December, 1956]
M.
R.
DHANDA
ET.
AL.
275
Preparation of the vaccine
(a) As the I. V. R. I. stock strain P52 which had hitherto been employed for
the production of H. S. Commercial broth and agar-wash vaccines, represented the
largest serological group and all the strains isolated from cases of haemorrhagic
septicaemia in bovines in this country subjected to immunological study appeared
to belong to one group, this strain was selected for the preparation of the vaccine in
the present study.
(b) With a view to securing information on several other important points having
a direct bearing on the production of a potent vaccine, investigations were carried
out at the I. V. R. I. and the following data were obtained:
Rajagopalan [1942] found that (i) a suspension of Past. septica in a concentra-
tion of 3,000 million organisms per c.c. in 2 per cent phosphate buffer pH. 7.6 was
completely sterilized when exposed for 5 minutes at 37ºC to the action of the follow-
ing disinfectants at the concentrations shown:
Lysot
Merthiolate
Alcohol
Formalin
0.11 per cent
1 in 22,500
12 per cent
0.5 per cent,
(ii) that in this respect cresol and phenol were inferior to lysol. Cresol took 24
minutes at a concentration of 0.15 per cent and phenol 10 minutes at a concentration
of 0.2 per cent, (iii) that carefully heat-killed (56ºC for 5 minutes) unpreserved
vaccine was antigenically better than heated and preserved, or a preserved vaccine,
(iv) that lysol and merthiolate significantly impaired the immunogenicity of the
vaccine, but alcohol and formalin did not, (v) that alcohol prevented the growth of
cocci and subtilis at a concentration of 7.5 per cent but not at 5 per cent, (vi) that
formalin prevented the growth of the organisms at 0.01 per cent but not at 0.001
per cent, however, growth of fungus was not prevented by formalin until a con-
centration of 0.5 per cent was reached, (vii) that vaccines made from broth cultures
sterilised by heat (56ºC for ½ hour) or by disinfectants were inferior to vaccines of the
same strength made from agar slants washed in buffer phosphate. If, however,
the broth culture was washed and resuspended in phosphate buffer before steriliza-
tion, the loss in potency did not occur. It appeared that the immunizing antigens
were impaired when sterilization was carried out by heat or disinfectants with the
organisms in contact with foreign proteins, (viii) that it was highly important to
ensure that the structure of the essential parts of the germs, particularly of their
surface layers did not suffer by the treatment adopted to kill them, and (ix) that a
vaccine made from a 24-hour agar culture was as potent as one of the same strength
from a 6-hour agar culture.
(c) Later, during 1949-1952 experiments were undertaken with a view to explore
the possibilities of evolving strains, which might be used for the production of living
vaccines.
(1) Passage of strains of Past. septica in developing chick embryo and on different media
Three virulent strains of Past. septica (Past. 52, 50 and 5) were serially passaged
for over two years in the developing chick embryo and on different media, viz.
plain agar with different pH values, bile agar and penicillin agar in order to see if any
of these treatments would reduce their virulence and render them suitable for vaccine
production.