NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
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growth of tetanus bacilli from it. This growth was also virulent, and killed guinea
pigs in small doses. (0. 1 c.c. of a four weeks' growth).
Both nibs, when taken out of the original test tube, were rusty, and on this
account I was rather surprised to obtain a growth of tetanus bacilli from either
of them, and I was also surprised to find that both cultures were virulent. The
spores on the first nib had been eleven years in a dried condition, and those on the
second nib 18 years, and no doubt they would have remained alive and virulent
for several more years.
Cases are on record where splinters of wood infected with tetanus spores and
kept for 2½ and 11 years were still capable of conveying the disease, but I have no
knowledge of any other authentic case in which the spores in a dried condition
remained alive and virulent for 18 years.
In the face of these facts it is only reasonable to infer that in the soil tetanus
spores might have an equally prolonged life, provided they are not directly exposed
to the sun's rays. From earth they find their way into the intestinal tracts of
horses, cattle, sheep, goats and other animals, attached to food, such as grass,
hay, roots, etc., and evidently in some cases into the intestinal tracts of man
adherent to uncooked vegetables. In these various intestinal regions the spores
find anaerobic conditions where they can germinate, and are then passed out
with the excreta, and again find their way to earth, roads, streets, and
stables, etc. In a vicious cycle of this kind their vitality and virulence would
depend on whether the conditions for development were favourable or the
reverse.
It is not to be wondered at, then, that tetanus germs are plentiful in nature.
The wonder is that cases of the disease are as rare as they are. This can be accounted
for only by the comparative non-virulence of the majority of strains if not connec-
ted with a case of tetanus.
When tetanus germs are isolated from garden earth, stable floors, and other
places, as a rule it requires a large dose to produce even local tetanus in a susceptible
animal such as a guinea pig but it sometimes happens that a virulent strain can
be obtained from the soil and other sources. As examples, I may mention that
on one occasion I found that it required 2 C.C. of a 10 days' culture to produce
l ocal tetanus i n a gui nea pi g; and on another occasi on ¼ C. C. of a 10 days' cul ture
proved fatal to a guinea pig. Both cultures had been isolated from earth but from
different localities. I have also worked with a tetanus culture of such virulence
that it was only necessary to dip the point of a surgical needle into it, and then
prick a guinea pig's skin to give the animal fatal tetanus. It will be seen from these
examples that there is a very wide variation in the virulence of different strains
of tetanus bacilli.
As far as indirect infection is concerned the horse is probably the most fertile
source. Some time ago a case was brought to my notice in which lint and a bandage
9
which had been for some days on a shelf in a stable were used to dress an open
boil on a man's arm, with the result that fatal tetanus set in a few days afterwards.
The lint and bandage used in this case were what remained from a dressing applied
to a horse's leg some time before, and after remaining exposed in the stable, where
they doubtless got contaminated with tetanus, they were applied to the patient's
arm without the knowledge of his medical attendant.
In this connection I might also mention that, when immunising horses with
snake venom in order to prepare antivenene, abscesses at the seat of injection of
the venom are of common occurrence. These abscesses, when opened, are likely
portals of entrance for the tetanus germs which horses harbour in their intestinal
tracts. I have had practical experience of four cases of tetanus in horses con-
tracted in this way. After these accidents I adopted the routine practice of giving
antivenene horses an occasional dose of antitetanic serum, and since then I have
not had a single case of tetanus in those horses.
In a tetanus-infected wound the spores which have gained an entrance and
found suitable local conditions germinate at the seat of infection. The bacilli
resulting from the growth of the spores remain localised at the seat of infection,
or in its immediate vicinity, and produce an extra-cellular toxine which is the direct
cause of the symptoms of tetanus.
The disease is what is known as an "intoxication process," i.e., it altogether
depends upon an extracellular poison elaborated by the causal micro-organism, and
in this respect it resembles diphtheria. When tetanus bacilli have been grown
in pure culture for a month (or less) at a temperature of 37.C., and the toxine
separated by filtration through a Pasteur Chamberland filter, every symptom of
tetanus can be produced in animals by injecting the germ-free toxine.
According to Ehrlich, tetanus toxine is made up of at least two toxine bodies,
viz., tetanospasmin and tetanolysin. Of these two bodies tetanospasmin is the
more important, as it has a strong affinity for nerve tissue, especially nerve centre
cells, and gives rise to the muscular spasms characteristic of the disease. Tetano-
lysin has some effect in breaking up red blood corpuscles, but it has not been proved
that this is of much account in a case of tetanus ; it is a negligible side issue.
Meyer and Ransome10 have demonstrated that the transmission of the toxine
to the central nervous system takes place by way of the motor nerves only. It is
absorbed by the end organs of the motor nerves at the seat of infection, and travels
by way of the axis cylinders to the susceptible nerve centres. These observers
also give it as their opinion that "the greater part of the period of incubation is
the expression of the time occupied in the conveyance of the toxine from the peri-
phery along the motor nerves to the susceptible centres, and that the actual move-
ment of the toxine takes place not in the lymphatics, but in the protoplasm of the
n e r v e s . "
c2
Within the last few years several experiments have been carried out which go
;to prove that part of the toxine may be absorbed by the lymph and blood channels,
and as a proof in this direction, generalised tetanus can be produced in animals by
injecting toxine into a vein.
Henri and Cernodeannu 11 are of the opinion that the toxine is conveyed by the
blood and lymph streams in all cases.
In any case it is certain that no symptoms of the disease appear until the toxine
produces its effects upon the cells of the nerve centres, brain, or spinal cord, or
b oth.
In acute generalised tetanus the brain centres are always affected, but in local
tetanus, which is common in experimental tetanus in guinea pigs, the toxine has
only reached to the section of the spinal cord supplying motor nerves to the seat
of infection, and when this is the case the disease is likely to become chronic and
last for weeks.
Tetanus toxine is easily destroyed by heat. A temperature of. 55°C. for 1½
hours renders it inert. A shorter time at 60°C., produces the same effect ; and a
temperature of 75° C. removes every trace of its toxic properties in 5 minutes
or less.
The pure spores of tetanus when freed from every trace of toxine are known as
" washed spores " and in this condition they can be injected into animals with-
out causing tetanus.
"Washed spores" can be obtained by filtering a pure tetanus culture, so as to
separate the toxine, and then washing the precipitate on the filter several times
with sterile normal saline solution, in order to remove every trace of toxine. Another
method is to heat a pure bouillon culture of tetanus in which the spores are well
formed at a temperature of 75°C. for 5 minutes.
Pure " washed spores " when obtained by either of these methods fail to con-
vey tetanus when injected into guinea pigs or other susceptible animals.
The phagocytes at the seat of injection are capable of dealing with pure tetanus
spores, and any which may escape phagocytosis get stowed or hidden away in the
local tissues, to remain there alive for months, or years, until conditions become
favourable for them to germinate, and grow out into toxine-producing
bacilli.
When virulent " washed tetanus spores " are injected with any materials
such as sterile sand, sterile powdered charcoal, or bacteria, which injure the tissues
and handicap phagocytes, the spores grow out and tetanus is the result. A non-
lethal dose of toxine mixed with washed spores produces fatal tetanus. In an
experiment of this kind the toxine acts by producing a negative chemiotactic in-
fluence on the phagocytes, and the spores get an opportunity of growing out into
bacilli. A tetanus infection can only take place when the spores germinate.
11
When quinine or lactic acid is mixed with washed tetanus spores and injected
into animals, tetanus sets in rapidly, and is more certainly fatal, because both
these substances destroy tissue at the seat of injection, and so favour the growth
of tetanus bacilli.
A few tetanus spores gaining entrance to a wound which had not been badly
contaminated with dirt and other micro-organisms would very likely get des-
- troyed by the phagocytic action which would immediately set in; but in a badly
contaminated wound, or one in which tissues were destroyed, or where anaerobic
conditions and feeble phagocytosis prevailed, tetanus would result.
In wounds infected with tetanus spores another result is possible, viz., a condi-
tion in which the spores remain latent or dormant, having escaped destruction by
the phagocytes, and at the same time not finding suitable conditions for germinat-
ing into bacilli—a latent infection in which for some reason or other the spores for
the time being failed to grow out into bacilli, and at the same time escaped
phagocytcsis.
In experimenting with tetanus spores, it is absolutely necessary to use pure
cultures, and it is advisable to use cultures which have had ample time to form
spores, and the strain should be virulent. A virulent strain grown at a temperature
of 37°C., for 3—4 weeks, and under strict anaerobic conditions should answer well.
Young bacilli with little or no toxine might not produce the disease, although
they would contain toxine locked up in their bodies (intracellular toxine). The
addition of dilute lactic acid or chloroform water would extract the toxine from
young bacilli or their spores, and when otherwise harmless young bacilli had been
steeped in either of these fluids the mixture would be toxic, and produce tetanus.
In a virulent strain of tetanus, the toxine produced when a culture has been
grown under the most favourable conditions, and for a month or six weeks, is fatal
to susceptible animals such as mice or guinea pigs in extremely minute doses.
I have grown a tetanus culture in which 1/1000 c . c . o f t h e t o x i n e f r e e f r o m
bacilli was sufficient to kill a medium sized guinea pig, and cases are on record
where a millionth of a c.c. was sufficient to kill mice, and weight for weight mice
are not so susceptible as guinea pigs.
Burnet12 states that weight for weight the animal most sensible to tetanus is
the horse. He also gives the following comparison of sensibilities culled from the
works of other authors : "If we express by the figure 1 the quantity lethal to
a given weight of horse, the quantity which kills the same weight is 2 in the case
of the guinea pig ; 4 in that of the dog ; 13 in that of the mouse ; 2,000 in that of the
rabbit ; and 200,000 in that of the fowl ; that is to say, the fowl is 200,000 times
more resistant to tetanus than the horse."
The earliest symptoms of tetanus in the horse are lockjaw, stiffness of the
neck, and difficulty in swallowing ; and in man, and monkeys, lockjaw is also one
12
of the earliest symptoms. We know that the horse is extremely susceptible,
probably one of the most susceptible animals to tetanus, and lockjaw being one of
the earliest symptoms in man and the horse, we might infer from this that man is
also very susceptible to tetanus.
Metchinkoff,13 some years ago, demonstrated that alligators and tortoises
are insensible to tetanus toxine. He injected large quantities into tortoises and
found that these animals remained well and retained the toxine in their bodies
for months.
The fowl can be rendered sensible to tetanus by placing it in a cold, chamber,
or making it stand in iced water; and some cold-blooded animals, such as frogs,
can be rendered susceptible by placing them in a chamber heated to a temperature
of 37°C.
When the symptoms of tetanus set in acutely, the death-rate is very high,
over 90 per cent., but when symptoms set in slowly, and the disease becomes to a
certain extent chronic, there are about 30 per cent. recoveries.
A form of tetanus known as "hydrophobic tetanus" following wounds on
the face has been described. In this form the incubation period is short, owing
to the fact that the toxine has only got a short distance to travel along the motor
nerves before it reaches the cells of the brain. Spasm of the throat, resembling
the spasms of hydrophobia, is an early symptom, hence the name "hydrophobic
tetanus."
Another form known as "visceral tetanus" has been described and recognised
by the French. In this form the infection originates in one of the internal organs.
It is extremely fatal and the disease runs a very rapid course, the symptoms being
somewhat masked and obscure, owing to the fact that the great splanchnic sym-
pathetic nervous system is involved. Cases of uterine tetanus which have been
known to follow criminal abortions would come under this heading.
McFarland14 refers to a case of tetanus reported by Kamen in which the intes-
tine of a patient dead of tetanus was found to be rich in tetanus bacilli. He gives
this as an example of cases in which tetanus may possibly occur without a wound
by absorption of the bacilli through abrasions in the mucous membrane. It is
conceivable that a few tetanus spores absorbed from the intestinal tract, and carried
by phagocytes to a part of the body where there was dead tissue ( e.g ., the site of a
quinine injection), would give rise to tetanus. Would this method of infection
account for some of the cases in which quinine had been given ?
13
CHAPTER III.
The effects of large doses of Quinine on small animals such as Rabbits
and Guinea Pigs.
In these experiments no attempt was made to follow up the excretion of
quinine from the system, or to enter minutely into its physiological effects. The
principal objects were—
(1) to arrive at a dose, which, without proving fatal, would produce
well marked visible effects;
(2) to arrive at some conclusion as to what the local effects of quinine
are when given hypodermically;
(3) to prove whether the local and general effects of quinine when given
hypodermically have any influence in favouring tetanus infections;
(4) to obtain information as to whether animals can withstand larger doses
by the stomach than hypodermically;
(5) to gain some information on the effects of quinine when given intra-
venously.
The salt of quinine used in almost all cases was the bihydrochloride, and in the
form of tabloids as prepared by Burroughs, Welcome & Co., London.
The tabloids were dissolved in sterile normal saline solution in the propor-
tion of 1 gr. to 1 c.c., a solution the acidity of which corresponds to 1.3 per cent.
combined sulphuric acid, or 1 per cent. combined hydrochloric acid. A solution
of this strength was chosen in order that the escharotic action might not be too
severe when injected hypodermically, or into muscles. The solution used when
the drug is administered hypodermically to malarial patients, is, I understand,
invariably much stronger, and on this account the local effects on the tissues at
the seat of injection must be vary pronounced.
It will be noticed that in a few of the experiments (see Tables I and VII) the
dilutions used were stronger, and in a few weaker than 1 gr. to 1 c.c. The objects
in using a stronger dilution were to ascertain whether absorption would be slower
or quicker—and whether the local effects would be to any appreciable extent more
pronounced than when weaker dilutions were used; and in the few intravenous
experiments in which weaker dilutions were used the object was to ascertain
whether more quinine could be tolerated when given by this method in weak
dilutions.
Rabbits and guinea pigs were the animals used. Both are susceptible to the
action of quinine, whether given hypodermically, intravenously, or by the stomach;
14
but they can stand fairly large doses without any apparent injury other than the
local effects when given hypodermically.
(a) quinine hypodermically.
When large doses of quinine are given hypodermically to rabbits and guinea
pigs, they rapidly come under its influence, and death takes place in a few hours.
A large dose for a guinea pig would be 1 grain per 150 grammes weight of animal.
One grain per 150 grammes of guinea pig is a certain lethal dose ; but I have
known 1 grain per 233 grammes to prove fatal in some cases and not in others, so
there is evidently an individual factor difficult to account for. On this account it
would be impossible to arrive at a minimum lethal dose for all cases, and to
express it in grains per grammes of guinea pig, and to say that this amount would
kill with absolute certainty.
Some years ago, when it was the practice to standardise antityphoid vaccine
by arriving at the minimum lethal dose for a guinea pig of a certain weight, a simi-
lar individual factor interfered with its accuracy, and to such an extent that the
method had to be abandoned and other more accurate methods of standardising,
such as counting the bacilli, adopted.
On the other hand it is possible to come somewhere near a minimum lethal
dose of quinine, which, without leaving much of a margin, would never fail to kill,
and such a dose is 1 grain per 150 grammes of guinea pig. I have never known
guinea pigs to survive this amount when given hypodermically, and in some cases
smaller doses have proved fatal.
It is easy to arrive at a dose which will produce well-marked visible effects
without killing the animal, and such a dose is 1 grain per 350 grammes of guinea
pig. This amount can be given hypodermically for several days in succession
without proving fatal, except perhaps in a few cases indirectly by the production of
severe sloughs at the seat of injection and the suppuration which accompanies them.
When large doses are given to guniea pigs, symptoms set in within 10 minutes.
The symptoms take the form of convulsive muscular spasms, which set in gradually
and soon become more pronounced and severe, and the animal dies asphyxia-
ted. For some time before death the animal lies in its cage, but slight twitching
of the muscles and limbs are apparent, and when disturbed these become more
severe. The rapidity and severity with which symptoms set in and prove fatal
depends on the amount of quinine given. When 1 grain per 150 grammes of
weight is given symptoms set in within ten minutes, and a fatal result may be ex-
pected any time between 4 and 24 hours. When larger doses are given symptoms
appear a few minutes earlier, and death takes place in all cases under 24 hours.
A glance at the experiments detailed in Table I will serve to explain all that is
necessary on the subject.
In Table II the results of four experiments with the bihydrochlorate of quinine
are given, and it will be seen that they differ in no material point from those of the
bihydrochloride used in all the other experiments, and on this account it was
deemed advisable to use only the latter salt of quinine, as it is the one most
generally used for hypodermic injections in cases of malaria.
The local effects were in all cases very evident. When the animals died before
a slough had time to form, the tissues at the seat of injection presented the ap-
pearance of having been burnt with pure carbolic acid. A white escharotic patch
was never absent, and one could trace exactly the extent to which the fluid had
been distributed. In those experiments in which the animals survived for a few
days or survived altogether, it was quite the exception for a local slough not to
form. These sloughs included the true skin, subcutaneous tissue, and deep fascia,
and exposed the muscles ; and in animals which survived it required weeks before
healing was complete.
In the few experiments recorded in Table III, in which the quinine was given
intramuscularly, the general and local effects differed in no way from the subcu-
taneous experiments, except that the animals appeared to suffer more pain. I
believe that the experience of malarial patients who have had intramuscular in-
jections of quinine is to the effect that the subsequent pain and discomfort are
worse than when given hypodermically.
In rabbits 6 grains per kilogramme of body weight is a certain lethal dose
when given hypodermically, and in some cases less would suffice. Here again
there is an individual factor difficult to account for. In. Table IV it will be seen
that in one experiment a 1,410 gramme rabbit survived for 4½ hours after receiving
10 grains ; whereas another rabbit weighing 1,500 grammes which received the
same amount of quinine died after 30 minutes. In another experiment a 1,500
gramme rabbit died 2½ hours after 8 grains, and a rabbit of the same weight showed
practically no symptoms after 6 grains.
Six grains per kilo. is a lethal dose for a rabbit when given hypodermically,
and in many cases 5 grs. per kilo. would suffice. Three grains per kilo. produces
symptoms of excitement without ever proving fatal.
When a lethal dose is given to a rabbit, symptoms set in after 10 or 15 minutes.
These symptoms always commence by quickened respiration, and, soon after, some
of the animals develop muscular twitchings and spasms, but not so marked as in
the case of guinea pigs. Before death they lie helpless for a variable period, and
die asphyxiated, the heart continuing to beat for some minutes after respiration
has ceased.
The local action on the tissues is the same as in guinea pigs, and when a non
-lethal dose is given a slough invariably forms, but much earlier than in the case of
guinea pigs.