“Alone unharmed of all who till the earthBy deadly serpents, dwells the Psyllian race.Potent as herbs their song; safe is their blood,Nor gives admission to the poison germE’en when the chant has ceased. Their home itselfPlaced in such venomous tract and serpent-throngedGained them this vantage, and a truce with death,Else could they not have lived. Such is their trustIn purity of blood, that newly bornEach babe they prove by test of deadly aspFor foreign lineage. So the bird of JoveTurns his new fledglings to the rising sun,And such as gaze upon the beams of dayWith eyes unwavering, for the use of heavenHe rears; but such as blink at Phœbus’ raysCasts from the nest. Thus of unmixed descentThe babe who, dreading not the serpent touch,Plays in his cradle with the deadly snake.”93
“Alone unharmed of all who till the earthBy deadly serpents, dwells the Psyllian race.Potent as herbs their song; safe is their blood,Nor gives admission to the poison germE’en when the chant has ceased. Their home itselfPlaced in such venomous tract and serpent-throngedGained them this vantage, and a truce with death,Else could they not have lived. Such is their trustIn purity of blood, that newly bornEach babe they prove by test of deadly aspFor foreign lineage. So the bird of JoveTurns his new fledglings to the rising sun,And such as gaze upon the beams of dayWith eyes unwavering, for the use of heavenHe rears; but such as blink at Phœbus’ raysCasts from the nest. Thus of unmixed descentThe babe who, dreading not the serpent touch,Plays in his cradle with the deadly snake.”93
“Alone unharmed of all who till the earthBy deadly serpents, dwells the Psyllian race.Potent as herbs their song; safe is their blood,Nor gives admission to the poison germE’en when the chant has ceased. Their home itselfPlaced in such venomous tract and serpent-throngedGained them this vantage, and a truce with death,Else could they not have lived. Such is their trustIn purity of blood, that newly bornEach babe they prove by test of deadly aspFor foreign lineage. So the bird of JoveTurns his new fledglings to the rising sun,And such as gaze upon the beams of dayWith eyes unwavering, for the use of heavenHe rears; but such as blink at Phœbus’ raysCasts from the nest. Thus of unmixed descentThe babe who, dreading not the serpent touch,Plays in his cradle with the deadly snake.”93
The only scientific conclusion to be drawn from the facts and statements that we have just set before the reader is that, under certain circumstances, man can unquestionably acquire the faculty of resisting intoxication by snake-venom, by conferring upon himself a veritableactive immunityby means of repeated inoculations of venom. We shall shortly see that the case is the same with regard to animals.
So long ago as the year 1887 it was shown by Sewall, in an important paper on “Rattlesnake-Venom,”94that it is possible to render pigeons gradually more resistant to the action of this venom by injecting them with doses at first very small, and certainly incapable of producing serious effects, and then with stronger and stronger doses. In this way, although these little animals are very sensitive, he succeeded in making them withstand doses ten times greater than the minimal lethal dose.
A little later Kaufmann95obtained the same result with the venom of French vipers. He did not, however, succeed in producing tolerance of doses more than two or three times greater than the lethal one.
In 1892, at the time of my first experiments with cobra-venom at Saigon,96I arrived at the conclusion that it was possible, bymeans of successive inoculations with heated venoms, to confer on animals a certain degree of resistance to doses invariably lethal to the controls.
From 1894 onwards, the investigations pursued simultaneously at the Paris Natural History Museum, by Phisalix and Bertrand, upon viper-venom, and at the Paris Pasteur Institute by myself, upon that of the cobra, and subsequently upon other venoms of various origins, led to much more definite results. These investigations show, on the one hand, that by vaccinating guinea-pigs or rabbits, and taking certain precautions, it is possible to confer upon these small animals a really strong immunity to venom; on the other hand, that animals vaccinated against cobra-venom are perfectly immune to doses of viper-venom or that of other snakes (Bungarus,Cerastes,Naja haje,Pseudechis) certainly lethal to the controls; and lastly, thatthe serum of the vaccinated animals contains antitoxic substances capable of transmitting the immunity to other animals.97
According to Phisalix and Bertrand, who, as we have stated, experimented only with viper-venom, the best method of vaccinating the guinea-pig consists in inoculating a dose of 0·4 milligramme of this venom heated for five minutes at 75° C., and, forty-eight hours afterwards, the same dose of non-heated venom. The latter is always lethal to the control guinea-pigs in from six to eight hours.
Vaccination against cobra-venom, which is much more toxic, is most surely effected by the method recommended by me, which consists in at first injecting small doses of this venom mixed with an equal quantity of a 1 per cent. solution of hypochlorite of lime. By degrees the quantity of venom is increased and that of the hypochlorite progressively diminished, and the injections arerepeated every three or four days, while attentively following the variations in the weight of the animals. The inoculations are suspended as soon as emaciation supervenes, and resumed when the weight becomes normal again. After four injections of chloridated venom the chloride is omitted, and a direct inoculation made with one-half the minimal lethal dose of pure venom; then, three or four days afterwards, the injection is increased to three-fourths of the minimal lethal dose; and finally, after the lapse of another three or four days, a lethal dose is injected.
If the animals prove resistant, the vaccination can thenceforth be pushed on rapidly, and the quantity of venom injected each time can be increased, testing the susceptibility of the organism by the variations in weight.
As a rule, three months are necessary for the vaccination of a rabbit against twenty lethal doses. In six months we can succeed in making it very easily withstand 100 lethal doses.
The serum of rabbits thus treated soon,i.e., after they have received from five to six lethal doses, exhibits antitoxic propertiesin vitro; these, however, are not very pronounced until after prolonged treatment. They gradually become just as intense as those observed in the case of animals vaccinated against diphtheria or tetanus.
In 1895 Fraser confirmed these results,98and on May 15 in that year exhibited before the Medico-Chirurgical Society of Edinburgh a rabbit vaccinated against a dose of cobra-venom fifty times lethal.
At once considering the possibility of obtaining serums highly antitoxic against snake-venoms, and of practical utility in the therapeutics of snake-bites, I prepared to vaccinate a certain number of large animals, horses and donkeys, in order to procure great quantities of active serum. I at first experienced some difficulties in providing myself with a sufficient store of venom. But thanks on the one hand to the obliging collaboration of some of my old pupilsor colleagues, and on the other to the valuable co-operation of the Colonial Governments of Indo-China, the French Settlements in India, and Martinique, I soon received poisonous snakes and dried venom in abundance.
Fig. 93.—Vaccinating a Horse against Venom at the Pasteur Institute at Lille.
Fig. 93.—Vaccinating a Horse against Venom at the Pasteur Institute at Lille.
After this I was not long in pushing the vaccination of a few horses until I made them resist, in a single injection, 2grammesof dry cobra-venom, a dose abouteighty times lethal; for I was able to satisfy myself that about 0·025 gramme of cobra-venom was sufficient to kill fresh horses in from twelve to twenty-four hours.
The immunisation of horses to this very high degree of tolerance of venom is not obtained without difficulties; many animals succumbin course of treatment from endocarditis or acute nephritis; in the case of others, each injection of venom leads to the formation of enormous aseptic abscesses, which have to be opened and drained. It may be said that on an average an interval ofsixteen monthsis necessary in order to obtain a serum sufficiently antitoxic.
Fig. 94.—Aseptically Bleeding a Horse Vaccinated against Venom in order to obtain Antivenomous Serum at the Pasteur Institute at Lille.
Fig. 94.—Aseptically Bleeding a Horse Vaccinated against Venom in order to obtain Antivenomous Serum at the Pasteur Institute at Lille.
When a horse is well vaccinated and tolerates without a reaction 2grammesof dry cobra-venom in a single subcutaneous injection, it may be bled on three consecutive occasions in the space of ten days, and in this way 20 litres of blood may be drawn from it (fig. 94).
The bleeding is arranged in the following manner:Twelve daysafter the last injection of venom the horse is bled for the first time to the extent of 8 litres; five days later it is bled for the second time to the extent of 6 litres; five days later still the third bleeding takes place, when 6 litres are again withdrawn.
The animal is then allowed to rest for three months and supplied with strengthening food, and during this period 2grammesof venom are again injected on two occasions at the end of a month, followed, a month and a half later, by the injection of 2 moregrammes. The antitoxic power of the serum is thus maintained approximately at the same standard.
The serum drawn off at each bleeding must be severely tested, which is done by gauging its antitoxic powerin vitro, when mixed with venom, and also its preventive effect.
An antivenomous serum may be considered to be utilisable when a mixture of 1 c.c. of serum with 0·001 gramme of cobra-venom produces no intoxicating effect in the rabbit, and when a preventive subcutaneous injection of 2 c.c. of serum into a rabbit of about 2 kilogrammes enables it to resist, two hours later, subcutaneous inoculation with 1 milligramme of venom.
Thepreventive powermay be very quickly tested by injecting a rabbit,in the marginal vein of the right earfor example, with 2 c.c. of serum, and injecting,five minutes afterwards,in the marginal vein of the left ear, 8 milligramme of venom. This dose of 1 milligramme generally kills the control rabbits in less than thirty minutes when introduced into the veins, and in from two to three hours when injected beneath the skin.
This rapid proof byintravenous injectionis extremely striking and demonstrative; it can be effected in public during a class or lecture in less than an hour, and enables an immediate estimate to be formed of the value of an antivenomous serum. When it is intended to adopt this method, it is essential to make use of a recent solution of venom, for solutions from a week to a fortnight old, although sterile, have already lost a large portion oftheir toxicity, and, if these be employed, the dose of venom calculated to kill the control animals in thirty minutes, for example, takes an hour or more to do so.
I always prepare my test solutions of venom in the following manner:—
Ten milligrammes of dry cobra-venom are weighed in a delicate balance. The venom is dissolved in 10 c.c. of 0·8 per cent. physiological salt solution, which takes a few minutes. When the venom is thoroughly dissolved it is transferred to a test-tube, which is immersed for three-quarters of an hour in a water-bath heated to + 72° C. In this way the non-toxic albumins are coagulated without modifying the neurotoxic substance. The solution is poured on to a filter of sterilised paper, and the clear liquid which is collected is immediately put up in glass phials, which are hermetically sealed, or in small sterilised bottles. Its toxicity is tested upon control animals, and it may be kept for five or six days if protected from light, or for several weeks in a refrigerator at about 0° C.
One-tenth of this solution corresponds exactly to 1 milligramme of dry venom.
As for the antivenomous serum, as soon as its antitoxic value has been ascertained by the methods that I have just described, and it has been separated from clots and red corpuscles by suitable decantation, it is portioned out, with the usual aseptic precautions, into small sterilised bottles of 10 c.c. capacity, without the addition of any antiseptic.
In order to ensure that it will keep for a long time, care is then taken to heat the hermetically sealed bottles in a water-bath at a temperature of 58° C. for one hour, and this operation is repeated for three days in succession.
Serum prepared in this way preserves its antitoxic power unimpaired for about two years,in all climates. I have had occasion at various times to receive bottles which had been sent eighteen months and two years previously to India and Indo-China,and I was able to show that their standard had not perceptibly deteriorated. It was only the appearance of the contained liquid that was slightly changed; it was discoloured, and when shaken small white flakes were seen floating through it. These flakes are not a sign of deterioration; they are composed of deposits of precipitated albumin. They can be partly dissolved again by violent shaking, or they may be separated before use by filtration through sterilised paper.
In a dry state, antivenomous serum may be kept for an almost indefinite period, in hermetically sealed glass tubes. In this condition it is usually divided into doses of 1 gramme, and when it is desired to make use of it, it is sufficient to dissolve a dose in 10 c.c. of water which has been boiled and allowed to cool, which takes two or three minutes. This solution is then injected beneath the skin, as though it were liquid serum.
The Pasteur Institute at Lille prepares in this way large quantities of antivenomous serum, which are sent all over the world to those countries in which poisonous snakes are most dangerous.
Recently, special laboratories for the production of this preparation have been instituted at Bombay and at Kasauli, in the Punjab, by Drs. G. Lamb and Semple; at Philadelphia, by Professor McFarland; at São-Paulo, in Brazil, by Dr. Vital Brazil; and at Sydney, by Dr. Tidswell.
Specificity and Polyvalence of Antivenomous Serums.—By means of a large number of experiments I have proved that snake-venoms, whatever their origin, contain two principal substances:neurotoxin, which exerts its effects upon the elements of the nervous system, andhæmorrhagin(Flexner and Noguchi), orproteolytic diastase, the effects of which remain exclusively local when the venom is introduced subcutaneously into the cellular tissue, but which produces coagulation of the blood when the venom is injected directly into the blood stream.
The venom ofColubridæin general is characterised by the constant predominence ofneurotoxin, to which it owes its extreme toxicity, which is especially intense in the case of cobra-venom. It contains no, or scarcely anyhæmorrhagin; for this reason the local symptoms of poisoning byColubrinevenom are almostnil. Thisneurotoxin, as we have seen, shows itself very highly resistant to heat.
The venom ofViperidæ, on the contrary, especially that ofLachesis, is characterised by the almost total absence ofneurotoxin, while its richness inhæmorrhaginis considerable. Consequently, heating for a few minutes at + 75° C. renders it almost entirely inactive, sincehæmorrhaginis very sensitive to heat.
Given venom of some kind or other, the origin of which is unknown, it is therefore possible to ascertain whether the snake from which it was extracted belonged to theColubridæorViperidæ, by determining its richness inneurotoxinresistant to heating at + 85° C.
CertainViperinevenoms, such as those of the EuropeanVipera berusandVipera aspis, the AfricanCerastesand AmericanCrotaluscontain at the same time a small proportion—varying greatly in amount according to the species—ofneurotoxin, and a much larger proportion ofhæmorrhagin. It is for this reason that these venoms, although greatly attenuated and deprived of their local action by heating, still remain toxic when injected in large doses into animals after having been heated to + 75° C.
On the other hand, someColubrinevenoms, such as those ofBungarus cæruleus, which are very rich inneurotoxin, contain a quantity of hæmorrhagin sufficient to differentiate their effects in appearance from those produced by cobra-venom, when they are injected, not beneath the skin, but directly into the veins. In this case their effects upon the blood are added to those of their neurotoxin.
It would seem, too, that the venoms of AustralianColubridæ(Hoplocephalus,Pseudechis) form a special group, which is richer inhæmorrhaginthan are those of theColubridæof the Old World.99
On studying, in the case of these various venoms, the actionin vitroandin vivoof a purelyantineurotoxicantivenomous serum, such as, for example, that of an animal vaccinated against cobra-venom heated to + 75° C., it is found that this serum has a very decided effect upon cobra-venom, and likewise upon that of snakes belonging to allied species (Naja bungarus,Naja haje), and that its action upon the other venoms is less in proportion as they contain lessneurotoxin. It prevents hæmolysisin vitro, and suppresses the effects of intoxication on the nervous system, but does not modify in any way the phenomena of coagulation or of proteolysis.
If this serum be made to actin vitroon thoseViperinevenoms that, when heated to + 75° C. and deprived of their hæmorrhagin, remain neurotoxic, like the venom of the common viper, it is found that it renders them entirely innocuous. Therefore, in the case of all species of poisonous snakes, and perhaps also in that of other poisonous animals (such as scorpions), it appears that theneurotoxicsubstance isone and the same, and always neutralisable by anantineurotoxicserum like that of animals vaccinated against cobra-venom.
Neurotoxinbeing the essentially active substance in venoms, and that to which the dangerous properties of poisonous snakes, as regards man and domestic animals, are especially due, it is the effects of this that it is most necessary to prevent. Consequently,the first quality that an antivenomous serum ought to exhibit, in order to be capable of being used in the therapeutics of poisoning, is the possession of anantineurotoxicpower as high as possible. This antineurotoxic power is easily obtained by employing cobra-venom for the fundamental immunisation of the horses destined for the production of the serum.
Antineurotoxicserum thus prepared shows itself perfectly capable of preventing all effects of intoxication from cobra-bites, which are much the most frequent in India. In the same way it shows itself quite sufficiently efficacious with regard toColubrineandViperinevenoms, the neurotoxic activity of which may cause death. But it does not possess any preventive action upon the local effects ofhæmorrhagin, to which the noxiousness of certainViperinevenoms—such as those ofLachesis—are almost exclusively due.
In countries in whichViperidæare very common, we must therefore not confine ourselves to vaccinating the animals that produce serum solely against theneurotoxinof cobra-venom, for instance; we must prepare these animals, after having immunised them to cobra-venom, by injecting them with progressively increasing doses of the various venoms derived from the snakes that are most frequently met with in the district.
Nothing, moreover, is easier than to train animals vaccinated against cobra-venom to tolerate strong doses of the venoms ofLachesis,Vipera russellii,Crotalus,Hoplocephalus, orPseudechis. In a few months we succeed in obtaining serums very active against these different venoms.
Utilising the horse as producer of antitoxin, I have prepared by this methodpolyvalentserums capable of preventing the local action ofViperinevenoms, and of suppressingin vitrotheir coagulant and proteolytic effects upon the blood.
Unfortunately, great as has been the kindness of the many persons who have most obligingly given me their assistance in the course of the fifteen years during which I have studied this question,I have found it impossible to procure sufficient quantities of venoms of various origins to furnish each country with the polyvalent serums corresponding to its particular needs. I have therefore been obliged to confine myself to preparing for the most partantineurotoxins, which I have been able to do, thanks to the abundant provision ofCobra- andBungarus-venoms, for which I am indebted to the liberality of the Government of the French Settlements in India, and to that of my pupils and friends who are at the present time in charge of the Colonial Laboratories of Indo-China. Moreover, the recent foundation of the Serum-Therapic Institutes of Bombay and Kasauli, Sydney, São-Paulo, and Philadelphia, to-day renders it very easy for each country to provide itself with antivenomous serum, either specific or polyvalent. Other institutes will doubtless be established for the purpose of extending the benefits of a method, the efficacy of which is sufficiently evident for its adoption to be incumbent upon all those who are concerned with safeguarding human existence.
It is difficult, in the present state of our knowledge on the subject of toxins and antitoxins, to determine the precise nature of the reactions that are produced in the living organism as the result of serum injected for the purpose of preventing the toxic action of venom.
I maintained, some years ago,100that the phenomenon in this case was a purely physiological one, which I considered to be proved by the fact that, if we mixin vitro, in determinate proportions, venom and antivenomous serum, and if we heat this mixture at 68° C. for half an hour, the injection of the heated mixture kills animals as if they were inoculated with venom alone, although with a considerable retardation. I concluded from this that, in all probability, antitoxic serum does not modify the toxin with which it is mixed, but that it confines itself to displaying a parallel and opposite action by preventing the noxious effects. I therefore supposed that no chemical combination is produced between these two substances, or, at least, that the combination effected is very unstable.
My experiments were subsequently repeated by Martin and Cherry,101who showed that the results as stated above were perfectly correct when the mixture of venom and antitoxin was heated less than ten minutes after it had been made, but that, if the heatingdid not take place until twenty or thirty minutes later, the toxicity of the venom no longer reappeared.
On the other hand, the admirable researches of Kyes and Sachs, and subsequently those of Morgenroth, pursued under the direction of Ehrlich at the Laboratory of Experimental Therapy at Frankfort, have proved the readiness of venom to enter into chemical combination with certain elements of normal serums, in particular withlecithin, a combination which results in the formation ofhæmolysingand non-toxiclecithides, theneurotoxinbeing left free.
It therefore seemed impossible to deny the existence of a chemical reaction between the venom and the serum, which was until quite recently considered as proved. We shall see presently that this is not the case. But let us first endeavour to determine the laws that govern the neutralisation of variable quantities of venom by antivenomous serum.
If, in a series of test-tubes, we bring the same quantity of cobra-venom (e.g., 0·00005 gramme, a dose which is invariably lethal to the mouse in two hours) into contact with progressively increasing quantities of an antivenomous serum (e.g., 0·01 c.c., 0·02 c.c., &c., up to 0·1 c.c.), and, after thirty minutes of contact, inject these different mixtures subcutaneously into a series of mice, we find that all those that have received the mixtures containing less than 0·05 c.c. of serum succumb after variable intervals, while all the rest survive. It is evident that, under these conditions, the serum experimented upon has shown itself capable of neutralisingin vitro, in a dose of 0·05 c.c., 5 centimilligrammes of venom.
The same serum should therefore neutralise 1 milligramme of venom in a dose of 1 c.c., that is to say, that this mixture injected into a mouse ought to be entirely innocuous. Experiments show, however, that in reality it is necessary to mix 1·2 c.c. of serum with 1 milligramme of venom in order that the inoculated mouse may not succumb.
This proves that, in the initial mixture of 0·00005 gramme of venom + 0·05 c.c. of serum, there remained an exceedingly smallquantity of non-neutralised venom, and that this quantity of venom in a free state was insufficient to cause the death of the animal, or even any apparent malaise. When multiplied by twenty, however, it becomes capable of producing toxic effects; it is for this reason that, when it is desired to inoculate a mouse with twenty times the lethal dose of 0·00005 grammeneutralised, it is necessary to mix with this twenty times lethal dose a dose of seruma little largerthan twenty times that which renders 0·00005 gramme of venom innocuous to the mouse, that is to say, 1·2 c.c.
If, instead of making use of themouseas test animal, we employ therabbit, it is found that the same serum, in a dose of 0·75 c.c., neutralises 0·001 gramme of venom sufficiently for the mixture to be innocuous when inoculated. It is clear that, in this mixture, the whole of the venom was not neutralised by the serum, but the small quantity left free is incapable of producing harmful effects.
By this method of employing mixtures of the same dose of venom with variable quantities of antivenomous serum, we are therefore enabled to determine with the greatest exactness the antitoxic powerin vitroof each specimen of serum. But it must not be forgotten thatthe result obtained applies only to the species of animal into which the mixtures were injected.
I have already stated (Chapter VIII.) that a fairly close parallelism exists between theneurotoxicaction of venoms and theirhæmolyticaction, and I have established that, in order that the sensitive red blood-corpuscles may be dissolved under the influence of venom, it is indispensable that the reaction take place in the presence of normal serum, since venoms have no effect upon red corpuscles freed from serum by several successive washings and centrifugings.
Preston Kyes has explained this phenomenon very well by showing that the venom combines with the lecithins in the serum, or with those contained in the stroma of the corpuscle, so as to constitute a hæmolysinglecithide.
The knowledge of this fact enables us to determine, by meansof a very neat and simple method, and with a sufficient degree of accuracy for practical purposes, the antitoxic power of an antivenomous serum by measuring itsantihæmolyticpower.102
To this end it is sufficient to cause variable doses of serum to act on a given quantity of defibrinated horse- or rat-blood, to which a constant dose of venom is then added. We employ, for example, a 5 per cent. dilution of defibrinated horse-blood, which is portioned out in doses of 1 c.c. into a series of test-tubes. To each of these tubes in succession is added a progressively increasing quantity of the serum for titration, starting with 0·01 c.c., and continuing with 0·02 c.c., 0·03 c.c., &c., up to 0·1 c.c. A control tube receives no serum. There are then introduced into all the tubes 1 decimilligramme of venom and 0·2 c.c. of normal horse-serum, deprived of alexin by previous heating for half an hour at 58° C. At a temperature of about 16° C. hæmolysis commences to manifest itself in the control tube in from fifteen to twenty minutes. It takes place in the other tubes with a retardation which varies with the dose of serum added. Tubes are to be noticed in which it does not occur even after the lapse of a couple of hours.
Experience shows that we may consider as good for therapeutic use serums which, in a dose of 0·05 c.c., completely prevent hæmolysis by 1 decimilligramme ofColubrinevenom, such as that of Cobra, Krait, &c., and those that in a dose of 0·7 c.c., prevent hæmolysis by 1 milligramme of the venom ofLachesisorVipera berus.
By a method calculated upon the foregoing, it is likewise possible to measure theantihæmorrhagicactivity of an antivenomous serum, for the parallelism existing between theantineurotoxicandantihæmolyticactions of serums occurs again, as I have been able to establish in conjunction with Noc, between theantihæmorrhagicandantiproteolyticaction of the same serums.
Now, theantiproteolyticaction is easily determined by means of a series of test-tubes containing the same quantity of 20 per cent. gelatinisedbouillon, rendered imputrescible by the addition of a small quantity of thymol. The gelatine being kept liquid in the incubating stove, a progressively increasing quantity of serum is poured into each tube. The same dose of venom, say 1 milligramme, is then added in each case. The tubes are placed in the stove for six hours at 36° C. They are then withdrawn and immersed in a bath of cold water. Those in which the gelatine solidifies are noted, and thus we establish the dose of antivenomous serum that inhibits the proteolysis of this substance.
These different methods of control enable us to verify the activity of antivenomous serums with great exactness, without the necessity of having recourse to experiments upon animals.
In a very important memoir on the reconstitution of the toxins from a mixture oftoxin+antitoxin, J. Morgenroth103has shown that the venom, after being naturalised by the antivenomous serum, can be dissociated from its combination by means of a method which consists in adding to the latter a small quantity of hydrochloric acid.
Previous experiments by Kyes had established:—
(1) That antivenomous serum, the antitoxic action of which is so manifest when it is mixedin vitrowith cobra-venom, remains entirely inert when brought into contact with the combinationlecithin+venom, that is to say, withcobra-lecithide.
(2) That the addition of lecithin to a neutral combination ofvenom+antivenomousserum does not set the venom free again, and that under these conditions nolecithideis formed.
If, in a neutral mixture ofcobra-hæmolysinandantitoxinwe could succeed in dissociating the two constituent elements, and in then making thecobra-hæmolysincombine with thelecithin,we should have a toxin and antitoxin side by side; for the reasons indicated above, this toxin (lecithide) and antitoxin (antivenomousserum) would be no longer capable of combining; but the toxin (lecithide), thanks to its hæmolytic properties, could easily be demonstrated.
It is precisely this desideratum that J. Morgenroth has succeeded in realising, by means of hydrochloric acid, which renders it possible to dissociate the neutral mixture,toxin+antitoxin, into its constituent elements, and then to obtain alecithide.
Experiments show that the quantity of lecithide thus restored absolutely corresponds to that of the cobra-hæmolysin originally added to the antitoxin, and that the antitoxin set free is not injured by the hydrochloric acid, even after twenty-four hours of contact. It is sufficient to add the quantity of soda or of ammonia necessary for the neutralisation of the acid, in order to see the antitoxin reappear in its original strength.
It is therefore possible, by causing hydrochloric acid (in a solution not stronger than 3 per cent.) to act on a neutral mixture of cobra-hæmolysin (toxin) and antitoxin, to set the former at liberty in the form oflecithide, to withdraw the latter from the action of the antitoxin, and to demonstrate its presence, owing to its hæmolytic properties.
It has been found by Kyes and Sachs that, under the influence of hydrochloric acid, cobra-hæmolysin becomes resistant to heat to such an extent that it is not destroyed even by prolonged heating at 100° C.
If to a neutral mixture of toxin + antitoxin we add a small quantity of hydrochloric acid, and then heat the mixture at 100° C., the antitoxin being in this case destroyed, we shall recover the whole of the toxin originally employed.
Therefore, as was shown by me so long ago as 1894, if the mixture of toxin + antitoxin produces a chemical combination between the two substances, this combination is unstable, and can be effectively broken up into these two constituent elements by various influences.
In all countries the remedies recommended for the bites of poisonous snakes are innumerable, and native pharmacopœias abound in so-called infallible recipes.
Pliny himself wrote on this subject as follows:—
“For poisonous bites, it is customary to employ a liniment made of fresh sheep-droppings, cooked in wine. Rats cut in two are also applied; these animals possess important properties, especially at the epoch of the ascension of the stars, seeing that the number of a rat’s fibres wax and wane with the moon.
“Of all birds, those that afford most assistance against snakes are vultures. The black ones are the weaker. The odour of their feathers when burnt puts snakes to flight. Provided with a vulture’s heart one need not fear encounters with snakes, and can also defy the wrath of wild beasts, robbers, and princes.
“Cock’s flesh, applied while still warm, neutralises the venom of snakes. The brains of the bird, swallowed in wine, produce the same effect. The Parthians, for this purpose, make use of chicken’s brains. The fresh flesh of the pigeon and the swallow, and owls’ feet burned, are good against snake-bites.
“If one has been bitten by a snake or by any venomous animal, another method of cure is to take salt fish and wine from time to time, so as to vomit in the evening. This remedy is chieflyefficacious against the bite of theChalcis,Cerastes,Seps,ElapsandDipsas.”
In Equatorial America, and especially in India, a multitude of plants are credited with marvellous properties, which they possess only in the imagination of the snake-charmers or medicine-men by whom they are employed. None of them stand the test of experiment, any more than the more or less compound drugs, numbers of specimens of which from all sources have passed through my hands.
It cannot, however, be denied that certain chemical substances, of well-defined composition, are very useful, not as physiological antidotes to venoms, but as agents for their modification or destruction in the poisoned wounds, when they have not yet been absorbed. In this waypermanganate of potash,chromic acid,chloride of gold, and thealkaline hypochlorites, especiallyhypochlorite of lime, may be extremely useful under many circumstances.
Permanganate of potashwas recommended in 1881 by Professor de Lacerda,104of Rio de Janeiro, as the result of experiments made by him with venoms of Brazilian snakes. When a few cubic centimetres of a 1 per cent. solution of permanganate of potash are quickly injected into the actual wound caused by the bite and around the point of inoculation, there can be no doubt that the venom not yet absorbed is destroyed. When mixedin vitrowith venom, permanganate renders the latter innocuous.
Here, however, it is a case of actual destruction by direct contact. If we inject a lethal dose of venom into the right thigh of an animal, for example, and several cubic centimetres of permanganate solution into different parts of the body, or beneath the skin of the left thigh, neither the general intoxication nor the local effects of the venom are modified.
The same may be said with regard tochromic acid(1 per cent.solution), recommended by Kaufmann105for the bite of the common viper.
No other effect is produced by a 1 per cent. solution ofchloride of gold, or thealkaline hypochlorites, which I have shown to possess a strong oxidising action on the different venoms, even on those that are most rapidly diffusible, such as cobra-venom (see Chapter V.). They possess, however, owing to their slight causticity, the advantage of not producing severe local disorders, and in this respect they are to be preferred.
The chemical reagent most to be recommended ishypochlorite of lime, in a fresh solution of 2 grammes per cent., and containing about 90 c.c. of chlorine per 100 grammes. It immediately and surely destroys the venom by simple contact, and the chlorine gas that it gives off, owing to its great diffusibility, acts at a fairly long distance from the point of inoculation on the venom which is already beginning to be absorbed.
Professor Halford, of Melbourne, advises the direct injection into the patient’s veins of from 10 to 20 drops of ammonia, diluted with an equal quantity of distilled water. This is a means of reviving nervous excitability in certain subjects at the commencement of intoxication; but torpor soon reappears, and, if the dose of venom inoculated is sufficient to cause death, a fatal ending takes place notwithstanding. Experimentally the effects of ammonia arenil.
No better results are obtained by injections of strychnine, as recommended by Dr. Mueller, in Australia. Moreover, the statistics published by Raston Huxtable106positively condemn this therapeutic method. They show that, in 426 cases of snake-bite, out of 113 treated by strychnine 15 proved fatal, the ratio of mortality being 13·2 per cent., while the 313 cases not treated by strychnine only resulted in 13 deaths, or a mortality of 4·1 per cent.
In the case of animals intoxicated by venom, injections of strychnine, morphia, nicotine, or curare in small doses always prove ineffective; they even considerably assist the progress of the intoxication and hasten death. The use of these drugs in the case of human beings should therefore be absolutely forbidden.
It appears, on the other hand, that alcohol and coffee, or tea, absorbed by ingestion, are very often beneficial. Indeed, it was long ago observed that the swallowing of alcohol until symptoms of drunkenness appear retards or diminishes the phenomena of torpor and paresis that precede the ultimate phase of the intoxication. Its use may therefore be recommended when it is impossible to have recourse to the only treatment really specific that modern science places in our hands—antivenomous serum-therapy. It is important, however, to state that,when serum is used, alcohol must be forbidden. The latter hinders the effects of the former.
In practice, the rational treatment of the bite of a venomous snake must be directed towards:—
(1) Preventing the absorption of the venom.
(2) Neutralising, by the injection of a sufficient quantity of antitoxic serum, the effects of the venom already absorbed.
In order to prevent the absorption of the venom introduced into the wound, the first precaution to be taken is to compress the bitten limb by means of a ligature of some kind, such as a handkerchief, as close as possible to the bite, and between it and the base of the limb. The ligature must be tightly twisted, and, by compressing the tissues around the bite, an attempt should be made to squeeze out the venom that may have been introduced into them. The expulsion of the poison should be hastened, either by making an incision 2 or 3 cm. in length and 1 cm. in depth in the direction taken by the fangs of the reptile and also parallel to the axis of the bitten member, or by sucking the wound hard.
The ligature on the limb should not be applied for more than half an hour; if it were kept on longer it would interfere with the circulation to a dangerous degree, and would certainly injure thevitality of the tissues. The period in question also usually affords sufficient time for taking the patient to a place where help can be obtained, and for the preparation of everything necessary for his subsequent treatment.
The wound should then be freely washed with a fresh 2per cent.solution ofhypochlorite of lime, or with a 1 in 1,000 solution ofchloride of gold. In default of hypochlorite of lime or chloride of gold, eithereau de Javel, diluted with tepid water to a strength of 1 in 10, or a 1 per cent. solution of permanganate of potash, may be employed. These reagents should be made to penetrate as deeply as possible into the tissues, and a few cubic centimetres of them should even be injected with a Pravaz syringe into the punctures caused by the bite and all round them.
The wound being then covered with a damp dressing by means of compresses saturated with hypochlorite of lime, or at least with pure alcohol, the next thing to be done is to prepare to apply the serum-therapic treatment in order to arrest the general intoxication, if this has already commenced to take effect, or to prevent it from setting in.
For the employment of serum it is necessary to be in possession of a sterilisable syringe of the capacity of 10 c.c., similar to those used in the treatment of diphtheria.
If the life of the patient be not immediately in danger, care should first be taken to have the syringe boiled, or at least to rinse it out with boiling water, making sure that the piston fits tightly, and that the syringe itself is in good working order.
Should a syringe of 10 c.c. not be available, any kind of Pravaz syringe, previously washed out with boiling water, may be employed, but in this case the use of so small an instrument renders it necessary to give several painful injections.
The entire contents of a bottle of serum (10 c.c. of liquid serum, or 1 gramme of dry serum dissolved in 10 c.c. of boiled water) should be injected into the subcutaneous areolar tissue of the abdomen, on the right or left side. There is no advantage inmaking the injection at the actual spot bitten; the serum is best and most rapidly absorbed when injected into the loose tissues of the abdominal wall (fig. 95).