Identification of ovotransferrin as an acute phase protein in chickens

Abstract

Inflammation is homeostatic process associated with a variety of cellular injuries resulting from infections, toxicosis, and physical trauma. The studies on inflammation in avian species are limited. To understand the inflammation-induced changes, 4-wk-old male broiler chickens were subjected to experimental inflammation by a subcutaneous injection of croton oil (inflammatory) with changes in serum measured over time and were compared with birds treated similarly with olive oil (injected control). Croton oil treatment significantly elevated serum interleukin (IL)-6 concentrations and heterophil counts by 6 and 16 h postinjection, respectively, which returned to the basal levels of controls at 16 and 24 h, respectively. Croton oil treatment affected the serum protein profiles of chickens as assessed by SDS-PAGE and densitometric analyses. Compared with olive oil-injected or noninjected chicken sera, there were increases in the density of protein bands corresponding to molecular weights (MW) of 42, 65, 200, and 219 kDa and decreases in bands corresponding to 49 kDa (serum albumin) and a 56-kDa protein in chickens treated with croton oil. Most of these changes were evident at 24 h and lasted through 48 h. The protein band corresponding to 65 kDa was further characterized using two-dimensional gel electrophoresis and N-terminal sequence analyses. A sequence similarity search in the Genbank database using the first 22 amino acids yielded a complete homology with chicken ovotransferrin. Western blot analysis using antichicken serum transferrin or antichicken ovotransferrin antibodies also confirmed the 65-kDa protein band to be ovotransferrin. Under nonreducing conditions, the ovotransferrin standard also showed an apparent MW corresponding to 65 kDa, like the serum transferrin. The serum ovotransferrin was found to be glycosylated using a glycoprotein stain. Although the significance of ovotransferrin in avian inflammation is not clear, these results show that it is a major acute phase protein (APP) in chickens.