This study investigated proteomic changes in bronchoalveolar lavage fluid (BALF) of beef calves to identify alterations related to development of naturally occurring bovine respiratory disease. BALF was collected from 162 healthy beef calves soon after weaning and transportation. Two-dimensional gel electrophoresis and mass spectrometric analysis revealed calves that later developed pneumonia had significantly lower levels of anti-inflammatory proteins including annexin A1, RAGE-binding protein, apolipoprotein-A, heat shock protein beta-1 and thioredoxin, but higher levels of antioxidant and pro-inflammatory proteins such as immunoglobulin light chain variable region, cyclophilin A, serum albumin precursor and glutathione S-transferase P.
Difference in gel electrophoresis-based analysis further showed lower levels of annexin A1, annexin A2, peroxiredoxin I, calycyphosin, superoxide dismutase, macrophage capping protein and dihydrodiol dehydrogenase 3 in the calves that later developed pneumonia. Differences in annexin levels were partially confirmed by Western blot analysis.
In healthy calves, immunohistochemistry revealed cytoplasmic expression of annexin A1 in surface epithelium of large airways, tracheobronchial submucosal glands, and goblet cells, and to a lesser degree in small airways but not in alveolar epithelium. Flow cytometry and immunocytochemistry labeled annexin A1 in blood and bronchoalveolar lavage neutrophils, monocytes, macrophages and lymphocytes. Annexin A2 expression was detected in surface epithelium of small airways, some mucosal lymphocytes, and endothelium, with weak expression in large airways, tracheobronchial submucosal glands and alveolar epithelium. For both proteins, the level of expression was similar in tissues collected 5 days after intrabronchial challenge with M. haemolytica compared to that from sham-inoculated calves.
A sandwich ELISA for annexin A1 was developed. For use with BALF, the working range was 0.3-317 ng/ml and the sensitivity was 0.8 ng/ml. The coefficient of variation of intra-assay and the between assays was less than 20%.
Together, these findings reveal annexins A1 and A2 as promising biomarkers of susceptibility to BRD in healthy at-risk calves. Further, the anti-inflammatory and pro-resolving functions of these proteins suggest roles in the pathogenesis of bacterial pneumonia of feedlot cattle. / Natural Sciences and Engineering Council (NSERC), Ontario Cattlemen’s Association, Ontario Ministry of Agriculture and Food and Ontario Veterinary College Fellowship Program
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OGU.10214/7427 |
| Date | 28 August 2013 |
| Creators | Senthilkumaran, Chandrika |
| Contributors | Caswell, Jeff |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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