The optimal production of P. haemolytica leukotoxin in the culture supernatant of a fluid medium is
dependent on a number of factors. The leukotoxin has to be produced by using a strain that is known
for its ability to produce high quantities of leukotoxin, inoculated into the most suitable type of medium
at the correct culture density containing the necessary supplements and harvested after a certain
growth period. The volume in which it is produced may also have an influence. Two different
procedures are described to produce the leukotoxin in 5 to 15-ℓ quantities in RPMI 1640 medium.
The first method used to produce leukotoxin is one that has been repeatedly described since the presence
of the leukotoxin was first established in 1978. Using this method seven batches of leukotoxin
were produced in litre quantities with leukotoxin activity ranging from 23-67 u/mℓ. The seed culture
inoculum is prepared in brain heart infusion broth, which is centrifuged before the organisms are inoculated
into RPMI 1640 medium containing 3,5% foetal calf serum and incubated for only 1 h in a
fermenter, after, which the leukotoxin is harvested.
An improved alternative method was devised which yielded higher levels of leukotoxin activity by
utilising the ability of the P. haemolytica organisms to grow and produce leukotoxin during the logarithmic
growth phase in a fermenter. A seed culture harvested in the log phase was prepared in brain
heart infusion broth by means of a series of cultures and inoculated into RPMI 1640 containing 3,5%
foetal calf serum. Three hours of active growth were allowed during which the leukotoxin was measured
by its biological activity and an ELISA assay, and the increase in cell mass by means of the optical
density every 30 min. The average leukotoxin biological activity measured 260 u/mℓ and by means of
the ELISA test the leukotoxin concentration measured 315 u/ℓ which is a substantial increase in
leukotoxin production. In comparison the average optical density only measured 0,469 at 650 nm.
Previous findings were substantiated that the highest cell density was not reflected in the highest
leukotoxin activity. It is possible to induce high levels of leukotoxin secretion in submerged cultures
with RPMI1640 medium containing foetal calf serum in the controlled environment of a fermenter in
large enough quantities for use as a vaccine by the improved preparation of the seed culture inoculum.
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