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No. 44.

■-i SCIENTIFIC MEMOIRS

^ OFFICERS OF THE MEDICAL AND SANITARY DEPARTMENTS

OF THE

GOVERNMENT OF INDIA '

The Preparation of a Safe and
Efficient Antirabic Vaccine

LiEUT.-CoLONEL SiR D. SEMPLE, Kt., M.D., D.P.H., R.A.M.C. {ReiiraT)
Director, Central Mesearch Institute, Kasauli

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SCIENTIFIC MEMOIRS

OFFICERS OF THE MEDICAL AND SANITARY DEPARTMENTS

OF THE

GOVERNMENT OF INDIA

The Preparation of a Safe and
Efficient Antirabic Vaccine

LiEiTT.-CoLONEL SiR D. SEMPLE, Kt., M.D., D.P.H., R.A.M.C. {Retired}

JJirector, Central Research Institute, Kasauli

Issued under the authority of the Governmext of India by the
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CONTKNTS.

CHAPTER I.

Page.

Iktkoductios 1

CHAPTER II.

The Effect of Heat on Rabies Virus 6

CHAI»TER III.

The Effect of Caebouc Acid ox Rabies Virus ........ &

CHAPTER IV.

The Effect of Normal Saltke Solutiox on Rabies Virus :

1- At Room Temperature ........... 14^

2. At a Temperature of 37°C ........... 15

CHAPTER V.

The Lustuxisation of Animals with Rabies Virus killed by Carbolic Acid, and their

SUBSEQUENT RESISTANCE TO THE SUBDURAL INOCULATION OF LeTHAL DOSES OF

"Street Virus," ant) also of "Fixed Virus." ....... 18

CHAPTEU VI.

The Evidence of Immunhty in the Serum of Animals treated with Rabies Virus
killed by Carbolic Acid ........... 26

CHAPTER VII.

The advantages of having a safe and efficient Antieabic Vaccine which could be

sent to wherever it is required foe the treatment of patients .... 29

CHAPTER VIII.

SuiniARY OF Conclusions 32

The Preparation of a Safe and Efficient
Antirabic Vaccine.

CHAPTER I.

Iiiti'udnctioii.

T^HE two cardinal factors to be taken into consideration in the preparation and
-*- administration of vaccines whether for prophylactic or curative purposes are,
safety and efficiency. "V^Tien Pasteur first appUed his method of antirabic treatment
to man these were the two points upon which he concentrated his attention, with
the result that he firmly established the principles of antirabic treatment now
carried out in almost every civilised country where rabies is found.

In the practice of conferring a specific immunity on man and animals by ino-
culating them with dead bacterial vaccines, the question of safety, or free-
dom from any risk of communicating the disease which the vaccine is intended to
prevent is foreclosed, and the main points to be taken into consideration are a
proper system of dosage, and the interspacing of doses ; but when living vaccines
are used the question assumes a difierent aspect.

It is now well established that dead bacterial vaccines are efficient in
producing an immunising response in man and animals ; a response which in most
cases it would not be justifiable to attempt to produce with living bacteria. On
this account it is now almost the universal custom in the prophylactic and cura-
tive vaccination of infectious diseases in man to make use of killed cultures of the
causal micro-orgam'sms. No person would be justified in using a living staphylo-
coccus, or a living streptococcus vaccine, when dead vaccines prepared from these
germs answer every purpose.

In the immunisation of animals it is not an uncommon practice to begin by
using dead bacterial cultures, and then go on to living cultures ; but in man it is
exceptional to use living microbes either for prophylactic or ciirative purposes,
although in some cases it has been done with perfect safety, e.g., in the prophylaxis
of cholera and tjrphoid fever. In some cases an attenuated hving vaccine is used
as a prophylactic by communicating a modified form of the disease, e.g., vaccinia
to protect against small-pox in man ; but in this particular case the person inocula-
ted can afiord to allow the virus to multiply without running any risks, and more-
over, the multipHcation of the hving element in this vaccine is no doubt the strong-

B 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 subciitaneously, 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 hydi'ophobia 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 whicli established a degree of immunity suffi-
cient to render the subsequent injection of hving and virident virus also quite safe.

Sometime after Pasteur inaugurated his system, Hogyes 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 ^^^us which has never been ciiltivated
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 " (Hogyes' method) of treatment
take it for granted that a rabies toxine separate from the living virus does not exist ;

and those who advocate the " dried cord method " (Pasteur's method) keep in
view the possibihty 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 hkely 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 ?

^Yith 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 wthout
lessening their immunising and keeping properties.^ On the other hand, a low
degree of heat, any temperature below 60°C, appHed from 15 to 30 mmutes does
not materially diminish the immunising properties of a bacterial vaccine intended
for early use ; but after heatmg it is always necessary to add an antiseptic when
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, carbohc 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.

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 tem^jerature 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.^

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 tcmpcratiu-e 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 imimpaired, 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 coimtry
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 vaccme 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 Vaccine? — The
Lancet, August U, 1909.

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 immimised 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,^ 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 uss. "

The same author also mentions that, " Otto Heller ui his work on protective
inoculation against rabies (Jena, 1906) gives the experience of others in the
use of virus killed in various ways ; e.gr. , 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 brams 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.

CIIAI»TKIt 11.

The Effiit of ll<>;it on Itahics Vims.

TABLE 1.

It luis long been known that rabies virus is easily destroyed by heat. This
is a fact which has been used to prove tliat 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 liigh temperature before they are destroyed, and
that many of them require a l)oiling temperature before they are even injured, and
a few kinds require boiling for one or two hours ))efore their destruction is finally
accomplished.

The very fact that rabies \irus 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 ^ 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 pent le steriliser par une chauffage de 5 minutes a 48°,
de une heure a 50° (Celli), de quelques minutes a 60° (Roux), de trente minutes a
52-58° (Hogyes)." 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
minutes at 60X' (Roux) ; and 30 minutes at 52° to 58°C (Hogyes).

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 ni.inutes
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. c. sterile normal saline solution, and then passed
through a fine wnre 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

' Millie— L'Ktiulc ICxporimcntalc do Li Rage, 1909, pp. 1(15-100.

was also inoculated subdurally with 0"2 c.c. of the 5 per cent, virus emulsion before
heating ; and this rabbit and the one inoculated with 0'2 c.c. of the virus after
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.

Experi7nents 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-
lated, and
method of in-
oculation.

Result.

Remarks.

Per cent.

55°C for 30 minutes.

Rabbit .

0-2 c.c. subdural-

ly.

Remained well.

55°C for 15 minutes.

Ditto.

50°C for 30 minutes.

Ditto.

50°C for 15 minutes.

Ditto.

50°C for 15 minutes.

Ditto.

45°C for 15 minutes.

Ditto.

Rabies, 7th day.

7
Control.

Not heated

Ditto.

Rabies, 7th day.

A 5 per cent, dilution of fixed rabies virus in normal saline solution is destroyed at a temperature of SCC in 15 minutes, but not
in the same time at 45''C.

(IIAPTEK III.

The Effen ut Ciihitlic Acid on Kalncs Vims.

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 vafcine, 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 use 1 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 temperatiu"e 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"2.5 c.c. of the carbolised virus emulsion.

Healthy full grown animals were used in all these experiments. The animals
were anaesthetised, 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 n.
This table gives the results of nine experiments with 1 per cent, dilutions
of virus in 0'5 per cent, carbolic acid normal saUne solution when the mixture

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. carboUc acid
when the niLxture had been kept for 24 hours at room temperature. It will be seen
that under these conditions 1 per cent, rabies virus in O'o per cent. carboUc acid
retains its vhulence unimpaired up to 12 days, and is not destroyed alter 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 hoius at least.

TABLE II.

Experinients to test the effect of carbolic acid on '" fixed rabies virus " when kept at room terri'

perature (about 2T C).

Percentaef

Percen-

Time and tem-

Animal
inocu-

Amount in-

No. of
experi-

of ■• fixed

virus " in

normal

tage of
carbolic

perature at
which kept be-
fore being used.

oculated, anc
method of

Result. Remarks.

ment. saline solu
t:on used.

acid
added.

lated.

inoculation.

Per cent

Per cent.

1 1

0-5 .

2-t hours, room Rabbit

0-2 c.c. sub-

Rabies, 7th day.

temp., about

durally.

20°C.

' (-5 .

48 hours, room
temp., about
20°C.

Ditto.

0-5 .

3 days, room

Ditto.

temp., about

20°C.

"'•5 .

4 days, room
temp., about
20°C.

Ditto.

C-5 .

5 days, room
temp., about
20''C.

Ditto.

0-5 .

10 days, room
temp., about
20°C.

Ditto.

0-5 .

12 days, room
temp., about
20°C.

Ditto.

C-5 ,

20 days, room
temp., about
20°C.

Ditto.

Rabies, lOth day.

(•5 .

24 hours, room
temp., about
20°C.

Ditto.

Rabies, 7th day.

A 1 per c€Dt. dilation of " fi\e(i rabies virus" in nom.al valine solution can juivive for at Ua;t 20 days
acid at room temperature (about 20''C).

0*5 per cent, carbolic

c 2

TABLE III.

In tlie 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 percent, 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 percent.)
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 efiects 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 2T C).

Xo. of

Percentage
ot -nxed

Percen-
tage of

Time and tem-
perature at

Animal

Amount in-
oculated and
method of

pxneri- *'™'* " '"

carbolic

which kpi)t

inocu-

Result.

Remarks.

ment. sBline ■••olu-
tioD used.

acid
added.

before being
used.

lated.

inoculation.

Per cent.

24 hours, room

Rabbit

0-2 c.c, sub-

Rabies, 7th day.

temperature,

durally.

about 20°C.

48 hours, room
temperature,
about 20°C.

Ditto.

3 days, room
temjjerature,
about 20°C.

Ditto.

4 days, room
temperature,
about 20°C.

Ditto.

Rabies, 8th day.

6 days, room
temperature,
about 20^C.

Ditto.

Remained well.

9 daj-s, room
temperature,
about 20°C.

Ditto.

Ditto .

Ditto.

12 days, room
temperature,
about 20°C.

Ditto.

24 hours, room
temperature,
about 20°C.

Ditto.

24 houi-8, room
temperature,

abi'iut 20-C.

Ditto.

A 2 pfT cent, ('jlution of " ttxaX rahii-s vfriiR " in nnrmni eiilinc solution can purvivp for nt loast 4 (lays, but not for tf cUvs in 1 ner
cent. rnrtKtlic ncld at room temperature (about 20°C) ; but a 4 per cent, la destroyeU in 24 liours in 2 per cent, carbolic acid at room
(Cmpcraturc,

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. Marie^ states that rabies virus in 1 per cent, dilutions is
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

ture [about 2)° C).

vJien Jcept at room tempera-

Xo. of
experi-
ment.

Perceiita -e
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
inoculated,

and Result.
method of |
inoculation.

Remarks.

Per cent.

24 hours, at room

Rabbit

0-2 c.c, sub-

1
Rabies, 7th day.

temperatiu-e,
about 20=C.
48 hours, at room

Ditto.

durally.
Ditto

Ditto.

temperature,
about 21) °C.
3 days, at room

Ditto.

Ditto

Ditto.

temperature,
about 20°C.
4 days, at room

Ditto.

Ditto

Rabies, 8th day.

temperature,
about 20°v'.
6 days, at room

Ditto.

Ditto

Remained well.

i
6

temperature,
about 20°C.
9 days, at room

Ditto.

Ditto

Ditto.

temperature,
about 20°C.
12 days, at room
temperature,
about 20°C.

Ditto.

Ditto

Ditto.

A 1 pe cent, dilution of " fixed rabies \1rus " in normal saline solution can survive for at least 4 days, but not for 6 daysjin 1 per
cent, carbolic acid at room temperature (about 20-C).

\_ ' Marie— L'Etude E.xperimentale de la Rage, 1909, page 125.

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 37°C. 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 37T. It will be seen from the experiments wliicli follow
that the killing power of carbolic acid on rabies virus is also increased at a tem-
perature of 37°C. In this series of twelve experiments, four were carried out with
0*5 per cent, carbolic acid added to 2 per cent., 1 percent., and 0'5 per cent, dilu-
tions of rabies virus ; and eight with 1 percent, carbolic acid added to 1 percent.,
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 37 °C 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 37°C even when the virus is tested
in 0'5 per cent, dilutions ; and that 1 percent, carbolic acid kills a 4 per cent, dilu-
tion of rabies virus in 24 hours at this temperature (37°C).

The result of this last experiment showed that it was reasonable to expect
that 1 per cent, carbolic acid at a temperature of 37°C would probably kill stronger
preparations of the vii'us, 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 37 °C.

TABLE V.

Experiments to test the effect of carbolic acid on '' fixed rabies virus

perature of ,>/°C for 21 hours.

when kept at a ten}-

Xo. of

Pctcentaitc
of ••nvcd

Percen-
tage of

Time and
temperature

Animal

Amount

inoculated,

and

method

experi-

v.rus *• iD
normal

carbolic

at wliicli

inocu-

Result.

Remarks.

ment.

saline sulu-

acid

kept before

lated.

t.on used.

added.

being used.

of inocu-
lation.

Per cent.

0-5

24 hours at 37°C

Rabbit

0-25 c.c.

Rabies, 9th day.

0-5

Ditto.

0-5

Ditto.

Rabies, 7th day.

0-5

Ditto.

Rabies, 8th day.

Ditto.

Remained well.

Ditto.

A 2 per cent.. I per tent., or i per eent. dilution of '" flxrti rabies virus " in 05 per cent, rarbulic acid normal saline solution i. not
destroyed In 21 Iiours at a temperature of S7°C ; but a 4 per cent, dilution in 1 per cent, carbolic acid i» dretroycd in 24 hours at a
temperature of ST^C.

TABLE VI.

In the series of six experiments recorded in this table the rabicidal pioperties
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 volmne 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 " ifhen kept at a tem-
perature of 37°C for 24 hours.

Percentage

Percen-

Time and

Amount

So. of

v.rus " iu

tage of

temperature at

Animal

inoculated,

experi-
ment.

normal
saline
solution

carbolic
acid

wliich kept
before being

inocu-
lated.

and method
of inocu-

Result.

Rem.\rks.

used.

added.

used.

lation.

Per cent.

[ 1

24 hours at
37°C.

Rabbit

0 -25 0.0.,
subdurally.

Remained «ell.

Ditto.

8 per cent, dilutions of " flxed rabies virus " in normal saline solution are killed in 1 per cent, carbolic acid in 24 hours at a tem-
peratiue of 37° C.

CHAPTER IV.

The Effect of IVoiiiial Saline Solution on Itahies ^ irns.

Before proceeding to demonstrate the immunising properties of a 4 percent,
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) appUed 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^ on the effect of incubation at 37°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 1-5 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.

' Lamb and McKendrick — Observations on Rabies. Scientific Memoirs by Officers of the Medical
and Sanitary Tcpartments cf the Government of India, Xo. 36, pp. 27-28.

TABLE VII.

Experiments to test the effect of normal saline solution on " fixed rabies virus " when kept at room

temperature (about 2'j°C).

No. of
experi-
ment

Fercenta-e

of " fl-xed

vims" in

normal saline

solut on

used.

Time and

temperature at

which kept

before being

used.

Animal
inoculated.

Amount

inoculated,

and method of

inoculation.

Result. Remarks.

Per cent.

24 hours, room tem-
perature, about 20°
C.

Rabbit

0-2 c.c. sub-
duraUy.

Rabies, 7th day.

48 hours, room tem-
perature, about 20°
C.

Ditto.

3 days, room tempera-
ture, about 20° C.

Ditto.

4 days, room tempera-
ture, about 20° C.

Ditto.

11 days, room tem-
perature, about 20°
C.

Ditto.

15 days, room tem-
perature, about 20°
C.

Ditto.

Rabies, 9th day.

A 1 per cent, dilution of " fixed virus " in normal saline solution is not destroyed in 15 days nlien kept at room temperature (about
20° C).

3.— .\t a Teiiiiierature 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. ; and that a 0"5 per cent, dilution of virus after similar treatment produced
rabies in rabbits on the 8th day after subdural inoculation with 0'3 c. o.

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.

It is evident from these results that a 1 in 200 dilution of rabies virus in nor.iial
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 05 per cent, carbolic acid at room temperature for another 24 hours.

Xo. of
experi-
ment.

Dilution
of '' fixed
virus " in
normal
saline
solution.

Treatment to

which it was

subjected before

Ijeing used.

Animal
inoculated.

Amount
inoculated,
and method
of inoculation.

Result.

REM;UIKS.

1 in 100

24 hours at 37°0, then
24 hours at room
temperature, and
finally 24 hours in
i per cent, carbolic
acid at room tem-
perature.

Rabbit

0-3 c.c. sub-
durally.

Rabies, 7th day.

lin200

Ditto.

Rabies, 8th day.

Ditto.

4
Control.

Ditto.

24 hours at 37°C .

Ditto.

Rabies, 7th day.

1 per cent, anti i per cent, dilution* of " flxe4 rabies virus '* in normal saline solution are not destroyed when kept for 24 hours at
a temperature of 37°C, then for 'U hours at room temperature, followed by another 24 hours in 05 per cent^ carbolic arid 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,(500 — 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. fi'om the 1 in 200
dilution after being kept for 1, 2, 4, and 24 hours in the incubator at 37 °C, and a

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 sahne 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 heft at a
temperature of 37 °C for various periods up to 21 hours.

Percen-

tage of

Time and

Amount

No. of

" fixed

temperature at

Animal

inoculn ted.

experi-

virus "

which kept

^i jri ■ ■ 1 1 mx

inoculated.

i 1.1 v.' ^ *A Lilu ^ w Vi y

and method of

Result.

Remarks.

ment.

in normal

saline
solution.

before being
used.

inoculation.

1 in 200

Used wlien made up

Rabbit .

0-2 c.c. sub-
durally.

Rabies, 7th '
day.

Controls of
experi-
ments 3,

Ditto.

0-3 c.c. sub-
duraUy.

Ditto.

4, 5 and 6.

Ditto.

1 hour at 37°C

Ditto.

2 hours at 37°C

Ditto.

4 hours at 37°C

Ditto.

24 hours at 37°C .

Ditto.

1 in 1,600

Used when made u p

Ditto.

Control of
experi-
ments 8,
9, 10, 11
and 12.

Ditto.

1 hour at 37°C

Ditto.

2 hours at 37°C

Ditto.

4 hours at 37°C

Ditto.

24 hours at 37°C .

Ditto.

Rabies, 11th day.

Ditto.

Rabies, 10th day.

A 1 in 200, and a 1 in 1,600 dilations of " fixed rabies virus " in normal saline solution are ncit destroyed when kept
ture of 37°C for 24 hours.

at a tempera-

(IIAl»TER V.

The Iniiiiiiiiisatioii of Aiiiiiiiils with It.ibics Virus killed liy Carbolic
A<-i(l. and tlicir NiihseqiU'iii lt(>si>taii(-e to the Subdural IncM-ula-
tion ot Lethal Doses of •• Stieet 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.

Nitscy 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 \Trus 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

Kitsch — Wiener klinische Wochenschrift. No. 2C of I'JOl.

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 fixing 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 im.munity, 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 siu-e that very few (if any) patients
after undergoing an ordinary com-se of antirabic treatment at a Pasteur Institute
would survive the subdural inoculation of living rabies virus, although they had
acquired sufficient immimity 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 subdm-al 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 li^Tng 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 rem^ained 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 senmi
had separated it was mixed with an equal volume of diluted Uving 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.

Altogether 12 experiments and 12 control experiments with seriun 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 seriun 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 treatm.ent 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.

Tw' o control rabbits were inoculated subdvu-ally with the same \'lrus ; one
with 015 c.c. of 1 in 500 dilution, and the other 015 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 \drus used
to immimise these monkeys remained well.

The results of this series of experiments prove that monkeys can be highly
immimised against rabies by the hypodermic inoculation of dead rabies virus,
when the virus has been killed and preserved in carbolic acid.

TABLE I.

1st Series of Immunising Experiments.

Experiments on monkeys to test tJie immunising properties o'; 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.

Total quantity

Xo. of

Duration of

of 4 per cent.

experiment.

treatment.

dead rabies virus
injected sub-

Test to whioli subjected.

Result.

[Remarks.

cutaneously.

■

1. Monkey

One injection
daily for 24

50 C.C.

15 days after completion Remained well,
of treatment, was tre-

days.

phined and inoculated
subdurally witli 0-2 c.c.
of 1 in 500 dilution of
2nd passage " street
virus ' ' fresh from a
rabbit's medulla.

2. Monkey

Ditto.

3. Rabbit. Con-

Trephined and inocu-

Rabies, 1 1th day.

trol of test

•

lated subdurally with

virus.

0-15 c.c. of 1 in 500
dilution of the same
virus used to test the
two monkeys.

4. Rabbit, 2nd

, ,

Same as Xo. 3 experi-

Rabies, 12th day.

control of test

ment, with the excep-

virus.

tion that 1 in 1,000
dilution of test virus
was used.

5. Rabbit. Con-

Trephined and inocu-

Remained well.

trol of immu-

lated subdurally with

nising virus.

1
1

0-2 c.c. of the virus used
for immunising Xos. 1
and 2 monkeys.

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 iromunised. 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 O'o per cent, carbolic acid
normal saline solution.

The smallest dose given was 1 c.c. and the largest Zh c.c, and the increase
from 1 c.c. at the outset to 3i 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-

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 GOO 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 fuiious
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 scries 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 SERrES 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 " flxM virus " in 1 per cent . carbolic acid normal
saline solution to a temperature of 3!°C. for 24 hours ; it was then diluted uith an equal volume
of normal saline solution, which gave 4 per cent, virus in G'o pier cent, carbolic acid.

Total quantity

No. of
experiment.

Duration of
treatment.

of 4 per cent.

dead rabies virus

injected sub-

cutaneou^ly.

Test to which subjected.

Result. Eeiiakks.

1. Monkey

One injection
daily lor 24
days.

60 c.c.

15 daj's 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.

3. Monkey. Con-

Trci)hined and inocu-

Rabies, 10th day.

trol of test

lated with 0-3 c.c. of

virus.

same virus used for ex-
periments 1 and 2.

4. Rabbit. 2n(l

, .

Same as above, but only

Ditto.

control of test

0-2 c.c. used.

virus.

5. Rabbit. Con-

■ ■

Trephined and inocu-

Remained well.

trol of iramu-

lated subdurally with

nbinj; virus.

0-2 c.c. of the virus
used for immunising
Xos. 1 and 2 monkeys.

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
in O'o per cent. carboHc acid normal sahne solution, and were given one dose daily
for 28 days. The doses varied from li 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 dogs to test the rabicidal properties of the serum on " fixed
virus."

The serum iu both cases gave a complete rabicidal efEect when mixed with
equal parts of a 1 in 300 dilution of " fixed vii'us," 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 \Trus, 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.

TABLE III.

3rd Series of Immunising Experiments.

ExperimaUs on dogs to test the immunising properties of dead rabies virus. The virus was killed
by exposing 071 S 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 O'o per cent, carbolic acid.

I<tt;il quantity

—

\o. or

Duration of

of 4 piT ii-nt.

experiment.

treatment.

dead rabies virus
injecti-d ^ul>-
cutaneuualy.

Tost to wliicli subjected.

Result.

Remarks.

1. Dog, weight

One injection

100 c.c. .

24 days after completion

Remained well.

24 |l)s., full

daily for 28

of treatment, was tre-

fliown.

days.

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.

2. Puppy (log,

Ditto.

1)0 c.c. .

Ditto.

Rabies, 29th day.

weight 13 lbs.

3. Control dog.

Trephined and inocu-

Rabies, 10th day.

weight 14 lbs.

lated subdurally with

full grown.

0-3 c.c. of 1 in 600
dilution of same virus
used to test Xos. 1 and
2 dog.s.

4. Rabbit. Con-

Trephined and inocu-

Remained well.

trol of immu-

lated subdurally with

nising virus.

0-2 c.c. of the virus
used for immunising
Xos. 1 and 2 dogs.

4th Series of Immunising Experiments.

The experiments in this series were carried out on rabbits. Two healthy full
grown rabbits each weighing 2J- kilogrammes were immunised with 2 per cent,
dilutions of dead rabies virus in 0*5 per cent, carbohc acid normal saline solution.
Thev received one hypodermic injection daUy 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 vacciae 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 I in 200 dilution of "fixed virus "
and kept for 2 hours at a temperature of 37°C.

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 I in 200 dilution of 2nd passage ' 'street

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 0'2o
c.c. of this virus used to immunise the two rabbits remained weD.

The results of this series of experiments show that rabbits can be immimised
with hvpodermic 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
tliis degree of immunity.

The test appUed 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.

Ex'periments on rabbits to test the immunising properties of dead rabies virus. The virus used wa$
hilled btj 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 ; 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.

Total quantity

No. of experi-
ment.

Duration of
treatment.

of 2 per cent.

dead rabies virus

injected sub-

cutaneously.

Test to which subjected.

Results.

Resiabks.

One injection

36 c.c.

25 da3-s after completion

Remained well.

The test ap-

daily for 24

of treatment was tre- i

plied to

days.

phined and inoculated '
subdurally with 0-25 c.c.

these two
experiments

ofl in 200 dilution of

(Nos. 1 and

2nd passage ' ' street

2) was a

virus " fresh from a

severe one,

rabbit's medulla.

as the virus
used was

Ditto

Ditto ditto

Rabies, 16th day.

diluted only
to 1 in 200.

3. Control of

, ,

Trephined and inoculated

Rabies, 10th day.

test \Trus.

subdurally w ith 0-25 c.c.
of 1 in 400 dilution of
same virus used for
experiments 1 and 2.

4. Control of

Trephined and inoculated

Remained well.

immunising

subdurally with 0-25

yirus.

c.c. of virus used for
the immunising experi-
ments 1 and 2.

E •!

(II A PIER VI.

The KvnIi'iKc of Iiniiiiiiiity in tlio Seniiii of Aiiiiii.ils tifutcd with
Kabics Virus killed by laibolit' Acid.

SERIES V.
Experiments to test the rabicidal pro?erties of the serum of animals immunised

WITH DEAD R^VBIES VIRUS.

This series of experiments s:ives 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 lY.

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 voliune of 1 in 200 or 1 in 300 dilution
of hving 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 en living rabies virus.

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
saKne 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 ^ 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, bat not during or immediately after treat-
ment.

Lamb and McKendrick,^ in 1908, tested the serum of patients whom they had
treated by Hogyes' 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 hving " 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, RemUnger, 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.

' Marie — L'Etudo Experimentale de la Rage 1909, page 172.

• Lamb and McKendrick — Scientific Memoii-s of Officers of the Medisal and Sanitary Departments of
the Government of India, Xo. 36, pp. 12_lo.

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 tith, 1908, p. 1611.

TABLE V.

Kabicidal properties of serum.

Exferiments on rabbits to test the rabicidal properties of the serum of animals immunised with

dead rabies virus.

TiDK- wiiicli
V t. . . had rlapsfd
>u. ol tx- iTiimratiui. ..I ^^er com-
peniuiutand dcaJ rabu;. virus ,^.^4 ^j
ammulmi- , used lor reatnient 1
inunised. Immunising. I when serum
I ' was tested, j
1 '

Z. Monkey.

4 per cent, rabies | 11 days
virus in 0*5 .

Scr cent, carbo-
cacid. i

£. Monkey. { Ditto.
8. Monkey. ' Ditto

4. Monkey.

5. Dog

Ditto

6. Dog

Ditto

7. Babbit .

8. Babbit .

2 per cent.

virus in 0"5 per
cent carbolic
acid.

Ditto

11 days
14 days

14 days
11 days

23 days

11 days

23 days

8 days

25 days
8 daj-s
23 days

rropurliuii t>i siTiiiu uuil

living virus tested, also

time and temperature at

wbicb kept.

Equal parts serum and 1
in :{(H> "fixed virus,"
kept 2 hours at 37^0.

Tt-st applied to
mixture of serum

and virus. i

BesuU.

Babbit inocula-
ted subdurally
with 0*2 c.c.

ditto.

Equal parts serum and 1
in 20U 1st passage luiman
virus, mixed and kept 2
hours at 37"C.

Equal parts serum and 1
in 300 " fixed virus "
kept 2 hours at 37*C.

Equal parts serum and 1
in 3U0 2nd passage jac-
kal's virus mixed and
kept for 2 hours at ST^C.

Equal parts scrum and 1
in 300 " fixed virus "
mixeJ and kept for 2
hours at ST^C.

Equal parts serum and 1
in 300 2nd passage jac-
kal's virus, mixed and
kept 2 hours at 37°C.

Equal parts serum and 1
in 200 " fixed virus,"
kept 2 hours at GT'^C.

Ditto
Ditto
Ditto

ditto,
ditto,
ditto

Ditto

Ditto.

Ditto
Ditto

Ditto

Ditto
Ditto
Ditto

Bcmained
well.

Bab i es,
10th day

Bab i e s,
17tbday

I Bemaiued

well.

Ditto.

Bab i e s,
15th day.

A control rabbit inoculated
subdurally with 0'2 c.c.
equal parts normal mon-
key's serum and 1 in 300
" fixed virus." kept for
2 Iioursat 37''Cdeveloped
rabies on the 7th day.

Ditto.

ditto.

A control rabbit inoculated
subdurally with 02 c.c.
equal parts normal mon-
key's scrum and 1 in 200
1st passage human virus
kept for 2 hours at 37°C
developed rabies on the
]Ot/i day.

Ditto.

ditto.

A control rabbit inoculated
subdurally witli 0'2 c.c.
equal parts normal dog's
serum and 1 in 300 "fixed
viru5 ' ' kept for 2 liours
at37*C developed rabies
on the 7th day.

A rontrol rabbit inoculated
subdurally with 0"2 c.c.
equal parts normal dog's
scrum and 1 in 300 2nd
passage jackal's virus
kept 2 hours at 37°C
developed rabies on the
11th day.

Remained A control rabbit inoculated
well. subdurally witli 0'2 c.c.

equal parts normal dog's
serum and 1 in 300
'■fixed virus" kept for 2
hours at :?7T developed
rabies on the 7th day.

Bab i e s, A control rabbit inoculated
15th day. subdurally with 0'2 c.c.
equal parts normal dog's
scrum and 1 in 300 2nd
passage ackal's virus,
kept lor 2 hours at 37*^0
developed rabies on tlie
11th day.

Bemained A control rabbit inoculated
well. subdurally witli 0*2 c.c.

equal parts normal rab-
bit's scrum and 1 in 200
" fixed " virus kept 2
hoTirs at37°C developed
rabies on tlie 7th day.

Ditto.

Bab i e 8,
10th

day.

Ditto.

ditto.

Ditto.

All these experiments with tlie serum from immunised rabbits, dogs, and monkeys, were controlled witli serum from normal rab-
bits, dogs, and monkeys, and treated exactly tlie same after being mixed with the same dilutions of viruH as the serum which was
being tebted. AU the control rabbits cuatracted rabies after the usual incubation period accoidin^ to the virus used.

tH4l»TER VII.

Tlie ii(IViiiita«tes of Ihiviiio- a safe and effinent Aiitirabic Vaciine
wliitli could be sent to wherever it is re(|uired 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 sUghtly 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. That it should be an efficient vaccine, i.e., capable of giving rise to an

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 appHed 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

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 ij 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 ; wh}- 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 cm. 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 j)er cent, cord emulsion.^ 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 Hogyes' 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 today to a
less diluted virus, such as a 1 in 500 ; 1 in 200 ; or even 1 per cent, dilutions at some
Institutes.

' Harvey and McKendrick — Scientific Memoirs by the OflBcers of the Medical and Sanitary Depart-
ments of the Government of India, Xo. 30, p. 19.

It will thus be seen that in the ' ' dried cord ' ' method, treatment is commenced
by a dead virus, and finally a virulent virus is used. In the ' ' dilution method
treatment is commenced by a very small quantity of a living virulent virus, and
increasing its amount as treatment advances.

Equally good results are claimed for both methods and their modifications ;
but with these results I am not concerned in the present paper which deals solely
with the safety and efficiency of a dead virus. The fact that living vaccine is
used in both methods would preclude the possibility of sending it to a distance for
the treatment of patients without running serious risks of interfering with its
efficiency ; whereas a dead carbohsed vaccine could well be sent to any centre where
treatment could be carried out without running any risks of injuring its properties.
The experiments recorded in this paper in which a dead carbolised rabies^virus was
used to immunise monkeys, dogs and rabbits, prove that it is a vaccine which can
be relied upon to produce a high degree of immunity ; and knowing that it is a
dead vaccine we can dismiss any doubts as to the possibility of its producing the
disease which it is intended to prevent. It is also less expensive than any other
method, and is easily carried out.

A vaccine prepared from the brain and medulla of ' ' fixed virus ' ' rabbits would
contain more of the active element (virus) than a vaccine prepared from the spinal
cords, and on this account its immimising properties would be greater than
cord vaccine, and moreover the brain and medulla of a rabbit would furnish a much
larger amount of vaccine than the spinal cord.

One Central Pasteur Institute in addition to carrying out treatment could
supply the whole of India with a vaccine ready to be administered to patients at
other centres. By this means patients would be saved long and expensive
journeys ; and most important of all, they would come under early treatment, and
a treatment free from risks.

CIIAPTEK VIII.

Niiiiiiiiiny <»f Conclusions.

1. Rabies virus is easily killed by heat. A temperature of 50''C is sufficient to
kill a 5 per cent, dilution of " fixed virus " in 15 minutes.

2. Rabies virus can remain alive and virulent for several days in norm.al saline
solution.

A 1 per cent, dilution of " fixed virus " in normal saline solution remains
alive and virulent for at least 15 days at room temperature (about 20°C). Dilu-
tions of 1 in 200, and 1 in 1,600 of " fixed virus " in normal saline are not killed
in 24 hours at a temperature of 37°C, and not even injured in any way in 4 hours
fit this temperature. Dilutions of 1 per cent, rabies virus in normal saline solution
are not killed 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.

3. Rabies virus can survive for a longer time in carbolic acid at room tempera-
ture (about 20°C) than at a temperature of 37 °C. It is also more resistant to
carbolic acid at both these temperatures than most non-spore forming bacteria.

4. A one per cent, dilution of rabies virus in 0"5 per cent, carbolic acid normal
saline solution remains alive and virulent for at least 20 days at room temperature
(about 20°C). One per cent, and 2 per cent, dilutions of rabies virus in 1 per
cent, carbolic acid normal saline solution remam alive and virulent for at least 4
days at room temperature (about 20''C).

5. Dilutions of 4 per cent, and 8 per cent, rabies virus in 1 per cent, carbolic
acid normal saline solution are killed in 24 hours at a temperature of 37°C.
Rabies virus killed by this method and then diluted with an equal volume of sterile
normal saline solution is a safe and efficient antirabic vaccine.

6. Animals such as monkeys, dogs, and rabbits can be highly immimised by
hypodermic injections of a rabies vaccine killed and preserved in carbolic acid. Of
8 animals, viz., 4 monkeys, 2 dogs, and 2 rabbits, immunised with a 4 per cent dead
virus in 0*5 per cent, carboHc acid, all the monkeys, one dog, and one rabbit
survived the subdural inoculation of lethal doses of virulent living virus ; and the
one dog and one rabbit which did not survive this test showed a longer incubation
period than the controls.

7. The serum of animals immunised with rabies vaccine killed and preserved
in carbolic acid gives a well marked rabicidal action on living virulent virus.

8. A rabies vaccine prepared from the brain and medulla of ' ' fixed virus
rabbits, and then killed and preserved in carbolic acid is a safe and efficient vaccine
in the immunisation of animals, as proved by experiments on monkeys, dogs, and
rabbits ; and judging from the results of these experiments it should also prove a
safe and efficient antirabic vaccine for the prophylactic treatment of persons bitten
by rabid animals.

CALCUTTA

SOPEEINTENDENI GOVERNMENT FEINTING, INDIA

8, HASTINGS STBEET

t IT •■:

RC Semple, (Sir) David
^g The preparation of a

g, safe and efficient
antirabic vaccine

Biological
8t Medical

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