Effect of Eimeria Acervulina Infection on Cell-Specific Xanthine Oxidase (XO) and Inducible NO Synthase (iNOS) Activities and Duodenal Protein Tyrosine Nitration (NTp) in Chickens

Intracellular generation of nitric oxide (NO) and superoxide anion (Oˉ₂) during pro-inflammatory stress can result in the formation of 3’-nitrotyrosine proteins (NTp) that correlate with alteration in protein function and metabolic impairment. Our objective was to determine the cell-specific relationship between XO (an Oˉ₂ -generating enzyme), iNOS (a NO-generating enzyme), and NTp antigens in the chicken duodenum during Eimeria acervulina infection. Ross broilers at 21 d of age were either infected orally with 3 × 10⁵ oocysts (INF) or non-infected (NOI). At d 1, 3, 6, 7, and 10 post-infection (PI) sets of 4 NOI and 4 INF birds were euthanized and blood and tissue samples collected for biochemical analysis and immunofluorescence quantification of antigens (pixel density/cell from quantitative digital image analysis; Q-dia) representative of XO, iNOS, and NTp as stratified between epithelial cells (ECs; Cytokeratin-18⁺) or infiltrating immune cells (IICs, Myeloperoxidase⁺). Compared to NOI, there was a reduction in BW gain in INF (P < 0.01) starting at d 6. Plasma concentrations of nitrate + nitrite (NO_x, estimate of systemic NO production) was increased in INF at d 6 and 7 (P < 0.01 vs. NOI) but normalized by d 10 PI. At the peak of infection (d 6), increases in duodenal homogenate iNOS activity were observed (P < 0.01 vs. NOI). Homogenate XO activities in INF were greater at d 6 PI in liver (P < 0.05) and at d 3 and 6 PI in ileum (P < 0.05) but not duodenum. In contrast to homogenate iNOS and XO activities, Q-dia, as assessed by cell type, demonstrated increases (P < 0.05) in INF in iNOS (d 3, 6, and 7) and XO (d 1, 3, 6 and 7) proteins in ECs. In IICs, XO was not detected while iNOS was highly expressed. Cell pixel densities associated with NTp in ECs were greater in INF than in NOI (P < 0.01) at d 1, 3, 6, and 7 PI but returned to NOI levels at d 10 PI. The maximum increases in pixel densities in ECs of INF birds for NTp were observed on d 6 PI (13-fold vs. NOI) and for XO on d 3 PI (5-fold). Data indicate that Eimeria acervulina infection in chickens results in protein nitration in duodenal ECs and that ECs themselves are capable of generating the molecular signals that alone or in cooperation with IICs may ultimately lead to protein nitration.