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Genetic Susceptibility to Infectious Diseases Linked to NRAMP1 Gene in Farm Animals

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Comparative genomics is playing a pivotal role in the genetic dissection of complex traits such as infectious diseases resistance. Using mouse models of infection, natural resistance associated macrophage protein 1 (Nramp1) was shown to have a critical role in innate resistance to infection with unrelated intracellular pathogens including Mycobacterium bovis, Leishmania donovani and Salmonella typhimurim. Nramp1 is a member of an ancient family of genes having orthologs among mammals, birds, invertebrates and plants. A role for NRAMP1 in host defense against microbial infection was demonstrated in two major zoonotic diseases of livestock: salmonellosis in chicken and brucellosis in cattle. In both cases disease susceptibility is inherited as a complex trait and polymorphisms within NRAMP1 contribute clearly to the risk and the progression of infection.

Introduction

Under natural conditions, the host response to infection in farm animals is multifactorial and involves the complex interaction between two genomes (the host and the pathogen) and the environment. It had been long observed that diseases rarely occur in all members of animal populations exposed to pathogens: studies of resistance to Salmonella pullorum in poultry and Brucella suis in swine confirmed a major role of the host genetic background in the expression of the disease.1,2 Natural disease resistance refers to the inherent capacity of an animal to resist disease when exposed to pathogens, without prior exposure or immunization.3 Although some of the observed variation in natural resistance to infection is related to environmental factors, a significant component of variation appears to be heritable and, therefore, stably passed from parent to offspring.

The understanding of the complex host response to infection in domestic animals and other mammalian species has advanced considerably through the use of mouse models of infection. The laboratory mouse is well known to have a broad range of host susceptibility to human pathogens (reviewed in Ref. 4). The development of genome resources and technologies combined with classical genetics contributed to the successful identification of several host resistance genes in laboratory mice including the gene encoding for Nramp1 (natural resistance associated macrophage protein 1).5

One powerful approach to unravel the genetic determinants involved in host resistance to infection in agriculturally important animal species is the use of comparative genomics. Despite traditional disease control measures, losses attributable to infectious diseases continue to impede the livestock industries. An alternative approach to enhancing animal health management systems is to increase the overall level of genetic resistance at herd and population levels by using selective breeding programs. Comparative genomics involves initially the mapping and the identification of genes for resistance to infectious disease in model organisms such as the mouse. Candidate chromosomal regions and/or orthologous genes are then tested for association to susceptibility to related infections in other animal species.6

Comparative Genomics

Comparative mapping was based for the past 20 years on genetic maps created through the integration of data obtained from visible phenotypes and traditional mapping techniques, such as restriction fragment length polymorphisms (RFLPs), fluorescence in situ hybridization (FISH), genetic studies of somatic cells, and pedigree analysis of interspecific crosses (reviewed in Ref. 7). More recently, information obtained from additional types of markers that amplify coding sequences and genes, microsatellite markers and single nucleotide polymorphisms (SNPs), has increased the density of these maps.8 The first indication that genes reside in similar region of the genome in different mammalian species was provided with the observation that the albino and pink-eye dilution mutations are closely linked in the mouse, rat and rabbit genomes7. In 1984, Nadeau and Taylor9 compared the chromosomal locations of orthologous genes (83 loci) in mouse and human using mouse linkage and human cytogenetic data. They predicted about 180 syntenic regions (defined as regions in which a series of loci occur in the same order on a single chromosome in both species), a number that was confirmed in a more recent study using more than 3600 orthologous loci to define about 200 regions of conserved synteny.10 A more comprehensive analysis of conserved synteny was reported using the draft sequences of the human and mouse genomes.11 The comparison of the entire mouse and human genome sequences was not based only on human-mouse gene pairs but also on a large set of orthologous landmarks (558,000 orthologous landmarks). Each genome could be parsed into 342 conserved syntenic segments.11

Comparative mapping offers many possibilities, such as the identification of candidate disease genes and modulators of disease susceptibility, the characterization of the genetic basis of complex disorders, the identification of novel genes, transcription factors, and regulatory elements. For years, agriculturally important animal populations have been genetically improved through selective breeding.12 Most of this selection was based on visible phenotypes without knowing the molecular basis of the trait in question.12 The human and mouse genome projects have paved the way for the development of genome resources and technologies in farm animals. Dense genetic maps and large-insert libraries (YAC and BAC) have been generated for all main farm animals.13,14 Radiation hybrid panels and collection of expressed sequence tags (ESTs) are available for pig, cow and chicken.13,14 However the number of genes mapped in farm animals is still small compared to humans and mice. Comparative mapping is currently an important component of farm animal genome programs and as more farm animal genomes are sequenced, comparative genomics will be exploited fully towards the identification of genes that control key economical and/or biological important traits.

Nramp1

Numerous success stories have emerged using comparative genomics between human and mice.8 The success of comparative genomics in studying host resistance to infection in animal species other than mice and humans has been well illustrated with the gene Nramp1.15,16 Nramp1 was positionaly cloned a decade ago as the gene underlying the Bcg/Lsh/Ity locus located on mouse chromosome 1.5 The Bcg/Lsh/Ity locus was known to control the exponential growth rate in the early phase of infection of unrelated intracellular pathogens such as different Mycobacterium species (including the Bacille Calmette-Guérin, BCG), Leishmania donovani as well as Salmonella typhimurium.17-19 A polymorphism within Nramp1 abolished the protein's function and consists in a glycine replacement by an aspartic acid residue at position 169 of the protein.20 Mice homozygous for the wild type allele, Nramp1G169, are highly resistant to BCG, Leishmania donovani and Salmonella typhimurium, whereas mice with the mutated allele, NrampD169, are highly susceptible.5 Further proof that Nramp1 was the gene underlying the Bcg/Lsh/Ity locus came when the functional Nramp1 gene was disrupted by homologous recombination in resistant mice which then became very susceptible to infection with BCG, Leishmania donovani and Salmonella typhimurium.21 Additionally, susceptible mice were rendered resistant to BCG and Salmonella typhimurium by transfer of the resistance allele, further confirming the identity of Nramp1 with the phenotypic resistance to Salmonella typhimurium.22 Nramp1 codes for a phosphoglycoprotein that is recruited to the phagosomal membrane early during an infection.23,24 Nramp1 functions as a pH-dependent manganese transporter and has pleiotropic effects on various effectors of the immune system to facilitate bacterial killing.24

The identification of Nramp1 and its function opened a whole new field in the area of host resistance to intracellular pathogens. Because of the critical role of Nramp1 in the mouse model of mycobacterial, Leishmania and Salmonella infections, several homologues of the mouse Nramp1 were investigated with respect to resistance to various intracellular pathogens causing major diseases in important agricultural species.

Chicken Genomics, Salmonella Infection, and NRAMP1

The chicken has been an important animal model in immunology and has led to several fundamental discoveries.14 During the past decade, there has been a concerted international effort to produce a molecular map of the chicken genome.2531 The consensus map comprises 39 pairs of chromosomes that are subdivided into eight pairs of cytologically distinct macrochromosomes, 2 sex chromosomes and 30 pairs of cytologically indistinguishable microchromosomes. The resulting map contains 1889 loci and spans ˜3800 cM.30 Although the physical size of the chicken genome is roughly 3 times smaller (˜1200Mb) than mammalian genomes, the recombination genetic map is comparable to that of most mammals. Comparative maps between chicken, man and mouse show a considerable amount of chromosomal conservation.30,32,33 With the development of genomic technologies, the chicken is becoming an excellent model for the study of host resistance to infection.

Salmonella are ubiquitous Gram-negative, facultative intracellular bacteria that replicate in macrophages and neutrophils of the reticuloendothelial systems of numerous animal species, including humans, laboratory and domestic animals, livestock and birds. Several Salmonella serotypes including Salmonella typhimurium and Salmonella enteritidis infect a broad spectrum of hosts. Other serotypes such as Salmonella typhi and Salmonella paratyphi in humans, Salmonella dublin in cattle and Salmonella gallinarum in birds are host specific. In humans, Salmonella cause two major types of infection, a systemic disease (typhoid fever) caused by Salmonella typhi, and a gastrointestinal disease (salmonellosis) caused by Salmonella enteritidis or Salmonella typhimurium. Typhoid fever is still a major disease in endemic areas of the world where access to clean water is limited. There are over 21 million cases of typhoid fever reported annually worldwide and 200 000 deaths associated with untreated infection.34 Salmonellosis caused by the ingestion of Salmonella-contaminated poultry products is one of the most common causes of food poisoning in humans.35 There has been a resurgence of salmonellosis in North America and Europe with an estimated number of salmonellosis of 1.4 million per year in the United States alone.34

In chickens, host specific Salmonella such as Salmonella gallinarum and Salmonella pullorum cause a systemic disease with high mortality rates in birds of all ages.36 Salmonella enteritidis and Salmonella typhimurium infections in young chickens cause also a major disease characterized by severe clinical signs of diarrhea and dehydration with high mortality rates. In adult chickens, Salmonella typhimurium and Salmonella enteritidis infections do not cause significant disease or mortality and birds can carry the bacteria for several weeks without presenting any clinical signs, which constitutes an insidious risk for public health.37

Chickens have been studied for more than 50 years for their resistance and susceptibility to Salmonella infection because of their central importance in the poultry industry worldwide.37 Many factors are known to affect the susceptibility of chickens to Salmonella infection, including age, Salmonella strains, sanitary conditions at the farms, as well as the genetic background of the animals. Host genetic factors clearly influence the epidemiology of Salmonella infection in chickens. Genetic regulation of chicken resistance to Salmonella infection was reported initially by Bumstead and Barrow.38 A survey of inbred and partially inbred lines of chicken showed significant differences in mortality following both oral and intramuscular challenge of newly hatched chicks with Salmonella typhimurium.38 Lines W1, 61 and N were highly resistant to Salmonella typhimurium with more than 70% survival during the course of infection, whereas lines C, 72 and 15I were highly susceptible with 70% to 100% mortality rates. Resistance to infection extended to other Salmonella serotypes in these chicken lines: lines resistant to Salmonella typhimurium were also resistant to infection with Salmonella gallinarum, Salmonella pullorum and Salmonella enteritidis, and chicken lines susceptible to Salmonella typhimurium were also susceptible to the other Salmonella serotypes. In susceptible chickens, mortality occurred early during the course of infection (within 7 days post-inoculation) and host differences in susceptibility to Salmonella infection in chicken have been shown to correlate with the bacterial load in the reticuloendothelial organs.36,39 Significantly higher numbers of Salmonellae were isolated from the spleen and liver of susceptible chickens compared to the resistant chickens, suggesting that resistance to salmonellosis in chicken is related to a greater ability of the reticuloendothelial system to control bacterial proliferation during the early stage of infection, resembling the phenotype described in mouse Nramp1 mutant. Segregation analysis using resistant W1 and susceptible C chickens showed that resistance to infection is dominant and inherited as a complex trait, not associated with the major histocompatibility complex or maternal factors.38 An effect of the genetic background in host resistance to infection with Salmonella was also seen in adult chickens.40 In this case, susceptible birds survived the infection but produced fewer eggs and presented higher degree of egg contamination.

Chicken NRAMP1 clones were identified from chicken genomic DNA and cDNA libraries using a mouse Nramp1 partial cDNA as hybridization probe.41 Chicken NRAMP1 encodes a polypeptide of 555 amino acid residues that shows a strong homology with mammalian Nramp1 including mouse and humans (83% and 81% similarity respectively). The overall structure and particularly the position and sequence of the 12 putative transmembrane domains, the two N-linked glycosylation sites and the consensus transport motif are highly conserved, suggesting the functional importance of these regions among phylogenetically distinct species. Chicken NRAMP1 was mapped using linkage analysis and fluorescence in situ hybridization to chromosome 7q13 within a syntenic linkage group including three known genes (the mitochondrial NADH-coenzyme Q reductase gene, NDUFS1; the T cell surface antigen, CD28 and the elongation factor 1, EF1B) that have been conserved in the mouse (chromosome 1) and human (chromosome 2) genomes.41,42

Analysis of NRAMP1 expression in a variety of chicken tissues has demonstrated that the highest expression of chicken NRAMP1 is in spleen and thymus with much lower expression in liver and lung.43 Fractionation of spleen cells into adherent (macrophage-enriched) and non-adherent (lymphocyte enriched) subpopulations show that the expression of NRAMP1 is enhanced in the adherent cell compartment as previously demonstrated in mice.5 A search for regulatory sequences in the chicken NRAMP1 promoter revealed a number of consensus motifs associated with binding of trans-acting nuclear factors implicated in myeloid differentiation corresponding to elements known to bind constitutively expressed transcription factors.43 Additional sequence motifs associated with inflammatory response or lymphokine-inducible gene expression was detected in the promoter region. The presence of PU.1 and PEA binding sites is in agreement with the restricted expression of chicken, mouse and human NRAMP1 mRNA in haematopoietic cells (macrophages and granulocytes). The overall sequence homology between the promoter region of the chicken and mouse NRAMP1 genes is 41% and is 36% between chicken and human NRAMP1. More interestingly, several of the consensus elements (PU box; interferon γ responsive element and binding sites for NF-IL6 and NF-kB) are present in chicken, mouse and human NRAMP1 promoters.16,43,44 The conservation of genomic organization of mammalian and avian NRAMP1 proteins is striking. The chicken NRAMP1 gene spans about 5 kb and contains 15 exons. Except for a smaller range of intron lengths, the overall structure of the chicken gene proved to be very similar (number of exons, exon sizes and splicing junctions) to those of mouse and human Nramp1. The high degree of sequence and structure conservation between chicken and mouse NRAMP1, the presence of similar regulatory elements within the promoter regions of the two homologous genes, and similar tissue expression supports the concept that the NRAMP1 protein exerts similar roles in vivo both in mice and birds.

Nucleotide sequence analyses of the coding portion of NRAMP1 in susceptible (72, 15I and C) and resistant (W1, 61 and N) chicken lines revealed eleven sequence variants within NRAMP1 cDNA.16 Almost all of these sequence variants (10 out of 11) resulted in silent mutations or conservative changes that were detected both in resistant and susceptible chicken lines. Only one sequence variant corresponding to a G → A transition at position 696, resulting in a non-conservative Arg223→Gln223 within the predicted TM5-6 interval was specific to the susceptible line C. A positively charged Arg residue was found at the equivalent position in most mammalian NRAMP1 proteins (human, sheep, horse, pig, dog, rat and rabbit) with the exception of the mouse (His) and cow (Gln).16,23,45

The role of NRAMP1 in resistance of chickens to Salmonella infection was tested by linkage analysis using a backcross chicken panel that consisted in 425 progeny, derived from resistant line W1 and susceptible line C. One day old progeny were infected with 103 CFUs Salmonella typhimurium intramuscularly and mortality rate was recorded for a period of 15 days.16 In this model, W1 chickens were highly resistant to infection (3% mortality was observed 15 days post inoculation) whereas C chickens were highly susceptible (89% mortality for the same time period). The overall mortality rate in the 425 (W1 X C)F1 X C backcross progeny was intermediate (35%) between those of resistant W1 and susceptible C lines.16 Mortality rate in line C occurred in two phases: an early phase (day 1-7) where most animals died from infection (about 80% of the deaths occurred during this period) and a late phase (day 8-15) where the mortality rate was much lower. The major effect of NRAMP1 was seen early after infection (7 days) when the mortality rate of homozygous CC chickens (27%) was twice the one observed in CW1 heterozygote progeny (13%). Using linkage analysis, NRAMP1 and the adjacent chromosomal region was linked to resistance to infection with Salmonella typhimurium in chickens (Likelihood ratio test of 9.44 p = 0.00213) using the Cox proportional hazards model for the period covering the first 7 days post-infection. A second region of the chicken genome located on microchromosome E41W17 and harboring the host resistance gene Toll-like receptor 4 (TLR4) was shown to be linked to disease susceptibility in the same model of infection.16,46 The impact of TLR4 on resistance to infection during the first 7 days post infection is similar to that observed with NRAMP1 (Likelihood ratio test of 10.2, p = 0.00138). The interaction between NRAMP1 and TLR4 was examined by dividing the backcross progeny into four two-locus genotypes at NRAMP1 and TLR4.46 The group NRAMP1CW1-TLR4CW1 presented the highest survival rate at day 7 post-infection (93%) compared to the progeny carrying NRAMP1CC-TLR4CC genotypes (58%). The two other groups (NRAMP1CC-TLR4CW1 and NRAMP1CW1-TLR4CC) presented intermediate survival rates (69% and 73% respectively). These data were consistent with the previous observation in mice that mutation within Nramp1 affects susceptibility to infection with Salmonella typhimurium one or two days earlier than mutation within Tlr4.47,48 Individually, NRAMP1CW1 accounted for 17% of the early differential resistance to infection and TLR4CW1 was found to account for 21% of the phenotype in the chickens tested. A genome scan performed on the same animal panel clearly showed that the chromosomal regions surrounding NRAMP1 and TLR4 had a major impact on susceptibility of chickens to Salmonella typhimurium infection (unpublished data).

In experimental Salmonella enteritidis infection in mice, Nramp1 was shown to exert an effect not only during the innate phase of the host response but also in controlling bacterial clearance during the late phase of infection.49 In young chickens, NRAMP1 was also associated with the early host response to infection with Salmonella enteritidis as measured by bacterial load in the spleen within a week post infection.50,51 In adult chickens, NRAMP1 was shown to have a dramatic impact on the risk of spleen contamination four weeks post infection suggesting an additional role for NRAMP1 in the control of resistance to Salmonella carrier state in chickens52. The mechanism by which NRAMP1 affects bacterial clearance in chickens is not clearly elucidated however studies performed in mice have shown that in addition to its specific role as a cation transporter,24 Nramp1 has been associated with regulation of macrophage activation as measured by production of nitric oxide, IL-1, INF-γ and MHC class II expression and Th1/Th2 differentiation.53,54

Bovine Genomics, Brucella abortus Infection and NRAMP1

Recent developments in the construction and comparison of mammalian genome maps combined with efficient tools such as whole genome radiation hybrids (RH) and large fragment clone libraries (BACs) have advanced considerably the progress made on the identification and characterization of disease candidate genes in cattle.55,56 Several evidences will be reviewed here supporting the concept that the bovine homologue of mouse Nramp1 is a major gene controlling natural resistance to infection with Brucella abortus in cattle.57 Brucella abortus, an ubiquitous facultative intracellular Gram-negative bacteria, causes a severe disease in cattle known as brucellosis. The disease is characterised by abortions, birth of weak or nonviable offspring, and infertility in both males and females. Brucella abortus infection caused an estimated annual loss of US$35 million to the livestock industries until it was brought under control and virtually eliminated in the late 1990's in the USA. The disease adversely affects the export trade of livestock by imposing quarantine on infected herds, requesting vigorous testing and restricting trade from high risk geographical areas. In the USA, the federal brucellosis eradication program has been in effect since 1934. This program, based on vaccination and elimination of domestic reservoirs, has cost over US$2 billion from 1951 to present. In USA and Canada, brucellosis is a notifiable disease and reportable to the local health authority. According to the Food and Agriculture Organization (FAO), the World Health Organization (WHO) and the Office International des Epizooties (OIE), brucellosis is still a serious economical problem for livestock and a major public health hazard for humans in several countries. A recent survey directed by these organisms reported that out of 174 countries evaluated for brucellosis occurrence, 19 had never encountered the disease, 15 had completely eradicated the disease and 140 countries still had infected livestock and human populations.

In humans, the three closely related species Brucella melitensis, Brucella abortus and Brucella suis are the etiologic agents of brucellosis. One important source of transmission in humans is the ingestion of contaminated milk and dairy products. The clinical disease is often difficult to diagnose because of non-specific clinical signs and an insidious onset: patients infected with Brucella spp. may present an acute fever, or a chronic or localized infection. Antibiotics (usually doxycycline and rifampin for 6 weeks) have proven to be generally effective, but recurrence of the disease is not uncommon, and foci of infection have been documented to persist for decades in some patients. Public health strategies to protect humans from brucellosis have virtually always relied on the elimination of diseased animals.

Using bovine brucellosis as a prototype intracellular disease model, studies aimed at defining the genetic basis for resistance to Brucella abortus in cattle began in the late 1970's.58 Although resistance to infection was associated in some studies with a simple mode of inheritance, it was most frequently associated with a model supporting the effect of several genes. This apparent discrepancy may be explained by the fact that studies aimed at demonstrating a genetic basis for natural disease resistance in domestic animals are highly dependent on methodology, including the nature of the exposure to the pathogen, the method used to assess resistance, and the type of genetic analysis used. In classical breeding studies, natural resistance to Brucella was demonstrated to be improved by simple mass selection in one generation of selective breeding.58 The frequency of natural resistance to brucellosis in challenged unvaccinated cattle was 20% (30/150). Breeding a naturally resistant bull to naturally resistant cows increased the frequency of natural resistance in their progeny to 58.6% (17/29). The genetic analysis of these crosses was consistent with a model of resistance to infection involving two or more genes.

The host response to brucellosis was analysed experimentally in a recent study using a standardized dose of Brucella abortus to maximize differences observed between resistant and susceptible animals, while approximating a natural exposure. Unvaccinated and previously unexposed sexually mature bulls or heifers at mid-term gestation (150±30 days) were challenged with the standardized discriminating challenge inoculum of 106 CFUs of Brucella abortus strain S2308, scored for the outcome of parturition, and quantitative cultures were collected from tissues and secretions 3-5 months post inoculation. Resistant cows did not abort, and no Brucella organisms were cultured from the cow or calf. Resistant bulls were similarly culture negative for Brucella in semen and at slaughter. In this system, natural resistance to Brucella infection in the cow was shown to correlate with macrophage, function, T-cell response and specific immunoglobulin allotypes (Table 1).

Table 1. Characteristics of macrophages, T cells and antibody responses in cattle naturally resistant or susceptible to Brucella abortus.

Table 1

Characteristics of macrophages, T cells and antibody responses in cattle naturally resistant or susceptible to Brucella abortus.

The expression of anti-LPS immunoglobulin IgG2a allotypes in resistant cattle was significantly different from the IgG2a A1 allotype predominating (p <0.05) in Brucella abortus susceptible cattle59,60 while previous studies have disputed the role if any that antibodies play in natural resistance to Brucella abortus in cattle61,62. No association of resistance with BoLA class I alleles was demonstrated; however, mammary and monocyte-derived macrophages from resistant cows and bulls were significantly more active than macrophages obtained from susceptible cows and bulls in terms of (1) respiratory burst activity in response to opsonized Brucella abortus;63 and (2) ability to control replication of Brucella abortus.6466 The differential response between macrophages from resistant and susceptible cattle was similar to that observed in mice strains carrying Nramp1G169 or Nramp1D169 in response to infection with BCG, Leishmania donovani, Mycobacterium paratuberculosis, and Salmonella typhimurium.6769 In addition to their capacity to restrict the intracellular replication of Brucella abortus, macrophages isolated from resistant cattle were able to restrict the growth of BCG and Salmonella dublin significantly better than macrophages obtained from susceptible cattle.66 The brucellacidal capacity of macrophages in vitro predicted with >80% accuracy disease susceptibility in vivo.

These striking similarities between bovine macrophage function in resistance to brucellosis and murine macrophage function in resistance to Salmonella, Mycobacterium and Leishmania supported the hypothesis that a bovine homolog of the murine Nramp1 gene is a major player in determining resistance to brucellosis. Consequently, the bovine homolog of mouse Nramp1, designated bovine NRAMP1 was cloned and the cDNA sequenced.57 The DNA sequences of the murine Nramp1, human NRAMP1 and bovine NRAMP1 show a remarkable degree of conservation. The predicted amino acid similarity between mouse Nramp1 and bovine NRAMP1 is 87% and 89% between human NRAMP1 and bovine NRAMP1.44,57 Bovine NRAMP1 encodes for a 60 KDa protein predominantly expressed in macrophages of the reticuloendothelial system. Specific characteristics of the mouse Nramp1 and human NRAMP1 proteins including N terminus SH3 motif, four PKC phosphorylation sites, and a 'binding protein dependent transport system inner membrane component signature' are also present in the bovine NRAMP1 protein.5,57,70 Additionally, binding motifs for regulation of specific tissue expression of bovine NRAMP1 are conserved in the 5' untranslated region from position -257 to -80, 5' to start the codon. These include protein binding sites for IRE, NF-IL6, PU.1, PEA-3, W element and NF-κB. In cattle, NRAMP1 was mapped to bovine chromosome 2 within the linkage group on mouse chromosome 1 and human chromosome 2q harbouring Nramp1/NRAMP1.7175

Cattle and several other animal species including humans, rats, chickens, bovine, bison, water buffalo, sheep, red deer, and moose possess a glycine residue at position 169 of the Nramp1 protein arguing in favour of the importance of this amino acid in protein function. Two sequence variants within bovine NRAMP1 have been identified and associated with disease resistance. These sequence variants are located within the 3' untranslated portion of bovine NRAMP1 and show a complex mutation pattern consisting of a T to G substitution at position 1782 and a variation in the number of GT dinucleotide repeats in a (GT)n microsatellite at position 1907.76,77 The variation in microsatellite length ranged from (GT)13 to (GT)16 and showed significant association with resistance to bovine brucellosis in SSCA analyses (Table 2). A (GT)n microsatellite was also detected in the 3' UTR of NRAMP1 isolated from bison, water buffalo, goat, sheep, red deer, white-tailed deer, fallow deer and moose, however the T to G substitution at position 1782 was present only in cattle76,78 (J.W.T. unpublished data). The two alleles observed for the T to G substitution in cattle were designated 1782T(GT)11 and 1782G(GT)10 and shown to segregate with resistance to brucellosis in 10 unrelated animals as well as within one family studied (J.W.T., unpublished data).

Table 2. Association of bovine NRAMP1 SSCA polymorphism with bovine brucellosis-resistant and -susceptible phenotypes.

Table 2

Association of bovine NRAMP1 SSCA polymorphism with bovine brucellosis-resistant and -susceptible phenotypes.

The impact of the (GT)n sequence variant at position 1907 on NRAMP1 function was studied using an experimental system in which bovine NRAMP1 under the regulatory control of its own promoter was transfected into murine RAW264.7 macrophage cell (Nramp1D169).79 The polymorphism within the microsatellite in the 3' untranslated region critically affects the expression of bovine NRAMP1 and the control of in vitro replication of Brucella abortus but not Salmonella dublin. A role for microsatellites in the regulation of gene expression has previously been proposed and is thought to be mediated through changes in the α-helical phasing between transcription factor binding domains.8082 These binding domains flank the sequence repeat and are found in a specific phase along the α-helix that allows their transcription factor ligands to bind the DNA in a cooperative fashion. Any alteration in the length of the repeat sequence may affect the cooperative binding of the transcription factors needed for optimal expression of the gene. These observations suggest that resistance to Brucella abortus infection in cattle may be attributable to the transcriptional regulation of NRAMP1 mediated through the length of the 3' UTR poly (GT) repeat sequence.

Although the bovine NRAMP1 gene is one of the major candidate gene controlling natural resistance to brucellosis, it does not determine resistance and susceptibility to infection with Mycobacterium bovis in cattle.83 This finding is in agreement with recent studies in the mouse that have failed to establish a role for Nramp1 in protection against virulent Mycobacterium tuberculosis.84,85 However, in humans, genetic variants at NRAMP1 contribute clearly to the risk and the progression of mycobacterial infections in humans, although it accounts for only a modest proportion of the overall genetic component of natural resistance to tuberculosis.8688

Conclusions

Infectious diseases remain among the most important burdens on human and animal health notwithstanding antibiotic therapy and immunization that have contributed substantially to the their control. One fundamental aspect of infectious disease pathogenesis is the identification of host genes that play a critical role in determining the outcome of host-pathogen interactions. Studies in important agricultural animals have demonstrated the role of such heritable factors in governing the susceptibility to infectious diseases. Identification of the molecular basis of host resistance to pathogens offers the possibility to improve disease resistance of breeding stock through marker-assisted selection.12,13 It is clear that any form of resistance to disease is relative rather than absolute due to the complex nature of the host pathogen interaction. Application of marker-assisted selection will be limited to genes of moderate to large effect until the host response to a specific infection is fully dissected.12 Thus, breeding animals to increase their level of natural resistance is not expected to completely prevent infectious diseases. However, the increased level of natural resistance conferred by selective breeding would be expected to reduce morbidity and economic losses caused by infectious diseases. The emerging genomic resources for farm animals coupled with new technologies for gene transfer will eventually facilitated the genetic improvement of agricultural important animals.14,73,8993

Acknowledgments

Judith Caron is the recipient of a Canadian Institutes for Health Research (CIHR) fellowship. Garry Adams is supported by the Texas Agricultural Experiment Station Projects No. 6194 and 8409, USDA NRICGP Project No. 2002-35204-11624, and DHHS/PHS/NIH-1 RO1 A144170-01A1. Danielle Malo is a member of the federal Networks of Centres of Excellence- the Canadian Bacterial Diseases Network (CBDN) and a scholar of CIHR and an International Research Scholar of the Howard Hughes Medical Institute (HHMI).

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