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The effects of polymorphisms in 7 candidate genes on resistance to Salmonella Enteritidis in native chickens
GENETICS The effects of polymorphisms in 7 candidate genes on resistance to Salmonella Enteritidis in native chickens R. Tohidi,* I. B. Idris,*1 J. Malar Panandam,* and M. Hair Bejo† *Department of Animal Science, Faculty of Agriculture, and †Department of Veterinary Pathology and Microbiology, Veterinary Faculty, Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia ABSTRACT Salmonella enterica serovar Enteritidis infection is a common concern in poultry production for its negative effects on growth as well as food safety for humans. Identification of molecular markers that are linked to resistance to Salmonella Enteritidis may lead to appropriate solutions to control Salmonella infection in chickens. This study investigated the association of candidate genes with resistance to Salmonella Enteritidis in young chickens. Two native breeds of Malaysian chickens, namely, Village Chickens and Red Junglefowl, were evaluated for bacterial colonization after Salmonella Enteritidis inoculation. Seven candidate genes were selected on the basis of their physiological role in immune response, as determined by prior studies in other genetic lines: natural resistance-associated protein 1 (NRAMP1), transforming growth factor β3 (TGFβ3), transforming growth factor β4 (TGFβ4), inhibitor of apoptosis protein 1 (IAP1), caspase 1 (CASP1), lipo-
polysaccharide-induced tumor necrosis factor (TNF) α factor (LITAF), and TNF-related apoptosis-inducing ligand (TRAIL). Polymerase chain reaction-RFLP was used to identify polymorphisms in the candidate genes; all genes exhibited polymorphisms in at least one breed. The NRAMP1-SacI polymorphism correlated with the differences in Salmonella Enteritidis load in the cecum (P = 0.002) and spleen (P = 0.01) of Village Chickens. Polymorphisms in the restriction sites of TGFβ3-BsrI, TGFβ4-MboII, and TRAIL-StyI were associated with Salmonella Enteritidis burden in the cecum, spleen, and liver of Village Chickens and Red Junglefowl (P < 0.05). These results indicate that the NRAMP1, TGFβ3, TGFβ4, and TRAIL genes are potential candidates for use in selection programs for increasing genetic resistance against Salmonella Enteritidis in native Malaysian chickens.
Key words: candidate gene, Salmonella Enteritidis, genetic resistance, native chicken 2013 Poultry Science 92:900–909 http://dx.doi.org/10.3382/ps.2012-02797
INTRODUCTION
eases, particularly in the case of salmonellosis (Lamont, 1998; Brogden et al., 2003; Reddy et al., 2004). Molecular approaches have provided tools to study the genetic composition of individuals. Using molecular data in selection programs increases the accuracy of selection (Dekkers and Hospital, 2002). This approach offers substantial ways of detecting molecular markers simply by using candidate genes. Single nucleotide polymorphism detection techniques have been used in association studies (Dearlove, 2002). Polymerase chain reaction-RFLP has been used to identify SNP in candidate genes responsible for a variety of physiological functions in poultry (Kramer et al., 2003; Liu and Lamont, 2003; Malek et al., 2004; Ye et al., 2006; Ahmed, 2010). A key step in validating candidate genes is to conduct association studies in multiple populations (Kramer et al., 2003). The natural resistance-associated protein 1 (NRAMP1) gene has been identified as a candidate gene that controls resistance to Salmonella Enteritidis in poultry (Liu et al., 2003). The NRAMP1 is produced in intracellular vesicular membranes; in the
A primary concern in animal husbandry is potential pre- and postslaughter pathogenic contamination that endangers human health and reduces net profit of producers. Salmonella enterica serovar Enteritidis is a major cause of food poisoning in the United States as well as in most European and developing countries (Schroeder et al., 2005; de Jong and Ekdahl, 2006; WHO, 2007). Some cases of antibiotic-resistant strains of Salmonella Enteritidis have been observed, leading to concerns about residual antibiotics in the bodies of treated poultry (Ebel and Schlosser, 2000; White et al., 2001; Johnson et al., 2005). Selection programs focus on increasing immune response and antimicrobial defense to provide improved genetic resistance over dis©2013 Poultry Science Association Inc. Received September 24, 2012. Accepted December 9, 2012. 1 Corresponding author: [email protected]
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ASSOCIATION OF CANDIDATE GENES WITH SALMONELLA
presence of pathogens, it is transferred to the pathogen’s membrane. The NRAMP1 functions by removing iron ions from macrophages, and in this way, it influences the growth of intracellular pathogens (Barton et al., 1999). The NRAMP1 gene polymorphisms were previously associated with immune traits in chickens (Hu et al., 2011). Transforming growth factor β (TGFβ) is a family of regulatory molecules that inhibit the proliferation of lymphocytes. In addition, they have antiinflammatory effects depending on the time and amount of expression. The TGFβ can promote cells to migrate, reside, and develop in different locations (Halder et al., 2005). The association of the TGFβ3 gene with bacterial load in the cecum has been reported (Malek and Lamont, 2003). The mechanism of avian TGFβ4 is similar to mammalian TGFβ1, with 82% amino acid identity to human TGFβ1 (Pan and Halper, 2003). The TGFβ genes serve well as molecular markers in marker-assisted selection programs because these polymorphisms are related to the growth of the spleen (Li et al., 2003). The caspase (CASP) family of proteases is essential for apoptosis and cytokine maturation. This family includes 14 members, of which CASP1 and CASP11 interfere with the proinflammatory cytokine process (Wang and Lenardo, 2000; Fan et al., 2005). During Salmonella infection, the invasion protein B induces macrophage apoptosis by binding and activating CASP1 (Hersh et al., 1999). The CASP1 activation results in the maturation of interleukin (IL) 1β and IL18 cytokines (Wang and Lenardo, 2000). However, new studies found that CASP1 kills intracellular bacteria independent of IL1β and IL18, by using pyroptosis as an efficient mode of microbe elimination (Miao et al., 2010). Therefore, CASP1 has an important role in the ability of the innate immune system to control Salmonella infections. Another protein family, the inhibitor of apoptosis proteins (IAP), inhibits the process of apoptosis, but it is restricted by IAP antagonists. Thus, apoptosis is modulated by these 3 factors: IAP, IAP antagonists, and caspases (Wei et al., 2008). Chicken IAP1 has been mapped to chromosome 1, and its sequence has 85% homology to the human IAP gene (Goodenbour et al., 2004). This gene encodes a 68-kDa protein that binds and inhibits caspase molecules (Roy et al., 1997; You et al., 1997; Deveraux and Reed, 1999). The IAP1-SNP in chickens were associated with Salmonella Enteritidis burden in the spleen and antibody responses in young chicks (Kaiser and Lamont, 2002; Zhou and Lamont, 2003). Tumor necrosis factor α (TNFα) is a cytokine that is excreted by many cells, including B-cells, T-cells, macrophages, natural killer (NK) cells, Kupffer cells, glial cells, and adipocytes in the presence of lipopolysaccharide (LPS) to cause inflammation, cell proliferation, differentiation, and apoptosis (Tracey and Cerami, 1993; Baud and Karin, 2001). Lipopolysaccharide-induced TNFα factor (LITAF) attaches to the TNFα
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promoter region and regulates the expression of TNFα (Stucchi et al., 2006). The exposure of chicken macrophages to Escherichia coli, Salmonella Typhimurium, and Eimeria maxima increased LITAF mRNA expression (Hong et al., 2006). Polymorphisms were observed within the intronic region of chicken LITAF by Malek et al. (2004). The TNF-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death and cytotoxic activity of CD4+ T-cells. The expression of TRAIL is necessary to activate IL2- or IL15-activated NK cells (Kayagaki et al., 1999). In normal cells, TRAIL suppresses the autoimmune response by blocking cell cycle progression (Song et al., 2000). Chicken TRAIL cDNA is 1,134-bp long and shares 54.4% identity with human TRAIL (Abdalla et al., 2004). The TRAIL mRNA is expressed in cells related to both the innate and adaptive immune systems (Falschlehner et al., 2009). A limited number of studies have investigated the association of candidate genes with immune responses in native chickens. Native Malaysian chickens are unique because they have historically been raised in a tropical area with a high probability of spreading Salmonella spp. The objective of this study was to investigate the association between polymorphisms in the NRAMP1, TGFβ3, TGFβ4, CASP1, IAP1, LITAF, and TRAIL genes and resistance to Salmonella Enteritidis in native Malaysian chickens.
MATERIALS AND METHODS Experimental Birds Two hundred 1-d-old Village Chickens and Red Junglefowl chicks (100 per breed) from 3 hatches were used in this study. The Village Chickens were derived from a flock established in 2008 by the Malaysian Agriculture Research and Development Institute. The founder parents had been collected from different villages and allowed to mate randomly. The Red Junglefowl chicks were obtained from a private farm. The parental stock were vaccinated against Newcastle disease and tested weekly to confirm the absence of Salmonella spp. After hatching, the chicks were wing-tagged, chosen randomly, and housed in wire-bottom cages. Swab samples were taken from the cloaca of all chicks to confirm the absence of Salmonella. To screen for the presence of Salmonella Enteritidis, the sterile swab collection from the cecal content was enriched in selenite broth (Oxoid, Basingstoke, UK) for 24 h at 37°C. Each enrichment culture was then plated on XLT4 agar (Oxoid) plates and incubated for 24 h at 37°C. The chicks were given access to water and antibiotic-free commercial starter broiler food ad libitum, and were monitored at least twice daily for mortality during the experiment. All procedures were undertaken according to the guidelines of the Institutional Animal Care and Use Committee.
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Table 1. Primer sets for PCR-RFLP assay of 7 candidate genes1
Gene
Accession number
NRAMP1
AY072001
TGFβ3
X60091
TGFβ4
AF459837
LITAF
AI979890
Caspase1
AF031351
IAP1
AF221083
TRAIL
AF537189
PCR product (bp)
Primer sequence F: 5′-GGCGTCATCCTGGGCTGCTAT-3′ R: 5′-AGACCGTTGGCGAAGTCATGC-3′ F: 5′-CGGCCTGGAAATCAGCATAC-3′ R: 5′-GAAGCAGTAGTTGGTATCCAG-3′ F: 5′-GGGGTCTTCAAGCTGAGCGT-3′ R: 5′-TTGGCAATGCTCTGCATGTC-3′ F: 5′-TGAGTTGCCCTTCCTGT-3′ R: 5′-CAGAGCATCAACGCAAA-3′ F: 5′-CCATGCTTGGGCTCTCAGTG-3′ R: 5′-GGTCCCGCAGATCCCAGTG-3′ F: 5′-TCACCATCTCTACGTTCCAT-3′ R: 5′-CATTGAAACTTGGTTGGTCT-3′ F: 5′-GTAAATTAGAGCCTCATCA-3′ R: 5′-CACCTCAGTTCCTCCGA-3′
Annealing temperature (°C)/time
Reference
801
64 to 60°C/45 s
Liu et al., 2003
1,078
64 to 54°C/45 s
Malek and Lamont, 2003
240
64 to 54°C/45 s
Kramer et al., 2003
497
54°C/30 s
Malek et al., 2004
1,070
64 to 54°C/45 s
Liu and Lamont, 2003
394
64 to 54°C/45 s
Liu and Lamont, 2003
786
59 to 51°C/45 s
Malek and Lamont, 2003
1NRAMP1 = natural resistance-associated protein 1; TGF = transforming growth factor; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; IAP1 = inhibitor of apoptosis protein 1; TRAIL = TNF-related apoptosis-inducing ligand. F = forward; R = reverse.
Salmonella Challenge and Examination One day after hatching, the chicks were intraesophageally inoculated with 0.1 mL of Luria-Bertani broth containing 1 × 107 cfu/bird of Salmonella Enteritidis phage type 13a (nalidixic acid-resistant). The concentration of bacteria was confirmed by serial plate dilution of the inoculum. All chicks were killed on d 7 postinoculation by cervical dislocation, and the organs (cecal content, spleen, and liver) were removed and homogenized. One gram of each sample was diluted in 10 mL of buffered peptone water (Oxoid), followed by homogenization using a Heidolph Diax 600 homogenizer (Heidolph, Schwabach, Germany). The homogenate (100 μL) was plated in duplicate on XLT4 agar containing 100 μg/mL of nalidixic acid (Sigma, St. Louis, MO) and incubated at 37°C for 24 h. An agglutination test using Salmonella polyvalent agglutinating sera (Remel Europe Ltd., Dartford, UK) was used to confirm the presence of Salmonella colonies. For agglutination tests, 20 μL of antiserum was added to a glass slide and mixed with Salmonella Enteritidis culture. A positive reaction was seen as visible agglutination. The number of Salmonella Enteritidis colonies was counted using a Galaxy 230 colony counter (Newstar Environmental Co., Roswell, GA).
DNA Isolation and PCR-RFLP Genomic DNA was extracted from blood collected in EDTA or from the breast muscle of dead chickens using a DNeasy mini kit (Qiagen, Hilden, Germany). The primer sets used to amplify the fragments of the NRAMP1, TGFβ3, TGFβ4, CASP1, IAP1, LITAF, and TRAIL genes (Table 1) have been previously described (Liu et al., 2003; Malek and Lamont, 2003; Kramer et al., 2003; Liu and Lamont, 2003; Malek et al., 2004). The PCR was performed in a 25-μL master mix containing 50 to 100 ng of chicken genomic DNA, 0.1 µM primer (each), 200 µM dNTP, 1.5 units of Taq DNA
polymerase (Promega, Madison, WI), 5 µL of 5 × PCR buffer, and 1.5 mM MgCl2. The reaction conditions for the amplification of LITAF fragments were similar to those described previously (Malek et al., 2004). A touch-down program was used to amplify TGFβ3, TGFβ4, CASP1, and IAP1 with an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 45 s, annealing at 64°C to 54°C for 45 s, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. A similar touch-down program was used to amplify NRAMP1 and TRAIL fragments with annealing temperatures 64 to 60°C and 59 to 51°C, respectively. Polymorphisms of candidate genes were screened using restriction enzymes (New England BioLabs Inc., Ipswich, MA). The PCR product of the TGFβ3 gene was digested with BsrI (10 U) at 65°C overnight. The SacI (10 U), MboII (1.5 U), BglI (10 U), Hsp92II (5 U), HinfI (7 U), and StyI (5 U) were used to digest the PCR products of NRAMP1, TGFβ4, IAP1, CASP1, LITAF, and TRAIL genes, respectively, at 37°C overnight. The digested products were separated on 2% agarose gel with ethidium bromide for 2 h at 80 V and visualized under UV light.
Statistical Analysis Population genetics measurements, including allelic and genotypic frequencies and deviation from HardyWeinberg equilibrium (HWE), were calculated using PopGene software V. 1.31 (Yeh et al., 1999). The Student’s t-test was used to compare the overall mean bacterial load in each organ between the 2 breeds. Two GLM were developed to analyze the association between genotypes and Salmonella Enteritidis load in the cecum, spleen, and liver using the SAS program V. 9.1 (SAS Institute Inc., 2004). The first model (model 1) included only one gene, whereas the second model (model 2) included 2 genes based on their physiological
ASSOCIATION OF CANDIDATE GENES WITH SALMONELLA
relationship to estimate the interaction between different genotypes: model 1: Yijkl = μ + genotypei + sexj + hk + gene × sexij + eijkl, where Yijkl is the bacterial count (cfu/mL) on the lth individual of the ith genotype from the jth sex and in the kth hatchery (h), and eijkl is the random environmental residual effects. The μ is the overall mean, gene and sex are fixed effects, and h is random effect. The Salmonella Enteritidis counts were transformed to their natural logarithms to improve the normality of the data. In model 2, the genes included TGFβ3/TGFβ4, IAP1/TRAIL, and IAP1/CASP1. Model 2: Yijklm = μ + genei + genej + sexk + hl + genei × genej + eijklm. The proportion of phenotypic variation that was explained by genotypic variation for each gene was calculated as below (Kramer et al., 2003): phenotypic variation (%) = 100 × (squared correlation of model 1 − squared correlation of model 3), where model 3: Yijk = μ + sexi + hj + eijk. The phenotypic variation was calculated when the effect of the gene was significant.
RESULTS Allelic Diversity The results of sequence analysis indicated the presence of SNP in native Malaysian chickens. All SNP characterized by PCR-RFLP assays were compatible with previous reports (Kramer et al., 2003; Liu et al., 2003; Liu and Lamont, 2003; Malek and Lamont, 2003; Malek et al., 2004). However, the genotype T/T was not observed for CASP1 in the Village Chickens and Red Junglefowl following the digestion of 1,070bp PCR products by Hsp92II restriction endonuclease (Figure 1). For LITAF, sequencing of the PCR products showed an A/T SNP in a HinfI site located within an intronic region in Village Chickens. The digested products include fragment sizes of 450 and 48 bp for one allele (T), and 393, 57, and 48 bp for the other allele (A). The Red Junglefowl was homozygous T/T for this locus. The results of PCR-RFLP assay showed that generally all candidate genes had no deviation from HardyWeinberg equilibrium in both breeds (Table 2). For Village Chickens, the minimum and maximum allelic frequency differences belonged to TGFβ3 and CASP1, respectively. The minimum allelic differences for Red
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Junglefowl belonged to TGFβ4 and the maximum was for the CASP1 loci. The observed heterozygosity for most loci was moderate to high in both Village Chickens and Red Junglefowl chickens.
Association of Candidate Genes with Salmonella Enteritidis Response All birds in the control group were free of Salmonella Enteritidis at the end of the experiment. Twenty percent of the infected Red Junglefowl died over the course of the experiment. Chickens that died were not included in the association study. The overall mean Salmonella Enteritidis burden in the spleen was significantly higher in Red Junglefowl than in Village Chickens (P < 0.01). However, no difference in Salmonella Enteritidis burden was seen for the cecum and liver (Table 3). Significant effects of the genotype on the Salmonella Enteritidis load were observed for NRAMP1, TGFβ3, TGFβ4, and TRAIL (Tables 4, 5, and 6). The C/C genotype of NRAMP1 was correlated with a higher Salmonella Enteritidis burden in the cecum and spleen of Village Chickens (P < 0.01). The effect of genotypic variation on phenotypic variation was 35 and 13% for the cecum and spleen Salmonella Enteritidis burden, respectively. The C/T mutation in the NRAMP1-exonic region did not significantly affect the response to Salmonella Enteritidis in the liver. For TGFβ3, the heterozygote A/C had the highest Salmonella Enteritidis load in the cecum, spleen, and liver compared with the other 2 genotypes (P < 0.01). The proportion of phenotypic variation that is demonstrated by genotypic variation was between 10 and 30% for the 2 breeds. A polymorphism in the exonic region of TGFβ4 was associated with Salmonella Enteritidis burden in the spleen and liver of Red Junglefowl; the C/C genotype indicated the highest Salmonella Enteritidis load (P < 0.05). The observed SNP explained 14 and 11% of the phenotypic variation for spleen and liver bacterial burden, respectively. For TRAIL, a silent mutation at position 82 bp of this gene was associated with the cecum, spleen, and liver bacterial load in Village Chickens and Red Junglefowl. The A/A genotype exhibited the lowest bacterial load in both breeds. The A/G SNP explained 8 to 13% of phenotypic variation for both breeds. The other candidate genes did not show any significant effect on the response to Salmonella Enteritidis challenge. Several interactions between genes in this study were analyzed; however, only the interaction between IAP1 and TRAIL in Village Chickens with Salmonella Enteritidis burden in spleen was significant (P < 0.05).
DISCUSSION In this study, the impact of polymorphisms in 7 candidate genes on Salmonella Enteritidis colonization in the cecum, spleen, and liver of native Malaysian chickens was investigated. The identified SNP were found
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Figure 1. Polymerase chain reaction-RFLP pattern for 7 candidate genes including (a) transforming growth factor (TGF) β3, (b) TGFβ4, (c) lipopolysaccharide-induced tumor necrosis factor (TNF) α factor (LITAF), (d) caspase 1 (CASP1), (e) inhibitor of apoptosis protein 1 (IAP1), and (f) TNF-related apoptosis-inducing ligand (TRAIL).
in both intronic and exonic regions, similar to those previously reported (Kramer et al., 2003; Liu et al., 2003; Liu and Lamont, 2003; Malek and Lamont, 2003; Malek et al., 2004). The level of heterozygosity for most of the candidate genes assayed in this study was high, probably a result of random mating. Nevertheless, an indication of genetic drift was observed for some genes, particularly in the Red Junglefowl. The natural habitat of this breed is tropical jungle with limited populations. The similarity of SNP, both in composition and position, between the 2 breeds is likely the result of the Village Chickens having descended from Red Junglefowl (Crawford, 1990). Significant associations between some of the candidate gene polymorphisms and the Salmonella Enteritidis load were observed. All the candidate genes surveyed in the current study were chosen because of their role in the immune system. The
chickens in this study were not inbred and were selected randomly. We found the NRAMP1 polymorphism showed a significant association with Salmonella Enteritidis burden in the cecum; this result was in agreement with the reports by Kramer et al. (2003). In this study, the homozygous C/C genotype was related to the highest Salmonella Enteritidis load; however, there is inconsistency among the results of different studies. Liu et al. (2003) described that the allele C had a lower Salmonella Enteritidis load in chicken spleen than did allele T. Liu et al. (2003) did not observe a significant effect of the C/T SNP on cecum Salmonella Enteritidis burden. Different lines or breeds may manifest different reactions to infections. The NRAMP1 has an important role in innate immunity, and the cecum is the first line of defense against colonization by Salmonella (Gruenheid et al., 1997; Berndt et al., 2007).
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Table 2. The genotypic frequencies and test for deviation from Hardy-Weinberg equilibrium (HWE) for Village Chickens and Red Junglefowl1 Observed genotypic frequency2 Gene
Chicken type
CASP1 IAP1 LITAF NRAMP1 TGFβ3 TGFβ4 TRAIL
Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl
Expected genotypic frequency
11
12
22
0.87 0.85 0.10 0.26 0.01 0.00 0.55 0.61 0.23 0.39 0.13 0.21 0.39 0.07
0.13 0.15 0.40 0.49 0.18 0.00 0.36 0.37 0.47 0.43 0.45 0.57 0.40 0.54
0.00 0.00 0.50 0.25 0.81 1.00 0.09 0.02 0.30 0.18 0.42 0.22 0.21 0.39
11
12
22
0.87 0.86 0.09 0.26 0.01 0.00 0.54 0.64 0.21 0.37 0.12 0.25 0.35 0.11
0.12 0.14 0.42 0.50 0.19 0.00 0.39 0.32 0.50 0.48 0.46 0.50 0.49 0.45
0.01 0.00 0.49 0.24 0.80 1.00 0.07 0.04 0.29 0.15 0.42 0.25 0.16 0.44
P-value for HWE test
0.53 0.56 0.57 0.85 0.78 0.00 0.49 0.41 0.56 0.37 0.85 0.28 0.08 0.11
1CASP1 = caspase 1; IAP1 = inhibitor of apoptosis protein 1; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; NRAMP1 = natural resistance-associated protein 1; TGF = transforming growth factor; TRAIL = TNF-related apoptosis-inducing ligand. 2The 1 for LITAF, TRAIL, TGFβ3, TGFβ4, interleukin (IL)2, interferon (IFN)γ, and IAP1 is equal to A and for NRAMP1, TLR4, TGFβ2, IgL, and CASP1 is equal to C. The 2 for TGFβ3 and TGFβ4 is equal to C, for TLR4, TRAIL, IL2, IFNγ, and IAP is equal to G, and for NRAMP1, LITAF, TGFβ2, IgL, and CASP1 is equal to T.
Therefore, the role of NRAMP1 in the cecal immune system is critical; NRAMP1 can accelerate the inflammatory response during Salmonella invasion (Valdez et al., 2009). The SNP identified in the current study was silent and may be linked to another gene sequence nearby. We also observed significant association of TGFβ3 and TGFβ4 polymorphisms with Salmonella Enteritidis colonization in this study. A neutral mutation in the TGFβ3 intron region was associated with bacterial colonization. This neutral mutation may be linked to a functional sequence in the genome (Rothschild and Soller, 1997). However, there was a conflict between the results of the 2 breeds. In the cecum, the C/C genotype of TGFβ3 showed the lowest bacterial burden for Village Chickens, whereas in Red Junglefowl, the A/A genotype exhibited the lowest Salmonella Enteritidis colonization. The results of the current study were consistent with those found by Kramer et al. (2003). The association between the TGFβ3 polymorphism and the spleen Salmonella Enteritidis load was not significant in the study by Malek and Lamont (2003), but it was significant for the cecum Salmonella Enteritidis load. A significant association between the TGFβ3-BsrI polymorphism and mortality between 14 and 42 d in broiler chickens was reported by Ye et al. (2006). For TGFβ4, no association was found between different genotypes and the cecum Salmonella Enteritidis load. However,
the bacterial burden in the spleen and liver was influenced by TGFβ4 genotypes. The TGFβ4 SNP changed the amino acid sequence, and that might affect protein function. It was demonstrated that the difference between different genetic lines in response to Salmonella infection in young chickens can be attributed to the developmental differences of the gut (Schokker et al., 2012). This may contribute to the inconsistency in the results of different studies or different lines of same study. Moreover, it has been documented that TGFβ gene polymorphisms are associated with antibody production in chickens (Zhou et al., 2003). Therefore, the observed association between TGFβ gene SNP in this study with Salmonella Enteritidis burden may be because of the effect of those SNP on antibody production. The results of this study introduce the TGFβ genes as potential candidate genes for future selection programs. For IAP1, the association between the G/A SNP and bacterial load in the cecum, spleen, and liver was not significant in the current study. The chicken IAP1 gene is expressed at a relatively high level in the intestine, spleen, thymus, bursa, and lungs (You et al., 1997). Here, the G/A SNP in the exonic region of the IAP1 gene was silent and had no effect on response to Salmonella Enteritidis. Previous studies showed Salmonella-infected macrophages are killed by a CASP1-dependent necrosis
Table 3. The overall mean ± SEM of Salmonella Enteritidis load (natural log cfu) in each organ for the 2 breeds Breed Village Junglefowl a,bCommon
Cecum 11.43 ± 11.24 ±
0.16a 0.08a
Spleen 8.82 ± 9.41 ±
0.09a 0.08b
superscripts in each column show nonsignificant differences.
Liver 8.77 ± 0.07a 8.90 ± 0.10a
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Table 4. Effect of 7 candidate genes on Salmonella Enteritidis load in the cecum of Village Chickens and Red Junglefowl1 Genotype2 (least squares means ± SE) Gene
Chicken type
NRAMP1 TGFβ3 TGFβ4 LITAF CASP1 IAP1 TRAIL
Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl
11 12.21 11.58 11.79 10.99 10.74 11.43 11.64 11.27 11.20 10.61 11.57 10.60 11.10
12
0.19a (18) 0.15a (23) 0.30a (7) 0.15b (15) 0.34a (9) 0.22a (9) Ua (2) — ± 0.23a (28) ± 0.10a (39) ± 0.40a (6) ± 0.20a (12) ± 0.47a (8) ± 0.15a (19)
± ± ± ± ± ± ±
11.37 10.91 11.89 11.66 11.39 11.11 10.55
± ± ± ± ± ± ±
10.94 11.11 11.37 11.11 11.28 11.22
± ± ± ± ± ±
0.16b (16) 0.21a (15) 0.23b (17) 0.14a (25) 0.32a (13) 0.13a (27) 0.62a (7) — 0.44a (6) 0.24a (6) 0.19a (17) 0.12a (21) 0.29ab (13) 0.13a (23)
Phenotypic variation (%)
22 10.76 ± 0.13b (4) — 11.04 ± 0.23b (12) 11.21 ± 0.29b (13) 11.63 ± 0.29a (15) 11.17 ± 0.21a (9) 11.27 ± 0.26a (27) — — — 11.28 ± 0.20a (14) 10.99 ± 0.17a (12) 11.85 ± 0.37b (14) 11.99 ± 0.20b (3)
35 — 25 30 — — — — — — — — 13 13
P-value 0.002 0.39 0.02 0.007 0.13 0.46 0.46 0.43 0.69 0.20 0.09 0.04 0.05
a,bCommon
superscripts in each row show nonsignificant differences (P > 0.05). = natural resistance-associated protein 1; TGF = transforming growth factor; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; CASP1 = caspase 1; IAP1 = inhibitor of apoptosis protein 1; TRAIL = TNF-related apoptosis-inducing ligand. The number of samples is shown in parentheses. For phenotypic variation, — means noncalculated. — shows unavailable data. U = unestimated. 2The 1 for LITAF, TRAIL, TGFβ3, TGFβ4, interleukin (IL)2, interferon (IFN)γ, and IAP1 is equal to A and for NRAMP1, TLR4, TGFβ2, IgL, and CASP1 is equal to C. The 2 for TGFβ3 and TGFβ4 is equal to C, for TLR4, TRAIL, IL2, IFNγ, and IAP1 is equal to G, and for NRAMP1, LITAF, TGFβ2, IgL, and CASP1 is equal to T. 1NRAMP1
mechanism (Brennan and Cookson, 2000). However, the statistical association between C/T SNP at position −368-bp flanking region of CASP1 gene and Salmonella Enteritidis burden was not able to be determined due to the absence of one genotype. For the common SNP, significant association with Salmonella Enteritidis loads in the cecum and spleen and with antibody levels was reported by Liu and Lamont (2003). In the current study, LITAF gene polymorphism did not show significant association with Salmonella Enteritidis load. The A/T substitution was identified in an intronic region. Therefore, it did not influence protein structure, and it might also not be linked to other func-
tional sequence. However, Red Junglefowl was homozygous T/T for the LITAF gene similar to a report by Malek et al. (2004). The TRAIL gene polymorphism was associated with Salmonella Enteritidis burden in the cecum, spleen, and liver. In other studies (Malek and Lamont, 2003; Ye et al., 2006), TRAIL gene polymorphism was associated with the cecum Salmonella Enteritidis burden and chicken early mortality (before 14 d of age). The A/G substitution in the exonic region did not change the amino acid sequence. Therefore, the results of the present study should be interpreted conservatively until this SNP can be analyzed in several populations.
Table 5. Effect of 7 candidate genes on Salmonella Enteritidis load in the spleen of Village Chickens and Red Junglefowl1 Genotype2 (least squares means ± SE) Gene
Chicken type
NRAMP1 TGFβ3 TGFβ4 LITAF CASP1 IAP1 TRAIL
Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl
a,bCommon
11 8.90 9.12 8.45 8.97 8.69 9.04 8.73
± ± ± ± ± ± ±
8.45 9.27 8.44 9.71 8.23 8.46
± ± ± ± ± ±
0.11a (45) 0.13a (26) 0.14ab (19) 0.15a (16) 0.19a (10) 0.34a (8) Ua (1) — 0.07a (70) 0.11a (42) 0.17a (10) 0.22a (13) 0.08a (29) 0.35a (4)
12 8.39 9.52 8.78 9.55 8.43 9.14 8.47
± ± ± ± ± ± ±
8.56 9.22 8.50 9.10 8.57 9.26
± ± ± ± ± ±
0.12b (29) 0.18a (17) 0.9a (40) 0.15b (22) 0.09a (34) 0.14a (28) 0.19a (13) — 0.15a (12) 0.28a (6) 0.09a (35) 0.14a (21) 0.08b (28) 0.14b (24)
22 8.58 ± 0.27ab (6) — 8.41 ± 0.13b (23) 9.43 ± 0.31ab (9) 8.54 ± 0.10a (32) 9.75 ± 0.20b (12) 8.50 ± 0.08a (56) — — — 8.50 ± 0.08a (49) 9.46 ± 0.17a (14) 8.43 ± 0.10ab (17) 9.39 ± 0.15b (20)
Phenotypic variation (%)
P-value
13 — 20 22 — 14 — — — — — — 18 17
0.01 0.06 0.03 0.02 0.40 0.04 0.81 — 0.52 0.88 0.91 0.06 0.02 0.05
superscripts in each row show nonsignificant differences (P > 0.05). = natural resistance-associated protein 1; TGF = transforming growth factor; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; CASP1 = caspase 1; IAP1 = inhibitor of apoptosis protein 1; TRAIL = TNF-related apoptosis-inducing ligand. The number of samples is shown in parentheses. For phenotypic variation, — means noncalculated. — shows unavailable data. U = unestimated. 2The 1 for LITAF, TRAIL, TGFβ3, TGFβ4, interleukin (IL)2, interferon (IFN)γ, and IAP1 is equal to A and for NRAMP1, TLR4, TGFβ2, IgL, and CASP1 is equal to C. The 2 for TGFβ3 and TGFβ4 is equal to C, for TLR4, TRAIL, IL2, IFNγ, and IAP1 is equal to G, and for NRAMP1, LITAF, TGFβ2, IgL, and CASP1 is equal to T. 1NRAMP1
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ASSOCIATION OF CANDIDATE GENES WITH SALMONELLA Table 6. Effect of 7 candidate genes on Salmonella Enteritidis load in the liver of Village Chickens and Red
Junglefowl1
Genotype2 (least squares means ± SE) Gene
Chicken type
NRAMP1 TGFβ3 TGFβ4 LITAF CASP1 IAP1 TRAIL
Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl
11 8.42 8.64 8.83 8.61 8.57 8.51 9.16
± ± ± ± ± ± ±
8.69 8.65 8.62 8.53 8.79 8.32
± ± ± ± ± ±
0.11a (45) 0.11a (23) 0.15ab (18) 0.13a (16) 0.24a (10) 0.20a (8) Ua (1) — 0.09a (63) 0.09a (38) 0.25a (7) 0.19a (11) 0.12a (30) 0.11a (3)
12 8.78 8.56 8.88 8.65 8.73 8.53 8.93
± ± ± ± ± ± ±
9.24 8.91 8.97 8.58 8.56 8.86
± ± ± ± ± ±
0.12a (29) 0.16a (17) 0.9a (38) 0.12a (21) 0.13a (32) 0.12a (26) 0.20a (13) — 0.26a (7) 0.27a (6) 0.12a (34) 0.12a (21) 0.15a (27) 0.12b (21)
22 8.80 8.16 8.48 8.81 8.81 9.11 8.85
± ± ± ± ± ± ±
8.68 8.78 8.73 8.68
± ± ± ±
0.27a (6) Ua (1) 0.13b (23) 0.20a (8) 0.13a (28) 0.16b (11) 0.10a (48) — — — 0.10a (48) 0.14a (13) 0.19a (15) 0.10ab (18)
Phenotypic variation (%)
P-value
— — 10 — — 11 — — — — — — — 8
0.85 0.68 0.03 0.85 0.66 0.01 0.85 — 0.25 0.76 0.10 0.45 0.43 0.02
a,bCommon
superscripts in each row show nonsignificant differences (P > 0.05). = natural resistance-associated protein 1; TGF = transforming growth factor; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; CASP1 = caspase 1; IAP1 = inhibitor of apoptosis protein 1; TRAIL = TNF-related apoptosis-inducing ligand. The number of samples is shown in parentheses. For phenotypic variation, — means noncalculated. — shows unavailable data. U = unestimated. 2The 1 for LITAF, TRAIL, TGFβ3, TGFβ4, interleukin (IL)2, interferon (IFN)γ, and IAP1 is equal to A and for NRAMP1, TLR4, TGFβ2, IgL, and CASP1 is equal to C. The 2 for TGFβ3 and TGFβ4 is equal to C, for TLR4, TRAIL, IL2, IFNγ, and IAP1 is equal to G, and for NRAMP1, LITAF, TGFβ2, IgL, and CASP1 is equal to T. 1NRAMP1
Several interactions between genes in this study were analyzed; however, only the interaction between IAP1 and TRAIL was significant. Interaction of genetic factors always exists in living organisms. Such epistatic interactions can exist among genes that are involved in similar functions (Ye et al., 2006). The TRAIL and IAP1 genes belong to the cell apoptotic system. However, understanding the cause of interaction needs analysis of gene expression and translation. The breed had a significant effect on the Salmonella Enteritidis load in the spleen. The bacterial counts for Red Junglefowl chickens were higher than those for Village Chickens. This result was expected because Red Junglefowl chickens live in a limited area and are in contact with few pathogens. Therefore, they are evolutionarily more susceptible to diseases. Moreover, inbreeding depression in limited populations is more prominent for viability traits (Falconer and MacKay, 1996). At the same time, the presence of gut microflora may interact with the genetic background of the animal to eliminate pathogens (Esworthy et al., 2010), and the systemic organs (spleen and liver) are free of natural microflora. Estimation of phenotypic variation revealed that distribution of genotypic variation through phenotypic variation was between 9 and 35%. It demonstrated that the effect of the observed SNP for some candidate genes on phenotypic variation was small and for others was moderate. The trait used as a resistance measurement was Salmonella Enteritidis load. It appeared that each gene had a small effect on resistance to Salmonella Enteritidis. Resistance to disease is a polygenic trait (Lamont, 2008); therefore, the total effect of all genes is important. Moreover, there are several other factors, such as invasion of Salmonella, the activity of immune system, and the age of the animal that influence resis-
tance to diseases (Kramer et al., 2003). Thus, the effect of one gene may not explain the whole phenotypic variation. The estimated heritability for resistance to Salmonella in published articles ranges from less than 0.05 to 0.32 (Berthelot et al., 1998; Beaumont et al., 1999; Girard Santosuosso et al., 2002). Thus, the calculated genotypic variation in this study is consistent with those estimates of heritability. All of the investigated genes are important in immune responses and most of them were associated with Salmonella Enteritidis load in at least one organ. The cecum as a first barrier against Salmonella Enteritidis colonization has an important role in resistance to Salmonella. Although the spleen is an immunological organ, the immune response of different organs to pathogens does not appear at the same time. Therefore, if the period of the experiment changes, the immune response of the infected organs may change. In conclusion, the main objectives of this study were to analyze the polymorphism status of immune candidate genes and their association with Salmonella Enteritidis resistance in 2 breeds of native Malaysian chickens. The results demonstrated that improvement of genetic resistance against Salmonella Enteritidis through marker-assisted selection is feasible. The candidate genes NRAMP1, TGFβ3, TGFβ4, and TRAIL contained a considerable proportion of phenotypic variations related to Salmonella Enteritidis resistance in the Village Chicken and Red Junglefowl populations. These results suggest several candidate genes that are involved in the mechanism of resistance to Salmonella Enteritidis and extend the knowledge of genetic resistance to diseases. However, practical implementation of these candidate genes will require further study of their roles in Salmonella Enteritidis resistance. The results of different studies show inconsistencies that can
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be attributed to the use of different strains of bacteria, populations, and environments. Thus, the result of any association study is applicable for that particular population under study. Future genomics studies utilizing the high-throughput technologies of high-density SNP genotyping, microarray technology, next-generation sequencing, and haplotype analysis will provide a better understanding of the mechanisms of immune gene responses to diseases in native Malaysian chickens.
ACKNOWLEDGMENTS This study was supported financially and technically by RUGS grant 01-01-10-0911RU from Universiti Putra Malaysia.
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ASSOCIATION OF CANDIDATE GENES WITH SALMONELLA Malek, M., and S. J. Lamont. 2003. Association of INOS, TRAIL, TGF-β2, TGF-β3, and IgL genes with response to Salmonella enteritidis in poultry. Genet. Sel. Evol. 35:S99–S111. Miao, E. A., I. A. Leaf, P. M. Treuting, D. P. Mao, M. Dors, A. Sarkar, S. E. Warren, M. D. Wewers, and A. Aderem. 2010. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria. Nat. Immunol. 11:1136– 1142. Pan, H., and J. Halper. 2003. Cloning, expression, and characterization of chicken transforming growth factor β4. Biochem. Biophys. Res. Commun. 303:24–30. Reddy, K. V., R. D. Yedery, and C. Aranha. 2004. Antimicrobial peptides: Premises and promises. Int. J. Antimicrob. Agents 24:536–547. Rothschild, M. F., and M. Soller. 1997. Candidate gene analysis to detect genes controlling traits of economic importance in domestic livestock. Probe 8:13–20. Roy, N., Q. L. Deveraux, R. Takahashi, G. S. Salvesen, and J. C. Reed. 1997. The c-IAP-1 and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J. 16:6914–6925. SAS Institute Inc. 2004. SAS/STAT 9.1 User’s Guide. SAS Institute, Cary, NC. Schokker, D., T. H. F. Peters, A. J. W. Hoekman, J. M. J. Rebel, and M. A. Smits. 2012. Differences in the early response of hatchlings of different chicken breeding lines to Salmonella enterica serovar Enteritidis infection. Poult. Sci. 91:346–353. Schroeder, C. M., A. L. Naugle, W. D. Schlosser, A. T. Hogue, F. J. Angulo, J. S. Rose, E. D. Ebel, W. T. Disney, K. G. Holt, and D. P. Goldman. 2005. Estimate of illnesses from Salmonella enteritidis in eggs, United States, 2000. Emerg. Infect. Dis. 11:113–115. Song, K., Y. Chen, R. D. Göke, A. Wilmen, C. Seidel, A. Göke, B. Hilliard, and Y. Chen. 2000. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J. Exp. Med. 191:1095–1104. Stucchi, A., K. Reed, M. O’Brien, S. Cerda, C. Andrews, A. Gower, K. Bushell, S. Amar, S. Leeman, and J. Becker. 2006. A new transcription factor that regulates TNF-α gene expression, LIT-
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polysaccharide-induced tumor necrosis factor (TNF) α factor (LITAF), and TNF-related apoptosis-inducing ligand (TRAIL). Polymerase chain reaction-RFLP was used to identify polymorphisms in the candidate genes; all genes exhibited polymorphisms in at least one breed. The NRAMP1-SacI polymorphism correlated with the differences in Salmonella Enteritidis load in the cecum (P = 0.002) and spleen (P = 0.01) of Village Chickens. Polymorphisms in the restriction sites of TGFβ3-BsrI, TGFβ4-MboII, and TRAIL-StyI were associated with Salmonella Enteritidis burden in the cecum, spleen, and liver of Village Chickens and Red Junglefowl (P < 0.05). These results indicate that the NRAMP1, TGFβ3, TGFβ4, and TRAIL genes are potential candidates for use in selection programs for increasing genetic resistance against Salmonella Enteritidis in native Malaysian chickens.
Key words: candidate gene, Salmonella Enteritidis, genetic resistance, native chicken 2013 Poultry Science 92:900–909 http://dx.doi.org/10.3382/ps.2012-02797
INTRODUCTION
eases, particularly in the case of salmonellosis (Lamont, 1998; Brogden et al., 2003; Reddy et al., 2004). Molecular approaches have provided tools to study the genetic composition of individuals. Using molecular data in selection programs increases the accuracy of selection (Dekkers and Hospital, 2002). This approach offers substantial ways of detecting molecular markers simply by using candidate genes. Single nucleotide polymorphism detection techniques have been used in association studies (Dearlove, 2002). Polymerase chain reaction-RFLP has been used to identify SNP in candidate genes responsible for a variety of physiological functions in poultry (Kramer et al., 2003; Liu and Lamont, 2003; Malek et al., 2004; Ye et al., 2006; Ahmed, 2010). A key step in validating candidate genes is to conduct association studies in multiple populations (Kramer et al., 2003). The natural resistance-associated protein 1 (NRAMP1) gene has been identified as a candidate gene that controls resistance to Salmonella Enteritidis in poultry (Liu et al., 2003). The NRAMP1 is produced in intracellular vesicular membranes; in the
A primary concern in animal husbandry is potential pre- and postslaughter pathogenic contamination that endangers human health and reduces net profit of producers. Salmonella enterica serovar Enteritidis is a major cause of food poisoning in the United States as well as in most European and developing countries (Schroeder et al., 2005; de Jong and Ekdahl, 2006; WHO, 2007). Some cases of antibiotic-resistant strains of Salmonella Enteritidis have been observed, leading to concerns about residual antibiotics in the bodies of treated poultry (Ebel and Schlosser, 2000; White et al., 2001; Johnson et al., 2005). Selection programs focus on increasing immune response and antimicrobial defense to provide improved genetic resistance over dis©2013 Poultry Science Association Inc. Received September 24, 2012. Accepted December 9, 2012. 1 Corresponding author: [email protected]
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presence of pathogens, it is transferred to the pathogen’s membrane. The NRAMP1 functions by removing iron ions from macrophages, and in this way, it influences the growth of intracellular pathogens (Barton et al., 1999). The NRAMP1 gene polymorphisms were previously associated with immune traits in chickens (Hu et al., 2011). Transforming growth factor β (TGFβ) is a family of regulatory molecules that inhibit the proliferation of lymphocytes. In addition, they have antiinflammatory effects depending on the time and amount of expression. The TGFβ can promote cells to migrate, reside, and develop in different locations (Halder et al., 2005). The association of the TGFβ3 gene with bacterial load in the cecum has been reported (Malek and Lamont, 2003). The mechanism of avian TGFβ4 is similar to mammalian TGFβ1, with 82% amino acid identity to human TGFβ1 (Pan and Halper, 2003). The TGFβ genes serve well as molecular markers in marker-assisted selection programs because these polymorphisms are related to the growth of the spleen (Li et al., 2003). The caspase (CASP) family of proteases is essential for apoptosis and cytokine maturation. This family includes 14 members, of which CASP1 and CASP11 interfere with the proinflammatory cytokine process (Wang and Lenardo, 2000; Fan et al., 2005). During Salmonella infection, the invasion protein B induces macrophage apoptosis by binding and activating CASP1 (Hersh et al., 1999). The CASP1 activation results in the maturation of interleukin (IL) 1β and IL18 cytokines (Wang and Lenardo, 2000). However, new studies found that CASP1 kills intracellular bacteria independent of IL1β and IL18, by using pyroptosis as an efficient mode of microbe elimination (Miao et al., 2010). Therefore, CASP1 has an important role in the ability of the innate immune system to control Salmonella infections. Another protein family, the inhibitor of apoptosis proteins (IAP), inhibits the process of apoptosis, but it is restricted by IAP antagonists. Thus, apoptosis is modulated by these 3 factors: IAP, IAP antagonists, and caspases (Wei et al., 2008). Chicken IAP1 has been mapped to chromosome 1, and its sequence has 85% homology to the human IAP gene (Goodenbour et al., 2004). This gene encodes a 68-kDa protein that binds and inhibits caspase molecules (Roy et al., 1997; You et al., 1997; Deveraux and Reed, 1999). The IAP1-SNP in chickens were associated with Salmonella Enteritidis burden in the spleen and antibody responses in young chicks (Kaiser and Lamont, 2002; Zhou and Lamont, 2003). Tumor necrosis factor α (TNFα) is a cytokine that is excreted by many cells, including B-cells, T-cells, macrophages, natural killer (NK) cells, Kupffer cells, glial cells, and adipocytes in the presence of lipopolysaccharide (LPS) to cause inflammation, cell proliferation, differentiation, and apoptosis (Tracey and Cerami, 1993; Baud and Karin, 2001). Lipopolysaccharide-induced TNFα factor (LITAF) attaches to the TNFα
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promoter region and regulates the expression of TNFα (Stucchi et al., 2006). The exposure of chicken macrophages to Escherichia coli, Salmonella Typhimurium, and Eimeria maxima increased LITAF mRNA expression (Hong et al., 2006). Polymorphisms were observed within the intronic region of chicken LITAF by Malek et al. (2004). The TNF-related apoptosis-inducing ligand (TRAIL) induces apoptotic cell death and cytotoxic activity of CD4+ T-cells. The expression of TRAIL is necessary to activate IL2- or IL15-activated NK cells (Kayagaki et al., 1999). In normal cells, TRAIL suppresses the autoimmune response by blocking cell cycle progression (Song et al., 2000). Chicken TRAIL cDNA is 1,134-bp long and shares 54.4% identity with human TRAIL (Abdalla et al., 2004). The TRAIL mRNA is expressed in cells related to both the innate and adaptive immune systems (Falschlehner et al., 2009). A limited number of studies have investigated the association of candidate genes with immune responses in native chickens. Native Malaysian chickens are unique because they have historically been raised in a tropical area with a high probability of spreading Salmonella spp. The objective of this study was to investigate the association between polymorphisms in the NRAMP1, TGFβ3, TGFβ4, CASP1, IAP1, LITAF, and TRAIL genes and resistance to Salmonella Enteritidis in native Malaysian chickens.
MATERIALS AND METHODS Experimental Birds Two hundred 1-d-old Village Chickens and Red Junglefowl chicks (100 per breed) from 3 hatches were used in this study. The Village Chickens were derived from a flock established in 2008 by the Malaysian Agriculture Research and Development Institute. The founder parents had been collected from different villages and allowed to mate randomly. The Red Junglefowl chicks were obtained from a private farm. The parental stock were vaccinated against Newcastle disease and tested weekly to confirm the absence of Salmonella spp. After hatching, the chicks were wing-tagged, chosen randomly, and housed in wire-bottom cages. Swab samples were taken from the cloaca of all chicks to confirm the absence of Salmonella. To screen for the presence of Salmonella Enteritidis, the sterile swab collection from the cecal content was enriched in selenite broth (Oxoid, Basingstoke, UK) for 24 h at 37°C. Each enrichment culture was then plated on XLT4 agar (Oxoid) plates and incubated for 24 h at 37°C. The chicks were given access to water and antibiotic-free commercial starter broiler food ad libitum, and were monitored at least twice daily for mortality during the experiment. All procedures were undertaken according to the guidelines of the Institutional Animal Care and Use Committee.
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Table 1. Primer sets for PCR-RFLP assay of 7 candidate genes1
Gene
Accession number
NRAMP1
AY072001
TGFβ3
X60091
TGFβ4
AF459837
LITAF
AI979890
Caspase1
AF031351
IAP1
AF221083
TRAIL
AF537189
PCR product (bp)
Primer sequence F: 5′-GGCGTCATCCTGGGCTGCTAT-3′ R: 5′-AGACCGTTGGCGAAGTCATGC-3′ F: 5′-CGGCCTGGAAATCAGCATAC-3′ R: 5′-GAAGCAGTAGTTGGTATCCAG-3′ F: 5′-GGGGTCTTCAAGCTGAGCGT-3′ R: 5′-TTGGCAATGCTCTGCATGTC-3′ F: 5′-TGAGTTGCCCTTCCTGT-3′ R: 5′-CAGAGCATCAACGCAAA-3′ F: 5′-CCATGCTTGGGCTCTCAGTG-3′ R: 5′-GGTCCCGCAGATCCCAGTG-3′ F: 5′-TCACCATCTCTACGTTCCAT-3′ R: 5′-CATTGAAACTTGGTTGGTCT-3′ F: 5′-GTAAATTAGAGCCTCATCA-3′ R: 5′-CACCTCAGTTCCTCCGA-3′
Annealing temperature (°C)/time
Reference
801
64 to 60°C/45 s
Liu et al., 2003
1,078
64 to 54°C/45 s
Malek and Lamont, 2003
240
64 to 54°C/45 s
Kramer et al., 2003
497
54°C/30 s
Malek et al., 2004
1,070
64 to 54°C/45 s
Liu and Lamont, 2003
394
64 to 54°C/45 s
Liu and Lamont, 2003
786
59 to 51°C/45 s
Malek and Lamont, 2003
1NRAMP1 = natural resistance-associated protein 1; TGF = transforming growth factor; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; IAP1 = inhibitor of apoptosis protein 1; TRAIL = TNF-related apoptosis-inducing ligand. F = forward; R = reverse.
Salmonella Challenge and Examination One day after hatching, the chicks were intraesophageally inoculated with 0.1 mL of Luria-Bertani broth containing 1 × 107 cfu/bird of Salmonella Enteritidis phage type 13a (nalidixic acid-resistant). The concentration of bacteria was confirmed by serial plate dilution of the inoculum. All chicks were killed on d 7 postinoculation by cervical dislocation, and the organs (cecal content, spleen, and liver) were removed and homogenized. One gram of each sample was diluted in 10 mL of buffered peptone water (Oxoid), followed by homogenization using a Heidolph Diax 600 homogenizer (Heidolph, Schwabach, Germany). The homogenate (100 μL) was plated in duplicate on XLT4 agar containing 100 μg/mL of nalidixic acid (Sigma, St. Louis, MO) and incubated at 37°C for 24 h. An agglutination test using Salmonella polyvalent agglutinating sera (Remel Europe Ltd., Dartford, UK) was used to confirm the presence of Salmonella colonies. For agglutination tests, 20 μL of antiserum was added to a glass slide and mixed with Salmonella Enteritidis culture. A positive reaction was seen as visible agglutination. The number of Salmonella Enteritidis colonies was counted using a Galaxy 230 colony counter (Newstar Environmental Co., Roswell, GA).
DNA Isolation and PCR-RFLP Genomic DNA was extracted from blood collected in EDTA or from the breast muscle of dead chickens using a DNeasy mini kit (Qiagen, Hilden, Germany). The primer sets used to amplify the fragments of the NRAMP1, TGFβ3, TGFβ4, CASP1, IAP1, LITAF, and TRAIL genes (Table 1) have been previously described (Liu et al., 2003; Malek and Lamont, 2003; Kramer et al., 2003; Liu and Lamont, 2003; Malek et al., 2004). The PCR was performed in a 25-μL master mix containing 50 to 100 ng of chicken genomic DNA, 0.1 µM primer (each), 200 µM dNTP, 1.5 units of Taq DNA
polymerase (Promega, Madison, WI), 5 µL of 5 × PCR buffer, and 1.5 mM MgCl2. The reaction conditions for the amplification of LITAF fragments were similar to those described previously (Malek et al., 2004). A touch-down program was used to amplify TGFβ3, TGFβ4, CASP1, and IAP1 with an initial denaturation at 95°C for 5 min, followed by 40 cycles of denaturation at 95°C for 45 s, annealing at 64°C to 54°C for 45 s, and extension at 72°C for 1 min, and a final extension at 72°C for 10 min. A similar touch-down program was used to amplify NRAMP1 and TRAIL fragments with annealing temperatures 64 to 60°C and 59 to 51°C, respectively. Polymorphisms of candidate genes were screened using restriction enzymes (New England BioLabs Inc., Ipswich, MA). The PCR product of the TGFβ3 gene was digested with BsrI (10 U) at 65°C overnight. The SacI (10 U), MboII (1.5 U), BglI (10 U), Hsp92II (5 U), HinfI (7 U), and StyI (5 U) were used to digest the PCR products of NRAMP1, TGFβ4, IAP1, CASP1, LITAF, and TRAIL genes, respectively, at 37°C overnight. The digested products were separated on 2% agarose gel with ethidium bromide for 2 h at 80 V and visualized under UV light.
Statistical Analysis Population genetics measurements, including allelic and genotypic frequencies and deviation from HardyWeinberg equilibrium (HWE), were calculated using PopGene software V. 1.31 (Yeh et al., 1999). The Student’s t-test was used to compare the overall mean bacterial load in each organ between the 2 breeds. Two GLM were developed to analyze the association between genotypes and Salmonella Enteritidis load in the cecum, spleen, and liver using the SAS program V. 9.1 (SAS Institute Inc., 2004). The first model (model 1) included only one gene, whereas the second model (model 2) included 2 genes based on their physiological
ASSOCIATION OF CANDIDATE GENES WITH SALMONELLA
relationship to estimate the interaction between different genotypes: model 1: Yijkl = μ + genotypei + sexj + hk + gene × sexij + eijkl, where Yijkl is the bacterial count (cfu/mL) on the lth individual of the ith genotype from the jth sex and in the kth hatchery (h), and eijkl is the random environmental residual effects. The μ is the overall mean, gene and sex are fixed effects, and h is random effect. The Salmonella Enteritidis counts were transformed to their natural logarithms to improve the normality of the data. In model 2, the genes included TGFβ3/TGFβ4, IAP1/TRAIL, and IAP1/CASP1. Model 2: Yijklm = μ + genei + genej + sexk + hl + genei × genej + eijklm. The proportion of phenotypic variation that was explained by genotypic variation for each gene was calculated as below (Kramer et al., 2003): phenotypic variation (%) = 100 × (squared correlation of model 1 − squared correlation of model 3), where model 3: Yijk = μ + sexi + hj + eijk. The phenotypic variation was calculated when the effect of the gene was significant.
RESULTS Allelic Diversity The results of sequence analysis indicated the presence of SNP in native Malaysian chickens. All SNP characterized by PCR-RFLP assays were compatible with previous reports (Kramer et al., 2003; Liu et al., 2003; Liu and Lamont, 2003; Malek and Lamont, 2003; Malek et al., 2004). However, the genotype T/T was not observed for CASP1 in the Village Chickens and Red Junglefowl following the digestion of 1,070bp PCR products by Hsp92II restriction endonuclease (Figure 1). For LITAF, sequencing of the PCR products showed an A/T SNP in a HinfI site located within an intronic region in Village Chickens. The digested products include fragment sizes of 450 and 48 bp for one allele (T), and 393, 57, and 48 bp for the other allele (A). The Red Junglefowl was homozygous T/T for this locus. The results of PCR-RFLP assay showed that generally all candidate genes had no deviation from HardyWeinberg equilibrium in both breeds (Table 2). For Village Chickens, the minimum and maximum allelic frequency differences belonged to TGFβ3 and CASP1, respectively. The minimum allelic differences for Red
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Junglefowl belonged to TGFβ4 and the maximum was for the CASP1 loci. The observed heterozygosity for most loci was moderate to high in both Village Chickens and Red Junglefowl chickens.
Association of Candidate Genes with Salmonella Enteritidis Response All birds in the control group were free of Salmonella Enteritidis at the end of the experiment. Twenty percent of the infected Red Junglefowl died over the course of the experiment. Chickens that died were not included in the association study. The overall mean Salmonella Enteritidis burden in the spleen was significantly higher in Red Junglefowl than in Village Chickens (P < 0.01). However, no difference in Salmonella Enteritidis burden was seen for the cecum and liver (Table 3). Significant effects of the genotype on the Salmonella Enteritidis load were observed for NRAMP1, TGFβ3, TGFβ4, and TRAIL (Tables 4, 5, and 6). The C/C genotype of NRAMP1 was correlated with a higher Salmonella Enteritidis burden in the cecum and spleen of Village Chickens (P < 0.01). The effect of genotypic variation on phenotypic variation was 35 and 13% for the cecum and spleen Salmonella Enteritidis burden, respectively. The C/T mutation in the NRAMP1-exonic region did not significantly affect the response to Salmonella Enteritidis in the liver. For TGFβ3, the heterozygote A/C had the highest Salmonella Enteritidis load in the cecum, spleen, and liver compared with the other 2 genotypes (P < 0.01). The proportion of phenotypic variation that is demonstrated by genotypic variation was between 10 and 30% for the 2 breeds. A polymorphism in the exonic region of TGFβ4 was associated with Salmonella Enteritidis burden in the spleen and liver of Red Junglefowl; the C/C genotype indicated the highest Salmonella Enteritidis load (P < 0.05). The observed SNP explained 14 and 11% of the phenotypic variation for spleen and liver bacterial burden, respectively. For TRAIL, a silent mutation at position 82 bp of this gene was associated with the cecum, spleen, and liver bacterial load in Village Chickens and Red Junglefowl. The A/A genotype exhibited the lowest bacterial load in both breeds. The A/G SNP explained 8 to 13% of phenotypic variation for both breeds. The other candidate genes did not show any significant effect on the response to Salmonella Enteritidis challenge. Several interactions between genes in this study were analyzed; however, only the interaction between IAP1 and TRAIL in Village Chickens with Salmonella Enteritidis burden in spleen was significant (P < 0.05).
DISCUSSION In this study, the impact of polymorphisms in 7 candidate genes on Salmonella Enteritidis colonization in the cecum, spleen, and liver of native Malaysian chickens was investigated. The identified SNP were found
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Figure 1. Polymerase chain reaction-RFLP pattern for 7 candidate genes including (a) transforming growth factor (TGF) β3, (b) TGFβ4, (c) lipopolysaccharide-induced tumor necrosis factor (TNF) α factor (LITAF), (d) caspase 1 (CASP1), (e) inhibitor of apoptosis protein 1 (IAP1), and (f) TNF-related apoptosis-inducing ligand (TRAIL).
in both intronic and exonic regions, similar to those previously reported (Kramer et al., 2003; Liu et al., 2003; Liu and Lamont, 2003; Malek and Lamont, 2003; Malek et al., 2004). The level of heterozygosity for most of the candidate genes assayed in this study was high, probably a result of random mating. Nevertheless, an indication of genetic drift was observed for some genes, particularly in the Red Junglefowl. The natural habitat of this breed is tropical jungle with limited populations. The similarity of SNP, both in composition and position, between the 2 breeds is likely the result of the Village Chickens having descended from Red Junglefowl (Crawford, 1990). Significant associations between some of the candidate gene polymorphisms and the Salmonella Enteritidis load were observed. All the candidate genes surveyed in the current study were chosen because of their role in the immune system. The
chickens in this study were not inbred and were selected randomly. We found the NRAMP1 polymorphism showed a significant association with Salmonella Enteritidis burden in the cecum; this result was in agreement with the reports by Kramer et al. (2003). In this study, the homozygous C/C genotype was related to the highest Salmonella Enteritidis load; however, there is inconsistency among the results of different studies. Liu et al. (2003) described that the allele C had a lower Salmonella Enteritidis load in chicken spleen than did allele T. Liu et al. (2003) did not observe a significant effect of the C/T SNP on cecum Salmonella Enteritidis burden. Different lines or breeds may manifest different reactions to infections. The NRAMP1 has an important role in innate immunity, and the cecum is the first line of defense against colonization by Salmonella (Gruenheid et al., 1997; Berndt et al., 2007).
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Table 2. The genotypic frequencies and test for deviation from Hardy-Weinberg equilibrium (HWE) for Village Chickens and Red Junglefowl1 Observed genotypic frequency2 Gene
Chicken type
CASP1 IAP1 LITAF NRAMP1 TGFβ3 TGFβ4 TRAIL
Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl
Expected genotypic frequency
11
12
22
0.87 0.85 0.10 0.26 0.01 0.00 0.55 0.61 0.23 0.39 0.13 0.21 0.39 0.07
0.13 0.15 0.40 0.49 0.18 0.00 0.36 0.37 0.47 0.43 0.45 0.57 0.40 0.54
0.00 0.00 0.50 0.25 0.81 1.00 0.09 0.02 0.30 0.18 0.42 0.22 0.21 0.39
11
12
22
0.87 0.86 0.09 0.26 0.01 0.00 0.54 0.64 0.21 0.37 0.12 0.25 0.35 0.11
0.12 0.14 0.42 0.50 0.19 0.00 0.39 0.32 0.50 0.48 0.46 0.50 0.49 0.45
0.01 0.00 0.49 0.24 0.80 1.00 0.07 0.04 0.29 0.15 0.42 0.25 0.16 0.44
P-value for HWE test
0.53 0.56 0.57 0.85 0.78 0.00 0.49 0.41 0.56 0.37 0.85 0.28 0.08 0.11
1CASP1 = caspase 1; IAP1 = inhibitor of apoptosis protein 1; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; NRAMP1 = natural resistance-associated protein 1; TGF = transforming growth factor; TRAIL = TNF-related apoptosis-inducing ligand. 2The 1 for LITAF, TRAIL, TGFβ3, TGFβ4, interleukin (IL)2, interferon (IFN)γ, and IAP1 is equal to A and for NRAMP1, TLR4, TGFβ2, IgL, and CASP1 is equal to C. The 2 for TGFβ3 and TGFβ4 is equal to C, for TLR4, TRAIL, IL2, IFNγ, and IAP is equal to G, and for NRAMP1, LITAF, TGFβ2, IgL, and CASP1 is equal to T.
Therefore, the role of NRAMP1 in the cecal immune system is critical; NRAMP1 can accelerate the inflammatory response during Salmonella invasion (Valdez et al., 2009). The SNP identified in the current study was silent and may be linked to another gene sequence nearby. We also observed significant association of TGFβ3 and TGFβ4 polymorphisms with Salmonella Enteritidis colonization in this study. A neutral mutation in the TGFβ3 intron region was associated with bacterial colonization. This neutral mutation may be linked to a functional sequence in the genome (Rothschild and Soller, 1997). However, there was a conflict between the results of the 2 breeds. In the cecum, the C/C genotype of TGFβ3 showed the lowest bacterial burden for Village Chickens, whereas in Red Junglefowl, the A/A genotype exhibited the lowest Salmonella Enteritidis colonization. The results of the current study were consistent with those found by Kramer et al. (2003). The association between the TGFβ3 polymorphism and the spleen Salmonella Enteritidis load was not significant in the study by Malek and Lamont (2003), but it was significant for the cecum Salmonella Enteritidis load. A significant association between the TGFβ3-BsrI polymorphism and mortality between 14 and 42 d in broiler chickens was reported by Ye et al. (2006). For TGFβ4, no association was found between different genotypes and the cecum Salmonella Enteritidis load. However,
the bacterial burden in the spleen and liver was influenced by TGFβ4 genotypes. The TGFβ4 SNP changed the amino acid sequence, and that might affect protein function. It was demonstrated that the difference between different genetic lines in response to Salmonella infection in young chickens can be attributed to the developmental differences of the gut (Schokker et al., 2012). This may contribute to the inconsistency in the results of different studies or different lines of same study. Moreover, it has been documented that TGFβ gene polymorphisms are associated with antibody production in chickens (Zhou et al., 2003). Therefore, the observed association between TGFβ gene SNP in this study with Salmonella Enteritidis burden may be because of the effect of those SNP on antibody production. The results of this study introduce the TGFβ genes as potential candidate genes for future selection programs. For IAP1, the association between the G/A SNP and bacterial load in the cecum, spleen, and liver was not significant in the current study. The chicken IAP1 gene is expressed at a relatively high level in the intestine, spleen, thymus, bursa, and lungs (You et al., 1997). Here, the G/A SNP in the exonic region of the IAP1 gene was silent and had no effect on response to Salmonella Enteritidis. Previous studies showed Salmonella-infected macrophages are killed by a CASP1-dependent necrosis
Table 3. The overall mean ± SEM of Salmonella Enteritidis load (natural log cfu) in each organ for the 2 breeds Breed Village Junglefowl a,bCommon
Cecum 11.43 ± 11.24 ±
0.16a 0.08a
Spleen 8.82 ± 9.41 ±
0.09a 0.08b
superscripts in each column show nonsignificant differences.
Liver 8.77 ± 0.07a 8.90 ± 0.10a
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Table 4. Effect of 7 candidate genes on Salmonella Enteritidis load in the cecum of Village Chickens and Red Junglefowl1 Genotype2 (least squares means ± SE) Gene
Chicken type
NRAMP1 TGFβ3 TGFβ4 LITAF CASP1 IAP1 TRAIL
Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl
11 12.21 11.58 11.79 10.99 10.74 11.43 11.64 11.27 11.20 10.61 11.57 10.60 11.10
12
0.19a (18) 0.15a (23) 0.30a (7) 0.15b (15) 0.34a (9) 0.22a (9) Ua (2) — ± 0.23a (28) ± 0.10a (39) ± 0.40a (6) ± 0.20a (12) ± 0.47a (8) ± 0.15a (19)
± ± ± ± ± ± ±
11.37 10.91 11.89 11.66 11.39 11.11 10.55
± ± ± ± ± ± ±
10.94 11.11 11.37 11.11 11.28 11.22
± ± ± ± ± ±
0.16b (16) 0.21a (15) 0.23b (17) 0.14a (25) 0.32a (13) 0.13a (27) 0.62a (7) — 0.44a (6) 0.24a (6) 0.19a (17) 0.12a (21) 0.29ab (13) 0.13a (23)
Phenotypic variation (%)
22 10.76 ± 0.13b (4) — 11.04 ± 0.23b (12) 11.21 ± 0.29b (13) 11.63 ± 0.29a (15) 11.17 ± 0.21a (9) 11.27 ± 0.26a (27) — — — 11.28 ± 0.20a (14) 10.99 ± 0.17a (12) 11.85 ± 0.37b (14) 11.99 ± 0.20b (3)
35 — 25 30 — — — — — — — — 13 13
P-value 0.002 0.39 0.02 0.007 0.13 0.46 0.46 0.43 0.69 0.20 0.09 0.04 0.05
a,bCommon
superscripts in each row show nonsignificant differences (P > 0.05). = natural resistance-associated protein 1; TGF = transforming growth factor; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; CASP1 = caspase 1; IAP1 = inhibitor of apoptosis protein 1; TRAIL = TNF-related apoptosis-inducing ligand. The number of samples is shown in parentheses. For phenotypic variation, — means noncalculated. — shows unavailable data. U = unestimated. 2The 1 for LITAF, TRAIL, TGFβ3, TGFβ4, interleukin (IL)2, interferon (IFN)γ, and IAP1 is equal to A and for NRAMP1, TLR4, TGFβ2, IgL, and CASP1 is equal to C. The 2 for TGFβ3 and TGFβ4 is equal to C, for TLR4, TRAIL, IL2, IFNγ, and IAP1 is equal to G, and for NRAMP1, LITAF, TGFβ2, IgL, and CASP1 is equal to T. 1NRAMP1
mechanism (Brennan and Cookson, 2000). However, the statistical association between C/T SNP at position −368-bp flanking region of CASP1 gene and Salmonella Enteritidis burden was not able to be determined due to the absence of one genotype. For the common SNP, significant association with Salmonella Enteritidis loads in the cecum and spleen and with antibody levels was reported by Liu and Lamont (2003). In the current study, LITAF gene polymorphism did not show significant association with Salmonella Enteritidis load. The A/T substitution was identified in an intronic region. Therefore, it did not influence protein structure, and it might also not be linked to other func-
tional sequence. However, Red Junglefowl was homozygous T/T for the LITAF gene similar to a report by Malek et al. (2004). The TRAIL gene polymorphism was associated with Salmonella Enteritidis burden in the cecum, spleen, and liver. In other studies (Malek and Lamont, 2003; Ye et al., 2006), TRAIL gene polymorphism was associated with the cecum Salmonella Enteritidis burden and chicken early mortality (before 14 d of age). The A/G substitution in the exonic region did not change the amino acid sequence. Therefore, the results of the present study should be interpreted conservatively until this SNP can be analyzed in several populations.
Table 5. Effect of 7 candidate genes on Salmonella Enteritidis load in the spleen of Village Chickens and Red Junglefowl1 Genotype2 (least squares means ± SE) Gene
Chicken type
NRAMP1 TGFβ3 TGFβ4 LITAF CASP1 IAP1 TRAIL
Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl
a,bCommon
11 8.90 9.12 8.45 8.97 8.69 9.04 8.73
± ± ± ± ± ± ±
8.45 9.27 8.44 9.71 8.23 8.46
± ± ± ± ± ±
0.11a (45) 0.13a (26) 0.14ab (19) 0.15a (16) 0.19a (10) 0.34a (8) Ua (1) — 0.07a (70) 0.11a (42) 0.17a (10) 0.22a (13) 0.08a (29) 0.35a (4)
12 8.39 9.52 8.78 9.55 8.43 9.14 8.47
± ± ± ± ± ± ±
8.56 9.22 8.50 9.10 8.57 9.26
± ± ± ± ± ±
0.12b (29) 0.18a (17) 0.9a (40) 0.15b (22) 0.09a (34) 0.14a (28) 0.19a (13) — 0.15a (12) 0.28a (6) 0.09a (35) 0.14a (21) 0.08b (28) 0.14b (24)
22 8.58 ± 0.27ab (6) — 8.41 ± 0.13b (23) 9.43 ± 0.31ab (9) 8.54 ± 0.10a (32) 9.75 ± 0.20b (12) 8.50 ± 0.08a (56) — — — 8.50 ± 0.08a (49) 9.46 ± 0.17a (14) 8.43 ± 0.10ab (17) 9.39 ± 0.15b (20)
Phenotypic variation (%)
P-value
13 — 20 22 — 14 — — — — — — 18 17
0.01 0.06 0.03 0.02 0.40 0.04 0.81 — 0.52 0.88 0.91 0.06 0.02 0.05
superscripts in each row show nonsignificant differences (P > 0.05). = natural resistance-associated protein 1; TGF = transforming growth factor; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; CASP1 = caspase 1; IAP1 = inhibitor of apoptosis protein 1; TRAIL = TNF-related apoptosis-inducing ligand. The number of samples is shown in parentheses. For phenotypic variation, — means noncalculated. — shows unavailable data. U = unestimated. 2The 1 for LITAF, TRAIL, TGFβ3, TGFβ4, interleukin (IL)2, interferon (IFN)γ, and IAP1 is equal to A and for NRAMP1, TLR4, TGFβ2, IgL, and CASP1 is equal to C. The 2 for TGFβ3 and TGFβ4 is equal to C, for TLR4, TRAIL, IL2, IFNγ, and IAP1 is equal to G, and for NRAMP1, LITAF, TGFβ2, IgL, and CASP1 is equal to T. 1NRAMP1
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ASSOCIATION OF CANDIDATE GENES WITH SALMONELLA Table 6. Effect of 7 candidate genes on Salmonella Enteritidis load in the liver of Village Chickens and Red
Junglefowl1
Genotype2 (least squares means ± SE) Gene
Chicken type
NRAMP1 TGFβ3 TGFβ4 LITAF CASP1 IAP1 TRAIL
Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl Village Chickens Junglefowl
11 8.42 8.64 8.83 8.61 8.57 8.51 9.16
± ± ± ± ± ± ±
8.69 8.65 8.62 8.53 8.79 8.32
± ± ± ± ± ±
0.11a (45) 0.11a (23) 0.15ab (18) 0.13a (16) 0.24a (10) 0.20a (8) Ua (1) — 0.09a (63) 0.09a (38) 0.25a (7) 0.19a (11) 0.12a (30) 0.11a (3)
12 8.78 8.56 8.88 8.65 8.73 8.53 8.93
± ± ± ± ± ± ±
9.24 8.91 8.97 8.58 8.56 8.86
± ± ± ± ± ±
0.12a (29) 0.16a (17) 0.9a (38) 0.12a (21) 0.13a (32) 0.12a (26) 0.20a (13) — 0.26a (7) 0.27a (6) 0.12a (34) 0.12a (21) 0.15a (27) 0.12b (21)
22 8.80 8.16 8.48 8.81 8.81 9.11 8.85
± ± ± ± ± ± ±
8.68 8.78 8.73 8.68
± ± ± ±
0.27a (6) Ua (1) 0.13b (23) 0.20a (8) 0.13a (28) 0.16b (11) 0.10a (48) — — — 0.10a (48) 0.14a (13) 0.19a (15) 0.10ab (18)
Phenotypic variation (%)
P-value
— — 10 — — 11 — — — — — — — 8
0.85 0.68 0.03 0.85 0.66 0.01 0.85 — 0.25 0.76 0.10 0.45 0.43 0.02
a,bCommon
superscripts in each row show nonsignificant differences (P > 0.05). = natural resistance-associated protein 1; TGF = transforming growth factor; LITAF = lipopolysaccharide-induced tumor necrosis factor (TNF) α factor; CASP1 = caspase 1; IAP1 = inhibitor of apoptosis protein 1; TRAIL = TNF-related apoptosis-inducing ligand. The number of samples is shown in parentheses. For phenotypic variation, — means noncalculated. — shows unavailable data. U = unestimated. 2The 1 for LITAF, TRAIL, TGFβ3, TGFβ4, interleukin (IL)2, interferon (IFN)γ, and IAP1 is equal to A and for NRAMP1, TLR4, TGFβ2, IgL, and CASP1 is equal to C. The 2 for TGFβ3 and TGFβ4 is equal to C, for TLR4, TRAIL, IL2, IFNγ, and IAP1 is equal to G, and for NRAMP1, LITAF, TGFβ2, IgL, and CASP1 is equal to T. 1NRAMP1
Several interactions between genes in this study were analyzed; however, only the interaction between IAP1 and TRAIL was significant. Interaction of genetic factors always exists in living organisms. Such epistatic interactions can exist among genes that are involved in similar functions (Ye et al., 2006). The TRAIL and IAP1 genes belong to the cell apoptotic system. However, understanding the cause of interaction needs analysis of gene expression and translation. The breed had a significant effect on the Salmonella Enteritidis load in the spleen. The bacterial counts for Red Junglefowl chickens were higher than those for Village Chickens. This result was expected because Red Junglefowl chickens live in a limited area and are in contact with few pathogens. Therefore, they are evolutionarily more susceptible to diseases. Moreover, inbreeding depression in limited populations is more prominent for viability traits (Falconer and MacKay, 1996). At the same time, the presence of gut microflora may interact with the genetic background of the animal to eliminate pathogens (Esworthy et al., 2010), and the systemic organs (spleen and liver) are free of natural microflora. Estimation of phenotypic variation revealed that distribution of genotypic variation through phenotypic variation was between 9 and 35%. It demonstrated that the effect of the observed SNP for some candidate genes on phenotypic variation was small and for others was moderate. The trait used as a resistance measurement was Salmonella Enteritidis load. It appeared that each gene had a small effect on resistance to Salmonella Enteritidis. Resistance to disease is a polygenic trait (Lamont, 2008); therefore, the total effect of all genes is important. Moreover, there are several other factors, such as invasion of Salmonella, the activity of immune system, and the age of the animal that influence resis-
tance to diseases (Kramer et al., 2003). Thus, the effect of one gene may not explain the whole phenotypic variation. The estimated heritability for resistance to Salmonella in published articles ranges from less than 0.05 to 0.32 (Berthelot et al., 1998; Beaumont et al., 1999; Girard Santosuosso et al., 2002). Thus, the calculated genotypic variation in this study is consistent with those estimates of heritability. All of the investigated genes are important in immune responses and most of them were associated with Salmonella Enteritidis load in at least one organ. The cecum as a first barrier against Salmonella Enteritidis colonization has an important role in resistance to Salmonella. Although the spleen is an immunological organ, the immune response of different organs to pathogens does not appear at the same time. Therefore, if the period of the experiment changes, the immune response of the infected organs may change. In conclusion, the main objectives of this study were to analyze the polymorphism status of immune candidate genes and their association with Salmonella Enteritidis resistance in 2 breeds of native Malaysian chickens. The results demonstrated that improvement of genetic resistance against Salmonella Enteritidis through marker-assisted selection is feasible. The candidate genes NRAMP1, TGFβ3, TGFβ4, and TRAIL contained a considerable proportion of phenotypic variations related to Salmonella Enteritidis resistance in the Village Chicken and Red Junglefowl populations. These results suggest several candidate genes that are involved in the mechanism of resistance to Salmonella Enteritidis and extend the knowledge of genetic resistance to diseases. However, practical implementation of these candidate genes will require further study of their roles in Salmonella Enteritidis resistance. The results of different studies show inconsistencies that can
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be attributed to the use of different strains of bacteria, populations, and environments. Thus, the result of any association study is applicable for that particular population under study. Future genomics studies utilizing the high-throughput technologies of high-density SNP genotyping, microarray technology, next-generation sequencing, and haplotype analysis will provide a better understanding of the mechanisms of immune gene responses to diseases in native Malaysian chickens.
ACKNOWLEDGMENTS This study was supported financially and technically by RUGS grant 01-01-10-0911RU from Universiti Putra Malaysia.
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