Serum cytokine profile of neonatal broiler chickens infected with Salmonella Typhimurium.

The avian immune system responds to Salmonella infection by expressing cytokines and chemokines. We hypothesized that the immune status of Salmonella Typhimurium (ST) challenged neonatal broilers would differ from the uninfected treatment. The objective of this experiment was to evaluate 12 cytokines. Day of hatch male chicks were randomly allocated into a control or ST challenged group. At day three of age, sterile diluent or 5.0 × 108 CFU of ST was given orally to each chick. Blood was obtained 24 h post challenge and serum separated for later analysis (n = 30 chicks/treatment). Significant (p ≤ 0.05) increases in pro-inflammatory cytokines-interleukin-6 (IL-6), IL-16, and IL-21; anti-inflammatory cytokines- IL-10; chemokines-regulated on activation, normal T cell expressed and secreted (RANTES), macrophage inflammatory protein-1ß (MIP-1ß), and MIP-3α; colony stimulating factors-macrophage colony-stimulating factor (M-CSF); and growth factors-vascular endothelial growth factor (VEGF) were observed in the serum of the challenged chicks when compared to the control. No significant differences were observed in IL-2, interferon gamma (IFNγ), and IFNα. These data indicate the detection of mucosal immune responses in broiler chickens following ST infection. The heightened levels of pro-inflammatory cytokines, chemokines, and colony stimulating factors align with known inflammatory mechanisms, like the influx of immune cells. However, the elevation of IL-10 was unexpected, due to its immunoregulatory properties. Notably, the rise in VEGF levels is compelling, as it suggests the possibility of tissue repair and angiogenesis in ST infected birds.

Unraveling frontiers in poultry health (part 1) - Mitigating economically important viral and bacterial diseases in commercial Chicken and Turkey production.

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.

Chicken-Specific Kinome Analysis of Early Host Immune Signaling Pathways in the Cecum of Newly Hatched Chickens Infected With Salmonella enterica Serovar Enteritidis.

Poultry is a major source of human foodborne illness caused by broad host range Salmonella serovars (paratyphoid), and developing cost-effective, pre-harvest interventions to reduce these pathogens would be valuable to the industry and consumer. Host responses to infectious agents are often regulated through phosphorylation. However, proteomic mechanisms of Salmonella acute infection biology and host responses to the bacteria have been limited concentrating predominately on the genomic responses of the host to infection. Our recent development of chicken-specific peptide arrays for kinome analysis of host phosphorylation-based cellular signaling responses provided us with the opportunity to develop a more detailed understanding of the early (4-24 h post-infection) host-pathogen interactions during the initial colonization of the cecum by Salmonella. Using the chicken-specific kinomic immune peptide array, biological pathway analysis showed infection with S. Enteritidis increased signaling related to the innate immune response, relative to the non-infected control ceca. Notably, the acute innate immune signaling pathways were characterized by increased peptide phosphorylation (activation) of the Toll-like receptor and NOD-like receptor signaling pathways, the activation of the chemokine signaling pathway, and the activation of the apoptosis signaling pathways. In addition, Salmonella infection induced a dramatic alteration in the phosphorylation events of the JAK-STAT signaling pathway. Lastly, there is also significant activation of the T cell receptor signaling pathway demonstrating the initiation of the acquired immune response to Salmonella infection. Based on the individual phosphorylation events altered by the early Salmonella infection of the cecum, certain conclusions can be drawn: (1) Salmonella was recognized by both TLR and NOD receptors that initiated the innate immune response; (2) activation of the PPRs induced the production of chemokines CXCLi2 (IL-8) and cytokines IL-2, IL-6, IFN-α, and IFN-γ; (3) Salmonella infection targeted the JAK-STAT pathway as a means of evading the host response by targeting the dephosphorylation of JAK1 and TYK2 and STAT1,2,3,4, and 6; (4) apoptosis appears to be a host defense mechanism where the infection with Salmonella induced both the intrinsic and extrinsic apoptotic pathways; and (5) the T cell receptor signaling pathway activates the AP-1 and NF-κB transcription factor cascades, but not NFAT.

From crypts to enteroids: establishment and characterization of avian intestinal organoids.

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 3ß 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.

Evaluation of Sodium Bisulfate on Reducing Salmonella Heidelberg Biofilm and Colonization in Broiler Crops and Ceca.

Salmonella Heidelberg (SH) on contaminated poultry causes economic and health risks to producers and consumers. We hypothesized that sodium bisulfate (SBS) would decrease SH biofilm on polyvinyl chloride (PVC) coupons and decrease the horizontal transfer of SH in broilers. Experiment 1: Salmonella Heidelberg biofilm was cultured with PVC coupons, which were treated with SBS at a pH of 3.5 for 10 min, 8 h, and 24 h. Experiment 2: Nine replicate pens per treatment were divided between two rooms. A seeder contact model was used to mimic a natural infection environment. Treatments consisted of tap water or sodium bisulfate in water at a pH of 3.5. Salmonella Heidelberg incidence and enumeration were measured in crops and ceca. Sodium bisulfate significantly reduced biofilm by 2.16 and 1.04 logs when treated for 8 and 24 h, respectively. Crop colonization was significantly decreased in trials 1 and 2 by 0.29 and 0.23 logs, respectively. Crop pH was significantly decreased in trial 2. Ceca colonization was significantly decreased in trial 1 by 0.39 logs. The results from the present study suggest that SBS may be administered to drinking water to decrease SH gut colonization and to reduce biofilm.

Determination of Antimicrobial Resistance Patterns in Salmonella from Commercial Poultry as Influenced by Microbiological Culture and Antimicrobial Susceptibility Testing Methods.

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.

Novel Models for Chronic Intestinal Inflammation in Chickens: Intestinal Inflammation Pattern and Biomarkers.

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.

Competitive Exclusion of Intra-Genus Salmonella in Neonatal Broilers.

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-1ß (IL-1ß), 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.

Altered expression of lactate dehydrogenase and monocarboxylate transporter involved in lactate metabolism in broiler wooden breast.

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.

Evaluation of the Effects of Pre-Slaughter High-Frequency Electrical Stunning Current Intensities on Lipid Oxidative Stability and Antioxidant Capacity in the Liver of Yangzhou Goose (Anser cygnoides domesticus).

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.

Dietary Factors as Triggers of Low-Grade Chronic Intestinal Inflammation in Poultry.

Inflammation is the reaction of the immune system to an injury; it is aimed at the recovery and repair of damaged tissue. The inflammatory response can be beneficial to the animal since it will reestablish tissue homeostasis if well regulated. However, if it is not controlled, inflammation might lead to a chronic response with a subsequent loss of tissue function. The intestine is constantly exposed to a number of environmental triggers that stimulate inflammation and lead to a reduction in performance. The diet and dietary components constitute consistent inflammatory triggers in poultry. Dietary components, such as anti-nutritional compounds, oxidized lipids, mycotoxins, and excess of soluble fiber or protein, are all capable of inducing a low-grade inflammatory response in the intestine of broilers throughout a 5-week grow-out period. We hypothesized that dietary factor-induced chronic intestinal inflammation is a key driver of the lower performance and higher incidence of intestinal problems observed in poultry production. Therefore, this review was aimed at exploring feed-induced chronic inflammation in poultry, the constituents of the diet that might act as inflammatory triggers and the possible effects of chronic intestinal inflammation on the poultry industry.

Effects of Bacillus subtilis and coccidial vaccination on cecal microbial diversity and composition of Eimeria-challenged male broilers.

In a companion study, the effects of dietary antibiotic alternative and coccidial vaccination on the growth performance of male broilers have been reported. In this paper, the effects of dietary probiotics and coccidial vaccination on diversity and composition of cecal microbiota were investigated using a 3 (diets) × 2 (vaccinated or non-vaccinated) factorial setting of treatments. Three diets, including a corn and soybean-meal control diet, an antibiotic diet (a control diet supplemented with bacitracin and salinomycin), and a probiotic diet (a control diet supplemented with Bacillus subtilis) were provided to broiler chicken from day 0 to 42. To simulate an Eimeria challenge in the field, all chicks were gavaged with a 20× dose of commercial coccidial vaccine containing live Eimeria oocysts on day 14. Cecal contents were collected on day 42. High-throughput sequencing of the 16S rRNA gene was used to determine microbial diversity and composition. Coccidial vaccination to broilers reduced bacterial diversity (Shannon index) of the cecal microbiota. There was a significant interaction between the dietary additive and coccidial vaccination on the observed bacterial species number. Diets supplemented with B. subtilis increased bacterial species of non-vaccinated broilers but decreased bacterial species of vaccinated broilers. In contrast, diets supplemented with antibiotics reduced bacterial species of broilers from both groups. Interactions between dietary additive and coccidial vaccination were also observed on microbial composition. Vaccinated broilers fed the B. subtilis diet exhibited the lowest Firmicutes percentage and highest Bacteroidetes percentage within the microbial community. In addition, vaccinated broilers fed the B. subtilis diet exhibited the highest Rikenella microfusus percentage. From this study, the coccidial vaccination on the day of hatch reduced the microbial diversity of broilers at a later age. The inclusion of B. subtilis-probiotics in the feed of vaccinated broilers may reduce microbial diversity in cecal content by increasing the proportion of a predominant bacterial species, R. microfusus, in the microbial community.

Evaluation of Alternative Euthanasia Methods of Neonatal Chickens.

Hatched male layer chicks are currently euthanized by maceration in the United States. Public concerns on the use of maceration have led to the search for alternative methods. We hypothesized that gas inhalation and low atmospheric pressure stunning (LAPS) are viable and humane alternatives to instantaneous mechanical destruction. The objective of this study was to evaluate the physiological and behavioral responses of recently hatched male layer chicks when subjected to carbon dioxide, nitrogen inhalation, or LAPS. The study consisted of seven treatments: breathing air (NEG), 25% carbon dioxide (CO2), 50% CO2, 75% CO2, 90% CO2, 100% nitrogen (N2), or LAPS. Ten day-of-hatch, male layer chicks were randomly assigned to each treatment, and each treatment was replicated on ten different days. A custom-made vacuum system was used to reduce air pressure inside the chamber from 100.12 kPa to 15.3 kPa for the LAPS treatment. Serum corticosterone and serotonin levels were measured using commercially available competitive enzyme linked immunosorbent assay (ELISA). Latencies to loss of posture and motionlessness were determined from video recordings. The 25% and 50% CO2 treatments were discontinued after the first replication, as the majority of the chicks recovered. The chicks in the negative (NEG) group had significantly higher levels of corticosterone than the other four euthanasia treatments. On the other hand, the serotonin levels of chicks in the NEG group was significantly lower when compared to the other four euthanasia treatments. The latencies to loss of posture and motionlessness of chicks exposed to 75% and 90% CO2 were significantly shorter than those in the LAPS and N2 inhalation treatments. These data suggest that the stress responses of chicks to the CO2, N2, and LAPS treatments do not differ among each other. However, the CO2 inhalation method was faster in inducing loss of posture and motionlessness in chicks than the LAPS and N2 inhalation treatments.

Depopulation of Caged Layer Hens with a Compressed Air Foam System.

During the 2014-2015 US highly pathogenic avian influenza (HPAI) outbreak, 50.4 million commercial layers and turkeys were affected, resulting in economic losses of $3.3 billion. Rapid depopulation of infected poultry is vital to contain and eradicate reportable diseases like HPAI. The hypothesis of the experiment was that a compressed air foam (CAF) system may be used as an alternative to carbon dioxide (CO2) inhalation for depopulating caged layer hens. The objective of this study was to evaluate corticosterone (CORT) and time to cessation of movement (COM) of hens subjected to CAF, CO2 inhalation, and negative control (NEG) treatments. In Experiment 1, two independent trials were conducted using young and spent hens. Experiment 1 consisted of five treatments: NEG, CO2 added to a chamber, a CO2 pre-charged chamber, CAF in cages, and CAF in a chamber. In Experiment 2, only spent hens were randomly assigned to three treatments: CAF in cages, CO2 added to a chamber, and aspirated foam. Serum CORT levels of young hens were not significantly different among the CAF in cages, CAF in a chamber, NEG control, and CO2 inhalation treatments. However, spent hens subjected to the CAF in a chamber had significantly higher CORT levels than birds in the rest of the treatments. Times to COM of spent hens subjected to CAF in cages and aspirated foam were significantly greater than of birds exposed to the CO2 in a chamber treatment. These data suggest that applying CAF in cages is a viable alternative for layer hen depopulation during a reportable disease outbreak.

Carbon Dioxide and Nitrogen Infused Compressed Air Foam for Depopulation of Caged Laying Hens.

Depopulation of infected poultry flocks is a key strategy to control and contain reportable diseases. Water-based foam, carbon dioxide inhalation, and ventilation shutdown are depopulation methods available to the poultry industry. Unfortunately, these methods have limited usage in caged layer hen operations. Personnel safety and welfare of birds are equally important factors to consider during emergency depopulation procedures. We have previously reported that compressed air foam (CAF) is an alternative method for depopulation of caged layer hens. We hypothesized that infusion of gases, such as carbon dioxide (CO2) and nitrogen (N2), into the CAF would reduce physiological stress and shorten time to cessation of movement. The study had six treatments, namely a negative control, CO2 inhalation, N2 inhalation, CAF with air (CAF Air), CAF with 50% CO2 (CAF CO2), and CAF with 100% N2 (CAF N2). Four spent hens were randomly assigned to one of these treatments on each of the eight replication days. A total of 192 spent hens were used in this study. Serum corticosterone and serotonin levels were measured and compared between treatments. Time to cessation of movement of spent hens was determined using accelerometers. The addition of CO2 in CAF significantly reduced the foam quality while the addition of N2 did not. The corticosterone and serotonin levels of spent hens subjected to foam (CAF, CAF CO2, CAF N2) and gas inhalation (CO2, N2) treatments did not differ significantly. The time to cessation of movement of spent hens in the CAF N2 treatment was significantly shorter than CAF and CAF CO2 treatments but longer than the gas inhalation treatments. These data suggest that the addition of N2 is advantageous in terms of shortening time to death and improved foam quality as compared to the CAF CO2 treatment.

Iron oxide nanozyme suppresses intracellular Salmonella Enteritidis growth and alleviates infection in vivo.

Rational:Salmonella Enteritidis (S. Enteritidis) is a globally significant zoonotic foodborne pathogen which has led to large numbers of deaths in humans and caused economic losses in animal husbandry. S. Enteritidis invades host cells and survives within the cells, causing resistance to antibiotic treatment. Effective methods of elimination and eradication of intracellular S. Enteritidis are still very limited. Here we evaluated whether a new intracellular antibacterial strategy using iron oxide nanozymes (IONzymes) exerted highly antibacterial efficacy via its intrinsic peroxidase-like activity in vitro and in vivo.Methods: The antibacterial activities of IONzymes against planktonic S. Enteritidis, intracellular S. Enteritidis in Leghorn Male Hepatoma-derived cells (LMH), and liver from specific pathogen free (SPF) chicks were investigated by spread-plate colony count method and cell viability assay. Changes in levels of microtubule-associated protein light chain 3 (LC3), a widely used marker for autophagosomes, were analyzed by immunoblotting, immunofluorescence, and electron microscopy. Reactive oxygen species (ROS) production was also assessed in vitro. High-throughput RNA sequencing was used to investigate the effects of IONzymes on liver transcriptome of S. Enteritidis-infected chicks. Results: We demonstrated that IONzymes had high biocompatibility with cultured LMH cells and chickens, which significantly inhibited intracellular S. Enteritidis survival in vitro and in vivo. In addition, co-localization of IONzymes with S. Enteritidis were observed in autophagic vacuoles of LMH cells and liver of chickens infected by S. Enteritidis, indicating that IONzymes mediated antibacterial reaction of S. Enteritidis with autophagic pathway. We found ROS level was significantly increased in infected LMH cells treated with IONzymes, which might enhance the autophagic elimination of intracellular S. Enteritidis. Moreover, orally administered IONzymes decreased S. Enteritidis organ invasion of the liver and prevented pathological lesions in a chicken-infection model. Non-target transcriptomic profiling also discovered IONzymes could change hepatic oxidation-reduction and autophagy related gene expressions in the S. Enteritidis infected chickens. Conclusion: These data suggest that IONzymes can increase ROS levels to promote the antibacterial effects of acid autophagic vacuoles, and thus suppress the establishment and survival of invading intracellular S. Enteritidis. As a result, IONzymes may be a novel alternative to current antibiotics for the control of intractable S. Enteritidis infections.

Immortalized cell lines for real-time analysis of circadian pacemaker and peripheral oscillator properties.

In the mammalian circadian system, cell-autonomous clocks in the suprachiasmatic nuclei (SCN) are distinguished from those in other brain regions and peripheral tissues by the capacity to generate coordinated rhythms and drive oscillations in other cells. To further establish in vitro models for distinguishing the functional properties of SCN and peripheral oscillators, we developed immortalized cell lines derived from fibroblasts and the SCN anlage of mPer2 (Luc) knockin mice. Circadian rhythms in luminescence driven by the mPER2::LUC fusion protein were observed in cultures of mPer2 (Luc) SCN cells and in serum-shocked or SCN2.2-co-cultured mPer2 (Luc) fibroblasts. SCN mPer2 (Luc) cells generated self-sustained circadian oscillations that persisted for at least four cycles with periodicities of ≈24 h. Immortalized fibroblasts only showed circadian rhythms of mPER2::LUC expression in response to serum shock or when co-cultured with SCN2.2 cells. Circadian oscillations of luminescence in mPer2 (Luc) fibroblasts decayed after 3-4 cycles in serum-shocked cultures but robustly persisted for 6-7 cycles in the presence of SCN2.2 cells. In the co-culture model, the circadian behavior of mPer2 (Luc) fibroblasts was dependent on the integrity of the molecular clockworks in co-cultured SCN cells as persistent rhythmicity was not observed in the presence of immortalized SCN cells derived from mice with targeted disruption of Per1 and Per2 (Per1(ldc) /Per2 (ldc) ). Because immortalized mPer2 (Luc) SCN cells and fibroblasts retain their indigenous circadian properties, these in vitro models will be valuable for real-time comparisons of clock gene rhythms in SCN and peripheral oscillators and identifying the diffusible signals that mediate the distinctive pacemaking function of the SCN.

Effects of neonatal alcohol exposure on vasoactive intestinal polypeptide neurons in the rat suprachiasmatic nucleus.

Neonatal alcohol exposure produces long-term changes in the suprachiasmatic nucleus (SCN) that are presumably responsible for disturbances in the light-dark regulation of circadian behavior in adult rats, including the pattern of photoentrainment, rate of re-entrainment to shifted light-dark cycles, and phase-shifting responses to light. Because SCN neurons containing vasoactive intestinal polypeptide (VIP) receive direct photic input via the retinohypothalamic tract and thus play an important role in the circadian regulation of the SCN clock mechanism by light, the present study examined the long-term effects of neonatal alcohol exposure on VIP neuronal populations within the SCN of adult rats. Male Sprague-Dawley rat pups were exposed to alcohol (EtOH; 3.0, 4.5, or 6.0 g/kg/day) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4-9 using artificial-rearing methods. At 2-3 months of age, animals from the suckle control (SC), GC, and EtOH groups were exposed to constant darkness (DD) and SCN tissue was harvested for subsequent analysis of either VIP mRNA expression by quantitative polymerase chain reaction (PCR) and in situ hybridization or of VIP-immunoreactive (ir) neurons using stereological methods. Neonatal alcohol exposure had no impact on VIP mRNA expression but dramatically altered immunostaining of neurons containing this peptide within the SCN of adult rats. The relative abundance of VIP mRNA and anatomical distribution of neurons expressing this transcript were similar among all control- and EtOH-treated groups. However, the total number and density of VIP-ir neurons within the SCN were significantly decreased by about 35% in rats exposed to alcohol at a dose of 6.0 g/kg/day relative to that observed in both control groups. These results demonstrate that VIP neuronal populations in the SCN are vulnerable to EtOH-induced insult during brain development. The observed alterations in SCN neurons containing VIP may have an impact upon clock responses to light input and thus contribute to the long-term effects of neonatal alcohol exposure on the photic regulation of circadian behavior.

Neonatal alcohol exposure differentially alters clock gene oscillations within the suprachiasmatic nucleus, cerebellum, and liver of adult rats.

BACKGROUND: In rats, alcohol exposure during the period of rapid brain growth produces long-term changes in the free-running period, photoentrainment and phase-shifting responses of the circadian rhythm in wheel-running behavior. To determine whether these alterations in circadian behavior are associated with permanent damage to the circadian timekeeping mechanism or reconfiguration of its molecular components, we examined the long-term effects of neonatal alcohol exposure on clock gene rhythms in the pacemaker located in the suprachiasmatic nucleus (SCN) and in other brain or peripheral tissues of adult rats. METHODS: Artificially reared male rat pups were exposed to alcohol (4.5 g/kg/d) or isocaloric milk formula (gastrostomy control; GC) on postnatal days 4 to 9. At 3 months of age, animals were exposed to constant darkness and then SCN, cerebellum, and liver tissue were harvested at 6-hour intervals for subsequent analysis of Period1 (Per1), Per2, Cryptochrome1 (Cry1), Bmal1, and Rev-erbalpha mRNA levels by quantitative PCR. RESULTS: In the SCN, cerebellum and liver, Per1, Per2, Cry1, Bmal1, and Rev-erbalpha expression oscillated with a similar amplitude (peak-to-trough differences of 2- to 9-fold) and phase in the suckle control (SC) and GC groups. These clock gene rhythms in control animals were marked by peak expression of Per1, Per2, Cry1, and Rev-erbalpha during the subjective day and of Bmal1 during the subjective night. The EtOH group was distinguished by altered rhythms in the expression of specific clock genes within the SCN, cerebellum and liver. In EtOH-treated rats, the SCN rhythm in Cry1 expression was strongly damped and the Per2 rhythms in the cerebellum and liver were phase-advanced such that peak expression occurred during the mid-subjective day. CONCLUSIONS: These results demonstrate alcohol exposure during the brain growth spurt alters the circadian regulation of some molecular components of the clock mechanism in the rat SCN, cerebellum, and liver. The observed alterations in the temporal configuration of essential "gears" of the molecular clockworks may play a role in the long-term effects of neonatal alcohol exposure on the regulation of circadian behavior.

Circadian regulation and function of voltage-dependent calcium channels in the suprachiasmatic nucleus.

Individual neurons within the suprachiasmatic nuclei (SCNs) are capable of functioning as autonomous clocks and generating circadian rhythms in the expression of genes that form the molecular clockworks. Limited information is available on how these molecular oscillations in individual clock cells are coordinated to provide for the ensemble rhythmicity that is normally observed from the entire SCN. Because calcium influx via voltage-dependent calcium channels (VDCCs) has been implicated in the regulation of gene expression and synchronization of rhythmicity across the population of SCN clock cells, we first examined the rat SCN and an immortalized line of SCN cells (SCN2.2) for expression and circadian regulation of different VDCC alpha1 subunits. The rat SCN and SCN2.2 cells exhibited mRNA expression for all major types of VDCC alpha1 subunits. Relative levels of VDCC expression in the rat SCN and SCN2.2 cells were greatest for L-type channels, moderate for P/Q- and T-type channels, and minimal for R- and N-type channels. Interestingly, both rat SCN and SCN2.2 cells showed rhythmic expression of P/Q- and T-type channels. VDCC involvement in the regulation of molecular rhythmicity in SCN2.2 cells was then examined using the nonselective antagonist, cadmium. The oscillatory patterns of rPer2 and rBmal1 expression were abolished in cadmium-treated SCN2.2 cells without affecting cellular morphology and viability. These findings raise the possibility that the circadian regulation of VDCC activity may play an important role in maintaining rhythmic clock gene expression across an ensemble of SCN oscillators.