α 1 agp

Birds were euthanized on days 1 and 42 of the experiment. On the first day of the experiment, 10 one-day-old chicks from each group were euthanized by decapitation to collect blood and liver tissue. The remaining 20 chickens in each group were euthanized on the 42nd D of the experiment. The livers (from all birds in each group) were immediately excised, rinsed of blood, and homogenized using a mechanical homogenizer in PBS containing 0.05% sodium azide and 0.5% Triton X-100 (pH 7.4). Liver samples were then sonicated for 10 min. All solid particles were removed by centrifugation of the homogenates at 12,000 × g for 10 min. Dichloromethane (0.4 mL) was added to 1 mL of supernatant to eliminate all lipid contaminants. This was followed by a second centrifugation for 10 min at 12,000 × g, after which the supernatant was removed and immediately assayed. ELISA kits for the determination of chicken CRP, Hp, α-1-AGP, SAA, TRF and TNF-α, IFN-γ, IL-2, and IL-10 were obtained from Wuhan Fine Biotech Co., Ltd. (East Lake High-tech Development District, Wuhan, Hubei Province, China). The ELISA experiments were performed according to the manufacturer's specifications. APP and cytokine content were expressed as ng/mL total protein.

ELISA kits specific for chicken CRP, Hp, α-1-AGP, SAA, and TRF (Wuhan Fine Biotech Co., Ltd., East Lake High-tech Development District, Wuhan, Hubei Province, China) were used to determine the levels of acute phase proteins in the serum, and ELISA kits specific for chicken TNF-α, IFN-γ, IL-2, and IL-10 (Wuhan Fine Biotech Co., Ltd., East Lake High-tech Development District, Wuhan, Hubei Province, China) were used to determine cytokine levels in the serum. Optical densities of kit standards and test samples were read at 450 nm using an ELISA plate reader (Benchmark Plus Microplate Spectrophotometer System with Incubator, Bio-Rad Laboratories, CA). All procedures were performed according to the manufacturer's instructions.