Age and Staphylococcus aureus Inoculation Route Differentially Alter Metabolic Potential and Immune Cell Populations in Laying Hens

Abstract

In 2018 and 2019, Staphylococcus aureus was isolated from multiple post-molt commercial laying hens with unusually high mortality. A challenge study was conducted to elucidate the role of S. aureus in this disease outbreak and the work herein represents the assessment of immunological responses in laying hens experimentally infected with S. aureus isolates from these cases. A total of 200 laying hens at 22 or 96 weeks of age (100/ age group) were assigned to 1 of 4 experimental inoculation groups (negative control, oral gavage, subcutaneous injection, or intravenous injection) after a 72 h acclimation period. Blood samples were taken prior to inoculation (baseline), 6 h post-inoculation (pi), 24 hpi, 3 dpi, and 7 dpi. Additional spleen samples to further assess systemic immunity were taken at baseline, 3 and 8 dpi. Metabolic phenotypes of peripheral blood mononuclear cells (PBMC) were isolated and assessed by Seahorse metabolic assay. Immune cell profiles in the spleen and PBMC were assessed by multicolor flow cytometry. At baseline, 96-week-old laying hens had 26.7% fewer PBMC-derived T cells compared to 22-week-old birds. Older hens had 28.9% increased helper T cell (TH) populations and 60.5% reduced γδ T cells (P = 0.03 and < 0.0001) which may contribute to variable clinical responses between age groups; however, no age-related differences in metabolic potential were observed. Metabolic outcomes showed that birds remained stressed from transport and re-housing past a 72 h acclimation period and through 24 h- 3 days post-inoculation. Inoculation with S. aureus generally reduced oxidative and glycolytic potentials compared to the control, with the greatest reductions observed in birds inoculated by intravenous injection (P < 0.05). Overall CD3+ T cell populations showed significant reductions in the intravenous group compared to other inoculation routes from 24 hpi to 7 dpi (23.6–39.0%; P ≤ 0.0001). These results suggest that age-related baseline differences in T cell populations and changes to T cell subpopulations and other immune cells due to inoculation route may have an additive effect on S. aureus- induced reductions in metabolic potential; however, further research linking metabolic potential and immune cell profiles is needed.

Identification and characterization of the causative agents of Focal Ulcerative Dermatitis in commercial laying hens

“…Staphylococci are ubiquitous in poultry farms and hatcheries and are natural inhabitants of the chicken skin and mucous membranes but have also been associated with secondary infections ( 12 ). The 16S metagenomics and isolation results in this study showed a high diversity of Staphylococcus species on skin and environmental samples from FUDS negative and control flocks as compared to the FUDS positive group.…”

“…Although little research has been done on the etiology of FUDS, a correlation between disease and S. hyicus abundance has been observed ( 7 ). The production losses due to Staphylococcus infections in poultry (egg laying or meat birds) have been associated with lameness, drop in egg production ( 9 , 12 ), increased mortality, and condemnation of carcasses at slaughter facilities ( 8 ).…”

Mitochondrial and Glycolytic Capacity of Peripheral Blood Mononuclear Cells Isolated From Diverse Poultry Genetic Lines: Optimization and Assessment

“…Studies using poultry cells, specifically, has included immortal cell culture lines, such as chicken macrophage-like cells (3), embryo fibroblast cells (4), and primary brain cells (5). More recent Seahorse optimization and disease research has been published using PBMCs isolated from humans (23,24) and human cell lines (25), and research using laying hen PBMCs within the Seahorse assay has been recently published by the current lab group (26), but no optimization of chicken PBMCs has been published. Peripheral blood mononuclear cells are, however, suitable and well-published cells to use in immune cell metabolic and disease research (25), and are an ideal option for Seahorse analysis.…”

Implementing real-time immunometabolic assays and immune cell profiling to evaluate systemic immune response variations to Eimeria challenge in three novel layer genetic lines

“…As a heterogenous population of immune cells including leukocytes, monocytes, thrombocytes, and erythrocytes ( 24 ), combining real-time metabolic assays with immune profiling by flow cytometry allows for the potential identification of cell populations responsible for observed metabolic shifts. This has been done previously in assessing hen responses to Staphylococcus aureus but has not been similarly applied to Eimeria infection or genetic line comparisons of Eimeria immune responses ( 25 ). The study objective was to evaluate immunometabolic shifts and underlying cell profiles in chicken PBMC isolated from legacy genetic lines identified as being resistant (Fayoumi M5.1) and susceptible (Leghorn Ghs6 and Ghs13) during Eimeria challenge.…”