NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
5
from the symptoms seen in experiments with cobra venom. Rogers 8 has shown
that death is due to respiratory paralysis of central origin as in the case of cobra
venom intoxication.
The minimum lethal dose for rabbits by intravenous injection was found to be
0.04 milligramme per kilo. The test dose now used to ascertain if either C. V.
serum or H. C. V. serum had any antitoxic effect for this venom was ten lethal
doses, namely, 0.4 milligramme per kilo.
It will be seen from the protocols (Table IV) that the rabbit which received
4 c.c. of either serum along with the test dose of poison died as quickly as the
control animal. It is, therefore, evident that neither C. V. serum nor H. C. V.
serum has any neutralising effect for the general action in vivo of the venom of
Bungarus cœruleus.
BUNGARUS FASCIATUS (BANDED KRAIT).—In a recent publication 9 I gave
a detailed account of the physiological actions of the venom of this species. It
was there shown that cases of intoxication with this poison can be divided into
three classes :
—
(a) Cases in which rapid death, due to intravascular thrombosis, follows
intravenous injection of large quantities of venom.
(b) Cases which present acute nervous symptoms and which terminate
fatally within two or three days after the injection of the poison. These
cases are indistinguishable, as far as symptoms are concerned, from
cases of cobra venom intoxication.
(c) Cases which run a chronic course and end fatally between the 6th and
12th day after the injection of the poison. Such cases are peculiar
to intoxication with this venom and present marked special symptoms.
They in no way resemble cases of chronic daboia poisoning. A his-
tological examination of the nervous system in these cases shows a
well marked primary degeneration of the cells of the central nervous
system.
Further, it was demonstrated in the paper cited that neither Calmette's
serum nor H. C. V. serum had any neutralising effect for this poison. We have,
therefore, now only to consider an observation made with C. V. serum and
Bungarus fasciatus venom.
This observation consisted in testing 4 c.c. of serum against three lethal
doses of venom by intravenous injection. A reference to Table V of the protocols
will at once show that the animal which received this mixture died after practically
the same interval of time as the control rabbit. We can, therefore, conclude that
C. V. serum has no antitoxic effect for the action of Bungarus fasciatus in vivo.
HOPLOCEPHALUS CURTUS (AUSTRALIAN TIGER SNAKE).—Tidswell 1 0 h a s
carefully tested the serum which he has prepared with the venom of this species
against the corresponding poison and has shown that 0.4 c.c. of serum was
6
sufficient to neutralise ten lethal doses for the rabbit, namely, 0.59 milligramme.
In the same paper he showed that Calmette's serum was quite inactive against the
venom of Hoplocephalus curtus. I have forwarded to Dr. Tidswell a supply of
the C. V. serum I have prepared, so that he may test it carefully against the
poisons of the various species of the poisonous snakes of Australia. The stock of
Hoplocephalus poison which I possess is so small that I am unable to undertake
any experiments in this direction. As, however, Calmette's serum has no
neutralising effect for this venom we may almost conclude from à priori reasons
that the pure C. V. serum will also be inactive.
ENHYDRINA VALAKADIEN (COMMON SEA SNAKE).—The physiological
action of the venom of this and other species of sea snake has been the subject of
recent investigation by Rogers.11 The symptoms produced by this poison may be
said to be identical with those seen in cobra venom intoxication. Death is due to
respiratory paralysis, the primary action of the poison being on the respiratory
centre in the medulla. Rogers also found post-mortem paralysis of the motor end
plates especially affecting the phrenic nerves. The toxicity of the poison of this
species is very much greater than the toxicity of cobra venom. As a preliminary
measure to testing the two antivenomous sera the minimum lethal dose of the
venom for rabbits by subcutaneous injection was determined. This was found
to be about 0.05 milligramme per kilo., a quantity about seven times less than
the corresponding minimum lethal dose of cobra venom.
The test dose now employed in both series of experiments was ten lethal
doses, namely, 0.5 milligramme per kilo.
The results obtained with C. V. serum are detailed in Table VI of the proto-
cols. From this table it will be seen that 4 c.c. of serum delayed death for a
short time; that 10 c.c. of serum had a still more marked effect in this direction,
and that 15 c.c. of serum prevented the fatal result. It would appear, therefore,
that a serum prepared with unmixed pure cobra venom has a certain neutralising
effect for the venom of one of the sea snakes, as far as its action in vivo is
concerned. This neutralising effect is, however, very slight. For it will be
remembered that 1 c.c. of C. V. serum neutralised 1.5 milligrammes of cobra
poison. In the case of the venom of Enhydrina valakadien we can calculate
that 1 c.c. of the same serum neutralised only 0.031 milligramme, an amount 50
times less than the quantity of cobra venom which was neutralised by the same
amount of serum. Allowing even for the greater toxicity of the sea snake venom,
it is evident that C.V. serum is far more active for its corresponding venom than
for that of Enhydrina valakadien. Further, although the amount of poison
injected by this snake is small, estimated by Rogers at about 10 milligrammes,
we can say that for therapeutic purposes cobra venom immune serum would be
of little or no value in cases of bites from Enhydrina valakadien.
The observations which Rogers has published with reference to the action of
7
Calmette's serum against the venom of Enhydrina valakadien are somewhat difficult
to comprehend. Thus, in one publication, 12 he states, that using only minimum
and slightly supra-minimal lethal doses of the poison he found that the serum
animals died in just about the same time as the controls. He, therefore, concluded
that Calmette's serum is of no use against this poison. Again, in a lecture
1 3
delivered a month later than this publication, he states, that in a series of experi-
ments in which ten lethal doses were employed Calmette's antivenomous serum
was found to have a certain neutralising effect for this poison, as it had also for
the venom of the king cobra. It is difficult to reconcile these two statements,
and as no details of the experiments performed are given in either instance it
is impossible to sift them critically with a view to ascertain where the error lies.
A single experiment was sufficient to show that H. C. V. serum has no
antitoxic action for the venom of Enhydrina valakadien. In this experiment the
animal which received 4 c.c. of serum along with the test dose, namely, ten lethal
doses, died even more quickly than the control animal ( vide Table VII of
protocols).
This experiment completes the observations made in vivo with the antiven-
omous sera and those venoms of the colubridæ which were available. We may
sum up the results as follows:—
(1) The serum of a horse immunised with pure cobra venom is strongly
antitoxic for the venom used in its preparation : when used in large quantity it has
a slight neutralising power for the venom of Enhydrina valakadien, one of the com-
mon sea snakes : further, it delays death in cases of intoxication with the venom
of the king cobra, a species belonging to the same genus as the cobra : it does
not, however, completely neutralise this poison even when used in large quantities.
The antitoxic effect against these two venoms is so slight that this serum would
be of little or no therapeutic use in cases of bites from these snakes. Finally, this
serum contains no antitoxic substances which are active against the venom of
either Bungarus cœruleus or Bungarus fasciatus.
(2) The serum of a horse immunised with the pure venom of Hoploce-
phalus curtus (Australian tiger snake) is strongly antitoxic for its corresponding
poison (Tidswell). It has no neutralising power for the poisons of any of the
Indian colubrine snakes against which it was tested, namely, Naia tripudians, Naia
bungarus, Bungarus cœruleus, and. Bungarus fasciatus, nor for the venom of a
common sea snake, Enhydrina valakadien.
We may now pass on to the consideration of some observations which were
made in vivo with the two antivenomous sera and the poisons of four species of
Viperidæ, three Indian and one American.
VIPERA RUSSELLII (DABOIA).—I have already shown 14 that, when this
venom is injected intravenously into an animal in sufficient quantity, rapid death,
the result of intravascular thrombosis, takes place. Further, I have also definitely
proved15 that neither Calmette's serum nor the serum prepared with the venom of
9
0.1 milligramme per kilo. of weight. It will be seen from the protocols (Table X)
that neither serum had any antitoxic effect as far as this action of the poison was
concerned.
In the second place, the sera were tested against the action of the venom on
the vaso-motor centre in the same way as has been described in the case of
daboia poison. A small preliminary non-lethal dose of poison was injected into
the marginal vein of one ear. Half an hour afterwards the mixture of serum
(4 c.c.) and venom (0.5 milligramme per kilo., an amount not exceeding 10 lethal
doses) was injected into the marginal vein of the other ear. This mixture had
been standing in vitro at laboratory temperature for half an hour. A reference to
the protocols (Table XI) will at once show that neither serum had any effect in
hindering this action. We can, therefore, conclude from these two series of
observations that neither C. V. serum nor H. C. V. serum has any antitoxic effect
for the general action in vivo of the venom of Echis carinata.
TRIMERESURUS GRAMINEUS (GREEN PIT VIPER).—I have not been able to
obtain sufficient venom from this small species of the rattlesnake family to enable
me to work out its physiological action in any detail. It was, however, ascer-
tained that fairly rapid death took place after an intravenous injection into a rabbit
of 2 milligrammes per kilo., and that no intravascular clotting could be demon-
strated. As this poison is able to coagulate citrate plasma in vitro, there is no doubt
a larger amount would have caused intravascular thrombosis. A dose of 1 milli-
gramme per kilo. failed to produce any symptoms.
The test dose now used for the purpose of ascertaining if either C. V. serum
or H. C. V. serum had any neutralising effect against the general action in vivo
of this poison was 2 milligrammes per kilo. In each instance 3 c.c. of serum
were used. It was found ( vide Table XII of protocols) that neither serum had any
antitoxic effect. The serum animals died after practically the same interval of time
as the control. In view of the fact that less than two lethal doses was used as the
test dose, we can conclude in general terms that neither serum had any neutralising
effect on the general action in vivo of the venom of Trimeresurus gramineus.
CROTALUS ADAMANTEUS (CALIFORNIAN RATTLESNAKE).—The mini-
mum lethal dose of this venom for a rabbit by intravenous injection was found to be
0.025 milligramme per kilo. An early symptom in all animals which received
more than this amount was hæmorrhage from the bowels. The animals which
lived long enough became very thin, emaciated and paralysed before death.
We have evidently, therefore, in these cases to deal both with a hæmorrhagic action
and with an action on the nervous system. In no experiment did I use a sufficient
quantity of poison to cause intravascular thrombosis, although this phenomenon is
well known to occur when a sufficient amount of venom is injected directly into
the circulation.
The amount of venom used to test the sera now under experiment was 0.5
C
10
milligramme per kilo. injected intravenously, namely, two lethal doses. In each
instance 4 c.c. of serum were tested against this dose. It will be seen from
the protocols (Table XIII) that the serum animals died in even a shorter time
than the control rabbit. We must, therefore, conclude that both C. V. serum and
H. C. V. serum are quite inactive against the general action in vivo of the venom
of Crotalus adamanteus.
These experiments complete the observations which were made with the two
antivenomous sera and those venoms of the Viperidæ which were available, as
far as the general actions of the poisons in vivo are concerned. We have seen
that both sera were tested against four venoms of this description and were found
in every instance to be quite inactive. It is unnecessary to comment further on
these results.
We may now pass on to the second part of our subject, that is, the considera-
tion of some observations which were made with the object of determining if the
antivenomous sera in question had any hindering effect on the hæmolytic action
in vitro of any of the venoms mentioned above.
It has been well known for many years that snake venoms possess a
marked power of breaking up living cells in general and the red blood corpuscles
in particular. This hæmolytic action can be demonstrated in most instances both
in vivo and in vitro. Quite recently, however, the work of Flexner and Noguchi 1 7
in America and of Preston Keyes and Sachs 18in Ehrlich's laboratory has
thrown a flood of light on the intimate mechanism of this particular action of snake
poisons. It is not my intention in the present communication to enter into any
details as regards this mechanism. It is sufficient for our purpose to mention the
fact that it has now been shown that venoms act only as amboceptors, and that
they require, in order to bring the red cells to solution, a complement of some
kind or other. This complement, as has been shown by Keyes and Sachs
in the case of cobra poison, may be either a serum complement or lecithin,
which is normally present in the red cells themselves and in serum. I have been
able to confirm these observations with cobra poison and have also extended them
with the other poisons mentioned above. I found that all these venoms act only
as amboceptors requiring complement to bring about the hæmolysis, but that in
the case of some of the venoms lecithin, either free or as a constituent of the red
cells, cannot take the place of serum complement. These observations, however,
will form the subject of another communication. For our present purpose it is
sufficient to recognise the facts, that complement is necessary for the hæmolytic
action of all venoms, and that lecithin cannot act as complement in the case of
some poisons. Therefore, to make the experiments now to be recorded uniform
throughout serum complement was used in all cases.
The red cells of the dog, as being very susceptible to hæmolysis by all
venoms, were chosen for the present observations. The blood was first gently
11
defibrinated : the red cells were then washed several times with salt solution
(0.85 per cent.), being centrifugalised between each washing. A 5 per cent.
suspension of the washed red cells was then made in sterile 0.85 per cent. salt
solution. It was found that these manipulations completely removed all serum
complement.
As a preliminary measure, it was necessary to determine the minimum com-
plete, or nearly complete, hæmolysing dose of each venom for 1 c.c. of the 5 per
cent. red cell suspension, when a fixed amount of serum complement was added.
The complement used was that of the dog, and the amount employed was 0.5 c.c.
of a two-fold dilution of fresh serum. Each tube thus contained—(1) Varying
amounts of venom dissolved in 0.85 per cent. salt solution : (2) 1 c.c. of a 5 per
cent. suspension of dog's washed red cells : (3) 0.5 c.c. of a two-fold dilution
of dog's fresh serum. The tubes were placed in the incubator (35º C) for one
hour and then in the ice chest over night. The results were then recorded.
Working in this way the hæmolysing value of each poison was accurately deter-
mined, and it was easy to fix the minimum amount of venom which could bring
about complete, or almost complete, hæmolysis of a fixed quantity of red cells.
This was the amount which was used as the test dose to determine whether
either C. V. serum or H. C. V. serum had any hindering effect on the hæmolytic
actions of the various poisons. The technique employed was as follows:
—
The test dose of poison dissolved in 0.85 per cent. salt solution was mixed
with varying amounts of the sera, and the mixtures were allowed to stand at
laboratory temperature for at least half an hour. To each tube were then added
1 c.c. of a 5 per cent. suspension of washed dog's red cells and 0.5 c.c. of a
two-fold dilution of fresh dog's serum. The preparations were then treated as
described above.
In the first place, it was determined that normal horse's serum had no
hindering effect on the hæmolysing action of any of the venoms. These
experiments need not be detailed.
In the second place, C. V. serum was tested in the manner described above.
A reference to the protocols (Table XIV) will show the results which were ob-
tained. It will be seen from this table that C. V. serum had a high neutralising
effect for cobra venom itself. The preparation containing 0.04 c.c. of serum
showed no hæmolysis, while the tube with 0.02 c.c. showed only a mere trace
of laking. We can, therefore, calculate that about 0.02 c.c. of serum was
able to neutralise almost completely the hæmolytic action of 0.025 milligramme
of cobra venom, the test dose used for these experiments. I have made a
number of similar experiments, using larger amounts of poison as a test dose, and
have always obtained results corresponding to that just mentioned. Further, it
will be seen from this table that C. V. serum prevented to a certain extent the
hæmolytic action of the venom of Bungarus cœruleus. A much greater quantity
C 2
12
of serum, however, was required to effect this than in the case of cobra venom.
Thus, it required 0.6 c.c. of serum to completely neutralise 0.025 milligramme
of this poison, an amount of serum 30 times as much as was necessary for the
same quantity of cobra venom. It is to be remembered also that in each instance
only a minimum complete hæmolysing amount of venum was used. Finally
it will be seen from this table, that C. V. serum had no hindering effect at all
on the hæmolysing action of the remaining eight venoms, the tubes containing
1 c.c. of serum showing as complete hæmolysis as the controls.
We may conclude from these observations that the specificity of C. V. serum
as regards this particular action of snake venoms is well marked, but not quite
absolute. The point of greatest interest and importance is that a serum prepared
with cobra venom has absolutely no hindering effect on the hæmolysing action
of the venom of the king cobra, a species, as I have mentioned, belonging to
the same genus as the cobra.
In the third place, H. C. V. serum was tested against the hæmolytic action
of each poison in exactly the same manner as has been described above. It
will be seen from the protocols (Table XV) that this serum was not so specific
in its action as C. V. serum Thus, it will be noted that it neutralised the venom
of Enhydrina valakadien rather better than it neutralised the poison with which it
had been prepared. Also, it will be seen that it had a marked hindering effect
on the action of cobra venom and on the action of the poison of Echis carinata.
It had no hindering effect on the hæmolysing action of the other six poisons
against which it was tested.
The results of these two series of observations appear to me to be of the
greatest importance. Thus, it is evident that the hæmolysing elements of venoms
differ from one another as markedly as do the elements which act on the central
nervous system. It cannot even be affirmed that this constituent of the poisons
of two species of the same genus are alike. For we have seen that a serum
prepared with cobra venom is very active against this poison but has no hinder-
ing effect on the hæmolytic action of the poison of the king cobra. In other words,
the hæmolysing amboceptors of these two venoms possess different haptophore
groups. On the other hand, the red cell receptor of the venom of a colubrine snake
may resemble that of the poison of a viperine species. For we have seen that the
serum prepared with the venom of Hoplocephalus curtus is active to a certain
extent for the poison of Echis carinata. We can, however, conclude that the
constituent of any one snake venom which acts as an amboceptor for the red
blood corpuscles differs, for the most part, from the similar constituent of any other
venom in not possessing exactly the same haptophore groups. There are, as we
have seen, exceptions to this rule, but no à p r i o r i r e s e m b l a n c e c a n b e t a k e n f o r
granted in venoms of species belonging to the same genus.
We can now pass on to the third and concluding part of the present inves-
13
tigation, that is, to the consideration of some observations which were made with
the view of ascertaining if either C. V. serum or H. C. V. serum had any hinder-
ing effect on the actions of the different poisons on the blood coagulability in
vitro. It will be convenient in this instance to consider these observations in
two groups, namely, (1) the observations made with the venoms which prevent
the clotting of citrate plasma which results on the addition of a soluble salt of
lime, and (2) the observations made with the venoms which rapidly clot citrate
plasma without the addition of any lime salt.
In the first group of poisons are the venoms of the cobra and of the king
cobra. In a previous communication19I showed that cobra poison had a
marked action on the coagulability of the blood plasma, and that this action
could be demonstrated in vitro with citrate plasma or oxalate plasma. If
cobra venom be added in suitable quantity to citrate plasma, the coagulation
which normally results from the addition of a suitable amount of a soluble
salt of lime is completely inhibited. If the quantity of poison added be too small
to completely prevent clotting, coagulation is delayed according to the amount
of venom added. In this paper I showed that 1 milligramme of cobra venom
was able to prevent completely the clotting of 2 c.c. of citrate plasma, which
normally resulted from the addition of a soluble salt of lime.
The poison of the king cobra has a similar action on the blood plasma as
that of cobra venom, that is to say, it inhibits or diminishes coagulability. This
action can also be demonstrated with citrate or oxalate plasma in vitro in the
manner I have already described. I found after a series of experiments that 0.8
milligramme of king cobra venom was the smallest amount which could com-
pletely inhibit the clotting of 2 c.c. of citrate plasma. The tube containing 0.6
milligramme of poison had a slight clot after standing twenty-four hours.
C. V. serum was now tested against this action of these two venoms. The
following was the method employed. The test dose of poison, namely, 1 milli-
gramme in each case, and varying amounts of serum were mixed in small test
tubes. The mixtures were allowed to stand at laboratory temperature for half
an hour. To each tube there was then added 2 c.c. of citrate plasma. The
preparations were left for two hours. Then there was run into each tube a quantity
of calcium chloride solution which clotted the control without venom in about
25 minutes. The results were noted at intervals. A reference to the protocols
(Table XVI) will show that C. V. serum was able to neutralise cobra venom,
as far as this action was concerned, but had little or no effect on king cobra
venom. Thus, the experiments show that 0.8 c.c. of serum completely,
and 0.6 c.c. nearly, neutralised 1 milligramme of cobra poison, and that
even 2 c.c. of serum were not able to neutralise the same amount of king
cobra venom. It is true that the tubes containing amounts of serum more
than 1.6 c.c. showed a trace of clot after 20 hours. There was evidently