NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
21
TABLE I.
1ST SERIES OF IMMUNISING EXPERIMENTS.
Experiments on monkeys to test the immunising properties of dead rabies virus. The virus was
killed by exposing an 8 per cent. dilution of " fixed virus " in 1 per cent. carbolic acid normal
saline solution to a temperature of 37°C for 24 hours ; it was then diluted with an equal
volume of normal saline solution, which gave 4 per cent. virus in 0.5 per cent. carbolic acid.
No. of
experiment.
Duration of
treatment.
Total quantity
of 4 per cent,
dead rabies virus
injected sub-
cutaneously.
Test to which subjected.
Result.
REMARKS.
1. Monkey . One injection
daily for 24
days.
50 c.c. . 15 days after completion
of treatment, was tre-
phined and inoculated
subdurally with 0.2 c.c.
of 1 in 500 dilution of
2nd passage " street
virus " fresh from a
rabbit's medulla.
Remained well.
2. Monkey .
Ditto.
Ditto.
Ditto.
Ditto.
3. Rabbit. Con-
trol of test
virus.
..
..
Trephined and inocu-
lated subdurally with
0.15 c.c. of 1 in 500
dilution of the same
virus used to test the
two monkeys.
Rabies, 11th day.
4. Rabbit, 2nd
control of test
virus.
..
..
Same as No. 3 experi-
ment, with the excep-
tion that 1 in 1,000
dilution of test virus
was used.
Rabies, 12th day.
5. Rabbit. Con-
trol of immu-
nising virus.
..
..
Trephined and inocu-
lated subdurally with
0.2 c.c. of the virus used
for immunising Nos. 1
and 2 monkeys.
Remained well.
2ND SERIES OF IMMUNISING EXPERIMENTS.
In this series two healthy full grown monkeys of a similar species to the two
used for the 1st series of experiments were immunised. Each weighed about 16
lbs.; and they had been in captivity for three months before being experimented on.
Both received the same treatment, viz., one hypodermic injection 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 3½ c.c., and the increase
from 1 c.c. at the outset to 3½ c.c. later on was gradual. No change was apparent
in the condition of their health during treatment.
Fourteen days after the completion of treatment samples of blood were taken
to test for rabicidal properties of the serum. The test applied to the serum of
these two monkeys was carried out in a manner similar to the test applied to the
serum of the two monkeys referred to in the 1st series of experiments, except that
a 1 in 200 dilution of 1st passage human virus, fresh from the medulla of a rabbit
which had died from subdural inoculation of a portion of the brain of a hydro-
22
phobia patient, was used. One monkey's serum showed a complete rabicidal
effect on an equal volume of 1 in 200 1st passage human virus ; and the other
monkey's serum an incomplete but marked effect.
On the fifteenth day after completion of treatment both monkeys were tre-
phined and inoculated subdurally with 0.3 c.c. of a 1 in 600 dilution of 1st passage
human virus freshly prepared from the medulla of a rabbit which had died from
rabies after the subdural inoculation of a portion of brain from a hydrophobia patient
.
Both monkeys remained well.
A control monkey inoculated subdurally with 0.3 c.c. of a 1 in 600 dilution
of the same virus used to test the two immunised monkeys developed furious
rabies on the 10th day, and died on the 13th day.
A control rabbit inoculated subdurally with 0.2 c.c. of a 1 in 600 dilution of
the same virus also developed rabies on the 10th day, the same period as the con-
trol monkey.
Another control rabbit inoculated subdurally with 0.2 c.c. of the virus used
to immunise the two monkeys remained well.
The results of this series of experiments are additional proof that monkeys can
be highly immunised by the hypodermic inoculation of dead rabies virus, when
the virus has been killed and preserved in carbolic acid.
TABLE II.
2ND SERIES OF IMMUNISING EXPERIMENTS.
Experiments on monkeys to test the immunising properties of dead rabies virus. The virus was
killed by exposing an 8 per cent. dilution of "fixed virus" in 1 per cent. carbolic acid normal
saline solution to a temperature of 37ºC. for 24 hours ; it was then diluted with an equal volume
of normal saline solution, which gave 4 per cent. virus in 0.5 per cent. carbolic acid.
No. of
experiment.
Duration of
treatment.
Total quantity
of 4 per cent.
dead rabies virus
injected sub-
cutaneously.
Test to which subjected.
Result.
REMARKS.
1. Monkey .
One injection
daily for 24
days.
60 c.c. .
15 days after completion
of treatment was tre-
phined and inoculated
subdurally with 0.3 c.c.
of 1 in 600 dilution of
1st passage human
virus, fresh from a
rabbit's medulla.
Remained well.
2. Monkey .
Ditto.
Ditto.
Ditto.
Ditto.
3. Monkey. Con-
trol of test
virus.
..
..
Trephined and inocu-
lated with 0.3 c.c. of
same virus used for ex-
periments 1 and 2.
Rabies, 10th day.
4. Rabbit. 2nd
control of test
virus.
..
..
Same as above, but only
0.2 c.c. used.
Ditto.
5. Rabbit. Con-
trol of immu-
nising virus.
..
..
Trephined and inocu-
lated subdurally with
0.2 c.c. of the virus
used for immunising
Nos. 1 and 2 monkeys.
Remained well.
23
3RD SERIES OF IMMUNISING EXPERIMENTS.
The experiments in this series were carried out on two dogs. One was a full
grown animal and weighed 24 lbs.; the other was a little more than half
grown (a puppy) and weighed 10 lbs. at the commencement of treatment, and
13 lbs. on completion.
Both were treated with hypodermic injections of 4 per cent. dead rabies virus
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 , a n d we r e g i v e n o n e d o s e d a i l y
for 28 days. The doses varied from 1½ c.c. to 4 c.c. in the larger dog, and from
1 c.c. to 3 c.c. in the smaller animal. The total amount of vaccine given to the
larger dog was 100 c.c., and to the smaller one 90 c.c.
On the 11th day after completion of treatment samples of blood were
taken from both clogs to test the rabicidal properties of the serum on " fixed
virus."
The serum in both cases gave a complete rabicidal effect when mixed with
equal parts of a 1 in 300 dilution of " fixed virus," and kept for 2 hours at a tem-
perature of 37°C.
On the 23rd day after completion of treatment the serum of both dogs was
again tested for its rabicidal effect, and this time on a 1 in 300 dilution of " street
virus" (2nd passage jackal's virus) with the result that although the effect was
incomplete it was marked in both cases. (See experiments 5 and 6 in 5th
series.)
In both these dogs either the rabicidal power of the serum was higher on the
11th day than on the 23rd day after completion of treatment, or possibly " fixed
virus " was more easily killed than 2nd passage jackal's virus.
Twenty-four days after completion of treatment both dogs were trephined
and inoculated subdurally with 0.3 c.c. of a 1 in 600 2nd passage jackal's virus
fresh from a rabbit's medulla. The large dog remained well, and the puppy dog
developed rabies on the 29th day. A control dog inoculated subdurally with
0.3 c.c. of 1 in 600 of the same virus used to test the two immunised dogs deve-
loped rabies on the 10th day. A control rabbit inoculated subdurally with 0.2
c.c. of the virus used to immunise the two dogs remained well.
The results of this series of experiments show that of two dogs treated with
dead rabies virus, one was so highly immunised as to be able to withstand the
subdural inoculation of a certain lethal dose of living rabies virus, and the other
almost succeeded in reaching this degree of immunity, as evidenced by the fact
that the incubation period after subdural inoculation was 29 days as compared
with 10 days in the control dog.
24
TABLE III.
3RD SERIES OF IMMUNISING EXPERIMENTS.
Experiments on dogs to test the immunising properties of dead rabies virus. The virus was killed
by exposing an 8 per cent. dilution of "fixed virus" in 1 per cent. carbolic acid normal saline
solution to a temperature of 37ºC. for 24 hours ; it was then diluted with an equal volume of
normal saline solution, which gave 4 p er cent. virus in 0.5 per cent. carbolic acid.
No. of
experiment.
Duration of
treatment.
Total quantity
of 4 per cent.
dead rabies virus
injected sub-
cutaneously.
Test to which subjected.
Result.
REMARKS
.
1. Dog, weight
24 lbs., full
grown.
One injection
daily for 28
days.
100 c.c. . 24 days after completion
of treatment, was tre-
phined and inoculated
subdurally with 0.3 c.c.
of 1 in 600 dilution 2nd
passage jackal's virus,
fresh from a rabbit's
medulla.
Remained well.
2. Puppy dog,
weight 13 lbs.
Ditto.
90 c.c. .
Ditto. .
Rabies, 29th day.
3. Control dog
weight 14 lbs.
full grown.
..
..
Trephined and inocu-
lated subdurally with
0.3 c.c. of 1 in 600
dilution of same virus
used to test Nos. 1 and
2 dogs.
Rabies, 10th day.
4. Rabbit. Con-
trol of immu-
nising virus.
..
..
Trephined and inocu-
lated subdurally with
0.2 c.c. of the virus
used for immunising
Nos. 1 and 2 dogs.
Remained well.
4TH SERIES OF IMMUNISING EXPERIMENTS.
The experiments in this series were carried out on rabbits. Two healthy full
grown rabbits each weighing 2½ kilogrammes were immunised with 2 per cent.
dilutions of dead rabies virus in 0.5 per cent. carbolic acid normal saline solution.
They received one hypodermic injection daily for 24 days. The doses varied
from 0.5 c.c. at the outset, to 2 c.c. later on, and the increase from the smaller to
the larger doses was gradual. The total amount of vaccine given to each animal
was 36 c.c.
On the 8th day after completion of treatment the serum of both animals gave
a complete rabicidal effect when mixed with a 1 in 200 dilution of "fixed virus"
and kept for 2 hours at a temperature of 37ºC.
On the 25th day the serum was again tested on a 1 in 200 dilution of "fixed
virus" when the rabicidal effect was found to be complete in one, and incomplete,
but marked in the other.
On the 25th day after completion of treatment both rabbits were trephined
and inoculated subdurally with 0.25 c.c. of a 1 in 200 dilution of 2nd passage "street
25
virus" fresh from the medulla of a rabbit dead from 2nd passage "street virus"
rabies. One rabbit remained well and the other (the one whose serum gave a
incomplete rabicidal effect on the 25th day) developed rabies on the 16th day.
A control rabbit inoculated subdurally with a 1 in 400 dilution (i.e., half the
strength) of the same virus used to test the two immunised animals developed
rabies on the 10th day. Another control rabbit inoculated subdurally with 0.25
c.c. of this virus used to immunise the two rabbits remained well.
The results of this series of experiments show that rabbits can be immunised
with hypodermic injections of dead rabies virus ; and of two rabbits immunised by
this method one attained a degree of immunity sufficient to withstand the subdural
inoculation of virulent rabies virus, and the other almost succeeded in attaining
this degree of immunity.
The test applied to these two rabbits was a very severe one, viz., the subdural
inoculation of 0.25 c.c. of a 1 in 200 dilution of virus. A more highly diluted virus
such as 1 in 600 or 1 in 1,000 would have been a fairer test.
TABLE IV.
4TH SERIES OF IMMUNISING EXPERIMENTS.
Experiments on rabbits to test the immunising properties of dead rabies virus. The virus used was
killed 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 21 hours ; it was then diluted with an equal
volume of normal saline solution, which gave 2 per cent. virus in 0.5 per cent. carbolic acid.
No. of experi-
ment.
Duration of
treatment.
Total quantity
of 2 per cent.
dead rabies virus
injected sub-
cutaneously.
Test to which subjected.
Results.
REMARKS.
1
One injection
daily for 24
days.
36 c.c.
.
25 days after completion
of treatment was tre-
phined and inoculated
subdurally with 0.25 c.c.
of 1 in 200 dilution of
2nd passage "street
virus" fresh from a
rabbit's medulla.
Remained well.
The test ap-
plied to
these two
experiments
(Nos. 1 and
2) was a
severe one,
as the virus
used was
diluted only
to 1 in 200.
2
Ditto
.
Ditto
.
Ditto ditto
. Rabies, 16th day.
3. Control of
test virus.
. .
..
Trephined and inoculated
subdurally with 0.25 c.c.
of 1 in 400 dilution of
same virus used for
experiments 1 and 2.
Rabies, 10th day.
4. Control of
immunising
virus.
. .
. .
Trephined and inoculated
subdurally with 0.25
c.c. of virus used for
the immunising experi-
ments 1 and 2.
Remained well.
E 2
26
CHAPTER VI.
The Evidence of Immunity in the Serum of Animals treated with
Rabies Virus killed by Carbolic Acid.
SERIES V.
EXPERIMENTS TO TEST THE RABICIDAL PROPERTIES OF THE SERUM OF ANIMALS IMMUNISED
WITH DEAD RABIES VIRUS.
This series of experiments gives the rabicidal properties of the serum of the
eight animals (four monkeys, two dogs, and two rabbits) immunised with 4 per
cent. dead virus in 0.5 per cent. carbolic acid normal saline solution referred to in
series I, II, III, and IV.
In each of these experiments blood was taken in glass capsules and put aside
at room temperature for 24 hours until the serum had separated. The serum was
then pipetted off and mixed with an equal volume of 1 in 200 or 1 in 300 dilution
of living rabies virus in normal saline solution as recorded opposite each experi-
ment. The mixture was then placed in an incubator at a temperature of 37°C
for two hours, after which it was re-mixed and 0.2 c.c. inoculated subdurally into
rabbits. In each case a control rabbit was inoculated subdurally with 0.2 c.c. of
an exactly similar mixture as regards the dilution of virus and kind of virus used,
except that the serum added was from a normal monkey, dog, or rabbit, as the case
might be and kept for the same time (2 hours) at 37°C.
On referring to the table (Series V) it will be seen that 12 experiments were
carried out with the serum from 8 immunised animals, and that in 7 of these experi-
ments the serum showed a complete rabicidal action, and in 5 an incomplete but
marked action.
It was a mistake perhaps, not to have filtered the rabies emulsions before
adding them to the serum to be tested. They were only passed through a fine wire
strainer to keep back coarse particles of fibrous tissue. However, the results as
they stand show a complete rabicidal action in 7, and a well marked action in 5.
In the 12 control experiments the animals developed rabies after an incuba-
tion period which corresponded to the normal incubation period of the virus used.
The results of these experiments prove that the serum of animals immunised
with rabies virus killed and preserved in carbolic acid has a well marked rabicidal
effect on living rabies virus.
27
When we examine the results recorded in Tables VII, VIII and IX, in which
the effects of normal saline solution on various dilutions of rabies virus at a tem-
perature of 37°C and at room temperature are given, it is evident that the normal
saline solution used to dilute the virus was not the cause of the rabicidal action at-
tributed to the serum in Series V.
In 1891 Babes demonstrated the fact that the serum of animals immunised
against rabies had a well marked rabicidal action on living rabies virus
in vitro.
Marie 1 mentions that Kraus and Kreissl have studied the rabicidal properties
of the serum of patients during and after completion of Pasteur's method of
antirabic treatment and found evidence of rabicidal properties 22 days after
the completion of treatment, but not during or immediately after treat-
ment.
Lamb and McKendrick, 2 in 1908, tested the serum of patients whom they had
treated by Högyes' dilution method of treatment, and failed to obtain any evi-
dence of rabicidal properties in the serum.
In 1903-04-05, when Director of the Pasteur Institute of India, I carried
out a series of experiments on the serum of patients after undergoing Pasteur's
method of treatment, and obtained evidence of well marked rabicidal effects a few
days after completion of treatment. In another series of experiments carried
out at the same time in which two ponies were highly immunised by com-
mencing with dead virus, and going on to living '' fixed virus," the serum was
so highly rabicidal that one volume added to two volumes of a 5 per cent.
dilution of fixed virus completely destroyed its virulence in ten minutes at room
temperature.³
Babes, Remlinger, Marie and other workers have obtained similar evidence
of the rabicidal properties of the serum of animals immunised with rabies
virus.
The serum of normal untreated man or animals does not furnish any evidence
of rabicidal action.
It is evident from these experiments that the rabicidal action of the serum
of animals treated with a rabies vaccine is one of the factors which indicate
immunity.
¹Marie—L'Etude Experimentale de la Rage 1909, page 172.
²Lamb and McKendrick—Scientific Memoirs of Officers of the Medical and Sanitary Departments of
the Government of India, No. 36, pp. 12—.15.
3 Semple—On the Preparation and Use of Antirabic Serum, and on the Rabicidal Properties of the
Serum of Patients after undergoing antirabic treatment.— Lancet, June 6th, 1908, p. 1611.
29
CHAPTER VII.
The advantages of having a safe and efficient Antirabic Vaccine
which could be sent to wherever it is required for the treatment
of patients.
Antirabic treatment is essentially a prophylactic treatment, but it differs
from the prophylactic treatment applied to other infectious diseases in that it is
carried out only when the patient has already been infected, and before the infec-
tion has had time to assume an active phase. On this account it is most important
when treatment is necessary, that it should be commenced as early as possible.
A person infected with rabies virus cannot afford to allow the virus to get a
good start in its growth, and then leisurely to make his way to a Pasteur Institute
for treatment.
In a country like India where rabies is so widely spread, and the distances
from a Pasteur Institute are great, a certain amount of delay is unavoidable. Those
who are slightly bitten on the limbs, or through clothing, can better afford delay
than those who are severely bitten, or bitten on the head, neck or face.
The fact that a large percentage of persons bitten by rabid animals escape
hydrophobia even without treatment, indicates that it does not require a very high
degree of immunity to prevent the virus from multiplying and giving rise to hydro-
phobia. Then again, the fact that equally good results are claimed for any and
every method of antirabic treatment, also indicates that a very high degree of im-
munity is not an absolute necessity in prophylactic treatment.
To my mind the two essential factors in any prophylactic vaccine are :
—
1. That it should be an absolutely safe vaccine.
2 . T h a t i t s h o u l d b e a n e f f i c i e n t v a c c i n e ,i
. e . ,
c a p a b l e o f g i v i n g r i s e t o a n
immunising response sufficient to prevent the onset of the disease for
which it is given.
The first of these factors deserves careful consideration when we are dealing
with a living vaccine, and especially a living rabies vaccine, where infection with
the living element in the vaccine would mean certain death to the patient.
As I have already stated, Pasteur, when he first applied antirabic treatment
to man, got over this difficulty by means of his " dried cord " method of treat-
ment. His well known method of obtaining " fixed virus " from the spinal cords of
rabbits only requires to be mentioned here. Suffice it to say that after a variable
number of passages of " street virus " (a virus of variable virulence) through a
series of rabbits, a virus of a constant and exalted virulence is invariably present in
the spinal cord (and also in the brain and medulla) of these animals. When the
cords are removed after death from " fixed virus " and dried over caustic potash
30
in glass jars, kept in a dark room at a temperature of 22°C the virus has lost all its
virulence in 14 days ; or in other words, it is then a dead virus. Cords which have
been dried for a lesser period than 14 days give very little evidence of virulence
after the 8th or 9th day, and have not lost anything in virulence until after the
3rd day. There are two opinions as to what happens to the virus in the cords
during this drying process. One opinion is, that the virus becomes attenuated,
and strictly in accordance with the time the cord has been dried ; and the other
is, that the virus is only diminished in amount and not attenuated.
It is a well known fact that germs which can be exalted in virulence can
also be attenuated in virulence, and that drying is one method of attenuation. It
is also accepted as a fact that rabies is caused by a germ of some kind, and a germ
which can be exalted in virulence ; why then should rabies virus be the exception
to a rule which is applicable to the causal organisms of other infectious diseases
in regard to its attenuation ?
As the subject is one about which there are sure to be differences of opinion
until it becomes possible to cultivate the causal organism of rabies in artificial media
we had better leave it with this explanation ; moreover, the acceptance of one
or other of these opinions does not materially concern the subject-matter of
this paper.
Pasteur commenced treatment by injecting vaccines prepared from cords
which had been dried for 14 days, and then from cords which had been dried for
13, 12, 11, 10 days, and so on down the scale to cords which had been dried for 3
days only. The dose for an adult consists of 1 c. m. of cord finely ground down
in 3 c.c. of a sterile fluid, such as normal saline solution, and injected subcutane-
ously ; it corresponds to about a 5 per cent. cord emulsion. 1 This method modified
in various directions is what is known as the " dried cord " method of treatment.
It has undergone many modifications since Pasteur's time, but the system is still
essentially that of Pasteur.
In the " dilution method " of treatment (also known as Högyes' method)
the vaccines are prepared direct from the fresh cord of a rabbit dead from " fixed
virus " rabies.
It is convenient to begin by making a 1 per cent. dilution of virus, and from
this to prepare any dilution required. The treatment by this method is commenced
by the injection of very weak dilutions, such as 1 in 6,000 at some Institutes, and 1
in 4,000, or even 1 in 2,000 in others ; and gradually going on from day to day to a
less diluted virus, such as a 1 in 500 ; 1 in 200 ; or even 1 per cent. dilutions at some
Institutes.
¹ H a r v e y a n d M c K e n d r i c k — S c i e n t i f i c M e m o i r s b y t h e O f f i c e r s o f t h e M e d i c a l a n d S a n i t a r y D e p a r t
- m e n t s o f t h e G o v e r n m e n t o f I n d i a , N o . 3 0 , p . 1 9 .