Abstract: Background: Natural resistance-associated macrophage protein-1 gene (NRAMP1) plays an important role in immune response against intracellular pathogens. Objective: This study was aimed at identifying NRAMP1 polymorphisms and their association with immune traits in Indonesian native chickens. Methodology: Genetic polymorphism of chickens was investigated using polymerase chain reaction-restriction fragment polymorphism. The concentrations of leukocytes and differentiation (heterophile, lymphocyte and monocyte) were assessed by the Giemsa method and immune traits were detected by followed clearance test. Results: The results showed that NRAMP1 was polymorphic in all native chickens. The CC genotype was significantly higher than CT and TT genotypes (p<0.05) in Sentul chickens resistant to Salmonella pullorum. Although, the concentrations of leukocytes and differentiation in chickens with all three of NRAMP1 genotypes (CC, CT and TT) were not statistically different, there was a significant correlation between different NRAMP1 genotypes and immune traits. Therfore, NRAMP1 is proposed to be a disease resistance candidate gene. Conclusion: However, this study should be validated in other chicken populations to evaluate the potential exploitation of NRAMP1 in selective breeding.
INTRODUCTION
Various breeding programs have been developed and implemented in an effort to improve the productivity of livestock commodities and current breeding programs which improve resistance of livestock to disease have become increasingly popular as an alternative method for increasing productivity while reducing production costs. In addition to economic concern, the study of the genetics of disease resistance is also driven by a greater probability of success as a result of significant progress in the field of molecular technology. Genetic approaches to animal disease resistance are very important, especially for production systems in developing countries because general maintenance systems are still traditional or semi-intensive and small-scale since vaccinations and treatments are difficult to disseminate and expensive.
Previous study on chickens focusing on immune reactions to disease has shown a strong genetic component associated with resistance1,2. Differences in disease resistance are also a genetic basis for selection in breeding3. Candidate gene approaches have been successfully used to detect genes important for disease resistance in chickens including major histocompatibility complex, caspase-1, inducible nitric oxide synthase, toll-like receptor 4 and natural resistance-associated macrophage proteins-1 (NRAMP1) genes2,4,5.
In chickens, a homologue of NRAMP1 has been mapped on chromosome seven which consists of a promoter region, 15 exons, 14 introns and flanking regions 5760 bp in length6. The NRAMP1 restricts microbial access to essential micronutrients such as Fe2+, Mn2+, Co2+ and Zn2+ within professional phagosomes7-10 and has been identified as a candidate gene that controls resistance to Salmonella enteritidis in poultry11. The NRAMP1 polymorphisms have been previously associated with certain immune traits in chickens12. For example, the CC genotype of NRAMP1 had been associated with a higher S. enteritidis burden in the cecum and spleen of Malaysian native chickens13. The NRAMP1 is mainly expressed in the liver, thymus and spleen in both female and male chickens14.
Until now, variations in NRAMP1 and their effect on disease have not been well investigated in Indonesian native chickens. Therefore, the objective of the present study was to identify the association of NRAMP1 polymorphisms with immune traits in Indonesian native chickens in order to provide a reference for disease-resistant chicken breeding by marker-based selection.
MATERIALS AND METHODS
Blood samples: Blood samples were collected from seven breeds of Indonesian native chickens (n = 265 total) including Kampung (n = 57), Sentul (n = 96), Broiler (n = 10), Pelung (n = 10), Merawang (n = 23), F1 Kampung×Broiler (KB, n = 30) and F2 KB×KB (n = 39). The DNA was extracted from blood samples at the Animal Molecular Genetics Laboratory, Faculty of Animal Science, Bogor Agricultural University, Indonesia.
DNA extraction: Genomic DNA was extracted from blood samples using phenol-chloroform method followed by ethanol precipitation15 and the DNA was dissolved in elution buffer. The quality of the total genomic extraction was assessed by 1% agarose gel electrophoresis.
DNA amplification: Polymerase Chain Reaction (PCR) was carried out using primers specific for a part of exon 11 (421 bp) of NRAMP1 (GenBank Accession No. AY072001): Forward 5’-CAATGAGACGGTGTCTGTGG-3’ and reverse 5’-CCCAGAAGAAATCTCCCTGC-3’. The PCR was carried out in a total reaction volume of 15 μL containing 0.5 μL of the genomic DNA template, 0.3 μL of each primer, 0.3 μL dNTPs, 1 μL MgCl2, 0.05 μL of Taq polymerase, 1.5 μL 10X reaction buffer and 10.85 μL of distillated water. Amplification was carried out with a GeneAmp® PCR 9700 System (Applied Biosystems, USA). Thermal cycling conditions consisted of predenaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 10 sec, annealing at 60°C for 20 sec and extension at 72°C for 30 sec the final extension step was at 72°C for 5 min. The DNA amplification products and a standard DNA ladder were separated on 1.5% agarose gels in 0.5X TBE buffer.
PCR-Restriction Fragment Length Polymorphism (RFLP) analysis: Genetic polymorphism of NRAMP1 was conducted by PCR-RFLP using a SacI restriction enzyme which recognizes and cleaves GAGCT|C sites. Amplification products were visualized on a 2% agoras gel containing 2.5 μL ethidium bromide and 0.5X TBE buffer (1 M tris-base, 0.9 M boric acid, 0.01 M EDTA, pH 8.0) with a DNA ladder as a standard size comparison. For enzymatic digestion and determination of RFLPs, PCR products (5 μL) were mixed with 0.3 μL of SacI, 1 μL distillated water and 0.7 μL tango buffer and then incubated at 37°C for 16 h. The digestion products were separated by horizontal electrophoresis (100 V, 40 min) in 2% agoras gel in 0.5X TBE and 2.5 μL ethidium bromide visualized on an ultraviolet-transiluminator.
Concentration of leukocytes and differentiation: The concentrations of leukocytes and differentiation were assessed by the Giemsa method16 as follows: 20 μL of chicken blood was dissolved in 380 μL of Turk solution (1 mL of 1% gentian violet in water, 1 mL glacial acetic acid and 100 mL distilled water) using a micropipet. The total number of leukocytes present was calculated by counting all viable cells present on four areas located in four corners of the room count under a light microscope (100X magnification) and then multiplying by 50 to determine the concentration of each millimeter cube3.
Clearance test: Immune traits were detected in blood samples using the clearance test17. This method was used to look at normal bacterial population growth compared that of populations given specific treatment. The treatment impact on bacterial growth was measured after incubating for 24-48 h at 35±1°C.
Genotype and allele frequencies: Genotype and allele frequencies were calculated according to the formula by Nei and Kumar18 using NRAMP1 genotyping data. Genotype frequency was calculated with the following formula:
Allele frequency was calculated with the following formula:
| Xii | = | Genotype frequency |
| Xi | = | Allele frequency |
| nii | = | Number of samples with ii genotype |
| nij | = | Number of samples with ij genotype |
| N | = | Total number of samples |
Statistical analysis: The NRAMP1 polymorphism and immune trait data were analyzed using the GLM procedure of SAS 9.1.3 software (SAS Institute, Cary, NC, USA). The following model was used:
Yij = μ+Gi+eij
where, Yij is the observation on immune traits, μ is the overall mean, Gi is the effect of the single nucleotide polymorphism genotypes and eij is the random residual effect.
RESULTS AND DISCUSSION
Genetic polymorphism of NRAMP1: All DNA samples from each chicken were amplified and the PCR products separated by agarose gel electrophoresis. Thus, the product was amplified with fine stability and specificity and could be cleavaged by the restriction enzyme directly. A 421 bp fragment of exon 11 from NRAMP1 in Indonesian native chicken samples is shown in Fig. 1.
The NRAMP1|SacI restriction analysis of the exon 11 region revealed three genotypic patterns in Indonesian native chickens and showed two alleles (C and T alleles) in all native chicken breeds examined. The restriction fragments included a single, uncut fragment of 421 bp (TT genotype), two fragments of 258 and 163 bp (CC genotype and three fragments of 421, 258 and 163 bp (CT genotype) (Fig. 2).
| Fig. 1: | NRAMP1 PCR product, M: DNA ladder and Lanes 1-8: Individual PCR products |
| Fig. 2: | SacI restriction pattern of exon 11 fragment of NRAMP1 by PCR-RFLP on 2% agarose gel, M: DNA ladder and Lanes 1-14: Individual PCR-RFLP products |
| Table 1: | Genotypes and allele frequencies of polymerase chain reaction-restriction fragment length polymorphism products from part of exon 11 of NRAMP1 |
| Table 2: | Correlation analysis between NRAMP1 genotypes and immune function in Sentul chickens |
The genotype and allele frequencies of exon 11 of NRAMP1 in all Indonesian native chicken breeds are presented in Table 1. The homozygous CC genotype was present in the highest frequency across all breeds. The distribution of the NRAMP1|SacI alleles of exon 11 is characterized by a higher frequency of the C allele compared to the T allele in most of the native chickens studied (Table 1).
The two genotypes found to in Kampung, Broiler, F1 KB and F2 KB×KB chickens were CC and CT (Table 1) CC, TT and CT genotypes were found in Sentul, Pelung and Merawang breeds. This genetic diversity can be caused by DNA sequence repeats sequences, insertions, deletions and/or recombinations between individuals, groups or populations18. The results show that the gene fragments of NRAMP1|SacI exon 11 in Indonesian native chickens were polymorphic because there were three genotypes found in each of fragments obtained and an allele frequency around 0.900.
Concentration of leukocytes and differentiation: Leukocyte concentrations in Sentul chickens with CC, CT and TT genotypes were not statistically different (Table 2). Leukocytes (white blood cells) are mobile parts of the immune system. After formation, white blood cells enter the bloodstream and go to the necessary parts of the body. Granular leukocytes consist of heterophiles, eosinophils and basophils; no granular leukocytes are monocytes or lymphocytes19. These four cell types (leukocyte, heterophile, lymphocyte and monocyte) have four important immune functions and are presented in Table 2.
The concentration of leukocytes in chickens with CC, CT and TT genotype20 were within the normal range 12-30×103 mm–3. These results indicate that the Sentul chicken with the third NRAMP1 genotype were not infected with the bacterium.
Resistance of Sentul chickens to S. pullorum: The CC genotype in Sentul chickens resistant to S. pullorum was significantly higher (p<0.05) than the CT or TT genotypes (Table 3).
| Table 3: | Association of NRAMP1 genotype in Sentul chickens resistant to Salmonella pullorum |
| Different letters in the same column means difference significantly (p<0.05) | |
According Tohidi et al.13 the homozygous CC genotype was related to the highest S. enteritidis load in Malasyian native chickens and red jungle fowl. The death rate of S. pullorum in Sentul chickens with the three genotypes ranged from 27-55%. The association of NRAMP1 genotype in Sentul chickens to S. pullorum resistance is presented in Table 3.
As a candidate disease resistance gene, NRAMP1 has been studied by a number of researchers throughout the world. Numerous studies have revealed a correlation between NRAMP1 and immune resistance to disease. Vidal et al.21 found that in mice NRAMP1 plays an important role during the early immune period when macrophages are activated by parasite antigens. Liu and Lamont11 found that a single nucleotide polymorphism of highly conserved sequences of chicken NRAMP1 is related to S. enteritidis vaccination and immune response to pathogenic attack.
CONCLUSION
The present study investigated the relationship between NRAMP1 polymorphisms of Indonesia native chicken and demonstrated that genetic mutation exon 11 of NRAMP1 effects the resistance of Sentul chickens to S. pullorum. Thus, NRAMP1 is a good candidate gene for disease resistant breeding.
ACKNOWLEDGMENT
The researchers thank and appreciate the Directorate General of Higher Education, Ministry of Education and Culture of The Republic of Indonesia for the financial support through a PMDSU (Scholarship No. 081/SP2H/PL/DIT.LITABMAS/VI/ 2014).