PS3-2. Bovine β-defensin and Cytokine Expression in Blood of Calves Diagnosed with Bovine Respiratory Disease by Lymphocyte or Neutrophil Concentration.

Abstract

Bovine respiratory disease (BRD) imposes substantial economic costs and risks to animal health in the U.S. beef industry. It's prevention remains a significant challenge due to the diversity in BRD-causing pathogens combined with antibiotic overuse. Defensins function as innate antimicrobial peptides with potent immunoregulatory functions. Pro-inflammatory cytokines produced by innate immune cells can promote acute phase protein production such as c-reactive protein (CRP). Characterizing β-defensins in relation to cytokine and CRP expression in the blood could identify biomarkers associated with the immune response to BRD. Therefore, the objectives of this study were to 1) characterize β-defensins expression in blood and 2) compare their interaction between pro-inflammatory cytokines and CRP expression of healthy and sick calves diagnosed with BRD. Blood was collected from post-weaned, crossbred beef calves (n = 9; average BW 278.1 ± 36 kg) at 0800h on D 0, 1, 2, and 3 for complete blood count (CBC), IL-6, IL-1β, CRP, TNFα, and DEFB403 gene expression analysis. BRD diagnosis was based on blood lymphocyte or neutrophil concentration where morbidity status was assigned (HEALTHY or SICK) through two separate analyses. In analysis one, morbidity status was ‘sick’ if lymphocytes ≥ 7.5 109/L, while concentrations < 7.5 109/L resulted in a 'healthy' status. In analysis two, morbidity status was 'sick' if neutrophils ≥ 4.0 109/L, while concentrations less than 4.0 109/L resulted in a 'healthy' status. Relative target gene expression was determined by qPCR using 2-ΔΔCt method. Data were analyzed using the GLIMMIX and PROC CANCORR procedures of SAS with significance declared at (P ≤ 0.05). No differences between sick and healthy calves were observed in IL-6, IL-1β, CRP, TNFα, or DEFB403 expression in animals diagnosed with BRD based on neutrophils (P ≥ 0.62). Similarly, IL-6, IL-1β, CRP, TNFα, and DEFB403 expression did not differ (P ≥ 0.41) for morbidity status based on lymphocytes. Canonical correlation analysis was used to investigate the multivariate association between inflammatory markers (IL-6, IL-1β, CRP, and TNFα) and DEFB403. Four canonical functions were extracted, and the overall multivariate test was significant (Wilks' Λ = 0.04, P < 0.01). Only the first canonical function was statistically significant (R2 = 0.94), whereas functions 2 - 4 did not indicate additional shared variance (R2 ≤ 0.26). The first inflammatory canonical variate was defined by IL-6 (R2 = 0.83), IL-1β (R2 = 0.71), CRP (R2 = 0.83), and TNFα (R2 = 0.59). The corresponding β-defensin variate was defined by DEFB403 (R2 = 0.99). These results indicate elevated pro-inflammatory markers are strongly correlated with increased DEFB403 expression, but neither differed between calves diagnosed with BRD based on lymphocytes or neutrophils. These findings suggest DEFB403 and systemic inflammation are related, but do not distinguish between lymphocyte- and neutrophil-defined BRD diagnosis in calves.