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Serotonin is a potent immunomodulatory neurohormone. Activities of the serotonergic and immune systems are often reported together in poultry studies with unidirectional analyses focused on serotonergic signaling mediating immune response. Considering serotonin's relevance across a range of immune-related poultry topics, elucidation of whether the immune system affects the serotonergic system can provide valuable insights into the bi-directionality of poultry neuroendocrine-immune interactions. The present study sought to determine whether selection for divergence in blood antibody titers to a non-pathogenic antigen coincides with parallel changes in the chicken's serotonergic system over the life of the bird. We utilized generations 49 and 50 male and female White Leghorn chickens (n = 10-12/chickens/line/sex/age) that have been selected long-term for high (HAS) or low (LAS) blood antibody response to intravenous sheep red blood cell injection. Because serotonin is predominantly produced in the gut and acts as an interkingdom signaling molecule, the first experiment sampled cecal tissue and luminal content samples from male and female HAS and LAS chickens at 293 days of age. In the second experiment, cecal tissue and luminal content, as well as plasma, were collected from HAS and LAS chickens at 28 and 56 days of age. Serotonin and its main metabolite, 5-hydroxyindoleacetic acid (5-HIAA), concentrations were determined in cecal tissue, luminal content, and plasma samples. Immunoglobulins IgA and IgY concentrations were analyzed in cecal luminal content or plasma, respectively. The HAS chickens had lower (p < 0.05) cecal IgA concentrations but higher (p < 0.05) plasma IgY concentrations than LAS chickens, suggesting a compensatory response within the intestinal tract following selection for high or low blood antibody titers. Serotonin and 5-HIAA concentrations in the ceca and plasma diverged (p < 0.05) in a pattern that mirrored ceca IgA and plasma IgY differences according to genetic line. The results from this study demonstrate that evolutionary selection pressure for humoral immune response in chickens causes responses in the serotonergic system at both enteric and systemic levels., Competing Interests: Disclosures The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

White Leghorn chickens from a common founder population have been divergently selected for high (HAS) or low (LAS) antibody responses to sheep red blood cells (SRBC) for 49 generations resulting in 2 diverse lines for this trait. Much has been studied in these two lines; however, the impact of these selection pressures on cytokine and chemokine expression is not fully understood. The purpose of this study is to determine if selection for antibody response to SRBC impacts cytokine and chemokine expression in peripheral blood leukocytes (PBL) and spleen from HAS and LAS chickens. Total RNA was isolated from PBL and spleen after which mRNA expression of cytokines (IL4, IL6, IL10, TGF-β4) and chemokines (CXCL8, CCL4) were determined by quantitative real-time RT-PCR (qRT-PCR). The data were analyzed using Student's t test comparing HAS and LAS (P < 0.05) and are reported as corrected 40-C T . PBL and spleen samples were analyzed separately. With respect to PBL, expression of IL6 was higher (P < 0.05) in PBL isolated from LAS chickens compared to those from the HAS line whereas there were no differences (P > 0.05) in IL4, IL10, CXCL8, CCL4, or TGF-β4. The cytokine and chemokine mRNA expression profiles were different in the spleen between the two lines. IL4 and CXCL8 expression were higher (P < 0.05) in spleen samples from HAS chickens than LAS. The expression of IL6, IL10, CCL4, or TGF-β4 in the spleens did not differ (P > 0.05) between the lines. The data indicate that selection for specific antibody responses to SRBC impacts the cytokine and chemokine expression profile in PBL and spleens but in different ways in HAS and LAS. These studies provide insight into the influence that selection pressures for antibody responses have on different immune response components, specifically cytokines and chemokines typically involved in the innate response., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Published by Elsevier Inc.)

Chicken and chicken products have been associated with foodborne pathogens such as Salmonella, Campylobacter, and Escherichia coli (E. coli). Poultry comprises an important segment of the agricultural economy (75 million birds processed as of 2019) in West Virginia (WV). The risk of pathogens on processed chickens has risen with the increased popularity of mobile poultry processing units (MPPUs). This study evaluated the microbial safety of broilers processed in a MPPU in WV. This study assessed aerobic plate counts (APCs), E. coli counts and the presence/absence of Salmonella and Campylobacter on 96 broiler carcasses following each MPPU step of scalding, eviscerating, and chilling. Samples were either chilled in ice water only (W) or ice water with 5 ppm chlorine (CW). The highest number of bacteria recovered from carcasses were APCs (4.21 log 10 CFU/mL) and E. coli (3.77 log 10 CFU/mL; P = 0.02). A total reduction of 0.30 (P = 0.10) and 0.63 (P = 0.01) log 10 CFU/mL for APCs and E. coli, respectively, occurred from chilling carcasses in CW. Overall, results show that E. coli, Salmonella, and Campylobacter were significantly (P < 0.05) reduced from the initial scalding to the chilling step. However, Salmonella frequency doubled (15.63-34.38%) after the evisceration step, indicating that washing carcasses after evisceration may be a critical control point in preventing cross-contamination by Salmonella. Proper chilling is also an important microbial mitigation step in MPPU processing. Results indicate that Campylobacter was more resistant to chilling than Salmonella. Campylobacter was not completely inactivated until carcasses were chilled in CW, whereas W was sufficient to reduce Salmonella on carcasses. The results led to the conclusion that although 5 ppm chlorine (Cl 2 ) achieved more bacterial reductions than water alone, the reductions were not always significant (P > 0.05). Further MPPU studies are needed to verify more effective chilling and processing strategies., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The cluster homolog of immunoglobulin-like receptors (CHIRs), previously known as the "chicken homolog of immunogloublin-like receptors," represents is a large group of transmembrane glycoproteins that direct the immune response. However, the full repertoire of putatively activating, inhibitory, or dual function CHIRA, CHIRB, and CHIRAB on chickens' immune responses is poorly understood. Herein, the study objective was to determine the genes encoding CHIR proteins and predict their function by searching canonical protein structure. A bioinformatics pipeline based on previous work was employed to search for the CHIRs from the newly updated broiler and layer genomes. The categorization into CHIRA, CHIRB, and CHIRAB types was assigned through motif searches, multiple sequence alignment, and phylogeny. In total, 150 protein-encoding genes on Chromosome 31 were identified as CHIRs. Gene members of each functional group (CHIRA, CHIRB, CHIRAB) were classified in accordance with previously recognized proteins. The genes were renamed to "cluster homolog of immunoglobulin-like receptors" (CHIRs) to allow for the naming of orthologous genes in other avian species. Additionally, expression analysis of the classified CHIRs across various reinforces their importance as immune regulators and activation in inflammatory tissues. Furthermore, over 1,000 diverse and rare CHIRs variants associated with differential Marek's disease response (P < 0.05) emphasize the impact of CHIRs on shaping avian immune responses in diverse contexts. The practical applications of these findings encompass advancing immunology, improving poultry health management, optimizing breeding programs for disease resistance, and enhancing overall animal health through a deeper understanding of the roles and functions of CHIRA, CHIRB, and CHIRAB types in avian immune responses., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Marek's disease virus (MDV), a naturally oncogenic, highly contagious alpha herpesvirus, induces a T cell lymphoma in chickens that causes severe economic loss. Marek's disease (MD) outcome in an individual is attributed to genetic and environmental factors. Further investigation of the host-virus interaction mechanisms that impact MD resistance is needed to achieve greater MD control. This study analyzed genome-wide DNA methylation patterns in 2 highly inbred parental lines 6 3 and 7 2 and 5 recombinant congenic strains (RCS) C, L, M, N, and X strains from those parents. Lines 6 3 and 7 2 , are MD resistant and susceptible, respectively, whereas the RCS have different combinations of 87.5% Line 6 3 and 12.5% Line 7 2 . Our DNA methylation cluster showed a strong association with MD incidence. Differentially methylated regions (DMRs) between the parental lines and the 5 RCS were captured. MD-resistant and MD-susceptible markers of DNA methylation were identified as transgenerational epigenetic inheritable. In addition, the growth of v-src DNA tumors and antibody response against sheep red blood cells differed among the 2 parental lines and the RCS. Overall, our results provide very solid evidence that DNA methylation patterns are transgenerational epigenetic inheritance (TEI) in chickens and also play a vital role in MD tumorigenesis and other immune responses; the specific methylated regions may be important modulators of general immunity., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Since the 1970s, 2 lines of White Leghorn chickens, HAS and LAS, have been continuously divergently selected for 5-day postinjection antibody titer to injection with sheep red blood cells (SRBC). Antibody response is a complex genetic trait and characterizing differences in gene expression could facilitate better understanding of physiological changes due to selection and antigen exposure. At 41 d of age, randomly selected HAS and LAS chickens, which had been coraised from hatch, were either injected with SRBC (HASI and LASI) or kept as the noninjected cohort (HASN and LASN). Five days later, all were euthanized, and samples collected from the jejunum for RNA isolation and sequencing. Resulting gene expression data were analyzed combining traditional statistics with machine learning to obtain signature gene lists for functional analysis. Differences in ATP production and cellular processes were observed in the jejunum between lines and following SRBC injection. HASN vs. LASN exhibited upregulation of ATP production, immune cell motility, and inflammation. LASI exhibits upregulation of ATP production and protein synthesis vs. LASN, reflective of what was observed in HASN vs. LASN. In contrast, no corresponding upregulation of ATP production was observed in HASI vs. HASN, and most other cellular processes appear inhibited. Without exposure to SRBC, gene expression in the jejunum indicates HAS generates more ATP than LAS, suggesting HAS maintains a "primed" system; and gene expression of HASI vs. HASN further suggests this basal ATP production is sufficient for robust antibody responses. Conversely, LASI vs. LASN jejunal gene expression implies a physiological need for increased ATP production with only minimal correlating antibody production. The results of this experiment provide insight into energetic resource needs and allocations in the jejunum in response to genetic selection and antigen exposure in HAS and LAS which may help explain phenotypic differences observed in antibody response., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

This study compares kinetic parameters of Salmonella and surrogate Enterococcus faecium in mash broiler feed during thermal inactivation. Two-gram samples of mash broiler feed were added into a filtered sample bag and inoculated with nalidixic acid (NaL, 200 ppm) resistant S. Typhimurium or Enterococcus faecium, followed by vacuum-packaging and heating in a circulated thermal water bath at 75°, 85°, and 95°C for 0 to 180 s. Counts of bacterial survival were analyzed on tryptic soy agar and bile esculin agar plus 200 ppm of NaL. Microbial data and thermal kinetic parameters (n = 8, Global-Fit and United States Department of Agriculture [USDA]-Integrated-Predictive-Modeling-Program software) were analyzed by JMP software. Heating mash broiler feed at 75°, 85°, and 95°C decreased (P < 0.05) Salmonella cell counts by >6 log 10 CFU/g after 180, 60, and 50 s, respectively. Heating E. faecium in feed at 75°, 85°, and 95°C for 180, 120, and 70 s achieved reductions of 3, 6, and >6.5 log 10 CFU/g, respectively. D-values of linear, Weibull models, and z-value of Salmonella at 75°, 85°, and 95°C were 1.8 to 11.2, 4.2 to 21.8, and 28.6 s, respectively, which were lower (P < 0.05) than those of E. faecium (3.7-18.1, 8.5-34.4, and 34.1 s). Linear with Tail, Linear with Tail and Shoulder, and Weibull with tail equations revealed that E. faecium were more resistant (P < 0.05) to heat than Salmonella as shown by longer "Shoulder-time" (26.5 vs. 16.2 s) and greater "Tail" effect (4.4-4.5 vs. 2.5-2.6 log 10 CFU/g). Results clearly suggested that E. faecium can be used as a surrogate for Salmonella to validate thermal inactivation during feed manufacture., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

MHCY is a candidate region for influencing immune responses in chickens. MHCY contains multiple specialized, polymorphic MHC class I loci along with loci belonging to 4 additional gene families. In this study, MHCY haplotypes were tested for association with cecal colonization after Campylobacter jejuni infection of a backcross [(Line 6 1 × Line N) × Line N] population derived from 2 White Leghorn research lines, Line 6 1 and Line N, that were previously shown to exhibit heritable differences in colonization. Samples were obtained for 51 birds challenged with 10 8 CFU Campylobacter jejuni at 3 wk of age. Viable C. jejuni in the ceca were enumerated 5 d postinfection and counts were log-transformed for analysis. Birds were assigned to either low or high colonization groups based on the individual count being below or above the mean bacterial count for all birds. The mean bacterial count of the low infection group differed significantly from the high infection group. Sex and MHCB haplotype had similar distributions within the 2 groups. Overall, 7 MHCY haplotypes were found to be segregating. Two were significantly associated with C. jejuni colonization. MHCY Y18 was associated with low colonization (P = 3.00 × 10 -5 ); whereas MHCY Y11a was associated with high colonization (P = 0.008). The MHCY haplotype impacted the mean bacterial count among all birds with MHCY Y18 having the lowest bacterial count compared with MHCY Y11a and all other MHCY (Y5, Y7, Y8, Y11b, and Y11c) haplotypes. These findings support further investigation of the contribution of chicken MHCY in resistance to Campylobacter colonization., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The chicken MHCY region contains members of several gene families including a family of highly polymorphic MHC class I genes that are structurally distinct from their classical class I gene counterparts. Genetic variability at MHCY could impart variability in immune responses, but robust tests for whether or not this occurs have been lacking. Here we defined the MHCY genotypes present in 2 sets of chicken lines selected for high or low antibody response, the Virginia Tech (VT) HAS and LAS, and the Wageningen University (WU) HA and LA lines. Both sets were developed under long-term bidirectional selection for differences in antibody responses following immunization with the experimental antigen sheep red blood cells. Lines in which selection was relaxed (VT HAR and LAR) or lacking (WU C) provided controls. We looked for evidence of association between MHCY genotypes and antibody titers. Chickens were typed for MHCY using a recently developed method based on a multilocus short tandem repeat sequence found across MHCY haplotypes. Five MHCY haplotypes were found segregating in the VT HAS and LAS lines. One haplotype was present only in HAS chickens, and another was present only in LAS chickens with distribution of the remaining 3 haplotypes differing significantly between the lines. In the WU HA and LA lines, there was a similar MHCY asymmetry. The control populations lacked similar asymmetries. These observations support the likelihood of MHCY genetics affecting heritable antibody responses and provide a basis for further investigations into the role of MHCY region genes in guiding immune responses in chickens., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The chicken bursa of Fabricius is a primary lymphoid tissue important for B-cell development. Our long-term goal is to understand the role of bursal microenvironment in an early B-cell differentiation event initiating repertoire development through immunoglobulin gene conversion in the chick embryo. We hypothesize that early bursal B-cell differentiation is guided by signals through cytokine receptors. Our theory is based on previous evidence for expression of the receptor tyrosine kinase superfamily members and interleukin receptors in unseparated populations of bursal B-cells and bursal tissue. Knowledge of the expressed genes that are responsible for B-cell differentiation is a prerequisite for understanding the bursal microenvironment's function. This project uses transcriptomic analysis to evaluate gene expression across early B-cell development. RNA-seq was performed with total RNA isolated from bursal B-cells at embryonic day (ED) 16 and ED 19 (n = 3). Approximately 90 million high-quality clean reads were obtained from the cDNA libraries. The analysis revealed differentially expressed genes involved in the Jak-STAT pathway, Wnt signaling pathway, MAPK signaling pathway, metabolic pathways including tyrosine metabolism, Toll-like receptor signaling pathway, and cell-adhesion molecules. The genes predicted to encode surface receptors, signal transduction proteins, and transcription factors identified in this study represent gene candidates for controlling B-cell development in response to differentiation factors in the bursal microenvironment., (© The Author(s) 2019. Published by Oxford University Press on behalf of Poultry Science Association.)

Nearly all genes presently mapped to chicken chromosome 16 (GGA 16) have either a demonstrated role in immune responses or are considered to serve in immunity by reason of sequence homology with immune system genes defined in other species. The genes are best described in regional units. Among these, the best known is the polymorphic major histocompatibility complex-B (MHC-B) region containing genes for classical peptide antigen presentation. Nearby MHC-B is a small region containing two CD1 genes, which encode molecules known to bind lipid antigens and which will likely be found in chickens to present lipids to specialized T cells, as occurs with CD1 molecules in other species. Another region is the MHC-Y region, separated from MHC-B by an intervening region of tandem repeats. Like MHC-B, MHC-Y is polymorphic. It contains specialized class I and class II genes and c-type lectin-like genes. Yet another region, separated from MHC-Y by the single nucleolar organizing region (NOR) in the chicken genome, contains olfactory receptor genes and scavenger receptor genes, which are also thought to contribute to immunity. The structure, distribution, linkages and patterns of polymorphism in these regions, suggest GGA 16 evolves as a microchromosome devoted to immune defense. Many GGA 16 genes are polymorphic and polygenic. At the moment most disease associations are at the haplotype level. Roles of individual MHC genes in disease resistance are documented in only a very few instances. Provided suitable experimental stocks persist, the availability of increasingly detailed maps of GGA 16 genes combined with new means for detecting genetic variability will lead to investigations defining the contributions of individual loci and more applications for immunogenetics in breeding healthy poultry., (© 2016 Poultry Science Association Inc.)

Background: Serum steroid assays play an important role in the clinical evaluation of a number of common endocrine disorders. Among various assays, tandem mass spectrometry (MS/MS) has being increasingly applied in clinical laboratories for its high sensitivity, specificity, and simultaneous multi-analyte quantitation capability. Our first generation isotope dilution steroid profile assay by HPLC-tandem MS/MS with a C-18 column allowed for the measurement of 9 steroids in 18 min employing a sample volume of 760 ul serum. We describe our second generation steroid profile assay which allows for the quantitation of 12 steroids simultaneously employing HPLC-MS/MS and isotope dilution tandem MS in 11 min. This method requires a sample volume of 200 microl., Methods: An API-5000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the PhotoSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing isotope dilution with deuterium labeled internal standard (IS) for each analyte. Two hundred microliters of serum were deproteinized by adding 300 microl of acetonitrile containing internal standards. After centrifugation, 450 microl of supernatant were diluted with 900 microl of water and 1000 microl aliquot were injected onto a C-8 column. After a 3 min wash the valve was activated to initiate the gradient elution program which eluted the steroids. Quantitation by MRM analysis was performed both in positive ion mode for 11 analytes and in negative ion mode for aldosterone. Within-day and between-day precision, reliability and accuracy of this method were assessed by correlation with other MS/MS and immunoassay methods and by recovery study., Results: Within-day CVs were <11.5% for all analytes tested and between-day CVs ranged from 3.5% to 12.2%. The results of the comparison study yield r values ranging between 0.908 and 0.999. Recovery ranged from 90% to 110%., Conclusions: This method can simultaneously measure 12 steroids in serum within 11 min with minimal sample preparation. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing and high throughput.