Spontaneous immunological activities in the target tissue of vitiligo-prone Smyth and vitiligo-susceptible Brown lines of chicken.

Introduction: Vitiligo is an acquired de-pigmentation disorder characterized by the post-natal loss of epidermal melanocytes (pigment-producing cells) resulting in the appearance of white patches in the skin. The Smyth chicken is the only model for vitiligo that shares all the characteristics of the human condition including: spontaneous post-natal loss of epidermal melanocytes, interactions between genetic, environmental and immunological factors, and associations with other autoimmune diseases. In addition, an avian model for vitiligo has the added benefit of an easily accessible target tissue (a growing feather) that allows for the repeated sampling of an individual and thus the continuous monitoring of local immune responses over time. Methods: Using a combination of flow cytometry and gene expression analyses, we sought to gain a comprehensive understanding of the initiating events leading to expression of vitiligo in growing feathers by monitoring the infiltration of leukocytes and concurrent immunological activities in the target tissue beginning prior to visual onset and continuing throughout disease development. Results: Here, we document a sequence of immunologically significant events, including characteristic rises in infiltrating B and αß T cells as well as evidence of active leukocyte recruitment and cell-mediated immune activities (CCL19, IFNG, GZMA) leading up to visual vitiligo onset. Examination of growing feathers from vitiligo-susceptible Brown line chickens revealed anti-inflammatory immune activities which may be responsible for preventing vitiligo (IL10, CTLA4, FOXP3). Furthermore, we detected positive correlations between infiltrating T cells and changes in their T cell receptor diversity supporting a T cell-specific immune response. Conclusion: Collectively, these results further support the notion of cell-mediated immune destruction of epidermal melanocytes in the pulp of growing feathers and open new avenues of study in the vitiligo-prone Smyth and vitiligo-susceptible Brown line chickens.

The Immuno-Suppressive Effects of Cyclic, Environmental Heat Stress in Broiler Chickens: Local and Systemic Inflammatory Responses to an Intradermal Injection of Lipopolysaccharide.

To assess effects of environmental heat stress (HS) on the local and systemic inflammatory responses to lipopolysaccharide (LPS), broilers were reared under thermoneutral (TN) or cyclic HS conditions. Thermoneutral temperatures followed commercial production settings, with HS broilers exposed to 35 °C for 14 h/day from 4 days onward. At 37 days, HS- and TN-broilers were assigned to either LPS (100 µg/mL) or endotoxin-free phosphate-buffered saline (PBS; vehicle) treatments, eight each to HS- and TN-LPS, four each to HS- and TN-PBS. Treatments were administered by intradermal injection of growing feather (GF) pulps; 10 µL/GF; 12 GF/broiler. Blood and GF were collected before and at 6 and 24 h post-injection to assess leukocyte population changes in GF-pulps and blood, reactive oxygen species (ROS) generation and cytokine expression in GF-pulps, and plasma concentrations of alpha-1 acid glycoprotein (AGP-1). HS-LPS broilers had lower (p ≤ 0.05) infiltration of heterophils and macrophages, ROS generation, and inflammatory cytokine expression in GF-pulps, and lacked the increases in heterophil, monocyte, and plasma AGP-1 concentrations observed in TN-LPS broilers. HS-broilers had similar or greater drops in blood lymphocytes 6 h post-LPS or -PBS injection, respectively, and lower baseline levels (p ≤ 0.05) of circulating T- and B-lymphocytes than TN-broilers. Results indicated that cyclic HS reduced the local and systemic acute inflammatory responses to LPS in broilers, likely impairing their innate defense against microbial infection.

Local and systemic inflammatory responses to lipopolysaccharide in broilers: new insights using a two-window approach.

The inflammatory response involves a complex interplay of local tissue activities designed to recruit leukocytes and proteins from the blood to the infected tissue. For egg-type chickens, we established the growing feather (GF) as an accessible tissue test site to monitor tissue responses to injected test-material. For commercial broilers, whose health depends to a large extent on innate immune system functions, the GF test system offers an important novel window to directly assess their natural defenses. This study was conducted to adapt the GF test system for use in broilers, and use it to simultaneously examine local (GF) and systemic (blood) inflammatory responses initiated by GF pulp injection of lipopolysaccharide (LPS). Specifically, GF of 12 male and 12 female, 5-week-old broilers were injected with LPS (16 GF/chicken; 1 µg LPS/GF). Blood and GF were collected at 0 (before), 6, and 24 h after GF injection. GF pulp was used to determine leukocyte-infiltration and gene-expression profiles, reactive-oxygen-species generation, and superoxide dismutase (SOD) activity. Blood was used to determine blood cell profiles and SOD activity. A time effect (P ≤ 0.05) was observed for most aspects examined. In GF, LPS injection resulted in heterophil and monocyte infiltration reaching maximal levels at 6 and 24 h, respectively. Reactive-oxygen-species generation, SOD activity, and mRNA levels of IL-1ß, IL-8, IL-6, IL-10, and cathelicidin B1 were elevated, whereas those of TNF-α, LITAF, SOD1, and SOD2 decreased after LPS injection. In blood, levels of heterophils and monocytes were elevated at 6 h, lymphocytes and RBC decreased at 6 h, and thrombocytes and SOD activity increased at 24 h. Assessment of LPS-induced activities at the site of inflammation (GF) provided novel and more relevant insights into temporal, qualitative, and quantitative aspects of inflammatory responses than blood. Knowledge generated from this dual-window approach may find direct application in identification of individuals with robust, balanced innate defenses and provide a platform for studying the effects of exogenous treatments (e.g., nutrients, probiotics, immunomodulators, etc.) on inflammatory responses taking place in a complex tissue.

75-kDa glucose-regulated protein (GRP75) is a novel molecular signature for heat stress response in avian species.

Glucose-regulated protein 75 (GRP75) was first characterized in mammals as a heat shock protein-70 (HSP70) family stress chaperone based on its sequence homology. Extensive studies in mammals showed that GRP75 is induced by various stressors such as glucose deprivation, oxidative stress, and hypoxia, although it remained unresponsive to the heat shock. Such investigations are scarce in avian (nonmammalian) species. We here identified chicken GRP75 by using immunoprecipitation assay integrated with LC-MS/MS, and found that its amino acid sequence is conserved with high homology (52.5%) to the HSP70 family. Bioinformatics and 3D-structure prediction indicate that, like most HSPs, chicken GRP75 has two principal domains (the NH2-terminal ATPase and COOH-terminal region). Immunofluorescence staining shows that GRP75 is localized predominantly in the avian myoblast and hepatocyte mitochondria. Heat stress exposure upregulates GRP75 expression in a species-, genotype-, and tissue-specific manner. Overexpression of GRP75 reduces avian cell viability, and blockade of GRP75 by its small molecular inhibitor MKT-077 rescues avian cell viability during heat stress. Taken together, this is the first evidence showing that chicken GRP75, unlike its mammalian ortholog, is responsive to heat shock and plays a key role in cell survival/death pathways. Since modern avian species have high metabolic rates and are sensitive to high environmental temperature, GRP75 could open new vistas in mechanistic understanding of heat stress responses and thermotolerance in avian species.

Isolated Rearing at Lactation Increases Gut Microbial Diversity and Post-weaning Performance in Pigs.

Environment and diet are two major factors affecting the human gut microbiome. In this study, we used a pig model to determine the impact of these two factors during lactation on the gut microbiome, immune system, and growth performance. We assigned 80 4-day-old pigs from 20 sows to two rearing strategies at lactation: conventional rearing on sow's milk (SR) or isolated rearing on milk replacer supplemented with solid feed starting on day 10 (IR). At weaning (day 21), SR and IR piglets were co-mingled (10 pens of 4 piglets/pen) and fed the same corn-soybean meal-dried distiller grain with solubles- and antibiotic-free diets for eight feeding phase regimes. Fecal samples were collected on day 21, 62, and 78 for next-generation sequencing of the V4 hypervariable region of the bacterial 16S rRNA gene. Results indicate that IR significantly increased swine microbial diversity and changed the microbiome structure at day 21. Such changes diminished after the two piglet groups were co-mingled and fed the same diet. Post-weaning growth performance also improved in IR piglets. Toward the end of the nursery period (NP), IR piglets had greater average daily gain (0.49 vs. 0.41 kg/d; P < 0.01) and average daily feed intake (0.61 vs. 0.59 kg/d; P < 0.01) but lower feed efficiency (0.64 vs. 0.68; P = 0.05). Consequently, IR piglets were heavier by 2.9 kg (P < 0.01) at the end of NP, and by 4.1 kg (P = 0.08) at market age compared to SR piglets. Interestingly, pigs from the two groups had similar lean tissue percentage. Random forest analysis showed that members of Leuconostoc and Lactococcus best differentiated the IR and SR piglets at weaning (day 21), were negatively correlated with levels of Foxp3 regulatory T cell populations on day 20, and positively correlated with post-weaning growth performance. Our results suggest that rearing strategies may be managed so as to accelerate early-life establishment of the swine gut microbiome to enhance growth performance in piglets.

Gene flow from weedy red rice (Oryza sativa L.) to cultivated rice and fitness of hybrids.

BACKGROUND: Gene transfer from weeds to crops could produce weedy individuals that might impact upon the evolutionary dynamics of weedy populations, the persistence of escaped genes in agroecosystems and approaches to weed management and containment of transgenic crops. The present aim was to quantify the gene flowrate from weedy red rice to cultivated rice, and evaluate the morphology, phenology and fecundity of resulting hybrids. Field experiments were conducted at Stuttgart and Rohwer, Arkansas, USA. Twelve red rice accessions and an imazethapyr-resistant rice (Imi-R; Clearfield) were used. RESULTS: Hybrids between Imi-R rice x red rice were 138-150 cm tall and flowered 1-5 days later than the rice parent, regardless of the red rice parent. Hybrids produced 20-50% more seed than the rice parent, but had equivalent seed production to the majority of red rice parents. Seeds of all hybrids were red, pubescent and dehisced at maturity. For the majority of hybrids, seed germination was higher than that of the red rice parent. The gene flowrate from red rice to rice was 0.01-0.2% and differed by red rice biotype. The hybrids had higher fecundity and potential competitive ability than the rice parent, and in some cases also the red rice parent. CONCLUSIONS: Red rice plants are vectors of gene flow back to cultivated rice and other weedy populations. The progeny of red rice hybrids from cultivated rice mother plants have higher chances of persistence than those from red rice mother plants. Gene flow mitigation strategies should consider this scenario.