGCTCAGAT CTCAGTGTTC TTGACTATAA AATGGGGACA 250 CCTACCTTGC AATGGTTGTA AATAAGACAC TTGAACGCAG AGCCTAGCAC 300 TTCAGATT 308 (2) INFORMATION FOR SEQ ID NO: 26: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 308 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 26: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GAAGAGGATC AGGAGAGGGG 50 GAGGACCTCG GGATGAGCTC CCACCAGGGC CTGGCCACAG GTGGGATGAG 100 TGGGCACAGT GGCTTGCCAG ACAAGGGTGT GTGTGTGGTC ACCCACAGCA 150 AGACGGAGAG GGAGTTCTGG AAGCCGGACA ACGTGAGTTC CATAGGGACC 200 TGCTGTTTCC TAGCTCAGAT CTCAGTGTTC TTGACTATAA AATGGGGACA 250 CCTACCTTGC AATGGTTGTA AATAAGACAC TAGAACGCAG AGCCTAGCAC 300 TTCAGATT 308 (2) INFORMATION FOR SEQ ID NO: 27: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 329 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 27: TCCCACCAAC GCTTCCTGTA TGGGCTTCCT GGAGAGGATC AGGAGGAGGG 50 GAGGACCTCG GGATGAACTC CCACCAGGGC CCGGCCACGG GTGGGATGAG 100 GTGTGTGTGT GTGTGTGTGT TGACCACAGT GGCCTGCCAG ACAAGGGTGT 150 GTCTGTGTGT GTGCGCGCGC ACACAGCAAG ATGGAGAGGG AATTCTGGAA 200 GCCGGCCAAG CCATAGGAGC CTGCTATTTC CTAGCTCAGA TCTTGGTATT 250 CTTGAGTATT AACTGGGGAC ACCTACCTTG CAATGGTTGT AAATAAGACA 300 CTTGAACGCA GAGCCTAGCA CTTCAGATT 329 (2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 548 amino acids (B) TYPE: peptide (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: Met Ser Gly Asp Thr Gly Pro Pro Lys Gln 5 10 Gly Gly Thr Arg Tyr Gly Being Be Ser 15 20 Pro Pro Pro Pro Glu Pro Gln Gl Wing Pr-a 25 30 Pro Gly Gly Thr Tyr Leu Ser Glu Lys He 35 40 Pro He Pro Asp Thr Glu Ser Gly Thr Phe 45 50 Ser Leu Arg Lys Leu Trp Wing Phe Thr Gly 55 60 Pro Gly Phe Leu Met Be He Wing Phe Leu 65 70 Asp Pro Gly Asn He Glu Be Asp Leu Gln 75 80 Ala Gly Ala Ala Ala Gly Phe Lys Leu Leu 85 90 Trp Val Leu Leu Trp Wing Thr Val Leu Gly 95 100 Leu Leu Cys Gln Arg Leu Ala Wing Arg Leu 105 110 Gly Val Val Thr Gly Lys Asp Leu Gly Glu 115 120 Val Cys His Leu Tyr Tyr Pro Lys Val Pro 125 130 Arg He Leu Leu Trp Leu Thr He Glu Leu 135 140 Ala He Val Gly Ser Asp Met Gln Glu Val 145 150 He Gly Thr Ala He Ala Phe Ser Leu Leu 155 160 Be Ala Gly Arg He 'Pro Leu Trp Gly Gly 165 170 Val Leu He Thr He Val Asp Ala Phe Phe 175 180 Phe Leu Phe Leu Asp Asn Tyr Gly Leu Arg 185 190 Lys Leu Glu Wing Phe Phe Gly Phe Leu He 195 200 Thr He Met Wing Leu Thr Phe Gly Tyr Glu 205 210 Tyr Val Val Wing Gln Pro Wing Gln Gly Wing 215 220 Leu Leu Gln Gly Leu Phe Leu Pro Ser Cys 225 230 Pro Gly Cys Gly Gln Pro Glu Leu Leu Gln 235 240 Ala Val Gly He He Gly Ala lie lie Met 245 250 Pro His Asn He Tyr Leu His Ser Ser Leu 255 260 Val Lys Ser Arg Glu Val Asp Arg Ser Arg 265 270 Arg Ala Asp He Arg Glu Ala Asn Met Tyr 275 280 Phe Leu He Glu Wing Thr He Ala Leu Ser 285 290 Val Ser Phe Leu lie Asn Leu Phe Val Met 295 300 Wing Val Phe Gly Gln Wing Phe Tyr Lys Gln 305 310 Thr Asn Gln Ala Ala Phe Asn He Cys Ala 315 320 Asn Ser Ser Leu Gln Asp Tyr Ala Pro lie 325 330 Phe Pro Arg Asn Asn Leu Thr Val Wing Val 335 340 Asp He Tyr Gln Gly Gly Val He Leu Gly 345 350 Cys Leu Phe Gly Pro Ala Ala Leu Tyr He 355 360 Trp Wing Val Gly Leu Leu Wing Wing Gly Gln 365 370 Be Ser Thr Met Thr Gly Thr Tyr Wing Gly 375 380 Gln Phe Val Met Glu Gly Phe Leu Lys Leu 385 390 Arg Trp Ser Arg Phe Ala Arg Val Leu Leu 395 400 Thr Arg Ser Cys Wing He Leu Pro Thr Val 405 410 Leu Leu Ala Val Phe Arg Asp Leu Arg Asp 415 420 Leu Ser Gly Leu Asn Asp Leu Leu Asn Val 425 430 Leu Gln Ser Leu Leu Leu Pro Phe Ala Val 435 440 Leu Pro He Leu Thr Phe Thr Ser Met Pro 445 450 Wing Leu Met Arg Glu Phe Wing Asn Gly Leu 455 460 Val Ser Lys Val He Thr Ser Ser He Met 465 470 Val Leu Val Cys Ala Val Asn Leu Tyr Phe 475 480 Val He Ser Tyr Val Pro Ser Leu Pro His 485 490 Pro Ala Tyr Phe Ser Leu Val Ala Leu Leu 495 500 Wing wing Wing Tyr Leu Gly Leu Thr Thr Tyr 505 510 Leu Val Trp Thr Cys Leu He Thr Gln Gly 515 520 Ala Thr Leu Leu Ala His Ser Ser His Gln 525 530 Arg Phe Leu Tyr Gly Leu Pro Glu Glu Asp 535 540 Gln Glu Lys Gly Arg Thr Ser sly 545 (2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 587 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) ) TYPE OF MOLECULE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29: GGGCTTCCTG AAGAGGATCA GGAGAAGGGG AGGACCTCGG GGATGAGCTC 50 CCACCAGGGC CTGGCCACGG GTGGGATGAG TGGGCACAGT GTGTGTGTGT GTGTGTGTGT GGCCTGTCAG 100 ACAAGGGTGT GTGAAGGCAG CAAGACAGAG 150 ACGGAGTTCT GGAAGCTGGC CAACGTGAGT TCCAGAGGGA CCTGTGTGTG 200 TGTGTGTGAC ACACTGGCCT GCCAGACAAG GGTGTGTGTG TGTGTGTGTG 250 TGTGTGTGTG TGTGCATGCA CAGCAAGACA GAGAGGGAGT TCTGOAAGCC 300 AGCCAACGTG AGTTCCATAG GGACCTGCTA TTTCCTAGCT CAGATCTCAG 350 TGTTCTTGAC TATAAAATGG GGACACCTAC CTTGGAATGG TTGTAAATAA 400 GACACTTGAA CGCAGAGCCT AGCACTTCAG ATTTAAAAAC AAAAGAATCA 450 TAATTCCAAA AGTTACTGAG CACTATCACA GGAGTGACCT GACAGACCCA 500 CCCAGTCCAG GGTGGGACCC AGGCTCCAAA CTGATTTAAA ATAAGAGTCT 550 GAAAATGCTA AATAAATGCT GTTGTGCTTA GTCCCCG (2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 585 nucleotides (B) TYPE, nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 30: GGGCTTCCTG AAGAGGATCA GGAGAAGGGG AGGACCTCGG GGATGAGCTC 50 CCACCAGGGC CTGGCCACGG GTGGGATGAG TGGGCACAGT GGCCTGTCAG 100 ACAAGGGTGT GTGTGTGTGT GTGTGTGTGT GAAGGCAGCA AGACAGAGAC 150 GGAGTTCTGG AAGCTGGCCA ACGTGAGTTC CAGAGGGACC TGTGTGTGTG 200 TGTGTGACAC ACTGGCCTGC CAGACAAGGG TGTGTGTGTG TGTGTGTGTG 250 TGTGTGTGTG TGCATGCACA GCAAGACAGA GAGGGAGTTC TGGAAGCCAG 300 CCAACGTGAG TTCCATAGGG ACCTGCTATT TCCTAGCTCA GATCTCAGTG 350 TTCTTGACTA TAAAATGGGG ACACCTACCT TGGAATGGTT GTAAATAAGA 400 CACTTGAACG CAGAGCCTAG CACTTCAGAT TTAAAAACAA AAGAATCATA 450 ATTCCAAAAG TTACTGAGCA CTATCACAGG AGTGACCTGA CAGACCCACC 500 CAGTCCAGGG TGGGACCCAG GCTCCAAACT GATTTAAAAT AAGAGTCTGA 550 AAATGCTAAA TAAATGCTGT TGTGCTTAGT CCCCG (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 149 nucleotides (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 31: GTGTGTGTGT GTGTGTGTGT ATGTGTGTNN NNNNNNNNNN ??? Sp > ??? N? 50 ?????? N ??? ?????????? ?????????? ????????? G TGTGTGTGT? 100 ?????????? ?? TSI ????? ? ??? GTGTGTG TGTGTGTGTG TGTGTGTGT 149

Claims (41)

1. CLAIMS 1. A method for screening animals for resistance or susceptibility to disease comprising the steps of: analyzing a genetic material of the animal for the presence of one or more specific genetic sequences associated with Nrampl of bovine whose sequences associated with susceptibility or resistance of an animal The disease was caused by intracellular parasites. The method of claim 1, wherein the disease caused by intracellular parasites is selected from the group consisting of brucellosis, tuberculosis, paratuberculosis and artiodactyls salmonellosis. 3. The method of claim 1, wherein the disease caused by intracellular parasites is selected from the group consisting of brucellosis, tuberculosis, paratuberculosis and salmonellosis of ungulates. 4. The method of claim 1, wherein the disease caused by intracellular parasites is selected from the group consisting of brucellosis, tuberculosis, paratuberculosis and salmonellosis of ruminants. 5. The method of claim 1, wherein the animal is an artiodactyl. 6. The method of claim 1, wherein the artiodactyl is an ungulate. 7. The method of claim 1, wherein the ungulate is a ruminant. The method of claim 7, wherein the ruminant is selected from the group consisting of bovine and bison. The method of claim 1, wherein a specific genetic sequence that is associated with Nrampl of bovine whose sequence is associated with the susceptibility of an animal to the disease caused by intracellular parasites, is selected from the sequence consisting of SEQ ID. US. 12, 13, 14 and 30. The method of claim 1, wherein a specific genetic sequence that is associated with Nrampl of bovine whose sequence is associated with the resistance of an animal to a disease caused by intracellular parasites, is selected of the group consisting of SEQ ID NOS. 11, 15, 29 and 31. The method of claim 1, wherein the genetic material of the animal is analyzed by PCR for the presence of one or more genetic sequences associated with Nrampl of bovine whose sequences are associated with susceptibility or resistance. from an animal to the disease caused by intracellular parasites. The method of claim 10, wherein the PCR uses at least one primer comprising a sequence selected from the group consisting of sequences identified by the sequence identification numbers 1 and 2. The method of claim 1 , wherein the genetic material of the animal is analyzed in order to detect the presence of one or more specific genetic sequences associated with Nrampl of bovine whose sequence is associated with susceptibility or resistance of an animal to the disease caused by intracellular parasites. The method of claim 1, wherein the genetic material of the animal is analyzed by ACSH in order to determine the presence of one or more specific genetic sequences associated with NRAMP1 whose sequence is associated with susceptibility or resistance of an animal to the disease of intracellular parasites. 15. A method for predicting the probability of an animal's susceptibility to the disease caused by intracellular parasites comprising: analyzing a genetic material of an animal for the presence of genetic sequences selected from the group consisting of the sequences identified by the numbers of identification of sequences 12-14 and 30, the presence of the sequence in sifu being associated with the susceptibility of an animal to the disease caused by intracellular parasites. 16. The method of claim 15, wherein the animal is an artiodactyl animal. 17. The method of claim 16, wherein the artiodactyl animal is an ungulate animal. 18. The method of claim 17, wherein the ungulate animal is a ruminant animal. 19. The method of claim 18, wherein the ruminant animal is selected from the group consisting of bovine and bison. 20. The method of claim 15, wherein the disease caused by intracellular parasites is selected from the group consisting of brucellosis, tuberculosis, paratuberculosis and arthiodactyly salmonellosis. The method of claim 15, wherein the disease caused by intracellular parasites is selected from the group consisting of bruceiosis, tuberculosis, paratuberculosis and salmonellosis of ungulates. 22. The method of claim 15, wherein the disease caused by intracellular parasites is selected from the group consisting of brucellosis, tuberculosis, paratuberculosis and salmonellosis of ruminants. 23. The method of claim 15, wherein the artiodactyl animal is an ungulate. The method of claim 15, wherein the genetic material of the animal is analyzed by PCR for the presence of the genetic Nrampl sequence of bovines. The method of claim 15, wherein the PCR uses at least one primer comprising a sequence selected from the group consisting of the sequences identified by the sequence identification numbers 1 and 2. 26. The method of claim 15, wherein the genetic material of the animal is analyzed by PCSH for the presence of the genetic sequence of NRAMP1 of bovines. 27. The method of claim 15, wherein the genetic material of the animal is analyzed by ACSH for the presence of the genetic sequence of NRAMP1 of bovines. 28. A method for predicting the susceptibility probability of an animal being resistant to the disease caused by intracellular parasites comprising: analyzing a genetic material from an animal for the presence of Nrampl genetic sequence selected from the group consisting of the sequences identified by the identification number of sequences 11, 15, 29 and 31, the presence of the sequence in situ is associated with the resistance of an animal to the disease caused by intracellular parasites. 29. The method of claim 28, wherein the animal is an artiodactyl animal. 30. The method of claim 29, wherein the animal artiodactyl is an ungulate animal. 31. The method of claim 30, wherein the unguided animal is a ruminant animal. 32. The method of claim 31, wherein the ruminant animal is selected from the group consisting of bovine and bison. 33. The method of claim 28, wherein the disease caused by intracellular parasites is selected from the group consisting of brucellosis, tuberculosis, paratuberculosis and artiodactyls salmonellosis. 34. The method of claim 28, wherein the disease caused by intracellular parasites is selected from the group consisting of brucellosis, tuberculosis, paratuberculosis and salmonellosis of ungulates. 35. The method of claim 28, wherein the disease caused by intracellular parasites is selected from the group consisting of brucellosis, tuberculosis, paratuberculosis and salmonellosis of ruminants. 36. The method of claim 28, wherein the animal is an artiodactyl animal. 37. The method of claim 36, wherein the artiodactyl animal is an ungulate. 38. The method of claim 28, wherein the genetic material of the animal is analyzed by PCR for the presence of the genetic sequence of Nrampl of bovines. 39. The method of claim 38, wherein the PCR uses at least one primer comprising a sequence selected from the group consisting of the sequences identified by the sequence identification numbers 1 and 2. 40. The method of the claim 28, where the genetic material of the animal is analyzed by PCSH for the presence of the genetic sequence of NRAMP1 of bovines. 41. The method of claim 28, wherein the genetic material of the animal is analyzed by ACSH for the presence of the genetic sequence of NRAMP1 of bovines.