NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
31
the material was obtained by heating a 0.1 per cent. solution, the filtrate which
had to be evaporated measured more than 90 c. c. Although in the latter case it
was divided between two platinum basins, the time occupied in the evaporation
process, which was done at 60° C., was very much longer than in the former
instance, namely 5 times longer.
We have here a possible explanation of the difference in the toxic power
of the two residues left after evaporation, namely, that the prolonged exposure
of the weaker filtrate to a temperature of 6o° C. had the effect of destroying
the toxicity of the venom, which toxicity, although weakened, had not been
completely destroyed by the initial heating at 75° C. for half an hour.
The two experiments, detailed below, distinctly show that the destruction
of the power of causing intravascular clotting took place before the evaporation
process was begun.
A solution of daboia venom of 0.1 per cent. strength was heated for half an
hour at 73° to 75° C. It was then carefully filtered. The filtrate was at once in-
jected intravenously into two rabbits.
Rabbit 18.—Weight 1,570 grammes. Received into marginal vein of ear
2 milligrammes per kilo. of heated poison.
No symptoms followed.
Rabbit 19.—Weight 1,650 grammes. Received into marginal vein of ear 4
milligrammes per kilo. of heated poison.
No symptoms followed.
This last rabbit, therefore, received an amount of venom, heated in 0.1 per
cent. solution, 40 times greater than the amount of unheated venom and 4 times
greater than the amount of venom heated in 1 per cent. solution, which can
produce extensive intravascular clotting.
It is evident, then, that the degree of concentration of the solution plays an
important part in determining whether the toxic power of daboia venom is com-
pletely destroyed by heat or not.
Now, it would have been expected that similar conclusions would have
been justified from the experiments which bear on the effect which heating
solutions of different concentration has on the substance which brings about the
deficiency in the blood coagulability. For it has been put forward by Martin of
Melbourne2 2
, that in all probability the substance which causes the intravas-
cular clotting and the substance which brings about the deficiency in the blood
coagulability are one and the same substance, as there is a decided parallelism
in the phenomena seen after the intravenous injection of viperine venom and of
Wooldridge's nucleo-proteids. While, on the one hand, however, our experi-
ments (Table VIII) show that there can be no possible doubt that heating a
solution of 0.1 per cent. strength completely destroys the power which daboia
32
venom has to produce a deficiency in the blood coagulability, on the other
hand, a closer study of the experiments detailed in series 3 (Table VII) leaves
a doubt in our minds as to whether or not a like result follows the heating of
a 1 p e r c e n t . s o l u t i o n . T h u s , w e h a v e s e e n t h a t m o n k e y s 1 7
a n d
1 8
s u f f e r e d
from a considerable œdema around the site of inoculation, and that on the
second day after the injection a slight diminution in the blood coagulation time
of about 1 mi nut e was not ed. Fur t her ,m o n k e y 1 9 ,
t h e a n i m a l w h i c h r e c e i v e d
the largest amount of the heated venom, died within 18 hours. In this case no
diminution in the blood coagulability was noted 12 hours before death, but no
estimation had been taken during those 12 hours. The fact that monkeys
17 and 18 did not show a marked diminution of blood coagulability is probably
due to an insufficient dose having been given. The fatal result in the case of
monkey 19 probably took place from embolism of the pulmonary arteries, the
emboli having their origin in thrombi in the right heart which were not of suffi-
cient size to cause rapid death, a case comparable, in fact, to that of rabbit 11.
On the whole, then, these experiments appear to point to the conclusion
that, while no great and appreciable diminution in the blood coagulability is
brought about by the substances which are left in solution after heating a
1 per cent, solution of daboia venom for half an hour at 73° C., still these substances
have not been completely destroyed in the same manner as when the solution
heated is of 0.1 per cent. strength. If such complete destruction did take place,
the only conclusion to be drawn would be that the substance which causes the
intravascular thrombosis is a different substance from that which brings about
the deficiency in the blood coagulability; and that, while the former is not
destroyed when a 1 per cent. solution of venom is heated for half an hour at
73° C., the latter is destroyed when heated in this way. Without further and
more delicate experimentation, however, which is at present in progress in this
laboratory, it is evident that it would be premature to form any conclusion.
When it is considered that heating a 0.1 per cent. solution of daboia
venom in the manner we employed both coagulates proteid substances and com-
pletely destroys the power of the venom to produce intravascular clotting, it is
natural to conclude that the substance which brings about the intravascular
clotting is a proteid coagulable by heat, and that the loss of this toxic element
is due to its being thrown out of solution by the heat. Now, we have seen that
the amount of proteid coagulated by heating a 1 per cent. solution for half an
hour at 73° C. is equal at least to the amount coagulated in a similar manner
from a 0.1 per cent. solution. In our experiments, in fact, a slightly greater
33
quantity of coagulated proteid was obtained by heat from a 1 per cent. solution
than from a 0.1 per cent. solution. Further, we have seen that the incoagulable
resi due got from t he 1 per cent . sol ut i on st i l l ret ai ns t he power of produci ng
intravascular clotting, while the similar residue got from the 0.1 per cent.
solution has completely lost this power. We can, therefore, only conclude that
the constituent of daboia venom which acts on the blood coagulability, what-
ever it may be, is not a proteid which is coagulated at 73° C., although
this temperature applied for half an hour can, when the solution is a dilute one,
viz., 0.1 per cent., completely destroy this toxic element.
Does daboia venom contain any toxic element similar to that
of cobra venom ?
We have seen above that one of the principal physiological actions of daboia
venom is its action on the blood, especially on the blood coagulability. We
may now, therefore, pass on to consider some experiments which were made
with a view of directly ascertaining if this venom contained any of the element
to which cobra poison owes its toxic power on the central nervous system.
It has been shown by Calmette 23 and other observers that cobra venom
when heated for half an hour at 73° C. still retains its toxic power, and that the
lethal dose of this heated venom is only slightly less than the lethal dose
of the original unheated venom. Now, experiments with daboia venom heated
in the same way (vide Table VI) show that 12 milligrammes per kilo. of such a
heated venom injected intravenously produce no symptoms in rabbits. When we
consider that 0.35 milligramme per kilo. of heated cobra poison, 24 a quantity
34 times less than this amount of heated daboia venom, is sufficient to kill a
rabbit, this observation alone would almost entitle us to conclude that daboia
venom contained no toxic albumose similar to that present in cobra poison,
which acts on the central nervous system.
With a view to definitely settle this question, two parallel series of experi-
ments were made with cobra and daboia venoms.
A 0.1 per cent, solution of each venom in distilled water was prepared.
These solutions were then heated in a water bath for half an hour at 73° C. A
copious coagulum was obtained in each case. This was carefully filtered off.
The filtrates were then evaporated to dryness over a water bath at 60° C. The
residue in each case was collected and thoroughly dried over lime. A 0.1 per
cent. solution was made, which, when necessary, was diluted tenfold. All injec-
tions were made subcutaneously in the inner side of the thighs of rats which
were carefully selected of about the same weight, i.e., 120 grammes. The
weights of the venom refer to the residue left after evaporation of the filtrate. E
The results were as follow :—
TABLE IX.
Experiments with residue of heated cobra poison.
Animal.
Amount of residue of filtrate
in millig rammes.
Result.
Rat 1
.
.
0 . 3
Died in 2 hours.
Rat 2 .
.
0.2
Died in 1½ hours.
Rat 3
0 . 1
Died in 3 hours.
Rat 4
. . .
0.09
D i e d i n 3 ½ h o u r s .
Rat 5 ..
0 . 0 8
Died in 2½ hours.
Rat 6 .
.
.
0 . 0 7
Died in 6½ hours.
Rat 7 .
.
0 . 0 6
Very ill : recovered.
Rat 8 .
.
0 . 0 5
Very ill : recovered.
Rat 9 . .
0.04
Died in 4 days.
It would appear, then, that 0.07 milligramme of this material is a certain
lethal dose for a rat.
TABLE X.
Experiments with residue of heated daboia poison.
A n i m a l .
Amount of residue of
filtrate in milligrammes.
Result.
Rat 1 .
.
1
No symptoms.
Ra t 2 .
2
No symptoms.
Rat 3
.
.
3
No symptoms.
Rat 4
.
.
0
N o s y m p t o m s .
Rat 5 .
.
10
No symptoms.
We have, therefore, shown that while 0.07 milligramme of the residue got from
the filtrate after heating cobra venom for half an hour at 73° C. is lethal for a rat,
such a large quantity as 10 milligrammes of the residue obtained in a similar man-
ner from daboia venom, viz., a quantity 150 times greater than the cobra venom
residue, produces no symptoms. This fact, taken in conjunction with the similar
observation made on the rabbit, proves conclusively that daboia venom is free
from, or contains only the merest trace of the principal toxic constituent of
cobra venom, a substance which is generally recognised to be a direct nerve
poison, acting as it does on the central nervous system.
On the other hand, it is more difficult to demonstrate that cobra venom
contains none of the substance present in daboia poison which causes the
35
intravascular clotting. It will be sufficient to detail a single experiment, among
many made by us, which would seem to point towards this conclusion.
Rabbit 20—Weight 1,500 grammes. Received intravenously 4.5 milli-
grammes of cobra venom. It died immediately the injection was completed.
A post-mortem examination made at once showed the heart to be beating vigor
ously. The blood throughout was quite liquid, and not a trace of clot could be
found. The blood when shed clotted solidly.
Now this rabbit received amount of cobra venom 45 times greater
than the quantity of daboia venom which can cause rapid death from extensive
intravascular clotting ( vide, Table III), and still not a sign of clot was observed
on careful post-mortem examination.
Although it has been shown by many observers, Cunningham, 2 5
K a n t h a c k ,
2 6
Stephens, 27 etc., that cobra venom " in vitro" has a considerable haemolytic
action, as well as the power to cause a deficiency in the blood coagulability, this
experiment,—an experiment which we repeated many times with the same
result—would seem to show that this venom contains no element similar to the
constituent of daboia poison which can cause intravascular clotting, which, as we
have seen, is such a remarkable result of the injection of that poison.
On this complicated and important question, however, experiments are at
present being carried on by one of us, the result of which will form the subject
of another paper.
Neutralising power of Calmette's serum tested with daboia venom.
We have now, in conclusion, to present a few experiments made with the
object of directly controlling. Calmette's statement, that his antivenomous serum
was protective against the venom of all snakes. 2 8
I n a p r e v i o u s p a p e r
2 9
w e
have shown that this serum possesses a considerable power of neutralising pure
cobra venom. We now tested it with pure daboia poison. When we consider
the method by which Calmette immunises his horses, along with the observa-
tions which we have detailed above, we would expect from à priori reasons to
find that the serum which is supplied from Lille would have little or no power
to neutralise pure daboia venom. For the venom which Calmette employs to
immunise his horses is a mixture of colubrine and viperine poisons, (80 per
cent. of the former), and the solution of this mixture is heated before injection for
half an hour at about 73° C. Such treatment, we have seen, destroys or greatly
weakens, according to the strength of solution heated, the principal element
to which daboia venom owes its toxicity, viz., the substance or substances
which cause intravascular coagulation or deficiency in the blood coagulability.
He is, therefore, injecting into his horses a poison which contains either none, or
very little, of the toxin for which he wishes to.procure an antitoxin, and we would
E2
36
therefore not expect to find such an antitoxin in the serum obtained from
these horses.
The following experiments were made with a view of directly testing
this hypothesis. It is to be regretted that these observations were not more
extensive, the reason for this being that the stock of fresh serum available
became exhausted.
Rabbits were the animals employed for our observations. We have seen
above (Table III) that 0.1 milligramme per kilo. of dried venom injected intra-
venously into a rabbit is the minimum dose, which will cause rapid death from
intravascular thrombosis. In the first four experiments now to be recorded, the
test dose we employed was ten times this amount, i e., 1 m i l l i g r a m m e p e r k i l o .
In a single experiment the test dose used was only two lethal doses, i e . ,
0 . 2
milligramme per kilo.
The dried venom was dissolved in normal saline solution, the strength of
the solution being 0.2 per cent. The test dose was then mixed " in vitro" with
varying amounts of serum. The mixture was allowed to stand at laboratory
temperature (about 25° C.) for half an hour. All injections were made into the
marginal vein of the ear. The serum was comparatively fresh serum, as it bore
the date 17th September 1900, and our experiments were made on the 27th
January 1901. It was obtained directly from M. Calmette, whom we take this
opportunity to thank for his courtesy in placing it at our disposal.
The results are tabulated as follows:—
TABLE XI.
Experiments to ascertain the neutralising power of Calmette's anti-
venomous serum when tested with pure daboia venom.
A n i m a l .
Amount of dried venom
in milligrammes per kilo.
of body weight.
Amount of serum
in cubic
centimetres.
Results.
Rabbit 21 .
.
.
1
1
Convulsions and death in 2 minutes.
Solid clotting throughout.
Rabbit 22.
.
1
2
Convulsions and death in 2 minutes
Solid clotting throughout.
Rabbit 23 .
. .
1
3
Convulsions and death in 1½ minutes.
Solid clotting throughout.
Rabbit 24 .
.
1
4
Convulsions and death in 1½ minutes.
Solid clotting throughout.
Rabbit 25 .
.
.
0.2 (two lethal
doses).
4
Convulsions and death in 1½ minutes.
Solid clotting throughout.
Rabbit 26 (Control)
.
0.2
Nil.
Convulsions and death in 2 minutes.
Solid clotting throughout.
We can, therefore, say that 4c.c . of Calmette's serum cannot neutralise
even such a small quantity of daboia venom as 0.2—0.075 milligramme = 0.125
37
milligramme, taking 0.075 milligramme as a maximum non-lethal dose for a
r a b b i t o f 1 k i l o . we i g h t ( T a b l e I I I ) . T h i s r e s u l t i s n o t e wo r t h y wh e n we
compare it with the result obtained with the same serum in the case of
pure cobra venom. For we have shown in a recent publication, 30t h a t 1 c . c . 0
this same sample of serum was able to neutralise 0.73 milligramme of cobra
venom, while these observations show that 1c.c. could not neutralise even
0.031 milligramme of daboia venom.
Our observations extend and confirm the single observation made by
Stephens,3 1
w h o s h o w e d t h a t C a l m e t t e ' s s e r u m h a d l i t t l e e f f e c t i n d e l a y i n g d e a t h
in guinea pigs when only a single lethal dose of daboia venom was used as a test
dose.
It would appear, therefore, justifiable to conclude, considering that daboia
venom contains none of the principal toxic albumose of cobra poison, that this
serum prepared by Calmette is of no therapeutic value in cases of bites from
Russell's viper.
Martin and Cherry,3 2
w o r k i n g w i t h t h e p o i s o n o fH
o p l o c e p h a l u s c u r t u s ,
showed that 1.5 c.c. of Calmette's serum was able to neutralise a test dose
consisting of 8 lethal doses for a rabbit, which Semple and one of us 33 calculated
was equivalent to showing that 1 c.c. was able to neutralise something be-
tween 0.12 and 0.18 milligrammes of this poison. In these experiments, how-
ever, the venom against which the serum was tested had been previously
heated, so as to remove the coagulable proteids. This heating, aslwe have
seen, must have at least greatly diminished, if it had not destroyed, the viperine
element of the poison. This result, therefore, when compared with our observ-
ations noted above, would point to the fact, that the poison of the Hoplocephalus
curtus is, as stated by Martin, not a pure viperine poison, but a mixture of
colubrine and viperine elements.
We may sum up the conclusions, which the observations detailed in this
paper would appear to justify, as follow :
—
1. Daboia venom owes its toxic property principally to its action on the
blood; it causes rapid death from extensive intravascular clotting, while in the
chronic cases a marked deficiency of the blood coagulability is observed.
To this deficiency are probably due the hæmorrhages and œdema, which are
such prominent symptoms in these cases.
2. Heating a weak solution of daboia venom, i.e., 0.1 per cent. solution, for
half an hour at 73°C. completely destroys its toxicity. When the solution is
more concentrated, i.e., 1 per cent., the power of causing intravascular throm-
bosis is not destroyed but only lessened.
3. Daboia venom contains no toxic element which has an action similar to
that of the toxic albumose, which is the chief toxic constituent of cobra
venom and which acts directly on the central nervous system. On the other
hand, cobra venom contains no substance which can cause intravascular
thrombosis.
4. Calmette's serum has no power to neutralise the toxin of daboia
venom..
References.
1. Smithsonian Contributions to Knowledge; Vols. XII and XXVI.
2. Proc. Roy. Soc., N.-S.-W., 1892, 1893 and 1895.
3. " Indian Snake-poisons : their nature and effect." W. H. Allen and Co.,
London, 1883.
4. Scientific Memoirs by Medical Officers of the Army of India : Part IX,
1895, and Part XI, 1898.
5. Scientific Memoirs by Medical Officers of the Army of India: Part XI,
1898.
6. Journal of Pathology and Bacteriology. Vol. V, p. 279. Loc. Cit., Vol.
VI, p. 273.
7. Journal of Pathology and Bacteriology. Vol. V, p. 279: Loc. Cit., Vol.
VI, p. 414.
8. Ann. de l'Inst. Pasteur, 1893,1894, 1897. Calmette, " Le venin des
Serpents", 1895.
9. Brit. Med. Journal, 1895, p. 1310, and 1896, p. 957.
10. Proc. Roy. Soc., Vols. XXI, XXII and XXIII.
11. " Notice sur le sérum antivenimeux et sur le traitment des morsures
de serpents," Lille, 1901.
12. Loc. Cit.
13. Clifford Allbutt's " System of Medicine," Vol. II, p. 809.
14. Loc. Cit.
15. Loc. Cit.
16. Clifford Allbutt's " System of Medicine," Vol. VI, p. 261.
17. Scientific Memoirs by Medical Officers of the Army of India : Part IX,
1895.
18. Brit. Med. Journal, July 29th, 1893, February 3rd, 1894. Lancet,
August 1st, 1898.
19. Brit. Med. Journal, July 14th, 1894 : Lancet, January 1st, 1896: January
3oth, 1897 : september 19th, 1896.
20. Clifford Allbutt's " System of Medicine", Vol. II, p. 809.
21. Clifford Allbutt's " System of Medicine", Vol. II, p. 809.
22. Cifford Allbutt's " System of Medicine", Vol. II, p. 809.
33. Loc. Cit.
24. Brit. Med. Journal, April 1st, 1899, p. 781.
25. Loc. Cit.
39
26. Loc. Cit.
27. Loc. Cit.
28. "Notice sur le sérum antivenimeux et sur le traitment des morsures
de serpents," Lille, 1901.
29. Lancet, June 15th, 1901.
30. Loc. Cit.
31. Journal of Pathology and Bacteriology, Vol. VI, p. 273.
32. Brit. Med. Journal, 1898, p. 1120.
33. Brit. Med. Journal, 1899, p. 781.
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