NAAS Score 2018

                   5.36

Declaration Format

Please download DeclarationForm and submit along with manuscript.

UserOnline

Free counters!

Previous Next

Cloning and Polymorphism of Disease Resistance SLC11A1 Gene in Pig (Sus scrofa): A Review

Bhanita Devi S. Laskar P. Borah P. K. Bharti
Vol 8(9), 36-48
DOI- http://dx.doi.org/10.5455/ijlr.20170912053011

Infectious diseases are responsible for major economic losses in livestock production. Using disease resistant breeds is of considerable importance in livestock species. Solute carrier family 11member 1 (SLC11A1) which is also called as Natural Resistance-Associated Macrophage Protein1 (NRAMP1), is one of the most potent candidate genes conferring host’s genetic resistance to various antigenically different intracellular pathogens. The gene controls the exponential growth of the bacteria during the early phase of infection. SLC11A1 have been identified and cloned in pigs and it is strongly expressed on macrophages and neutrophils following stimulation with lipopolysaccharide. The present review discuss the localization, function and molecular techniques used to study the cloning and sequencing of SLC11A1 gene polymorphism in pigs by using Restriction Fragment Length Polymorphism (PCR-RFLP) and Single Strand Conformation Polymorphism (PCR-SSCP) methods as the SLC11A1 gene plays a very important role due to its association with resistance/ susceptibility to various intracellular pathogens in human as well as in livestock species.


Keywords : Cloning NRAMP1 PCR-RFLP PCR-SSCP Pig Polymorphism SLC11A1

The livestock sector alone contributes nearly 28.5% of value of output at current prices of total value of output in Agriculture, Fishing and Forestry sector. The overall contribution of livestock sector in total Gross Domestic Product (GDP) is nearly 4.5% at current price (Annual Report, 2016-17). Infectious diseases in India causes annual loss mainly through morbidity, mortality, reduced fertility, production losses and inefficient feed utilization. Diseases in animals have numerous impacts such as loss of productivity, loss of income from activities using animal resources, prevention or control costs and suboptimal use of production potential (McInerney, 1996). According to 19th Livestock Census (2012), the total livestock population and the total pig population in the country has decreased by about 3.33% and 7.54%, respectively over the previous census. The total pig population in the country is 10.29 million which accounts for only 2.01% of the total livestock population (512.5 million). Susceptibility to the infectious diseases depends more or less on genetic component of the animals. Infectious diseases are difficult to cure and cause significant economic losses in livestock species. Selective breeding is used to increase resistance against infectious disease and it may prove to be an economical and sustainable practice. Genetic methods, such as selection of disease resistance in the pig, have not been widely used. Genetic resistance or susceptibility to diseases due to candidate gene polymorphisms could be used in selection and breeding programme for disease resistance in animals (Thomas and Joseph, 2012). In livestock, a number of candidate genes has been studied and selected on the basis of their association to resistance or susceptibility in certain other diseases and their known role in disease pathogenesis. These genes include solute carrier family 11 member 1 (SLC11A1), interferon gamma (IFN-γ), peptidoglycan recognition protein 1 (PGLYRP-1), Toll-like receptors (TLRs), Caspase associated recruitment domain 15 (CARD15), mannose binding lectin-1 (MBL-1), Nitric oxide synthase (NOS2) etc. Genome-wide association studies have attempted to confirm associations found and identify new genes involved in pathogenesis and susceptibility. Selection of disease resistance animal for breeding might be the prophylactic measure of first choice. Breeding stock could be chosen by marker-assisted selection (MAS) independently from environmental factors because of development of a genetic marker for disease resistance (Meuwissen and Van Arendonk, 1992). Molecular marker is a potential selection tool for disease resistance and susceptibility in animals. The completion of the porcine whole genome sequencing has provided powerful tools to identify the genome regions that harbor genes controlling disease or immunity (Zhao et al., 2012). Presently, many types of molecular markers have been utilized to detect the variation among individual and population. The Solute Carrier family 11member 1 (SLC11A1) is a proton-coupled divalent metal ion transporter which was previously known as Natural Resistance-Associated Macrophage Protein1 (NRAMP1). It is also involved in the innate immune response against pathogens of viral, bacterial and protozoan origin (Awomoyi, 2007).The NRAMP1was first isolated in the mouse as a candidate gene for the Bcg/Ity/Lsh locus that conferred genetic resistance/susceptibility of inbred mouse strains to infection by Mycobacteria, Salmonella, and Leishmania (Vidal et al., 1993). Belouchi et al. (1995) reported that the members of NRAMP gene family were first found in mouse and then Belouchi named these genes as the NRAMP1gene family. The SLC11A1 gene was originally recognized in the mouse as three loci Ity, Lsh, and Bcg and the latter proved to be the NRAMP1, which has been recently renamed as SLC11A1 (Vidal et al., 1996).Transgenic mice homozygous for engineered mutations in NRAMP1 have similar phenotypic effects on natural resistance to intracellular pathogens which indicates a critical role of NRAMP1 protein in pre-immune macrophage function (Vidal et al., 1995a). A naturally occurring Gly→Asp mutation at amino acid 169 of SLC11A1 makes mice as susceptible to Leishmania donovani, Salmonella typhimurium and Mycobacterium bovis as gene-disrupted mice (Vidal et al., 1995b). The amino-terminal domain of NRAMP1 binding to microtubules that could mediate microtubule-dependent phagosome and/or lysosome transport (Kishi et al., 1996). The NRAMP1 encodes phospho-glycoprotein with whole membrane and has the characteristics of transport activity and ion channel (Vidal et al., 1996). NRAMP1 acts by stabilizing the mRNA of genes associated with macrophage activation, thus accounting for the functional differences that have been attributed to these macrophage populations (Brown et al., 1997). NRAMP1 gene encodes a protein with 12 trans-membrane domains that localizes in the phagolysosome membrane, particularly in macrophages (Gruenheid et al., 1997). The whole sequence of NRAMP1 in swine encodes 539 amino acids, which has 87% similarity with human beings (Tuggle et al., 1997). A link was confirmed in vivo between iron metabolism and NRAMP1 function and established that excess iron hampers NRAMP1– encoded protein function and strongly argue for a role of NRAMP1 as an iron pump that depletes the phagosomal compartments of this nutrient, leading to starvation of the pathogen of this essential cation (Gomes and Appleberg, 1998).

NRAMP1 protein coding sequences and gene structures showed a high degree of conservation across species which suggested that the protein cannot tolerate significant alteration in primary structure (Dorschner and Phillips, 1999; Zhang et al., 2000).The movement of cations may actually occur in the opposite direction which results an increased concentration of iron into the phagolysosome and may favour bacterial killing by generating oxygen intermediates through the Fenton reaction (Goswami et al., 2001).The NRAMP protein has a pH dependent cation transport activity and they act as a transporter of divalent cations like  Fe2+ and Mn2+from the lumen of the phagolysosome towards the cytosol, thereby preventing acquisition of iron by intracellular pathogens (Wyllie et al., 2002; Forbes and Gros, 2003).  Polymor­phisms of the human NRAMP1 gene are sig­nificantly associated with pulmonary Mycobacterium avium complex infection (Tanaka et al., 2007), Crohn’s disease (Gazouli et al., 2008), rheumatoid arthritis (Ates et al., 2009), type 1 diabetes (T1D) (Yang et al., 2011).

Localization of SLC11A1 Gene

SLC11A1 localizes to membranes of late endosomes and lysosomes in mammals but not to early endosomes (Gruenheid et al., 1997; Searle et al., 1998) and suggests that it targets directly from the trans-Golgi network. NRAMP1mainly exists in reticulo-endothelial cells and organs such as outer white blood cells, spleen, and lung which can resist several intracellular pathogenic microorganisms (Blackwell, 1996). NRAMP1gene in swine is located on q23-26 of chromosome 15 (Sun et al.,1998) and the whole length of NRAMP1 in swine is about 15 kb which contains15 exons and 14 introns as human beings and mouse (Govoni et al.,1995; Marquet et al., 2000; Wu et al., 2007). It was reported that the whole sequence of NRAMP1in swine encodes 539 amino acids, which had 87% similarity with human beings (Tuggle et al., 1997). NRAMP1gene is specially expressed in phagocytic cells, such as macrophages neutrophilic granulocytes and outer blood cells.

Functions of SLC11A1 Gene

NRAMP1 functions as an H1-sensitive divalent cation transporter in the membrane of intact phagosomes In situ as they have the ability to transport metals because of their expression by both phagocytes and pathogens. NRAMP proteins appear to play a key role in the host–parasite interface in the microenvironment of the phagosome (Forbes and Gros, 2003). SLC11A1 is a divalent cation (Fe2+, Zn2+ and Mn2+) transporter (Goswami et al., 2001) and an antiporter that can flux divalent cations in either direction against a proton gradient. SLC11A1 in late endosomes/ lysosomes delivers divalent cations from the cytosol to the acidic compartment.

Cloning and Sequencing of SLC11A1 Gene in Pig

The cloning and sequencing of a full-length SLC11A1 gene cDNA was done and found that the entire protein coding region of the pig NRAMP1 gene which is corresponding to a mouse protein is known to cause susceptibility to infection by several different bacteria. Tuggle et al. (1997) reported that pig protein encoded within the gene is highly similar to the mouse and human NRAMP1 and new NRAMP1 sequence identified a 538 amino acid protein. The derived pig protein sequence had much higher identity to NRAMP1 from humans, cattle, and mice (87%, 88% and 85%, respectively) than to NRAMP2 from humans or mice (64% and 62%, respectively). Expression profile of pig NRAMP1 indicates that it is expressed in spleen which is a rich source of immune cells and may be expressed in other tissues at low levels. Their data strongly indicates that the newly cloned gene has a similar physiological function in pigs to that seen for mouse NRAMP1. It was revealed that the association of NRAMP1 to Salmonella infection in pigs can be tested from their information (Tuggle et al., 1997).

The cloning of the full-length cDNA for porcine NRAMP1suggest that over 85% identities in amino acid sequence to its congeners from humans, mice, cattle and sheep. Expression of porcine NRAMP1 mRNA was cell and tissue specific and was highest in macrophages. Investigation of the molecular mechanisms by which NRAMP1 is induced showed that Lipopolysaccharide(LPS) -induced expression in macrophages, neutrophils and peripheral blood mononuclear cells was time and dose dependent and was mediated primarily through Cluster of Differentiation (CD) -14. Induction of NRAMP1 required de novo protein synthesis and Mitogen-Activated Protein Kinases (MAPK) as essential factors. Blockage of either p38 or p42/44 MAPK pathways suppressed the expression of NRAMP1 to basal levels. Their findings suggest that bacterial infection and pro-inflammatory mediators induce NRAMP1 expression via activation of MAPK pathways. It was revealed that cloned porcine NRAMP1 cDNA and characterized tissue expression patterns and molecular mechanisms of induction of NRAMP1 by LPS, TNF-a and IL-1b and shown that LPS-induced NRAMP1 induction in macrophages is primarily dependent upon CD14 which also involves newly synthesized protein(s). It was reported that p38 and p42/44 MAPK cascades are involved in the induction of NRAMP1 expression in response to LPS, Tumor Necrosis Factor (TNF) -a, and Interleukin (IL)-1b. The relative significance of other protein kinases, such as PKC, protein kinase A, G proteins, and ceramide- activated protein kinase, in NRAMP1 induction remains to be elucidated (Zhang et al., 2000).

Devi et al. (2016) studied the genetic diversity of the SLC11A1 gene in Doom pigs of Assam especially on its evolution and differentiation within and among species, the partial sequence of the SLC11A1 gene was sequenced, characterized and compared with published sequences of pigs and other livestock species. The gene sequence of Doom pigs showed the highest sequence identity with EF200584.1 (exotic pig) and the lowest similarity AY368475.1 (large white strain 008). One single nucleotide polymorphism (SNP) was identified in the heterozygous sequence at the 736 bp position (A→G). The sequence showed the highest sequence identity (81.74%) with that of O. aries and the lowest similarity (39.12%) with B. bubalis (Mehsana breed), respectively. Phylogenetic analysis revealed that the Doom pig is more closely related to EU135795.1 (Chinese local pig) and EF200584.1 (Pig, Iowa State University). The sequencing and phylogenetic analysis of the SLC11A1 gene of Doom pigs could be used for genetic selection with disease-resistance varieties and upgradation of indigenous germplasm of domestic livestock.

The sequence analysis of the NRAMP1 gene in Tibetan, Gansu Black, Large White, Yorkshire, and Duroc pig breeds revealed the presence of 11 nucleotide variants in intronic regions, 2 nucleotide variants in the control region, 10 nucleotide variants and one deletion in the 3′ non-coding region and 15 nucleotide variants in the exons. Only 4 nucleotide variants resulted in amino acid changes. The Tibetan pig NRAMP1 protein harbors 10 transmembrane domains. The analysis also predicted 10 serine phosphorylation sites, 3 threonine phosphorylation sites, and 4 tyrosine phosphorylation sites in the NRAMP1 protein of Tibetan pigs. Predictions of the Tibetan pig NRAMP1 tertiary structure revealed 7 putative a -helices and 5 putative b-sheets. Predictions of the Yorkshire pig NRAMP1 protein revealed significant differences compared with the Tibetan pig NRAMP1 protein. The differences in transmembrane domains and tertiary structures of the NRAMP1 protein of the Tibetan and Yorkshire pig breeds could explain differences in the disease resistance of these two breeds (Wang et al., 2017).

PCR-RFLP to Detect Polymorphism of SLC11A1Gene

Mapping of NRAMP1 in the pig as potential candidate gene in controlling pig resistance to Salmonella infection was reported (Sun et al., 1998).  It was revealed that the primers from the pig cDNA to amplify a 1.6 kb fragment between exons 1 and 3. Pig-rodent somatic cell hybrid panel was used to map the NRAMP1 pig chromosome 15 (SSC15) with 100% probability and the regional assignment was SSC15q23-26 with 87% concordance. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker was developed by using the HinfI enzyme and three alleles were identified from a population including 11 breeds. Linkage analysis confirmed the physical assignment by using the PiGMaP reference families. Pig NRAMP1 was linked to SSC15 markers S0088, S0149 and S0284 (LOD > 3). A small population study revealed large allele frequency differences among tested breeds. An A allele is only observed in dam (white) lines whereas a similar exclusivity of the C allele was seen in sire (coloured) breeds.

Analysis of the variation at a number of genetic loci using PCR-RFLP in two local Romanian pig breeds, the Mangalitsa and Bazna was studied (Ciobanu et al., 2001). These breeds are part of a conservation programme in Romania and marker information may be useful in preserving a representative gene pool in the populations. Mangalitsa has a higher frequency of allele A (0.33), although allele B is the most common in both breeds. Allele B was the most prevalent and polymorphism was assessed at loci which are known to cause phenotypic variation, potentially involved in trait differences or putative candidate genes. The traits considered are disease resistance, growth, coat colour, meat quality and prolificacy. Even though the populations are small and the markers are limited to specific genes, significant differences in five of the ten characterized loci were reported. In some cases the observed allele frequencies were interesting in relation to gene function and the phenotype of the breed.

Yan et al., 2004, investigated the genetic variations in a 1.6 kb region spanning exon 1 and exon 3 of the porcine NRAMP1 gene by PCR-HinfI -RFLP in samples of 1347 individuals from 21 Chinese indigenous pig populations and 3 western pig breeds. Three alleles (A, B and C) and four genotypes (AA, BB, AB and BC) were detected. The differences in genotype and allele frequencies between Chinese indigenous pig populations and exotic pig breeds were found to be significant, while in general the differences in genotype and allele frequencies within the Chinese indigenous pig populations were not significant. The allele C was detected only in Duroc, Leping Spotted and Dongxiang Spotted pig and the two Chinese pig populations showed similar genotype and allele frequencies. Four Chinese Tibetan pig populations displayed genetic differentiation at the NRAMP1 gene locus. In addition, intron 5 of the NRAMP1 gene was isolated and characterized directly by sequencing the PCR products encompassing intron 5. The alignment of intron 5 of the porcine, human, equine and ovine NRAMP1 gene showed similarity of 45.38% between pig and human, 52.55% between pig and horse, 63.47% between pig and sheep, respectively. Wu et al. (2008) designed to study the association of polymorphism of NRAMP1 with some immune function and the production performance in Large White pig. The PCR-RFLP technique was applied to analyze the correlation between the polymorphisms of NRAMP1gene and immune function value of Polymorpho-nuclear Leukocytes (PMN) obtained by Nitroblue Tetrazolium (NBT) Reduction and effect of Cytotoxin in Monocyte and production performance in 165 Large White pigs. It was found that  one NdeI restriction locus in Large White pig and both values of PMN by NBT Reduction and effect of Cytotoxin in Monocyte in genotype BB were higher than those in genotype AB (P<0.05)  and the weight of 180-day-old pigs with genotype BB was higher than that with genotype AB (P<0.05). It was concluded that there was a significant correlation between different genotypes of NRAMP1gene and Immune function and production performance and it can be regarded as a candidate gene of disease resistance. It was also suggested that all those results provide valuable reference to further studies of pig disease resistance (Table 1).

Table 1: Restriction enzymes, polymorphism sites, genotypes, SNP identified and amino acid change in PCR-RFLP to detect polymorphism in SLC11A1 gene

S. No. Restriction Enzyme Polymorphism site Genotype SNP identified Amino acid changes References
1 HinfI  3 allele Sun et al. (1998)
2 HinfI            – A,B,C (Allele)         – Ciobanu et al. (2001)
3 HinfI Intron 5 AA, BB, AB and BC No SNPs Yan et al. (2004)
4 NdeI Intron 6 (483 bp) AA, AB and BB        – Wu et al. (2008)
5 SmaI Intron 1(738 bp position) GG, AG and AA A→G transition Liu et al. (2011)
6 SmaI One SNP at  intron 1 and one SNP at exon 1 CC, CT, TT, AA, AG and GG C>T and A>G   Ding et al. (2014)
7 SmaI 736 bp AA, AB and BB  A↔G transition Devi et al. (2015)

In Chinese indigenous pigs and exotic pigs one single nucleotide polymorphism (SNP) was found in intron 1 after digestion with SmaI and found A→G transition at 738 bp position (Liu et al., 2011). The three genotypes; GG genotype: 506 bp, 232 bp and 198 bp, AG genotype: 506 bp 430 bp and 232 bp and AA genotype: 505 bp and 430 bp were reported in Landrace pigs. They could establish an association of polymorphism of the SLC11A1 gene with the level of monocyte (p=0.010) and CD4¯CD8+ percentage (p = 0.041) and also found that the animals with AA genotype had significantly (p<0.05) higher monocyte and CD4¯CD8+ percentage than that of animals with GG and GA genotype monocyte. The T lymphocyte found at A→G transition site 738 in intron1 forms another restriction site for SmaI enzyme. It was suggested that the SLC11A1 could be used as a marker gene for genetic selection of disease susceptibility in pigs.

To evaluate the effects of NRAMP1 gene on immune capacity in pigs, tissue expression of NRAMP1 mRNA was observed by real time quantitative polymerase chain reaction (PCR) and the results revealed NRAMP1 expressed widely in nine tissues. One single nucleotide polymorphism (SNP) (ENSSSCG00000025058: g.130 C>T) in exon1 and one SNP (ENSSSCG00000025058: g.657 A>G) in intron1 region of porcine NRAMP1 gene were demonstrated by DNA sequencing and PCR-RFLP analysis. A further analysis of SNP genotypes associated with immune traits including white blood cell (WBC), granulocyte, lymphocyte, monocyte (MO), rate of cytotoxin in monocyte (MC) and CD4/CD8 T lymphocyte subpopulations in blood was carried out in four pig populations including Large White and three Chinese indigenous breeds (Wannan Black, Huai pig and Wei pig). The results showed that the SNP (ENSSSCG00000025058: g.130 C>T) was significantly associated with level of WBC % (p = 0.031), MO% (p = 0.024), MC% (p = 0.013) and CD4CD8+ T lymphocyte (p = 0.023). The other SNP (ENSSSCG00000025058: g.657 A>G) was significantly associated with the level of MO% (p = 0.012), MC% (p = 0.019) and CD4CD8+ T lymphocyte (p = 0.037).These results indicate that the NRAMP1 gene can be regarded as a molecular marker for genetic selection of disease susceptibility in pig breeding (Dinga et al., 2014).

The population of Doom pigs was found to be polymorphic in respect of SLC11A1 gene having 2 alleles with frequencies 0.8364 and 0.1636 for ‘A’ and ‘B’, respectively (Devi et.al., 2015). It was revealed that the genotypic frequencies to be 0.71, 0.26 and 0.03 for AA, AB and BB genotypes, respectively.

PCR-SSCP to Detect Polymorphism

PCR-SSCP method was used to analyze the NRAMP1 gene (exon 2 and intron 6) polymorphisms in Hezuo pig (Juan et al., 2010). It was aimed to get molecular genetics information and provided the scientific basis for molecular marker of disease resistance of Hezuo pig. The result of PCR-SSCP polymorphism examination of the NRAMP1 exon 2 from cloning and sequencing indicated that there were two polymorphic loci 62 T→C and 92 A→G and this mutation did not induce the changes of encoding amino acid being controlled by allelic genes A and B. There were three genotypes, namely AA, BB and AB in NRAMP1 exon 2. Frequency of A allele was 0.6519 which was found predominantly. The result of PCR-SSCP polymorphism examination of the NRAMP1 intron 6 from sequencing analysis showed that ten new nucleotide polymorphisms in intron 6, which were the single nucleotide substitution of C→T at the position of 99, 225, 304, 307, 313, A→G at the position of 105, C→A at the position of 190, T→G at the position of 298, T→C at the position of 306 and 295-296 bp was detected one insertion: AA and CC genotype -→T, BB genotype → G. Among the five genotypes, namely AA, AB, BB, AC and CC; allele B was found predominant. From the statistical results it was revealed that the NRAMP1 exon 2 SNPs sites in Hezuo pig was at Hardy-Weinberg disequilibrium (P < 0.05), PIC was intermediate polymorphism; NRAMP1 intron 6 SNPs sites in Hezuo pig was at Hardy-Weinberg equilibrium (P > 0.05), PIC was high polymorphism (Table 2).

 

Table 2: Restriction enzymes, polymorphism sites, genotypes, SNP identified and amino acid change in PCR-SSCP to detect polymorphism in SLC11A1 gene

S. No. Restriction Enzyme Polymorphism site Genotype SNP identified Amino acid changes References
1 Exon 2 AA, BB and AB Two polymorphic loci 62 T→C and 92 A→G No changes of  amino acid Juan et al. (2010)
2 Intron 6 AA, AB, BB, AC and CC C→T,A→G, C→A,T→G, T→C,one insertion: AA and CC genotype -→T, BB genotype → G. Juan et al. (2010)
3 Sma1 Intron 1 2 allele A «G transitions Tuggle et al. ( 2005)
4 NdeI 278-279 base sites CA-TG mutation (40polymorphis, 6 of which were reported to be located in exons and 34 in introns)   Wu et al. (2007)

NRAMP1gene as candidate genes for contributing to resistance in Salmonella cholera suis (SC) challenge in pigs was investigated and reported five NRAMP1 sequence differences polymorphisms and SNPs (Tuggle et al., 2004). The effects these polymorphisms have on resistance to infection were tested in two experimental disease studies. In study 1, results showed NRAMP1 genotypes are associated with decreased fecal bacterial load during infection (P values: < 0.0006 to < 0.06).  In the second study, many additional immune traits and spleen and liver bacterial counts were collected. The NRAMP1 genotypes were associated with bacterial count in liver (P < 0.05 and P < 0.0006) and with polymorpho-nuclear phagocytes (P < 0.003 to < 0.05).  These data indicate NRAMP1 gene variation may control, in part, response to Salmonella infection in pigs, and that these differences could be used to identify resistant animals. Tuggle et al. (2005) invented a method for determining improved innate immunity, disease resistance or performance in animals.

The method involves assays for a genetic difference in the NRAMP1 gene of the animal. Novel NRAMP1 sequence, assays and compositions for identifying the presence or absence of the alleles was provided and further two alleles in intron 1 upon Sma1 digestion were reported. It was reported that at A «G transitions, both the allele has G position at 737 and allele 7 also has A position at 737. The investigation of the porcine response to gastrointestinal infection with Salmonella enteric serovars Cholerae suis (narrow host range) and Typhimurium (broad host range) was also studied which revealed markedly different transcriptional profiles (Uthe et al., 2007). Identification of seven genes through suppression subtractive hybridization as up-regulated in the mesenteric lymph nodes at 24 h post-inoculation (p.i.) in serovar Cholerae suis-infected pigs (ARPC2, CCT7, HSPH1, LCP1, PTMA, SDCBP, VCP) and three genes in serovar Typhimurium-infected pigs (CD47/IAP, CXCL10, SCARB2) were analyzed by real-time PCR at 8 h, 24 h, 48 h, 7 days (d) and 21 d p.i. A comparison between the two Salmonella infections revealed significant differences in transcriptional induction early in the infection (8–24 h) for the serovar Typhimurium-infected pigs, whereas the serovar Cholerae suis-infected pigs exhibited significantly higher levels of gene expression at the later time points (48 h–21 d), except for HSPH1. A similar gene expression trend was observed for immune-related genes involved in innate immunity and the inflammatory T helper 1 (Th1) response. Initial repression of gene expression in the serovar Cholerae suis-infected pigs from 8 to 48 h p.i. (IFNG, IL12A, IL4, IL8 and CSF2) coincided with extended transcriptional activation throughout the 21 d infection (IFNG, INDO, SOCS1, STAT1, IL1B, IL6, IL8 and SLC11A1). The serovar Typhimurium-infected swine presented a more transient induction of immune-related genes (IFNG, INDO, IRF1, SOCS1, STAT1, IL1B, IL8, SLC11A1) early in the infection (24–48 h) followed by a significant repression of IL12A, IL12B, IL4, IL8 and CSF2. Collectively, these data reveal specific porcine genes with differences in gene expression kinetics that may be responsible for the variation in disease progression observed in swine infected with Typhimurium compared to Cholerae suis.

The polymorphism of the sixth intron eleven sino-foreign in swine was reported that this polymorphism site was caused by CA-TG mutation at 278-279 base sites of the Nramp1 gene, resulting in failure of reorganization of restriction enzyme sites by NdeI (Wu et al., 2007). The restriction enzyme sites of NdeI tend to be the same in gene frequency and genotypic frequency of each breed of NRAMP1 among different pig breeds. However, the correlation between gene polymorphism and disease resistance of NRAMP1 as function gene was not being reported. It was reported that the porcine SLC11A1 gene consist of 15 exons and 14 introns in pigs. All introns were sequenced and their nucleotide sequences were submitted to Gene Bank. The exon/intron boundaries were determined by comparing cDNA sequence with amplified genomic DNA sequences. Mutational analysis was performed on exonic and neighboring intronic region by denaturing high-performance liquid chromatography (DHPLC) and sequencing confirmation. Among the identified 40 polymorphisms, six were reported to be located in exons and 34 in introns. Two exonic polymorphisms are non-synonymous changes (D6H and V175I), three are synonymous changes (S23, G33 and I155), and one is in 3′ UTR.  They opined that the availability of the fine genomic organization and identification of the polymorphism facilitate the evaluation of the functional role of porcine SlC11A1 genes to disease resistance or susceptibility.

Conclusion

Breeding for genetic resistance is one of the promising ways to control the infectious diseases. Increase in host resistance is the most important method for controlling such diseases, but no breed is totally resistant. The total resistance to these diseases is the ultimate goal and its progress towards achievement could be enhanced through introgression of resistance genes to breeds with low resistance. There is need to explore more and more SNPs in our population and establish its association with diseases but it is also expected that all SNP-based studies should be carefully crafted and well-designed from the beginning so that the utility of that particular study will exist at sent for some time. Further studies are required in the area of identification of candidate gene polymorphisms associated with disease resistance/ susceptibility and inclusion of these markers in selection and breeding programmes.

References

  1. Annual Report. 2016-17. Department of Animal Husbandry, Dairying and Fisheries. Ministry Of Agriculture and Farmer’s Welfare. Government of India, Krishi Bhawan, New Delhi. Pp 162.
  2. Ates O, Dalyan L, Musellim B, Hatemi G, Türker H, Ongen G, Hamuryudan V, Topal- Sarikaya A. NRAMP1 (SLC11A1) gene polymorphisms that correlate with autoimmune versus infectious disease susceptibility in tuberculosis and rheumatoid arthritis. Int J Immunogenet. 2009; 36:15–19.
  3. Awomoyi A A. 2007. The human solute carrier family 11 member 1 protein (SLC11A1): linking infections, autoimmunity and cancer. FEMS Immunology and Medical Microbiology 49:324-329.
  4. Belouchi A, Cellier M, Kwan T, Saini H S, Leroux G and GrosP. The macrophage specific membrane protein NRAMP controlling natural to infections in mice has homologues expressed in the root system of plants. Plant Molecular Biology 29: 1181-1196.
  5. Blackwell J M.1996. Structure and function of the natural resistance- associated macrophage protein (NRAMP1), a candidate protein for infectious and autoimmune disease susceptibility. Molecular Medicine Today 2: 205-211.
  6. Brown D H, Lafuse W P and Zwilling B S. 1997. Stabilized expression of mRNA is associated with Mycobacterial resistance controlled by NRAMP1.Infection and Immunology 65:597-603.
  7. Ciobanu D C, Day A E, Nagy A, Wales R, Rothschild M F and Plastow G S. 2001. Genetic variation in two conserved local Romanian pig breeds using type 1 DNA markers. Genetic Selection Evolution 33: 417-432.
  8. Devi B, Laskar S, Borah P, Kalita D J, Zaman G U, Ferdoci A M and Hussain I. 2015. Polymorphism of SLC11A1 gene in Doom pigs of Asom. Indian Journal of Animal Science 85 (4): 404–406.
  9. Devi B, Laskar S, Borah P, Hussain I and Bharti P K. 2016.Sequencing and phylogenetic analysis of the SLC11A1 gene in pigs. Journal of Applied Animal Research 45: 494-497,
  10. Ding X, Zhanga X, Yang Y, Ding Y, Xue W, Meng Y, Zhu W and Yin Z.Polymorphism, Expression of Natural Resistance-associated Macrophage Protein 1 Encoding Gene (NRAMP1) and its Association with Immune Traits in Pigs. Asian-Australasian Journal of Animal Science 27(8): 1189–1195.
  11. Doschner M O and Phillips R B 1999.Comparative analysis of two NRAMP loci from rainbow trout DNA. Cell Biology 18 (7): 573-583.
  12. Forbes J R and Gros P. 2003. Iron, manganese, and cobalt transport by NRAMP1 (SLC11A1) and NRAMP2 (SLC11A2) expressed at the plasma membrane. Blood. 102:1884–1892.
  13. Gazouli M, Atsaves V, Mantzaris G, Economou M, Nasioulas G, Evangelou K, Archimandritis AJ, Anagnou NP. Role of functional polymorphisms of NRAMP1 gene for the development of Crohn’s disease. Inflamm Bowel Dis. 2008; 14:1323–1330.
  14. Gomes M S and Appelberg R. Evidence for a link between iron metabolism and Nramp1 gene function in innate resistance against Mycobacterium avium. Immunology. 95:165–168.
  15. Goswami T, Bhattacharjee A, Babal P, Searle S, Moore E, Li M and Blackwell J M.2001. Natural-resistance-associated macrophage protein 1 is an H+ bivalent cation antiporter. Biochemistry Journal. 354 (Pt 3): 511–519.
  16. Govoni G, Vidal S, Cellier M, Lepage P, Malo D. and Gros P. 1995. Genomic structure, promoter sequence and induction of expression of the mouse NRAMP1 gene in macrophages. 27: 9-19.
  17. Gruenheid S, Pinner E, Desjardins, M. 1997. Natural resistance to infection with intracellular pathogens: the NRAMP1 protein is recruited to the membrane of the phagosome. Journal of Experimental Medicine. 185 (4): 717-30.
  18. Juan-Juan Li., Xiao-jun Ma., Li-xian Wang., Xiao-li Zhang. and Xiao-fei Li. 2010. Polymorphism of the NRAMP1 gene intron 6 in Hezuopig. Journal of Gansu Agricultural University
  19. Kishi F, Yoshida T and Aiso S.1996.Location of NRAMP1 molecule on the plasma membrane and its association with microtubules. Molecular Immunology. 33(16):1241-6.
  20. Liu Y, Qiu X, Xu J, Hu Fang Y Li, Li H, Gong Y and Zhang Q. 2011.Association Analysis between the Polymorphism of the SLC11A1 gene and Immune Response Traits in Pigs. Asian Journal of Animal and Veterinary Advances. 6(6)580-586.
  21. Marquet S., LepageP, HudsonT J, MusserJ M and Schurr E. 2000. Complete nucleotide sequence and genomic structure of the human NRAMP1 gene region on chromosome region 2q35. Genome. 11(9):755-762.
  22. McInerney, John.1996. Old economics for new problems – livestock disease: Presidential address. 47(3): 295-314.
  23. Meuwissen, T H E, and van Arendonk, J A M 1992. Potential improvements in rate of genetic gain from marker assisted selection in dairy cattle breeding schemes. Journal of Dairy Science. 75: 1651-1659.
  24. Searle S, Bright N A, Roach T I A, Atkinson P G P, Barton CH, Meloen RH, Blackwell JM.1998. Localization of Nramp1 in macrophages: modulation with activation and infection. Journal of Cellular Science. 111:2855–2866.
  25. Sun H S, Wang L, Rothschild M F and Tuggle C K. Mapping of the natural resistance-associated macrophage protein 1 (NRAMP1) gene to pig chromosome 15. Animal Genetics. 29: 138-140.
  26. Tanaka G, Shojima J, Matsushita I, Nagai H, Kurashima A, Nakata K, Toyota E, Kobayashi N, Kudo K, Keicho N. Pulmonary Mycobacterium avium complex infection: Association with NRAMP1polymorphisms. Eur Respir J. 2007; 30:90–96.
  27. Thomas N and Joseph S.2012.Role of SLC11A1 Gene in Disease Resistance. Biotechnology in Animal Husbandry. 28 (1):99-106.
  28. Tuggle C K, Schmitz C B and Gingerich-Feil D. Rapid communication: Cloning of a pig full-length natural resistance associated macrophage protein (NRAMP1) cDNA. Journal of Animal Science.75: 277.
  29. Tuggle C K, Shi Xian-wei, Marklund L, Stumbaugh A and Stabel T J. 2004. Association of bacterial infection traits with geneticvariation at candidate genes for porcine disease resistance. Animal Industrial Report: AS 650, ASL R1952.
  30. Tuggle C K, Marklund L, Stabel T J, Mellencamp M A andStumbaugh A.2005.Genetic markers for screening animals for improved disease resistance (NRAMP).United States Patent, 6844159B2. http://ddr.nal.usda .gov/handle/10113/6983.
  31. Uthe J J, Royaee A, Lunney Joan K Stabel,  Thomas J Shu-Hong Zhao, Tuggle C K and Bearson S M. 2007.Porcine differential gene expression in response to Salmonella enteric serovarsCholeraesuis and Typhimurium. Molecular Immunology. 44:2900–2914.
  32. Vidal S M, Malo D V K. Skamene E and Gros P. 1993. Natural resistance to infection with intracellular parasites: isolation of a candidate for Bcg. 73: 469-48.
  33. Vidal S, Gros P and Skamene E. 1995a. Natural resistance to infection with intracellular parasites: molecular genetics identifies NRAMP1 as the Bcg/Ity/Lsh locus. Journal of Leukocyte Biology. 58 (4): 382-90.
  34. Vidal S, Tremblay M L, Govoni G, Gauthier S, Sebastiani G, Malo D, Skamene E, Olivier M, Jothy S and Gros P. 1995b. The Ity/Lsh/Bcg locus: natural resistance to infection with intracellular parasites is abrogated by disruption of the NRAMP1 Journal of Experimental Medicine. 182: 65.
  35. Vidal S M, Pinner E, Lepage P, Gauthier S and Gras P.1996. Natural resistance to intracellular infections: NRAMP1 encodes a membrane phospho-glycoprotein absent in macrophages from susceptible (NRAMP1 D169) mouse strains. Journal of Immunology.157 (8): 3559-68.
  36. Wang Z., Chen Q.,  Liao R.,  Zhang Z.,  Zhang X.,  Liu X.,  Zhu M.,  Zhang W., Xue M., Yang H.,  Zheng Y.,  Wang Q., and Y. Pan. 2017. Genome‐wide genetic variation discovery in Chinese Taihu pig breeds using next generation sequencing. Anim Genet. 48(1): 38–47.
  37. Wu Z F, Luo W H, Yang G F and Zhang X Q.2007.Genomic organization and polymorphisms detected by denaturing high-performance liquid chromatography of porcine SLC11A1 DNA Sequence. 18 (5): 327-333.
  38. Wu H, Cheng D, Wang L.2008.Association of polymorphism of NRAMP1 gene with immune function and production performance of large white pig. Journal of Genetics and Genomics. 35 (2): 91-95.
  39. Wyllie S, Seu P and Goss J A. 2002. The natural resistance-associated macrophage protein 1 SLC11A1 (formerly NRAMP1) and iron metabolism in macrophages. Microbiology and Infection.4: 351–359.
  40. Yan X M, Ren J, Ai H S, Ding N S, Gao J, Guo Y M, Chen C Y, Ma J W, Shu Q L and Huang L S. 2004.Genetic variation analysis and characterization of the fifth intron of porcine NRAMP1 Asian-Australasian Journal of Animal Science.17: 1183-1187.
  41. Yang JH, Downes K, Howson JM, Nutland S, Stevens HE, Walker NM, Todd JA. Evidence of association with type 1 diabetes in the SLC11A1 gene region. BMC Med Genet. 2011; 12:59.
  42. Zhang G L, Wu H, Ross C R, Minton J E and BlechaF.2000.Cloning of porcine NRAMP1 and its induction by lipopolysaccharide, tumor necrosis factor alpha, and interleukin-1: role of CD14 and mitogen- activated protein kinases. Infection and Immunity. 68 (3):1086-1093.
  43. Zhao S, Zhu M and Chen H.2012.Immunogenomics for identification of disease resistance genes in pigs: a review focusing on Gram-negative bacilli. Journal of Animal Science and Biotechnology. 3:34
Abstract Read : 91 Downloads : 15
Previous Next
Close