ANTIBODY-RESPONSE AGAINST PSEUDOMONAS-AERUGINOSA MEMBRANE-PROTEINS IN EXPERIMENTALLY INFECTED SHEEP

Shedded sheep inoculated epicutaneously with P. aeruginosa and then wetted experimentally by a sprinkler system, rapidly develop a green bacterial stain. This was associated with an outpouring of serous exudates onto the skin surface in the fleecerot lesion site. Histopathological analysis of dermatitic lesions revealed an infiltration of polymorphonuclear leucocytes into the dermis and the formation of a mosaic of microabscesses beneath the sloughed sheets of cornified epithelium. P. aeruginosa if present, was always localized as aggregates at the leading front of the seropurulent exudate and was never observed to invade the dermis. Animals that had been inoculated with P. aeruginosa but kept dry, showed no signs of dermatitis or serological reactivity against the inoculated bacterium. In contrast, sheep that had been inoculated and wetted, reacted serologically against P. aeruginosa whole cells in an enzyme-linked immunosorbent assay (ELISA). Eleven of 18 sheep were considered to be high-antibody responders and registered an ELISA ratio > 2.5 at one or more time points over the duration of the experiment (14 weeks). Analysis of ELISA reactivity of fleecerot sheep against fractionated cell envelope proteins of P. aeruginosa showed a preferential antibody response to outer (OMP) rather than inner (LMP) membrane proteins. Immunoblots revealed strong antibody activity against 2 major OMPs - Opr F and Opr H with apparent molecular masses of 39 and 21 kDa respectively. OMPs prepared from sarkosyl-resistant outer membrane vesicles were electrophoretically identical to OMPs prepared by a more rapid and efficient organic phase partitioning procedure (Chin and Dal, 1990). Although two other OMPs - Opr E (44 kDa) and Opr G (25 kDa) were seen in Coomassie blue-stained SDS-PAGE gels of P. aeruginosa OMPs, they were not reactive with sera from fleecerot affected sheep. It is likely that sheep with high levels of circulating serum antibody against major outer membrane proteins of P. aeruginosa may, in the event of a fleecerot episode, exude such antibodies onto the skin surface. This could provide a strategy for the control of ovine fleecerot by vaccination if highly conserved outer membrane proteins of P. aeruginosa were found to be protective.