S. J. Lamont

Candidate genes for resistance to Salmonella enteritidis colonization in chickens as detected in a novel genetic cross

Salmonellosis is a zoonotic disease that is problematic for both animal production and food safety. A novel genetic cross, named the Iowa Salmonella response resource population (ISRRP), was established to elucidate the genetic control of resistance to Salmonella enteritidis (SE) colonization in young chicks, to characterize unique resistance alleles, and to estimate gene interaction effects. Outbred broiler sires were mated with dams of diverse, highly inbred, light-bodied lines to produce an F(1) generation that was informative for all heterozygous alleles of the sires. Mating F(1) sires back to dams of the corresponding inbred line produced a backcross generation. To mimic the natural route of exposure and thus afford the opportunity to investigate mucosal immunity, pathogenic SE were inoculated into the esophagus of day-old chicks. After 1 week, the SE colonizing the cecal lumen and the spleen were enumerated. Candidate genes were selected for analysis based upon one of the two criteria. Functional candidates were genes with reported activity related to the tested traits. Positional candidates were genes mapped near microsatellites that were linked, in other phases of this project, with antibody levels to SE vaccine. Broiler sire alleles of the MHC class I, NRAMP1, PSAP, and IAP1 genes showed association with SE colonization in the F(1) generation of this novel disease resistance resource population.

Toll-like receptor gene expression in cecum and spleen of advanced intercross line chicks infected with Salmonella enterica serovar Enteritidis.

Understanding the changes in host gene expression that occur with bacterial infection will help to elucidate the basis of molecular genetic control of disease resistance. The effect of infecting chicks with Salmonella enterica serovar Enteritidis on the RNA expression level of Toll-like receptor (TLR) genes, and the correlation between TLR RNA expression level and bacterial burden in the cecum and spleen of young birds was studied. Chicks from two advanced intercross lines were either infected or mock infected with S. enteritidis at 1 day of age. The RNA expression levels of TLR2, TLR4 and TLR5 genes were assessed by quantitative reverse transcriptase-PCR (qRT-PCR) in cecum and spleen tissues harvested at one week post-infection. Infected chicks had significant upregulation of TLR2 RNA expression in spleen, TLR4 RNA expression in both cecum and spleen, and downregulation of TLR5 RNA expression in cecum. Bacterial burden of S. enteritidis in infected birds was not correlated with TLR RNA expression level. Infecting chicks with S. enteritidis caused an increase in TLR2, TLR4 and TLR5 RNA expression level in spleen in males but not in females. The effect of sex on response to S. enteritidis infection suggests a role for TLR signaling pathways in sex-based modulation of immune response to pathogens. High correlation between TLR2 and TLR4 mRNA expression level in cecum of S. enteritidis infected birds suggests coordinated regulation or simultaneous stimulation of these genes by S. enteritidis. In conclusion, this study clearly showed that young chicks respond to S. enteritidis infection by upregulating TLR2, TLR4 RNA expression. The downregulation of TLR5 RNA expression was observed in cecum by S. enteritidis infection, which might be beneficial to protect host cells from overstimulation by bacterial flagellin.

Genetic lines differ in Toll-like receptor gene expression in spleens of chicks inoculated with Salmonella enterica serovar Enteritidis

Toll-like receptors (TLR) recognize evolutionarily conserved molecular motifs (pathogen-associated molecular patterns) of infectious microbes and initiate innate immune response upon activation with relevant pathogens. This study investigated the acute effect of Salmonella Enteritidis challenge on TLR mRNA expression in cecum and spleen of birds from 3 distinct genetic lines. Chicks from broiler, Leghorn, and Fayoumi lines were inoculated or mock-inoculated with Salmonella Enteritidis. The mRNA expression levels of TLR2, TLR4, and TLR5 genes were assessed by quantitative reverse transcription-PCR of cecum and spleen tissue harvested at 2 or 18 h postinoculation (PI). There were no significant genetic line effects on TLR mRNA expression in spleen or cecum of mock-infected birds, or in the cecum of infected birds. Genetic line effect was significant (P < 0.05) on TLR mRNA expression in the spleen of Salmonella Enteritidis-infected birds. The Fayoumi line had higher TLR2 and TLR4 expression than Leghorn, higher TLR2 mRNA expression than broiler, and the broiler line had higher TLR5 expression than Leghorn and Fayoumi. In Salmonella Enteritidis-infected birds, the TLR2 expression in both cecum and spleen and TLR4 expression in spleen were significantly higher at 18 h PI than 2 h PI. The results demonstrate a significant genetic line effect on TLR expression in the spleen of Salmonella Enteritidis-infected birds, which may partly explain genetic variability in immune response to Salmonella Enteritidis.

RNA-seq analysis revealed novel genes and signaling pathway associated with disease resistance to avian influenza virus infection in chickens

Avian influenza virus (AIV) is a type A virus of the family Orthomyxoviridae. Avian influenza virus infection can cause significant economic losses to the poultry industry, and raises a great public health threat due to potential host jump from animals to humans. To develop more effective intervention strategies to prevent and control AIV infection in poultry, it is essential to elucidate molecular mechanisms of host response to AIV infection in chickens. The objective of this study was to identify genes and signal pathways associated with resistance to AIV infection in 2 genetically distinct highly inbred chicken lines (Fayoumi, relatively resistant to AIV infection, and Leghorn, susceptible to AIV infection). Three-week-old chickens were inoculated with 10(7) EID50 of low pathogenic H5N3 AIV, and lungs and trachea were harvested 4 d postinoculation. Four cDNA libraries (1 library each for infected and noninfected Leghorn, and infected and noninfected Fayoumi) were prepared from the lung samples and sequenced by Illumina Genome Analyzer II, which yielded a total of 116 million, 75-bp single-end reads. Gene expression levels of all annotated chicken genes were analyzed using CLC Genomics Workbench. DESeq was used to identify differentially expressed transcripts between infected and noninfected birds and between genetic lines (false discovery rate < 0.05 and fold-change > 2). Of the expressed transcripts in a total of 17,108 annotated chicken genes in Ensembl database, 82.44 and 81.40% were identified in Leghorn and Fayoumi birds, respectively. The bioinformatics analysis suggests that the hemoglobin family genes, the functional involvements for oxygen transportation and circulation, and cell adhesion molecule signaling pathway play significant roles in disease resistance to AIV infection in chickens. Further investigation of the roles of these candidate genes and signaling pathways in the regulation of host-AIV interaction can lead new directions for the development of antiviral drugs or vaccines in poultry.

Effects of dietary vitamin E type and level on lipopolysaccharide-induced cytokine mRNA expression in broiler chicks

Vitamin E modulates the immune response, in part by reducing inflammation. The bacterial component lipopolysaccharide (LPS) can induce an inflammatory response in chickens. The objective of this study was to evaluate immunomodulatory effects of dietary type and level of vitamin E on response of broilers to LPS. One-day-old broiler males (n=96) were placed in a vitamin E-type (synthetic, natural) × vitamin E level (22, 220 IU/kg)×LPS (LPS, saline) block design. At 22 d, LPS (or saline) was injected subcutaneously. Spleens were harvested for RNA isolation at 3 and 24 h postinjection. Relative levels of RNA expression were measured for the immune-related genes: avian β defensin 10 (AvBD10), interleukin 6 (IL6), interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), interleukin 10 and transforming growth factor- β1 (TGF-β1). Avian β defensin 10 and iNOS are innate antimicrobial proteins. Interleukin 6 and IFN-γ are pro-inflammatory cytokines, whereas interleukin 10 and transforming growth factor-β1 are anti-inflammatory cytokines. There were significantly higher splenic levels of IL6, IFN-γ, iNOS, and IL10 RNA expression at 3 h postinjection in chickens receiving LPS than in chickens 24 h post-LPS injection or saline-injected birds at either time. These data suggest that LPS induced an immune response that was regulated by both pro- and anti-inflammatory cytokines. Birds fed natural-type (versus synthetic) vitamin E had a significantly lower LPS-induced inflammatory response, as indicated by lower IL6 RNA expression levels, suggesting a protective effect from natural-type vitamin E when a chicken encounters a bacterial component.

Distinct lines of chickens express different splenic cytokine profiles in response to Salmonella Enteritidis challenge

Chicken meat and eggs contaminated with Salmonella result in economic losses in the poultry industry and potential human infection. Intestinal parasites have been shown to lead to a reduction in the utilization of nutrients and performance in poultry. This study provides insight into the immune responses used by hens of 3 genetically distinct chicken lines (broiler, Leghorn, and Fayoumi) in the presence and absence of Salmonella Enteritidis infection. Understanding the range of immune responses used by different lines in response to Salmonella Enteritidis may help the poultry industry genetically select birds that are more pathogen resistant. The splenic mRNA levels of several immune-related genes [IL-6, IL-8, IL-10, IL-18, macrophage inflammatory protein 1β, interferon (IFN)-γ, transforming growth factor β1, and regulated upon activation, normal T cell expressed, and secreted (RANTES)] were analyzed by quantitative PCR. Line, challenge, and their interaction were considered fixed effects. Line had a significant effect on the mRNA expression of RANTES (P < 0.02) and IFN-γ (P < 0.03). Broilers expressed significantly more splenic RANTES mRNA than Fayoumis, and significantly more splenic IFN-γ mRNA than Leghorns (P < 0.05). There was a significant interaction of genetic line and challenge on IL-18 (P < 0.02) and IL-6 (P < 0.01) mRNA expression. Although there was a significant interaction of genetic line and challenge for IL-18, Tukeys test analysis only showed differences at a suggestive level (P < 0.1). Bacterial challenge had a significant effect on IL-6 mRNA expression only within the Fayoumi line. Challenged Fayoumis expressed significantly less IL-6 mRNA than nonchallenged Fayoumis (P < 0.05). The observed differences in mRNA expression of selected cytokines support the concept that these distinct genetic lines utilize different immune responses at homeostasis and in response to Salmonella Enteritidis infection.

Effects of dietary fiber on cecal short-chain fatty acid and cecal microbiota of broiler and laying-hen chicks

This experiment was conducted to evaluate the effects of feeding dietary fiber on cecal short-chain fatty acid (SCFA) concentration and cecal microbiota of broiler and laying-hen chicks. The lower fiber diet was based on corn-soybean meal (SBM) and the higher fiber diet was formulated using corn-SBM-dried distillers grains with solubles (DDGS) and wheat bran to contain 60.0 g/kg of both DDGS and wheat bran from 1 to 12 d and 80.0 g/kg of both DDGS and wheat bran from 13 to 21 d. Diets were formulated to meet or exceed NRC nutrient requirements. Broiler and laying-hen chicks were randomly assigned to the high and low fiber diets with 11 replicates of 8 chicks for each of the 4 treatments. One cecum from 3 chicks was collected from each replicate: one cecum underwent SCFA concentration analysis, one underwent bacterial DNA isolation for terminal restriction fragment length polymorphism (TRFLP), and the third cecum was used for metagenomics analyses. There were interactions between bird line and dietary fiber for acetic acid (P = 0.04) and total SCFA (P = 0.04) concentration. There was higher concentration of acetic acid (P = 0.02) and propionic acid (P < 0.01) in broiler chicks compared to laying-hen chicks. TRFLP analysis showed that cecal microbiota varied due to diet (P = 0.02) and chicken line (P = 0.03). Metagenomics analyses identified differences in the relative abundance of Helicobacter pullorum and Megamonas hypermegale and the genera Enterobacteriaceae, Campylobacter, Faecalibacterium, and Bacteroides in different treatment groups. These results provide insights into the effect of dietary fiber on SCFA concentration and modulation of cecal microbiota in broiler and laying-hen chicks.

Differential immune-related gene expression in two genetically disparate chicken lines during infection by Eimeria maxima.

Two M5.1 and M15.2 B complex congenic lines of Fayoumi chickens were evaluated for body weight loss and faecal oocyst counts as parameters of avian coccidiosis. M5.1 chickens exhibited resistance to E. maxima compared with M15.2. To correlate the differential responses of the M5.1 and M15.2 lines to E. maxima infection with cellular immune responses, the expression levels of mRNAs encoding 14 immune-related molecules were measured by quantitative RT-PCR in intestinal intraepithelial lymphocytes (IELs) and splenocytes at 0, 3, 4, and 5 days following parasite infection. Intestinal IELs from M5.1 chickens expressed higher levels of transcripts encoding interferon gamma (IFNG), interleukin-lbeta (1L1B), IL6, IL8, IL12, IL15, IL17A, inducible nitric oxide synthase (iNOS), and lipopolysaccharide-induced tumour necrosis a factor (LITAF), and lower levels of mRNAs for IFNA, IL10, IL17D, NK-lysin (NKL), and tumour necrosis factor superfamily 15 (TNFSF15) at 3 days post infection, compared with the M15.2 line. In the spleen, E. maxima infection was associated with higher expression levels of IFNA, and IL15 and lower levels of IL6, IL17D, and IL12 in M5.1 compared to M15.2 birds. Using an intestinal IEL cDNA microarray, the differential dynamics of gene expression in the gut of M5.1 and M15.2 chickens following experimental coccidiosis were evident. In particular, the genes encoding lymphotactin and parathymosin were expressed at significantly higher levels in M5.1 compared with M15.2 line chickens. In conclusion, genetic determinants within the chicken major histocompatibility complex (MHC) B complex influence resistance to E. maxima infection by controlling the local and systemic expression of immune-related cytokine and chemokine genes.

Predicting allele frequencies in DNA pools using high density SNP genotyping data

To evaluate the ability to use DNA pools with the Illumina Infinium genotyping platform, two sets of gradient pools were created using two pairs of highly inbred chicken lines. Replicate pools containing 0%, 10%, 20%, 40%, 60%, 80%, 90% and 100% of DNA from line A vs. B or line C vs. D were created, for a total of 28 pools. All pools were genotyped for 12,046 SNPs. Three frequency estimation methods proposed in the literature (standard, heterozygote-corrected and normalized) were compared with three alternate methods proposed herein based on mean square error (MSE), bias and variance of estimated vs. true allele frequencies and the fit of regression of estimated on true frequencies. The three new methods had average square root MSE of 4.6%, 4.6% and 4.7% compared to 5.2%, 5.5% and 11.2% for the three literature methods. Average absolute biases of the literature methods were 2.4%, 2.7% and 8.2% compared to 2.4% for all new methods. Standard deviations of estimates were also smaller for the new methods, at 3.1%, 3.2% and 3.2% compared to 3.5%, 4.0% and 5.0% for previously reported methods. In conclusion, intensity data from the Illumina Infinium Assay can be efficiently used to estimate allele frequencies in pools, in particular using any of the new methods proposed herein.

High resolution, advanced intercross mapping of host resistance to Salmonella colonization.

Availability of a dense single-nucleotide polymorphism (SNP) map in chickens has allowed for whole-genome QTL mapping for disease resistance. In this study, two F8 advanced intercross lines of broiler by Fayoumi and broiler by Leghorn chickens, and a dense, genome-wide SNP panel were used to map genomic regions associated with host resistance to bacterial colonisation. One week after inoculation of day-old chicks with Salmonella enteritidis (SE), caecum and spleen tissues were collected to quantitate the bacterial load. Of 2733 genotyped SNPs, 875 were homozygous and the remaining SNPs with a minor allele frequency of > 0.2 were individually tested for association with SE burden by utilising a Chi-square log-likelihood test between models with and without SNP genotype. Using a Q-value of 25%, calculated utilising 1207 SNPs with Chi-square P-Value < 1.0 to control for false discovery, 21 SNPs identifying 19 genes were significantly associated with SE bacterial levels. Ten genes were in pathways associated with immune response to Salmonella (toll-like receptor signaling, apoptosis, and MAPK signaling), further supporting their involvement in host resistance pathways. In addition to identifying new candidate genes for bacterial resistance, the trait-associated SNPs may be useful in marker assisted selection programmes for disease resistance.