Odendaal M W, Du Plessies L
Bacterial Vaccine Development Unit, Onderstepoort Veterinary Institute, South Africa.
Onderstepoort J Vet Res. 2000 Sep;67(3):205-16.
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.
溶血曼氏杆菌白细胞毒素是在RPMI 1640培养基中进行深层培养的对数生长期产生的，无论是否添加胎牛血清或白蛋白。为了确定深层培养中小体积培养物中白细胞毒素的最佳产生模式，并确定其中涉及的一些参数，将两株高产白细胞毒素的溶血曼氏杆菌菌株在含有FCS或BSA的RPMI 1640培养基中培养。在6小时的时间段内，每小时同时测定每种菌株在含有每种添加剂的RPMI 1640培养基中的细胞生长和白细胞毒素产生能力。使用上述参数，同时比较了用每种培养物和添加剂制备的标准化种子培养接种物的三种稀释度的生长性能。不同的种子培养接种物稀释度对白细胞毒素产生的时间和量有一定影响。两株菌株在活跃生长4小时后均表现出白细胞毒素产生高峰。向两种分离物中添加白蛋白均使白细胞毒素水平略有升高，并且两者均表明白细胞毒素高峰与细胞浓度高峰无关。注意到不同溶血曼氏杆菌菌株产生白细胞毒素的能力存在明显的数量差异。菌株12296在4小时后从种子培养接种物的培养基（1/25）稀释度中产生了最佳白细胞毒素浓度，而菌株1/10在低（1/5）稀释度种子培养接种物中产生了相同浓度，这可能反映了第一株菌株的优越生产能力。然而，溶血曼氏杆菌的每个菌株似乎都有其自己特定的对数细胞生长和白细胞毒素产生模式。在RPMI 1640深层培养基中生长的溶血曼氏杆菌的细胞密度高峰不能用作最佳白细胞毒素水平的指标。
