Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils

Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 potential neutrophil reference genes (RPL19, GAPDH, ACTB, B2M, HPRT, G6PD, TFRC, PGK1, YWHAZ, SDHA and GYPC) for sheep under disease conditions of foot rot (FR) and with or without Se supplementation. Initial screening was based on gene expression level (<28 Cq cycles) and variability (SD < 1.5 Cq cycles) and excluded TFRC, GYPC and HPRT from further analysis. Expression stability of the remaining genes was evaluated using four software programs: geNorm, NormFinder, BestKeeper and the comparative delta Cq method. The neutrophil reference genes, G6PD, YWHAZ, GAPDH, RPL19 and SDHA, consistently ranked among the top five most stable genes under these experimental conditions. The SDHA gene expression was not stable in FR-diseased sheep receiving Se treatment and, thus, cannot be recommended as a reference gene. The commonly used genes, PGK1, ACTB and B2M, were not reliable reference genes, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple references genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes was SDHA/G6PD in healthy sheep and GADPH/YWHAZ in FR-diseased sheep.Keywords: Whole blood, Footrot, Normalization, Immune responses, Selenium, Real time PCR, Expression, Housekeeping gene

Higher whole-blood selenium is associated with improved immune responses in footrot-affected sheep

We reported previously that sheep affected with footrot (FR) have lower whole-blood selenium (WB-Se) concentrations and that parenteral Se-supplementation in conjunction with routine control practices accelerates recovery from FR. The purpose of this follow-up study was to investigate the mechanisms by which Se facilitates recovery from FR. Sheep affected with FR (n = 38) were injected monthly for 15 months with either 5 mg Se (FR-Se) or saline (FR-Sal), whereas 19 healthy sheep received no treatment. Adaptive immune function was evaluated after 3 months of Se supplementation by immunizing all sheep with a novel protein, keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity (DTH) skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. Innate immunity was evaluated after 3 months of Se supplementation by measuring intradermal responses to histamine 30 min after injection compared to KLH and saline, and after 15 months of Se supplementation by isolating neutrophils and measuring their bacterial killing ability and relative abundance of mRNA for genes associated with neutrophil migration. Compared to healthy sheep, immune responses to a novel protein were suppressed in FR-affected sheep with smaller decreases in FR-affected sheep that received Se or had WB-Se concentrations above 250 ng/mL at the time of the immune assays. Neutrophil function was suppressed in FR-affected sheep, but was not changed by Se supplementation or WB-Se status. Sheep FR is associated with depressed immune responses to a novel protein, which may be partly restored by improving WB-Se status (> 250 ng/mL)

Selenium Supplementation Restores Innate and Humoral Immune Responses in Footrot-Affected Sheep

Dietary selenium (Se) alters whole-blood Se concentrations in sheep, dependent upon Se source and dosage administered, but little is known about effects on immune function. We used footrot (FR) as a disease model to test the effects of supranutritional Se supplementation on immune function. To determine the effect of Se-source (organic Se-yeast, inorganic Na-selenite or Na-selenate) and Se-dosage (1, 3, 5 times FDA-permitted level) on FR severity, 120 ewes with and 120 ewes without FR were drenched weekly for 62 weeks with different Se sources and dosages (30 ewes/treatment group). Innate immunity was evaluated after 62 weeks of supplementation by measuring neutrophil bacterial killing ability. Adaptive immune function was evaluated by immunizing sheep with keyhole limpet hemocyanin (KLH). The antibody titer and delayed-type hypersensitivity skin test to KLH were used to assess humoral immunity and cell-mediated immunity, respectively. At baseline, FR-affected ewes had lower whole-blood and serum-Se concentrations; this difference was not observed after Se supplementation. Se supplementation increased neutrophil bacterial killing percentages in FR-affected sheep to percentages observed in supplemented and non-supplemented healthy sheep. Similarly, Se supplementation increased KLH antibody titers in FR-affected sheep to titers observed in healthy sheep. FR-affected sheep demonstrated suppressed cell-mediated immunity at 24 hours after intradermal KLH challenge, although there was no improvement with Se supplementation. We did not consistently prevent nor improve recovery from FR over the 62 week Se-treatment period. In conclusion, Se supplementation does not prevent FR, but does restore innate and humoral immune functions negatively affected by FR

Effect of Feeding Selenium-Fertilized Alfalfa Hay on Performance of Weaned Beef Calves

Selenium (Se) is an essential micronutrient in cattle, and Se-deficiency can affect morbidity and mortality. Calves may have greater Se requirements during periods of stress, such as during the transitional period between weaning and movement to a feedlot. Previously, we showed that feeding Se-fertilized forage increases whole-blood (WB) Se concentrations in mature beef cows. Our current objective was to test whether feeding Se-fertilized forage increases WB-Se concentrations and performance in weaned beef calves. Recently weaned beef calves (n = 60) were blocked by body weight, randomly assigned to 4 groups, and fed an alfalfa hay based diet for 7 wk, which was harvested from fields fertilized with sodium-selenate at a rate of 0, 22.5, 45.0, or 89.9 g Se/ha. Blood samples were collected weekly and analyzed for WB-Se concentrations. Body weight and health status of calves were monitored during the 7-wk feeding trial. Increasing application rates of Se fertilizer resulted in increased alfalfa hay Se content for that cutting of alfalfa (0.07, 0.95, 1.55, 3.26 mg Se/kg dry matter for Se application rates of 0, 22.5, 45.0, or 89.9 g Se/ha, respectively). Feeding Se-fertilized alfalfa hay during the 7-wk preconditioning period increased WB-Se concentrations (P[subscript Linear] < 0.001) and body weights (P[subscript Linear] = 0.002) depending upon the Se-application rate. Based upon our results we suggest that soil-Se fertilization is a potential management tool to improve Se-status and performance in weaned calves in areas with low soil-Se concentrations

Dietary Enrichment with 20% Fish Oil Decreases Mucus Production and the Inflammatory Response in Mice with Ovalbumin-Induced Allergic Lung Inflammation.

The prevalence of asthma has increased in recent decades, which may be related to higher dietary intake of (n-6) polyunsaturated fatty acids (PUFA) and lower intake of (n-3) PUFA, e.g., those contained in fish oil. The objective of this study was to determine if dietary PUFA enrichment decreases mucus production or the inflammatory response associated with ovalbumin (OVA)-induced allergic lung inflammation. Mice (n = 10/group) were fed control, 20% fish oil, or 20% corn oil enriched diets for a total of 12 weeks. At 8 and 10 weeks, mice were given an intraperitoneal injection of saline (10 control-fed mice) or OVA (30 remaining mice). Once at 10 weeks and on 3 consecutive days during week 12, mice were challenged by nebulizing with saline or OVA. Mice were euthanized 24 hours after the last challenge and blood was collected for plasma FA analysis. Bronchoalveolar lavage (BAL) fluid was collected to determine cell composition and Th2-type cytokine (IL-4, IL-13) concentrations. Periodic acid-Schiff (PAS) + mucus-producing cells and CD45+ inflammatory cell infiltrates in lung tissue were quantified using morphometric analysis. Relative abundance of mRNA for mucin (Muc4, Muc5ac, and Muc5b) and Th2-type cytokine (IL-4, IL-5, and IL-13) genes were compared with ß-actin by qPCR. Supplementation with either corn oil or fish oil effectively altered plasma FA profiles towards more (n-6) FA or (n-3) FA, respectively (P < 0.0001). Sensitization and challenge with OVA increased the proportion of neutrophils, lymphocytes, and eosinophils, and decreased the proportion of macrophages and concentrations of IL-13 in BAL fluid; increased the percentage of PAS+ mucus-producing cells and CD45+ inflammatory cell infiltrates in lung tissue; and increased gene expression of mucins (Muc4, Muc5ac, and Muc5b) and Th2-type cytokines (IL-5 and IL-13) in lung tissue of control-fed mice. Dietary PUFA reversed the increase in PAS+ mucus-producing cells (P = 0.003). In addition, dietary enrichment with fish oil attenuated the percentage of CD45+ inflammatory cell infiltrates in lung tissue, and increased Muc4 and Muc 5b gene expression compared with OVA-sensitized and challenged control mice. In conclusion, dietary enrichment with either (n-3) or (n-6) PUFA decreased mucus production in lung tissues of OVA-sensitized and challenged mice. More specifically, enrichment with dietary (n-3) PUFA decreased CD45+ inflammatory cell infiltrates, thus inducing potentially beneficial changes in lung tissue of OVA-sensitized and challenged mice

Effect of feeding selenium-fertilized alfalfa hay on performance of weaned beef calves.

Selenium (Se) is an essential micronutrient in cattle, and Se-deficiency can affect morbidity and mortality. Calves may have greater Se requirements during periods of stress, such as during the transitional period between weaning and movement to a feedlot. Previously, we showed that feeding Se-fertilized forage increases whole-blood (WB) Se concentrations in mature beef cows. Our current objective was to test whether feeding Se-fertilized forage increases WB-Se concentrations and performance in weaned beef calves. Recently weaned beef calves (n = 60) were blocked by body weight, randomly assigned to 4 groups, and fed an alfalfa hay based diet for 7 wk, which was harvested from fields fertilized with sodium-selenate at a rate of 0, 22.5, 45.0, or 89.9 g Se/ha. Blood samples were collected weekly and analyzed for WB-Se concentrations. Body weight and health status of calves were monitored during the 7-wk feeding trial. Increasing application rates of Se fertilizer resulted in increased alfalfa hay Se content for that cutting of alfalfa (0.07, 0.95, 1.55, 3.26 mg Se/kg dry matter for Se application rates of 0, 22.5, 45.0, or 89.9 g Se/ha, respectively). Feeding Se-fertilized alfalfa hay during the 7-wk preconditioning period increased WB-Se concentrations (P Linear<0.001) and body weights (P Linear = 0.002) depending upon the Se-application rate. Based upon our results we suggest that soil-Se fertilization is a potential management tool to improve Se-status and performance in weaned calves in areas with low soil-Se concentrations

The effect of Se-source and dosage on sheep requiring oxytetracyline treatment.

<p>Foot rot severity was assessed after 0, 20, 28, 40, and 60 wk of Se supplementation in ewes receiving no Se treatment, Na-selenate at a dosage rate of 8.95 mg Se/wk per ewe, or Na-selenite and Se-Yeast at 4.9, 14.7, or 24.5 mg Se/wk per ewe for 62 wk. If a sheep had one foot with a score of 4, or one foot with a score of 3 and a second foot with a score of 2 or greater, or if all 4 feet had FR, then 20 mg/kg oxytetracycline was administered subcutaneously (Liquamycin LA-200; Pfizer Animal Health, Exton, PA) at the 28- and 40-wk foot trimming sessions. No significant differences in parental oxytetracycline treatment were observed for individual treatment groups at 28 wk; however; more ewes with supranutritional Na-selenite treatment were treated with oxytetracyline than ewes receiving the lowest Na-selenite dosage (<i>P</i> = 0.05). At 40 wk, more ewes receiving Na-selenite at the highest dosage had to be treated with oxytetracycline than ewes receiving no Se, Na-selenite at both lower dosages, or Se-yeast at 14.5 mg/wk (all <i>P</i> ≤ 0.05).</p

BAL fluid cytology proportions for mice fed (n-3) or (n-6) PUFA enriched foods for 8 weeks and then sensitized and challenged with OVA for an additional 4 weeks compared with mice fed control food that were sensitized and challenged with saline (Negative Control) or OVA (Positive Control)¹.

<p>BAL fluid cytology proportions for mice fed (n-3) or (n-6) PUFA enriched foods for 8 weeks and then sensitized and challenged with OVA for an additional 4 weeks compared with mice fed control food that were sensitized and challenged with saline (Negative Control) or OVA (Positive Control)<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163819#t004fn001" target="_blank">¹</a>.</p