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.

Comparison of Gaseous and Water-Based Medium-Expansion Foam Depopulation Methods in Cull Sows.

The U.S. swine industry is currently inadequately prepared to counteract the increasing threat of high-consequence diseases. Although approved and preferred depopulation guidelines exist, ventilation shutdown (VSD+) is currently the only method being deployed during a state of emergency to depopulate large swine populations. However, the permitted use of VSD+ during constrained circumstances has been criticized due to raised swine welfare concerns. The objective of this study was to investigate the effectiveness of carbon dioxide gas (CO2), nitrogen gas (N2), compressed air foam (CAF), compressed nitrogen foam (CAF-N2) and aspirated foam (AF) during a 15-min dwell time on adult swine in an emergency depopulation situation. A small-scale trial using 12 sows per depopulation method showed the highest efficiency to induce cessation of movement for AF and CO2 (186.0 ± 48 vs. 202.0 ± 41, s ± SD). The ease of implementation and safety favored AF for further investigation. A large-scale field study using AF to depopulate 134 sows in modified rendering trailers showed a mean fill time of 103.8 s (SD: 5.0 s) and cessation of movement of 128.0 s (SD: 18.6 s) post filling. All sows were confirmed dead post-treatment for both trials. The implementation of AF in modified rendering trailers may allow for a safe and reliable method that allows for the expedient and mobile depopulation of both small and large numbers of sows during an emergency.

Evaluation of Euthanasia Methods on Behavioral and Physiological Responses of Newly Hatched Male Layer Chicks.

Newly hatched male layer chicks are considered as "by-products" in the egg industry and must be humanely euthanized at the hatchery. Instantaneous mechanical destruction (maceration) is the predominant euthanasia method applied in poultry hatcheries and is approved by the American Veterinary Medical Association (AVMA). However, maceration is not perceived by the public to be a humane means of euthanasia. The effects of alternative euthanasia methods, including carbon dioxide (CO2) or nitrogen (N2) inhalation, and a commercial negative pressure stunning system on behavioral and physiological responses of day-of-hatch male layer chicks, were evaluated in a field trial. Chick behaviors, including ataxia, loss of posture, convulsions, cessation of vocalization, and cessation of movement, were monitored. Serum hormones were assessed at the end of each of the alternative euthanasia treatments, including a control group allowed to breathe normal atmospheric air. The N2 method induced unconsciousness and death later than the CO2 and negative pressure methods, and increased serum corticosterone concentrations of neonatal chicks. Carbon dioxide inhalation increased serotonin concentrations as compared to controls, as well as the N2 and the negative pressure methods. The behavioral and physical responses observed in this study suggest that both CO2 inhalation and negative pressure stunning can be employed to humanely euthanize neonatal male layer chicks.

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.

Supplementing chestnut tannins in the broiler diet mediates a metabolic phenotype of the ceca.

As the demand for alternatives to antibiotic growth promoters (AGP) increases in food animal production, phytobiotic compounds gain popularity because of their ability to mimic the desirable bioactive properties of AGP. Chestnut tannins (ChT) are one of many phytobiotic compounds used as feed additives, particularly in South America, for broilers because of its favorable antimicrobial and growth promotion capabilities. Although studies have observed the microbiological and immunologic effects of ChT, there is a lack of studies evaluating the metabolic function of ChT. Therefore, the objective of this study was to characterize the cecal metabolic changes induced by ChT inclusion and how they relate to growth promotion. A total of 200 day-of-hatch broiler chicks were separated into 2 feed treatment groups: control and 1% ChT. The ceca from all the chicks in the treatment groups were collected on day 2, 4, 6, 8, and 10 after hatch. The cytokine mRNA quantitative RT-PCR was determined using TaqMan gene expression assays for IL-1B, IL-6, IL-8, IL-10, and interferon gamma quantification. The cytokine expression showed highly significant increased expressions of IL-6 and IL-10 on day 2 and 6, whereas the other proinflammatory cytokines did not have significantly increased expression. The results from the kinome array demonstrated that the ceca from birds fed with 1% ChT had significant (P < 0.05) metabolic alterations based on the number of peptides when compared with the control group across all day tested. The increased expression of IL-6 appeared to be strongly indicative of altered metabolism, whereas the increased expression of IL-10 indicated the regulatory effect against other proinflammatory cytokines other than IL-6. The ChT initiate a metabolic mechanism during the first 10 d in the broiler. For the first time, we show that a phytobiotic product initially modulates metabolism while also potentially supporting growth and feed efficiency downstream. In conclusion, a metabolic phenotype alteration in the ceca of chickens fed ChT may indicate the importance of enhanced broiler gut health.

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.

Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody-hapten complex: rapid and robust IgG response induced by a single subcutaneous injection.

Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90 µg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens.

Concentrations of antibiotic residues vary between different edible muscle tissues in poultry.

Antibiotics are used by veterinarians and producers to treat disease and improve animal production. The federal government, to ensure the safety of the food supply, establishes antibiotic residue tolerances in edible animal tissues and determines the target tissues (e.g., muscle) for residue monitoring. However, when muscle is selected as the target tissue, the federal government does not specify which type of muscle tissue is used for monitoring (e.g., breast versus thigh). If specific muscle tissues incorporate residues at higher concentrations, these tissues should be selected for residue monitoring. To evaluate this possibility in poultry, chickens were divided into four groups and at 33 days of age were dosed with enrofloxacin (Baytril), as per label directions, at either 25 ppm for 3 days, 25 ppm for 7 days, 50 ppm for 3 days, or 50 ppm for 7 days. Breast and thigh muscle tissues were collected from each bird (n = 5 birds per day per group) during the dosing and withdrawal period, and fluoroquinolone concentrations were determined. The results indicate higher overall enrofloxacin concentrations in breast versus thigh muscle for each treatment group (P < 0.05). These data indicate, at least for enrofloxacin, that not all muscle tissues incorporate antibiotics at the same concentrations. These results may be helpful to regulatory agencies as they determine what tissues are to be monitored to ensure that the established residue safety tolerance levels are not exceeded.

Pharmacological analysis of signal transduction pathways required for oxidative burst in chicken heterophils stimulated by a Toll-like receptor 2 agonist.

Toll-like receptors (TLRs) play an important role in the innate immune response of avian heterophils. We previously used the pharmacological inhibitors genistein, verapamil, chelerythrine, and pertussis toxin to investigate the upstream signaling events involved in TLR2-mediated oxidative burst in chicken heterophils. Only chelerythrine, a protein kinase C inhibitor, was found to significantly inhibit oxidative burst stimulated by the TLR2 agonist lipoteichoic acid (LTA). In the present study, we used selective pharmacological inhibitors to investigate the roles of phosphatidylinositol-3'-kinase (PI3-K), phospholipase C (PLC), calcium-dependent protein kinase C (PKC), extra-cellular signal regulated kinase (ERK), and nuclear translocation factor kappa B (NF-kappaB) on TLR2-mediated oxidative burst. U-73122 (a PLC inhibitor), wortmannin (a PI3-K inhibitor), PD 98059 (an ERK inhibitor), Gö 6976 (a PKC inhibitor) and Bay 11-7082 (a NF-kappaB inhibitor) significantly decreased LTA-stimulated oxidative burst in heterophils by 77%, 30%, 36%, 78%, and 61%, respectively. Activated TLR2 utilizes PI3-K, PLC, PKC, ERK, and NF-kappaB as signaling factors that mediate the oxidative burst of chicken heterophils.

Differential activation of signal transduction pathways mediating oxidative burst by chicken heterophils in response to stimulation with lipopolysaccharide and lipoteichoic acid.

Toll-like receptors (TLRs) have been previously shown to mediate oxidative burst in chicken heterophils. This study was conducted to begin to map the molecular pathways that regulate TLR-mediated oxidative burst. Peripheral blood heterophils from neonatal chicks were isolated and exposed to known inhibitors of signal transduction pathways for either 20 min (genistein, verapamil, or chelerythrine) or 120 min (pertussis toxin) at 39 degrees C. The cells were then stimulated for 30 min at 39 degrees C with Salmonella enteritidis lipopolysaccharide (LPS) or Staphylococcus aureus lipoteichoic acid (LTA). The heterophil oxidative burst was then quantitated by luminol-dependent chemiluminescence (LDCL). Genistein (a tyrosine kinase inhibitor), verapamil (a calcium channel blocker), chelerythrine (a protein kinase C inhibitor), and pertussis toxin (a G-protein inhibitor) significantly reduced LPS-stimulated oxidative burst in chicken heterophils by 34, 50, 63, and 51%, respectively. Although genistein had a statistically significant effect on reducing LPS-stimulated LDCL biologically it seems to play only a minor role within the oxidative burst pathway. Heterophils stimulated with the gram-positive TLR agonist, LTA, activated a different signal transduction pathway since chelerythrine was the only inhibitor that significantly reduced (72%) LTA-stimulated oxidative burst. These findings demonstrate that distinct signal transduction pathways differentially regulate the stimulation of oxidative burst in avian heterophils. Pertussis toxin-sensitive, protein kinase C-dependent, Ca(++)-dependent G proteins appear to regulate oxidative burst of avian heterophils stimulated with gram-negative agonist LPS; whereas, a protein kinase C-dependent signal transduction pathway plays the major role activating the oxidative burst of avian heterophils stimulated with gram-positive agonists. The distinct differences in the response of heterophils to these two agonists illustrate the specificity of TLRs to pathogen-associated molecular patterns (PAMP)s.

Functional comparison of heterophils isolated from commercial broiler chickens.

Heterophils from two pure lines (A and B) of commercial broiler chickens were isolated on days 1, 4, and 7 post-hatch to evaluate their ability to (1) phagocytose Salmonella enteritidis (SE) (2) degranulate when exposed to immune-IgG opsonized SE, and (3) produce an oxidative burst. On days 1 and 4, heterophils from line A were functionally more efficient compared to heterophils from line B (p<0.05). By 7 days post hatch, heterophil functions for both lines were comparable. To further study the inheritance of heterophil functional efficiency, F1 reciprocal crosses (line C=male Bxfemale A; line D=male Axfemale B) were evaluated for functional activity and compared with the immunologically efficient (A) and non-efficient (B) parent lines. Heterophils from line D had a more efficient heterophil function (p<0.05) when compared to heterophils from C. These results suggest that heterophil function and efficiency can be genetically transferred to progeny. Moreover they indicate that heterophil function is sex-associated and genetically controlled by the rooster since progeny of line A males maintained immunologically efficient characteristics whereas heterophils from the progeny of line B roosters remained immunologically inefficient. To our knowledge, this is the first report to describe a functional relationship between pure and F1 reciprocal crosses of broiler chickens with regard to heterophils and the innate immune response.

Inflammatory agonist stimulation and signal pathway of oxidative burst in neonatal chicken heterophils.

Heterophils are the predominant polymorphonuclear leukocytes (PMNs) in poultry. The oxidative burst of activated heterophils, which generates reactive oxygen species (ROS), is one of the first line cellular defenses against invading microorganisms. In this report, the oxidative response of heterophils from neonatal chicks to in vitro stimulation by various inflammatory agonists was investigated using a fluorescence microplate assay. Both non-opsonized formalin-killed Salmonella enteritidis and Staphylococcus aureus were able to stimulate heterophil oxidative burst. The phorbol myristate acetate (PMA) was the most potent stimulant for the chicken heterophil oxidative response, whereas, the bacterial cell surface components lipopolysaccharide (LPS) and lipoteichoic acid (LTA) were less effective. Protein kinase C (PKC) is an essential signaling component regulating heterophil oxidative response to stimulation by PMA, LPS, LTA and S. enteritidis. However, inhibition of PKC did not affect the oxidative response to stimulation by S. aureus, suggesting differential signaling pathway responsible for the activation of oxidative burst by Gram-negative S. enteritidis and Gram-positive S. aureus. Inhibition of mitogen activated protein (MAP) kinase p38 and extracellular response kinase (ERK) by SB 203580 and PD 098059, respectively, did not inhibit activated oxidative burst.

Identification of CpG oligodeoxynucleotide motifs that stimulate nitric oxide and cytokine production in avian macrophage and peripheral blood mononuclear cells.

Unmethylated CpG dinucleotides within specific flanking bases (referred to as CpG motif) are relatively abundant in bacterial DNA and are known to stimulate innate immune responses. In this study, synthetic CpG containing oligodeoxydinucleotides (CpG-ODNs) were evaluated for their ability to stimulate nitric oxide (NO), interleukin-1beta (IL-1beta), and interferon-gamma (IFN-gamma) production using an avian macrophage cell line (HD11) and peripheral blood mononuclear cells (PBMC). Results showed ODNs containing the CpG motif can activate the HD11cells and induce NO production. The optimal CpG-ODN motif for NO induction was GTCGTT. Increasing GTCGTT motifs in CpG-ODN significantly enhanced the stimulatory effect. Deviation of flanking bases of the CpG dinucleotide diminished the stimulatory activity. We also found CpG-ODN differentially stimulated expression of cytokine genes. The most active CpG motif for NO induction was also a strong stimulant for the IL-1beta gene expression in the HD11 cells, whereas different CpG motifs were found to induce IFN-gamma gene expression in PBMC.

Oxidative burst mediated by toll like receptors (TLR) and CD14 on avian heterophils stimulated with bacterial toll agonists.

Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) such as lipopolysaccharide (LPS) and lipoteichoic acid (LTA), which are found in the cell walls of gram-negative and gram-positive bacteria, respectively. This study was conducted to determine if TLRs are present on chicken heterophils and if these receptors mediate oxidative burst. Heterophils isolated from neonatal chicks were exposed to gram-negative Salmonella enteritidis (SE), gram-positive Staphylococcus aureus (SA), SE-LPS, and SA-LTA and the oxidative burst quantitated by luminol-dependent chemiluminescence. SE, SA, SE-LPS, and SA-LTA stimulated a significant increase in oxidative burst from heterophils. Furthermore, we measured the inhibitory effects of polyclonal antibodies on rat CD14, human TLR2 and TLR4 on the oxidative burst of heterophils when stimulated with LPS and LTA. The data suggest that TLR2 and TLR4 mediate LPS-stimulated oxidative burst while CD14 and TLR2 mediate LTA-stimulated oxidative burst in heterophils. This is the first report of PAMPs from gram-positive and gram-negative bacteria interacting with TLRs of avian heterophils.