Sequential development of antigens and toxins of Pasteurella haemolytica serotype 1 grown in cell culture medium

Abstract

Summary Pasteurella haemolytica was grown in nonsupplemented cell culture medium, or in medium supplemented with bovine serum albumin (bsa) for 24 hours. The production of leukotoxin (lkt) and endotoxin was sequentially evaluated, as were bacterial antigens associated with bacterial cell lysates and culture supernates. Supplementation of medium with bsa had no effect on bacterial growth curves; however, lkt activity was detected earlier and was greater in culture supernates from bsa-Supplemented media than from nonsupplemented medium. Leukotoxin antigen (105 kDa) was detected in culture supernates, using a monoclonal antibody, immunoblot analysis, and densitometry. The relative concentrations of lkt antigen were proportional to lkt activity. Endotoxin activity was initially lowest in the culture supernates from nonsupplemented medium, but increased during the incubation period, whereas endotoxin activity in bsa-supplemented culture supernates decreased with time in culture. In culture supernates from nonsupplemented medium, the number of antigenic bands identified by immunoblot analysis with hyperimmune anti-P haemolytica and densitometry was greater than in culture supernates from supplemented media. In bacterial lysates, a 95-kDa antigen was the major antigen detected, using the anti-lkt monoclonal antibody. The concentration of that antigen varied among lysates from nonsupplemented medium and bsa-supplemented media. Using hyperimmune anti-P haemolytica serum, minor differences were seen in the relative quantities of lysate-associated antigens dependent on time in culture and medium used. Among the major antigens seen, differences were most apparent for 150-, 100-, and 87-kDa antigens, whereas differences were not obvious for 42-40-, and 30-kDa antigens. In conclusion, at various times in culture, moderate differences were evident in P haemolytica antigens or toxins in bacterial lysates or culture supernates, and the presence of bsa in the medium altered antigenic profiles and toxin concentrations.