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Vaccination against Ranikhet Disease
the centrifuge was used instead of natural sedimentation. An effective gel
was thus obtained, the process requiring only 2 days as compared with 2 weeks
or more in the original procedure. Distilled water must be used throughout;
in the preparation of a large quantity of this gel, tap water was used once
and a product of entirely different physical and adsorptive properties was
obtained. The following is the procedure for preparing 250 c.c. of the gel:—
To distilled water at 70°C. sufficient concentrated ammonia is added to
make a 4 per cent solution. A filtered solution of 50 gm. Al 2 (SO 4 ) 3 .18H 2 0
in 150 c.c. of water at 65°C. is rapidly poured into the ammonia, and the
mixture shaken vigorously for 15 minutes. The gel is then centrifuged until
a well-packed sediment is obtained; the supernatant water-clear liquid is
poured off. The sediment is made up to 1000 c.c. with water, shaken and
recentrifuged ; this process is repeated five times. After the fifth decantation,
400 c.c. of 4 per cent ammonia at 70°C. is added to the sediment and the
mixture shaken for 15 minutes. It is then centrifuged, the supernatant
liquid poured off, and the sediment made up to 1000 c.c. with water. After
shaking, it is again centrifuged. The supernatant fluid is water clear until
about the ninth or tenth washing, when it becomes opalescent. When this
stage is reached the gel is washed once more and after centrifugation and
decantation, the sediment is made up to 250 c.c. with water and thoroughly
shaken with glass beads. The gel is standardized by determining the quantity
of A1 2 0 3 per c.c. which is accomplished by drying 5 c.c. in a crucible at 110°C.,
igniting, and weighing the residue. When the quantities given above are
used, different preparations contain from 21-25 mg. A1 2 0 3 per c.c."
To 1 c.c. of this gel 1 c.c. of an M/15 solution of acid potassium phosphate
(KH 2 PO 4 ) in distilled water is added, in order to attain a suitable pH. for
adsorption (pH6.6-6.8), and then 1 c.c. of the virus mixture. The whole
is mixed and placed in the refrigerator for 2½ hours. At no time must the
gel be frozen, otherwise it will disintegrate.
After this period the gel is again stirred and centrifuged at 3000 r.p.m.
for 30 minutes. The supernatant fluid is discarded, the deposit is made up
to double its volume with 1/1000 solution of formalin in distilled water and
stored at a predetermined temperature until mature.
The source of the virus has in all cases been the spleens of birds taken
on the 4th, 5th or 6th day after inoculation with stock virus (the inoculum
has consisted of 1 c.c. of 1/100 suspension of spleen, liver and kidney, pre-
served in 50 per cent glycerine). By the 4th to 6th day the birds are either
moribund or dead. It is probable that other tissues could be used for the
preparation of vaccine but as it was desired to keep the virus content as
constant as possible only one tissue (spleen) was employed. The tissue is
first sliced with scissors and then ground as finely as possible in a mortar,
broth being added gradually to produce a homogeneous suspension. This is
then passed through muslin and a Whatman No. 42 filter paper. The filtrate
is still markedly turbid. In the first test the proportion of spleen tissue to
broth was one gram to 20 c.c., the M.L.D. of the filtrate from this mixture
being less than 0.5 c.c. of a 1 x 10 -5 dilution ; in subsequent experiments the
proportion was one gram to 4 c.c, broth, the M.L.D. being 0.5 c.c. of a
1 ×10 -7 dilution.