Chronic heat stress, rather than Clostridium perfringens and/or Eimeria spp. infection, reshapes splenic and circulating T-lymphocyte subsets and mitogen responsiveness in broiler chickens

Heat stress is a growing threat to poultry health, and necrotic enteritis caused by Clostridium perfringens , predisposed by Eimeria spp. infection, has re-emerged after the withdrawal of antibiotic growth promoters. How heat stress shapes systemic cellular immunity in birds challenged with these pathogens is poorly understood. Using a 2 × 2 × 2 factorial design (cyclic heat stress × C. perfringens × Eimeria spp.; n = 7 per group), we characterized splenic and circulating leukocyte subsets by flow cytometry, splenocyte proliferation to concanavalin A (ConA) and lipopolysaccharide (LPS), and serum immunoglobulins in broiler chickens. Heat stress was the dominant source of variation for almost all T-cell phenotypes (η² up to 0.96), largely independently of infection. In the spleen, heat stress increased CD4 + , CD8 + , CD4 + CD8 + and CD4 + CD25 + cells, CD28 + CD8αβ + T cells (from 27.7% to 82.2%) and ChT1 + recent thymic emigrants, and reduced CD8αα + T cells, Bu1 + B cells and KUL01 + cells. In blood, it reduced TCRγδ + and Bu1 + lymphocytes and monocytes and shifted CD8 + T cells from the CD8αα to the CD8αβ isoform. Heat stress reduced ConA-induced but markedly increased LPS-induced proliferation, particularly of CD8 + cells, an effect attenuated by Eimeria spp., and increased serum IgA while decreasing IgY. Infections had fewer and smaller effects: Eimeria spp. increased splenic CD4 + CD25 + CD44 + cells and reduced mitogen responsiveness, and C. perfringens increased circulating KUL01 + MHCII bright monocytes, an effect blunted by heat stress. Chronic heat stress, rather than enteric infection, thus drives a coordinated remodelling of systemic T-cell compartments towards costimulation-competent CD8αβ + and CD4 + CD25 + phenotypes.