|Knowles, Donald - Don|
Submitted to: Veterinary Immunology and Immunopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/28/2010
Publication Date: 10/15/2010
Citation: Subramaniam, R., Dassanayake, R.P., Norimine, J., Brown, W.C., Knowles Jr, D.P., Srikumaran, S. 2010. Molecular cloning, characterization and in vitro expression of SERPIN B1 of bighorn sheep (Ovis canadensis) and domestic sheep (Ovis aries), and comparison with that of other species. Veterinary Immunology and Immunopathology. 137(3-4):327-331. Interpretive Summary: The bighorn sheep (BHS, Ovis canadensis) population in North America has dramatically declined over the years, from an estimated 2 million in the 1800s to less than 70,000 at present. Pneumonia is the disease that contributed the most to the decline of BHS. Mannheimia haemolytica is an important etiological agent of pneumonia in BHS, domestic sheep (DS, Ovis aries) and other ruminants. Leukotoxin produced by M. haemolytica is cytolytic to all subsets of leukocytes, but PMNs are the most susceptible subset. Leuotoxin-induced cytolysis and degranulation of PMNs are responsible for the acute inflammation and lung damage characteristic of this disease. Experimental inoculation of M. haemolytica results in rapid death of BHS, but not DS. The cellular and molecular mechanisms underlying the disease process are not clear. Serine Protease Inhibitor B1 (SERPIN B1), a member of the clade B serpins, is the most effective inhibitor of PMN derived proteases such as neutrophil elastase, cathepsin G and proteinase-3. Reagents (antibodies) were developed which recognize rSERPIN and these reagents will be used to help understand this important disease process in bighorn and domestic sheep.
Technical Abstract: Mannheimia haemolytica infection results in enhanced PMN-mediated tissue damage in the lungs of bighorn sheep (BHS) compared to that of domestic sheep (DS). SERPIN B1 is an inhibitor of PMN-derived serine proteases. It prevents lung tissue injury by inhibiting the serine proteases released as a result of PMN lysis and degranulation. It is conceivable that PMNs of BHS exhibit decreased quantity and/or activity of SERPIN B1 which results in enhanced tissue injury and decreased bacterial clearance in pneumonic lungs of BHS. The objective of this study was to clone and express SERPIN B1 of BHS and DS, and develop antibodies to facilitate quantification of SERPIN B1. The 1,134 bp cDNA of SERPIN B1 of BHS and DS encodes a polypeptide of 377 amino acids. SERPIN B1 of BHS and DS exhibits 100% identity at the nucleotide and amino acid levels. The amino acid sequence of ovine (BHS/DS) SERPIN B1 displays 69%, 71%, 74%, 78% and 80% identity with that of rats, dogs, mice, humans and horses, respectively. Ovine SERPIN B1 expressed in Escherichia coli was used to develop polyclonal antibodies in mice. Western blot analysis revealed the specificity of these antibodies for ovine rSERPIN B1.