S. Shini

Understanding stress-induced immunosuppression: Exploration of cytokine and chemokine gene profiles in chicken peripheral leukocytes

At present, the poultry meat and egg industry has gained a lot of ground, being viewed as a provider of a healthy alternative to red meat and other protein sources. If this trend is to be maintained, solutions must be found to improve resistance of chickens to disease, which often is weakened by stressful conditions. In poultry, stress-induced immunosuppression is manifested by failures in vaccination and increased morbidity and mortality of flocks. Currently, several modern cellular and molecular approaches are being used to explore the status of the immune system during stress and disease. It is likely that these new techniques will lead to the development of new strategies for preventing and controlling immunosuppression in poultry. Using quantitative reverse transcription-PCR assays, a broad spectrum of cytokine, chemokine, and their receptor genes can be quantified in birds and then be used as markers to assess the effects of stress on the immune system. Currently, we are investigating immune and endocrine interactions in the chicken, in particular the cells and molecules that are known to be involved in such interactions in mammals. We have evaluated the effects of corticosterone administration in drinking water on peripheral lymphocyte and heterophil cytokine and chemokine gene profiles. In particular, there seems to be effects on cytokine and chemokine mRNA expression levels in both lymphocytes and heterophils, especially expression of the proinflammatory cytokines interleukin (IL)-1beta, IL-6, and IL-18 and chemokines C-C motif, ligand 1 inflammatory (CCLi1); C-C motif, ligand 2 inflammatory (CCLi2); C-C motif, ligand 5 (CCL5); C-C motif, ligand 16 (CCL16); C-X-C motif ligand 1 inflammatory (CXCLi1); and C-X-C motif ligand 2 inflammatory (CXCLi2), which are initially upregulated and are potentially involved in modulating the adaptive immune response. A chronic treatment with corticosterone downregulates proinflammatory cytokines and chemokines, suggesting that the delayed effects of chronic stress can suppress the immune response. Messenger RNA expression levels of transforming growth factor-beta4 (TGF-beta4) are also upregulated in cortisosterone-treated birds. It appears that the balance between T-helper (Th) 1 and Th2/T regulatory cytokine production is altered in conditions associated with significant changes in plasma corticosterone concentration. Experiments are underway to decipher the cytokine and chemokine responses to vaccination and bacterial challenge on the background of stress-induced immunosuppression.

Effects of stress, mimicked by administration of corticosterone in drinking water, on the expression of chicken cytokine and chemokine genes in lymphocytes

In this study, we identify molecular mediators that participate in the regulation of the immune response during corticosterone-induced stress in chickens. At 7 weeks of age, 120 chickens were exposed for 1 week to corticosterone treatment. Cytokine and chemokine mRNA expression levels were evaluated in peripheral blood and splenic lymphocytes. Expression levels of interleukin (IL)-1β, IL-6, IL-18 and transforming growth factor (TGF)-β4 mRNA were significantly up-regulated in lymphocytes 3 h after first treatment with corticosterone. TGF-β4 and IL-18 remained elevated 1 week post-initial treatment. Compared with controls, corticosterone-treated birds showed greater expression levels of chemokine (CC) mRNA, particularly for CCLi2, CCL5 (RANTES), CCL16 and CXCLi1, in peripheral and splenic lymphocytes 3 h post-initial exposure. CCLi2 mRNA was highly expressed in splenocytes at all time-points. Administration of corticosterone significantly increased circulating corticosterone concentrations and decreased total lymphocyte counts at 3, 24 h and 1 week post-initiation of corticosterone treatment. There was a positive correlation between plasma corticosterone concentrations and CCL5 and CCL16 mRNA at 3 h post-initial administration. At 1 week post-initial treatment, corticosterone concentrations correlated positively with CCL5 and negatively with IL-18 mRNA level. Conditions associated with significant changes in corticosterone levels might therefore affect the immune response by increasing pro-inflammatory responses, leading to potential modulation of the Th1/Th2 balance.

Cytokine and chemokine gene expression profiles in heterophils from chickens treated with corticosterone

In chickens, corticosterone is the end-product of stress. However, the nature of the immune response to elevated plasma corticosterone concentrations at the molecular level has not yet been characterised. We recently demonstrated that exposure to corticosterone in drinking water for 1 week significantly upregulates mRNA expression levels for the pro-inflammatory interleukins (IL)-1β, IL-6, IL-18 and the pro-inflammatory chemokine CCLi2 in chicken lymphocytes, particularly 3 h after the treatment started. In the present study, we investigated cytokine and chemokine mRNA expression levels in circulating heterophils of chickens, and show that at 3 h post initial treatment with corticosterone in drinking water (20 mg/1L) the mRNA expression levels for IL-1β, IL-6, IL-10, IL-12α and IL-18 are upregulated. The mRNA expression levels for IL-6, IL-10 and IL-18 correlate with plasma corticosterone concentration and total heterophil counts. Corticosterone downregulated the expression levels of all pro-inflammatory cytokines at 24 h and 1 week post-treatments. Repeated treatment with corticosterone upregulated mRNA expression levels of transforming growth factor-β4 and the chemokine CCL16. These data indicate that cytokine and chemokine gene expression signatures in chicken heterophils can be altered during stress and therefore could be used as an indicator of stress.

Physiological responses of Australian Merino wethers exposed to high heat load

Twelve 9-mo-old Merino wethers (30.4 ± 3.2 kg of BW) were used in a crossover study to investigate the heat tolerance of Australian Merino sheep by testing their physiological responses to repeated heat loads that occurred during summer months. Wethers were randomly divided into 2 groups of 6 wethers each, housed individually in an environmental chamber, and subjected to 2 d of thermoneutral conditions (TNC) followed by either 7 d of TNC (maximum temperature of 24°C, minimum temperature of 16°C) or 7 d of hot conditions (maximum temperature of 38°C, minimum temperature of 28°C), and then 2 d of TNC. These treatments were applied in 2 replicates, with each replicate in a separate environmental chamber. Rectal temperature (RT) and respiration rate were measured daily at 0600, 0800, 1000, 1200, 1400, 1600, and 1800 h. Feed and water intakes were measured daily, and wethers were weighed on d 1 and 11. Blood samples were collected from each whether on d 2 and 6, and serum was assayed for concentrations of creatine, glucose, total protein, cholesterol, NEFA, calcium, sodium, and potassium. Exposure to a high ambient temperature resulted in an 0.8°C increase in RT (P < 0.001), an increase in respiration rate (P < 0.001) by 66 breaths/min, and a 2.7 L/d increase in water intake (P < 0.0001). Feed intake decreased by 22% (P < 0.0001), BW decreased by 5.2% (P < 0.03), and creatine concentration was reduced (P < 0.05). No differences (P > 0.05) between treatments were observed for any of the remaining serum variables. These results indicate that Australian Merino sheep were able to maintain RT within the normal range during exposure to a prolonged increase in heat and that they recovered quickly from the negative effect of heat stress within 2 d of conditions returning to TNC. It would appear that they have a high heat tolerance, and further studies are needed to examine the effects of a greater heat load to determine the temperature-humidity index thresholds for Australian Merino sheep.

Effect of various doses of injected selenium on performance and physiological responses of sheep to heat load

The aim of the study was to determine the effects of various doses of injected Se on the physiological responses of sheep to heat load. Fifteen 9-mo-old Australian Merino wethers (mean BW = 27.2 ± 2.1 kg) were randomly assigned to 1 of 3 treatments: 0 (control), 0.5, and 5 mg of Se, which was administered as a subcutaneous sodium selenate injection (5 mg/mL Se) on d 1, 8, and 15 of exposure to heat stress. The animals were housed individually in an environmental chamber and exposed to high temperature from 0700 to 1800 h (maximum = 38°C; minimum = 24°C) and to thermoneutral temperature from 1800 to 0700 h (maximum = 24°C; minimum = 20°C) for 21 d. Rectal temperature (RT) and respiration rate (RR) were measured daily at 0800, 1200, and 1600 h. Feed intake was measured daily, and sheep were weighed on d 1, 8, 15, and 21. Blood samples were collected on d 1 and 21. The 5 mg Se treatment decreased RT by 0.3°C (P = 0.02) and BW loss by 4.5% (P < 0.05) and increased eosinophil count (P < 0.05). There were no differences (P > 0.05) between treatments in RR and DMI, serum concentrations of glucose, total protein, cholesterol, and NEFA or in blood hematology variables. The findings of this study have important implications for the sheep industry. Further studies are warranted to elucidate the dynamics of Se on productivity and health during hot conditions.

Effects of housing conditions during the rearing and laying period on adrenal reactivity, immune response and heterophil to lymphocyte (H/L) ratios in laying hens

This study was conducted to evaluate the effect of early rearing conditions on physiological, haematological and immunological responses relevant to adaptation and long-term stress in white Leghorn hens with intact beaks housed in furnished cages (FC) or conventional cages (CC) during the laying period. Pullets were cage reared (CR) or litter floor reared (FR). From 16 to 76 weeks of age, hens were housed in FC (eight hens per cage) or in CC (three hens per cage). As measures of long-term stress at the end of the laying period, adrenal reactivity was quantified by assessing corticosterone responses to adrenocorticotropin challenge, and immune response was assessed by measuring antibody responses after immunization with sheep red blood cells (SRBC) and keyhole limpet haemocyanin (KLH). Heterophil to lymphocyte (H/L) ratio was employed as an indicator of stress. Rearing conditions significantly affected anti-SRBC titres (P < 0.0001) and tended to affect H/L ratios (P = 0.07), with the highest values found in FR hens. Layer housing affected H/L ratio (P < 0.01); the highest ratio was found in FR birds housed in FC during the laying period. This study shows that early rearing environment affects immunological indicators that are widely used to assess stress in laying hens. However, while results on H/L ratio indicated that FR birds experienced more stress particularly when they were housed in FC during the laying period, the immune responses to SRBC in FR hens was improved, indicating the opposite. This contradiction suggests that the effects on immune response may have been associated with pathogenic load due to environmental complexity in FR and FC hens rather than stress due to rearing system or housing system per se.

Effects of selenium and vitamin E on performance, physiological response, and selenium balance in heat-stressed sheep

Forty-two 7-mo-old Australian Merino wethers were used in a 50-d trial to investigate the effects of Se and vitamin E on the performance and physiological responses of heat-stressed sheep. Sheep were exposed to thermoneutral conditions (maximum = 24°C and minimum = 20°C) for 28 d followed by heat (maximum = 38°C and minimum = 28°C) for 22 d. Hot conditions were imposed between 0700 and 1800 h. Sheep were randomly allocated to diets containing 0.8 mg/kg Se (Sel-Plex), 150 mg/kg vitamin E, or 0.8 mg/kg Se and 150 mg/kg vitamin E for either the duration of the study (50 d) or from d 1 of the hot period until the end of the study. A control group that received no supplemental Se and vitamin E for the duration of the study was included. Feed intake was measured daily and sheep were weighed weekly. Blood samples were collected from all sheep before feeding on d 1, 21, and 49 for measurement of biochemical and enzymatic variables. The concentration of Se was determined in offered and refused feed, feces, urine, water, plasma, liver, and kidneys. Exposure to heat reduced ( < 0.05) DMI by 11.9%, ADG by 198 g, serum concentration of urea nitrogen and Se by 17.8%, and plasma total antioxidant status by 26.4%. During hot conditions, sheep receiving Se and vitamin E supplements for 50 d had reduced ( < 0.05) BW loss and elevated G:F compared to control sheep. Serum Se concentration and the plasma total antioxidant status were greatest in sheep receiving Se and vitamin E supplements for 50 d ( < 0.05). These results indicate that dietary supplementation with Se and vitamin E reduces the adverse effects of a high heat load. Additional studies are warranted to elucidate the mechanisms responsible for these effects.

The potential for probiotics to prevent reproductive tract lesions in free-range laying hens

A study was undertaken to investigate the ability of two commercial probiotics applied in free-range laying hens (from 18 to 22 weeks of age) in reducing the occurrence of reproductive tract pathologies, and improving hen health and performance. In all, 630 17-week-old brown layers were transferred to a freshly cleaned free-range laying facility, and randomly divided into three groups, with three replicates of 70 birds each. Both probiotics were administered in the drinking water (Groups 1 and 2) on a daily basis for 4 weeks, while Group 3 was left untreated. At 38 weeks of age, the results demonstrated that treatment with either probiotic significantly reduced the occurrence of reproductive tract pathologies (control vs probiotics, 33% vs 22% and 11%; P < 0.01), mortalities (control vs probiotics; 3.8% vs 1.5 and 1.9%; P < 0.01), and increased the performance of hens, for another 20 weeks post-treatments (hen day production for control vs probiotics 75% vs 90% and 94%; P < 0.01). Birds treated with probiotics maintained their bodyweight and egg weights at standard ranges, while untreated birds did not perform at this level. Although we were unable to show any effect on cloacal bacterial colonisation, the results of the present study provided some initial evidence that reproductive pathologies that often cause drops in egg production and sudden deaths of birds, can be reduced if free range hens are treated with a commercial probiotic before or during the onset of lay. The use of a probiotic benefits the health and performance status of hens, resulting in better hen welfare and significant economic gains to egg producers.

A review of diazepam and its use in the horse

Abstract The application of diazepam in clinical equine practice has come into favor in recent years. This review covers the relevant studies concerning the pharmacological properties, pharmacodynamics and pharmacokinetics of diazepam and its clinical use in the horse. As a benzodiazepinic drug diazepam potentates GABA-mediated inhibition in the CNS. It exerts its principal action in the brain stem reticular formation and produces hypnotic, sedative, anxiolytic, anticonvulsant and skeletal muscle-relaxant effects. However, Diazepam is largely used in equine practice in combination with ketamine and xylazine hydrochloride for induction of anesthesia and alone as a sedative/ataractic/neuroleptic. We aimed to present a general discussion of the relevant data from pharmacokinetic, pharmacodynamic and clinical studies and subsequently to evaluate their results. The discussion is based on our results, reported partially in previous publications including an evaluation of the literature. Available knowledge concerning the pharmacology of diazepam and its application in the horse is presented. The relative merits and demerits of a variety of agents intended for use in horses are discussed, with the aim of providing practitioners and clinicians with a wider selection of safe drugs for equine sedation and premedication.

Intron retention enhances gene regulatory complexity in vertebrates.

BackgroundWhile intron retention (IR) is now widely accepted as an important mechanism of mammalian gene expression control, it remains the least studied form of alternative splicing. To delineate conserved features of IR, we performed an exhaustive phylogenetic analysis in a highly purified and functionally defined cell type comprising neutrophilic granulocytes from five vertebrate species spanning 430 million years of evolution.ResultsOur RNA-sequencing-based analysis suggests that IR increases gene regulatory complexity, which is indicated by a strong anti-correlation between the number of genes affected by IR and the number of protein-coding genes in the genome of individual species. Our results confirm that IR affects many orthologous or functionally related genes in granulocytes. Further analysis uncovers new and unanticipated conserved characteristics of intron-retaining transcripts. We find that intron-retaining genes are transcriptionally co-regulated from bidirectional promoters. Intron-retaining genes have significantly longer 3′ UTR sequences, with a corresponding increase in microRNA binding sites, some of which include highly conserved sequence motifs. This suggests that intron-retaining genes are highly regulated post-transcriptionally.ConclusionsOur study provides unique insights concerning the role of IR as a robust and evolutionarily conserved mechanism of gene expression regulation. Our findings enhance our understanding of gene regulatory complexity by adding another contributor to evolutionary adaptation.