NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
80
requent sowings from the original flask, we get a series of cultivations
of decreasing virulence, which may furnish, as in the case of cultures
at 42°-43°, attenuated virus capable of preserving animals into which
it has been inoculated as a preventive, from mortal charbon. Repeated
cultures of the vaccines reproduce virus with attenuated properties and
perpetuate them. The filaments obtained in these conditions, in place of
being abundant and long as in normal cultures and matting themselves
into flocculi resembling cotton-wool, are more scanty in numbers and
shorter, and deposit themselves in grumous masses on the walls of the
receptacle. These bacilli, so altered in their form, easily give numerous
and resisting spores.
" Phenic acid is not the only agent which gives these results; with
bichromate of potash we may obtain analogous ones. A broth to which
has been added 1-1000-1-700 of bichromate of potash, will not cultivate
the virus, which dies quickly in such a medium. A weaker dose of
bichromate of potassium, 1-2000-1-5000°, allows of the development of
the bacillus; but, under these conditions it does not form spores, and soon
loses its virulence to such a degree that sown three days after the com-
mencement of the experiment, it gives a cultivation which kills rabbits
and guinea-pigs, but only a small percentage of sheep into which it is
inoculated; a cultivation prepared from a bichromate broth, after ten
days, still destroys rabbits and guinea-pigs, but does not kill sheep;
lastly, after a longer interval the cultures become innocuous even to
guinea-pigs. Smaller proportions of bichromate of potash retard the
formation of spores without absolutely preventing them. The bacilli
formed from the filaments which have undergone the action of bichro-
mate, form spores, which perpetuate their properties and assure their pre-
servation. Moreover, if the action of the bichromate has been prolonged,
the bacillus loses the property of forming spores. In this way, cultures
issuing from a flask at 1-2000, after the 8th day from the commence-
ment of the experiment, have never formed spores, and it has been the
same with all successive cultures issuing from it. These bacilli, in-
capable of forming spores, inoculated into guinea-pigs, destroy them in
from three to four days. A small drop of their blood sown in broth
gives an abundant cultivation of bacilli, which does not form spores;
they remain in the state of filaments, and after thirty or forty days they
perish. " The diminution of the virulence of the bacilli so modified by
means of antiseptics, is not a passing one; the cultivation does not
regain its virulence. The bacilli, so attenuated by the action of antiseptics
that they either do or do not form spores, preserve in repeated cultures a
lessened virulence. It appears, therefore, that the varieties of bacilli so
created are, if anything, more fixed in their new degree of virulence, as
the action which has modified them is exercised more quickly upon them.
81
" Other antiseptics exercise an analogous action to those of phenic
acid and bichromate of potash. On the other hand, the dose of the
antiseptic necessary to produce a determinate effect varies with the com-
position of the culture medium. Each of the varieties of the bacillus
has a special action upon the various species of animals. So that the
bacilli attenuated by means of bichromate of potassium, may destroy
sheep, or at least render them very ill, after which they are protected,
whilst they do not produce any appreciable effect upon guinea pigs and
rabbits, nor are they protected by the inoculation.
" In another series of experiments we have submitted the filaments
to the action of a chemical agent in the midst of a liquid where its deve-
lopment is not possible; we have caused an antiseptic dissolved in pure
water, which contains no nutritive element, to act upon the formed
bacillus.
" The filaments of a drop of charbonous blood placed in water
in which phenic acid 1-600 has been dissolved, perish rapidly ; we have
seen before that it lives and grows for a month in a nutritive medium
which contains the same proportion of phenic acid. In a phenicated
solution 1-900, the filaments remain alive for a very long time, as is
proved by the fact that cultivations may be made from them after several
month. During the whole time occupied in the experiment, they do
not form spores, and their virulence becomes enfeebled. So that the cul-
tivation of filaments which has remained for a month in contact with a
solution of phenic acid 1-900 kills rabbits and guinea pigs. A cultiva-
tion made after three months contact does not kill rabbits. Under
these circumstances, the loss of virulence is less rapid than is the case
when the bacillus vegetates in the presence of an antiseptic. But a
short time elapses between this diminution of virulence as regards
rabbits and the death of the cultivation.
" The essential condition in the attenuation of the virulence of the
virus of charbon, either by the method of cultivating at 42°-43° C., or by
the employment of antiseptics, is the absence of spores in the filaments
submitted to the prolonged action of the air, heat, or various chemical
agents. The spore is the form in which resistance or permanence of
virulence is most marked. It preserves, so to speak, from the action of
the surrounding media, the properties of the filament which has given it
birth. Despite this resistance to external agents, the spore may be modi-
fied and attenuated in its virulence like the filament itself.
" Well formed spores of the filaments, fifteen or twenty days old, are
placed in contact with 2 % solution of sulphuric acid, and exposed at
a temperature of 35° C. in closed tubes, which are frequently shaken
to ensure thorough contact of the spores with the acid. Every two days a
small quantity of these spores is sown in broth (veal) rendered slightly
G
82
alkaline. The cultivation so obtained during the first days kills rabbits
and guinea pigs; the cultivation made on the fourteenth day only kills
a small percentage of the guinea pigs into which it is inoculated. The
bacilli, etc., so obtained form numerous spores, and preserve their attenu-
ated virulence in successive cultures.
"But it is worthy of remark that cultures issuing from spores
treated with sulphuric acid, which have lost their virulence as regards
rabbits have not done so as regards sheep, and destroy them in the pro-
portion of 7 to 10. This fact, and the analogous ones already quoted,
shows that each species of animal has a particular receptivity for each
of the races of virus which we have been able to prepare by artifices of
cultivation.
" The diminution of the virulence of the spores and lastly their death
under the action of sulphuric acid occurs, if anything, more rapidly as
the temperature is more elevated and the acid more concentrated, and
more slowly as the temperature is lower, and the acid solution more
diluted." Zagari has attenuated the virus, by making it live in a broth
which has cultivated the microbe of fowl cholera, and Pavone by culti-
vating it in a broth which had already cultivated the bacillus of typhus.
In the same manner of working, we may also cite the progressive
attenuation which Manfredi has obtained by cultivation in a medium
containing a certain proportion of fatty material.
The bacillus anthracis sown in a medium composed of gelatine or
nutritive jelly containing an emulsion 1/3 of its volume of fatty matter
develops well and liquefies the gelatine. When more than one-third of
fatty matter is present, the cultivation is slower and the bacillus forms
long filaments with few spores; it ceases to liquefy the gelatine. When
two-thirds of fatty matter is present, development does not occur. Si-
multaneously the virulence of the culture diminishes to its disappearing
point; this attenuation depends upon the fatty matter introduced into
the medium, for it augments and diminishes with the proportion of this
material; but temperature and time have, at the same time, a great in-
fluence. So in the cultures of Manfredi, the virulence disappeared in
two or three days at 37° C., in twenty or thirty days at 28°-30° C., in
twenty-five to forty-five days at 19°-20° C. On the tenth day at
28°-30°, the cultivation killed all the guinea pigs, and the majority of
rabbits inoculated; at the twentieth day it did not kill rabbits or large
guinea pigs; beyond the twenty-fifth day it only killed mice; at the
thirtieth day it lost all virulence. The attenuated bacilli preserve their
degree of virulence when placed in ordinary media; more or less it has
been noticed that in return cultures, a slight increase of virulence which
has been only slightly attenuated occurs, and that there is a slight
83
diminution in those very much attenuated. These return cultures re-
gain the property of liquefying gelatine, but they liquefy it more slowly
than normal cultures. According to M. Duclaux, the attenuation so
obtained is explained by the fact that the fatty matter employed in
oxidysing impoverishes the medium in oxygen, saponifies and becomes
acid.
Various means of attenuation.—-M. Roux studying the action of
the air and heat on the spores of charbon has recognized that they
do not perish for a very long time at 70°, if they are protected from
the air; and that they may after a time undergo a certain amount of
attenuation before they perish. He has also found that under the same
conditions as regards temperature, they die more quickly when they are
heated in contact with the air, and that before perishing they undergo
diminution in virulence.
M. Arloing has observed that the action of sun-light attenuates
cultivations of the virus; and that it may be utilized in the prepara-
tion of the vaccines, if we are careful to allow it to act on transparent
cultivations distributed in thin layers at the bottom of flat flasks. MM.
Apostoli and Laquerriere have demonstrated that the action of a
galvanic current may, according to its intensity, either kill the virus or
simply attenuate it.
The virus of charbon may be also attenuated by its passage through
various organisms. In this way, the virus appears to become attenuated
according to certain experimentalists in a certain measure, by passing
through the organism of the rat, white mice, rodents, frog, etc. It
has also been thought that in a general way, the virus is attenuated
in passing through the organism of an animal which is refractory,
that of the fowl, pigeon, white rat, frog, etc. But it has been demonstra-
ted that it does not attenuate in passing through the pigeon or rat
(Metchnikoff) nor the fowl (Perroncito) nor the frog (Nuttal); it has
also been observed that the virus of charbon cultivated from pigeon to
pigeon becomes more virulent to the pigeon, fowl, guinea pig and rabbit.
Metchnikoff has observed that the bacillus cultivated in the
blood of sheep previously rendered refractory, becomes in a certain
measure attenuated. Sadowsky after inoculating charbon into the
dog believed that he recognized some attenuation of the virus, but
that when cultivated afterwards in broth it resumed its virulence.
Malm has drawn the following conclusions from his experiments;
the bacillus lived in the dog, an animal more or less constantly re-
fractory to it, far from losing its virulence it increases it; the same
phenomenon of increase of virulence is also observed in bacilli which
have been passed through rabbits previously rendered refractory by
G 2
84
protective inoculation; the bacillus in passing through a refractory
animal, increases in virulence and this increase occurs in the œdema
following the subcutaneous inoculation, in the blood, and in the spleen
after intravenous inoculation; in dogs which are not refractory the
exaltation of virulence is irregular: a previous injection, either subcuta-
neous or intravenous, increases the natural immunity of the dog so as to
render it absolute; dogs resist intravenous injection less than subcuta-
neous. Moreover, the bacillus does not appear to become attenuated
by long residence in the organism of the frog (Lubarsch) ; it increases
in passing through the ox and dog ; also by being cultivated in a series
of fowls of ascending age (Roux and Chamberland) by passing through
the pigeon (Metchnikoff) as we have already seen ; it increases rather
than attenuates by passing through the organism of a vaccinated animal
—the rabbit for instance. M. Nocard having injected the bacillus into
the teat of a goat previously protected by inoculation has noticed that
the animal did not become ill, although the bacillus multiplied in the udder,
and he was able to obtain virulent milk for more than two months, but
during the whole of this time the animal was in no way affected, To
recapitulate then, numerous modes of procedure permit of attenuations
of the virus; but at the present time two only are followed in the
preparation of vaccines destined for protective inoculation, and these are
those of M. Pasteur and M. Chauveau. We are able, by the methods
of Pasteur and Chauveau, to obtain virus of varying degrees of attenua-
tion capable of reproducing themselves with their newly acquired pro-
perties ; some do not destroy the sheep, but still destroy the rabbit;
others do not kill the rabbit, but are still fatal to the guinea pig, whilst
others again do not kill the guinea pig, but destroy mice. We may
gradually return these races of virus to their original virulence by cul-
tivating them after a certain method of procedure, by inoculating
animals which are still susceptible to their action, the mouse, and then
animals more and more resistant, the young guinea pig, then the
adult guinea pig, then the rabbit, and then the sheep. We may obtain
vaccines which, inoculated without any great danger into animals, confer
immunity upon them.
Pasteur's method of vaccination against Anthrax.— Charbon
virus, attenuated by M. Pasteur's method, allows of our conferring
immunity at once by a single inoculation, or progressively by several
inoculations with vaccines less and less attenuated.
Method of operating-—(Condensed from Chamberland's " Le Char-
bon et la Vaccination charbonneuse.") In practice, vaccination is
performed at twice, at an interval of 12 to 15 days between the inocu-
lations; we inoculate to commence with a weak vaccine (premier vaccin)
which only confers an incomplete immunity ; and afterwards at an
85
interval of 12 or 15 days we inoculate a stronger vaccine (deuxieme
vaccin), which completes the immunity. It is customary to make these
two inoculations at different points on the body, the parts selected in
the sheep being for the first inoculation, the inside of the right thigh,
and for the second the inside of the left thigh, whilst in oxen the one
is made in front of and the other behind the shoulder. In the case of
horses, the inoculation is made in front of the shoulder in saddle horses,
so that the point may not be in the way of the saddle.
Full grown sheep of all ages, lambs even when very young, ewes
more or less advanced in pregnancy, some in the point of lambing, have
been vaccinated without any untoward consequences. Still accidents
have sometimes been reported as occurring in young lambs, when the
same vaccine used at the same time in full grown sheep produced only
a more or less slight disease, but not death. With the vaccines now in
use even very young lambs can be vaccinated without inconvenience.
The vaccination of ewes on the point of lambing has also given rise
to some accidents. Several times abortions have been reported to us. We
thought at first that these accidents ought to be attributed more to the
shaking which the animals had undergone in being turned over on to
their backs in order to present the internal aspect of the thigh to the
operator, but it may also be possible that the fever from which the ewe
suffered may have directly induced abortion. According to researches,
which have recently been made in collaboration with M. I. Straus, it
appears that, contrary to the view hitherto held, bacilli introduced into
the mother, sometimes pass into the fœtus. It must be asked, then,
whether the cases of abortion which have been reported are not due to the
vaccine bacilli of the mother, bacilli which she can withstand without
serious results, passing into the fœtus, which being less resistant, has
succumbed and has consequently been expelled. However this may be,
it is better to vaccinate ewes when they are not pregnant, or at least
during the earlier periods of gestation, except in urgent cases, as, for
example, when the disease is raging in a flock.
It may be asked whether lambs born of vaccinated ewes, are them-
selves vaccinated. This question is not completely cleared up, and
experiments are being made with regard to it. Everything leads us to
believe that a large number of the lambs, if not all, do not enjoy
immunity, so that for the present it is necessary to vaccinate lambs when
there is reason to fear that they may be exposed to the spontaneous
disease.
Spring ought, whenever possible, to be chosen for the operation, for
the disease being rare at that time, we avoid, at least in the great
majority of cases, the combined action of the vaccine and of the spon-
86
taneous disease. Morever, recently inoculated animals are better able
to resist the disease which rages generally in summer and autumn. In
oxen slight œdema sometimes follows the puncture: such swelling dis-
appears spontaneously and should not be interfered with. Cows are
also liable to suffer from œdema or even from perceptible fever after the
inoculation; the quantity of milk diminishes ; it is better therefore to
vaccinate cows when they are giving little or no milk, and an additional
reason why this is advisable is, that it has not been proved to be abso-
lutely impossible for the vaccine bacillus to enter into the milk. All
that has been said respecting pregnant ewes applies equally to cows in
that condition.
The following are the instructions given for the guidance of veteri-
nary surgeons in using the vaccine by M. Boutroux :—
Method of operation.—sheep and goats.—The vaccine is sent out in
tubes closed with caoutchouc corks, containing a sufficient quantity for the
vaccination of 1, 2 or 300 sheep or 50, 100, or 150 cows, oxen or horses,
the quantity used in the inoculation of the larger animals being practi-
cally double that for small ruminants. It is labelled, " premier vaccin"
and " deuxieme vaccin." This liquid is to be introduced in a determined
dose under the skin of the animals, and for this purpose we employ a
Pravaz' syringe. The syringe must first be filled with the liquid. In
order to accomplish this we first remove the fine wire from the hollow
of the needle, and adjust the needle on the canula; the tube of vaccine
is now well shaken so as to thoroughly mix the contents; unless this
is done, the vaccine bacilli settle to the bottom of the tube, and there is
considerable risk of drawing up several syringefuls of liquid containing
few or no bacilli, whilst later we get a large number, with the result
that some of the vaccinations will have a negative result. After shak-
ing well then, the cork is removed from the tube, and the liquid is as-
pirated into the syringe by gently raising the piston. If the syringe is
in proper order, it completely fills with liquid, leaving only a very small
bubble of air under the piston, but it frequently happens that the sucker
has become dry, or that the needle is not properly adjusted on the canula
and then the liquid does not completely fill the syringe and a large bubble
of air remains under the piston. In this case we must readjust the
needle on the canula and reject the liquid into the tube, as it is abso-
lutely necessary to have the syringe quite full, to the exclusion of all
air. We perform the same manœuvre two or three times until the
poiston becomes moistened, and if the needle is well fitted on to
the canula, the syringe fills completely. This condition is, as has
been already noted, indispensable. In cases where the piston has
become very dry and allows air to pass, we may boil it in water, leaving
it to cool in the vessel in which it has been boiled until moderately
87
warm, and then aspirating two or three syringes full of the water in
order to cause the piston to swell. Water which has not been boiled must
not be used for this purpose. It is of course of the utmost importance
to employ antiseptic precautions in so far as regards the employment of a
perfectly clean and sterilized syringe. We must also be extremely
careful that we use every expedient to prevent the entrance of germs
into the vaccine whilst aspirating it into the syringe. The syringe
then being perfectly clean and filled with the vaccine completely, we
turn the little nut which is at the top of the stem until it reaches the
division marked I on the stem. Then an assistant seizes the sheep or
goat to be vaccinated and presents the inner aspect of the thigh to the
operator by turning the animal on its back in a sitting attitude. The
operator now introduces the needle under the skin, towards the middle
of the right thigh and presses the piston until the nut touches the
syringe. The inoculation of the first animal is thus made. The
syringe is now removed and the nut on the piston turned until it stands
at 2. The second sheep is now inoculated and so on. One Pravaz'
syringe holds enough for 8 sheep. The syringe is filled again and the
process repeated. With a little practice 150 to 200 sheep can be
vaccinated in an hour. After an interval of 15 days we inoculate them
in the same way with the " dexuieme vaccin " in the left thigh.
Oxen and Horses.—We employ the same vaccine for these animals
as for sheep and goats, but we introduce it in double the doses, so that
one syringeful is only sufficient for four animals. In place of making the
inoculation at the thigh we make it behind the shoulder, in oxen and
horses. The skin of oxen being somewhat difficult to pierce, it is necessary
to be careful to hold the needle exactly in the axis of the syringe,
so as not to break it off. It is also a good plan to take a fold of skin in
the fingers of the left hand to facilitate the introduction of the needle.
The same needle which serves for the inoculation of sheep may also be
used for oxen, but it is advisible to use a stronger one for large animals.
M. Chamberland says:—The vaccine liquid introduced under the skin
ought to be pure in order to produce its proper effect, that is to say, it
ought not to be contaminated with any foreign organism derived from
the serial dust, morsels of wool or straw, or scraps of dung, etc., sometimes
found on animals, for if such impurities get mixed with the vaccine they
may give rise to other diseases, such as septicæmia, suppuration, etc. At
other times these impurities may, as M. Pasteur has shown, produce no
apparent effect, but may hinder the action of the vaccine. In order to ob-
tain the highest possible degree of this condition of purity, the syringes
are boiled after each operation, and put together again, in order to prevent
the small quantity of liquid which has soaked into the piston or remain-
ed below from giving rise to new organisms; with regard to the tube