Tissue Protein Nitration and Peripheral Blood Endotoxin Activity are Indicative of the Severity of Systemic Organ Compromise in Naturally-Occurring Clinical Cases of Bacterial Mastitis in Holstein Dairy Cows

Abstract Text: The objective of this survey study was to determine a relationship between the intensity of tissue protein tyrosine nitration measured in samples of mammary gland, liver, pancreas and lung compared to endotoxin (LPS) activity estimated in blood.  Blood was collected from nine multiparous Holstein cows upon confirmation by the State Diagnostic Laboratory of Maryland of mastitis and the relevant causative pathogen.  In addition, control blood was collected from 17 healthy animals (cows and steers).  Blood LPS activity (BLA) was estimated using an autologous neutrophil chemiluminescence-based assay (EAA™, Spectral Diagnostics, Inc., Toronto, Canada) and expressed as a ratio of chemiluminescence of blood samples without and with an added reference quantity of LPS.  In accordance with an approved USDA Animal Care Committee protocol, mastitis animals were subsequently euthanized and tissue collected for immunohistochemical (IHC) quantification (pixel density from digital image analysis) of antigens representative of protein tyrosine nitration (pNT) and inducible nitric oxide synthase (iNOS).  Resolved pixel densities for pNT and iNOS were compared to a standardized panel of similar tissues previously obtained from healthy animals. Estimated BLA was higher (P < 0.01) in cows diagnosed with clinical mastitis than in healthy cows (0.354 ± 0.068 vs. 0.058 ± 0.010).  All mastitic cows presented IHC evidence of pNT and iNOS in the designated lobulo-alveolar mammary tissues from both infected and noninfected quarters and peripheral presence of pNT and iNOS in liver, pancreatic islets, and lung alveolae and bronchiolar epithelial cells (P < 0.03 vs. control).  Across the spectrum of BLA levels, correlation assessments suggested that the greatest levels of tissue pNT were associated with the higher levels of BLA. Given the known relationship between the presence of pNT in pathologic tissues and organ dysfunction, the data here suggest that mastitis generates perturbations in peripheral organs essential to proper metabolic and pulmonary function. 

Keywords: mastitis, endotoxin, protein tyrosine nitration