Investigation of a bighorn sheep strain of Pasteurella trehalosi: Novel characteristics and potential relevance to protection.

摘要

A strain of Pasteurella trehalosi serotype 10 (E _CO-100), originally isolated from a bighorn sheep that had succumbed to pneumonic pasteurellosis during an epizootic, was investigated and compared to well characterized strains of P. trehalosi serotype 10 and the related pathogen Mannheimia haemolytica serotype 1. Attempts were made to generate a mutant strain for use as an intranasal vaccine; however, immunological and regulatory concerns led to a shift in the focus of the project. Leukotoxin is a major virulence factor and the leukotoxin A gene (lktA) from E_CO-100 was sequenced and found to be identical at the amino acid level to a published sequence of 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 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. In contrast, supernatant from E_CO-100 exhibited a sharp decline in toxicity to undetectable levels after mid-logarithmic growth. Investigation of this anomaly revealed high protease activity in the culture supernatant of E_CO-100 compared to another P. trehalosi serotype 10 and an M. haemolytica serotype 1 using a commercial kit with a porcine gelatin/bovine albumin substrate matrix. This protease was not substrate specific as it was shown to degrade both homologous and heterologous leukotoxin, as well as collagen, which is an important pulmonary structural protein. Protease activity could be neutralized by bighorn sera in a titratable manner. The ability to neutralize protease was associated with low pneumonic lung scores in bighorns from an experimental challenge trial (r = 0.5, P = 0.1) suggesting that this previously unidentified protease may be an important protective antigen. The presence of fibrinogen-binding proteins in the culture supernatant of E_CO-100 was also observed. Collagenase and fibrinogen-binding proteins produced by P. trehalosi are previously unreported and may contribute to the pathogenesis of the characteristically fibrinous pneumonia associated with infection by E_CO-100 in bighorn sheep.