NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
11
TABLE IV.
This presents a series of experiments carried out with 1 per cent. dilutions of
rabies virus in 1 per cent. carbolic acid normal saline solution when kept at room
temperature for various periods.
The results of this series show that a 1 per cent. dilution of virus retains its
virulence unimpaired for 3 days in 1 per cent. carbolic acid at room temperature
but begins to lose it after 4 days, and is killed in 6 days, therefore a vaccine prepared
by this method would require 6 days, and it would then have to be diluted with an
equal volume of normal saline solution so as to reduce the amount of carbolic acid
to a workable percentage for antirabic treatment. This would give 0.5 per cent.
dead rabies virus in 0.5 per cent. carbolic acid after waiting for 6 days. A vaccine
prepared by this method would be too dilute for efficient treatment, so the method
was discarded.
An efficient rabies vaccine should contain at least 2 per cent. rabies virus, and not
more than 0.5 per cent carbolic acid when administered to patients. To obtain
such a vaccine by killing the virus in carbolic acid at room temperature in 24 hours
without having to add too large a percentage of carbolic acid in the first instance
would be impossible. Marie1 s t a t e s t h a t r a b i e s v i r u s i n 1 p e r c e n t . d i l u t i o n s i s
not destroyed in 2.5 per cent. carbolic acid in 24 hours at room temperature.
TABLE IV.
Experiments to test the effect of carbolic acid on " fixed rabies virus " when kept at room tempera-
ture (about 20° C).
No. of
experi-
ment.
Percentage
of " fixed
virus " in
n o r m a l s a l i n e s o l u -
tion used.
Percen-
tage of
carbolic
acid
added.
Time and tem-
perature at
which kept
before being
used.
Animal
inocu-
lated.
Amount
inoculated,
and
method of
inoculation.
Result.
REMARKS.
Per cent. Per cent.
1
1
1
24 hours, at room
temperature,
about 20°C.
Rabbit 0.2 c.c., sub-
durally.
Rabies, 7th day.
2
1
1
48 hours, at room
temperature,
about 20°C.
Ditto.
Ditto .
Ditto.
3
1
1
3 days, at room
temperature,
about 20°C.
Ditto. Ditto .
Ditto.
4
1
1
4 days, at room
temperature,
about 20°C.
Ditto.
Ditto . Rabies, 8th day.
5
1
1
6 days, at room
temperature,
about 20°C.
Ditto.
Ditto . Remained well.
6
1
1
9 days, at room
temperature,
about 20°C.
Ditto.
Ditto .
Ditto.
7
1
1
12 days, at room
temperature,
about 20°C.
Ditto.
Ditto .
Ditto.
A 1 pe cent. dilution of " fixed rabies virus " in normal saline solution can survive for at least 4 days, but not for 6 days in 1 per
cent. carbolic acid at room temperature (about 20°C).
1 Marie—L'Etude Experimentale de la Rage, 1909, page 125.
12
TABLE V.
Owing to the results obtained from the experiments given in Tables II, III,
and IV with dilutions of rabies virus in various percentages of carbolic acid,
it was necessary to try the killing effects of carbolic acid on rabies virus at a
temperature of 37ºC. It is a well known fact that rabies virus can survive for a
prolonged period in glycerine at room temperature, but is quickly killed in gly-
cerine at a temperature of 37ºC. It will be seen from the experiments which follow
that the killing power of carbolic acid on rabies virus is also increased at a tem-
perature of 37ºC. In this series of twelve experiments, four were carried out with
0.5 per cent. carbolic acid added to 2 per cent., 1 per cent., and 0.5 per cent. dilu-
tions of rabies virus ; and eight with 1 per cent. carbolic acid added to 1 per cent.,
2 per cent., and 4 per cent. dilutions of rabies virus. All these carbolised emul-
sions of virus were kept for 24 hours at a temperature of 37ºC before being used
to inoculate rabbits subdurally with 0.25 c.c.
On referring to the table it will be seen that 0.5 per cent. carbolic acid does not
kill rabies virus in 24 hours at a temperature of 37ºC even when the virus is tested
in 0.5 per cent. dilutions ; and that 1 per cent. carbolic acid kills a 4 per cent. dilu-
tion of rabies virus in 24 hours at this temperature (37ºC).
The result of this last experiment showed that it was reasonable to expect
that 1 per cent. carbolic acid at a temperature of 37ºC would probably kill stronger
preparations of the virus, and as this point was of practical importance in the
preparation of a stronger vaccine it was necessary to investigate still further the
killing properties of carbolic acid at a temperature of 37ºC.
TABLE V.
Experiments to test the effect of carbolic acid on "fixed rabies virus" when kept at a tem-
perature of 37ºC for 24 hours.
No. of
experi-
ment.
Percentage
of "fixed
virus" in normal
saline solu-
tion used.
Percen-
tage of
carbolic
acid
added.
Time and
temperature
at which
kept before
being used.
Animal
inocu-
lated.
Amount
inoculated,
and
method
of inocu-
lation.
Result.
REMARKS.
Per cent. Per cent.
1
2
0.5
24 hours at 37°C Rabbit 0.25 c.c.
Rabies, 9th day.
2
2
0.5
Ditto.
Ditto.
Ditto.
Ditto.
3
1
0.5
Ditto.
Ditto.
Ditto.
Rabies, 7th day.
4
½
0.5
Ditto.
Ditto.
Ditto.
Rabies, 8th day.
5
1
1
Ditto.
Ditto.
Ditto.
Remained well.
6
2
1
Ditto.
Ditto.
Ditto.
Ditto.
7
4
1
Ditto.
Ditto.
Ditto.
Ditto.
8
4
1
Ditto.
Ditto.
Ditto.
Ditto.
9
4
1
Ditto.
Ditto.
Ditto.
Ditto.
10
4
1
Ditto.
Ditto.
Ditto.
Ditto.
11
4
1
Ditto.
Ditto.
Ditto.
Ditto.
12
4
1
Ditto.
Ditto.
Ditto.
Ditto.
A 2 p e r c e n t . , 1 p e r c e n t . , o r ½ p e r c e n t . d i l u t i o n o f " f i x e d r a b i e s v i r u s " i n 0 . 5 p e r c e n t . c a r b o l i c a c i d n o r ma l s a l i n e s o l u t i o n i s n o t
destroyed in 24 hours at a temperature of 37°C ; but a 4 per cent. dilution in 1 per cent. carbolic acid is destroyed in 24 hours at a
temperature of 37°C.
13
TABLE VI.
In the series of six experiments recorded in this table the rabicidal properties
of 1 per cent. carbolic acid at a temperature of 37°C were tested on 8 per cent.
dilutions of rabies virus, with the result that in every instance the virus was
killed in 24 hours. A vaccine prepared by this method and then diluted with an
equal volume of sterile normal saline solution becomes 4 per cent. rabies virus in
0 . 5 per cent. carbolic acid normal saline solution ; a very suitable strength of virus
for antirabic treatment, containing sufficient carbolic acid to prevent any chance
of subsequent contamination when stored for use after being made up.
The safety of such a vaccine is beyond dispute ; so the next point to decide was,
is it efficient in conferring immunity against the subdural inoculation of lethal
doses of living rabies virus in animals, such as monkeys, dogs, and rabbits ? The
immunising experiments recorded further on furnish the answer to this question.
TABLE VI.
Experiments to test the effect of carbolic acid on "fixed rabies virus" when kept at a tem-
perature of 37°C for 24 hours.
No. of
experi-
ment.
Percentage
o f " f i x e d
virus" in
normal saline
solution
used.
Percen-
tage of
carbolic
acid
added.
Time and
temperature at
which kept
before being
used.
Animal
inocu-
lated.
Amount
inoculated,
and method
of inocu-
lation.
Result.
REMARKS.
Per cent. Per cent.
1
8
1
24 hours at
37°C.
Rabbit 0.25 c.c.,
subdurally.
Remained well.
2
8
1
Ditto.
Ditto.
Ditto.
Ditto.
3
8
1
Ditto.
Ditto.
Ditto.
Ditto.
4
8
1
Ditto.
Ditto.
Ditto.
Ditto.
5
8
1
Ditto.
Ditto.
Ditto.
Ditto.
6
8
1
Ditto.
Ditto.
Ditto.
Ditto.
8 per cent. dilutions of "fixed rabies virus" in normal saline solution are killed in 1 per cent. carbolic acid in 24 hours at a tem-
perature of 37° C.
14
CHAPTER IV.
The Effect of Normal Saline Solution on Rabies Virus.
Before proceeding to demonstrate the immunising properties of a 4 per cent.
dilution of dead rabies virus in 0.5 per cent. carbolic acid, it is necessary to prove
the effect of normal saline solution on rabies virus at room temperature, and also
at a temperature of 37°C as some of the tests of immunity (such as the rabicidal
properties of the serum) applied to the animals treated with dead rabies virus
depend upon this point, viz., whether dilutions of rabies virus are or are not quickly
killed in normal saline solution.
The experiments of Lamb and McKendrick 1 o n t h e e f f e c t o f i n c u b a t i o n a t 3 7°C
on 1 in 200, and 1 in 1,600 dilutions of rabies virus in normal saline soultion for
various periods up to 24 hours show that the virus is partially destroyed after from
2 to 4 hours, and completely destroyed after 24 hours. My own experiments on
this point failed to confirm those of Lamb and McKendrick (see Table IX).
1.—At Room Temperature.
TABLE VII.
The experiments in this table were carried out with a 1 per cent. dilution of
" fixed virus " in normal saline solution, when kept at room temperature (about
20°C) for various periods up to 15 days.
All the animals inoculated subdurally with 0.2 c.c. of the virus kept under
these conditions for 1, 2, 3, 4, and 11 days developed rabies on the 7th day, proving
that no diminution of virulence had taken place up to that time. One animal
was inoculated subdurally with 0.2 c.c. of the virus after it had been kept for 15
days, and rabies developed on the 9th day.
These results prove that rabies virus retains its virulence unimpaired in 1 per
cent. dilutions in normal saline solution at room temperature for 11 days at least,
and is not destroyed in similar dilutions in 15 days.
1 Lamb and McKendrick—Observations on Rabies. Scientific Memoirs by Officers of the Medical
and Sanitary Departments of the Government of India, No. 36, pp. 27-28.
15
TABLE VII.
Experiments to test the effect of normal saline solution on " fixed rabies virus " when kept at room
temperature (about 20°C).
No. of
experi-
ment.
Percentage of " fixed v i r u s " i n
normal saline
solution used.
Time and
temperature at
which kept
before being
used.
Animal
inoculated.
Amount
inoculated,
and method of
inoculation.
Result.
REMARKS.
Per cent.
1
1
24 hours, room tem-
perature, about 20°C
.
Rabbit . 0.2 c.c. sub-
durally.
Rabies, 7th day.
2
1
48 hours, room tem-
perature, about 20°C
.
Ditto.
Ditto.
Ditto.
3
1
3 days, room tempera-
ture, about 20° C.
Ditto.
Ditto.
Ditto.
4
1
4 days, room tempera-
ture, about 20° C.
Ditto.
Ditto.
Ditto.
5
1
11 days, room tem-
perature, about 20°C
.
Ditto.
Ditto.
Ditto.
6
1
15 days, room tem-
perature, about 20°C
.
Ditto.
Ditto.
Rabies, 9th day.
A 1 per cent. dilution of " fixed virus " in normal saline solution is not destroyed in 15 days when kept at room temperature (about
20º C).
2.—At a Temperature of 37°C.
TABLE VIII.
The object of this series of experiments was to test the effects of 1 per cent.
and 0.5 per cent. dilutions of rabies virus in normal saline solution when kept for
24 hours at a temperature of 37°C, then for 24 hours at room temperature, and
finally for 24 hours in 0.5 per cent. carbolic acid at room temperature. The results
show that a 1 per cent. dilution of virus when subjected to this treatment,
produced rabies in a rabbit on the 7th day after subdural inoculation with 0.3
c . c . ; a n d t h a t a 0 . 5 p e r c e n t . d i l u t i o n o f v i r u s a f t e r s i m i l a r t r e a t m e n t p r o d u c e d
rabies in rabbits on the 8th day after subdural inoculation with 0.3 c. c.
A control rabbit inoculated subdurally with 0.3 c.c. of 1 in 200 virus kept
for 24 hours at 37°C only, developed rabies on the 7th day.
D
16
It is evident from these results that a 1 in 200 dilution of rabies virus in normal
saline solution is not destroyed in 24 hours at a temperature of 37°C; and that it
can still further survive the effects of 0.5 per cent. carbolic acid for another 24
hours at room temperature.
TABLE VIII.
Experiments to test the effect of normal saline solution on " fixed rabies virus " when kept for 24
hours at 37°C, followed by 0.5 per cent. carbolic acid at room temperature for another 24 hours.
No. of
experi-
ment.
Dilution
of " fixed
virus " in
normal
saline
solution.
Treatment to
which it was
subjected before
being used.
Animal
inoculated.
Amount
inoculated,
and method
of inoculation.
Result.
REMARKS.
1
1 in 100 24 hours at 37°C, then
24 hours at room
temperature, and
finally 24 hours in
½ per cent. carbolic
acid at room tem-
perature.
Rabbit . 0.3 c.c. sub-
durally.
Rabies, 7th day.
2
1 in 200
Ditto.
Ditto.
Ditto.
Rabies, 8th day.
3
Ditto.
Ditto.
Ditto.
Ditto.
Ditto.
4
Ditto.
24 hours at 37°C .
Ditto.
Ditto.
Rabies, 7th day.
Control.
1 p e r c e n t . a n d ½ p e r c e n t . d i l u t i o n s o f " f i x e d r a b i e s v i r u s " i n n o r ma l s a l i n e s o l u t i o n a r e n o t d e s t r o y e d wh e n k e p t f o r 2 4 h o u r s a t
a temperature of 37°C, then for 24 hours at room temperature, followed by another 24 hours in 0.5 per cent. carbolic acid at room
temperature.
TABLE IX.
The results recorded in Table VIII clearly indicate that a temperature of
37°C. for 24 hours has no effect in diminishing the virulence of either a 1 per
cent. or a 0.5 per cent. dilution of rabies virus in normal saline solution.
In order to leave no possible doubt on this point it was decided to carry out
similar experiments, but with higher dilutions kept for various periods up to 24
hours at a temperature of 37°C. Two dilutions in normal saline solution were
prepared from the medulla of a rabbit a few hours after the animal had died
from fixed virus—one dilution 1 in 200, and the other 1 in 1,600—and both were
placed in the incubator at a temperature of 37°C. Two control rabbits were
inoculated subdurally with these dilutions immediately they were made up;
one with 0.2 c.c. of the 1 in 200 dilution, and the other with 0.3 c.c. of the 1 in
1,600 dilution; and both animals developed rabies on the 7th day.
A series of rabbits were inoculated subdurally with 0.3 c.c. from the 1 in 200
dilution after being kept for 1, 2, 4, and 24 hours in the incubator at 37°C, and a
17
similar series with 0.3 c.c. from the 1 in 1,600 dilution also kept for 1, 2, 4, and 24
hours in the incubator at 37°C. The rabbits inoculated with the 1 in 200 virus
kept for 1, 2, 4, and 24 hours at 37°C all developed rabies on the 7th day ; and
those inoculated with the 1 in 1,600 virus kept for 1, 2, and 4 hours also developed
rabies on the 7th day.
Two rabbits inoculated with the 1 in 1,600 virus kept for 24 hours at 37°C
developed rabies, one on the 10th day, and the other on the 11th day.
The results of this series of experiments prove that dilutions of 1 in 200 rabies
virus in normal saline solution are not destroyed in 24 hours at a temperature
of 37°C ; and that dilution of 1 in 1,600 loses nothing of its virulence when kept
for 4 hours at a temperature of 37°C, but has lost a little of its virulence although
not destroyed at the end of 24 hours.
TABLE IX.
Experiments to test the effect of normal saline solution on "fixed rabies virus" when kept at a
temperature of 37°C for various periods up to 24 hours.
No. of
experi-
ment.
Percen-
tage of
"fixed
virus"
in normal
saline
solution.
Time and
temperature at
which kept
before being
used.
Animal
inoculated.
Amount
inoculated,
and method of
inoculation.
Result.
REMARKS.
1
1 in 200
Used when made up
Rabbit . 0.2 c.c. sub-
durally.
Rabies, 7th
day.
Controls of
experi-
ments 3,
4, 5 and 6.
2
Ditto.
Ditto.
Ditto.
0.3 c.c. sub-
durally.
Ditto.
3
Ditto.
1 hour at 37°C
.
Ditto.
Ditto.
Ditto.
4
Ditto.
2 hours at 37°C
.
Ditto.
Ditto.
Ditto.
5
Ditto.
4 hours at 37°C
.
Ditto.
Ditto.
Ditto.
6
Ditto.
24 hours at 37°C .
Ditto.
Ditto.
Ditto.
7
1 in 1,600 Used when made up Ditto.
Ditto.
Ditto.
Control of
experi-
ments 8,
9, 10, 11
and 12.
8
Ditto.
1 hour at 37°C
.
Ditto.
Ditto.
Ditto.
9
Ditto.
2 hours at 37°C
Ditto.
Ditto.
Ditto.
10
Ditto.
4 hours at 37°C
.
Ditto.
Ditto.
Ditto.
11
Ditto.
24 hours at 37°C .
Ditto.
Ditto.
Rabies, 11th day.
12
Ditto.
Ditto.
Ditto.
Ditto.
Rabies, 10th day.
A 1 in 200, and a 1 in 1,600 dilutions of "fixed rabies virus" in normal saline solution are not destroyed when kept at a tempera-
ture of 37°C for 24 hours.
D 2
18
CHAPTER V.
The Immunisation of Animals with Rabies Virus killed by Carbolic
Acid, and their Subsequent Resistance to the Subdural Inocula-
tion of Lethal Doses of " Street Virus," and also of " Fixed Virus."
The vaccines used in these immunising experiments were prepared from the
brain and medulla of rabbits dead from " fixed virus " rabies. The dilutions of
living virus used to test for immunity were prepared from the medulla. In no case
was a vaccine or a test virus prepared from the spinal cord, as the cord contains
less virus than the brain and medulla, and besides the virus in the cord may not be
so evenly distributed as it is in the brain and medulla.
Nitsch1
has shown that in " fixed virus " rabies the brain and medulla are
more virulent (i.e., contain more virus) than the spinal cord, and this has been
confirmed by other workers.
In three of the four series of experiments here recorded, the virus was killed
by exposing 8 per cent. dilutions in 1 per cent. carbolic acid normal saline solution
to a temperature of 37°C for 24 hours. An equal volume of sterile normal
saline solution was then added, and the mixture put aside until it was required
for use. This gave a 4 per cent. dead virus in 0.5 per cent. carbolic acid.
In one series of experiments (Table IV, 4th series) on rabbits, the virus used
was a 2 per cent. dilution in 0.5 per cent. carbolic acid, and killed in the same way
as described above, viz., by exposing a 4 per cent, dilution of fixed virus in 1 per
cent. carbolic acid normal saline solution to a temperature of 37°C for 24 hours
and then diluting with an equal volume of sterile normal saline solution.
The animals subjected to treatment were monkeys, dogs, and rabbits. In
all cases the vaccine was injected hypodermically on the sides of the abdomen,
and as far as possible a fresh site was selected each time. One dose of vaccine was
given daily,—a small dose to begin with, and gradually increased as treatment
advanced. During the treatment the animals remained in good health, and as
they were well fed and attended to they increased in weight. In no case were
any local or general effects noticed. Ordinary aseptic precautions were observed
in administering the daily doses of vaccine.
The length of time which elapsed after the vaccine was prepared and before
it was used varied from ten days to three weeks, and in no case was the vaccine
used until it had been proved dead by the result of the subdural inoculation of a
sample into a rabbit. As none of the rabbits which had been inoculated sub-
durally with 8 per cent. virus kept for 24 hours in 1 per cent. carbolic acid at a
temperature of 37°C developed rabies, it may be accepted as proved that virus
N i t s c h — W i e n e r k l i n i s c h e W o c h e n s c h r i f t . N o . 2 6 o f 1 9 0 4 .
19
which has been subjected to this treatment is invariably killed. This opinion is
based on the results of some 20 experiments.
The test of immunity applied to the animals after treatment consisted in the
subdural inoculation of living virulent rabies virus ; and previous to this the serum
was tested for its rabicidal properties. The results of these experiments are given
in the tables which follow.
It is necessary to explain that the subdural inoculation of living rabies virus
is a very severe test of immunity, and that it requires a very high degree of immunity
to withstand this test. In the practical application of antirabic treatment, it is
not necessary to confer such a high degree of immunity in order to prevent infec-
tion from the bites of rabid animals. I am sure that very few (if any) patients
after undergoing an ordinary course of antirabic treatment at a Pasteur Institute
would survive the subdural inoculation of living rabies virus, although they had
acquired sufficient immunity to prevent the onset of hydrophobia after being
bitten by rabid animals.
The fact that it is possible to immunise even a small percentage of animals
treated with a rabies vaccine against the subsequent subdural inoculation of a
living rabies virus is sufficient proof that such a vaccine is efficient.
It is a well known fact that no matter what method we adopt in immunising
animals with rabies virus, only a percentage of them will be able to withstand the
subdural inoculation of living virus. In the immunising experiments recorded.
in this paper, 8 animals were treated with dead virus, and afterwards trephined
and inoculated subdurally with living virus, with the result that 6 remained well,
and 2 developed rabies. The 2 which developed rabies, developed it at a later
period than the control animals, proving that they were more resistant than the
controls.
It is worthy of note that all the monkeys (four) treated with dead vaccine
survived the subdural inoculation of living virulent virus. This result in itself is
sufficient proof that the vaccine used was efficient, and there can be no manner of
doubt about its safety.
The other test of immunity applied to the 8 animals treated with a dead
rabies vaccine was the rabicidal properties of the serum.
Samples of blood were taken (in some cases once, and in other cases twice)
before the animals were trephined for subdural inoculation, and when the serum
had separated it was mixed with an equal volume of diluted living rabies virus,
and the mixture placed in the incubator at a temperature of 37°C for 2 hours.
After remaining for 2 hours at a temperature of 37°C, the mixture was removed,
remixed, and 0.2 c.c. inoculated subdurally into rabbits.
In every case a control rabbit was inoculated with a similar preparation, ex-
cept that the serum of a normal untreated animal of the same species as the immu-
nised animal was used.
20
Altogether 12 experiments and 12 control experiments with serum were carried
out as described above, with the result that evidence of complete rabicidal power
was present in 7, and in the remaining 5 there was evidence of considerable rabi-
cidal power, as the animals developed rabies at a later period than the control
animals in which normal serum was used. The virus dilutions used in these
rabicidal experiments were passed through a fine wire strainer to keep back any
coarse particles of fibrous tissue which might be present, and in no case was the
virus passed through a filter of any kind.
1ST SERIES OF IMMUNISING EXPERIMENTS.
In this series of experiments, two healthy full grown monkeys each
weighing about 15 lbs. were immunised. They were the species of common brown
monkey found in the hills in the neighbourhood of Kasauli, and had been in cap-
tivity for three or four months before being experimented on. Each received
one inoculation daily for 24 days of a 4 per cent. dilution of dead rabies virus in
0.5 per cent. carbolic acid normal saline solution. The smallest dose given was
1 c.c. and the largest 3 c.c., and the increase from 1 to 3 c.c. was gradual. The
total amount of vaccine injected was 50 c.c.
Both animals remained in good health during treatment and gained a little
in weight.
Eleven days after completion of treatment, samples of blood were taken,
and the serum tested for its rabicidal power on " fixed virus." One monkey's
serum showed a complete rabicidal effect on an equal volume of 1 in 300 dilution
of " fixed virus," when the mixture of serum and virus was kept for 2 hours at a
temperature of 37°C, and the other monkey's serum an incomplete but marked
rabicidal effect. (See experiments 1 and 2 in 5th series.)
Fifteen days after completion of treatment both monkeys were trephined and
inoculated subdurally with 0.2 c.c. of a 1 in 500 dilution of 2nd passage " street
virus," fresh from a rabbit's medulla a few hours after death from 2nd passage
" street virus."
Both monkeys remained well.
Two control rabbits were inoculated subdurally with the same virus ; one
with 0.15 c.c. of 1 in 500 dilution, and the other 0.15 c.c. of 1 in 1,000 dilution.
The former developed rabies on the 11th day, and the latter on the 12th day.
Another control rabbit inoculated subdurally with 0.2 c.c. of the virus used
to immunise these monkeys remained well.
The results of this series of experiments prove that monkeys can be highly
immunised against rabies by the hypodermic inoculation of dead. rabies virus,
when the virus has been killed and preserved in carbolic acid.