Heurísticas en el razonamiento probabilístico en la implementación de la actividad sin el

The natural-resistance-associated macrophage protein 1 (NRAMP1) gene plays an important role in protection against several intracellular pathogens and was first identified in mice by positional cloning (Vidal et al. 1993). It was earlier denoted as the Bcg/Lsh/Ity locus because of its role in resistance/susceptibility to Mycobacterium bovis strain bacilli Callmette-Guerin, Leishmania donovani and Salmonella typhimurium infections (Skamene 1994). NRAMP1, based on its function as a divalent cation-proton antiporter, has been renamed as solute carrier family 11a member 1 (SLC11A1, Goswami et al. 2001). SLC11A1, by virtue of pleiotropic effects on macrophage activation, has potential for antimicrobial defences (including anti- viral), anti-tumour defences and autoimmunity (Blackwell et al 2000). Its effects include generation of antimicrobial hydroxyl radicals and modulation of TNFα, interleukin-1β, MHC class II molecules, chemokine KC, nitric oxide and nitric oxide synthetase.

A natural glycine→aspartate mutation at amino acid 169 of the murine SLC11A1 renders mice susceptible to Leishmania donovani, Salmonella typhimurium and Mycobacterium bovis (Vidal et al. 1995). The SLC11A1 gene is evolutionarily conserved in many phylogenetically distant organisms like mammals, birds, insects, worms, plants, fungi and bacteria (Cellier et al. 1996). A fragment of the ovine SLC11A1 was first isolated by screening of a sheep cosmid library (Pitel et al. 1994) and was later assigned to chromosome 2q41→q42 by in situ hybridization (Pitel et al. 1995).

2.4.1 Structure and polymorphism

Sequencing of ovine SLC11A1 cDNA cloned by reverse transcription polymerase chain reaction (RT-PCR) of RNA from macrophages yielded a sequence of 2083 nucleotides (GenBank accession # AF005380, Matthews and Crawford 1998). The open reading frame (exclusive of stop codon TGA) was 1644 nucleotides long, with the predicted protein being 548 amino acids long. The SLC11A1 amino acid sequence in sheep was found to be ≈98% similar to that in cattle and deer. A 2172-bp full length cDNA sequence of the sheep SLC11A1 gene was also reported by a different research group (GenBank accession #

U70255, Bussmann et al. 1998). The lengths of open reading frame and predicted protein and were similar to those reported by Matthews and Crawford (1998). While the in-frame initiator codon followed a 134-nucleotide 5’ untranslated region, the TGA termination codon was located downstream from glycine 548 (nucleotide 1778).

The exon-intron boundaries of the sheep SLC11A1 gene were determined by PCR

amplification, cloning and complete or partial sequencing of introns (Bussmann et al. 1998). The ovine gene possessed 15 exons. The exon-intron organization was found to be perfectly conserved among human, mouse and sheep genes, although intron sizes varied from species to species. The complete sheep SLC11A1 gene was predicted to span up to 10.5 kb of genomic DNA.

The sheep SLC11A1 protein was predicted to be 59.4 kDa in molecular weight and its structure to be in agreement with the putative murine, human, bovine and chicken SLC11A1 protein (Bussmann et al. 1998). The sheep protein is highly hydrophobic with 12 putative transmembrane domains, two potential amino-linked glycosylation sites and four protein kinase C phosphorylation sites. Sequence alignment of the bovine, human, mouse and chicken SLC11A1 predicted proteins, in comparison with the sheep sequence, exhibited identities of 98%, 89%, 86% and 68%, respectively.

Cell-specific expression of sheep SLC11A1 mRNA in various organs and cell types was studied employing RT-PCR (Bussmann et al. 1998). SLC11A1 mRNA could be detected in liver, spleen and lung, but not in the lymph node and heart. Also, an increase in SLC11A1 expression was observed in alveolar macrophages subsequent to activation with live Salmonella abortusovis. It was concluded that the SLC11A1 gene in sheep, as in other mammals, expressed specifically in the reticulo-endothelial system, macrophages in particular.

Two polymorphic microsatellite markers were identified close to the sheep SLC11A1 gene (Pitel et al. 1996). A (TG)n repeat (OVINRA1, EMBL accession # X89268) was found to be located in the 3’ untranslated region, while a second (GT)n repeat (OVINRA2, EMBL accession # X89269), associated with a short interspersed repetitive element (SINE), was in the same 40 kilo bp cosmid as the SLC11A1 gene. The two microsatellites were found to be genetically linked. Eight and four alleles could be detected for the two microsatellites respectively in 129 two-generation families sired by 15 rams. While the (TG)n repeat is absent in the 3’ region of human and mouse SLC11A1 genes, there are two such repeats in the bovine SLC11A1 gene (Matthews and Crawford 1998). While eight OVINRA1 alleles were detected in 15 unrelated New Zealand Romney sheep (Matthews and Crawford 1998), seven and four alleles were found at the same locus in two Australian Merino flocks

(Reddacliff et al. 2005). In highly structured populations of wild sheep, nine and three alleles were found at OVINRA1 and OVINRA2 loci, respectively (Worley et al. 2006).

2.4.2 Association with disease resistance

Studies in mice indicated SLC11A1 to be associated with natural resistance/susceptibility to intracellular infections (Vidal et al. 1993, 1995; Puliti et al. 1995; Leclercq et al. 1996). Controversy exists with regard to the role of SLC11A1 in susceptibility to clinical

tuberculosis in humans. There were indications for positive (Bellamy 1999; Awomoyi et al. 2002) as well as negative (Shaw et al. 1997; Soborg et al. 2002) associations of alleles at the locus with susceptibility to tuberculosis. A recent review of literature in this area, by meta- analysis, concluded that polymorphisms at SLC11A1 loci were not associated with

susceptibility to tuberculosis in subjects of European descent, while SLC11A1 gene polymorphisms significantly influenced susceptibility to the disease in Asian and African subjects (Li et al. 2006). Further, it has been reported that SLC11A1 gene polymorphisms would influence progression to severe forms of pulmonary tuberculosis, rather than

influencing susceptibility to the disease (Zhang et al. 2005).

A few studies investigated the association of polymorphisms in SLC11A1 gene with disease resistance/susceptibility in cattle and sheep. A preliminary study aimed at association between SLC11A1 alleles and susceptibility to ovine PTB (Beard et al. 1999). No evidence of the glycine→aspartate mutation at amino acid 169 of SLC11A1 (that was implicated in susceptibility of mice to intracellular parasites including mycobacteria, Vidal et al. 1995) was found in the investigated PTB-effected as well as unaffected sheep . A recent study on

Australian Merinos revealed possible associations of 162 and 160 bp alleles at OVINRA1 microsatellite locus within the SLC11A1 gene with susceptibility and resistance, respectively, to clinical PTB (Reddacliff et al. 2005).

SLC11A1 protein was found to play an important role in T-cell reactions combating bovine tuberculosis. High-level expression of SLC11A1 protein was detected in peripheral blood cells and granulomas of Mycobacterium bovis-infected cattle (Estrada-Chavez et al. 2001; Pereira-Suarez et al. 2006). However, no associations could be detected between

resistance/susceptibility to Mycobacterium bovis infection and polymorphism in the SLC11A1 gene in cattle (Barthel et al. 2000). Studies have also been undertaken to

SLC11A1 gene and resistance to infection with Brucella abortus, a facultative intracellular pathogen. Alleles (GT)13 and (GT)14-16 at the microsatellite locus were implicated with resistance and susceptibility, respectively, to Brucella abortus infection in Holstein Friesian cattle (Adams and Templeton 1998). However, the (GT)13 allele, even in homozygous condition, was found to be incapable of protecting Indian Zebu and their crosses with Bos taurus cattle against brucellosis (Kumar et al. 2005). Also, a recent study in Caucasian Spaniards found no difference in SLC11A1 allelic frequencies between brucellosis-positive and healthy individuals (Bravo et al. 2006).

2.5 OVINE IFN-γ GENE

Interferons (IFN) constitute a class of cytokines having an important role in immune responses. Apart from having direct antiviral effects (Lane et al. 1988), they are known to induce expression of cell-surface class II MHC molecules and increase the activity of natural killer cells and the anti-microbicidal activity of macrophages and neutrophils (Wallach et al. 1982; Metcalf 1987). IFN-γ differs from IFN-α and IFN-β in its biochemical and biological properties (Hovanessian 1985) and plays a central role in induction and modulation of immune responses (Young and Hardy 1995).

IFN-γ is produced by T cells in response to stimulation with an antigen or a mitogen (Havell et al. 1982). More specifically, it is produced by TH1 subset of T cells, rather than by the TH2 subset (Mosmann and Coffman 1987). IFN-γ secretion by antigen-sensitized

lymphocytes could be considered as a good measure of T-cell activity (Rothel et al. 1990).

Ovine IFN-γ was detected for the first time by bioassay and found to possess properties similar to those of murine and human IFN- γ (Entrican et al. 1989). Ovine IFN-γ was found to be functionally cross-reactive with the bovine IFN- γ, indicating structural similarity of the ovine and bovine molecules (Rothel et al. 1990). The gene encoding the ovine IFN-γ was cloned by RT-PCR of mRNA from stimulated lymphocytes (McInnes et al. 1990; Radford et al. 1991). It was later mapped to 3q23 by in situ hybridization (Goldammer et al. 1996).

2.5.1 Structure and polymorphism

The ovine IFN-γ gene was cloned and sequenced by two independent groups (McInnes et al. 1990; Radford et al. 1991). cDNA was obtained by RT-PCR of lymphocyte mRNA,

93% identical to the bovine one. The cDNA sequence had a putative signal sequence coding for 20 amino acids, followed by sequence for a mature IFN-γ molecules spanning 145 amino acids. Complete sequence of the gene was also reported (Crawford and McEwan 1998). It was 4842 nucleotides long, with four exons spanning 114, 69, 183 and 135 nucleotides, respectively. A total of 36 (34 in the non-coding and two in the coding regions) single nucleotide polymorphisms (SNP) and two insertions/deletions were identified in the gene.

A diallelic tetranucleotide microsatellite [o(IFN)-γ] of the form (GTTT)5/6 was identified in the ovine IFN-γ gene (Schmidt et al. 1996). PCR amplification of the larger and shorter alleles resulted in 128 and 124 bp long fragments, respectively. Several studies investigated polymorphism at this locus. While 128 and 124 bp length alleles were identified in the majority of studies (Crawford and McEwan 1998; Dukkipati et al. 2005; Reddacliff et al. 2005; Sayers et al. 2005b), alleles of sizes 126 and 130 bp were found in Soay sheep

(Coltman et al. 2001). Also, a 122 bp allele was found fixed in a large structured population of wild sheep (Worley et al. 2006). It was also revealed that in New Zealand Romney-

Coopworth crosses, the larger allele had ‘G’ 49 bp downstream of the microsatellite (referred to as haplotype A) and the smaller allele had ‘A’ at the corresponding position (referred to as haplotype B; Crawford and McEwan 1998). Similar haplotypes were reported to exist in Texel sheep, while two additional haplotypes, C (with ‘A’ at the corresponding position in the larger allele) and D (with ‘G’ at the corresponding position in the smaller allele), were identified in Suffolks (Sayers et al. 2005b).

A different microsatellite (OarKP6) of the form (AC)n was identified in a BAC containing exon 1 sequence of the ovine IFN-γ gene (Paterson and Crawford 2000). Seven alleles were observed in 170 unrelated sheep belonging to five breeds. No recombination was observed between the OarKP6 and o(IFN)-γ microsatellites. However, there were no subsequent reports on the polymorphism of OarKP6 microsatellite in sheep. Ovine IFN-γ exon 3 sequence polymorphism in 310 sheep (Merino, Corriedale, Romney, Poll Dorset and cross- bred) was studied employing SSCP analysis (Zhou et al. 2007). Five unique SSCP patterns, corresponding to five different alleles, *01 to *05 (GenBank accession # DQ311095-

DQ311099), were identified. Alleles *01 and *02 were most common, together accounting for 86% of the allelic population.

2.5.2 Association with disease resistance

The ovine IFN-γ gene received increasing attention because of its association with nematode resistance in both domestic and feral sheep breeds. Independent full genome scans carried out on flocks at the CSIRO and AgResearch (Beh et al. 1998; Crawford 1998) revealed three to six putative QTL related to FEC. A large effect was detected on the ‘q’ arm of Chromosome 3, near the positional candidate gene IFN-γ. Subsequently, alleles at the o(IFN)-γ

microsatellite locus were found to be significantly (P<0.05) associated with

resistance/susceptibility to gastrointestinal nematodiasis in sheep belonging to two divergent nematode selection lines at AgResearch, New Zealand (Crawford and McEwen 1998). The larger (128 bp) and smaller (124 bp) alleles at the locus were more frequent in the nematode- resistant and susceptible lines, respectively. Association of alleles at this locus with FEC was also reported in feral Soay sheep (Coltman et al. 2001). However, the smaller 126 bp allele (rather than the larger 128 bp allele linked to nematode resistance in domestic sheep) was found to be associated with reduced FEC in lambs as well as yearlings. Also, the 126 bp allele was associated with increased T. circumcincta-specific antibodies in lambs.

Two other studies examined the association of this microsatellite locus with nematode

resistance in domestic sheep. While no significant influence of the alleles at the locus on FEC could be detected in New Zealand Romneys (Dukkipati et al. 2005) and Irish Suffolks

(Sayers et al. 2005b), the 124 bp allele was found to be associated with resistance to nematode infection in Irish Texel sheep (Sayers et al. 2005b).

Association of genotypes at the o(IFN)-γ microsatellite locus on susceptibility to natural ovine PTB in two Australian Merino sheep flocks was also investigated (Reddacliff et al. 2005). There were no consistent findings for the effect of IFN-γ genotype on

resistance/susceptibility to PTB. Genotype 128/128 was found to be significantly (P<0.01) associated with clinical signs of the disease in flock B. However, no such association of genotype with phenotype was evident in flock A. In flock A, allele 124 was ranked high for association with severe disease, but in flock B, allele 128 was ranked higher.

2.6 OTHER LOCI FOUND TO BE ASSOCIATED WITH SUSCEPTIBILITY TO