NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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The cultivation of the microbe in vacuo has succeeded with others,
in the presence of inert gases. Galtier has essayed cultivations and has
drawn the following conclusions from his experiments :—
Cultures made with blood, juice from the tumor or with any other
product succeed when made in beef broth, gelatine or jelly. They de-
velop well at the ordinary temperature of the laboratory, that is, at from
15° to 25°. They develop more rapidly in a stove regulated to 30°-37°C.
They develop in vacuo, also in the presence of air when the broth is in
deep layers and the seed is inoculated by deep puncture of the gelatine.
Culture in broth in vacuo at 37°C increase very rapidly, after 20 hours
the broth has become slightly lactescent. The preparations contain in-
numerable motile, round or ovoid microbes and others in the form of
bâtonnets of various lengths. Later on a white colored deposit forms
at the bottom of the tubes, and the broth becomes very limpid; when the
tube is agitated the deposit becomes suspended as a white cloud of fine
dust. These cultures, even those which have not been made in vacuo or
in the stove, cause the disease when inoculated into the guinea pig.
Cultivated in gelatine and jelly, the microbe gives rise to the formation
of gases; gelatine is liquified in three days, and in from 20 to 22 days
all the medium is nearly completely liquid, the microbes forming white
masses which fall to the bottom of the tube. The microbe of charbon
symptomatique which MM. Arloing, Cornevin and Thomas have named
" Bacterium Chauvœi " in recognition of the services which M. Chau-
veau so kindly rendered them during the period of their studies, and the
classification of which is still not certainly determined, is a bacillus or a
bacterium which stains with the dyes used in the preparation of various
bacteria. It resists the action of acids and alkalies; acetic and more
especially formic acid clears it. The pulp scraped from a section of the
tissues of the tumor and mounted, shows numerous microbes easily seen
without the aid of any stain. But preparations previously stained show
the microbe best. The microbe may be stained with gentian violet
fuchsine, methylene blue, etc.; it stains well with Löffler's blue
(potass 3—10,000, alcoholic solution of methyl blue I) ; with hydro-
alcoholic solutions and especially with aniline solutions of gentian violet,
fuchsine, etc.; it resists decolorization by Gram's solution; it is not decolo-
rized, when heated in balsam, when it has been stained with aniline.
We may obtain beautiful stained specimens, with the serosity or virulent
liquids, or with scrapings or pulp from diseased organs, as well as from
diseased tissues previously hardened in alcohol, by using the above
mentioned stains.
Slips or cover glasses furnished with pulp culture, or sections are
treated as for staining the bacteridium of Davaine " Bacillus anthracis ;"
leave them for ten to twenty minutes in the coloring material, then
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wash, dehydrate, clear and mount in balsam. In such preparations we
distinguish easily the peculiarities of form assumed by the microbe; those
which are in the form of rods appear uniformly colored throughout their
extent, etc.
Resistance of the microbe to physical and chemical agents.—
The virus resists dessication; we may obtain a very virulent material by
drying the pulp of a charbonous tumor at 35° C. This resistance may be
attributed to the resistance of the spores, as in anthrax. The dried virus
may preserve its properties for years, and is more resistant to physical
or chemical agents than is the fresh virus.
Very intense cold has no apparent effect upon the virus, which may
be exposed to a temperature of—130°C without undergoing either
sterilization or even attenuation. Fresh virus sealed in a tube may be
kept for thirty minutes in a stove at 65° C without losing its activity;
at a temperature of 70° C it is sterilized in two hours and twenty
minutes; in two hours at 80° and in twenty minutes at 100° C. The
addition of boiling water in the proportion of three parts to one of the
virus does not sterilize it, whilst fresh material enclosed in a tube and
left in water at 100° is sterilized in twenty minutes. Virus dried at 35° C
bears a more elevated temperature; previously moistened or mixed with
water and placed in the stove it withstands a temperature of 85° for six
hours without undergoing more than a slight attenuation; heated for
the same time at 90,° 100° and 105° C it undergoes more marked atten-
nation without losing its virulence, which is only destroyed by six
hours' exposure to 110°C. The same virus dried and placed in a tube
and put into boiling water is sterilized only after two hours' exposure.
In a medium saturated with watery vapour heating to 110° C for fifteen
minutes has sterilized the fresh virus contained in tubes, fresh and even
dried virus previously mixed with water and spread over fragments
of tissue: the dried virus added to water and enclosed in tubes has re-
quired heating for thirty-five minutes. The fresh virus, falling into
a large quantity of water, becomes attenuated by such dilution; when
it is mixed with a small quantity of still water, the microbes fall to the
bottom of the vessel, and the superior layers are innocuous.
Putrefaction does not destroy the microbes rapidly in buried carcass-
es. They have been found to be virulent six months after putrefaction
has commenced, but innoculation has failed to produce the disease when
carried out thirty months after putrefaction has started, but this does not
prove that the microbes have been destroyed. The microbe may live in
company with others; we have produced at the same time charbon
symptomatique and septicæmia, charbon symptomatique and anthrax;
the germ is not destroyed by admixture with the ferments of rennet nor
that of ammoniacal fermentation.
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All these facts lead us to the conclusion that it may be preserved
in the soil, and direct experiments have established the fact. Feser has
caused the disease by injecting the microbes found on the plants from
marshy pastures of the Alps, where the disease is rife. Gotti caused the
disease by injecting the water, in which had been washed earth from
San Arcangelo, where the disease had been prevalent, under the skin
of young oxen It is apparent, therefore, that the germs may be
preserved in the soil or on its surface, and this explains how it is that
animals which live in places- where these germs exist, may ingest
tbem.
The virus offers great resistance to those chemical agents which we
designate baetericides; it is only sterilized, especially when dry, by
the most energetic of these. Solutions of potash, lime, sulphate of iron,
sodium chloride, arsenious acid, chloride of zinc, turpentine, sulphurous
acid, etc., are useless for purposes of disinfection. The agents which
may be recommended for this purpose are:— phenic acid iu aqueous
solution 2-4%; salicyiic acid l-2°/ 0 ; nitrate of silver 1 -2.°/ 0 ; sulphate
o£ copper 1-5; perchloiide of mercury 1-2 p. 1,000, etc.
Animals susceptible to the disease.—Chaibon symptomatique is
nearly entirely confined in its attacks to the bovine species; it has only
exceptionally been witnessed in young sheep and solipeds. It is
transmissable by inoculation to oxen, sheep, goat, and guinea pig. The
carnivoia, pig, rabbit and bird are refractory. The disease does not
appear to be transmitted to man.
The large and small ruminants are the most susceptible to the
disease, although, as has been stated, oxen do notexhibit the same
receptivity at all ages. The spontaneous malady is very rare iu calves
before the age of six months, and it is rather a difficult matter to induce
it in suckliug calves. Well marked and characteristic cases have,
however, been noted by veterinarians in young calves, which had already
been submitted to a vegetable diet. This qnasi-im-a±uu'\iy which youn^
calves enjoy appears to be due partly to heredity transmitted by an
already immune mother, and more particularly to the mode of alimentation
special to the age, as well as to the condition of the tissues and humors
during that age. Experimenting with animals of various breeds, we
have been able to inject six drops of fresh virus of a very active charac-
ter into the muscles of the young with impunity, whilst animals more
advanced in age succumbed in the proportion of 90°/ o under similar con-
ditions. These inoculations, however, sometimes give rise to lameness
and elevation of temperature iu certain of the calves; and by employ-
ing larger doses we cause death. Six or eight drops may destroy
calves of less than sixteen days of age ; it requires from ten to twenty
drops to kill calves aged from fifteen days to three months. The
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feeble receptivity of the calf is very evident from its behaviour to the
intravenous injection of strong virus and the subcutaneous injection of
attenuated virus which give to the adult animal a mild attack and confer
immunity, whilst the calf has no protection conferred upon it by the pro-
ceeding. The receptivity of young calves increases as they become more
entirely herbivorous. The influence of diet is still further shown by what
occurs in the young guinea pig, which is very susceptible to the disease,
because it becomes herbivorous very early after birth.
Oxen from localities where charbon symptomatique is enzootic
are not apt to contract the disease after they have passed the age
of four years.—This has been demonstrated both by clinical observation
and by experiment. But this privilege is not enjoyed by adult animals,
which have been raised in regions where the disease is not endemic ; these
animals contract the disease at all ages when they are inoculated, or when
they are introduced into localities where it is rife. Immunity is then only
acquired by animals (adult) which have been reared in localities where the
disease is endemic ; and this immunity appears to result from successive
inoculations, either with weak doses of the virus, or with germs which have
become attenuated in external media which occur spontaneously during
the youth of the animals. It may be likened to what occurs when we
inoculate small doses of the virus or attenuated virus Young cattle living
in an infected area may ingest or otherwise introduce into the system
variable doses of germs more or less attenuated. Some in this way become
inoculated with sufficiently large doses to provoke a fatal attack ; whilst
others, by receiving either attenuated virus or a small dose of the
virulent, only contract a mild form of the disorder which may pass
unperceived and confer on them relative immunity, which may be in-
creased by further spontaneous inoculations, and become more or less
absolute at the age of four or five years, or even before.
The receptivity of the sheep is very marked; and, although it seldom
contracts the disease spontaneously, it may be easily infected by inocula-
tion. As to the goat, it may also contract the disease after inoculation,
but less surely than does the sheep. Lastly, the guinea pig may be em-
ployed as a soil for the culture of the virus. Its receptivity is far from
equal to that of the ox and sheep; the virus tends to decrease in viru-
lence when it is passed from pig to pig, and to such an extent does this
weakening process occur that after a time we obtain such an attenuated
form as to be capable of conferring immunity without danger.
The horse and ass, at adult age, are generally refractory, resisting
inoculations and only presenting local lesions as the result of them.
Clinical observation has shown or led us to suppose that foals are more
susceptible and may even contract a grave form of the disease.
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Amongst the refractory animals, one may be rendered susceptible to
the disease, and this is the rabbit. When we associate the virus of
charbon symptomatique with another microbe, especially the micrococcus
prodigiosus (Roger), we may induce the disease in this animal by inocu-
lating the mixture. The " prodigiosus " favours infection by the action
of its soluble products, which modify the general state of the animal,
when they pass into the circulation. This is proved by the fact that the
receptivity is more especially increased when the prodigiosus is injected
directly into the circulation. The receptivity created is evanescent, dis-
appearing in twenty-four hours. This fact is interesting, as it shows us
that a microbe, inoffensive in itself, may create a passing receptivity to a
morbific one in subjects ordinarily refractory; and that it is not im-
possible that animals more or less refractory may in practice be rendered
susceptible to a grave form of the disease.
In addition to all this the microbe of charbon symptomatique, which
is a ferment liable to induce butyric fermentation in the majority of
sugars and of several neutral hydrocarbons (Arloing) secretes soluble mat-
ters (Roger) which favour its development. Hence, when it is inoculated
into the anterior chamber of the eye of a rabbit, it causes the disease and
death, although this is not the case when it is inoculated into a muscle
or under the skin. The soluble substances secreted in the eye modify the
general health of the animal and create receptivity; the absence of pha-
gocytes in the aqueous humour explains the development of the microbe
in the eye. More than this, serosity obtained by filtration of virulent
material injected in doses varying from 1 to 5 C C. into the vein of a
rabbit, temporarily abolish its natural immunity and render an inocula-
tion into the muscles successful; the predisposition so created does not
last more than twenty-four hours. M. Roux has demonstrated, as we shall
see later on, that such serosity has vaccinal properties If the prodigio-
sus and the substances of bacillus chauvœi produce temporary predisposi-
tion in the rabbit, this arises from a momentary weakening due to the
action of these substances, which render the animal more or less ill, and
consequently less resistant for a time. This explanation of the phenomenon
appears so probable that MM. Nocard and Roux, by enfeebling the
vitality of a muscle with acetic acid, potash, alcohol, etc., or by produc-
ing a traumatism, or a contusion, have been able to render the rabbit
susceptible to the disease, and the virus has become intensified and more
active to the normal rabbit. Galtier himself has succeeded in inducing
charbon symptomatique by inoculation in the rabbit which he had weak-
ened by feeding it on watery food and by submitting it to the action
of cold. These facts are interesting from a clinical point of view, as they
show us clearly some of the predisposing causes of the disease.
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Modes of Contagion.—Influence of the dose and the mode of pene-
tration of the virus.—In a general way, the grave form of the disease
is only produced by a more or less large dose of the virus, which varies
according to the manner and the seat of its introduction, and of course
the degree of its activity; punctures with a lancet are not usually suc-
cessful in inducing the disease for the reason that they do not permit of
the introduction of the virus in sufficient quantity nor to a sufficient
depth; the inoculated virus must be placed in the subcutaneous tissue
if we wish to be certain of its producing its effects, at the same time
certain regions are not favourable for this method of inoculation, if we
wish to induce a fatal form of the disease. Thus, the tumors do not
develop below the knee and hock, nor in the tail: and the virus inocu-
lated in doses of one to seven drops at the extremity of the tail determines
neither local nor general disturbance. Injected in doses of ten drops it
causes elevation of temperature; injected in very large doses (two gram-
mes) it gives rise to a local engorgement, fever, loss of appetite, rise of
temperature, and confers immunity. If injected at twenty centimetres
below the root of the tail it is more dangerous, and may, exceptionally it
is true, cause death in from three to four days, the virus taken up by the
lymphatics or blood vessels is transported to other parts of the body and
there produces at some part a fatal tumor. In proportion as we approach
the base of the tail the chances of general infection and the formation of
symptomatic tumors increase, but do not equal those following inoculation
into the thigh, quarter, or neck. The tail then possesses a feeble recep-
tivity, which increases as we approach the root; this is due to the low
temperature and the density of this region.
The most favourable seats for inoculation are those rich in muscle, or
in which connective tissue is abundant and lax, and the temperature
fairly high. The virulent material for inoculation may be extracted from
various sources: from all the lesions and products reputed active as well as
from cultivated microbes. In order that we may be certain of the
result, material from the ganglia, organs rich in microbes, and especially
from the tumor, should be employed.
Virus taken from the tumor may be used either in the fresh or dried
state (dessicated at 35°C.) The material to be employed is obtained by
triturating fragments of tissue taken from the blackest portion of the
tumor, in a glass or mortar; to this is added sterilized water, and the
whole is shaken in a flask and filtered through linen; the filtered
material serves very well for inoculation. The dried material is obtained
by submitting the pulp of the muscle invaded by the tumor to rapid
dessication, selecting those portions of it which have undergone most
morbid change. The method adopted is first to mince the material; add
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sterilized water; bruise in a mortar or shake well in a flask; filter and
spread the material on plates and place in a stove regulated to 35°C.
After complete evaporation and dessication, a bright reddish layer of
material remains, which is to be reduced to powder and may be kept
in a flask dry and in a dark place. Virus so prepared may be kept for
more than five years. When we desire to use this virus for inoculation,
it is necessary to add a little sterilized water to it and triturate in a
mortar and filter through calico; the virulent liquid thus obtained may
be increased in virulence by adding a drop of lactic acid.
Inoculation with a lancet, as has been already said, is not a satis-
factory method with this virus; it does not produce any local lesion, or
tumor when made in the ear of a guinea pig; although it may induce a
fatal attack of the disease. When this occurs the animal dies in about three
days; at the autopsy we find infarcts in the muscles of various regions,
and congestion with tumefaction of the lymphatic ganglia. The most
certain method of inoculation is the subcutaneous, the injection into the
subcutaneous connective or the intra muscular tissue. The effects of the
inoculation vary according to the dose employed and the region selected.
The dose of the virus injected should generally be large; two or three
drops injected under the skin of the quarter, shoulder, etc., suffice to in-
duce fatal charbon in the sheep and oxen, whilst a similar quantity
injected into a region rich in cellular tissue, such as the ear, extremity
of the tail or limbs, is insufficient to do so. When the injection of virus
has been made into the thigh of a guinea pig, the part becomes swollen and
painful in a few hours; movement is impossible, and death ensues in from
twenty-four to forty-eight hours. At the autopsy we find very extensive
red coloured œdema of the connective tissue of the abdominal wall; a char-
bonous tumor at the seat of inoculation, with swelling, dark or black
colour of the muscles, and a very marked smell of lactic acid pervading
the parts. When a large dose of the virus is injected into the subcu-
taneous connective tissue or the muscles of an animal endowed with great
receptivity, such as the sheep and ox, grave pathological changes occur
and death results. If the injection has been made in the subcutaneous
connective tissue, the lesions appear in the connective tissue and ganglia,
encroaching little on the muscles; œdema, which is hot, firm increases
rapidly and becomes crepitant in certain parts. When the injection has
been made into the muscle, a true crepitant tumor is rapidly evolved
analogous to that of spontaneous charbon, and accompanied at times by
tumors in other regions.
The subcutaneous injection of medium doses (1/10 of a drop of pulp)
produces effects similar to those following inoculation with the lancet;
when successful the local lesions are nil or little marked, but muscular
B
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lesions, a tumor more or less distant from the seat of inoculation, follow-
ing the same course as those evolved during the course of the spontane-
ous disease, makes its appearance and is followed by death.
The injection of less active virus, or of a very small dose of active
virus is followed either by no result, or by the appearance of slight symp-
toms, such as fever, loss of appetite, and dulness, without any local
lesion ; this slight derangement, however, gives immunity to the inoculat-
ed animal. In certain cases, however, where the animal inoculated is
more than ordinarily susceptible, which we are unable to foresee, a viru-
lent and mortal attack of the disease may occur from a very minute dose
of the virus.
The introduction of relatively large doses of virus directly into the
veins is usually well borne. The intravenous injection of doses varying
from 3/10 to 5 drops up to 6 C. C. of filtered muscular juice induces in
oxen, sheep and goats a mild form of the disease (unaccompanied by
tumors) with dulness, elevation of temperature, etc., lasting for two or
three days and followed by immunity. Whilst practising intravenous
injection it is of the greatest importance to avoid inoculating the periven-
ous tissue, as, should this accident occur, it will in all probability be follow-
ed by a virulent form of the disease. Sometimes intravenous injection
with large doses, or with active virus, or when the animal is a very suscep-
tible one, may be followed by a virulent form of the disease with all its
symptoms.
Moreover, when very large doses are injected, the animal may suc-
cumb before the appearance of the tumor and without muscular infarcts ;
in these cases the ganglia are virulent. How are we to explain the
reason why intravenous injection does not produce the disease with its
characteristic tumor ? Beyond the fact that a certain dose of the virus
may be destroyed by the blood, we have reason to believe that if the
microbes multiply in that fluid, and we know they do since it becomes
virulent, they are retained by the vascular endothelium, which prevents
their extending into the connective tissue, and hence the tumors are not
formed. It is certain that if we permit them to emigrate from the
vessels, they give rise to a tumor and cause death. Thus it happens, if
during the slight malady which follows intravenous injection, we provoke
a small hæmorrhage in any region, a tumor develops and the animal
perishes. It appears, then, that in order to cause a tumor and induce a
grave form of the disease, the microbe must gain exit from the vessels
and undergo certain changes in the connective tissues. It appears also
that there are two phases in the evolution of the disease—one the multipli-
cation of the microbe in the blood, and the other intoxication when the
microbe passes into the connective tissue; the virus remains in part and