Genetic architecture of body weight, carcass, and internal organs traits of Ghanaian local chickens.

Information on the genetic architecture of the production traits of indigenous African chicken is limited. We performed a genome-wide association study using imputed Affymetrix Axiom® 600K SNP-chip genotypes on 1,113 chickens from three agroecological zones of Ghana. After quality control, a total of 382,240 SNPs remained. Variance components and heritabilities for some growth, carcass and internal organ traits were estimated. The genetic and phenotypic correlations among these traits were also estimated. The estimated heritabilities of body weight at week 22 (BW22), average daily gain (ADG), dressed weight, breast weight, thigh weight, wing weight, drumstick weight, and neck weight were high and ranged from 0.50 to 0.69. Estimates of heritabilities for head weight, shank weight, and gizzard weight were moderate (0.31-0.35) while those of liver weight, back weight, dressing percentage, and heart weight were low (0.13-0.21). The estimated heritabilities of dressed weight, breast weight, wing weight, drumstick weight, neck weight, shank weight, and gizzard weight, corrected for BW22, were moderate (0.29-0.38), while the remaining traits had low heritability estimates (0.13-0.21). A total of 58 1-Mb SNP windows on chromosomes 1, 2, 4, 5, 6, 7, 8, 9, 13, 18, and 33 each explained more than 1% of the genetic variance for at least one of these traits. These genomic regions contained many genes previously reported to have effects on growth, carcass, and internal organ traits of chickens, including EMX2, CALCUL1, ACVR1B, CACNB1, RB1, MLNR, FOXO1, NCARPG, LCORL, LAP3, LDB2, KPNA3, and CAB39L. The moderate to high heritability estimates and high positive genetic correlations suggest that BW22, ADG, dressed weight, breast weight, thigh weight, wing weight, drumstick weight, and neck weight could be improved through selective breeding.

Genomic Regions and Candidate Genes Affecting Response to Heat Stress with Newcastle Virus Infection in Commercial Layer Chicks Using Chicken 600K Single Nucleotide Polymorphism Array.

Heat stress results in significant economic losses to the poultry industry. Genetics plays an important role in chickens adapting to the warm environment. Physiological parameters such as hematochemical parameters change in response to heat stress in chickens. To explore the genetics of heat stress resilience in chickens, a genome-wide association study (GWAS) was conducted using Hy-Line Brown layer chicks subjected to either high ambient temperature or combined high temperature and Newcastle disease virus infection. Hematochemical parameters were measured during three treatment phases: acute heat stress, chronic heat stress, and chronic heat stress combined with NDV infection. Significant changes in blood parameters were recorded for 11 parameters (sodium (Na+, potassium (K+), ionized calcium (iCa2+), glucose (Glu), pH, carbon dioxide partial pressure (PCO2), oxygen partial pressure (PO2), total carbon dioxide (TCO2), bicarbonate (HCO3), base excess (BE), and oxygen saturation (sO2)) across the three treatments. The GWAS revealed 39 significant SNPs (p < 0.05) for seven parameters, located on Gallus gallus chromosomes (GGA) 1, 3, 4, 6, 11, and 12. The significant genomic regions were further investigated to examine if the genes within the regions were associated with the corresponding traits under heat stress. A candidate gene list including genes in the identified genomic regions that were also differentially expressed in chicken tissues under heat stress was generated. Understanding the correlation between genetic variants and resilience to heat stress is an important step towards improving heat tolerance in poultry.

LncIRF1 promotes chicken resistance to ALV-J infection.

The pathogenesis of avian leukosis virus subgroup J (ALV-J) is complex and our understanding of it is limited. Based on our previous research, we explored the relationship between ALV-J infection and regulatory factor 1&7 (IRF1 and IRF7), interferon beta (IFNß), and the newly identified long noncoding RNA IRF1 (LncIRF1). LncIRF1 is 1603 nt and exists in the cytoplasm and nucleus. After the occurrence of ALV-J infection, the expression levels of LncIRF1, IRF1, IRF7, and IFNß varied in different chicken tissues. In DF1 cell lines of chicken embryo fibroblast cells (DF1 cells) the expression levels of LncIRF1, IRF7, IRF1, and IFNß increased when ALV-J infection. Similarly, after LncIRF1 overexpression and the ALV-J challenge, the expression levels of IRF1, IRF7, and IFNß increased, while increased LncIRF1 inhibited the proliferation of DF1 cells. Interference with LncIRF1 did not affect IRF1, IRF7, and IFNß. However, expression levels of IRF1, IRF7, and IFNß decreased due to LncIRF1 interference after the ALV-J challenge. An assay of the RNA-binding domain abundant in apicomplexans indicated that most of the proteins bound to LncIRF1 are related to cell proliferation and viral replication and these proteins also interact with IRF1, IRF7, and IFNß. We suggest that LncIRF1 plays an important immunomodulatory role in the anti-ALV-J response. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03773-y.

Implementing real-time immunometabolic assays and immune cell profiling to evaluate systemic immune response variations to Eimeria challenge in three novel layer genetic lines.

Introduction: Evaluating differences in immune responses to Eimeria spp. between poultry genetic lines could be valuable for understanding favorable traits to address coccidiosis, a costly poultry disease. The objective was to compare peripheral blood mononuclear cell (PBMC) immunometabolism and composition during Eimeria challenge in three distinct and highly inbred genetic lines; Leghorn Ghs6, Leghorn Ghs13, and Fayoumi M5.1. Methods: At hatch, 180 chicks (60/ line) were placed in wire-floor cages (10 chicks/cage) and fed a commercial diet. Baseline PBMC were isolated on d21 (10 chicks/line) and 25 chicks/line were inoculated with 10X Merck CocciVac®-B52 (Kenilworth, NJ), creating 6 genetic line × Eimeria groups total. Chicks were euthanized on 1, 3, 7, and 10d post-inoculation (pi; 5 chicks/ line × Eimeria group) for PBMC isolation with body weight and feed intake recorded throughout. Immunometabolic assays to determine PBMC ATP production profiles and glycolytic activity were implemented along with flow cytometric immune cell profiling. Genetic line × Eimeria challenge, and line´challenge fixed effects were analyzed using the MIXED procedure (SAS 9.4; P ≤ 0.05). Results and Discussion: Before inoculation, M5.1 chicks had 14.4-25.4% greater average daily gain (ADG) with 19.0-63.6% increased monocyte/macrophage+, Bu-1+ B cell, and CD3+ T cell populations compared to both Ghs lines (P < 0.0001) but similar immunometabolic phenotype. The Eimeria main effect reduced ADG by 61.3% from 3-7dpi (P = 0.009) except in M5.1 chicks, where no ADG difference due to challenge was found. At 3dpi, Eimeria-challenged M5.1 chicks had 28.9 and 33.2% reduced PBMC CD3+ T cells and CD3+CD8α+ cytotoxic T cells than unchallenged chicks, suggesting early and preferential recruitment from systemic circulation to tissues local to Eimeria challenge (i.e., intestine; P ≤ 0.01). Both Ghs lines displayed 46.4-49.8% T cell reductions at 10dpi with 16.5-58.9% recruitment favoring underlying CD3+CD4+ helper T cells. Immunometabolic responses in Eimeria-challenged Ghs6 and Ghs13 chicks were characterized by a 24.0-31.8% greater proportion of ATP from glycolysis compared to unchallenged counterparts at 10dpi (P = 0.04). These results suggest that variable T cell subtype recruitment timelines in addition to altered systemic immunometabolic requirements may work synergistically to determine favorable immune responses to Eimeria challenge.

Chicken pituitary transcriptomic responses to acute heat stress.

BACKGROUND: Poultry production is vulnerable to increasing temperatures in terms of animal welfare and in economic losses. With the predicted increase in global temperature and the number and severity of heat waves, it is important to understand how chickens raised for food respond to heat stress. This knowledge can be used to determine how to select chickens that are adapted to thermal challenge. As neuroendocrine organs, the hypothalamus and pituitary provide systemic regulation of the heat stress response. METHODS AND RESULTS: Here we report a transcriptome analysis of the pituitary response to acute heat stress. Chickens were stressed for 2 h at 35 °C (HS) and transcriptomes compared with birds maintained in thermoneutral temperatures (25 °C). CONCLUSIONS: The observations were evaluated in the context of ontology terms and pathways to describe the pituitary response to heat stress. The pituitaries of heat stressed birds exhibited responses to hyperthermia through altered expression of genes coding for chaperones, cell cycle regulators, cholesterol synthesis, transcription factors, along with the secreted peptide hormones, prolactin, and proopiomelanocortin.

The immune cell landscape and response of Marek's disease resistant and susceptible chickens infected with Marek's disease virus.

Genetically resistant or susceptible chickens to Marek's disease (MD) have been widely used models to identify the molecular determinants of these phenotypes. However, these prior studies lacked the basic identification and understanding of immune cell types that could be translated toward improved MD control. To gain insights into specific immune cell types and their responses to Marek's disease virus (MDV) infection, we used single-cell RNA sequencing (scRNAseq) on splenic cells from MD resistant and susceptible birds. In total, 14,378 cells formed clusters that identified various immune cell types. Lymphocytes, specifically T cell subtypes, were the most abundant with significant proportional changes in some subtypes upon infection. The largest number of differentially expressed genes (DEG) response was seen in granulocytes, while macrophage DEGs differed in directionality by subtype and line. Among the most DEG in almost all immune cell types were granzyme and granulysin, both associated with cell-perforating processes. Protein interactive network analyses revealed multiple overlapping canonical pathways within both lymphoid and myeloid cell lineages. This initial estimation of the chicken immune cell type landscape and its accompanying response will greatly aid efforts in identifying specific cell types and improving our knowledge of host response to viral infection.

Effects of maternal methionine supplementation on the response of Japanese quail (Coturnix coturnix japonica) chicks to heat stress.

This study investigated the hypothesis that methionine supplementation of Japanese quail (Coturnix coturnix japonica) hens can reduce the effects of oxidative stress and improve the performance of the offspring exposed to heat stress during growth. For that, the quail hens were fed with three diets related to the methionine supplementation: methionine-deficient diet (Md); diet supplemented with the recommended methionine level (Met1); and diet supplemented with methionine above the recommended level (Met2). Their chicks were identified, weighed, and housed according to the maternal diet group from 1 to 14 d of age. On 15 d of age, chicks were weighed and divided into two groups: thermoneutral ambient (constant temperature of 23 °C) and intermittent heat stress ambient (daily exposure to 34 °C for 6 h). Methionine-supplemented (Met1 and Met2) hens had higher egg production, better feed conversion ratio, higher hatchability of total and fertile eggs, and offspring with higher body weight. Supplemented (Met1 and Met2) hens showed greater expression of glutathione synthase (GSS) and methionine sulfoxide reductase A (MSRA) genes, greater total antioxidant capacity, and lower lipid peroxidation in the liver. The offspring of hens fed the Met2 diet had lower death rate (1 to 14 d), higher weight on 15 d of age, weight gain, and better feed conversion ratio from 1 to 14 d of age. Among chicks reared under heat stress, the progeny of methionine-supplemented hens had higher weight on 35 d, weight gain, expression of GSS, MSRA, and thermal shock protein 70 (HSP70) genes, and total antioxidant capacity in the liver, as well as lower heterophil/lymphocyte ratio. Positive correlations between expression of glutathione peroxidase 7 (GPX7) and MSRA genes in hens and offspring were observed. Our results show that maternal methionine supplementation contributes to offspring development and performance in early stages and that, under conditions of heat stress during growth, chicks from methionine-supplemented hens respond better to hot environmental conditions than chicks from nonsupplemented hens. Supplementation of quail hens diets with methionine promoted activation of different metabolic pathways in offspring subjected to stress conditions.

The deficiency of nutrients such as methionine in the diet of birds is affecting fertility rate, egg production, egg weight, and progeny weight. In addition, the maternal environment influences gene expression through epigenetic mechanisms, where the conditions experienced by the parental generation during embryonic development can produce effects on the progeny. This study investigates how methionine supplementation in the diet of quail hens can reduce the effects of oxidative stress and improve the performance of progeny subjected to heat stress during growth. For that, the quail hens were fed with diets containing three different levels of methionine; and their chicks were created (15 on 35 d of age) into thermoneutral and/or intermittent heat stress ambient. It was observed that methionine supplementation in the quail hens had a positive effect on mortality during the initial phase and greater weight gain in the progeny growth phase. In addition, genetic inheritance was observed through the positive correlation between the expression of genes (maternal and progeny) related to oxidative stress. The results show that methionine supplementation in the maternal diet contributes to the development and performance of the progeny when subjected to heat stress during the growth phase.

Genetic Analyses of Response of Local Ghanaian Tanzanian Chicken Ecotypes to a Natural Challenge with Velogenic Newcastle Disease Virus.

Newcastle disease is a devastating poultry disease that often causes significant economic losses in poultry in the developing countries of Africa, Asia, as well as South and Central America. Velogenic Newcastle disease virus (NDV) outbreaks are associated with high mortalities, which can threaten household livelihoods, especially in the rural areas, and lead to loss of high-quality proteins in the form of meat and eggs, as well as household purchasing power. In this study, we exposed unvaccinated Ghanaian and Tanzanian chickens of six local ecotypes to velogenic NDV strains, measured NDV response traits, sequenced their DNA on a genotyping-by-sequencing platform, and performed variance component analyses. The collected phenotypes included: growth rates (pre- and post-exposure); lesion scores (gross lesion severity) in the trachea, proventriculus, intestine, and cecal tonsils; natural antibody levels; anti-NDV antibody levels at 7 days post exposure (dpe); tear and cloacal viral load at 2, 4, and 6 dpe; and survival time. Heritability estimates were low to moderate, ranging from 0.11 for average lesion scores to 0.36 for pre-exposure growth rate. Heritability estimates for survival time were 0.23 and 0.27 for the Tanzanian and Ghanaian ecotypes, respectively. Similar heritability estimates were observed when data were analyzed either separately or combined for the two countries. Survival time was genetically negatively correlated with lesion scores and with viral load. Results suggested that response to mesogenic or velogenic NDV of these local chicken ecotypes could be improved by selective breeding. Chickens that are more resilient to velogenic NDV can improve household livelihoods in developing countries.

Gene expression profiling of RIP2-knockdown in HD11 macrophages - elucidation of potential pathways (gene network) when challenged with avian pathogenic E.coli (APEC).

BACKGROUND: Receptor interacting serine/threonine kinase 2 (RIP2), ubiquitous in many tissue/cell types, is the key regulator of immune and inflammatory responses for many diseases, including avian pathogenic E. coli (APEC), which causes a wide variety of localized or systemic infections. However, the molecular mechanisms by which RIP2 drives its transcriptional program to affect immune and inflammatory response upon APEC infection remains poorly understood. RESULTS: In this study, RNA-seq and bioinformatics analyses were used to detect gene expression and new direct/indirect RIP2 targets in the treatments of wild type HD11 cells (WT), RIP2 knockdown cells (shRIP2), APEC stimulation cells (APEC), and RIP2 knockdown cells combined with APEC infection (shRIP2 + APEC). The results revealed that a total of 4691 and 2605 differentially expressed genes (DEGs) were screened in shRIP2 + APEC vs. APEC and shRIP2 vs. WT, respectively. Functional annotation analysis showed that apoptosis, MAPK, p53, Toll-like receptor, and Nod-like receptor signaling pathways were involved in APEC-induced RIP2 knockdown HD11 cells. By analyzing the enriched pathway and gene networks, we identified that several DEGs, including HSP90AB1, BID, and CASP9 were targeted by RIP2 upon APEC infection. CONCLUSION: As a whole, this study can not only provide data support for constructing gene networks of RIP2 knockdown with APEC challenge but also provide new ideas for improving the immune and inflammatory response.

Transcriptional Regulation of RIP2 Gene by NFIB Is Associated with Cellular Immune and Inflammatory Response to APEC Infection.

Avian pathogenic E. coli (APEC) can cause localized or systemic infection, resulting in large economic losses per year, and impact health of humans. Previous studies showed that RIP2 (receptor interacting serine/threonine kinase 2) and its signaling pathway played an important role in immune response against APEC infection. In this study, chicken HD11 cells were used as an in vitro model to investigate the function of chicken RIP2 and the transcription factor binding to the RIP2 core promoter region via gene overexpression, RNA interference, RT-qPCR, Western blotting, dual luciferase reporter assay, CHIP-PCR, CCK-8, and flow cytometry assay following APEC stimulation. Results showed that APEC stimulation promoted RIP2 expression and cells apoptosis, and inhibited cells viability. Knockdown of RIP2 significantly improved cell viability and suppressed the apoptosis of APEC-stimulated cells. Transcription factor NFIB (Nuclear factor I B) and GATA1 (globin transcription factor 1) binding site was identified in the core promoter region of RIP2 from -2300 bp to -1839 bp. However, only NFIB was confirmed to be bound to the core promoter of RIP2. Overexpression of NFIB exacerbated cell injuries with significant reduction in cell viability and increased cell apoptosis and inflammatory cytokines levels, whereas opposite results were observed in NFIB inhibition treatment group. Moreover, RIP2 was up-regulated by NFIB overexpression, and RIP2 silence mitigated the effect of NFIB overexpression in cell apoptosis, inflammation, and activation of NFκB signaling pathways. This study demonstrated that NFIB overexpression accelerated APEC-induced apoptosis and inflammation via up-regulation of RIP2 mediated downstream pathways in chicken HD11 cells.

Response of three local chicken ecotypes of Ghana to lentogenic and velogenic Newcastle disease virus challenge.

This study was carried out to assess the response of three Ghanaian local chicken ecotypes to LaSota (lentogenic) and virulent field strains of Newcastle disease virus (NDV). Local chickens sampled from the Interior Savannah (IS), Forest (FO) and Coastal Savannah (CS) agro-ecological zones were bred and their offspring were challenged with LaSota NDV at 4 weeks of age. The LaSota challenge was replicated four times with different chicken groups. A total of 1438 chicks comprising 509 Coastal Savannah, 518 Forest and 411 Interior Savannah ecotypes were used. Pre- and post-challenge anti-NDV antibody titre levels were determined via ELISA assays. A second trial was conducted by introducing sick birds infected with virulent NDV to a flock of immunologically naïve chickens at 4 weeks old. Body weights were measured pre- and post-infection. Sex of the chickens was determined using a molecular method. In both trials, there was no significant difference among ecotypes in body weight and growth rate. In the LaSota trial, anti-NDV antibody titre did not differ by ecotype or sex. However, there was a positive linear relationship between body weight and antibody titre. In the velogenic NDV trial, survivability and lesion scores were similar among the three ecotypes. This study confirms that a relatively high dose of LaSota (NDV) challenge has no undesirable effect on Ghanaian local chicken ecotypes. All three Ghanaian local chicken ecotypes were susceptible to velogenic NDV challenge. Resistance to NDV by Ghanaian local chickens appears to be determined more by the individual's genetic makeup than by their ecotype.

Differential immunological response detected in mRNA expression profiles among diverse chicken lines in response to Salmonella challenge.

Salmonella enterica serovar Enteritidis is a bacterial pathogen that contributes to poultry production losses and human foodborne illness. The bacterium elicits a broad immune response involving both the innate and adaptive components of the immune system. Coordination of the immune response is largely directed by cytokines. The objective of the current study was to characterize the expression of a select set of cytokines and regulatory immune genes in three genetically diverse chicken lines after infection with S. Enteritidis. Leghorn, Fayoumi and broiler day-old chicks were orally infected with pathogenic S. Enteritidis or culture medium. At 2 and 18 h postinfection, spleens and ceca were collected and mRNA expression levels for 7 genes (GM-CSF, IL2, IL15, TGF-ß1, SOCS3, P20K, and MHC class IIß) were evaluated by real-time quantitative PCR. Genetic line had a significant effect on mRNA expression levels of IL15, TGF-ß1, SOCS3 and P20K in the spleen and on P20K and MHC class IIß in the cecum. Comparing challenged vs. unchallenged birds, the expression of SOCS3 and P20K mRNA were significantly higher in the spleen and cecum, while MHC class IIß mRNA was significantly lower in spleen. Combining the current RNA expression results with those of previously reported studies on the same samples reveals distinct RNA expression profiles among the three genetic chicken lines and the 2 tissues. This study illustrates that these diverse genetic lines have distinctively different immune response to S. Enteritidis challenge within the spleen and the cecum.

Effect of prenatal ambient temperature on the performance physiological parameters, and oxidative metabolism of Japanese quail (Coturnix coturnix japonica) layers exposed to heat stress during growth.

A strategy to mitigate the negative effects of stress on animals is to enhance their ability to beneficially respond to stressful conditions. This study aimed to assess whether prenatal ambient temperature influences the response of Japanese quail (Coturnix coturnix japonica) chicks to environmental challenges during growth. The experiment was conducted in a 2 × 2 factorial arrangement: two temperature conditions for the mothers (thermoneutral and heat stress by continuous exposure to 32 °C) and two offspring ambient temperature conditions (thermoneutral and heat stress by intermittent exposure to 34 °C for 6 h/day from 15 to 35 days of age). Heat stress in mothers led to lower laying rate, egg mass, expression of methionine sulfoxide reductase A (MSRA) gene, and antioxidant capacity as well as higher chick mortality rate (1-15 days of age). Maternal heat stress led to lower weight gain and total antioxidant capacity and higher feed conversion ratio. Maternal temperature × Offspring temperature interaction effects were observed on carbonylated protein content and HSP70, GSS, and MSRA gene expression. It was observed that, for chicks hatched from heat-stressed mothers, exposure to heat stress led to higher carbonylated protein content and HSP70 expression than exposure to thermoneutral conditions. Maternal heat stress was also responsible for increasing GSS expression in chicks grown under thermoneutral conditions. Chicks hatched from non-stressed mothers and subjected to heat stress had higher MSRA expression compared to chicks maintained in a thermoneutral environment. Our results show that, although maternal heat stress had no negative effects on performance or oxidative metabolism of offspring grown under thermoneutral conditions, it was associated with lower performance and higher protein oxidation in offspring exposed to heat stress during growth. These results could be due in part to alterations in the expression of genes related to antioxidant capacity.

Comparison of cellular immune responses to avian influenza virus in two genetically distinct, highly inbred chicken lines.

Low pathogenicity avian influenza causes mild disease involving the respiratory, gastrointestinal, and reproductive systems of wild and domestic birds. Avian influenza research often emphasizes the effect of the virus genetics on disease, but the influence of host genetics on resistance to infection is not well understood. The genetic determinants of enhanced resistance to influenza can be explored by using genetically distinct, highly inbred chicken lines that differ in susceptibility to influenza. In this study, we compared the mucosal cellular immune responses between the relatively resistant Fayoumi M43 chicken line and the relatively susceptible Leghorn GB2 chicken line after challenging with low pathogenicity avian influenza virus (LPAIV) H6N2. The birds were inoculated at 21 days of age with 107 50 % egg infective dose (EID50) LPAIV H6N2 via nasal and tracheal routes in two separate experiments. Clinical signs were recorded, tracheal swabs were collected to measure viral titer, and tracheas and lungs were harvested for flow cytometric analysis of macrophage, B cell, and T cell populations at 4 days post-infection (dpi) (Experiments 1 and 2) and 6 dpi (Experiment 2). Blood and tears were also collected at 7 and 14 dpi (Experiment 1) to measure antibody levels. Compared to both the non-challenged Fayoumis and the relatively susceptible Leghorn chickens, relatively resistant Fayoumi chickens challenged with LPAIV demonstrated enhanced MHC class I expression on antigen-presenting cells and increased macrophage, B cell, and T cell frequencies in the trachea, which were associated with reduced tracheal viral titers at 4 dpi. In contrast, MHC class I expression and immune cell frequencies in the trachea were not different between challenged Leghorns and non-challenged Leghorns. Furthermore, Leghorns shed higher virus titers in their trachea compared to Fayoumis. Challenged Fayoumis and Leghorns both produced AIV-specific IgY detected in the serum and tears, but AIV-specific IgA was not detected in the tears. In this study, we provide new insight into immune mechanisms of enhanced resistance to avian influenza in chickens, which may lead to improved vaccination strategies and breeding programs.

Distinct transcriptomic response to Newcastle disease virus infection during heat stress in chicken tracheal epithelial tissue.

Newcastle disease (ND) has a great impact on poultry health and welfare with its most virulent (velogenic) strain. In addition, issues exacerbated by the increase in global temperatures necessitates a greater understanding of the host immune response when facing a combination of biotic and abiotic stress factors in poultry production. Previous investigations have revealed that the host immune response is tissue-specific. The goal of this study was to identify genes and/or signaling pathways associated with immune response to NDV (Newcastle disease virus) in the trachea, an essential organ where NDV replicate after the infection, by profiling the tissue specific transcriptome response in two genetically distinct inbred chicken lines when exposed to both abiotic and biotic stressors. Fayoumis appear to be able to respond more effectively (lower viral titer, higher antibody levels, immune gene up-regulation) and earlier than Leghorns. Our results suggest NDV infection in Fayoumis appears to elicit proinflammatory processes, and pathways such as the inhibition of cell viability, cell proliferation of lymphocytes, and transactivation of RNA, more rapidly than in Leghorns. These differences in immune response converge at later timepoints which may indicate that Leghorns eventually regulate its immune response to infection. The profiling of the gene expression response in the trachea adds to our understanding of the chicken host response to NDV infection and heat stress on a whole genome level and provides potential candidate genes and signaling pathways for further investigation into the characterization of the time-specific and pathway specific responses in Fayoumis and Leghorns.

Role of the chicken oligoadenylate synthase-like gene during in vitro Newcastle disease virus infection.

The enzyme 2'-5' oligoadenylate synthase (OAS) is one of the key interferon-induced antiviral factors that act through inhibition of viral replication. In chickens, there is a single well-characterized OAS gene, oligoadenylate synthase-like (OASL) that has been shown to be upregulated after infection with various viruses. However, a deeper understanding of how chicken OASL acts against viral infection is still necessary. In this study, we tested the hypothesis that OASL short interfering RNA (siRNA)-mediated knockdown would decrease the host gene expression response to the Newcastle disease virus (NDV) by impacting antiviral pathways. To assess our hypothesis, a chicken fibroblast cell line (DF-1) was infected with the NDV (LaSota strain) and OASL expression was knocked down using a specific siRNA. The level of NDV viral RNA in the cells and the expression of interferon response- and apoptosis-related genes were evaluated by quantitative PCR at 4, 8, and 24 h postinfection (hpi). Knockdown of OASL increased the level of NDV viral RNA at 4, 8, and 24 hpi (P < 0.05) and eliminated the difference between NDV-infected and noninfected cells for expression of interferon response- and apoptosis-related genes (P > 0.05). The lack of differential expression suggests that knockdown of OASL resulted in a decreased response to NDV infection. Within NDV-infected cells, OASL knockdown reduced expression of signal transducer and activator of transcription 1, interferon alfa receptor subunit 1, eukaryotic translation initiation factor 2 alpha kinase 2, ribonuclease L, caspase 8 (CASP8) and caspase 9 (CASP9) at 4 hpi, CASP9 at 8 hpi, and caspase 3, CASP8, and CASP9 at 24 hpi (P < 0.05). We suggest that the increased NDV viral load in DF-1 cells after OASL knockdown was the result of a complex interaction between OASL and interferon response- and apoptosis-related genes that decreased host response to the NDV. Our results provide comprehensive information on the role played by OASL during NDV infection in vitro. Targeting this mechanism could aid in future prophylactic and therapeutic treatments for Newcastle disease in poultry.

Genetic resistance to avian pathogenic Escherichia coli (APEC): current status and opportunities.

Infections with avian pathogenic Escherichia coli (APEC) can be extremely detrimental to poultry health and production. Investigating host genetic variation could identify the biological mechanisms that control resistance to this pathogen and allow selection for improved resistance in experimental and commercial poultry populations. In this review, the current knowledge of how host genetics contributes to APEC resistance and future opportunities that would benefit the understanding or application of genetic resistance are discussed. Phenotypes, such as antibody responses, lesion scores, and mortality, revealed that genetic background impacts APEC resistance and interacts with other factors including the environment and challenge conditions. Experiments have used divergent selection for APEC-specific antibody levels to facilitate genetic studies, estimated heritabilities in relevant traits, detected quantitative trait loci using microsatellites, and made associations with sequence variation in the major histocompatibility complex, which collectively suggest that improving APEC resistance through selection is feasible, although genetic control is partial, complex, and highly polygenic. Additionally, functional genomics techniques have identified antimicrobial responses, toll-like receptor and cytokine signalling, and the cell cycle as central pathways in the host response to APEC challenge. Opportunities for future research are discussed, including the expansion of existing lines of research and the application of new technologies that are relevant to the study of host genetics and APEC. This review closes with prospective strategies for improvement of host genetic resistance to APEC.

Transcriptome Response of Liver and Muscle in Heat-Stressed Laying Hens.

Exposure to high ambient temperature has detrimental effects on poultry welfare and production. Although changes in gene expression due to heat exposure have been well described for broiler chickens, knowledge of the effects of heat on laying hens is still relatively limited. In this study, we profiled the transcriptome for pectoralis major muscle (n = 24) and liver (n = 24), during a 4-week cyclic heating experiment performed on layers in the early phase of egg production. Both heat-control and time-based contrasts were analyzed to determine differentially expressed genes (DEGs). Heat exposure induced different changes in gene expression for the two tissues, and we also observed changes in gene expression over time in the control animals suggesting that metabolic changes occurred during the transition from onset of lay to peak egg production. A total of 73 DEGs in liver were shared between the 3 h heat-control contrast, and the 4-week versus 3 h time contrast in the control group, suggesting a core set of genes that is responsible for maintenance of metabolic homeostasis regardless of the physiologic stressor (heat or commencing egg production). The identified DEGs improve our understanding of the layer's response to stressors and may serve as targets for genetic selection in the future to improve resilience.

Transcriptional response of blood leukocytes from turkeys challenged with Salmonella enterica serovar Typhimurium UK1.

Non-typhoidal Salmonella is one of the most common causes of bacterial foodborne disease and consumption of contaminated poultry products, including turkey, is one source of exposure. Minimizing Salmonella colonization of commercial turkeys could decrease the incidence of Salmonella-associated human foodborne illness. Understanding host responses to these bacteria is critical in developing strategies to minimize colonization and reduce food safety risk. In this study, we evaluated bacterial load and blood leukocyte transcriptomic responses of 3-week-old turkeys challenged with the Salmonella enterica serovar Typhimurium (S. Typhimurium) UK1 strain. Turkeys (n = 8/dose) were inoculated by oral gavage with 108 or 1010 colony forming units (CFU) of S. Typhimurium UK1, and fecal shedding and tissue colonization were measured across multiple days post-inoculation (dpi). Fecal shedding was 1-2 log10 higher in the 1010 CFU group than the 108 CFU group, but both doses effectively colonized the crop, spleen, ileum, cecum, colon, bursa of Fabricius and cloaca without causing any detectable clinical signs in either group of birds. Blood leukocytes were isolated from a subset of the birds (n = 3-4/dpi) both pre-inoculation (0 dpi) and 2 dpi with 1010 CFU and their transcriptomic responses assayed by RNA-sequencing (RNA-seq). At 2 dpi, 647 genes had significant differential expression (DE), including large increases in expression of immune genes such as CCAH221, IL4I1, LYZ, IL13RA2, IL22RA2, and ACOD1. IL1ß was predicted as a major regulator of DE in the leukocytes, which was predicted to activate cell migration, phagocytosis and proliferation, and to impact the STAT3 and toll-like receptor pathways. These analyses revealed genes and pathways by which turkey blood leukocytes responded to the pathogen and can provide potential targets for developing intervention strategies or diagnostic assays to mitigate S. Typhimurium colonization in turkeys.

Mitochondrial and Glycolytic Capacity of Peripheral Blood Mononuclear Cells Isolated From Diverse Poultry Genetic Lines: Optimization and Assessment.

Cellular metabolic preference is a culmination of environment, nutrition, genetics, and individual variation in poultry. The Seahorse XFe24 analyzer was used to generate foundational immune cellular metabolic data in layer, broiler, and legacy genetic strains using fresh chicken peripheral blood mononuclear cells (PBMCs). Baseline mitochondrial respiration [oxygen consumption rate (OCR)] and glycolytic activity [extracellular acidification rate (ECAR)] were determined in modern commercial laying hen (Bovans White) and broiler (Ross 308) lines, as well as the highly inbred lines of Iowa State University (L8, Fayoumi M-15.2, Spanish, Ghs-6), partially inbred broiler line, and advanced intercrosses of broiler by Fayoumi M-15.2 and broiler by Leghorn lines. Commercial broiler vs. Bovans layer and unvaccinated vs. vaccinated Bovans layer immune cell metabolic potential were compared following an in-assay pathway inhibitor challenge. Titrations consistently showed that optimal PBMC density in laying hens and broilers was 3 million cells per well monolayer. Assay media substrate titrations identified 25 mM glucose, 1 mM glutamine, and 1 mM sodium pyruvate as the optimal concentration for layer PBMCs. Pathway inhibitor injection titrations in Bovans layers and broilers showed that 0.5 µM carbonyl cyanide-4 phenylhydrazone (FCCP) and 1 µM oligomycin were optimal. Baseline OCR and ECAR were significantly affected by genetic line of bird (p < 0.05), with the dual-purpose, L8 inbred line showing the highest OCR (mean 680 pmol/min) and the partially inbred broiler line showing the greatest ECAR (mean 74 mpH/min). ECAR metabolic potential tended to be greater in modern layers than broilers (p < 0.10), indicating increased ability to utilize the glycolytic pathway to produce energy. OCR was significantly higher in vaccinated than unvaccinated hens (p < 0.05), while baseline ECAR values were significantly lower in vaccinated Bovans laying hens, showing increased oxidative capacity in activated immune cells. These baseline data indicate that different genetic strains of birds utilized the mitochondrial respiration pathway differently and that modern commercial lines may have reduced immune cell metabolic capacity compared with legacy lines due to intense selection for production traits. Furthermore, the Seahorse assay demonstrated the ability to detect differences in cellular metabolism between genetic lines and immune status of chickens.