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Chronic low-grade inflammation is a hallmark of aging, aka “inflammaging”, which is linked to a wide range of age-associated diseases. Immune dysfunction increases disease susceptibility, and increases morbidity and mortality of aging. Innate immune cells, including monocytes, macrophages and neutrophils, are the first responders of host defense and the key mediators of various metabolic and inflammatory insults. Currently, the understanding of innate immune programming in aging is largely fragmented. Here we investigated the phenotypic and functional properties of innate immune cells in various peripheral tissues of young and aged mice under normal and endotoxic conditions. Under the steady state, aged mice showed elevated pro-inflammatory monocytes/macrophages in peripheral blood, adipose tissue, liver, and colon. Under lipopolysaccharide (LPS)-induced inflammatory state, the innate immune cells of aged mice showed a different response to LPS stimulus than that of young mice. LPS-induced immune responses displayed differential profiles in different tissues and cell types. In the peripheral blood, when responding to LPS, the aged mice showed higher neutrophils, but lower pro-inflammatory monocytes than that in young mice. In the peritoneal fluid, while young mice exhibited significantly elevated pro-inflammatory neutrophils and macrophages in response to LPS, aged mice exhibited decreased pro-inflammatory neutrophils and variable cytokine responses in macrophages. In the adipose tissue, LPS induced less infiltrated neutrophils but more infiltrated macrophages in old mice than young mice. In the liver, aged mice showed a more robust increase of pro-inflammatory macrophages compared to that in young mice under LPS stimulation. In colon, macrophages showed relatively mild response to LPS in both young and old mice. We have further tested bone-marrow derived macrophages (BMDM) from young and aged mice, we found that BMDM from aged mice have impaired polarization, displaying higher expression of pro-inflammatory markers than those from young mice. These data collectively suggest that innate immunity in peripheral tissues is impaired in aging, and the dysregulation of immunity is tissue- and cell-dependent. Our findings in the rodent model underscore the complexity of aging immunity. Further investigation is needed to determine whether the immune profile observed in aged mice is applicable in age-associated diseases in humans.

In the past decade, the withdrawal of antibiotics used as growth promoters (AGP) has increased some poultry industry challenges, such as the rise of intestinal diseases. Experts advocate that AGPs improve performance due to the modulation of the intestinal microbiota, with resulting anti-inflammatory effects. However, the impact and interactions of AGPs with the host intestinal immune system are still unknown, which represents issues in developing effective alternatives for AGPs. Therefore, this study was aimed at better understanding the potential mechanism of action of bacitracin used as AGP and its impacts on the intestinal immune system. Ninety day-of-hatch chickens were randomly assigned to two treatments with three repetitions of fifteen birds, a control (CNT) group with a corn/soybean meal standard diet, and a control diet supplemented with 50 g/ton of feed of bacitracin (BMD). The cytokines’ and chemokines’ production (IFN-α, IFN-γ, IL-16, IL-10, IL-21, IL-6, M-CSF, MIP-3α, MIP-1β, VEGF and CCL-5) were assessed in the jejunum and ileum at 14, 21, 28 and 36 days of age by using a chicken-specific cytokine/chemokine peptide ELISA array. Broilers with BMD supplementation were found to have a lower production of IL-16, IFN-γ, M-CSF, IL-21, MIP-1β and VEGF in the jejunum at 14 d. However, from 21 through 36 days, the effect of BMD on cytokine production in the jejunum was negligible except for CCL-5, which was reduced at D36. In the ileum, BMD effects on the cytokine profile started at 28 d, when BMD-supplemented broilers showed a reduced IL-6 production level. At day 36, BMD reduced IL-16 and MIP-3α production but increased VEGF concentration in the ileum tissue. The present study demonstrated that the use of bacitracin as an AGP modulates the small intestine immune system, especially in the first phase of the broiler’s life (up to 14 days). Moreover, BMD anti-inflammatory effects include not only innate immunity but also seemed to influence the development of the adaptive immune response as seen by the decreased production of IL-21 and IL-16. These results demonstrate that a commonly used AGP in broiler feed had a local anti-inflammatory effect.

ABSTRACT: This symposium offered up-to-date perspectives on field experiences and the latest research on significant viral and bacterial diseases affecting poultry. A highlight was the discussion on the use of enteroids as advanced in vitro models for exploring disease pathogenesis. Outcomes of this symposium included identifying the urgent need to improve the prevention and control of avian influenza by focusing research on vaccine effectiveness. In this regard, efforts should focus on enhancing the relatedness of vaccine antigen to the field (challenge) virus strain and improving immunogenicity. It was also revealed that gangrenous dermatitis could be controlled through withholding or restricting the administration of ionophores during broiler life cycle, and that administration of microscopic polymer beads (gel) based-live coccidia vaccines to chicks could be used to reduce necrotic enteritis-induced mortality. It was emphasized that effective diagnosis of re-emerging Turkey diseases (such as blackhead, fowl cholera, and coccidiosis) and emerging Turkey diseases such as reoviral hepatitis, reoviral arthritis, Ornithobacterium rhinotracheale infection, and strepticemia require complementarity between investigative research approaches and production Veterinarian field approaches. Lastly, it was determined that the development of a variety of functionally-specific enteroids would expedite the delineation of enteric pathogen mechanisms and the identification of novel vaccine adjuvants.

ABSTRACT: Intestinal organoids (IO), known as “mini-guts”, derived from intestinal crypts, are self-organizing three-dimensional (3D) multicellular ex vivo models that recapitulate intestine epithelial structure and function and have been widely used for studying intestinal physiology, pathophysiology, molecular mechanisms of host-pathogen interactions, and intestinal disease in mammals. However, studies on avian IO are limited and the development of long-term cultures of IO model for poultry research is lacking. Therefore, the objectives of this study were to generate crypt-derived organoids from chicken intestines and to optimize conditions for cell growth and enrichments, passages, and cryopreservation. Crypts were collected from the small intestines of birds at embryonic d-19 and ceca from layer and broiler chickens with ages ranging from d 1 to 20 wk, embedded in a basement membrane matrix, and cultured with organoid growth media (OGM) prepared in house. The crypt-derived organoids were successfully grown and propagated to form 3D spheres like structures that were cultured for up to 3 wk. Organoids were formed on d one, budding appeared on d 3, and robust budding was observed on d 7 and beyond. For cryopreservation, dissociated organoids were resuspended in a freezing medium. The characteristics of IO upon extended passages and freeze-thaw cycles were analyzed using reverse transcription (RT)-PCR, immunoblotting, and live cell imaging. Immunoblotting and RT-PCR using E-cadherin (the marker for epithelial cells), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5, the marker for stem cells), chromogranin A (the marker for enteroendocrine cells), lysozyme (the marker for Paneth cells), and mucin (the biomarker for goblet cells) confirmed that IO were composed of heterogeneous cell populations, including epithelial cells, stem cells, enteroendocrine cells, Paneth cells, and goblet cells. Furthermore, OGM supplemented with both valproic acid and CHIR99021, a glycogen synthase kinase 3β inhibitor and a histone deacetylase inhibitor, increased the size of the avian IO (P < 0.001). To the best of our knowledge, this is the first comprehensive report for establishing long-term, organoid culture models from small intestines and ceca of layer and broiler chickens. This model will facilitate elucidation of the mechanisms impacting host-pathogen interactions, eventually leading to the discovery of pathogen intervention strategies in poultry.

ABSTRACT: Wooden breast (WB) results in significant losses to the broiler industry due to reductions in meat quality. While the etiology of WB is unknown, it is believed to be associated with localized hypoxia and decreased lactate levels in skeletal muscles, indicating the presence of altered lactate metabolism in WB. We hypothesized that the expression levels of the major signaling molecules that control lactate metabolism, including lactate dehydrogenases (LDHA and LDHB) and monocarboxylate transporters (MCT1 and MCT4), were altered in WB. Therefore, the objectives of this study were to evaluate whether there were changes in mRNA and protein levels of LDHA, LDHB, MCT1, and MCT4 in WB compared to normal breast (NB) muscles. Biochemical analysis for LDH enzyme activity in NB and WB muscles was studied. MicroRNA375 (miR-375) expression, known to be inversely associated with LDHB protein expression in human cells, was also investigated. The level of LDHA mRNA was 1.7-fold lower in WB tissues than in NB tissues (P < 0.0001). However, the LDHA protein levels were similar in WB and NB tissues. In contrast, the levels of LDHB mRNA and protein were 8.4-fold higher (P < 0.002) and 13.6-fold higher (P < 0.02) in WB than in NB tissues, respectively. The level of miR-375 was not different between WB and NB muscles. The specific LDH isoenzyme activity that converted lactate to pyruvate was 1.8-fold lower in WB compared to NB tissues (P < 0.01). The level of MCT1 mRNA was 2.3-fold higher in WB than those in NB muscles (P < 0.02). However, this upregulation was not observed with MCT1 protein expression levels. The expression levels of MCT4 mRNA and protein were elevated 2.8-fold (P < 0.02) and 3.5-fold (P < 0.004) in WB compared to NB tissues, respectively. Our current findings suggest the potential roles of LDHB and MCT4 on lactate metabolism and provide a unique molecular elucidation for altered lactate homeostasis in WB muscles of broilers.

For poultry producers, chronic low-grade intestinal inflammation has a negative impact on productivity by impairing nutrient absorption and allocation of nutrients for growth. Understanding the triggers of chronic intestinal inflammation and developing a non-invasive measurement is crucial to managing gut health in poultry. In this study, we developed two novel models of low-grade chronic intestinal inflammation in broiler chickens: a chemical model using dextran sodium sulfate (DSS) and a dietary model using a high non-starch polysaccharide diet (NSP). Further, we evaluated the potential of several proteins as biomarkers of gut inflammation. For these experiments, the chemical induction of inflammation consisted of two 5-day cycles of oral gavage of either 0.25mg DSS/ml or 0.35mg DSS/ml; whereas the NSP diet (30% rice bran) was fed throughout the experiment. At four times (14, 22, 28 and 36-d post-hatch), necropsies were performed to collect intestinal samples for histology, and feces and serum for biomarkers quantification. Neither DSS nor NSP treatments affected feed intake or livability. NSP-fed birds exhibited intestinal inflammation through 14-d, which stabilized by 36-d. On the other hand, the cyclic DSS-treatment produced inflammation throughout the entire experimental period. Histological examination of the intestine revealed that the inflammation induced by both models exhibited similar spatial and temporal patterns with the duodenum and jejunum affected early (at 14-d) whereas the ileum was compromised by 28-d. Calprotectin (CALP) was the only serum protein found to be increased due to inflammation. However, fecal CALP and Lipocalin-2 (LCN-2) concentrations were significantly greater in the induced inflammation groups at 28-d. This experiment demonstrated for the first time, two in vivo models of chronic gut inflammation in chickens, a DSS and a nutritional NSP protocols. Based on these models we observed that intestinal inflammation begins in the upper segments of small intestine and moved to the lower region over time. In the searching for a fecal biomarker for intestinal inflammation, LCN-2 showed promising results. More importantly, calprotectin has a great potential as a novel biomarker for poultry measured both in serum and feces.

Recently, a P-deficient diet caused rickets in commercial chicks within three days. This study aimed to investigate the duration of onset of rickets in chicks. Data were collected from 3–11 day old chicks raised on 88 commercial farms. Male day-old Arbor Acres Plus broilers (n = 450) were studied in three trials, with three to four treatments each. Each treatment used one of the following crumbled feeds: control feed (calcium (Ca): phosphorus (P)-1.41), slightly high Ca:P feed (SHCa:P, Ca:P-2.69), high Ca:P ratio, P deficient feed (HCa:P, Ca:P-3.08), and HCa:P feed plus 1.5% dicalcium phosphate (HCa:P + DP). Each treatment had three replicates with 15 birds each. Rickets was induced by HCa:P, and cured by HCa:P + DP, confirmed by gross anatomy, gait score, serum P concentration and growth performance. Lameness was not found in control groups, whereas, observed in the HCa:P groups as early as day 2.7 on commercial farms and day 3 in experimental farm. Serum P was reduced in HCa:P (p < 0.01). Bodyweight and feed intake started decreasing at day 3 on commercial farms and in all trials (p < 0.01). The duration of onset of hypophosphatemic rickets in broiler chicks fed HCa:P crumbled feed is approximately three days.

Monitoring antimicrobial resistance of foodborne pathogens in poultry is critical for food safety. We aimed to compare antimicrobial resistance phenotypes in Salmonella isolated from poultry samples as influenced by isolation and antimicrobial susceptibility testing methods. Salmonella isolates were cultured from a convenience sample of commercial broiler ceca with and without selective broth enrichment, and resistance phenotypes were determined for 14 antimicrobials using the Sensititre® platform and a qualitative broth breakpoint assay. The broth breakpoint method reported higher resistance to chloramphenicol, sulfisoxazole, and the combination of trimethoprim and sulfamethoxazole, and lower resistance to streptomycin as compared to the Sensititre® assay in trial one. Selective enrichment of samples containing Salmonella in Rappaport-Vassiliadis broth reported lowered detectable resistance to amoxicillin/clavulanic acid, ampicillin, azithromycin, cefoxitin, ceftriaxone, nalidixic acid, and meropenem, and increased resistance to streptomycin and tetracycline than direct-plating samples in trial one. Using matched isolates in trial two, the Sensititre® assay reported higher resistance to chloramphenicol and gentamicin, and lower resistance to nalidixic acid as compared to the broth breakpoint method. These results suggest methodology is a critical consideration in the detection and surveillance of antimicrobial resistance phenotypes in Salmonella isolates from poultry samples and could affect the accuracy of population or industry surveillance insights and intervention strategies.

Salmonellosis is a zoonotic infection caused by Salmonella enterica serotypes contracted from contaminated products. We hypothesized that competitive exclusion between Salmonella serotypes in neonatal broilers would reduce colonization and affect the host immune response. Day of hatch broilers were randomly allocated to one of six treatment groups: (1) control, which received saline, (2) Salmonella Kentucky (SK) only on day 1 (D1), (3) Salmonella Typhimurium (ST) or Salmonella Enteritidis (SE) only on D1, (4) SK on D1 then ST or SE on day 2 (D2), (5) ST or SE on D1 then SK on D2, and (6) SK and ST or SE concurrently on D1. Salmonella gut colonization and incidence were measured from cecal contents. Livers and spleens were combined and macerated to determine systemic translocation. Relative mRNA levels of interleukin-1β (IL-1β), IL-6, IL-10, IL-18, and gamma interferon (IFN-γ) were measured in cecal tonsils and liver to investigate local and systemic immune responses. When a serotype was administered first, it was able to significantly reduce colonization of the following serotype. Significant changes were found in mRNA expression of cytokines. These results suggest competitive exclusion by Salmonella enterica serotypes affect local and systemic immune responses.

Limited research has been performed to evaluate the effects of high-frequency electrical stunning (ES) methods on the lipid oxidative stability of the meat goose livers. This study was conducted to evaluate the effects of high-frequency-ES current intensities on lipid oxidative stability and antioxidant capacity in the liver of Yangzhou goose (Anser cygnoides domesticus). Forty 92-day-old male Yangzhou geese were randomly divided into five treatments (n = 8). Geese were not stunned (control) or exposed to ES for 10 s with alternating current (AC) at 500 Hz in a water bath. Current intensities were set at 30 V/20 mA (E30V), 60 V/40 mA (E60V), 90 V/70 mA (E90V), or 120 V/100 mA (E120V), respectively. The malondialdehyde level at day 0 was the highest in 120 V (p < 0.05). Antioxidant enzymes’ activity on day 2 was the highest in E60V. The 1, 1-diphenyl-2-picrylhydrazyl free radical (DPPH·) elimination ability was lower in the E120V than that in the E60V at two days and four days postmortem (p < 0.05). A combination of 60 V/40 mA/ 500 Hz/ 10 s per bird could be applied in the ES of Yangzhou geese to improve the lipid oxidative stability and antioxidant capacity in the livers.