title: Novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep: characteristics and potential relevance to protection.
abstract: A strain of Pasteurella trehalosi serotype 10, E(CO)-100, isolated from a bighorn sheep that had succumbed to pneumonic pasteurellosis during an epizootic, was compared to well-characterized strains of P. trehalosi serotype 10 and Mannheimia haemolytica serotype 1. The gene for leukotoxin A (lktA) from E(CO)-100 was sequenced and found to be identical on an amino acid basis to a published sequence for lktA from P. trehalosi serotype 10. However, the toxic activity in culture supernatant measured over time for E(CO)-100 was quite different from reference strains. Typically, the ability of the supernatant to lyse target cells increases over time corresponding to the logarithmic growth of the organism, peaks at mid to late phase, then declines gradually. Supernatant from E(CO)-100 exhibited a sharp decline in toxicity after mid-logarithmic growth to undetectable levels. Investigation of this anomaly using a commercial kit with a porcine gelatin/bovine albumin substrate matrix revealed high protease activity in the supernatant of this strain compared to another P. trehalosi serotype 10 and to a M. haemolytica serotype 1. Protease activity was also visualized using gelatin based zymogram gels. This protease was not substrate specific as it was shown to degrade leukotoxin. Activity was neutralized by bighorn sera in a titratable manner. There was an association between the ability to neutralize protease and low pneumonic lung scores in bighorn sheep experimentally challenged with E(CO)-100 (r=0.5, P=0.1). This previously unidentified protease may be an important protective antigen in vaccines designed to prevent pneumonic pasteurellosis resulting from P. trehalosi in bighorn sheep.
authors.affiliation: Department of Pathobiology, Ontario Veterinary College, University of Guelph, Ont., N1G 2W1, Guelph, Canada. hmcneil@uoguelph.ca
authors.forename: Heather J
authors.initials: HJ
authors.lastname: McNeil
authors.forename: Patricia E
authors.initials: PE
authors.lastname: Shewen
authors.forename: Reggie Y C
authors.initials: RY
authors.lastname: Lo
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Conlon
authors.forename: Michael W
authors.initials: MW
authors.lastname: Miller
mesh_terms: D000818:Animals; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D005098:Exotoxins / Q000235:genetics / Q000378:metabolism; D015151:Immunoblotting / Q000662:veterinary; D016978:Mannheimia haemolytica / Q000201:enzymology; D010325:Pasteurella / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D012766:Pasteurellosis, Pneumonic / Q000382:microbiology* / Q000473:pathology; D017422:Sequence Analysis, DNA; D012757:Sheep Diseases / Q000382:microbiology* / Q000473:pathology; D034581:Sheep, Bighorn; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000662:veterinary
chemical_list: D004269:DNA, Bacterial; D005098:Exotoxins; C018579:leukotoxin; D010450:Endopeptidases