Expression of Inflammatory and Cell Death Program Genes and Comet DNA Damage Assay Induced by Escherichia coli in Layer Hens.

Modern methods of industrial poultry and egg production systems involve stressful practices that stimulate Escherichia coli (E. coli) activity causing endotoxic shock. This investigation was conducted to evaluate the expression of pro-inflammatory cytokines and cell death program genes and DNA damage induced by E. coli in the brain and liver tissues of laying hens. A total of two hundred and ten H&N brown layer hens with 20 week age, were used in this research. First, preliminary experiments were designed (60 hens in total) to establish the optimal exposure dose of E. coli and to determine the nearest time of notable response to be used in the remainder studies of this research. At 35-wk of age, 150 hens were randomly assigned into 2 groups with 3 replicates of 25 birds each; the first group was injected in the brachial wing vein with 107 E. coli colony/hen, while the second group was injected with saline and served as a control. The body temperature and plasma corticosterone concentration were measured 3 hr after injection. Specimens of liver and brain were obtained from each group and the gene expression of p38 mitogen-activated protein kinase, interlukin-1β (IL-1β), tumor necrosis factor alpha (TNF-α), Bax, and caspase-3 genes were measured by quantitative real-time PCR. DNA damage in the brain and liver tissues were also measured by comet assay. Hens treated with E. coli showed significant (P<0.05) increase of body temperature and plasma corticosterone (42.6°C and 14.5 ng/ml, respectively) compared to the control group (41.1°C and 5.5 ng/ml, respectively). Additional remarkable over-inflammation gene expression of p38, IL-1β and TNF-α.genes were also detected in the brain (2.2-fold, 2.0-fold and 3.3-fold, respectively) and the liver (2.1-fold, 1.9-fold and 3.0-fold, respectively) tissues of the infected chickens. It is also important to note that hens injected with E. coli showed an increase in DNA damage in the brain and liver cells (P<0.05). These results were synchronized with activating cell death program since our data showed significant high expression of Bax gene by 2.8- and 2.7-fold and caspase-3 gene by 2.5- and 2.7-fold in the brain and liver tissues of infected chickens, respectively (P<0.05). In conclusion, the current study indicates that E. coli injection induces inflammatory physiological response and triggers cell death program in the brain and liver. Our results provide more understanding to endotoxic shock by E. coli in chickens at cellular level. Further studies are required to confirm if such responses are destructive or protective to set the means through which a chicken mounts a successful defense against avian pathogenic E. coli

Propolis alleviates the negative effects of heat stress on egg production, egg quality, physiological and immunological aspects of laying Japanese quail.

The present work was carried out to investigate the effects of dietary propolis supplementation to laying Japanese quail (Coturnix coturnix japonica) on egg production, egg quality, physiological and immunological aspects under heat stress conditions. A total of 200, 21-day-old, Japanese quail females were distributed equally into standard wired cages in two identical environmentally-controlled rooms (10 cages per room, 10 birds per cage). From 29-70 d of age, the quail birds in the first room remained at a normal temperature of 24°C (C group), whereas the quail birds in the second room were kept under heat stress at 35°C (HS group). Each group was further assigned to 2 propolis subgroups (5 cages per subgroup); one of them received a basal diet without propolis supplementation (-PR subgroup), while, the other received 1 g propolis/ kg basal diet (+PR subgroup). In the present study, performance and egg production of laying quail were significantly (P<0.001) impaired by HS treatment and improved by the PR treatment. Similarly, the negative and positive effects of HS and PR, respectively, were appeared on the egg shell thickness and yolk index. Stress indicators in laying quail were significantly (P<0.001) increased by HS, while, PR significantly (P<0.05) moderated these levels in the HS+PR group when compared to the HS-PR quail group. In addition to the positive impact of PR on the plasma levels of calcium, phosphorus, and albumin, it also normalized the plasma levels of alanine aminotransferase and cholesterol in the heat-stressed quail birds. Moreover, the quail birds in the HS groups expressed lower immunological aspects than those in the C group, while, the addition of propolis to the diets enhanced the immune status of laying quail birds under HS conditions. These results strongly suggest that dietary propolis supplementation could be a successful attempt to maintain the performance and egg production of laying Japanese quail at convenient levels under heat stress conditions

Effect of <i>E</i>. <i>coli</i> on the relative expression of protein kinase <i>p38</i> gene in the brain and liver tissues of chickens.

<p>^a,b Mean values within tissue with unlike superscript letters are significantly different (P<0.05).</p

Effect of <i>E</i>. <i>coli</i> on the relative expression of cell death program genes <i>Bax</i> (A) and <i>caspase-3</i> (B) in brain and liver tissues of chickens.

<p>^a,b Mean values within tissue with unlike superscript letters are significantly different (P<0.05).</p

Details of primers used for real-time PCR quantitative analysis.

<p>Details of primers used for real-time PCR quantitative analysis.</p

Visual score of DNA damage in brain and liver tissues of chicken treated with <i>E</i>. <i>coli</i> using comet assay.

<p>Visual score of DNA damage in brain and liver tissues of chicken treated with <i>E</i>. <i>coli</i> using comet assay.</p

Expression of Inflammatory and Cell Death Program Genes and Comet DNA Damage Assay Induced by <i>Escherichia coli</i> in Layer Hens

<div><p>Modern methods of industrial poultry and egg production systems involve stressful practices that stimulate <i>Escherichia coli</i> (<i>E</i>. <i>coli</i>) activity causing endotoxic shock. This investigation was conducted to evaluate the expression of pro-inflammatory cytokines and cell death program genes and DNA damage induced by <i>E</i>. <i>coli</i> in the brain and liver tissues of laying hens. A total of two hundred and ten H&N brown layer hens with 20 week age, were used in this research. First, preliminary experiments were designed (60 hens in total) to establish the optimal exposure dose of <i>E</i>. <i>coli</i> and to determine the nearest time of notable response to be used in the remainder studies of this research. At 35-wk of age, 150 hens were randomly assigned into 2 groups with 3 replicates of 25 birds each; the first group was injected in the brachial wing vein with 10⁷ <i>E</i>. <i>coli</i> colony/hen, while the second group was injected with saline and served as a control. The body temperature and plasma corticosterone concentration were measured 3 hr after injection. Specimens of liver and brain were obtained from each group and the gene expression of <i>p38</i> mitogen-activated protein kinase, interlukin-1β (<i>IL-1β</i>), tumor necrosis factor alpha (<i>TNF-α</i>), <i>Bax</i>, and <i>caspase-3</i> genes were measured by quantitative real-time PCR. DNA damage in the brain and liver tissues were also measured by comet assay. Hens treated with <i>E</i>. <i>coli</i> showed significant (P<0.05) increase of body temperature and plasma corticosterone (42.6°C and 14.5 ng/ml, respectively) compared to the control group (41.1°C and 5.5 ng/ml, respectively). Additional remarkable over-inflammation gene expression of <i>p38</i>, <i>IL-1β</i> and <i>TNF-α</i>.genes were also detected in the brain (2.2-fold, 2.0-fold and 3.3-fold, respectively) and the liver (2.1-fold, 1.9-fold and 3.0-fold, respectively) tissues of the infected chickens. It is also important to note that hens injected with <i>E</i>. <i>coli</i> showed an increase in DNA damage in the brain and liver cells (P<0.05). These results were synchronized with activating cell death program since our data showed significant high expression of <i>Bax</i> gene by 2.8- and 2.7-fold and <i>caspase-3</i> gene by 2.5- and 2.7-fold in the brain and liver tissues of infected chickens, respectively (P<0.05). In conclusion, the current study indicates that <i>E</i>. <i>coli</i> injection induces inflammatory physiological response and triggers cell death program in the brain and liver. Our results provide more understanding to endotoxic shock by <i>E</i>. <i>coli</i> in chickens at cellular level. Further studies are required to confirm if such responses are destructive or protective to set the means through which a chicken mounts a successful defense against avian pathogenic <i>E</i>. <i>coli</i>.</p></div

Comprehensive growth performance, immune function, plasma biochemistry, gene expressions and cell death morphology responses to a daily corticosterone injection course in broiler chickens

<div><p>The massive meat production of broiler chickens make them continuously exposed to potential stressors that stimulate releasing of stress-related hormones like corticosterone (CORT) which is responsible for specific pathways in biological mechanisms and physiological activities. Therefore, this research was conducted to evaluate a wide range of responses related to broiler performance, immune function, plasma biochemistry, related gene expressions and cell death morphology during and after a 7-day course of CORT injection. A total number of 200 one-day-old commercial Cobb broiler chicks were used in this study. From 21 to 28 d of age, broilers were randomly assigned to one of 2 groups with 5 replicates of 20 birds each; the first group received a daily intramuscular injection of 5 mg/kg BW corticosterone dissolved in 0.5 ml ethanol:saline solution (CORT group), while the second group received a daily intramuscular injection of 0.5 ml ethanol:saline only (CONT group). Growth performance, including body weight (BW), daily weight gain (DG), feed intake (FI) and feed conversion ratio (FC), were calculated at 0, 3 and 7 d after the start of the CORT injections. At the same times, blood samples were collected in each group for hematological (TWBC’s and H/L ratio), T- and B-lymphocytes proliferation and plasma biochemical assays (total protein, TP; free triiodothyronine hormone, fT₃; aspartate amino transaminase, AST; and alanine amino transaminase, ALT). The liver, thymus, bursa of Fabricius and spleen were dissected and weighed, and the mRNA expression of insulin-like growth factor 1 gene (IGF-1) in liver and cell-death-program gene (caspase-9) in bursa were analyzed for each group and time; while the apoptotic/necrotic cells were morphologically detected in the spleen. From 28 to 35 d of age, broilers were kept for recovery period without CORT injection and the same sampling and parameters were repeated at the end (at 14 d after initiation of the CORT injection). In general, all parameters of broiler performance were negatively affected by the CORT injection. In addition, CORT treatment decreased the plasma concentration of fT₃ and the mRNA expression of hepatic IGF-1. A significant increase in liver weight accompanied by an increase in plasma TP, AST and ALT was observed with CORT treatment, indicating an incidence of liver malfunction by CORT. Moreover, the relative weights of thymus, bursa and spleen decreased by the CORT treatment with low counts of TWBC’s and low stimulation of T & B cells while the H/L ratio increased; indicating immunosuppressive effect for CORT treatment. Furthermore, high expression of caspase-9 gene occurred in the bursa of CORT-treated chickens, however, it was associated with a high necrotic vs. low apoptotic cell death pathway in the spleen. Seven days after termination of the CORT treatment in broilers, most of these aspects remained negatively affected by CORT and did not recover to its normal status. The current study provides a comprehensive view of different physiological modulations in broiler chickens by CORT treatment and may set the potential means to mount a successful defense against stress in broilers and other animals as well.</p></div

Details of primers used for real-time PCR quantitative analysis.

<p>Details of primers used for real-time PCR quantitative analysis.</p