NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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been applied and has succeeded in giving vaccines with septic virus—the
virus of charbon symptomatique, etc.
Toussaint, recognizing that his first explanation of vaccination by
heated blood was erroneous, and having observed that filtered blood did
not confer immunity, discovered at the same time the method of attenu-
ation by the employment of an antiseptic. He conferred immunity
by treating blood which had been defibrinated and passed through a
linen or paper filter, with diluted phenic acid, one hundred grammes of
blood to 1 or 1½ grammes of phenic acid dissolved in 5 cubic centi-
metres of alcohol and placed in 100 grammes of water.
The method of attenuation by the employment of an antiseptic
was afterwards applied by MM. Chamberland and Roux to cultures of the
bacilli : by the employment of a solution of phenic acid or bichromate
of potash, they succeeded in attenuating the virus of cultures, and
forming, as we shall see later, races of attenuated virus.
Attenuation by culture in the presence of air at 42°-43° C. —
M. Pasteur, after observing the immunity of the sheep and ox to
charbon, after they had been vaccinated with a culture of the virus
of fowl cholera; after observing that sheep resisted the malady con-
tracted by the ingestion of spores and acquired immunity; after
noting that the cow frequently resists spontaneous or inoculated
charbon and becomes refractory, applied the method which had served
him for the attenuation of fowl cholera to the virus of charbon, and
succeeded in attenuating it. Profiting by his experience with fowl
cholera virus and also from the results obtained by Toussaint with
heat, he invented his method of attenuation by the combined action
of heat and air upon cultivated bacilli.
The bacillus, cultivated under ordinary conditions, multiplies
for from 24 to 48 hours by scission, then it forms spores, which are
henceforth little modified by contact with the air, and preserve all
their virulence for a long time. But the bacillus, in its filamentous
form, and so long as it only multiplies by scission, is attenuated by
the action of the oxygen of the air ; it suffices then to submit it to
the action of the oxygen of the air, and to cause it to develop so that it
cannot form spores; by this method of proceeding we are enabled to
obtain the microbe of charbon in the most diverse degrees of virulence,
from that of a virulence sufficient to destroy the animals which offer
the greatest resistance to that which is almost, if not entirely, innocuous,
passing through all the various degrees imaginable. The virus, in
its various degrees of attenuation, may reproduce itself in new cultures
which preserve the attenuated virulence, whilst that attenuated by
heat at 55° C. does not retain its attenuation in cultures in which
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it is sown. Amongst these attenuated bacilli of various degrees of
virulence, it becomes easy to find one which, when inoculated into animals,
only gives them a mild and benign attack of the disease, and confers
upon them immunity against the non-attenuated virus.
At temperatures the most elevated compatible with the culture of
the bacillus, varying with the medium used, they do not form spores.
They do not grow at 45° C., but will at 42°-43° C., and at 425° C.
they do not form spores in cultures made in flasks where the liquid is
deep enough. Koch says that they do so when cultivated on plates.
The bacilli, cultivated in chicken broth, either neutral or feebly
alkaline, in contact with the air, at a constant temperature of be-
tween 42°-43° C., do not even after a very long time form spores.
Left at that temperature, in contact with the air, the microbe, after
being multiplied by seission and forming a filamentous mycelium,
becomes rapidly of less virulence. During the first days it passes
through various degrees of attenuation. Each of these various con-
ditions of virulence, more or less attenuated, may be reproduced by
culture.
Each attenuated microbe constitutes a vaccine against the superior
microbe; and it is easy to discover, in these successive viruses, vaccines
capable of producing charbonous fever in animals without destroying
them, and to confer such immunity on them as to preserve them
from the mortal malady. Once attenuated, the microbe preserves its
attenuation in the new cultures, and is sown in fresh media maintained
at a less elevated temperature; it then produces spores of an attenuated
virulence, from whence are produced again bacilli equally attenuated.
To recapitulate, the bacillus cultivated at 42°-43° C. in contact
with the air, becomes progressively attenuated ; each state of virulence
is reproduced by culture, preserving its proper virulence ; attenuated
virus may produce spores similarly attenuated when placed in a me-
dium the temperature of which is 33°-35° C. The spores which they
produce are attenuated like the bacilli, and keep better. Each attenuated
bacillus may be considered as a vaccine to the one superior to it in viru-
lence ; it gives a benign form of disease, when sufficiently attenuated,
and confers protection against a graver attack : in this manner, by
inoculating animals, first with a very attenuated virus, and then with
a stronger one, we are enabled to confer upon them immunity against
very virulent charbon without running any, or scarcely any, appreciable
danger of causing serious illness, or destroying them. At the end of
12 or 13 days, the bacillus, cultivated and maintained in contact with
the air at 42°-43° C., loses its virulence as regards the adult guinea-
pig, and when inoculated into it does not kill it, whilst it lives and
may be cultivated and reproduce itself in this inoffensive condition. At
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the end of 31 days it only kills young mice ; and at the end of a month
and a half it entirely loses its virulence. The microbe of charbon may be
attenuated without any marked change in its morphological characters ;
it may retain this attenuation in cultures, produce attenuated spores, give
only a benign form of disease, and confer immunity. The attenuated
virus presents a thinner, shorter, and more divided form of bacillus ;
and the spores are very small. The virus attenuated by exposure to the
air at a temperature of 42° to 43° C. is less abundant, and forms upon
the sides of the flasks a uniform deposit, whilst the virulent virus
forms a flooculent, snowy mass, made up of long filaments. When
we sow attenuated spores, however, we obtain filaments forming
flocculi. The attenuated bacilli and their spores are less stable, and
perish after some months. The attenuation obtained by this method
is due, according to M. Pasteur, to oxygen : he has recognized the fact
that the action of oxygen varies with the temperature and with the
culture medium ; he also recognizes the influence of the temperature
for he has seen the guinea-pig resist for five or six days inoculations
made with a virus heated to 42°-43° C. in closed tubes. The virus
attenuated by ten minutes' rapid heating at 55° C. does not preserve
its attenuation in cultures in the same way as those attenuated at
42°-43° C.; this is not due to a difference in the mode of action, but
to the duration of the action of the temperature. The virus of charbon
of a very attenuated virulence, may regain it progressively, so long as
it has not been killed outright, by successive cultures upon animals
which are very susceptible and very young, then upon older animals,
then upon animals less susceptible. If we commence upon guinea-pigs
of 1, 2, 3, 4, 5, 10, 20, 30 days old, then pass on to those several
months old, then to the sheep, for example, we resuscitate the viru-
lence; in this return to virulence we may, by cultivating virus at the
various stages of the process, produce vaccines of the various degrees of
activity. In the practice of protective inoculation, according to M.
Pasteur's method, we employ successively two vaccines of different
degrees of attenuation, commencing with a very attenuated virus, ob-
tained by exposing culture at 42.5° C. for 15 or 20 days, and following
this up with a second vaccine less attenuated, derived from cultures
exposed to the air at 42.5° C. for 10 or 12 days.
In the vaccines prepared according to M. Pasteur's method just
described, the properties acquired are not absolutely fixed. After a
series of successive generations, or during the prolonged inactivity of
an attenuated culture, the virulence may become modified, increase or
decrease, and hence it becomes necessary to return from time to time to
the commencement of the operations, and produce fresh vaccines in
order to be certain of the results. We must never lose sight of the
fact that charbon vaccines regain their virulence when they have
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developed in a sheep or other animal, and have accidentally destroyed
it after inoculation. After the publication of the results which we have
set forth, the method of attenuation instituted by M. Pasteur was
tested outside his laboratory; and these tests were fully confirmative
(Feltz, Perroncito, etc., etc.); to-day the method is applied in various
bacteriological institutes.
Attenuation in the soil.—M. Feltz, in testing the facts published
by M. Pasteur and his pupils, after having observed that the virus
cultivated in broth at 42°-43° C. becomes progressively attenuated,
whilst it is not sensibly attenuated below that temperature, has, re-
marked that rabbits resist the attenuated virus better than guinea-pigs;
that rabbits inoculated with a virus insufficiently attenuated for them,
die,—some rapidly, others in from 8 to 10 days,—and that amongst
these latter some die of charbon, presenting few microbes and showing
hæmorrhagic spots on the gastro-intestinal mucous membrane, capillary
emboli formed of bacilli, whilst others die after having passed through
charbon, without presenting microbes even in the hæmorrhagic spots
on the intestine. He concluded from these observations that in the
case of spontaneous recovery, there is destruction and elimination of
the virus by the digestive apparatus. He observed that earth rendered
charbonous loses its virulence in time ; that nature accomplishes at-
tenuation of the virus in the soil analogous to that which is artificially
produced in laboratories, and that it is in this way that we may explain
the greater or less virulence of epizooties of charbon. The observations
made by Feltz of the attenuation of the virus of charbon in the soil
enable us to explain not only the variable gravity of epizooties of
charbon, but also the more or less marked resistance of certain animals
to the infection. It becomes certain, thanks to these observations,
that animals may become vaccinated more or less completely in pastures
by introducing into the system attenuated spores.
Attenuation by compressed oxygen.—M. Chauveau, having re-
cognized the possibility of attenuating the effect of virulent inocula-
tions by the employment of very small doses of the virus, after having
demonstrated the fact that it is possible by this proceeding to increase
the immunity of Barbary sheep, turned his attention to the attenuation
of the virus of charbon. In 1882 he experimented with the procedure of
Toussaint (heat) ; he gained his object and realized the explanation and
development which it admitted of ; he decided the conditions, the
degree and duration of the heating necessary to attenuate charbonous
blood ; he showed by means of a more or less prolonged heating that
we may obtain virus of various degrees of attenuation as by M. Pasteur's
method. He advised, for practical purposes, that defibrinated blood
be heated for fifteen minutes at 50° C. to produce a first vaccine (premier
vaccin faible), and for nine or ten minutes to produce the second
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vaccine (vaccin fort), and that we should inoculate animals first with the
weak and later with the strong vaccines, allowing some days' interval
between the inoculations as is done in M. Pasteur's method.
M. Chauveau besides demonstrated in 1883 that heat may be used
to attenuate cultures of the virus rapidly; that cultures developed
in 20 hours at 42°-43° C. may be attenuated in this way very quickly
in 3 hours at 47° C. they become so attenuated as to be innocuous
to the adult guinea-pig; that we may produce attenuation without the
aid of oxygen ; that we may produce attenuated cultures capable of form
ing a stock and producing batonnets or rods and filaments equally
attenuated, from the spores developed in it. By means of heat, M.
Chauveau has been able to obtain an attenuated virus capable of trans-
mitting its attenuation to the successive cultures which it serves to sow.
It suffices in order to do this, firstly, to leave the cultures in broth sown
with a drop of charbonous blood for 20 hours at a temperature of
42.5° C.; secondly, to place them after this time for three hours at a tem-
perature of 47° C. to lessen its virulence ; thirdly, to expose the culture so
heated, or a broth sown with it, to a temperature of 35°-37° for from five
to seven hours to allow of the formation of spores; fourthly, to heat the
spore-bearing culture for a period of from one to one and a half hours at
80° C. The spores, already attenuated in a certain measure before this
last heating, are still further attenuated to such a degree as to be innocu-
ous to the sheep excepting in very exceptional cases; they are, at the
same time, able to germinate in new media and to produce a mycelium
attenuated like themselves. In order to obtain vaccines by this method
of procedure, it is sufficient to make cultures in bulk, the first two as we
have already described, and the third by sowing the attenuated virus in
a large receptacle containing a considerable quantity of broth, assuring
its rapid multiplication by allowing filtered air to gain access to the
medium of culture; lastly, in the fourth, we give definite attenuation by
a fresh heating. In order to do this and to ensure an equal attenuation
of all the spores, we place the cultivation in tubes of 10 cubic centi-
metres capacity; seal them and keep them plunged for an hour in water
heated to 84° C. for the first vaccine, and at 82° C. for the second.
M. Chauveau attributes the attenuation so obtained to the action of
heat. He decides that in these cases oxygen has little, if anything, to
do with the attenuation, and moreover states that it takes place more
rapidly in vacuo than in the presence of air ; that in M. Pasteur's
procedure heat also plays the principal part. However, M. Chauveau
realizes that the fixity of the attenuated virulence produced by heat is
less than that produced by M. Pasteur's method, and that the attenua-
tion produced by the combined action of air and a less elevated temper-
ature (42.5°), but acting for a considerable time, is transmitted and
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maintained better in future cultivations. That which the microbes have
taken a longer time to acquire, they preserve better.
Having abandoned the procedure of attenuation by rapid heating
as inferior in its results to the method of attenuation slowly by the
combined and prolonged action of air and heat, M. Chauveau afterwards
obtained results of greater scientific interest and greater practical value
by cultivating the virus in contact with compressed oxygen. P. Bert
had demonstrated that oxygen at high pressure, which respects the vital-
ity of the spores, kills the adult microbes, and M. Chauveau asked him-
self whether this gas, maintained at a pressure less than that at which it
kills, might not comport itself as an agent of attenuation. He found
that oxygen under pressure, and compressed air, could attenuate cultures
of the virus and render them fit for vaccination. Air under moderate
pressure augments the virulence of the microbes as regards the guinea-
pig and sheep; under a little greater pressure it increases the virulence
for the guinea-pig, but attenuates it for the sheep; lastly, under a
pressure near that which arrests vegetation (13 atmospheres), cultures
at 35° C. are sufficiently attenuated to not kill the sheep, but they still
destroy the guinea-pig.
In this way, after having made four successive generations of a
culture, leaving each generation for three weeks at a pressure of 8
atmospheres of air at a temperature of 38° C., M. Chauveau observed
that the last generation, which still destroyed the guinea-pig, did not
kill the sheep; a new culture being sown with this fourth generation
and four new successive generations being obtained at a pressure of 9
atmospheres, we may obtain graduated vaccines of different intensity
progressively decreasing. By employing pure oxygen, we may obtain
the same result with a pressure five times less.
After these preparatory trials, M. Chauvea udecided upon the fol-
lowing modus faciendi to obtain charbon vaccine: sow several flasks
with ordinary virus, and enclose them in a well-closed metallic re-
ceptacle; substitute pure oxygen for air, and accumulate it in the
receiver up to a pressure of 2½ atmospheres, place the receptacle in a
stove regulated to a temperature of 35°-36° C., and leave it to from
15 to 30 days, taking care to maintain the oxygen at a pressure of 2½ ;
after the 15th day take seed from some of the flasks to fertilize large
flasks in which at first a small quantity of broth is placed; leave these
new cultures at the ordinary pressure of the air, and after several weeks
add sterilized broth in order to dilute them. We obtain, in this manner,
cultivations of various degrees of attenuation. We may confer im-
munity on the sheep by inoculating him with two drops of this diluted
fluid, and on the ox by five drops.
In this manner, then, the attenuation obtained by cultivation under
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compressed oxygen transmits itself to cultures of the second generation
made at 36°-37° C. and under a normal pressure, and the cultivations
attenuated in this manner may confer immunity upon the ox and sheep
More than this, the blood of a guinea-pig killed by this attenuated virus
will be vaccinal for the sheep.
The virus of charbon attenuated by cultivation under compressed
oxygen can confer immunity by a single inoculation. It is as in-
nocuous as that of M. Pasteur; it preserves its acquired properties during-
several months,—for two months at least. However, its fixity is not
absolute; after two months it has been found to regain a dangerous
virulence. At the same time fixity of the attenuated virulence is not pos-
sessed by the vaccines prepared by M. Pasteur. All the microbes of:
the same cultivation do not necessarily or simultaneously undergo the
same attenuation; certain of them may retain sufficient activity to kill
or to return to their primitive type. Besides the attenuation which it
produces, compressed oxygen has the effect of weakening the toxicity of
cultivations, whilst leaving them their vaccinal properties.
In the more recent researches of M. Chauveau in 1889, by submitting
new cultures sown with old ones attenuated in the same way, to the
action of compressed oxygen, it has been found that the virus may be
despoiled of its virulence, whilst preserving its power of conferring im-
munity ; that attenuated to such a degree as not to kill the guinea-pig,
(an animal most sensitive to charbon), it may be still cultivated and
produce vaccine which is effectual without eudangering the lives of the
animals inoculated; that it may, lastly, be reinvested with its virulence
by cultivating it in broth to which the blood of the guinea-pig has been
added, and placing it in rarefied air.
If the blood added to the broth has been taken from a guinea-pig,
the bacillus recovers the property of mortally infecting first a mouse
and the newly-born guinea-pig, then adult guinea-pigs and rabbits.
Arrived at this stage, the virus, in course of reconstitution, vaccinates
small ruminants, being incapable of killing them. In order that it
may attain this activity, we should cultivate it in broth to which
the blood of the sheep has been added, the bacillus which is obtained is
fatal to rodents. The types obtained in the ascending series may be
reproduced with great constancy.
Attenuation by age.—In a cultivation of bacillus anthracis left to
itself, the virulence disappears at the end of a longer or shorter period
varying with certain intrinsic and extrinsic conditions which are not well
understood. Whilst the virulence diminishes, the bacilli lose their power
of vegetating and die. At a given time there remain in the liquid a few
bacilli having still, but not intact, their virulence and power of vegetating.
At this stage an inoculation performed in the ordinary way with the usual
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quantity may be insufficient to betray the pathogenic activity still in-
herent to the cultivation. In order to reveal it, it becomes necessary
to employ the method of cultivating it afresh or inoculating with a
strong dose. In a cultivation, the various bacilli have not all the same
virulence nor the same power of vegetating; time changes the weaker
ones first, and by degrees the cultivation becomes poorer in microbes
which are virulent and capable of growing; but if at a given time
inoculation with a small dose is insufficient to betray the vestiges of
virulence which remain, cultivation allows of our growing a generation
of virulent bacilli. Analogous phenomena are produced in cultivations
experimentally attenuated ; and certainly we ought, in the study of
the influences capable of restoring the virulence, to take account of a
spontaneous selection of the virulent agents in the cultures of restoration.
An ordinary simple cultivation may suffice to restore the virulence to an
old culture (Arloing). Perroncito has succeeded in obtaining a charbon
vaccine, capable of conferring immunity on the ox by a single inocu-
lation, by cultivating the virus for five days, and then exposing the
cultures to air heated to 20°-25°.
Attenuation by Antiseptics. — MM. Chamberland and Roux
have, after Toussaint, attenuated the virus of charbon by the employ-
ment of antiseptic agents; they have been able to constitute races of
attenuated virus by the employment of a solution of phenic acid, or of
bichromate of potassium. These are the principal facts to be derived from
their researches :—" The addition of 1/400 phenic acid to veal broth stops
all multiplication of the virus. Moreover, after a sojourn of 48 hours in
such a medium, the bacillus has ceased to live ; it gives no development
when sown in neutralized veal broth. If the proportion of phenic acid
is not greater than 1/600, 1/800, 1/1200, the bacillus lives and multiplies;
and, even after it has been maintained for a long time in contact with
the antiseptic, it easily reproduces itself, when it is placed in a suitable
nutritive medium. In this way, after six months the bacilli have
remained alive in liquids at 1/800 and 1/1200 of phenic acid. If the
dose of antiseptic is stronger, the bacillus dies more rapidly ; in a flask
at 1/500 all life had ceased at the end of five months.
" It is sufficient to place 1/800 phenic acid in the culture liquid to
prevent the formation of germs. The bacillus dies in this medium with-
out producing spores. When the dose of phenic acid is "weaker, 1/1200 for
example, the filaments form spores.
The culture issuing from bacilli which have lived for twelve days
in broth to which 1/600 phenic acid has been added, is virulent to guinea-
pigs and rabbits. The cultivation from the same bacilli, after two days,
kills neither guinea-pigs nor rabbits. The action of the antiseptic has
resulted in the diminution of the virulence of the bacilli. If we make