The influence of Mannheimia haemolytica A1 seed culture inoculum cell density on the production of leukotoxin in submerged culture supernatant

Abstract

Mannheimia haemolytica leukotoxin is produced during the logarithmic growth phase in submerged culture in RPMI 1640 medium with and without the addition of foetal calf serum or albumin . In order to establish a pattern of optimal leukotoxin production in small volumes in submerged cultures and to define some parameters involved, two high leukotoxin producing Mannheimia haemolytica strains were grown in RPMI 1640 medium containing either FCS or BSA. The cell growth and leukotoxin production abilities of each strain were determined concomitantly every hour in RPMI 1640 medium containing each of the additives over a time period of 6 h. The growth performance of three dilutions of a standardized seed culture inoculum prepared with each of the cultures and additives were simultaneously compared with each other using the above parameters. The different seed culture inoculum dilutions had a definite effect on the time and quantity of leukotoxin production. Both strains demonstrated peak leukotoxin production after 4 h of active growth. The addition of albumin to both isolates gave slightly increased leukotoxin levels, and both showed that the peak leukotoxin was not associated with peak cell concentration . Obvious quantitative differences in the ability of different M. haemolytica strains to produce leukotoxin were noted. Strain 12296 produced optimal leukotoxin concentration from the medium (1/25) dilution of the seed culture inoculum after 4 h, whereas strain 1/10 produced the same concentration with the low (1/5) dilution seed culture inoculum, possibly reflecting the superior production ability of the first strain. However, each strain of M. haemolytica appeared to have its own specific logarithmic cell growth and leukotoxin production pattern. The peak cell density of M. haemolytica grown in submerged RPMI 1640 culture medium cannot be used as an indication of optimal leukotoxin levels.