Lindsay H. Sumners

Immune responses to dietary β-glucan in broiler chicks during an Eimeria challenge

Escalating consumer concerns regarding pathogen resistance have placed the poultry industry under mounting pressure to eliminate the use of chemotherapeutic agents as feed additives. One possible alternative receiving increased attention is the use of immunomodulators such as β-glucan. A study was conducted to investigate the effects of a yeast-derived β-glucan (Auxoferm YGT) on broiler chick performance, lesion scores, and immune-related gene expression during a mixed Eimeria infection. Day-old chicks were fed diets containing 0, 0.02, or 0.1% YGT. On d 8 posthatch, one-half of the replicate pens were challenged with a mixed inoculum of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Measurements were taken and samples collected on d 4, 10, 14, and 21 posthatch. Dietary supplementation had no effect on performance or mortality. On d 14, 3 birds per pen (n = 24/treatment) were scored for intestinal coccidia lesions. Gross lesion severity was significantly reduced in birds supplemented with 0.1% YGT. On d 10, inducible nitric oxide synthase (iNOS) expression was downregulated in the jejunum of challenged birds fed 0.1% YGT. Expression of iNOS in the ileum was downregulated in the nonchallenged birds, but upregulated in the challenged birds fed 0.1% YGT on d 14. Interleukin (IL)-18 was upregulated in the jejunum of 0.1% YGT-treated birds. Interferon (IFN)-γ expression was decreased in challenged and nonchallenged birds fed 0.1% YGT. The IL-4 expression was downregulated in the nonchallenged birds with 0.1% YGT diet supplementation. The IL-13 and mucin-1 levels were also reduced due to β-glucan supplementation. Mucin-2 expression was increased in the nonchallenged birds, but decreased in the infected birds fed 0.1% YGT. These results suggest that although Auxoferm YGT at doses of 0.02 and 0.1% does not influence performance, it significantly reduces lesion severity and is capable of altering immune-related gene expression profiles, favoring an enhanced T helper type-1 cell response during coccidiosis.

Quantity of glucose transporter and appetite-associated factor mRNA in various tissues after insulin injection in chickens selected for low or high body weight.

Chickens from lines selected for low (LWS) or high (HWS) body weight differ by 10-fold in body weight at 56 days old with differences in food intake, glucose regulation, and body composition. To evaluate if there are differences in appetite-regulatory factor and glucose transporter (GLUT) mRNA that are accentuated by hypoglycemia, blood glucose was measured, and hypothalamus, liver, pectoralis major, and abdominal fat collected at 90 days of age from female HWS and LWS chickens, and reciprocal crosses, HL and LH, at 60 min after intraperitoneal injection of insulin. Neuropeptide Y (NPY) and receptor (NPYR) subtypes 1 and 5 mRNA were greater in LWS compared with HWS hypothalamus (P < 0.05), but greater in HWS than LWS in fat (P < 0.05). Expression of NPYR2 was greater in LWS than HWS in pectoralis major (P < 0.05). There was greater expression in HWS than LWS for GLUT1 in hypothalamus and liver (P < 0.05), GLUT2 in fat and liver (P < 0.05), and GLUT9 in liver (P < 0.05). Insulin was associated with reduced blood glucose in all populations (P < 0.05) and reduced mRNA of insulin receptor (IR) and GLUT 2 and 3 in liver (P < 0.05). There was heterosis for mRNA, most notably NPYR1 (-78%) and NPYR5 (-81%) in fat and GLUT2 (-70%) in liver. Results suggest that NPY and GLUTs are associated with differences in energy homeostasis in LWS and HWS. Reduced GLUT and IR mRNA after insulin injection suggest a compensatory mechanism to prevent further hypoglycemia.

Expression of Toll-like receptors and antimicrobial peptides during Eimeria praecox infection in chickens

Intestinal colonization of avian species by Eimeria parasites results in the enteric disease, coccidiosis. A study was carried out to assess the immunologic effects of Eimeria praecox infection on the gut of infected chickens. In Experiment 1, birds were orally gavaged with 50,000 E. praecox oocysts; in Experiment 2, an infection dosage of 500,000 E. praecox oocysts was used. Duodenal and jejunal intestinal sections were sampled consecutively on days 1-7 post-infection. Intestinal expression of innate immune gene transcripts was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Analysis of relative gene expression in Experiment 1 revealed an increase (P<0.05) in duodenal Toll-like receptor (TLR)3 expression on days 4 and 6 post-infection. TLR15 expression was significantly decreased in the duodenum of infected birds on day 2, and significantly increased on day 6 post-infection. In Experiment 2, TLR3 was significantly downregulated in the duodenum on day 7 post-infection; however, no significant results were observed in terms of TLR15 expression. TLR4 also exhibited decreased expression (P<0.05) on day 7 post-infection in both intestinal sections. Regarding antimicrobial peptide expression; in the first experiment, expression of liver-expressed antimicrobial peptide-2 (LEAP-2) in infected birds was significantly decreased in the duodenum on days 3 and 4, and in the jejunum on day 4. Similarly, Experiment 2 resulted in depression of LEAP-2 (P<0.05) on days 3-5 in the duodenum. In Experiment 1, cathelicidin antimicrobial peptide (CATHL3) was downregulated (P<0.05) in the jejunum of infected chickens on day 3 post-infection; however, CATHL3 results were non-significant in Experiment 2. Based on the differing results observed in each experiment, it was concluded that both TLR and antimicrobial peptide expression, and thus immunity may be dependent on infection load.

Chickens from lines selected for high and low body weight show differences in fatty acid oxidation efficiency and metabolic flexibility in skeletal muscle and white adipose tissue.

Objective:The Virginia lines of chickens have resulted from more than 55 generations of artificial selection for low (LWS) or high (HWS) juvenile body weight. We hypothesized that the relative hyperphagia and greater body weight in juvenile HWS chickens are associated with altered fatty acid oxidation efficiency and metabolic flexibility in tissues associated with energy sensing and storage, and relative cellular hypertrophy in white adipose tissue.Methods:Hypothalamus, liver, pectoralis major, gastrocnemius, abdominal fat, clavicular fat and subcutaneous fat were collected from the juvenile (56–65 days old) LWS and HWS chickens for metabolic, gene expression and histological assays.Results:The HWS chickens had reduced fatty acid oxidation efficiency in abdominal fat (P<0.0001) and reduced rates of oxidation in abdominal fat and gastrocnemius (P<0.0001) as compared with the LWS. There was reduced citrate synthase activity in white adipose tissue (P<0.0001) and greater metabolic inflexibility in skeletal muscle (P=0.006) of the HWS compared with the LWS. Greater pyruvate dehydrogenase kinase 4 (PDK4) and forkhead box O1A (FoxO1) mRNA were found in skeletal muscle and white adipose tissue of 56-day-old HWS than LWS. Expression of peroxisome proliferator-activated receptor γ (PPARγ) in all adipose tissue depots was greater (P<0.05) in LWS than in HWS chickens. The HWS chickens had larger (P<0.0001) and fewer (P<0.0001) adipocytes per unit area than the LWS.Conclusion:Compared with the LWS, the HWS chickens have impaired metabolic flexibility and fatty acid oxidation efficiency due to greater pyruvate dehydrogenase activity to accommodate the influx of acetyl-CoA from fatty acid oxidation in skeletal muscle and adipose tissue. These metabolic adaptations can be linked to differences in gene expression regulation, adipocyte cellularity and body composition between the lines, which may provide valuable insight into metabolic disorders in other species.

Molecular characterization and immunological roles of avian IL-22 and its soluble receptor IL-22 binding protein

As a member of the interleukin (IL)-10 family, IL-22 is an important mediator in modulating tissue responses during inflammation. Through activation of STAT3-signaling cascades, IL-22 induces proliferative and anti-apoptotic pathways, as well as antimicrobial peptides (AMPs), that help prevent tissue damage and aid in its repair. This study reports the cloning and expression of recombinant chicken IL-22 (rChIL-22) and its soluble receptor, rChIL22BP, and characterization of biological effects of rChIL-22 during inflammatory responses. Similar to observations with mammalian IL-22, purified rChIL-22 had no effect on either peripheral blood mononuclear cells (PBMCs) or lymphocytes. This was due to the low expression of the receptor ChIL22RA1 chain compared to ChIL10RB chain. rChIL-22 alone did not affect chicken embryo kidney cells (CEKCs); however, co-stimulation of CEKCs with LPS and rChIL-22 enhanced the production of pro-inflammatory cytokines, chemokines and AMPs. Furthermore, rChIL-22 alone stimulated and induced acute phase reactants in chicken embryo liver cells (CELCs). These effects of rChIL-22 were abolished by pre-incubation of rChIL-22 with rChIL22BP. Together, this study indicates an important role of ChIL-22 on epithelial cells and hepatocytes during inflammation.

Chickens from lines artificially selected for juvenile low and high body weight differ in glucose homeostasis and pancreas physiology

Artificial selection of White Plymouth Rock chickens for juvenile (day 56) body weight resulted in two divergent genetic lines: hypophagic low weight (LWS) chickens and hyperphagic obese high weight (HWS) chickens, with the latter more than 10-fold heavier than the former at selection age. A study was designed to investigate glucose regulation and pancreas physiology at selection age in LWS chickens and HWS chickens. Oral glucose tolerance and insulin sensitivity tests revealed differences in threshold sensitivity to insulin and glucose clearance rate between the lines. Results from real-time PCR showed greater pancreatic mRNA expression of four glucose regulatory genes (preproinsulin, PPI; preproglucagon, PPG; glucose transporter 2, GLUT2; and pancreatic duodenal homeobox 1, Pdx1) in LWS chickens, than HWS chickens. Histological analysis of the pancreas revealed that HWS chickens have larger pancreatic islets, less pancreatic islet mass, and more pancreatic inflammation than LWS chickens, all of which presumably contribute to impaired glucose metabolism.

Changes in expression of an antimicrobial peptide, digestive enzymes, and nutrient transporters in the intestine of E. praecox-infected chickens

Coccidiosis is a major intestinal disease of poultry, caused by several species of the protozoan Eimeria. The objective of this study was to examine changes in expression of digestive enzymes, nutrient transporters, and an antimicrobial peptide following an Eimeria praecox challenge of chickens at days 3 and 6 post-infection. Gene expression was determined by real-time PCR and analyzed by one-way ANOVA. In the duodenum, the primary site of E. praecox infection, a number of genes were downregulated at both d3 and d6 post-infection. These genes included liver expressed antimicrobial peptide 2 (LEAP2), the cationic (CAT1), anionic (EAAT3), and L-type (LAT1) amino acid transporters, the peptide transporter PepT1 and the zinc transporter ZnT1. Other transporters were downregulated either at d3 or d6. At both d3 and d6, there was downregulation of B(o)AT and CAT1 in the jejunum and downregulation of LEAP2 and LAT1 in the ileum. LEAP2, EAAT3, and ZnT1 have been found to be downregulated following challenge with other Eimeria species, suggesting a common cellular response to Eimeria.

Immunological responses to Clostridium perfringens alpha-toxin in two genetically divergent lines of chickens as influenced by major histocompatibility complex genotype

Chickens genetically selected for low (LA) or high (HA) antibody response to SRBC displayed a correlated change in MHC, so that LA chickens were 96% B13 and HA chickens were 96% B21. The LA line appears to be less susceptible to invasion by extracellular pathogens, whereas HA chickens are more resistant to infection by intracellular organisms. Resistance to Clostridium perfringens is one instance in which the lines do not follow their established trend of pathogen susceptibility, where during a clinical outbreak of necrotic enteritis, B21B21 genotypes experienced significantly less mortality than B13B13 genotypes. A study was carried out to assess immunological differences between LA and HA lines during exposure to C. perfringens α-toxin. Peripheral blood mononuclear cells were isolated from each genetic line, cultured with or without lipopolysaccharide (4 h), and exposed to varying concentrations of α-toxin (1; 10; 100; and 1,000 U/L) for 2 and 4 h. Evaluation of cellular proliferation, percentage of cytotoxicity, and immunological gene expression was carried out in a series of experiments. Cells isolated from HA chickens had significantly increased proliferation than those from LA chickens at low toxin levels (1 and 10 U/L) and significantly decreased proliferation at high toxin levels (100 and 1,000 U/L). Following exposure to lipopolysaccharide, the percentage of cytotoxicity was higher for LA than HA cells. In both assays, HA cells displayed superior performance following lipopolysaccharide-stimulation. Gene expression analysis of immune transcripts by quantitative real-time PCR revealed significantly upregulated expression of interferon (IFN)-γ, interleukin (IL)-8, IL-13 (2 h), IL-15, and CXCLi1 (4 h) in HA than LA chickens. Cells isolated from the LA line displayed significantly elevated expression of IL-2, IL-10, IL-13 (4 h), IL-16, IL-18, inducible nitric oxide synthase (iNOS), CXCLi1 (2 h), and lipopolysaccharide-induced tumor necrosis factor-α factor (LITAF) compared with the HA line. Clearly, these 2 genetic lines display highly divergent immune responses in regards to C. perfringens toxin exposure.

Delayed feeding after hatch caused compensatory increases in blood glucose concentration in fed chicks from low but not high body weight lines

This experiment used 2 lines of chickens that have been selected 54 generations for either low (LWS) or high (HWS) 8-wk BW from the same founder population, sublines (HWR and LWR) in which selection was relaxed in generation 43 in the selected lines, and crosses (HL and LH) made from generation 54 of HWS and LWS. For 8-wk BW, the difference between lines LWS and HWS in generation 54 was approximately 10-fold, whereas for the relaxed contemporary lines they were approximately 7-fold. Three trials were designed to measure developmental, nutritional, and genetic aspects of blood glucose homeostasis during the first 2 wk posthatch. In trial 1, we measured BW, whole blood glucose (BG), and weights (relative to BW) of liver, pancreas, and yolk sac of chicks fed from day of hatch to d 15. In trial 2, we compared those traits in chicks feed-delayed 72 h posthatch and in chicks without feed delay. In trial 3, we evaluated the effect of a 16-h fast on BW and BG on d 3, 8, and 15. There were higher levels of BG in HWS than LWS, and males than females in the fed state. Delayed access to feed for 72 h after hatch was associated with a dramatic reduction in BG. Feeding triggered a compensatory response whereby LWS displayed greater BG but smaller pancreases (% BW; d 15), compared with the controls. There were maternal effects for BW in both fed and fasted states and the reciprocal crosses exhibited heterosis for BG in the fasted state. These results show that chickens selected for high or low BW differ in BG regulation during the early posthatch period.

Delayed access of low body weight-selected chicks to food at hatch is associated with up-regulated pancreatic glucagon and glucose transporter gene expression.

Chickens selected for low (LWS) and high (HWS) juvenile body weight (BW) for 55 generations differ in BW by 10-fold at selection age. High (HWR) and low (LWR) body weight-relaxed lines have been random-bred since the 46th generation. Our objective was to evaluate the developmental and nutritional regulation of pancreatic mRNA abundance of pancreatic and duodenal homeobox 1 (PDX1), preproinsulin (PPI), preproglucagon (PPG), and glucose transporter 2 (GLUT2). At day of hatch (DOH) and days 1, 3, 7, and 15 (D1, 3, 7 and 15, respectively), pancreas was collected and real time PCR was performed in Experiment 1. In Experiment 2, HWS and LWS were fed or delayed access to food for 72 h post-hatch, and pancreas collected at D15. There was an interaction of line and age for GLUT2 (P=0.001), PPI (P<0.0001), PPG (P=0.034), and PDX1 (P<0.0001). Expression was greater in chicks from LWR and LWS than HWR and HWS. There was an interaction of line and nutrition on PPG (P<0.0001) and GLUT2 (P=0.001) mRNA, where expression was similar among chicks that were fed but greater in LWS than HWS when chicks were delayed access to food. Thus, the first two weeks is important for maturation of pancreatic endocrine function. Long-term selection for BW is associated with differences in pancreas development, and delaying access to food at hatch may have persisting effects on glucose regulatory function.