NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
2
est point in favour of its efficiency. In the other two examples given, viz., in the
prophylaxis of cholera and typhoid fever in man, in which living vaccines have been
injected subcutaneously, a multiplication is the last thing to be desired, and as
far as we know it does not take place when these vaccines are given subcutaneously ;
but even so, it is now the custom in the prophylaxis of typhoid and cholera
to use only dead bacterial cultures.
When we apply these facts to antirabic treatment, the subject assumes an
aspect worthy of careful consideration, because the multiplication of a living rabies
virus intended as a prophylactic vaccine would mean hydrophobia and death to
the person inoculated.
Pasteur recognised this danger, and provided against its occurrence by com-
mencing treatment with a dead virus, followed by an attenuated living virus. By
this means he was able to commence the immunisation of his patients with a vac-
cine which was perfectly safe and which established a degree of immunity suffi-
cient to render the subsequent injection of living and virulent virus also quite safe.
Sometime after Pasteur inaugurated his system, Högyes of Buda-Pesth in-
troduced a method of antirabic treatment by injecting high dilutions of living and
virulent virus to begin with, gradually going on to lower dilutions. This method
has been adopted by several Pasteur Institutes, and has been reported upon favour-
ably by some observers. It has the merit of being easier to carry out, and is less
expensive than the treatment advocated by Pasteur and his followers ; but whether
it is the best and safest method that science can devise in the absence of exact
knowledge about the causal micro-organism of rabies, is a disputed point.
We know that rabies is due to a living virus which has never been cultivated
outside the tissues of man or animals suffering from the disease ; we also know
that many of the symptoms present in rabies resemble those present in cases of
tetanus.
In tetanus the symptoms depend upon extra-cellular toxines elaborated by
the tetanus bacillus ; but whether any or all of the symptoms of rabies depend
upon extra-cellular toxines elaborated by the causal micro-organism in a manner
somewhat similar to those of tetanus is a subject about which we know practically
nothing, and we are not likely to gain much information on this point until it be-
comes possible to cultivate the causal micro-organism of rabies in artificial media,
and to experiment with pure cultures separate from the nerve tissue of animals
dead of the disease.
In the meantime it only remains to follow the lines laid down by Pasteur, but
modified in accordance with the experience gained from recent advances in our
knowledge of prophylactic inoculations in other diseases.
Those who advocate the " dilution method " (Högyes' method) of treatment
take it for granted that a rabies toxine separate from the living virus does not exist ;
3
and those who advocate the " dried cord method " (Pasteur's method) keep in
view the possibility of the existence of a toxine in the nerve centres (brain and
spinal cord), the principal seat of the virus and where it multiplies. Moreover,
there is another point which influences the selection of a method of treatment, viz.,
the attenuation of rabies virus.
In the dried cord method of preparing a vaccine, attenuation of the virus is
supposed to take place from day to day, and strictly in accordance with the length
of time the cord has been dried under fixed conditions ; but here, again, the oc-
currence of attenuation is denied by some and firmly believed in by others. It
would serve no useful purpose to enumerate the arguments for and against these
different views. The cultivation of the virus in artificial media (should this ever
become possible) is the source from which the gaps in our knowledge about rabies
virus are likely to be filled in and until this has been accomplished the views of
workers on rabies are sure to differ widely.
Setting aside these two methods of preparing an antirabic vaccine, viz., the
" dried cord method " and the " dilution method," in both of which a living virus
is used, are there any other possible methods of preparing a safe and efficient
vaccine ?
As I have already stated dead vaccines have been found safe and efficient in.
the prophylaxis of other diseases, why should not similar methods apply to rabies ?
With the object of throwing some light upon this important question the ex-
periments upon which this paper is based were carried out. In a word, my object
was to ascertain whether animals can be as highly immunised with a dead virus
as with a living virus and if this is possible it would naturally follow that such a
vaccine would be safe and efficient, two most important and essential properties
in an antirabic vaccine, or in any other vaccine.
We know that heat is a damaging agent to use in killing bacterial vaccines
when it is necessary to store them for some time before use ; and that carbolic
acid in weak dilutions is a reliable agent for sterilising bacterial vaccines without
lessening their immunising and keeping properties. 1 On the other hand, a low
degree of heat, any temperature below 60°C, applied from 15 to 30 minutes does
not materially diminish the immunising properties of a bacterial vaccine intended
f o r e a r l y u s e ; b u t a f t e r h e a t i n g i t i s a l wa y s n e c e s s a r y t o a d d a n a n t i s e p t i c wh e n
the vaccine has to be stored, or sent out for use.
In those vaccines which are easily killed in a small percentage of an antisep-
tic, such as from 0.5 to 1 per cent. carbolic acid, it is unnecessary to use heat at
all, because it would not do away with the necessity of afterwards adding an anti-
Semple & Matson : On the Preparation and Keeping Properties of Antityphoid Vaccines.— The
Lancet, August 14, 1909.
4
septic to preserve the vaccine, and to guard against any subsequent contamina-
tion. In preparing a dead antirabic vaccine it was necessary to make a choice
between these two methods, viz., heat followed by an antiseptic, or an antiseptic
only.
In the experiments which follow it will be seen that the rabies virus is easily
killed by heat, and that a temperature of 50°C for 15 minutes is sufficient to des-
troy it. It will also be seen that it is more resistant to carbolic acid at room tem-
perature than most non-spore forming bacteria ; but at a temperature of 37°C it
is easily killed in 1 per cent. carbolic acid.
Of these two methods of killing a vaccine, viz., by heat, and by carbolic acid,
carbolic acid is the one which produces least change in the vaccine. 1
When heat is employed we make use of an agent of the effect of which we know
nothing whatever, except that it kills the living element, and that bacterial vac-
cines killed by heat retain their immunising properties for a shorter period than
similar vaccines killed by carbolic acid.
For these reasons it was decided to experiment with a vaccine prepared by
killing the virus in 8 per cent. dilutions in normal saline solution to which had been
added 1 per cent. carbolic acid. Twenty-four hours at a temperature of 37°C was
found to be sufficient to kill the virus in 8 per cent, dilutions when 1 per cent.
carbolic acid had been added. After 24 hours the killed vaccine was diluted
with an equal volume of sterile normal saline solution, and kept at room temper-
ature for use when required. This gave a 4 per cent. dilution of virus in 0.5 per
cent. carbolic acid normal saline solution a dilution suitable for treatment pur-
poses, or which could be still more diluted if considered necessary.
In a preparation of this kind the vaccine will keep for months, and retain its
immunising properties unimpaired, provided that it is kept in a moderately cool
place screened from light. That it is a safe and efficient vaccine in the immunisa-
tion of animals the experiments recorded in this paper afford ample proof.
It also possesses the great advantage that it could be sent from the labora-
tory where it is prepared to places at a distance where any medical man with a good
knowledge of the administration of vaccines could carry out the treatment of
patients bitten by rabid animals. When we realise what this means in a country
like India, where rabies is so widely scattered and of common occurrence, the ad-
vantages of such a system in providing a safe and efficient vaccine are enormous.
Instead of having a number of Pasteur Institutes scattered over the country it
would be necessary to have only a Central Institute where the vaccine could be
prepared and sent to other centres where treatment could be carried out. The
expenses connected with these centres would be very small, long and costly
Semple & Matson : On the Preparation and Keeping Properties of Antityphoid Vaccines.— The
Lancet, August 14, 1909.
5
journeys would be avoided, and most important of all, the patients would come
under early treatment, and a treatment free from risks.
In the preparation of a dead rabies vaccine it is necessary to know what are
the effects on living virus of agents such as heat and carbolic acid which could be
used in killing the virus ; and in testing the rabicidal properties of the serum of
animals immunised with a rabies vaccine it is necessary to know what are the
effects of normal saline solution (the fluid used to prepare dilutions) on the virus
dilutions used for testing the serum. The experiments recorded in Tables I to IX
answer these questions. The experiments recorded in Series I to V prove that
animals, such as monkeys, dogs, and rabbits, can be highly immunised with a dead
rabies vaccine, killed and preserved in carbolic acid.
Other workers have also obtained evidence of the immunising properties
of dead rabies virus.
Poor,1 recording his own experiments with heated virus, in a recent article
(September 1910) mentions, that, " Fermi as the result of his experiments recom-
mended the use of dead virus only ; his plan being to kill the virus with carbolic
acid, and store the emulsions for future use. "
The same author also mentions that, " Otto Heller in his work on protective
inoculation against rabies (Jena, 1906) gives the experience of others in the
use of virus killed in various ways ; e.g., heat, and by glycerine ; and his own
experiments in the use of virus killed by grinding according to the method of
McFadyen." Heller used the brains of rabbits after death from " fixed virus "
infection.
1 Poor—The Immunising Properties of Killed Rabies Virus. Collected Studies from the Research
Laboratory, Department of Public Health, City of New York. Vol. IV. Issued September 1910.
6
CHAPTER II.
The Effect of Heat On Rabies Virus.
TABLE I.
It has long been known that rabies virus is easily destroyed by heat. This
is a fact which has been used to prove that the causal organism of rabies (what-
ever it may eventually turn out to be) cannot be a spore-forming organism, for we
know that all spores require a high temperature before they are destroyed, and
that many of them require a boiling temperature before they are even injured, and
a few kinds require boiling for one or two hours before their destruction is finally
accomplished.
The very fact that rabies virus is so sensitive to heat puts the causal organism
out of the category of spore-forming germs. This sensitiveness to heat of a virus
about the nature of which we know so little would make one hesitate before using
heat as an agent in the preparation of a dead vaccine, a vaccine in which an extra-
cellular toxine may possibly be an important factor in its immunising properties.
Marie 1 states that the destruction of the virus begins at a temperature of 45°C,
and that 24 hours' exposure to this temperature completes its destruction. Marie
also mentions the following results of the effects of heat on rabies virus obtained
by other workers : " On peut le stériliser par une chauflfge de 5 minutes à 48°,
de une heure à 50° (Celli), de quelques minutes à 60° (Roux), de trente minutes à
52.58° (Högyes)." It will be seen that there is a wide range of difference in the
examples given, viz., from 5 minutes at 48°C to 1 hour at 50°C (Celli) ; several
mi nutes at 60°C (Roux) ; and 30 mi nutes at 52° to 58°C (Högyes).
The results of my own experiments on this point are given in Table 1. On
referring to this table it will be seen that a 5 per cent. emulsion of fixed virus from
the medulla of a rabbit is destroyed when heated in a water bath at a temperature
of 50°C in 15 minutes, and that a similar emulsion is not even injured in 15 minutes
at a temperature of 45°C. In the preparation of 5 per cent. emulsions used
in the experiments referred to in Table 1, one gramme was taken from the medulla
of a rabbit soon after it had died from " fixed virus " rabies ; it was ground up
with a pestle and mortar in 20 c. sterile normal saline solution, and then passed
through a fine wire strainer to remove any rough particles of fibrous tissue, etc.
A few c. c. were then transferred to several sterile test tubes, and heated in a water
bath at the temperatures, and for the times specified in the table. A few minutes
after removal from the water bath, 0.2 c.c. was used to inoculate healthy full grown
rabbits subdurally, with the results recorded in Table 1. A control rabbit
Marie—L'Etude Experimentale de la Rage, 1909, pp. 105-106.
7
was also inoculated subdurally with 0 . 2 c.c. of the 5 per cent. virus emulsion before
h e a t i n g ; a n d t h i s r a b b i t a n d t h e o n e i n o c u l a t e d wi t h 0 . 2 c . c . o f t h e v i r u s a f t e r
heating at 45°C for 15 minutes both developed rabies on the seventh day. Five
other rabbits inoculated subdurally with a similar amount of the same virus, but
heated either at a temperature of 55°C or 50°C for 30 or 15 minutes, remained
well.
The results of these experiments show that a 5 per cent. emulsion of fixed virus
in normal saline solution is destroyed at a temperature of 50°C in 15 minutes, but
not at a temperature of 45°C in the same time.
TABLE I.
Experiments to test the effects of heat on " fixed rabies virus" when emulsified in normal
saline solution.
No. of
experi-
ment.
Percen-
tage of
" fixed
virus "
in normal
saline solu-
tion used.
Time and tempera-
ture at which heated
before being used.
Animal
inoculated.
Amount inocu-
laced, and
method of in-
oculation.
Result.
REMARKS.
Per cent.
1
5
55°C for 30 minutes .
Rabbit
. 0.2 c.c. subdural-
ly.
Remained well.
2
5
55°C for 15 minutes .
Ditto.
Ditto.
Ditto.
3
5
50°C for 30 minutes .
Ditto.
Ditto.
Ditto.
4
5
50°C for 15 minutes.
Ditto.
Ditto.
Ditto.
5
5
50°C for 15 minutes .
Ditto.
Ditto.
Ditto.
6
5
45°C for 15 minutes.
Ditto.
Ditto.
Rabies, 7th day.
7
5
Not heated .
.
Ditto.
Ditto.
Rabies, 7th day.
Control.
A 5 per cent. dilution of fixed rabies virus in normal saline solution is destroyed at a temperature of 50°C in 15 minutes, but not
in the same time at 45°C.
8
CHAPTER III.
The Effect of Carbolic Acid on Rabies Virus.
The fact that carbolic acid destroys rabies virus has never been doubted, but
in order to kill the virus for the purpose of making a vaccine, the dilution of carbo-
lic acid, the dilution of virus to use, the temperature to which the mixture should
be exposed and the time limit of exposure require careful consideration. The
less the change produced in any vaccine used for immunising purposes
the better. This is a principle to be kept in mind when the object is to obtain a
dead but otherwise unaltered and efficient vaccine. It is necessary that the active
principle and original properties of the vaccine, once it has been rendered safe for
use, should be interfered with as little as possible, and as so much depends upon
the immunising properties of a rabies vaccine the utmost care should be taken in
its preparation.
In order to place the subject on a sound footing, it was first of all necessary to
experiment with various dilutions of rabies virus to which had been added various
percentages of pure carbolic acid, the mixtures of virus and carbolic acid being
kept for variable periods either at room temperature, or a temperature of 37°C.
The rabies virus used was " fixed virus " taken fresh from the medulla of rabbits
after death from " fixed virus " rabies.
A portion of the medulla was removed a few hours after death, weighed, then
ground up in a mortar with sterile normal saline solution to which had been added
the percentage of carbolic acid to be tested. The emulsions were strained through
a fine wire strainer, and filled into bottles which were then capped with rubber,
and finally exposed to whatever temperature it was desired to test. By this means
it was easy to obtain any dilution of virus, and in any percentage of carbolic acid
desirable ; it was also easy to remove a sample for experimental purposes from
any of the bottles by puncturing the rubber cap with the needle of a syringe and
withdrawing the piston.
The test applied in all cases was the subdural inoculation of a rabbit with
0.2, or 0.25 c.c. of the carbolised virus emulsion.
Healthy full grown animals were used in all these experiments. The animals
were anæsthetised, trephined and the amount specified injected under the dura-
mater. The first symptom of paralysis was taken as the first sign of rabies,
although in some cases it might have been possible to arrive at a diagnosis by
taking into consideration other and earlier symptoms, such as tremors of the head,
when the animals were disturbed.
TABLE II
This table gives the results of nine experiments with 1 per cent. dilutions
of virus in 0.5 per cent. carbolic acid normal saline solution when the mixture
9
had been kept for various periods up to 20 days at room temperature; also the
result of one experiment with 0.5 per cent. rabies virus in 0.5 per cent. carbolic acid
when the mixture had been kept for 24 hours at room temperature. It will be seen
that under these conditions 1 per cent. rabies virus in 0.5 per cent. carbolic acid
retains its virulence unimpaired up to 12 days, and is not destroyed after 20 days,
although it had by that time lost somewhat in virulence, as shown by a 10 days'
incubation period when a rabbit was inoculated subdurally, instead of 7 days, the
normal period after which a rabbit shows symptoms of paralysis when inoculated
subdurally with "fixed virus."
In experiment 9 it will be seen that 0.5 per cent. virus in 0.5 per cent. carbolic
acid retains its virulence unimpaired at room temperature for 24 hours at least.
TABLE II.
Experiments to test the effect of carbolic acid on "fixed rabies virus" when kept at room, tem-
perature (about 20º C).
No. of
experi-
ment.
Percentage of "fixed
virus" in
normal
saline solu-
tion used.
Percen-
tage of
carbolic
acid
added.
Time and tem-
perature at
which kept be-
fore being used.
Animal
inocu-
lated.
Amount in-
oculated, and
method of
inoculation.
Result.
REMARKS.
Per cent. Per cent.
1
1
0.5 . 24 hours, room
temp., about
20ºC.
Rabbit 0.2 c.c. sub-
durally.
Rabies, 7th day.
2
1
0.5 . 48 hours, room
temp., about
20ºC.
Ditto.
Ditto.
Ditto.
3
1
0.5 . 3 days, room
temp., about
20ºC.
Ditto.
Ditto.
Ditto.
4
1
0.5 . 4 days, room
temp., about
20ºC.
Ditto.
Ditto.
Ditto.
5
1
0.5 . 5 days, room
temp., about
20ºC.
Ditto.
Ditto.
Ditto.
6
1
0.5 . 10 days, room
temp., about
20ºC.
Ditto.
Ditto.
Ditto.
7
1
0.5 . 12 days, room
temp., about
20ºC.
Ditto.
Ditto.
Ditto.
8
1
0.5 . 20 days, room
temp., about
20ºC.
Ditto.
Ditto.
Rabies, 10th day.
9
½
0.5 . 24 hours, room
temp., about
20ºC.
Ditto.
Ditto.
Rabies, 7th day.
A 1 per cent. dilution of "fixed rabies virus" in normal saline solution can survive for at least 20 days in 0.5 per cent. carbolic
acid at room temperature (about 20ºC).
10
TABLE III.
In the nine experiments recorded in this table, seven were carried out with
2 per cent. dilutions of virus in 1 per cent. carbolic acid ; and two with 4 per cent
dilutions of virus in 2 per cent. carbolic acid, when the mixtures had been kept at
room temperature for various periods.
The results show that a 2 per cent. dilution of virus retains its virulence un-
impaired in 1 per cent. carbolic acid at room temperature for 3 days, but begins to
lose it after 4 days, and is killed in 6 days ; and that a 4 per cent. dilution of
virus in 2 per cent. carbolic acid is killed in 24 hours at room temperature.
From these results it is evident that the amount of carbolic acid (2 per cent.)
necessary to kill a 4 per cent. dilution of rabies virus in 24 hours at room tempera-
ture is too large an amount to be retained in a vaccine intended for antirabic
treatment ; on this account it was decided to test the effects of a reduction in
the percentage of virus, and not to increase the carbolic acid beyond 1 per cent.
(see Table IV).
TABLE III.
Experiments to test the effect of carbolic acid on "fixed rabies virus" when kept at room tem-
perature (about 29° C ).
No. of
experi-
ment.
Percentage
of "fixed
virus
" in
normal
saline solu-
tion used.
Percen-
tage of
carbolic
acid
added.
Time and tem-
perature at
which kept
before being
used.
Animal
inocu-
lated.
Amount in-
oculated and
method of
inoculation.
Result.
REMARKS.
Per cent. Per cent.
1
2
1
24 hours, room
temperature,
about 20°C.
Rabbit 0.2 c.c., sub-
durally.
Rabies, 7th day.
2
2
1
48 hours, room
temperature,
about 20°C.
Ditto.
Ditto.
Ditto.
3
2
1
3 days, room
temperature,
about 20°C.
Ditto.
Ditto.
Ditto.
4
2
1
4 days, room
temperature,
about 20°C.
Ditto.
Ditto.
Rabies, 8th day.
5
2
1
6 days, room
temperature,
about 20°C.
Ditto.
Ditto.
Remained well.
6
2
1 9 days, room
temperature,
about 20°C.
Ditto.
Ditto.
Ditto.
7
2
1
12 days, room
temperature,
about 20°C.
Ditto.
Ditto.
Ditto.
8
4
2
24 hours, room
temperature,
about 20°C.
Ditto.
Ditto.
Ditto.
9
4
2
24 hours, room
temperature,
about 20°C.
Ditto.
Ditto.
Ditto.
A 2 per 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) ; but a 4 per cent. is destroyed in 24 hours in 2 per cent. carbolic acid at room
t e m p e r a t u r e .